Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

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Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

The Epithelial-Mesenchymal Transition Factor SNAIL Paradoxically Enhances Reprogramming

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Juli J. Unternaehrer

2014-11-01

Full Text Available Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs entails a mesenchymal to epithelial transition (MET. While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD of the epithelial-to-mesenchymal transition (EMT factor SNAI1 (SNAIL paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency.

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.

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Kamerkar, Sushrut; LeBleu, Valerie S; Sugimoto, Hikaru; Yang, Sujuan; Ruivo, Carolina F; Melo, Sonia A; Lee, J Jack; Kalluri, Raghu

2017-06-22

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic Kras G12D , a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

NF-κB activation impairs somatic cell reprogramming in ageing.

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Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

2015-08-01

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Context-dependent interpretation of the prognostic value of BRAF and KRAS mutations in colorectal cancer

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Popovici, Vlad; Budinska, Eva; Bosman, Fred T; Tejpar, Sabine; Roth, Arnaud D; Delorenzi, Mauro

2013-01-01

The mutation status of the BRAF and KRAS genes has been proposed as prognostic biomarker in colorectal cancer. Of them, only the BRAF V600E mutation has been validated independently as prognostic for overall survival and survival after relapse, while the prognostic value of KRAS mutation is still unclear. We investigated the prognostic value of BRAF and KRAS mutations in various contexts defined by stratifications of the patient population. We retrospectively analyzed a cohort of patients with stage II and III colorectal cancer from the PETACC-3 clinical trial (N = 1,423), by assessing the prognostic value of the BRAF and KRAS mutations in subpopulations defined by all possible combinations of the following clinico-pathological variables: T stage, N stage, tumor site, tumor grade and microsatellite instability status. In each such subpopulation, the prognostic value was assessed by log rank test for three endpoints: overall survival, relapse-free survival, and survival after relapse. The significance level was set to 0.01 for Bonferroni-adjusted p-values, and a second threshold for a trend towards statistical significance was set at 0.05 for unadjusted p-values. The significance of the interactions was tested by Wald test, with significance level of 0.05. In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of poor survival only in subpopulations involving microsatellite stable and left-sided tumors, with higher effects than in the whole population. There was no evidence for prognostic value in microsatellite instable or right-sided tumor groups. We found that BRAF was also prognostic for relapse-free survival in some subpopulations. We found no evidence that KRAS mutations had prognostic value, although a trend was observed in some stratifications. We also show evidence of heterogeneity in survival of patients with BRAF V600E mutation. The BRAF mutation represents an additional risk factor only in some subpopulations of colorectal cancers, in

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

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Fiorella Guadagni

2012-01-01

Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

Local epigenetic reprogramming induced by G-quadruplex ligands

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Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-11-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

Detection of EGFR and KRAS mutations in fine-needle aspirates stored on Whatman FTA cards: is this the tool for biobanking cytological samples in the molecular era?

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da Cunha Santos, Gilda; Liu, Ni; Tsao, Ming-Sound; Kamel-Reid, Suzanne; Chin, Kayu; Geddie, William R

2010-12-25

The aims of this study were to compare the quality of DNA recovered from fine-needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin-fixed paraffin-embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. Analyses were successful with all FNAs and lung cancer-derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer-derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

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Delun Huang

2018-01-01

Full Text Available The regulatory process of naïve-state induced pluripotent stem cell (iPSC generation is not well understood. Leukemia inhibitory factor (LIF-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3 is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs. However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs, and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

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Page Grier P

2009-04-01

Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

The phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-dependent Tup1 conversion (PIPTC) regulates metabolic reprogramming from glycolysis to gluconeogenesis.

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Han, Bong-Kwan; Emr, Scott D

2013-07-12

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans.

KRAS detection in colonic tumors by DNA extraction from FTA paper: the molecular touch-prep.

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Petras, Melissa L; Lefferts, Joel A; Ward, Brian P; Suriawinata, Arief A; Tsongalis, Gregory J

2011-12-01

DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is usually more degraded and contains more polymerase chain reaction (PCR) inhibitors than DNA isolated from nonfixed tissue. In addition, the tumor size and cellular heterogeneity found in tissue sections can often impact testing for molecular biomarkers. As a potential remedy to this situation, we evaluated the use of Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA for use in a real-time PCR-based KRAS mutation assay. Eleven colon tumor samples were collected by making a cut into the fresh tumor and applying the Whatman FTA paper to the cut surface. Matched FFPE tissue blocks from these tumors were also collected for comparison. KRAS mutation analysis was carried out using the Applied Biosystems 7500 Fast Real-time PCR System using 7 independent custom TaqMan PCR assays. Of the 11 colon tumors sampled, 6 were positive for KRAS mutations in both the Whatman FTA paper preparations and corresponding FFPE samples. Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA have many advantages including ease of use, intrinsic antimicrobial properties, long storage potential (stability of DNA over time), and a faster turnaround time for results. Extracted DNA should be suitable for most molecular diagnostic assays that use PCR techniques. This novel means of DNA preservation from surgical specimens would benefit from additional study and validation as a dependable and practical technique to preserve specimens for molecular testing.

Safety and efficacy of the addition of simvastatin to panitumumab in previously treated KRAS mutant metastatic colorectal cancer patients.

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Baas, Jara M; Krens, Lisanne L; Bos, Monique M; Portielje, Johanneke E A; Batman, Erdogan; van Wezel, Tom; Morreau, Hans; Guchelaar, Henk-Jan; Gelderblom, Hans

2015-09-01

Panitumumab has proven efficacy in patients with metastatic or locally advanced colorectal cancer patients, provided that they have no activating KRAS mutation in their tumour. Simvastatin blocks the mevalonate pathway and thereby interferes with the post-translational modification of KRAS. We hypothesize that the activity of the RAS-induced pathway in patients with a KRAS mutation might be inhibited by simvastatin. This would theoretically result in increased sensitivity to panitumumab, potentially comparable with tumours with wild-type KRAS. A Simon two-stage design single-arm, phase II study was designed to test the safety and efficacy of the addition of simvastatin to panitumumab in colorectal cancer patients with a KRAS mutation after failing fluoropyrimidine-based, oxaliplatin-based and irinotecan-based therapy. The primary endpoint of this study was the proportion of patients alive and free from progression 11 weeks after the first administration of panitumumab, aiming for at least 40%, which is comparable with, although slightly lower than, that in KRAS wild-type patients in this setting. If this 40% was reached, then the study would continue into the second step up to 46 patients. Explorative correlative analysis for mutations in the KRAS and related pathways was carried out. One of 14 patients was free from progression at the primary endpoint time. The median progression-free survival was 8.4 weeks and the median overall survival status was 19.6 weeks. We conclude that the concept of mutant KRAS phenotype expression modulation with simvastatin was not applicable in the clinic.

The Phosphatidylinositol 3,5-Bisphosphate (PI(3,5)P2)-dependent Tup1 Conversion (PIPTC) Regulates Metabolic Reprogramming from Glycolysis to Gluconeogenesis*

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Han, Bong-Kwan; Emr, Scott D.

2013-01-01

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans. PMID:23733183

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer

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Masayuki Hiraki

2015-01-01

Full Text Available KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR signaling may cause dysregulation of microRNA (miRNA and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS and v-akt murine thymoma viral oncogene homolog 1(AKT1, key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK and phosphoinositide 3-kinase (PI3K/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

Targeting the PI3K signaling pathway in KRAS mutant colon cancer

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Hong, Suntaek; Kim, SoYoung; Kim, Hye Youn; Kang, Myunghee; Jang, Ho Hee; Lee, Won-Suk

2015-01-01

Metastatic colorectal cancer (CRC) patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are resistant to monoclonal antibody that targets the epidermal growth factor receptor such as cetuximab. BKM120 targets phosphatidylinositide-3-kinase (PIK3CA), but it is unknown whether BKM120 can reverse cetuximab resistance in KRAS mutant CRC. Human CRC cell lines with KRAS mutations (DLD-1, HCT116, and LoVo) were used to test the effect of cetuximab, BKM120, and cetuximab plus BKM120 on cell proliferation in vitro and in vivo. BKM120 reduced cell proliferation in a concentration-dependent manner in the LoVo (PI3KCA wild type) as well as the HCT116 and DLD1 cells (that carry a PI3KCA mutation). BKM120 only inhibited ERK phosphorylation in LoVo cells (PIK3CA wild type), but not in DLD1 or HCT116 cells at a concentration of 1 μmol/L. Treatment with cetuximab and BKM120 significantly reduced the growth of xenograft tumors originating from KRAS mutant cells compared with cetuximab alone (P = 0.034). BKM120 may overcome cetuximab resistance in colon cancer cells with KRAS mutation

Targeting Lipid Metabolic Reprogramming as Anticancer Therapeutics

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Cha, Ji-Young; Lee, Ho-Jae

2016-01-01

Cancer cells rewire their metabolism to satisfy the demands of growth and survival, and this metabolic reprogramming has been recognized as an emerging hallmark of cancer. Lipid metabolism is pivotal in cellular process that converts nutrients into energy, building blocks for membrane biogenesis and the generation of signaling molecules. Accumulating evidence suggests that cancer cells show alterations in different aspects of lipid metabolism. The changes in lipid metabolism of cancer cells c...

Combination of siRNA-directed Kras oncogene silencing and arsenic-induced apoptosis using a nanomedicine strategy for the effective treatment of pancreatic cancer.

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Zeng, Linjuan; Li, Jingguo; Wang, Yong; Qian, Chenchen; Chen, Yinting; Zhang, Qiubo; Wu, Wei; Lin, Zhong; Liang, Jianzhong; Shuai, Xintao; Huang, Kaihong

2014-02-01

The synergetic inhibitory effects on human pancreatic cancer by nanoparticle-mediated siRNA and arsenic therapy were investigated both in vitro and in vivo. Poly(ethylene glycol)-block-poly(L-lysine) were prepared to form siRNA-complexed polyplex and poly(ethylene glycol)-block-poly(DL-lactide) were prepared to form arsenic-encapsulated vesicle, respectively. Down-regulation of the mutant Kras gene by siRNA caused defective abilities of proliferation, clonal formation, migration, and invasion of pancreatic cancer cells, as well as cell cycle arrest at the G0/G1 phase, which substantially enhanced the apoptosis-inducing effect of arsenic administration. Consequently, co-administration of the two nanomedicines encapsulating siRNA or arsenic showed ideal tumor growth inhibition both in vitro and in vivo as a result of synergistic effect of the siRNA-directed Kras oncogene silencing and arsenic-induced cell apoptosis. These results suggest that the combination of mutant Kras gene silencing and arsenic therapy using nanoparticle-mediated delivery strategy is promising for pancreatic cancer treatment. Treatment of pancreatic cancer remains a major challenge. These authors demonstrate a method that combines a siRNA-based Kras silencing with arsenic delivery to pancreatic cancer cells using nanoparticles, resulting in enhanced apoptosis induction in the treated cells. © 2013.

Beyond KRAS mutation status: influence of KRAS copy number status and microRNAs on clinical outcome to cetuximab in metastatic colorectal cancer patients

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Mekenkamp Leonie JM

2012-07-01

Full Text Available Abstract Background KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR antibodies in metastatic colorectal cancer (mCRC. Novel predictive markers are required to further improve the selection of patients for this treatment. We assessed the influence of modification of KRAS by gene copy number aberration (CNA and microRNAs (miRNAs in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. Methods Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n = 17 or poor (n = 17 progression-free survival (PFS upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. Results Copy number loss of the KRAS locus was observed in the tumour of 5 patients who were all good responders including patients with a KRAS mutation. Copy number gains in two wild-type KRAS tumours were associated with a poor PFS. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good PFS. In wild-type KRAS patients, miRNA expression did not correlate with PFS in a multivariate model. Conclusions Our results indicate that the assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of mCRC eligible for anti-EGFR therapy.

High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

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Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

2015-01-01

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

PKM2-dependent metabolic reprogramming in CD4+ T cells is crucial for hyperhomocysteinemia-accelerated atherosclerosis.

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Lü, Silin; Deng, Jiacheng; Liu, Huiying; Liu, Bo; Yang, Juan; Miao, Yutong; Li, Jing; Wang, Nan; Jiang, Changtao; Xu, Qingbo; Wang, Xian; Feng, Juan

2018-06-01

Inflammation mediated by activated T cells plays an important role in the initiation and progression of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis in ApoE -/- mice. Homocysteine (Hcy) activates T cells to secrete proinflammatory cytokines, especially interferon (IFN)-γ; however, the precise mechanisms remain unclear. Metabolic reprogramming is critical for T cell inflammatory activation and effector functions. Our previous study demonstrated that Hcy regulates T cell mitochondrial reprogramming by enhancing endoplasmic reticulum (ER)-mitochondria coupling. In this study, we further explored the important role of glycolysis-mediated metabolic reprogramming in Hcy-activated CD4 + T cells. Mechanistically, Hcy-activated CD4 + T cell increased the protein expression and activity of pyruvate kinase muscle isozyme 2 (PKM2), the final rate-limiting enzyme in glycolysis, via the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin signaling pathway. Knockdown of PKM2 by small interfering RNA reduced Hcy-induced CD4 + T cell IFN-γ secretion. Furthermore, we generated T cell-specific PKM2 knockout mice by crossing LckCre transgenic mice with PKM2 fl/fl mice and observed that Hcy-induced glycolysis and oxidative phosphorylation were both diminished in PKM2-deficient CD4 + T cells with reduced glucose and lipid metabolites, and subsequently reduced IFN-γ secretion. T cell-depleted apolipoprotein E-deficient (ApoE -/- ) mice adoptively transferred with PKM2-deficient CD4 + T cells, compared to mice transferred with control cells, showed significantly decreased HHcy-accelerated early atherosclerotic lesion formation. In conclusion, this work indicates that the PKM2-dependent glycolytic-lipogenic axis, a novel mechanism of metabolic regulation, is crucial for HHcy-induced CD4 + T cell activation to accelerate early atherosclerosis in ApoE -/- mice. Metabolic reprogramming is crucial for Hcy-induced CD4 + T cell inflammatory activation. Hcy activates

Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

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Wang, Xiao-Song; Shankar, Sunita; Dhanasekaran, Saravana M.; Ateeq, Bushra; Sasaki, Atsuo T.; Jing, Xiaojun; Robinson, Daniel; Cao, Qi; Prensner, John R.; Yocum, Anastasia K.; Wang, Rui; Fries, Daniel F.; Han, Bo; Asangani, Irfan A.; Cao, Xuhong; Li, Yong; Omenn, Gilbert S.; Pflueger, Dorothee; Gopalan, Anuradha; Reuter, Victor E.; Kahoud, Emily Rose; Cantley, Lewis C.; Rubin, Mark A.; Palanisamy, Nallasivam; Varambally, Sooryanarayana; Chinnaiyan, Arul M.

2011-01-01

Using an integrative genomics approach called Amplification Breakpoint Ranking and Assembly (ABRA) analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, specific knock-down of which, attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 MAPK pathways. This is the first report of a gene fusion involving Ras family suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. PMID:22140652

Fast-ball sports experts depend on an inhibitory strategy to reprogram their movement timing.

Science.gov (United States)

Nakamoto, Hiroki; Ikudome, Sachi; Yotani, Kengo; Maruyama, Atsuo; Mori, Shiro

2013-07-01

The purpose of our study was to clarify whether an inhibitory strategy is used for reprogramming of movement timing by experts in fast-ball sports when they correct their movement timing due to unexpected environmental changes. We evaluated the influence of disruption of inhibitory function of the right inferior frontal gyrus (rIFG) on reprogramming of movement timing of experts and non-experts in fast-ball sports. The task was to manually press a button to coincide with the arrival of a moving target. The target moved at a constant velocity, and its velocity was suddenly either increased or decreased in some trials. The task was performed either with or without transcranial magnetic stimulation (TMS), which was delivered to the region of the rIFG. Under velocity change conditions without TMS, the experts showed significantly smaller timing errors and a higher rate of reprogramming of movement timing than the non-experts. Moreover, TMS application during the task significantly diminished the expert group's performance, but not the control group, particularly in the condition where the target velocity decreases. These results suggest that experts use an inhibitory strategy for reprogramming of movement timing. In addition, the rIFG inhibitory function contributes to the superior movement correction of experts in fast-ball sports.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

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Yanagihara Kazuyoshi

2009-06-01

Full Text Available Abstract Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS, which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20 of gastric cancer cell lines (8–18-fold amplification and 4.7% (4/86 of primary gastric tumors (8–50-fold amplification. KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

International Nuclear Information System (INIS)

Mita, Hiroaki; Yanagihara, Kazuyoshi; Fujita, Masahiro; Hosokawa, Masao; Kusano, Masanobu; Sabau, Sorin Vasile; Tatsumi, Haruyuki; Imai, Kohzoh; Shinomura, Yasuhisa; Tokino, Takashi; Toyota, Minoru; Aoki, Fumio; Akashi, Hirofumi; Maruyama, Reo; Sasaki, Yasushi; Suzuki, Hiromu; Idogawa, Masashi; Kashima, Lisa

2009-01-01

Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

OpenAIRE

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler Jr, Richard E.; Lalani, Alshad S.; Dent, Paul

2017-01-01

ABSTRACT The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFR...

EGFR and KRAS mutation coexistence in lung adenocarcinomas

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Vitor Manuel Leitão de Sousa

2015-04-01

Full Text Available Lung cancer is one of the most common causes of cancer deaths. The development of EGFR targeted therapies, including monoclonal antibodies and tyrosine kinase inhibitors have generated an interest in the molecular characterization of these tumours. KRAS mutations are associated with resistance to EGFR TKIs. EGFR and KRAS mutations have been considered as mutually exclusive. This paper presents three bronchial-pulmonary carcinomas, two adenocarcinomas and one pleomorphic sarcomatoid carcinoma, harboring EGFR and KRAS mutations. Case 1 corresponded to an adenocarcinoma with EGFR exon 21 mutation (L858R and KRAS codon 12 point mutation (G12V; case 2, a  mucinous adenocarcinoma expressed coexistence of EGFR exon 21 mutation (L858R and KRAS codon 12 point mutation (G12V; and case 3 a sarcomatoid carcinoma with EGFR exon 19 deletion – del 9bp and KRAS codon 12 point mutation (G12C - cysteine. Based on our experience and on the literature, we conclude that EGFR and KRAS mutations can indeed coexist in the same bronchial-pulmonary carcinoma, either in the same histological type or in different patterns. The biological implications of this coexistence are still poorly understood mainly because these cases are not frequent or currently searched. It is therefore necessary to study larger series of cases with the two mutations to better understand the biological, clinical and therapeutic implications.

A new scintillation proximity assay-based approach for the detection of KRAS mutations

Energy Technology Data Exchange (ETDEWEB)

Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

2016-04-01

KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

A new scintillation proximity assay-based approach for the detection of KRAS mutations

International Nuclear Information System (INIS)

Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee

2016-01-01

KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125 I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

Mutations of the KRAS oncogene in endometrial hyperplasia and carcinoma.

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Wiesława Niklińska

2009-05-01

Full Text Available The aim of this study was to examine the prevalence and clinicopathological significance of KRAS point mutation in endometrial hyperplasia and carcinoma. We analysed KRAS in 11 cases of complex atypical hyperplasia and in 49 endometrial carcinomas using polymerase chain reaction associated with restriction fragment length polymorphism (PCR-RFPL. Point mutations at codon 12 of KRAS oncogene were identified in 7 of 49 (14,3% tumor specimens and in 2 of 11 (18,2% hyperplasias. No correlation was found between KRAS gene mutation and age at onset, histology, grade of differentiation and clinical stage. We conclude that KRAS mutation is a relatively common event in endometrial carcinogenesis, but with no prognostic value.

KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells.

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Yu-Lin Lin

Full Text Available Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC, but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V by small interfering RNAs (siRNA and overexpressed in KRAS-wild-type CRC cells (COLO320DM by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR. In KRAS-wild-type CRC cells (COLO320DM, KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1 downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V, KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.

Discovery and progress of direct cardiac reprogramming.

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Kojima, Hidenori; Ieda, Masaki

2017-06-01

Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate

OpenAIRE

Ward, Patrick S.; Thompson, Craig B.

2012-01-01

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria, but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-ass...

Myocardial KRAS(G12D) expression does not cause cardiomyopathy in mice.

Science.gov (United States)

Dalin, Martin G; Zou, Zhiyuan; Scharin-Täng, Margareta; Safari, Roghaiyeh; Karlsson, Christin; Bergo, Martin O

2014-02-01

Germ-line mutations in genes encoding components of the RAS/mitogen-activated protein kinase (MAPK) pathway cause developmental disorders called RASopathies. Hypertrophic cardiomyopathy (HCM) is the most common myocardial pathology and a leading cause of death in RASopathy patients. KRAS mutations are found in Noonan and cardio-facio-cutaneous syndromes. KRAS mutations, unlike mutations of RAF1 and HRAS, are rarely associated with HCM. This has been attributed to the fact that germ-line KRAS mutations cause only a moderate up-regulation of the MAPK pathway. Highly bioactive KRAS mutations have been hypothesized to cause severe cardiomyopathy incompatible with life. The aim of this study was to define the impact of KRAS(G12D) expression in the heart. To generate mice with endogenous cardiomyocyte-specific KRAS(G12D) expression (cKRAS(G12D) mice), we bred mice with a Cre-inducible allele expressing KRAS(G12D) from its endogenous promoter (Kras2(LSL)) to mice expressing Cre under control of the cardiomyocyte-specific α-myosin heavy chain promoter (αMHC-Cre). cKRAS(G12D) mice showed high levels of myocardial ERK and AKT signalling. However, surprisingly, cKRAS(G12D) mice were born in Mendelian ratios, appeared healthy, and had normal function, size, and histology of the heart. Mice with cardiomyocyte-specific KRAS(G12D) expression do not develop heart pathology. These results challenge the view that the level of MAPK activation correlates with the severity of HCM in RASopathies and suggests that MAPK-independent strategies may be of interest in the development of new treatments for these syndromes.

Nicotine promotes initiation and progression of KRAS-induced pancreatic cancer via Gata6-dependent dedifferentiation of acinar cells in mice.

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Hermann, Patrick C; Sancho, Patricia; Cañamero, Marta; Martinelli, Paola; Madriles, Francesc; Michl, Patrick; Gress, Thomas; de Pascual, Ricardo; Gandia, Luis; Guerra, Carmen; Barbacid, Mariano; Wagner, Martin; Vieira, Catarina R; Aicher, Alexandra; Real, Francisco X; Sainz, Bruno; Heeschen, Christopher

2014-11-01

Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting

Higher prevalence of KRAS mutations in colorectal cancer in Saudi ...

African Journals Online (AJOL)

We studied retrospectively tumor samples of 83 Saudi metastatic CRC patients for KRAS mutations in codon 12 and codon 13, to evaluate the relevance of KRAS mutation positive colorectal cancers with metastatic sites. KRAS mutation was observed in 42.2% (35/83) patients with CRC. The most common mutations were in ...

KRAS mutation: should we test for it, and does it matter?

Science.gov (United States)

Roberts, Patrick J; Stinchcombe, Thomas E

2013-03-10

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. Previously, lung cancer was simplistically divided into non-small-cell lung cancer (NSCLC) and small-cell lung cancer. However, in the last decade, we have gone from a simplistic binary system of classifying lung cancer to defining NSCLC on the basis of molecular subsets. KRAS mutations represent the most common molecular change in NSCLC. The presence of KRAS mutation has been shown to be associated with a poor prognosis in NSCLC, but this is of little clinical utility. More important is determining the clinical utility of KRAS mutational analysis for predicting benefit of chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), anti-EGFR monoclonal antibodies, or other novel therapeutics. Current data does not support the routine use of KRAS mutational analysis for predicting chemotherapy benefit. Additionally, there was significant interest in using KRAS status to select patients for EGFR TKI and anti-EGFR monoclonal antibodies. However, the EGFR mutational status has demonstrated significant predictive value in the selection of patients for EGFR TKI therapy and is now the preferred test. An association between KRAS mutational status and benefit of anti-EGFR monoclonal antibodies has not been demonstrated in NSCLC. Here we review, in the context of NSCLC, the underlying biology of KRAS mutations, the predictive value of KRAS mutations for therapeutic intervention, and the integration of KRAS mutational testing into our current clinical paradigms.

The cellular memory disc of reprogrammed cells.

Science.gov (United States)

Anjamrooz, Seyed Hadi

2013-04-01

The crucial facts underlying the low efficiency of cellular reprogramming are poorly understood. Cellular reprogramming occurs in nuclear transfer, induced pluripotent stem cell (iPSC) formation, cell fusion, and lineage-switching experiments. Despite these advances, there are three fundamental problems to be addressed: (1) the majority of cells cannot be reprogrammed, (2) the efficiency of reprogramming cells is usually low, and (3) the reprogrammed cells developed from a patient's own cells activate immune responses. These shortcomings present major obstacles for using reprogramming approaches in customised cell therapy. In this Perspective, the author synthesises past and present observations in the field of cellular reprogramming to propose a theoretical picture of the cellular memory disc. The current hypothesis is that all cells undergo an endogenous and exogenous holographic memorisation such that parts of the cellular memory dramatically decrease the efficiency of reprogramming cells, act like a barrier against reprogramming in the majority of cells, and activate immune responses. Accordingly, the focus of this review is mainly to describe the cellular memory disc (CMD). Based on the present theory, cellular memory includes three parts: a reprogramming-resistance memory (RRM), a switch-promoting memory (SPM) and a culture-induced memory (CIM). The cellular memory arises genetically, epigenetically and non-genetically and affects cellular behaviours. [corrected].

Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells

Science.gov (United States)

Bennett Saidu, Nathaniel Edward; Bretagne, Marie; Mansuet, Audrey Lupo; Just, Pierre-Alexandre; Leroy, Karen; Cerles, Olivier; Chouzenoux, Sandrine; Nicco, Carole; Damotte, Diane; Alifano, Marco; Borghese, Bruno; Goldwasser, François; Batteux, Frédéric; Alexandre, Jérôme

2018-01-01

KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF. PMID:29507676

Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells.

Science.gov (United States)

Bennett Saidu, Nathaniel Edward; Bretagne, Marie; Mansuet, Audrey Lupo; Just, Pierre-Alexandre; Leroy, Karen; Cerles, Olivier; Chouzenoux, Sandrine; Nicco, Carole; Damotte, Diane; Alifano, Marco; Borghese, Bruno; Goldwasser, François; Batteux, Frédéric; Alexandre, Jérôme

2018-02-06

KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF.

Nuclear Reprogramming in Mouse Primordial Germ Cells: Epigenetic Contribution

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Massimo De Felici

2011-01-01

Full Text Available The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occur in vivo under still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly defined in vitro culture conditions, when PGCs are transformed into embryonic germ (EG cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.

Reprogramming tumor-infiltrating dendritic cells for CD103+CD8+ mucosal T cell differentiation and breast cancer rejection

Science.gov (United States)

Wu, Te-Chia; Xu, Kangling; Banchereau, Romain; Marches, Florentina; Yu, Chun I; Martinek, Jan; Anguiano, Esperanza; Pedroza-Gonzalez, Alexander; Snipes, G. Jackson; O’Shaughnessy, Joyce; Nishimura, Stephen; Liu, Yong-Jun; Pascual, Virginia; Banchereau, Jacques; Oh, Sangkon; Palucka, Karolina

2014-01-01

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory T helper 2 (iTh2) cells and protumor inflammation. Here we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells, and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DC via the ligation of dectin-1, enabling the DC to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL12p70, and to favor the generation of T helper 1 (Th1) cells. DC activated via dectin-1, but not those activated with TLR-7/8 ligand or poly IC, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+CD8+ mucosal T cells accumulate in the tumors thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+CD8+ mucosal T cells elicited by reprogrammed DC can reject established cancer. Thus, reprogramming tumor-infiltrating DC represents a new strategy for cancer rejection. PMID:24795361

Distinct Clinicopathological Patterns of Mismatch Repair Status in Colorectal Cancer Stratified by KRAS Mutations.

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Wenbin Li

Full Text Available In sporadic colorectal cancer (CRC, the BRAFV600E mutation is associated with deficient mismatch repair (MMR status and inversely associated with to KRAS mutations. In contrast to deficient MMR (dMMR CRC, data on the presence of KRAS oncogenic mutations in proficient MMR (pMMR CRC and their relationship with tumor progression are scarce. We therefore examined the MMR status in combination with KRAS mutations in 913 Chinese patients and correlated the findings obtained with clinical and pathological features. The MMR status was determined based on detection of MLH1, MSH2, MSH6 and PMS2 expression. KRAS mutation and dMMR status were detected in 36.9% and 7.5% of cases, respectively. Four subtypes were determined by MMR and KRAS mutation status: KRAS (+/pMMR (34.0%, KRAS (+/dMMR (2.9%, KRAS (-/pMMR (58.5% and KRAS (-/dMMR (4.6%. A higher percentage of pMMR tumors with KRAS mutation were most likely to be female (49.0%, proximal located (45.5%, a mucinous histology (38.4%, and to have increased lymph node metastasis (60.3%, compared with pMMR tumors without BRAFV600E and KRAS mutations (36.0%, 29.3%, 29.4% and 50.7%, respectively; all P < 0.01. To the contrary, compared with those with KRAS(-/dMMR tumors, patients with KRAS(+/dMMR tumors demonstrated no statistically significant differences in gender, tumor location, pT depth of invasion, lymph node metastasis, pTNM stage, and histologic grade. This study revealed that specific epidemiologic and clinicopathologic characteristics are associated with MMR status stratified by KRAS mutation. Knowledge of MMR and KRAS mutation status may enhance molecular pathologic staging of CRC patients and metastatic progression in CRC can be estimated based on the combination of these biomarkers.

Reprogramming cells with synthetic proteins

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Xiaoxiao Yang

2015-06-01

Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

Reprogramming cells with synthetic proteins.

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Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

2015-01-01

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

Reprogramming cells with synthetic proteins

Science.gov (United States)

Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

2015-01-01

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

Boosters and barriers for direct cardiac reprogramming.

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Talkhabi, Mahmood; Zonooz, Elmira Rezaei; Baharvand, Hossein

2017-06-01

Heart disease is currently the most significant cause of morbidity and mortality worldwide, which accounts for approximately 33% of all deaths. Recently, a promising and alchemy-like strategy has been developed called direct cardiac reprogramming, which directly converts somatic cells such as fibroblasts to cardiac lineage cells such as cardiomyocytes (CMs), termed induced CMs or iCMs. The first in vitro cardiac reprogramming study, mediated by cardiac transcription factors (TFs)-Gata4, Tbx5 and Mef2C-, was not enough efficient to produce an adequate number of fully reprogrammed, functional iCMs. As a result, numerous combinations of cardiac TFs exist for direct cardiac reprogramming of mouse and human fibroblasts. However, the efficiency of direct cardiac reprogramming remains low. Recently, a number of cellular and molecular mechanisms have been identified to increase the efficiency of direct cardiac reprogramming and the quality of iCMs. For example, microgrooved substrate, cardiogenic growth factors [VEGF, FGF, BMP4 and Activin A], and an appropriate stoichiometry of TFs boost the direct cardiac reprogramming. On the other hand, serum, TGFβ signaling, activators of epithelial to mesenchymal transition, and some epigenetic factors (Bmi1 and Ezh2) are barriers for direct cardiac reprogramming. Manipulating these mechanisms by the application of boosters and removing barriers can increase the efficiency of direct cardiac reprogramming and possibly make iCMs reliable for cell-based therapy or other potential applications. In this review, we summarize the latest trends in cardiac TF- or miRNA-based direct cardiac reprogramming and comprehensively discuses all molecular and cellular boosters and barriers affecting direct cardiac reprogramming. Copyright © 2017 Elsevier Inc. All rights reserved.

Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

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Federico González

2013-03-01

Full Text Available Induced pluripotent stem cells (iPSCs hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs. Additional mechanistic studies uncover a direct role of the homologous recombination (HR pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

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Simona Lamba

2014-09-01

Full Text Available KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

Understanding the molecular mechanisms of reprogramming

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Krause, Marie N. [Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla 92037, CA (United States); University Hospital of Würzburg, Department of Pediatrics, 2 Josef-Schneiderstrasse, 97080 Würzburg (Germany); Sancho-Martinez, Ignacio [Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla 92037, CA (United States); Centre for Stem Cells and Regenerative Medicine, King' s College London, 28th Floor, Tower Wing, Guy' s Hospital, Great Maze Pond, London (United Kingdom); Izpisua Belmonte, Juan Carlos, E-mail: belmonte@salk.edu [Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla 92037, CA (United States)

2016-05-06

Despite the profound and rapid advancements in reprogramming technologies since the generation of the first induced pluripotent stem cells (iPSCs) in 2006[1], the molecular basics of the process and its implications are still not fully understood. Recent work has suggested that a subset of TFs, so called “Pioneer TFs”, play an important role during the stochastic phase of iPSC reprogramming [2–6]. Pioneer TFs activities differ from conventional transcription factors in their mechanism of action. They bind directly to condensed chromatin and elicit a series of chromatin remodeling events that lead to opening of the chromatin. Chromatin decondensation by pioneer factors progressively occurs during cell division and in turn exposes specific gene promoters in the DNA to which TFs can now directly bind to promoters that are readily accessible[2, 6]. Here, we will summarize recent advancements on our understanding of the molecular mechanisms underlying reprogramming to iPSC as well as the implications that pioneer Transcription Factor activities might play during different lineage conversion processes. - Highlights: • Pioneer transcription factor activity underlies the initial steps of iPSC generation. • Reprogramming can occur by cis- and/or trans- reprogramming events. • Cis-reprogramming implies remodeling of the chromatin for enabling TF accessibility. • Trans-reprogramming encompasses direct binding of Tfs to their target gene promoters.

Optimal ROS Signaling Is Critical for Nuclear Reprogramming

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Gang Zhou

2016-05-01

Full Text Available Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS signaling, we sought to determine whether there was a role of ROS signaling in nuclear reprogramming. We examined ROS production during the reprogramming of doxycycline (dox-inducible mouse embryonic fibroblasts (MEFs carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM] into induced pluripotent stem cells (iPSCs. ROS generation was substantially increased with the onset of reprogramming. Depletion of ROS via antioxidants or Nox inhibitors substantially decreased reprogramming efficiency. Similarly, both knockdown and knockout of p22phox—a critical subunit of the Nox (1–4 complex—decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, our data indicate that ROS signaling is activated early with nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency.

Reprogramming to pluripotency can conceal somatic cell chromosomal instability.

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Masakazu Hamada

Full Text Available The discovery that somatic cells are reprogrammable to pluripotency by ectopic expression of a small subset of transcription factors has created great potential for the development of broadly applicable stem-cell-based therapies. One of the concerns regarding the safe use of induced pluripotent stem cells (iPSCs in therapeutic applications is loss of genomic integrity, a hallmark of various human conditions and diseases, including cancer. Structural chromosome defects such as short telomeres and double-strand breaks are known to limit reprogramming of somatic cells into iPSCs, but whether defects that cause whole-chromosome instability (W-CIN preclude reprogramming is unknown. Here we demonstrate, using aneuploidy-prone mouse embryonic fibroblasts (MEFs in which chromosome missegregation is driven by BubR1 or RanBP2 insufficiency, that W-CIN is not a barrier to reprogramming. Unexpectedly, the two W-CIN defects had contrasting effects on iPSC genomic integrity, with BubR1 hypomorphic MEFs almost exclusively yielding aneuploid iPSC clones and RanBP2 hypomorphic MEFs karyotypically normal iPSC clones. Moreover, BubR1-insufficient iPSC clones were karyotypically unstable, whereas RanBP2-insufficient iPSC clones were rather stable. These findings suggest that aneuploid cells can be selected for or against during reprogramming depending on the W-CIN gene defect and present the novel concept that somatic cell W-CIN can be concealed in the pluripotent state. Thus, karyotypic analysis of somatic cells of origin in addition to iPSC lines is necessary for safe application of reprogramming technology.

Epigenetic reprogramming in the porcine germ line

DEFF Research Database (Denmark)

Matzen, Sara Maj Hyldig; Croxall, Nicola; Contreras, David A.

2011-01-01

BACKGROUND: Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next...... an increased proportion of cells in G2. CONCLUSIONS: Our study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic...... DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding...

FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

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Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

2014-01-01

Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

Coexistence of K-ras mutations and HPV infection in colon cancer

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Tezol Ayda

2006-05-01

Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

Totipotency, Pluripotency and Nuclear Reprogramming

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Mitalipov, Shoukhrat; Wolf, Don

Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

KRAS oncogene in lung cancer: focus on molecularly driven clinical trials

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Emmanuelle Kempf

2016-03-01

Full Text Available KRAS mutations are the most frequent molecular abnormalities found in one out of four nonsmall cell lung cancers (NSCLC. Their incidence increases in cases of adenocarcinoma, smokers and Caucasian patients. Their negative value in terms of prognosis and responsiveness to both standard chemotherapy and targeted therapies remains under debate. Many drugs have been developed specifically for KRAS-mutated NSCLC patients. Direct inhibition of RAS activation failed to show any clinical efficacy. Inhibition of downstream targets of the mitogen-activated protein kinase (MEK pathway is a promising strategy: phase II combinations of MEK 1/2 kinase inhibitors with chemotherapy doubled patients’ clinical outcomes. One phase III trial in such a setting is ongoing. Double inhibition of MEK and epidermal growth factor receptor proteins is currently being assessed in early-phase trials. The association with mammalian target of rapamycin pathway inhibition leads to non-manageable toxicity. Other strategies, such as inhibition of molecular heat-shock proteins 90 or focal adhesion kinase are currently assessed. Abemaciclib, a cyclin-dependent kinase 4/6 inhibitor, showed promising results in a phase I trial, with a 54% disease control rate. Results of an ongoing phase III trial are warranted. Immunotherapy might be the next relevant step in KRAS-mutated NSCLC management due to the high burden of associated mutations and neo-antigens.

Insights into significance of combined inhibition of MEK and m-TOR signalling output in KRAS mutant non-small-cell lung cancer.

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Broutin, Sophie; Stewart, Adam; Thavasu, Parames; Paci, Angelo; Bidart, Jean-Michel; Banerji, Udai

2016-08-23

We aimed to understand the dependence of MEK and m-TOR inhibition in EGFR(WT)/ALK(non-rearranged) NSCLC cell lines. In a panel of KRAS(M) and KRAS(WT) NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively. GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, PTOR compared with maximal m-TOR+MEK inhibition. However, GI caused by the combination was significantly greater in the KRAS(M) cell lines (79% vs 61%, P=0.017). m-TOR inhibition was more critical to GI than MEK inhibition in EGFR(WT)/ALK(non-rearranged) NSCLC cells. The combination of MEK and m-TOR inhibition was most effective in KRAS(M) cells.

Asymmetric Reprogramming Capacity of Parental Pronuclei in Mouse Zygotes

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Wenqiang Liu

2014-03-01

Full Text Available It has been demonstrated that reprogramming factors are sequestered in the pronuclei of zygotes after fertilization, because zygotes enucleated at the M phase instead of interphase of the first mitosis can support the development of cloned embryos. However, the contribution of the parental pronucleus derived from either the sperm or the oocyte in reprogramming remains elusive. Here, we demonstrate that the parental pronuclei have asymmetric reprogramming capacities and that the reprogramming factors reside predominantly in the male pronucleus. As a result, only female pronucleus-depleted (FPD mouse zygotes can reprogram somatic cells to a pluripotent state and support the full-term development of cloned embryos; male pronucleus-depleted (MPD zygotes fail to support somatic cell reprogramming. We further demonstrate that fusion of an additional male pronucleus into a zygote greatly enhances reprogramming efficiency. Our data provide a clue to further identify critical reprogramming factors in the male pronucleus.

Colorectal cancer patients with low abundance of KRAS mutation may benefit from EGFR antibody therapy.

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Shaorong Yu

Full Text Available Epidermal growth factor receptor monoclonal antibody was approved for treatment of metastatic colorectal cancer patients carrying KRAS wild type DNA. However, recent studies showed that patients with KRAS G13D mutation may benefit from EGFR antibody therapy. In this study we tried to explore whether the abundance of KRAS mutation could affect the efficacy of EGFR antibody therapy. We firstly established a PNA-PCR method which could calculate the percentage of KRAS mutation in total DNA and proved its ability on 47 colorectal cancer samples bearing KRAS mutations. Then we analyzed the correlation between the abundance of KRAS mutations and efficacy of EGFR antibody therapy in another 35 metastatic colorectal cancer patients. We proved that PNA-PCR assay could calculate the abundance of KRAS mutation and the percentage of mutant DNA in tumor cells varied a lot (10.8%∼98.3% on the 47 colorectal cancer patients. The efficacy of EGFR antibody correlated with the abundance of KRAS mutations: in the KRAS mutation less than 30% group, the disease control rate was 44.4% (4/9; the disease control rate of 30∼80% group was 5.6% (1/18 and the >80% group was 12.5% (1/8 (P = 0.038. In summary, our study showed that PNA-PCR method could easily detect the percentage of KRAS mutation in tumor cells and colorectal cancer patients with low abundance of KRAS mutation might benefit from EGFR antibody therapy.

Re: Epigenetics of Cellular Reprogramming

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Fehmi Narter

2016-12-01

Full Text Available EDITORIAL COMMENT Cells have some specific molecular and physiological properties that act their functional process. However, many cells have an ability of efficient transition from one type to another. This ability is named plasticity. This process occurs due to epigenetic reprogramming that involves changes in transcription and chromatin structure. Some changes during reprogramming that have been identified in recent years as genomic demethylation (both histone and DNA, histone acetylation and loss of heterochromatin during the development of many diseases such as infertility and cancer progression. In this review, the authors focused on the latest work addressing the mechanisms surrounding the epigenetic regulation of various types of reprogramming, including somatic cell nuclear transfer, cell fusion and transcription factor- and microRNA-induced pluripotency. There are many responsible factors such as genes, cytokines, proteins, co-factors (i.e. vitamin C in this local area network. The exact mechanisms by which these changes are achieved and the detailed interplay between the players responsible, however, remain relatively unclear. In the treatment of diseases, such as infertility, urooncology, reconstructive urology, etc., epigenetic changes and cellular reprogramming will be crucial in the near future. Central to achieving that goal is a more thorough understanding of the epigenetic state of fully reprogrammed cells. By the progress of researches on this topic, new treatment modalities will be identified for these diseases.

Lack of evidence for KRAS oncogenic mutations in triple-negative breast cancer

International Nuclear Information System (INIS)

Sánchez-Muñoz, Alfonso; Gallego, Elena; Luque, Vanessa de; Pérez-Rivas, Luís G; Vicioso, Luís; Ribelles, Nuria; Lozano, José; Alba, Emilio

2010-01-01

Mutational analysis of the KRAS gene has recently been established as a complementary in vitro diagnostic tool for the identification of patients with colorectal cancer who will not benefit from anti-epidermal growth factor receptor (EGFR) therapies. Assessment of the mutation status of KRAS might also be of potential relevance in other EGFR-overexpressing tumors, such as those occurring in breast cancer. Although KRAS is mutated in only a minor fraction of breast tumors (5%), about 60% of the basal-like subtype express EGFR and, therefore could be targeted by EGFR inhibitors. We aimed to study the mutation frequency of KRAS in that subtype of breast tumors to provide a molecular basis for the evaluation of anti-EGFR therapies. Total, genomic DNA was obtained from a group of 35 formalin-fixed paraffin-embedded, triple-negative breast tumor samples. Among these, 77.1% (27/35) were defined as basal-like by immunostaining specific for the established surrogate markers cytokeratin (CK) 5/6 and/or EGFR. KRAS mutational status was determined in the purified DNA samples by Real Time (RT)-PCR using primers specific for the detection of wild-type KRAS or the following seven oncogenic somatic mutations: Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val and Gly13Asp. We found no evidence of KRAS oncogenic mutations in all analyzed tumors. This study indicates that KRAS mutations are very infrequent in triple-negative breast tumors and that EGFR inhibitors may be of potential benefit in the treatment of basal-like breast tumors, which overexpress EGFR in about 60% of all cases

Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

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Malyshev, Igor; Malyshev, Yuri

2015-01-01

Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity. PMID:26366410

Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

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Pavithra eViswanath

2016-03-01

Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

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Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

Fluctuating levels of reprogramming factor expression in cultured ...

African Journals Online (AJOL)

Although human undifferentiated keratinocytes (HUKs) can be reprogrammed to become induced pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the endogenous expression of reprogramming factors in cultured HUKs is not clear at different stages. In this ...

Alterations in the K-ras and p53 genes in rat lung tumors

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Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E. [Inhalation Toxicology Research Institute, Albuquerque, NM (United States)] [and others

1997-06-01

Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

K-RAS and N-RAS mutations in testicular germ cell tumors

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Bekir Muhammet Hacioglu

2017-05-01

Full Text Available Testicular cancer is a relatively rare tumor type, accounting for approximately 1% of all cancers in men. However, among men aged between 15 and 40 years, testicular cancer is the most commonly diagnosed malignancy. Testicular germ cell tumors (TGCTs are classified as seminoma and non-seminoma. The RAS oncogene controls several cellular functions, including cell proliferation, apoptosis, migration, and differentiation. Thus, RAS signaling is important for normal germ cell development. Mutations of the Kirsten RAS (K-RAS gene are present in over 20% of all cancers. RAS gene mutations have also been reported in TGCTs. We investigated K-RAS and N-RAS mutations in seminoma and non-seminoma TGCT patients. A total of 24 (55% pure seminoma cases and 19 (45% non-seminoma cases were included in the study. K-RAS and N-RAS analyses were performed in our molecular pathology laboratory, using K-RAS and N-RAS Pyro Kit 24 V1 (Qiagen. In total, a RAS mutation was present in 12 patients (27%: 7 seminoma (29% and 5 non-seminoma cases (26% [p = 0.55]. A K-RAS mutation was present in 4 pure seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63], and an N-RAS mutation was observed in 4 seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63]. Both, K-RAS and N-RAS mutations were present in two patients: one with seminoma tumor and the other with non-seminoma tumor. To date, no approved targeted therapy is available for the treatment of TGCTs. The analysis of K-RAS and N-RAS mutations in these tumors may provide more treatment options, especially in platinum-resistant tumors.

c-Raf in KRas Mutant Cancers: A Moving Target.

Science.gov (United States)

McCormick, Frank

2018-02-12

Therapies for KRas cancers remain a major clinical need. In the current issue of Cancer Cell, Sanclemente and coworkers in Mariano Barbacid's group validate c-Raf as a prime target for these cancers. c-Raf ablation caused regression of advanced KRas G12V /Trp53 tumors, without obvious systemic toxicity and without affecting MAPK signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

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Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

The prevalence and prognostic significance of KRAS mutation subtypes in lung adenocarcinomas from Chinese populations

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Zheng DF

2016-02-01

Full Text Available Difan Zheng,1,2,* Rui Wang,1,2,* Yang Zhang,1,2 Yunjian Pan,1,2 Xinghua Cheng,3 Chao Cheng,1,2 Shanbo Zheng,1,2 Hang Li,1,2 Ranxia Gong,1,2 Yuan Li,2,4 Xuxia Shen,2,4 Yihua Sun,1,2 Haiquan Chen1–3,51Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, 2Department of Oncology, Shanghai Medical College, Fudan University, 3Shanghai Chest Hospital, Shanghai Jiao Tong University, 4Department of Pathology, Fudan University Shanghai Cancer Center, 5Institutes of Biomedical Sciences, Fudan University, Shanghai, People’s Republic of China*These authors contributed equally to this workBackground: We performed this retrospective study to identify the prevalence of KRAS mutation in Chinese populations and make a comprehensive investigation of the clinicopathological features of KRAS mutation in these patients.Patients and methods: Patients from 2007 to 2013 diagnosed with primary lung adenocarcinoma who received a radical resection were examined for KRAS, EGFR, HER2, BRAF mutations, and ALK, RET, and ROS1 fusions. Clinicopathological features, including sex, age, tumor–lymph node–metastasis stage, tumor differentiation, smoking status, histological subtypes, and survival information were analyzed.Result: KRAS mutation was detected in 113 of 1,368 patients. Nine different subtypes of KRAS mutation were identified in codon 12, codon 13, and codon 61. KRAS mutation was more frequently found in male patients and former/current smoker patients. Tumors with KRAS mutation had poorer differentiation. Invasive mucinous adenocarcinoma predominant and solid predominant subtypes were more frequent in KRAS mutant patients. No statistical significance was found in relapse-free survival or overall survival between patients with KRAS mutation and patients with other mutations.Conclusion: In Chinese populations, we identified KRAS mutation in 8.3% (113/1,368 of the patients with lung adenocarcinoma. KRAS mutation defines a molecular subset of

Deterministic direct reprogramming of somatic cells to pluripotency.

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Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

2013-10-03

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

Zfp296 is a novel, pluripotent-specific reprogramming factor.

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Gerrit Fischedick

Full Text Available Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs. However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs, indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296 led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell-specific conserved region 4 (CR4, and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells.

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

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Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

2010-11-01

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

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Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

2017-06-05

Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

KRAS Mutation and Epithelial-Macrophage Interplay in Pancreatic Neoplastic Transformation.

Science.gov (United States)

Bishehsari, Faraz; Zhang, Lijuan; Barlass, Usman; Preite, Nailliw; Turturro, Sanja; Najor, Matthew S; Shetuni, Brandon B; Zayas, Janet P; Mahdavinia, Mahboobeh; Abukhdeir, Abde M; Keshavarzian, Ali

2018-05-14

Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased i) ductal to acinar gene expression ratios, ii) epithelial cells proliferation, and iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a pro-tumorigenic phenotype in macrophages. Altered macrophages decreased epithelial Pigment Epithelial Derived Factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) as well as in our human PDA specimens. Epithelium-macrophage cross talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a pro-tumorigenic phenotype in macrophages, in turn augmenting neoplastic growth. This article is protected by copyright. All rights reserved. © 2018 UICC.

Marine Corps Budgetary Reprogramming Effectiveness

Science.gov (United States)

2015-03-01

infrastructure (Appropriations Act of Congress, 2008). The environmental restoration is a transfer account controlled by the DOD. Usually in the case of...at an average just over 11 percent and the Marine Corps encircle the backend of the DOD portion of reprogramming with the Marine Corps reprogramming...blue force tracker (BFT), radio systems, high mobility multipurpose wheeled vehicle (HMMWV), medium tactical vehicle replacement (MTVR), and

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

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Nicolas Piton

2015-01-01

Full Text Available KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27% and specificity (64% in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100% and specificity (100% in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

Science.gov (United States)

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

KRAS biomarker testing disparities in colorectal cancer patients in New Mexico

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Alissa Greenbaum

2017-11-01

Full Text Available Introduction: American Society of Clinical Oncology (ASCO guidelines recommend that all patients with metastatic colorectal cancer (mCRC receive KRAS testing to guide anti-EGFR monoclonal antibody treatment. The aim of this study was to assess for disparities in KRAS testing and mutational status. Methods: The New Mexico Tumor Registry (NMTR, a population-based cancer registry participating in the National Cancer Institute’s Surveillance, Epidemiology and End Results program, was queried to identify all incident cases of CRC diagnosed among New Mexico residents from 2010 to 2013. Results: Six hundred thirty-seven patients were diagnosed with mCRC from 2010–2013. As expected, KRAS testing in Stage 4 patients presented the highest frequency (38.4%, though testing in stage 3 (8.5%, stage 2 (3.4% and stage 1 (1.2% was also observed. In those with metastatic disease, younger patients (≤ 64 years were more likely to have had testing than patients 65 years and older (p < 0.0001. Patients residing in urban areas received KRAS testing more often than patients living in rural areas (p = 0.019. No significant racial/ethnic disparities were observed (p = 0.66. No significant differences were seen by year of testing. Conclusion: Age and geographic disparities exist in the rates of KRAS testing, while sex, race/ethnicity and the year tested were not significantly associated with testing. Further study is required to assess the reasons for these disparities and continued suboptimal adherence to current ASCO KRAS testing guidelines. Keywords: Oncology, Health sciences, Clinical genetics

Computational analysis of KRAS mutations: implications for different effects on the KRAS p.G12D and p.G13D mutations.

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Chih-Chieh Chen

Full Text Available BACKGROUND: The issue of whether patients diagnosed with metastatic colorectal cancer who harbor KRAS codon 13 mutations could benefit from the addition of anti-epidermal growth factor receptor therapy remains under debate. The aim of the current study was to perform computational analysis to investigate the structural implications of the underlying mutations caused by c.38G>A (p.G13D on protein conformation. METHODS: Molecular dynamics (MD simulations were performed to understand the plausible structural and dynamical implications caused by c.35G>A (p.G12D and c.38G>A (p.G13D. The potential of mean force (PMF simulations were carried out to determine the free energy profiles of the binding processes of GTP interacting with wild-type (WT KRAS and its mutants (MT. RESULTS: Using MD simulations, we observed that the root mean square deviation (RMSD increased as a function of time for the MT c.35G>A (p.G12D and MT c.38G>A (p.G13D when compared with the WT. We also observed that the GTP-binding pocket in the c.35G>A (p.G12D mutant is more open than that of the WT and the c.38G>A (p.G13D proteins. Intriguingly, the analysis of atomic fluctuations and free energy profiles revealed that the mutation of c.35G>A (p.G12D may induce additional fluctuations in the sensitive sites (P-loop, switch I and II regions. Such fluctuations may promote instability in these protein regions and hamper GTP binding. CONCLUSIONS: Taken together with the results obtained from MD and PMF simulations, the present findings implicate fluctuations at the sensitive sites (P-loop, switch I and II regions. Our findings revealed that KRAS mutations in codon 13 have similar behavior as KRAS WT. To gain a better insight into why patients with metastatic colorectal cancer (mCRC and the KRAS c.38G>A (p.G13D mutation appear to benefit from anti-EGFR therapy, the role of the KRAS c.38G>A (p.G13D mutation in mCRC needs to be further investigated.

Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

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Budniatzky, Inbar; Gepstein, Lior

2014-04-01

Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

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Yun, Jihye; Mullarky, Edouard; Lu, Changyuan; Bosch, Kaitlyn N; Kavalier, Adam; Rivera, Keith; Roper, Jatin; Chio, Iok In Christine; Giannopoulou, Eugenia G; Rago, Carlo; Muley, Ashlesha; Asara, John M; Paik, Jihye; Elemento, Olivier; Chen, Zhengming; Pappin, Darryl J; Dow, Lukas E; Papadopoulos, Nickolas; Gross, Steven S; Cantley, Lewis C

2015-12-11

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations. Copyright © 2015, American Association for the Advancement of Science.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis

DEFF Research Database (Denmark)

Tran, Phuoc T; Shroff, Emelyn H; Burns, Timothy F

2012-01-01

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer...

Detection of TET2, KRAS and CBL variants by Next Generation ...

African Journals Online (AJOL)

Dilara Fatma Akin

2015-10-01

Oct 1, 2015 ... sarcoma viral oncogene homolog (KRAS), and Casitas B-cell ... AML by screening hot-spot exons of TET2, KRAS, and CBL using Next Generation Sequencing ... Methods: Eight patients who were diagnosed with pediatric AML at Losante ..... mutations in pre-leukemic stem cells in acute myeloid leukemia.

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

International Nuclear Information System (INIS)

Plowman, Sarah J.; Arends, Mark J.; Brownstein, David G.; Luo Feijun; Devenney, Paul S.; Rose, Lorraine; Ritchie, Ann-Marie; Berry, Rachel L.; Harrison, David J.; Hooper, Martin L.; Patek, Charles E.

2006-01-01

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmΔ4A/tmΔ4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras -/- (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmΔ4A/tmΔ4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmΔ4A/tmΔ4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmΔ4A/tmΔ4A and K-ras -/- ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras -/- cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmΔ4A/tmΔ4A ES cells were more resistant to etoposide-induced apoptosis than K-ras -/- cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice

Reprogramming Cells for Brain Repair

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Randall D. McKinnon

2013-08-01

Full Text Available At present there are no clinical therapies that can repair traumatic brain injury, spinal cord injury or degenerative brain disease. While redundancy and rewiring of surviving circuits can recover some lost function, the brain and spinal column lack sufficient endogenous stem cells to replace lost neurons or their supporting glia. In contrast, pre-clinical studies have demonstrated that exogenous transplants can have remarkable efficacy for brain repair in animal models. Mesenchymal stromal cells (MSCs can provide paracrine factors that repair damage caused by ischemic injury, and oligodendrocyte progenitor cell (OPC grafts give dramatic functional recovery from spinal cord injury. These studies have progressed to clinical trials, including human embryonic stem cell (hESC-derived OPCs for spinal cord repair. However, ESC-derived allografts are less than optimal, and we need to identify a more appropriate donor graft population. The cell reprogramming field has developed the ability to trans-differentiate somatic cells into distinct cell types, a technology that has the potential to generate autologous neurons and glia which address the histocompatibility concerns of allografts and the tumorigenicity concerns of ESC-derived grafts. Further clarifying how cell reprogramming works may lead to more efficient direct reprogram approaches, and possibly in vivo reprogramming, in order to promote brain and spinal cord repair.

Performance and cost efficiency of KRAS mutation testing for metastatic colorectal cancer in routine diagnosis: the MOKAECM study, a nationwide experience.

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Hélène Blons

Full Text Available Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM was granted to analyze reproducibility and costs.96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists.This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0.The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment.

Performance and cost efficiency of KRAS mutation testing for metastatic colorectal cancer in routine diagnosis: the MOKAECM study, a nationwide experience.

Science.gov (United States)

Blons, Hélène; Rouleau, Etienne; Charrier, Nathanaël; Chatellier, Gilles; Côté, Jean-François; Pages, Jean-Christophe; de Fraipont, Florence; Boyer, Jean-Christophe; Merlio, Jean Philippe; Morel, Alain; Gorisse, Marie-Claude; de Cremoux, Patricia; Leroy, Karen; Milano, Gérard; Ouafik, L'houcine; Merlin, Jean-Louis; Le Corre, Delphine; Aucouturier, Pascaline; Sabourin, Jean-Christophe; Nowak, Frédérique; Frebourg, Thierry; Emile, Jean-François; Durand-Zaleski, Isabelle; Laurent-Puig, Pierre

2013-01-01

Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs. 96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists. This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0. The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment.

Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

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Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

2017-09-16

Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

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Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

2015-01-01

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

Personalized treatment for advanced colorectal cancer: KRAS and beyond

International Nuclear Information System (INIS)

Patel, Gargi Surendra; Karapetis, Christos S

2013-01-01

Targeted therapies have improved the survival of patients with advanced colorectal cancer (CRC). However, further improvements in patient outcomes may be gained by the development of predictive biomarkers in order to select individuals who are most likely to benefit from treatment, thus personalizing treatment. Using the epidermal growth-factor receptor (EGFR) pathway, we discuss the existing and potential predictive biomarkers in clinical development for use with EGFR-targeted agents in metastatic CRC. The data and technological issues surrounding such biomarkers as expression of EGFR or its family members or ligands, KRAS-, NRAS-, and BRAF-mutation status, PI3K/PTEN expression, and imaging and clinical biomarkers, such as rash and hypomagnesemia, are summarized. Although the discovery of KRAS mutations has improved patient selection for EGFR-targeted treatments, further biomarkers are required, especially for those patients who exhibit KRAS mutations rather than the wild-type gene

Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

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Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

2014-01-01

Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.

Heterogeneity of Osteosarcoma Cell Lines Led to Variable Responses in Reprogramming

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Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

2014-01-01

Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture. PMID:25170299

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

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Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

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Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2018-02-01

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Molecular analysis of p53 and K-ras in lung carcinomas of coal miners

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Sarkar, F.H.; Li, Y.W.; Vallyathan, V. [Wayne State University, Detroit, MI (United States). School of Medicine, Dept. of Pathology

2001-10-01

Thirty-three cases of non-small cell lung cancers (NSCLC) from the archives of National Coal Workers' Autopsy Study were studied for mutational alterations in p53 and K-ras using PCR-SSCP, DNA sequencing and PCR-oligonucleotide probe hybridization techniques. Mutations of the p53 were observed in 4 smokers (19%) and one in a never smoker (8%). Two polymorphisms in smokers were detected at codon 213, a common site for sequence variation. Among the smokers the p53 mutations were in the heavy smokers. In never smokers there was only a single p53 mutation and two K-ras mutations. In never smokers the frequency of K-ras mutations was similar (17%) in smokers, but one never smoker had two K-ras mutations. Mutations of p53 were more frequent in adenocarcinomas (27%) and they were AT-GC transitions. There were two large cell undifferentiated carcinomas with p53 mutation and one with a K-ras mutation. Two of the 16 squamous cell carcinomas were positive for p53 mutation, while no K-ras mutations were found in this group. The results of these preliminary studies indicate a moderately different mutational spectrum of p53 and K-ras in coal miners independent of cigarette smoking. The mutational spectrum observed in this study of coal miners with heavy cigarette smoking history suggest a protective effect of coal mine dust in preventing abnormal mutations induced by chemical carcinogens in cigarette smoke or reactive oxygen species.

Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

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Jang, Hyunbum; Abraham, Sherwin J; Chavan, Tanmay S; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I; Nussinov, Ruth; Gaponenko, Vadim

2015-04-10

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of a novel oncogenic K-ras mutation in colon cancer

International Nuclear Information System (INIS)

Akagi, Kiwamu; Uchibori, Ryosuke; Yamaguchi, Kensei; Kurosawa, Keiko; Tanaka, Yoichiro; Kozu, Tomoko

2007-01-01

Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity

Metabolic reprogramming: a cancer hallmark even warburg did not anticipate.

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Ward, Patrick S; Thompson, Craig B

2012-03-20

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

The value of KRAS mutation testing with CEA for the diagnosis of pancreatic mucinous cysts

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Kadayifci, Abdurrahman; Al-Haddad, Mohammad; Atar, Mustafa; Dewitt, John M.; Forcione, David G.; Sherman, Stuart; Casey, Brenna W.; Fernandez-del Castillo, Carlos; Schmidt, C. Max; Pitman, Martha B.; Brugge, William R.

2016-01-01

Background and aims: Pancreatic cyst fluid (PCF) CEA has been shown to be the most accurate preoperative test for detection of cystic mucinous neoplasms (CMNs). This study aimed to assess the added value of PCF KRAS mutational analysis to CEA for diagnosis of CMNs. Patients and methods: This is a retrospective study of prospectively collected endoscopic ultrasonography (EUS) fine-needle aspiration (FNA) data. KRAS mutation was determined by direct sequencing or equivalent methods. Cysts were classified histologically (surgical cohort) or by clinical (EUS or FNA) findings (clinical cohort). Performance characteristics of KRAS, CEA and their combination for detection of a cystic mucinous neoplasm (CMN) and malignancy were calculated. Results: The study cohort consisted of 943 patients: 147 in the surgical cohort and 796 in the clinical cohort. Overall, KRAS and CEA each had high specificity (100 % and 93.2 %), but low sensitivity (48.3 % and 56.3 %) for the diagnosis of a CMN. The positivity of KRAS or CEA increased the diagnostic accuracy (80.8 %) and AUC (0.84) significantly compared to KRAS (65.3 % and 0.74) or CEA (65.8 % and 0.74) alone, but only in the clinical cohort (P < 0.0001 for both). KRAS mutation was significantly more frequent in malignant CMNs compared to histologically confirmed non-malignant CMNs (73 % vs. 37 %, P = 0.001). The negative predictive value of KRAS mutation was 77.6 % in differentiating non-malignant cysts. Conclusions: The detection of a KRAS mutation in PCF is a highly specific test for mucinous cysts. It outperforms CEA for sensitivity in mucinous cyst diagnosis, but the data does not support its routine use. PMID:27092317

Individualized therapies in colorectal cancer: KRAS as a marker for response to EGFR-targeted therapy

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Li Kuiyuan

2009-04-01

Full Text Available Abstract Individualized therapies that are tailored to a patient's genetic composition will be of tremendous value for treatment of cancer. Recently, Kirsten ras (KRAS status has emerged as a predictor of response to epidermal growth factor receptor (EGFR targeted therapies. In this article, we will discuss targeted therapies for colorectal cancers (CRC based on EGFR signaling pathway and review published data about the potential usefulness of KRAS as a biological marker for response to these therapies. Results from relevant studies published since 2005 and unpublished results presented at national meetings were retrieved and summarized. These studies reflected response (or lack of response to EGFR-targeted therapies in patients with metastatic CRC as a function of KRAS status. It has become clear that patients with colorectal cancer whose tumor has an activating mutation in KRAS do not respond to monoclonal antibody therapies targeting EGFR. It should now become a standard practice that any patients being considered for EGFR targeted therapies have their tumors tested for KRAS status and only those with wild-type KRAS being offered such therapies.

Evaluation of K-ras and p53 expression in pancreatic adenocarcinoma using the cancer genome atlas.

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Liming Lu

Full Text Available Genetic alterations in K-ras and p53 are thought to be critical in pancreatic cancer development and progression. However, K-ras and p53 expression in pancreatic adenocarcinoma have not been systematically examined in The Cancer Genome Atlas (TCGA Data Portal. Information regarding K-ras and p53 alterations, mRNA expression data, and protein/protein phosphorylation abundance was retrieved from The Cancer Genome Atlas (TCGA databases, and analyses were performed by the cBioPortal for Cancer Genomics. The mutual exclusivity analysis showed that events in K-ras and p53 were likely to co-occur in pancreatic adenocarcinoma (Log odds ratio = 1.599, P = 0.006. The graphical summary of the mutations showed that there were hotspots for protein activation. In the network analysis, no solid association between K-ras and p53 was observed in pancreatic adenocarcinoma. In the survival analysis, neither K-ras nor p53 were associated with both survival events. As in the data mining study in the TCGA databases, our study provides a new perspective to understand the genetic features of K-ras and p53 in pancreatic adenocarcinoma.

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

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Barbara Ziegler

2011-11-01

Full Text Available This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip. Biochip hybridization identified 17 (21% samples to carry a KRAS mutation of which 16 (33% were adenocarcinomas and 1 (3% was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

A high-fat diet activates oncogenic Kras and COX2 to induce development of pancreatic ductal adenocarcinoma in mice.

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Philip, Bincy; Roland, Christina L; Daniluk, Jaroslaw; Liu, Yan; Chatterjee, Deyali; Gomez, Sobeyda B; Ji, Baoan; Huang, Haojie; Wang, Huamin; Fleming, Jason B; Logsdon, Craig D; Cruz-Monserrate, Zobeida

2013-12-01

Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and the accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice. We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1-Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated extracellular-regulated kinase, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs. Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs. In mice, an HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis and development of PanINs and PDAC. This

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

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Pathan AAK

2016-05-01

Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the �

Chemical compound-based direct reprogramming for future clinical applications

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Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

2018-01-01

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

KRAS (G12D Cooperates with AML1/ETO to Initiate a Mouse Model Mimicking Human Acute Myeloid Leukemia

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Shanmin Zhao

2014-01-01

Full Text Available Background/Aims: It has been demonstrated that KRAS mutations represent about 90% of cancer-associated mutations, and that KRAS mutations play an essential role in neoplastic transformation. Cancer-associated RAS mutations occur frequently in acute myeloid leukemia (AML, suggesting a functional role for Ras in leukemogenesis. Methods: We successfully established a mouse model of human leukemia by transplanting bone marrow cells co-transfected with the K-ras (G12D mutation and AML1/ETO fusion protein. Results: Mice transplanted with AML/ETO+KRAS co-transduced cells had the highest mortality rate than mice transplanted with AML/ETO- or KRAS-transduced cells (115d vs. 150d. Upon reaching a terminal disease stage, EGFP-positive cells dominated their spleen, lymph nodes, peripheral blood and central nervous system tissue. Immunophenotyping, cytologic analyses revealed that AML/ETO+KRAS leukemias predominantly contained immature myeloid precursors (EGFP+/c-Kit+/Mac-1-/Gr-1-. Histologic analyses revealed that massive leukemic infiltrations were closely packed in dense sheets that effaced the normal architecture of spleen and thymus in mice transplanted with AML1/ETO + KRAS co-transduced cells. K-ras mRNA and protein expression were upregulated in bone marrow cells of the K-ras group and AML1/ETO + Kras group. The phosphorylation of MEK/ERK was significantly enhanced in the AML1/ETO + Kras group. The similar results of the AML1/ETO + Nras group were consistent with those reported previously. Conclusion: Co-transduction of KrasG12D and AML1/ETO induces acute monoblastic leukemia. Since expression of mutant K-ras alone was insufficient to induce leukemia, this model may be useful for investigating the multi-step leukemogenesis model of human leukemia.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

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Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

2017-10-01

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

BAG3-dependent expression of Mcl-1 confers resistance of mutant KRAS colon cancer cells to the HSP90 inhibitor AUY922.

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Wang, Chun Yan; Guo, Su Tang; Croft, Amanda; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

2018-02-01

Past studies have shown that mutant KRAS colon cancer cells are susceptible to apoptosis induced by the HSP90 inhibitor AUY922. Nevertheless, intrinsic and acquired resistance remains an obstacle for the potential application of the inhibitor in the treatment of the disease. Here we report that Mcl-1 is important for survival of colon cancer cells in the presence of AUY922. Mcl-1 was upregulated in mutant KRAS colon cancer cells selected for resistance to AUY922-induced apoptosis. This was due to its increased stability mediated by Bcl-2-associated athanogene domain 3 (BAG3), which was also increased in resistant colon cancer cells by heat shock factor 1 (HSF1) as a result of chronic endoplasmic reticulum (ER) stress. Functional investigations demonstrated that inhibition of Mcl-1, BAG3, or HSF1 triggered apoptosis in resistant colon cancer cells, and rendered AUY922-naïve colon cancer cells more sensitive to the inhibitor. Together, these results identify that the HSF1-BAG3-Mcl-1 signal axis is critical for protection of mutant KRAS colon cancer cells from AUY922-induced apoptosis, with potential implications for targeting HSF1/BAG3/Mcl-1 to improve the efficacy of AUY922 in the treatment of colon cancer. © 2017 Wiley Periodicals, Inc.

KRAS exon 2 mutations influence activity of regorafenib in an SW48-based disease model of colorectal cancer.

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Camaj, Peter; Primo, Stefano; Wang, Yan; Heinemann, Volker; Zhao, Yue; Laubender, Ruediger Paul; Stintzing, Sebastian; Giessen-Jung, Clemens; Jung, Andreas; Gamba, Sebastian; Bruns, Christiane Josephine; Modest, Dominik Paul

2015-01-01

To investigate the impact of KRAS mutation variants on the activity of regorafenib in SW48 colorectal cancer cells. Activity of regorafenib was evaluated in isogenic SW48 KRAS wild-type (WT) and mutant cells. Subcutaneous xenografts (KRAS WT and G12C mutant variants) in NOD/SCID mice were analyzed to elucidate the effect of regorafenib treatment in vivo. Compared with KRAS WT cells, all mutant variants seemed associated with some degree of resistance to regorafenib-treatment in vitro. In vivo, activation of apoptosis (TUNEL) and reduction of proliferation (Ki67) after treatment with regorafenib were more pronounced in KRAS WT tumors as compared with G12C variants. In SW48 cells, exon 2 mutations of the KRAS gene may influence antitumor effects of regorafenib.

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

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Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

2015-01-01

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

The higher level of complexity of K-Ras4B activation at the membrane.

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Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2016-04-01

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. © FASEB.

Genome-wide transcriptional reprogramming under drought stress

KAUST Repository

Chen, Hao

2012-01-01

Soil water deficit is one of the major factors limiting plant productivity. Plants cope with this adverse environmental condition by coordinating the up- or downregulation of an array of stress responsive genes. Reprogramming the expression of these genes leads to rebalanced development and growth that are in concert with the reduced water availability and that ultimately confer enhanced stress tolerance. Currently, several techniques have been employed to monitor genome-wide transcriptional reprogramming under drought stress. The results from these high throughput studies indicate that drought stress-induced transcriptional reprogramming is dynamic, has temporal and spatial specificity, and is coupled with the circadian clock and phytohormone signaling pathways. © 2012 Springer-Verlag Berlin Heidelberg. All rights are reserved.

Loss of RASSF1A Expression in Colorectal Cancer and Its Association with K-ras Status

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Dan Cao

2013-01-01

Full Text Available Background. The RAS-association domain family 1 A (RASSF1A is a classical member of RAS effectors regulating cell proliferation and apoptosis. Loss of RASSF1A expression may shift the balance towards a growth-promoting effect without the necessity of activating K-ras mutations. Its potential association with K-ras mutations in colorectal cancer (CRC is unclear. Methods. RASSF1A expression was examined in normal mucosa, adenoma, and tumor tissues of colon and rectum, respectively. We examined the association of RASSF1A expression, mutations of K-ras, and EGFR status in 76 primary CRCs. The relationship between clinicopathological characteristics and RASSF1A expression was also analyzed. Results. RASSF1A expression level decreased progressively in normal mucosa, adenoma and, tumor tissues, and the loss of RASSF1A expression occurred more frequently in tumor tissues. Of 76 primary CRCs, loss of RASSF1A expression and/or K-ras mutations were detected in 77% cases. Loss of RASSF1A expression was more frequent in K-ras wild-type than in mutation cases (63% versus 32%, . Conclusions. Our study indicates that loss of RASSF1A may be involved in pathogenesis of CRC, its expression was found predominantly in K-ras wild-type CRCs, suggesting that it may be another way of affecting RAS signaling, in addition to K-ras mutations.

A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution

OpenAIRE

Grace R. Anderson; Peter S. Winter; Kevin H. Lin; Daniel P. Nussbaum; Merve Cakir; Elizabeth M. Stein; Ryan S. Soderquist; Lorin Crawford; Jim C. Leeds; Rachel Newcomb; Priya Stepp; Catherine Yip; Suzanne E. Wardell; Jennifer P. Tingley; Moiez Ali

2017-01-01

Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tiss...

The higher level of complexity of K-Ras4B activation at the membrane

Science.gov (United States)

Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S.; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2016-01-01

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5′-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.—Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. PMID:26718888

Linking incomplete reprogramming to the improved pluripotency of murine embryonal carcinoma cell-derived pluripotent stem cells.

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Gang Chang

Full Text Available Somatic cell nuclear transfer (SCNT has been proved capable of reprogramming various differentiated somatic cells into pluripotent stem cells. Recently, induced pluripotent stem cells (iPS have been successfully derived from mouse and human somatic cells by the over-expression of a combination of transcription factors. However, the molecular mechanisms underlying the reprogramming mediated by either the SCNT or iPS approach are poorly understood. Increasing evidence indicates that many tumor pathways play roles in the derivation of iPS cells. Embryonal carcinoma (EC cells have the characteristics of both stem cells and cancer cells and thus they might be the better candidates for elucidating the details of the reprogramming process. Although previous studies indicate that EC cells cannot be reprogrammed into real pluripotent stem cells, the reasons for this remain unclear. Here, nuclei from mouse EC cells (P19 were transplanted into enucleated oocytes and pluripotent stem cells (P19 NTES cells were subsequently established. Interestingly, P19 NTES cells prolonged the development of tetraploid aggregated embryos compared to EC cells alone. More importantly, we found that the expression recovery of the imprinted H19 gene was dependent on the methylation state in the differential methylation region (DMR. The induction of Nanog expression, however, was independent of the promoter region DNA methylation state in P19 NTES cells. A whole-genome transcriptome analysis further demonstrated that P19 NTES cells were indeed the intermediates between P19 cells and ES cells and many interesting genes were uncovered that may be responsible for the failed reprogramming of P19 cells. To our knowledge, for the first time, we linked incomplete reprogramming to the improved pluripotency of EC cell-derived pluripotent stem cells. The candidate genes we discovered may be useful not only for understanding the mechanisms of reprogramming, but also for deciphering the

Patient and tumor characteristics and BRAF and KRAS mutations in colon cancer, NCCTG/Alliance N0147.

Science.gov (United States)

Gonsalves, Wilson I; Mahoney, Michelle R; Sargent, Daniel J; Nelson, Garth D; Alberts, Steven R; Sinicrope, Frank A; Goldberg, Richard M; Limburg, Paul J; Thibodeau, Stephen N; Grothey, Axel; Hubbard, Joleen M; Chan, Emily; Nair, Suresh; Berenberg, Jeffrey L; McWilliams, Robert R

2014-07-01

KRAS and BRAF (V600E) mutations are important predictive and prognostic markers, respectively, in colon cancer, but little is known about patient and clinical factors associated with them. Two thousand three hundred twenty-six of 3397 patients in the N0147 phase III adjuvant trial for stage III colon cancer completed a patient questionnaire. Primary tumors were assessed for KRAS and BRAF (V600E) mutations and defective mismatch repair (dMMR) status. Logistic regression models and categorical data analysis were used to identify associations of patient and tumor characteristics with mutation status. All statistical tests were two-sided. KRAS (35%) and BRAF (V600E) (14%) mutations were nearly mutually exclusive. KRAS mutations were more likely to be present in patients without a family history of colon cancer and never smokers. Tumors with KRAS mutations were less likely to have dMMR (odds ratio [OR] = 0.21; 95% confidence interval [CI] = 0.15 to 0.31; P characteristics are associated with KRAS and BRAF (V600E) mutations. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Advances in reprogramming somatic cells to induced pluripotent stem cells.

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Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

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Drosten, Matthias; Simón-Carrasco, Lucía; Hernández-Porras, Isabel; Lechuga, Carmen G; Blasco, María T; Jacob, Harrys K C; Fabbiano, Salvatore; Potenza, Nicoletta; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

2017-02-01

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras G12V oncogene sequences. Germline expression of H-Ras G12V or K-Ras G12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras G12V elicited papillomas and hematopoietic tumors, K-Ras G12V induced lung tumors and gastric lesions. Pulmonary expression of H-Ras G12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras G12V but not K-Ras G12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras G12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR. ©2016 American Association for Cancer Research.

Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

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Perets, Ruth; Greenberg, Orli; Shentzer, Talia; Semenisty, Valeria; Epelbaum, Ron; Bick, Tova; Sarji, Shada; Ben-Izhak, Ofer; Sabo, Edmond; Hershkovitz, Dov

2018-05-01

Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers

Alteration of strain background and a high omega-6 fat diet induces earlier onset of pancreatic neoplasia in EL-Kras transgenic mice.

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Cheon, Eric C; Strouch, Matthew J; Barron, Morgan R; Ding, Yongzeng; Melstrom, Laleh G; Krantz, Seth B; Mullapudi, Bhargava; Adrian, Kevin; Rao, Sambasiva; Adrian, Thomas E; Bentrem, David J; Grippo, Paul J

2011-06-15

Diets containing omega-6 (ω-6) fat have been associated with increased tumor development in carcinogen-induced pancreatic cancer models. However, the effects of ω-6 fatty acids and background strain on the development of genetically-induced pancreatic neoplasia is unknown. We assessed the effects of a diet rich in ω-6 fat on the development of pancreatic neoplasia in elastase (EL)-Kras(G12D) (EL-Kras) mice in two different backgrounds. EL-Kras FVB mice were crossed to C57BL/6 (B6) mice to produce EL-Kras FVB6 F1 (or EL-Kras F1) and EL-Kras B6 congenic mice. Age-matched EL-Kras mice from each strain were compared to one another on a standard chow. Two cohorts of EL-Kras FVB and EL-Kras F1 mice were fed a 23% corn oil diet and compared to age-matched mice fed a standard chow. Pancreata were scored for incidence, frequency, and size of neoplastic lesions, and stained for the presence of mast cells to evaluate changes in the inflammatory milieu secondary to a high fat diet. EL-Kras F1 mice had increased incidence, frequency, and size of pancreatic neoplasia compared to EL-Kras FVB mice. The frequency and size of neoplastic lesions and the weight and pancreatic mast cell densities in EL-Kras F1 mice were increased in mice fed a high ω-6 fatty acid diet compared to mice fed a standard chow. We herein introduce the EL-Kras B6 mouse model which presents with increased frequency of pancreatic neoplasia compared to EL-Kras F1 mice. The phenotype in EL-Kras F1 and FVB mice is promoted by a diet rich in ω-6 fatty acid. Copyright © 2010 UICC.

Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

DEFF Research Database (Denmark)

Ardighieri, Laura; Zeppernick, Felix; Hannibal, Charlotte G

2014-01-01

There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced......), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST-implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST-implant pairs. Wild-type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11...... SBT/APST-implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST-implant (non-invasive and invasive) pairs (p identical KRAS or BRAF...

Galectin-3 mediates cross-talk between K-Ras and Let-7c tumor suppressor microRNA.

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Ran Levy

Full Text Available BACKGROUND: Galectin-3 (Gal-3 and active (GTP-bound K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60% of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50% of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1 induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.

Epigenetic reprogramming of breast cancer cells with oocyte extracts

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Kumari Rajendra

2011-01-01

Full Text Available Abstract Background Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis. Results We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts. Conclusions This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.

Detection and Analysis of EGFR and KRAS Mutations 
in the Patients with Lung Squamous Cell Carcinomas

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Hui ZHANG

2015-10-01

Full Text Available Background and objective Activating mutations in epidermal growth factor receptor (EGFR and KRAS are important markers in non-small cell lung cancer. However, EGFR and KRAS gene mutations in lung squamous cell carcinoma are rarely reported. The aim of this study was to analyze EGFR and KRAS gene mutation rate and their relationship with clinical features in patients with lung squamous cell carcinomas. Methods A total of 139 patients undergoing treatment for naïve lung squamous cell carcinomas with tumor tissue samples available for testing were recruited. EGFR and KRAS mutation statuses of the tumor samples were detected using a mutant enriched liquid chip. Results Of the 139 cases of lung squamous cell carcinoma, EGFR mutations were detected in 25 cases (18%, KRAS mutations were detected in 7 cases (5%, and the presence of both EGFR and KRAS mutations was detected in 1 case (0.7%. EGFR mutations occurred more often in females than in males (33.3% vs 16.5% and in patients that never smoked than in those who smoke (29.6% vs 16.1%. However, the difference did not reach statistical significance (P>0.05. No significant differences were observed in age, stage, and different biopsy type. KRAS mutations occurred more often in males than in females (5.5% vs 0%, but the difference did not reach statistical significance (P>0.05. No significant differences were observed in age, stage, different biopsy type, and smoking status (P>0.05. Conclusion EGFR and KRAS mutations were low in lung squamous cell carcinomas, and had no significant correlation with clinical features. Before using tyrosine kinase inhibitor targeted therapy, EGFR and KRAS mutations should be detected in patients with lung squamous cell carcinomas.

MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

KAUST Repository

Latrasse, David; Jé gu, Teddy; Li, Huchen; Zé licourt, Axel de; Raynaud, Cé cile; Legras, Sté phanie; Gust, Andrea; Samajova, Olga; Veluchamy, Alaguraj; Rayapuram, Naganand; Ramirez Prado, Juan Sebastian; Kulikova, Olga; Colcombet, Jean; Bigeard, Jean; Genot, Baptiste; Bisseling, Ton; Benhamed, Moussa; Hirt, Heribert

2017-01-01

Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

KAUST Repository

Latrasse, David

2017-07-06

Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

Science.gov (United States)

Bihl, Michel P; Hoeller, Sylvia; Andreozzi, Maria Carla; Foerster, Anja; Rufle, Alexander; Tornillo, Luigi; Terracciano, Luigi

2012-03-01

Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

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Kristensen Lasse

2012-11-01

Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

DNA replication is an integral part of the mouse oocyte's reprogramming machinery.

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Bingyuan Wang

Full Text Available Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1 and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

Glis family proteins are differentially implicated in the cellular reprogramming of human somatic cells.

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Lee, Seo-Young; Noh, Hye Bin; Kim, Hyeong-Taek; Lee, Kang-In; Hwang, Dong-Youn

2017-09-29

The ground-breaking discovery of the reprogramming of somatic cells into pluripotent cells, termed induced pluripotent stem cells (iPSCs), was accomplished by delivering 4 transcription factors, Oct4, Sox2, Klf4, and c-Myc, into fibroblasts. Since then, several efforts have attempted to unveil other factors that are directly implicated in or might enhance reprogramming. Importantly, a number of transcription factors are reported to retain reprogramming activity. A previous study suggested Gli-similar 1 (Glis1) as a factor that enhances the reprogramming of fibroblasts during iPSC generation. However, the implication of other Glis members, including Glis2 and Glis3 (variants 1 and 2), in cellular reprogramming remains unknown. In this study, we investigated the potential involvement of human Glis family proteins, including hGlis1-3, in cellular reprogramming. Our results demonstrate that hGlis1, which is reported to reprogram human fibroblasts, promotes the reprogramming of human adipose-derived stromal cells (hADSCs), indicating that the reprogramming activity of Glis1 is not cell type-specific. Strikingly, hGlis3 promoted the reprogramming of hADSCs as efficiently as hGlis1. On the contrary, hGlis2 showed a strong negative effect on reprogramming. Together, our results reveal clear differences in the cellular reprogramming activity among Glis family members and provide valuable insight into the development of a new reprogramming strategy using Glis family proteins.

The impact of KRAS mutations on VEGF-A production and tumour vascular network

International Nuclear Information System (INIS)

Figueras, Agnès; Arbos, Maria Antonia; Quiles, Maria Teresa; Viñals, Francesc; Germà, Josep Ramón; Capellà, Gabriel

2013-01-01

The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis

Tumour gene expression predicts response to cetuximab in patients with KRAS wild-type metastatic colorectal cancer.

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Baker, J B; Dutta, D; Watson, D; Maddala, T; Munneke, B M; Shak, S; Rowinsky, E K; Xu, L-A; Harbison, C T; Clark, E A; Mauro, D J; Khambata-Ford, S

2011-02-01

Although it is accepted that metastatic colorectal cancers (mCRCs) that carry activating mutations in KRAS are unresponsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, a significant fraction of KRAS wild-type (wt) mCRCs are also unresponsive to anti-EGFR therapy. Genes encoding EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are promising gene expression-based markers but have not been incorporated into a test to dichotomise KRAS wt mCRC patients with respect to sensitivity to anti-EGFR treatment. We used RT-PCR to test 110 candidate gene expression markers in primary tumours from 144 KRAS wt mCRC patients who received monotherapy with the anti-EGFR antibody cetuximab. Results were correlated with multiple clinical endpoints: disease control, objective response, and progression-free survival (PFS). Expression of many of the tested candidate genes, including EREG and AREG, strongly associate with all clinical endpoints. Using multivariate analysis with two-layer five-fold cross-validation, we constructed a four-gene predictive classifier. Strikingly, patients below the classifier cutpoint had PFS and disease control rates similar to those of patients with KRAS mutant mCRC. Gene expression appears to identify KRAS wt mCRC patients who receive little benefit from cetuximab. It will be important to test this model in an independent validation study.

Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion.

Directory of Open Access Journals (Sweden)

Hyun Sik Jang

Full Text Available Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.

Blockade of the IL-6 trans-signalling/STAT3 axis suppresses cachexia in Kras-induced lung adenocarcinoma.

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Miller, A; McLeod, L; Alhayyani, S; Szczepny, A; Watkins, D N; Chen, W; Enriori, P; Ferlin, W; Ruwanpura, S; Jenkins, B J

2017-05-25

Lung cancer is the leading cause of cancer death worldwide, and is frequently associated with the devastating paraneoplastic syndrome of cachexia. The potent immunomodulatory cytokine interleukin (IL)-6 has been linked with the development of lung cancer as well as cachexia; however, the mechanisms by which IL-6 promotes muscle wasting in lung cancer cachexia are ill-defined. In this study, we report that the gp130 F/F knock-in mouse model displaying hyperactivation of the latent transcription factor STAT3 via the common IL-6 cytokine family signalling receptor, gp130, develops cachexia during Kras-driven lung carcinogenesis. Specifically, exacerbated weight loss, early mortality and reduced muscle and adipose tissue mass were features of the gp130 F/F :Kras G12D model, but not parental Kras G12D mice in which STAT3 was not hyperactivated. Gene expression profiling of muscle tissue in cachectic gp130 F/F :Kras G12D mice revealed the upregulation of IL-6 and STAT3-target genes compared with Kras G12D muscle tissue. These cachectic features of gp130 F/F :Kras G12D mice were abrogated upon the genetic normalization of STAT3 activation or ablation of IL-6 in gp130 F/F :Kras G12D :Stat3 -/+ or gp130 F/F :Kras G12D :Il6 -/- mice, respectively. Furthermore, protein levels of the soluble IL-6 receptor (sIL-6R), which is the central facilitator of IL-6 trans-signalling, were elevated in cachectic muscle from gp130 F/F :Kras G12D mice, and the specific blockade of IL-6 trans-signalling, but not classical signalling, with an anti-IL-6R antibody ameliorated cachexia-related characteristics in gp130 F/F :Kras G12D mice. Collectively, these preclinical findings identify trans-signalling via STAT3 as the signalling modality by which IL-6 promotes muscle wasting in lung cancer cachexia, and therefore support the clinical evaluation of the IL-6 trans-signalling/STAT3 axis as a therapeutic target in advanced lung cancer patients presenting with cachexia.

Overcoming reprogramming resistance of Fanconi anemia cells

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Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

Optimizing the multimodal approach to pancreatic cyst fluid diagnosis: developing a volume-based triage protocol.

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Chai, Siaw Ming; Herba, Karl; Kumarasinghe, M Priyanthi; de Boer, W Bastiaan; Amanuel, Benhur; Grieu-Iacopetta, Fabienne; Lim, Ee Mun; Segarajasingam, Dev; Yusoff, Ian; Choo, Chris; Frost, Felicity

2013-02-01

The objective of this study was to develop a triage algorithm to optimize diagnostic yield from cytology, carcinoembryonic antigen (CEA), and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) testing on different components of a single pancreatic cyst fluid specimen. The authors also sought to determine whether cell block supernatant was suitable for CEA and KRAS testing. Fifty-four pancreatic cysts were triaged according to a volume-dependent protocol to generate fluid (neat and supernatant) and cell block specimens for cytology, comparative CEA, and KRAS testing. Follow-up histology, diagnostic cytology, or a combined clinicopathologic interpretation was recorded as the final diagnosis. There were 26 mucinous cystic lesions and 28 nonmucinous cystic lesions with volumes ranging from 0.3 mL to 55 mL. Testing different components of the specimens (cell block, neat, and/or supernatant) enabled all laboratory investigations to be performed on 50 of 54 cyst fluids (92.6%). Interpretive concordance was observed in 17 of 17 cases (100%) and in 35 of 40 cases (87.5%) that had multiple components tested for CEA and KRAS mutations, respectively. An elevated CEA level (>192 ng/mL) was the most sensitive test for the detection of a mucinous cystic lesion (62.5%) versus KRAS mutation (56%) and "positive" cytology (61.5%). KRAS mutations were identified in 2 of 25 mucinous cystic lesions (8%) in which cytology and CEA levels were not contributory. A volume-based protocol using different components of the specimen was able to optimize diagnostic yield in pancreatic cyst fluids. KRAS mutation testing increased diagnostic yield when combined with cytology and CEA analysis. The current results demonstrated that supernatant is comparable to neat fluid and cell block material for CEA and KRAS testing. Copyright © 2012 American Cancer Society.

A wide reprogramming of histone H3 modifications during male meiosis I in rice is dependent on the Argonaute protein MEL1.

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Liu, Hua; Nonomura, Ken-Ichi

2016-10-01

The roles of epigenetic mechanisms, including small-RNA-mediated silencing, in plant meiosis largely remain unclear, despite their importance in plant reproduction. This study unveiled that rice chromosomes are reprogrammed during the premeiosis-to-meiosis transition in pollen mother cells (PMCs). This large-scale meiotic chromosome reprogramming (LMR) continued throughout meiosis I, during which time H3K9 dimethylation (H3K9me2) was increased, and H3K9 acetylation and H3S10 phosphorylation were broadly decreased, with an accompanying immunostaining pattern shift of RNA polymerase II. LMR was dependent on the rice Argonaute protein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which is specifically expressed in germ cells prior to meiosis, because LMR was severely diminished in mel1 mutant anthers. Pivotal meiotic events, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes, were also disrupted in this mutant. Interestingly, and as opposed to the LMR loss in most chromosomal regions, aberrant meiotic protein loading and hypermethylation of H3K9 emerged on the nucleolar organizing region in the mel1 PMCs. These results suggest that MEL1 plays important roles in epigenetic LMR to promote faithful homologous recombination and synapsis during rice meiosis. © 2016. Published by The Company of Biologists Ltd.

Mouse model of proximal colon-specific tumorigenesis driven by microsatellite instability-induced Cre-mediated inactivation of Apc and activation of Kras.

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Kawaguchi, Yasuo; Hinoi, Takao; Saito, Yasufumi; Adachi, Tomohiro; Miguchi, Masashi; Niitsu, Hiroaki; Sasada, Tatsunari; Shimomura, Manabu; Egi, Hiroyuki; Oka, Shiro; Tanaka, Shinji; Chayama, Kazuaki; Sentani, Kazuhiro; Oue, Naohide; Yasui, Wataru; Ohdan, Hideki

2016-05-01

KRAS gene mutations are found in 40-50% of colorectal cancer cases, but their functional contribution is not fully understood. To address this issue, we generated genetically engineered mice with colon tumors expressing an oncogenic Kras(G12D) allele in the context of the Adenomatous polyposis coli (Apc) deficiency to compare them to tumors harboring Apc deficiency alone. CDX2P9.5-G22Cre (referred to as G22Cre) mice showing inducible Cre recombinase transgene expression in the proximal colon controlled under the CDX2 gene promoter were intercrossed with Apc (flox/flox) mice and LSL-Kras (G12D) mice carrying loxP-flanked Apc and Lox-Stop-Lox oncogenic Kras(G12D) alleles, respectively, to generate G22Cre; Apc(flox/flox); Kras(G12D) and G22Cre; Apc(flox/flox); KrasWT mice. Gene expression profiles of the tumors were analyzed using high-density oligonucleotide arrays. Morphologically, minimal difference in proximal colon tumor was observed between the two mouse models. Consistent with previous findings in vitro, Glut1 transcript and protein expression was up-regulated in the tumors of G22Cre;Apc (flox/flox) ; Kras(G12D) mice. Immunohistochemical staining analysis revealed that GLUT1 protein expression correlated with KRAS mutations in human colorectal cancer. Microarray analysis identified 11 candidate genes upregulated more than fivefold and quantitative PCR analysis confirmed that Aqp8, Ttr, Qpct, and Slc26a3 genes were upregulated 3.7- to 30.2-fold in tumors with mutant Kras. These results demonstrated the validity of the G22Cre; Apc(flox/flox) ;Kras (G12D) mice as a new mouse model with oncogenic Kras activation. We believe that this model can facilitate efforts to define novel factors that contribute to the pathogenesis of human colorectal cancer with KRAS mutations.

An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations

DEFF Research Database (Denmark)

Liu, Yuliang; Guðnason, Haukur; Li, Yiping

2014-01-01

Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidity and mortality worldwide. CRC is curable if diagnosed at an early stage. Mutations in the oncogene KRAS play a critical role in early development of CRC. Detection of activated KRAS is of diagnostic...

KRAS Testing for Anti-EGFR Therapy in Advanced Colorectal Cancer: An Evidence-Based and Economic Analysis.

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2010-01-01

In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT: www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST CANCER: An Evidence-Based and Economic AnalysisEpidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based and Economic AnalysisK-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic Analysis. The objective of this systematic review is to determine the predictive value of KRAS testing in the treatment of metastatic colorectal cancer (mCRC) with two anti-EGFR agents, cetuximab and panitumumab. Economic analyses are also being conducted to evaluate the cost-effectiveness of KRAS testing. CONDITION AND TARGET POPULATION Metastatic colorectal cancer (mCRC) is usually defined as stage IV disease according to the American Joint Committee on Cancer tumour node metastasis (TNM) system or stage D in

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

International Nuclear Information System (INIS)

Davies, Janine M; Trembath, Dimitri; Deal, Allison M; Funkhouser, William K; Calvo, Benjamin F; Finnegan, Timothy; Weck, Karen E; Tepper, Joel E; O'Neil, Bert H

2011-01-01

KRAS mutations may predict poor response to radiotherapy. Downstream events from KRAS, such as activation of BRAF, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of KRAS and BRAF mutation status with p-AKT and p-ERK and outcomes in RC. Pre-radiotherapy RC tumor biopsies were evaluated. KRAS and BRAF mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry. Of 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were KRAS WT, 95% were BRAF WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. KRAS WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by KRAS status. KRAS mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS

The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer

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Nygaard, Anneli Dowler; Garm Spindler, Karen-Lise; Pallisgaard, Niels

2013-01-01

BACKGROUND: Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality. New agents targeting the epidermal growth factor system are emerging, but only a subgroup of the patients will benefit from the therapy. Cell free DNA (cf......DNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Patients with newly diagnosed, advanced NSCLC eligible....... RESULTS: The study included 246 patients receiving a minimum of 1 treatment cycle, and all but four were evaluable for response according to RECIST. Forty-three patients (17.5%) presented with a KRAS mutation. OS was 8.9 months and PFS by intention to treat 5.4 months. Patients with a detectable plasma...

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts.

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Gupta, Manoj K; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F; Windmueller, Rebecca; Wagers, Amy J; Kulkarni, Rohit N

2015-10-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. ©AlphaMed Press.

A Blueprint for a Synthetic Genetic Feedback Controller to Reprogram Cell Fate.

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Del Vecchio, Domitilla; Abdallah, Hussein; Qian, Yili; Collins, James J

2017-01-25

To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Optical reprogramming with ultrashort femtosecond laser pulses

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Uchugonova, Aisada; Breunig, Hans G.; Batista, Ana; König, Karsten

2015-03-01

The use of sub-15 femtosecond laser pulses in stem cell research is explored with particular emphasis on the optical reprogramming of somatic cells. The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through the ectopic expression of defined transcription factors. Conventional approaches utilize retro/lenti-viruses to deliver genes/transcription factors as well as to facilitate the integration of transcription factors into that of the host genome. However, the use of viruses may result in insertional mutations caused by the random integration of genes and as a result, this may limit the use within clinical applications due to the risk of the formation of cancer. In this study, a new approach is demonstrated in realizing non-viral reprogramming through the use of ultrashort laser pulses, to introduce transcription factors into the cell so as to generate iPS cells.

A comparison of non-integrating reprogramming methods

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Schlaeger, Thorsten M; Daheron, Laurence; Brickler, Thomas R; Entwisle, Samuel; Chan, Karrie; Cianci, Amelia; DeVine, Alexander; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen S; Brookes, Emily; Cherry, Anne B C; Demirbas, Didem; Tsankov, Alexander M; Zon, Leonard I; Rubin, Lee L; Feinberg, Andrew P; Meissner, Alexander; Cowan, Chad A; Daley, George Q

2015-01-01

Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. PMID:25437882

Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients.

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Papadopoulou, Eirini; Tsoulos, Nikolaos; Tsirigoti, Angeliki; Apessos, Angela; Agiannitopoulos, Konstantinos; Metaxa-Mariatou, Vasiliki; Zarogoulidis, Konstantinos; Zarogoulidis, Pavlos; Kasarakis, Dimitrios; Kakolyris, Stylianos; Dahabreh, Jubrail; Vlastos, Fotis; Zoublios, Charalampos; Rapti, Aggeliki; Papageorgiou, Niki Georgatou; Veldekis, Dimitrios; Gaga, Mina; Aravantinos, Gerasimos; Karavasilis, Vasileios; Karagiannidis, Napoleon; Nasioulas, George

2015-10-01

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor ( EGFR ) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18-21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.

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Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-06-26

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.

Algorithm for cellular reprogramming.

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Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

2017-11-07

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

Developmental Programming of Adult Disease: Reprogramming by Melatonin?

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Tain, You-Lin; Huang, Li-Tung; Hsu, Chien-Ning

2017-02-16

Adult-onset chronic non-communicable diseases (NCDs) can originate from early life through so-called the "developmental origins of health and disease" (DOHaD) or "developmental programming". The DOHaD concept offers the "reprogramming" strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs.

A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution

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Grace R. Anderson

2017-07-01

Full Text Available Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS/PIK3CA double-mutant colorectal cancers (CRCs with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.

Reprogramming cancer cells: a novel approach for cancer therapy or a tool for disease-modeling?

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Yilmazer, Açelya; de Lázaro, Irene; Taheri, Hadiseh

2015-12-01

Chromatin dynamics have been the major focus of many physiological and pathological processes over the past 20 years. Epigenetic mechanisms have been shown to be reshaped during both cellular reprogramming and tumorigenesis. For this reason, cancer cell reprogramming can provide a powerful tool to better understand both regenerative and cancer-fate processes, with a potential to develop novel therapeutic approaches. Recent studies showed that cancer cells can be reprogrammed to a pluripotent state by the overexpression of reprogramming transcription factors. Activation of transcription factors and modification of chromatin regulators may result in the remodeling of epigenetic status and refueling of tumorigenicity in these reprogrammed cancer cells. However, studies focusing on cancer cell reprogramming are contradictory; some studies reported increased tumor progression whereas others showed that cellular reprogramming has a treatment potential for cancer. In this review, first, the current knowledge on the epigenetic mechanisms involved during cancer development and cellular reprogramming will be presented. Later, different reports and key factors about pluripotency-based reprogramming of cancer cells will be reviewed in detail. New insights will be provided on cancer biology and therapy in the light of cellular reprogramming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming

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Cheng-Xu Delon Toh

2016-06-01

Full Text Available Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.

Impact of KRAS, BRAF and PI3KCA mutations in rectal carcinomas treated with neoadjuvant radiochemotherapy and surgery

International Nuclear Information System (INIS)

Derbel, Olfa; La Fouchardière, Christelle de; Wang, Qing; Desseigne, Françoise; Rivoire, Michel; Meeus, Pierre; Peyrat, Patrice; Stella, Mattia; Martel-Lafay, Isabelle; Lemaistre, Anne-Isabelle

2013-01-01

Conventional treatment for locally advanced rectal cancer usually combines neoadjuvant radiochemotherapy and surgery. Until recently, there have been limited predictive factors (clinical or biological) for rectal tumor response to conventional treatment. KRAS, BRAF and PIK3CA mutations are commonly found in colon cancers. In this study, we aimed to determine the mutation frequencies of KRAS, BRAF and PIK3CA and to establish whether such mutations may be used as prognostic and/or predictive factors in rectal cancer patients. We retrospectively reviewed the clinical and biological data of 98 consecutive operated patients between May 2006 and September 2009. We focused in patients who received surgery in our center after radiochemotherapy and in which tumor samples were available. In the 98 patients with a rectal cancer, the median follow-up time was 28.3 months (4–74). Eight out of ninety-eight patients experienced a local recurrence (8%) and 17/98 developed distant metastasis (17%). KRAS, BRAF and PIK3CA were identified respectively in 23 (23.5%), 2 (2%) and 4 (4%) patients. As described in previous studies, mutations in KRAS and BRAF were mutually exclusive. No patient with local recurrence exhibited KRAS or PIK3CA mutation and one harbored BRAF mutation (12.5%). Of the seventeen patients with distant metastasis (17%), 5 were presenting KRAS mutation (29%), one BRAF (5%) and one PIK3CA mutation (5%). No relationship was seen between PIK3CA, KRAS or BRAF mutation and local or distant recurrences. The frequencies of KRAS, BRAF and PIK3CA mutations in our study were lower than the average frequencies reported in colorectal cancers and no significant correlation was found between local/distant recurrences and KRAS, BRAF or PIK3CA mutations. Future studies with greater number of patients, longer follow-up time and greater power to predict associations are necessary to fully understand this relationship

Epigenetic reprogramming in mammalian species after SCNT-based cloning.

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Niemann, Heiner

2016-07-01

The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. Copyright © 2016 Elsevier Inc. All rights reserved.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

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Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

Cell reprogramming modelled as transitions in a hierarchy of cell cycles

International Nuclear Information System (INIS)

Hannam, Ryan; Annibale, Alessia; Kühn, Reimer

2017-01-01

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings. (paper)

Association of folate intake, dietary habits, smoking and COX-2 promotor -765G>C polymorphism with K-ras mutation in patients with colorectal cancer.

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Kamal, Manal M; Youssef, Omar Z; Lotfy, Ahmed N; Elsaed, Eman T; Fawzy, May M T

2012-09-01

Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G>C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, -765G>C. The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) -765G>C polymorphism were determined by PCR-RFLP. The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96±51.3 ng/ml) than the negative group (216.6±166.4 ng/ml), (P<0.01). Higher folate levels were found in males than females (median=173 ng/ml and 85 ng/ml; respectively, P=0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P=0.0018) followed by gender (P=0.036) contributed significantly in the discrimination between patients prone to develop K-ras

KRAS early testing: consensus initiative and cost-effectiveness evaluation for metastatic colorectal patients in an Italian setting.

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Carlo Barone

Full Text Available KRAS testing is relevant for the choice of the most appropriate first-line therapy of metastatic colorectal cancer (CRC. Strategies for preventing unequal access to the test should be implemented, but their relevance in the practice is related to economic sustainability. The study adopted the Delphi technique to reach a consensus on several topics. Issues related to execution of KRAS testing were identified by an expert's board and proposed to 108 Italian oncologists and pathologists through two subsequent questionnaires. The emerging proposal was evaluated by decision analyses models employed by technology assessment agencies in order to assess cost-effectiveness. Alternative therapeutic strategies included most commonly used chemotherapy regimens alone or in combination with cetuximab or bevacizumab. The survey indicated that time interval for obtaining KRAS test should not exceed 15 days, 10 days being an optimal interval. To assure the access to proper treatment, a useful strategy should be to anticipate the test after radical resection in patients at high risk of relapse. Early KRAS testing in high risk CRC patients generates incremental cost-effectiveness ratios between 6,000 and 13,000 Euro per quality adjusted life year (QALY gained. In extensive sensitivity analyses ICER's were always below 15,000 Euro per QALY gained, far within the threshold of 60,000 Euro/QALY gained accepted by regulatory institutions in Italy. In metastatic CRC a time interval higher than 15 days for result of KRAS testing limits access to therapeutic choices. Anticipating KRAS testing before the onset of metastatic disease in patients at high risk does not affect the sustainability and cost-effectiveness profile of cetuximab in first-line mCRC. Early KRAS testing may prevent this inequality in high-risk patients, whether they develop metastases, and is a cost-effective strategy. Based on these results, present joined recommendations of Italian societies of

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma.

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McFadden, David G; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K; Song, Xiaoling; Pirun, Mono; Santiago, Philip M; Kim-Kiselak, Caroline; Platt, James T; Lee, Emily; Hodges, Emily; Rosebrock, Adam P; Bronson, Roderick T; Socci, Nicholas D; Hannon, Gregory J; Jacks, Tyler; Varmus, Harold

2016-10-18

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

Science.gov (United States)

McFadden, David G.; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K.; Song, Xiaoling; Pirun, Mono; Santiago, Philip M.; Kim-Kiselak, Caroline; Platt, James T.; Lee, Emily; Hodges, Emily; Rosebrock, Adam P.; Bronson, Roderick T.; Socci, Nicholas D.; Hannon, Gregory J.; Jacks, Tyler; Varmus, Harold

2016-01-01

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity. PMID:27702896

Metabolic Reprogramming During Multidrug Resistance in Leukemias

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Raphael Silveira Vidal

2018-04-01

Full Text Available Cancer outcome has improved since introduction of target therapy. However, treatment success is still impaired by the same drug resistance mechanism of classical chemotherapy, known as multidrug resistance (MDR phenotype. This phenotype promotes resistance to drugs with different structures and mechanism of action. Recent reports have shown that resistance acquisition is coupled to metabolic reprogramming. High-gene expression, increase of active transport, and conservation of redox status are one of the few examples that increase energy and substrate demands. It is not clear if the role of this metabolic shift in the MDR phenotype is related to its maintenance or to its induction. Apart from the nature of this relation, the metabolism may represent a new target to avoid or to block the mechanism that has been impairing treatment success. In this mini-review, we discuss the relation between metabolism and MDR resistance focusing on the multiple non-metabolic functions that enzymes of the glycolytic pathway are known to display, with emphasis with the diverse activities of glyceraldehyde-3-phosphate dehydrogenase.

Cellular Reprogramming Employing Recombinant Sox2 Protein

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Marc Thier

2012-01-01

Full Text Available Induced pluripotent stem (iPS cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT and Sox2 (Sox2-TAT proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

KRAS as a predictor of poor prognosis and benefit from postoperative FOLFOX chemotherapy in patients with stage II and III colorectal cancer.

Science.gov (United States)

Deng, Yanhong; Wang, Li; Tan, Shuyun; Kim, George P; Dou, Ruoxu; Chen, Dianke; Cai, Yue; Fu, Xinhui; Wang, Lei; Zhu, Jun; Wang, Jianping

2015-08-01

The KRAS gene frequently mutates in colorectal cancer (CRC). Here we investigated the prognostic and predictive role of KRAS mutation in patients with stage II or III CRC. A consecutive cohort of patients with stage II or III CRC from a single center database was studied. The association between KRAS status, adjuvant FOLFOX therapy, and 3-year disease-free survival (3-y DFS) was analyzed. Of our 433 patients, 166 (38.3%) exhibited the KRAS mutation. Among the 190 patients who did not receive adjuvant therapy, those with KRAS mutation tumors had a worse 3-y DFS (hazard ratio [HR], 1.924; 95% confidence interval [CI], 1.078-3.435; P = 0.027). Among patients who received adjuvant chemotherapy, KRAS mutation was not correlated with worse 3-y DFS (HR, 1.083; 95% CI, 0.618-1.899; P = 0.781). Adjuvant chemotherapy improved 3-y DFS only among patients with KRAS mutant tumors (78.0% vs 69.2%) on multivariate analysis adjusted for age, stage, grade, site, vessel invasion, and carcinoembryonic antigen level (HR, 0.454; 95% CI, 0.229-0.901; P = 0.024). In contrast, there was no benefit of adjuvant chemotherapy in the KRAS wild-type group (84.3% vs 82.0%). KRAS mutation indicates poor prognosis. FOLFOX adjuvant chemotherapy benefits patients with stage II or III colorectal cancer with KRAS mutant tumors and is worth further investigation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

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Nathalie Swales

Full Text Available AIMS/HYPOTHESIS: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3. In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. METHODS: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. RESULTS: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. CONCLUSIONS/INTERPRETATION: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

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Finley, Lydia W.S.; Carracedo, Arkaitz; Lee, Jaewon; Souza, Amanda; Egia, Ainara; Zhang, Jiangwen; Teruya-Feldstein, Julie; Moreira, Paula I.; Cardoso, Sandra M.; Clish, Clary B.; Pandolfi, Pier Paolo; Haigis, Marcia C.

2011-01-01

Summary Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression. PMID:21397863

KRAS and BRAF mutations in anal carcinoma

DEFF Research Database (Denmark)

Serup-Hansen, Eva; Linnemann, Dorte; Høgdall, Estrid

2015-01-01

the frequency and the prognostic value of KRAS and BRAF mutations in a large cohort of patients with anal cancer. One hundred and ninety-three patients with T1-4N0-3M0-1 anal carcinoma were included in the study. Patients were treated with curative (92%) or palliative intent (8%) between January 2000...

[miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

Science.gov (United States)

Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

Cancer stemness in Apc- vs. Apc/KRAS-driven intestinal tumorigenesis.

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Mehrnaz Ghazvini

Full Text Available Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in Apc/KRAS tumours, they appear to be very rare (<10(-6 in the Apc-mutant adenomas. In contrast, the Lin(-CD24(hiCD29(+ subpopulation of adenocarcinoma cells appear to be enriched in CSCs with increased levels of active β-catenin. Expression profiling analysis of the CSC-enriched subpopulation confirmed their enhanced Wnt activity and revealed additional differential expression of other signalling pathways, growth factor binding proteins, and extracellular matrix components. As expected, genes characteristic of the Paneth cell lineage (e.g. defensins are co-expressed together with stem cell genes (e.g. Lgr5 within the CSC-enriched subpopulation. This is of interest as it may indicate a cancer stem cell niche role for tumor-derived Paneth-like cells, similar to their role in supporting Lgr5(+ stem cells in the normal intestinal crypt. Overall, our results indicate that oncogenic KRAS activation in Apc-driven tumours results in the expansion of the CSCs compartment by increasing ®-catenin intracellular stabilization.

New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

NARCIS (Netherlands)

Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

2000-01-01

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

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Bektik, Emre; Dennis, Adrienne; Pawlowski, Gary; Zhou, Chen; Maleski, Danielle; Takahashi, Satoru; Laurita, Kenneth R; Deschênes, Isabelle; Fu, Ji-Dong

2018-05-04

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFP high iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFP low cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

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Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

2017-06-01

Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

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Van Laethem Jean-Luc

2010-05-01

Full Text Available Abstract Background With the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas. Methods Formalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers. Results EGFR immunoreactivity was present in 36/43 (83.7% of anal canal and in 20/24 (83.3% of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23, but could be identified in 4 of 24 tonsil tumours. From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified. Conclusion EGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified

Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

International Nuclear Information System (INIS)

Van Damme, Nancy; Pauwels, Patrick; Peeters, Marc; Deron, Philippe; Van Roy, Nadine; Demetter, Pieter; Bols, Alain; Dorpe, Jo Van; Baert, Filip; Van Laethem, Jean-Luc; Speleman, Franki

2010-01-01

With the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas. Formalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers. EGFR immunoreactivity was present in 36/43 (83.7%) of anal canal and in 20/24 (83.3%) of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23), but could be identified in 4 of 24 tonsil tumours. From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified. EGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified. This indicates that EGFR and K-RAS mutation analysis is not

Evaluation of EGFR, KRAS and BRAF gene mutations in renal cell carcinoma

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Omer Bayrak

2014-08-01

Full Text Available A subset of renal cell carcinoma (RCC patients has been shown to respond to anti-EGFR therapy. As KRAS and BRAF mutations are associated with poor response to anti-EGFR therapy in some cancers, it has been suggested that screening for KRAS and BRAF mutations in RCC may be a promising strategy to identify patients who might respond to EGFR-targeted therapy. The aim of this study was to investigate the mutation status of EGFR, KRAS and BRAF in RCC patients. Renal tumors and normal renal samples from forty-eight patients who underwent radical or partial nephrectomy for kidney cancer were used in this study. Histological classification of the tumors was performed according to International Union against Cancer (UICC / American Joint Committee on Cancer (AJCC classification. Seventeen patients (48% had clear-cell RCC, 7 (20% had chromophobe RCC, and 11 patients (32% had papillary RCC. DNA isolated from the samples was subjected to melting curve mutation analysis for EGFR, BRAF and KRAS using ABI-3130 DNA sequencer. DNA sequencing analysis of RCC samples, when compared with morphologically normal matched regions, did not show any exon mutations. Our results do not support the notion that EGFR, KRAS and BRAF might be mutated in RCC. Normal 0 false false false TR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:TR; mso-fareast-language:EN-US;}

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

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Xu, Shenyuan; Long, Brian N.; Boris, Gabriel H.; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A.

2017-11-10

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

Neutral evolution of drug resistant colorectal cancer cell populations is independent of their KRAS status.

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Krastan B Blagoev

Full Text Available Emergence of tumor resistance to an anti-cancer therapy directed against a putative target raises several questions including: (1 do mutations in the target/pathway confer resistance? (2 Are these mutations pre-existing? (3 What is the relative fitness of cells with/without the mutation? We addressed these questions in patients with metastatic colorectal cancer (mCRC. We conducted an exhaustive review of published data to establish a median doubling time for CRCs and stained a cohort of CRCs to document mitotic indices. We analyzed published data and our own data to calculate rates of growth (g and regression (d, decay of tumors in patients with CRC correlating these results with the detection of circulating MT-KRAS DNA. Additionally we estimated mathematically the caloric burden of such tumors using data on mitotic and apoptotic indices. We conclude outgrowth of cells harboring intrinsic or acquired MT-KRAS cannot explain resistance to anti-EGFR (epidermal growth factor receptor antibodies. Rates of tumor growth with panitumumab are unaffected by presence/absence of MT-KRAS. While MT-KRAS cells may be resistant to anti-EGFR antibodies, WT-KRAS cells also rapidly bypass this blockade suggesting inherent resistance mechanisms are responsible and a neutral evolution model is most appropriate. Using the above clinical data on tumor doubling times and mitotic and apoptotic indices we estimated the caloric intake required to support tumor growth and suggest it may explain in part cancer-associated cachexia.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

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Phuoc T Tran

Full Text Available KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

Mitochondrial Spare Respiratory Capacity Is Negatively Correlated with Nuclear Reprogramming Efficiency

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Yan, Zhou; Al-Saaidi, Rasha Abdelkadhem; Fernandez Guerra, Paula

2017-01-01

Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (i......, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level...

Mitochondrial Spare Respiratory Capacity Is Negatively Correlated With Nuclear Reprogramming Efficiency

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Zhou, Yan; Al-Saaidi, Rasha Abdelkadhem; Guerra, Paula Fernandez

2017-01-01

Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (i......, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level...

Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis

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Kun-Ming Rau

2016-04-01

Full Text Available Mutations on epidermal growth factor receptor (EGFR of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5% were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6% were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy.

Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

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Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

2012-01-01

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self

Genomic stability during cellular reprogramming: Mission impossible?

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Joest, Mathieu von; Búa Aguín, Sabela; Li, Han, E-mail: han.li@pasteur.fr

2016-06-15

The generation of induced pluripotent stem cells (iPSCs) from adult somatic cells is one of the most exciting discoveries in recent biomedical research. It holds tremendous potential in drug discovery and regenerative medicine. However, a series of reports highlighting genomic instability in iPSCs raises concerns about their clinical application. Although the mechanisms cause genomic instability during cellular reprogramming are largely unknown, several potential sources have been suggested. This review summarizes current knowledge on this active research field and discusses the latest efforts to alleviate the genomic insults during cellular reprogramming to generate iPSCs with enhanced quality and safety.

Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

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Peng-Cheng Kong

Full Text Available The birthrate following round spermatid injection (ROSI remains low in current and evidence suggests that factors in the germinal vesicle (GV cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI, but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer

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Vallejo, Adrian; Perurena, Naiara; Guruceaga, Elisabet

2017-01-01

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identifica...

A high level of liver-specific expression of oncogenic KrasV12 drives robust liver tumorigenesis in transgenic zebrafish

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Anh Tuan Nguyen

2011-11-01

Human liver cancer is one of the deadliest cancers worldwide, with hepatocellular carcinoma (HCC being the most common type. Aberrant Ras signaling has been implicated in the development and progression of human HCC, but a complete understanding of the molecular mechanisms of this protein in hepatocarcinogenesis remains elusive. In this study, a stable in vivo liver cancer model using transgenic zebrafish was generated to elucidate Ras-driven tumorigenesis in HCC. Using the liver-specific fabp10 (fatty acid binding protein 10 promoter, we overexpressed oncogenic krasV12 specifically in the transgenic zebrafish liver. Only a high level of krasV12 expression initiated liver tumorigenesis, which progressed from hyperplasia to benign and malignant tumors with activation of the Ras-Raf-MEK-ERK and Wnt–β-catenin pathways. Histological diagnosis of zebrafish tumors identified HCC as the main lesion. The tumors were invasive and transplantable, indicating malignancy of these HCC cells. Oncogenic krasV12 was also found to trigger p53-dependent senescence as a tumor suppressive barrier in the pre-neoplastic stage. Microarray analysis of zebrafish liver hyperplasia and HCC uncovered the deregulation of several stage-specific and common biological processes and signaling pathways responsible for krasV12-driven liver tumorigenesis that recapitulated the molecular hallmarks of human liver cancer. Cross-species comparisons of cancer transcriptomes further defined a HCC-specific gene signature as well as a liver cancer progression gene signature that are evolutionarily conserved between human and zebrafish. Collectively, our study presents a comprehensive portrait of molecular mechanisms during progressive Ras-induced HCC. These observations indicate the validity of our transgenic zebrafish to model human liver cancer, and this model might act as a useful platform for drug screening and identifying new therapeutic targets.

EGFR, HER-2 and KRAS in canine gastric epithelial tumors: a potential human model?

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Rossella Terragni

Full Text Available Epidermal growth factor receptor (EGFR or HER-1 and its analog c-erbB-2 (HER-2 are protein tyrosine kinases correlated with prognosis and response to therapy in a variety of human cancers. KRAS mediates the transduction of signals between EGFR and the nucleus, and its mutation has been identified as a predictor of resistance to anti-EGFR drugs. In human oncology, the importance of the EGFR/HER-2/KRAS signalling pathway in gastric cancer is well established, and HER-2 testing is required before initiating therapy. Conversely, this pathway has never been investigated in canine gastric tumours. A total of 19 canine gastric epithelial neoplasms (5 adenomas and 14 carcinomas were retrospectively evaluated for EGFR/HER-2 immunohistochemical expression and KRAS mutational status. Five (35.7% carcinomas were classified as intestinal-type and 9 (64.3% as diffuse-type. EGFR was overexpressed (≥ 1+ in 8 (42.1% cases and HER-2 (3+ in 11 (57.9% cases, regardless of tumour location or biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type (80% than in the diffuse-type (11.1%, p = 0.023. KRAS gene was wild type in 18 cases, whereas one mucinous carcinoma harboured a point mutation at codon 12 (G12R. EGFR and HER-2 may be promising prognostic and therapeutic targets in canine gastric epithelial neoplasms. The potential presence of KRAS mutation should be taken into account as a possible mechanism of drug resistance. Further studies are necessary to evaluate the role of dog as a model for human gastric cancer.

Fbxw7 Deletion Accelerates KrasG12D-Driven Pancreatic Tumorigenesis via Yap Accumulation.

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Zhang, Qiang; Zhang, Yaqing; Parsels, Joshua D; Lohse, Ines; Lawrence, Theodore S; Pasca di Magliano, Marina; Sun, Yi; Morgan, Meredith A

2016-11-01

Pancreatic cancers driven by KRAS mutations require additional mutations for tumor progression. The tumor suppressor FBXW7 is altered in pancreatic cancers, but its contribution to pancreatic tumorigenesis is unknown. To determine potential cooperation between Kras mutation and Fbxw7 inactivation in pancreatic tumorigenesis, we generated P48-Cre;LSL-Kras G12D ;Fbxw7 fl/fl (KFC fl/fl ) compound mice. We found that KFC fl/fl mice displayed accelerated tumorigenesis: all mice succumbed to pancreatic ductal adenocarcinoma (PDA) by 40 days of age, with PDA onset occurring by 2 weeks of age. PDA in KFC fl/fl mice was preceded by earlier onset of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions, and associated with chromosomal instability and the accumulation of Fbxw7 substrates Yes-associated protein (Yap), c-Myc, and Notch. Using KFC fl/fl and FBXW7-deficient human pancreatic cancer cells, we found that Yap silencing attenuated growth promotion by Fbxw7 deletion. Our data demonstrate that Fbxw7 is a potent suppressor of Kras G12D -induced pancreatic tumorigenesis due, at least in part, to negative regulation of Yap. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cocktail of chemical compounds robustly promoting cell reprogramming protects liver against acute injury

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Yuewen Tang

2017-02-01

Full Text Available Abstract Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.

Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function▿

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Plowman, Sarah J.; Ariotti, Nicholas; Goodall, Andrew; Parton, Robert G.; Hancock, John F.

2008-01-01

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades. PMID:18458061

Role of [18F]FDG PET in prediction of KRAS and EGFR mutation status in patients with advanced non-small-cell lung cancer

International Nuclear Information System (INIS)

Caicedo, Carlos; Garcia-Velloso, Maria Jose; Vigil Diaz, Carmen; Richter Echevarria, Jose Angel; Lozano, Maria Dolores; Labiano, Tania; Lopez-Picazo, Jose Maria; Gurpide, Alfonso; Perez Gracia, Jose Luis; Zulueta, Javier

2014-01-01

The tumour molecular profile predicts the activity of epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). However, tissue availability and tumour heterogeneity limit its assessment. We evaluated whether [ 18 F]FDG PET might help predict KRAS and EFGR mutation status in NSCLC. Between January 2005 and October 2011, 340 NSCLC patients were tested for KRAS and EGFR mutation status. We identified patients with stage III and IV disease who had undergone [ 18 F]FDG PET/CT scanning for initial staging. SUVpeak, SUVmax and SUVmean of the single hottest tumour lesions were calculated, and their association with KRAS and EGFR mutation status was assessed. A receiver operator characteristic (ROC) curve analysis and a multivariate analysis (including SUVmean, gender, age and AJCC stage) were performed to identify the potential value of [ 18 F]FDG PET/CT for predicting KRAS mutation. From 102 patients staged using [ 18 F]FDG PET/CT, 28 (27 %) had KRAS mutation (KRAS+), 22 (22 %) had EGFR mutation (EGFR+) and 52 (51 %) had wild-type KRAS and EGFR profiles (WT). KRAS+ patients showed significantly higher [ 18 F]FDG uptake than EGFR+ and WT patients (SUVmean 9.5, 5.7 and 6.6, respectively; p 18 F]FDG uptake between EGFR+ patients and WT patients. ROC curve analysis for KRAS mutation status discrimination yielded an area under the curve of 0.740 for SUVmean (p 18 F]FDG uptake than WT patients, as assessed in terms of SUVpeak, SUVmax and SUVmean. A multivariate model based on age, gender, AJCC stage and SUVmean might be used as a predictive marker of KRAS mutation status in patients with stage III or IV NSCLC. (orig.)

Looking into the Black Box: Insights into the Mechanisms of Somatic Cell Reprogramming

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Jeffrey L. Wrana

2011-01-01

Full Text Available The dramatic discovery that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs, by the expression of just four factors, has opened new opportunities for regenerative medicine and novel ways of modeling human diseases. Extensive research over the short time since the first iPSCs were generated has yielded the ability to reprogram various cell types using a diverse range of methods. However the duration, efficiency, and safety of induced reprogramming have remained a persistent limitation to achieving a robust experimental and therapeutic system. The field has worked to resolve these issues through technological advances using non-integrative approaches, factor replacement or complementation with microRNA, shRNA and drugs. Despite these advances, the molecular mechanisms underlying the reprogramming process remain poorly understood. Recently, through the use of inducible secondary reprogramming systems, researchers have now accessed more rigorous mechanistic experiments to decipher this complex process. In this review we will discuss some of the major recent findings in reprogramming, pertaining to proliferation and cellular senescence, epigenetic and chromatin remodeling, and other complex cellular processes such as morphological changes and mesenchymal-to-epithelial transition. We will focus on the implications of this work in the construction of a mechanistic understanding of reprogramming and discuss unexplored areas in this rapidly expanding field.

Endogenous retinal neural stem cell reprogramming for neuronal regeneration

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Romain Madelaine

2017-01-01

Full Text Available In humans, optic nerve injuries and associated neurodegenerative diseases are often followed by permanent vision loss. Consequently, an important challenge is to develop safe and effective methods to replace retinal neurons and thereby restore neuronal functions and vision. Identifying cellular and molecular mechanisms allowing to replace damaged neurons is a major goal for basic and translational research in regenerative medicine. Contrary to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system, including retina. This regenerative process depends on endogenous retinal neural stem cells, the Müller glial cells. Following injury, zebrafish Müller cells go back into cell cycle to proliferate and generate new neurons, while mammalian Müller cells undergo reactive gliosis. Recently, transcription factors and microRNAs have been identified to control the formation of new neurons derived from zebrafish and mammalian Müller cells, indicating that cellular reprogramming can be an efficient strategy to regenerate human retinal neurons. Here we discuss recent insights into the use of endogenous neural stem cell reprogramming for neuronal regeneration, differences between zebrafish and mammalian Müller cells, and the need to pursue the identification and characterization of new molecular factors with an instructive and potent function in order to develop theurapeutic strategies for eye diseases.

Cell fate reprogramming by control of intracellular network dynamics

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Zanudo, Jorge G. T.; Albert, Reka

Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

Molecular testing guidelines for lung adenocarcinoma: Utility of cell blocks and concordance between fine-needle aspiration cytology and histology samples

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Heymann, Jonas J.; Bulman, William A.; Maxfield, Roger A.; Powell, Charles A.; Halmos, Balazs; Sonett, Joshua; Beaubier, Nike T.; Crapanzano, John P.; Mansukhani, Mahesh M.; Saqi, Anjali

2014-01-01

Background: Lung cancer is a leading cause of mortality, and patients often present at a late stage. More recently, advances in screening, diagnosing, and treating lung cancer have been made. For instance, greater numbers of minimally invasive procedures are being performed, and identification of lung adenocarcinoma driver mutations has led to the implementation of targeted therapies. Advances in molecular techniques enable use of scant tissue, including cytology specimens. In addition, per recently published consensus guidelines, cytology-derived cell blocks (CBs) are preferred over direct smears. Yet, limited comparison of molecular testing of fine-needle aspiration (FNA) CBs and corresponding histology specimens has been performed. This study aimed to establish concordance of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma (KRAS) virus homolog testing between FNA CBs and histology samples from the same patients. Materials and Methods: Patients for whom molecular testing for EGFR or KRAS was performed on both FNA CBs and histology samples containing lung adenocarcinoma were identified retrospectively. Following microdissection, when necessary, concordance of EGFR and KRAS molecular testing results between FNA CBs and histology samples was evaluated. Results: EGFR and/or KRAS testing was performed on samples obtained from 26 patients. Concordant results were obtained for all EGFR (22/22) and KRAS (17/17) mutation analyses performed. Conclusions: Identification of mutations in lung adenocarcinomas affects clinical decision-making, and it is important that results from small samples be accurate. This study demonstrates that molecular testing on cytology CBs is as sensitive and specific as that on histology. PMID:24987443

Molecular testing guidelines for lung adenocarcinoma: Utility of cell blocks and concordance between fine-needle aspiration cytology and histology samples

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Jonas J. Heymann

2014-01-01

Full Text Available Background: Lung cancer is a leading cause of mortality, and patients often present at a late stage. More recently, advances in screening, diagnosing, and treating lung cancer have been made. For instance, greater numbers of minimally invasive procedures are being performed, and identification of lung adenocarcinoma driver mutations has led to the implementation of targeted therapies. Advances in molecular techniques enable use of scant tissue, including cytology specimens. In addition, per recently published consensus guidelines, cytology-derived cell blocks (CBs are preferred over direct smears. Yet, limited comparison of molecular testing of fine-needle aspiration (FNA CBs and corresponding histology specimens has been performed. This study aimed to establish concordance of epidermal growth factor receptor (EGFR and Kirsten rat sarcoma (KRAS virus homolog testing between FNA CBs and histology samples from the same patients. Materials and Methods: Patients for whom molecular testing for EGFR or KRAS was performed on both FNA CBs and histology samples containing lung adenocarcinoma were identified retrospectively. Following microdissection, when necessary, concordance of EGFR and KRAS molecular testing results between FNA CBs and histology samples was evaluated. Results: EGFR and/or KRAS testing was performed on samples obtained from 26 patients. Concordant results were obtained for all EGFR (22/22 and KRAS (17/17 mutation analyses performed. Conclusions: Identification of mutations in lung adenocarcinomas affects clinical decision-making, and it is important that results from small samples be accurate. This study demonstrates that molecular testing on cytology CBs is as sensitive and specific as that on histology.

K-ras mutations in sinonasal cancers in relation to wood dust exposure

International Nuclear Information System (INIS)

Bornholdt, Jette; Vogel, Ulla; Husgafvel-Pursiainen, Kirsti; Wallin, Håkan; Hansen, Johnni; Steiniche, Torben; Dictor, Michael; Antonsen, Annemarie; Wolff, Henrik; Schlünssen, Vivi; Holmila, Reetta; Luce, Danièle

2008-01-01

Cancer in the sinonasal tract is rare, but persons who have been occupationally exposed to wood dust have a substantially increased risk. It has been estimated that approximately 3.6 million workers are exposed to inhalable wood dust in EU. In previous small studies of this cancer, ras mutations were suggested to be related to wood dust exposure, but these studies were too limited to detect statistically significant associations. We examined 174 cases of sinonasal cancer diagnosed in Denmark in the period from 1991 to 2001. To ensure uniformity, all histological diagnoses were carefully reviewed pathologically before inclusion. Paraffin embedded tumour samples from 58 adenocarcinomas, 109 squamous cell carcinomas and 7 other carcinomas were analysed for K-ras codon 12, 13 and 61 point mutations by restriction fragment length polymorphisms and direct sequencing. Information on occupational exposure to wood dust and to potential confounders was obtained from telephone interviews and from registry data. Among the patients in this study, exposure to wood dust was associated with a 21-fold increased risk of having an adenocarcinoma than a squamous cell carcinoma compared to unexposed [OR = 21.0, CI = 8.0–55.0]. K-ras was mutated in 13% of the adenocarcinomas (seven patients) and in 1% of squamous cell carcinomas (one patient). Of these eight mutations, five mutations were located in the codon 12. The exact sequence change of remaining three could not be identified unambiguously. Among the five identified mutations, the G→A transition was the most common, and it was present in tumour tissue from two wood dust exposed adenocarcinoma patients and one patient with unknown exposure. Previously published studies of sinonasal cancer also identify the GGT → GAT transition as the most common and often related to wood dust exposure. Patients exposed to wood dust seemed more likely to develop adenocarcinoma compared to squamous cell carcinomas. K-ras mutations were detected

Prognostic significance of anti-p53 and anti-KRas circulating antibodies in esophageal cancer patients treated with chemoradiotherapy

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Blanchard, Pierre; Quero, Laurent; Pacault, Vincent; Schlageter, Marie-Helene; Baruch-Hennequin, Valerie; Hennequin, Christophe

2012-01-01

P53 mutations are an adverse prognostic factor in esophageal cancer. P53 and KRas mutations are involved in chemo-radioresistance. Circulating anti-p53 or anti-KRas antibodies are associated with gene mutations. We studied whether anti-p53 or anti-KRas auto-antibodies were prognostic factors for response to chemoradiotherapy (CRT) or survival in esophageal carcinoma. Serum p53 and KRas antibodies (abs) were measured using an ELISA method in 97 consecutive patients treated at Saint Louis University Hospital between 1999 and 2002 with CRT for esophageal carcinoma (squamous cell carcinoma (SCCE) 57 patients, adenocarcinoma (ACE) 27 patients). Patient and tumor characteristics, response to treatment and the follow-up status of 84 patients were retrospectively collected. The association between antibodies and patient characteristics was studied. Univariate and multivariate survival analyses were conducted. Twenty-four patients (28%) had anti-p53 abs. Abs were found predominantly in SCCE (p = 0.003). Anti-p53 abs were associated with a shorter overall survival in the univariate analysis (HR 1.8 [1.03-2.9], p = 0.04). In the multivariate analysis, independent prognostic factors for overall and progression-free survival were an objective response to CRT, the CRT strategy (alone or combined with surgery [preoperative]) and anti-p53 abs. None of the long-term survivors had p53 abs. KRas abs were found in 19 patients (23%, no difference according to the histological type). There was no significant association between anti-KRas abs and survival neither in the univariate nor in the multivariate analysis. Neither anti-p53 nor anti-KRas abs were associated with response to CRT. Anti-p53 abs are an independent prognostic factor for esophageal cancer patients treated with CRT. Individualized therapeutic approaches should be evaluated in this population

Prognostic significance of anti-p53 and anti-KRas circulating antibodies in esophageal cancer patients treated with chemoradiotherapy.

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Blanchard, Pierre; Quero, Laurent; Pacault, Vincent; Schlageter, Marie-Helene; Baruch-Hennequin, Valerie; Hennequin, Christophe

2012-03-26

P53 mutations are an adverse prognostic factor in esophageal cancer. P53 and KRas mutations are involved in chemo-radioresistance. Circulating anti-p53 or anti-KRas antibodies are associated with gene mutations. We studied whether anti-p53 or anti-KRas auto-antibodies were prognostic factors for response to chemoradiotherapy (CRT) or survival in esophageal carcinoma. Serum p53 and KRas antibodies (abs) were measured using an ELISA method in 97 consecutive patients treated at Saint Louis University Hospital between 1999 and 2002 with CRT for esophageal carcinoma (squamous cell carcinoma (SCCE) 57 patients, adenocarcinoma (ACE) 27 patients). Patient and tumor characteristics, response to treatment and the follow-up status of 84 patients were retrospectively collected. The association between antibodies and patient characteristics was studied. Univariate and multivariate survival analyses were conducted. Twenty-four patients (28%) had anti-p53 abs. Abs were found predominantly in SCCE (p = 0.003). Anti-p53 abs were associated with a shorter overall survival in the univariate analysis (HR 1.8 [1.03-2.9], p = 0.04). In the multivariate analysis, independent prognostic factors for overall and progression-free survival were an objective response to CRT, the CRT strategy (alone or combined with surgery [preoperative]) and anti-p53 abs. None of the long-term survivors had p53 abs. KRas abs were found in 19 patients (23%, no difference according to the histological type). There was no significant association between anti-KRas abs and survival neither in the univariate nor in the multivariate analysis. Neither anti-p53 nor anti-KRas abs were associated with response to CRT. Anti-p53 abs are an independent prognostic factor for esophageal cancer patients treated with CRT. Individualized therapeutic approaches should be evaluated in this population.

Prognostic significance of anti-p53 and anti-KRas circulating antibodies in esophageal cancer patients treated with chemoradiotherapy

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Blanchard Pierre

2012-03-01

Full Text Available Abstract Background P53 mutations are an adverse prognostic factor in esophageal cancer. P53 and KRas mutations are involved in chemo-radioresistance. Circulating anti-p53 or anti-KRas antibodies are associated with gene mutations. We studied whether anti-p53 or anti-KRas auto-antibodies were prognostic factors for response to chemoradiotherapy (CRT or survival in esophageal carcinoma. Methods Serum p53 and KRas antibodies (abs were measured using an ELISA method in 97 consecutive patients treated at Saint Louis University Hospital between 1999 and 2002 with CRT for esophageal carcinoma (squamous cell carcinoma (SCCE 57 patients, adenocarcinoma (ACE 27 patients. Patient and tumor characteristics, response to treatment and the follow-up status of 84 patients were retrospectively collected. The association between antibodies and patient characteristics was studied. Univariate and multivariate survival analyses were conducted. Results Twenty-four patients (28% had anti-p53 abs. Abs were found predominantly in SCCE (p = 0.003. Anti-p53 abs were associated with a shorter overall survival in the univariate analysis (HR 1.8 [1.03-2.9], p = 0.04. In the multivariate analysis, independent prognostic factors for overall and progression-free survival were an objective response to CRT, the CRT strategy (alone or combined with surgery [preoperative] and anti-p53 abs. None of the long-term survivors had p53 abs. KRas abs were found in 19 patients (23%, no difference according to the histological type. There was no significant association between anti-KRas abs and survival neither in the univariate nor in the multivariate analysis. Neither anti-p53 nor anti-KRas abs were associated with response to CRT. Conclusions Anti-p53 abs are an independent prognostic factor for esophageal cancer patients treated with CRT. Individualized therapeutic approaches should be evaluated in this population.

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

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Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

Reprogramming stem cells is a microenvironmental task

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Bissell, Mina J; Inman, Jamie

2008-10-14

That tumor cells for all practical purposes are unstable and plastic could be expected. However, the astonishing ability of the nuclei from cells of normal adult tissues to be reprogrammed - given the right embryonic context - found its final truth even for mammals in the experiments that allowed engineering Dolly (1). The landmark experiments showed that nuclei originating from cells of frozen mammary tissues were capable of being reprogrammed by the embryonic cytoplasm and its microenvironment to produce a normal sheep. The rest is history. However, whether microenvironments other than those of the embryos can also reprogram adult cells of different tissue origins still containing their cytoplasm is of obvious interest. In this issue of PNAS, the laboratory of Gilbert Smith (2) reports on how the mammary gland microenvironment can reprogram both embryonic and adult stem neuronal cells. The work is a follow-up to their previous report on testis stem cells that were reprogrammed by the mammary microenvironment (3). They demonstrated that cells isolated from the seminiferous tubules of the mature testis, mixed with normal mammary epithelial cells, contributed a sizable number of epithelial progeny to normal mammary outgrowths in transplanted mammary fat pads. However, in those experiments they were unable to distinguish which subpopulation of the testis cells contributed progeny to the mammary epithelial tree. The current work adds new, compelling, and provocative information to our understanding of stem cell plasticity. Booth et al. (2) use neuronal stem cells (NSCs) isolated from WAP-cre/R26R mice combined with unlabeled mammary epithelial cells that subsequently are implanted in cleared mammary fat pads. In this new microenvironment, the NSCs that are incorporated into the branching mammary tree make chimeric glands (Fig. 1) that remarkably can also express the milk protein {beta}-casein, progesterone receptor, and estrogen receptor {alpha}. Remarkably, the

Epigenetic Reprogramming of Lineage-Committed Human Mammary Epithelial Cells Requires DNMT3A and Loss of DOT1L

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Jerrica L. Breindel

2017-09-01

Full Text Available Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment.

Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations

DEFF Research Database (Denmark)

Holck, Susanne; Bonde, Jesper; Pedersen, Helle

2016-01-01

Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ...... detectable increases in phosphorylation of ERK (pERK), we stained biopsies from 36 CRC patients with activating mutations in the BRAF gene (BRAFV600E: BRAF(m)), the KRAS gene (KRAS(m)) or in neither (BRAF/KRAS(n)) with this optimized method. Staining was scored in blind-coded specimens by two observers....... Staining of stromal cells was used as a positive control. BRAF(m) or KRAS(m) tumors did not show higher staining scores than BRAF/KRAS(n) tumors. Although BRAFV600E staining occurred in over 90% of cancer cells in all 9 BRAF(m) tumors, 3 only showed staining for pERK in less than 10% of cancer cell nuclei...

Association of folate intake, dietary habits, smoking and COX-2 promotor-765G > C polymorphism with K-ras mutation in patients with colorectal cancer

International Nuclear Information System (INIS)

Kamal, M.M.; Youssef, O.Z.; Lotfy, A.N.; Elsaed, E.T.; Fawzy, M.M.T.

2012-01-01

Background: Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G > C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. Aim of work: To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, —765G > C. Subjects and methods: The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) —765G > C polymorphism were determined by PCR-RFLP. Results: The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96 ± 51.3 ng/ml) than the negative group (216.6 ± 166.4 ng/ml), (P < 0.01). Higher folate levels were found in males than females (median = 173 ng/ml and 85 ng/ml; respectively, P = 0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P = 0.0018) followed by gender (P = 0

External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds

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Bellon, Ellen; Ligtenberg, Marjolijn J.L.; Tejpar, Sabine; Cox, Karen; de Hertogh, Gert; de Stricker, Karin; Edsjö, Anders; Gorgoulis, Vassilis; Höfler, Gerald; Jung, Andreas; Kotsinas, Athanassios; Laurent-Puig, Pierre; López-Ríos, Fernando; Hansen, Tine Plato; Rouleau, Etienne; Vandenberghe, Peter; van Krieken, Johan J.M.

2011-01-01

The use of epidermal growth factor receptor–targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild-type KRAS tumors by the European Medicines Agency since 2008, based on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors. In an effort to ensure optimal, uniform, and reliable community-based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use their own preferred method for histological evaluation, DNA isolation, and mutation analysis. In this study, we analyze the setup of the KRAS scheme. We analyzed the advantages and disadvantages of the regional scheme organization by analyzing the outcome of genotyping results, analysis of tumor percentage, and written reports. We conclude that only 70% of laboratories correctly identified the KRAS mutational status in all samples. Both the false-positive and false-negative results observed negatively affect patient care. Reports of the KRAS test results often lacked essential information. We aim to further expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization. PMID:21441573

DMPD: Cellular reprogramming by gram-positive bacterial components: a review. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 16885502 Cellular reprogramming by gram-positive bacterial components: a review. Bu...(.csml) Show Cellular reprogramming by gram-positive bacterial components: a review. PubmedID 16885502 Title... Cellular reprogramming by gram-positive bacterial components: a review. Authors

Comparison of EGFR and KRAS Status between Primary Non-small Cell Lung Cancer and Corresponding Metastases: A Systematic Review and meta-analysis

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Chengbo HAN

2010-09-01

Full Text Available Background and objective Epidermal growth factor receptor (EGFR and KRAS status were particularly critical for the choice of first-line targeted therapy of non-small cell lung cancer (NSCLC, while the primary tumor and metastases might be different in the EGFR and KRAS gene status. The aim of this pooled analysis is to compare EGFR and KRAS status in matching primary NSCLC and metastases and further to guide clinical practice. Methods Systematic computerized searches of the Pubmed and Medline databases (up to May 10, 2010 meeting specified search criteria were performed, followed by a further screening according to inclusive and exclusive criteria. Results Fourteen articles were selected into the final meta-analysis with paired primary and metastatic cases of 598. Expression level of EGFR protein and mutation frequency of KRAS gene in primary tumors were higher than that in metastases, relative risk (RR=1.13 (95%CI: 0.98-1.31, P=0.09 and RR=1.39 (95%CI: 0.95-2.03, P=0.09, respectively. EGFR gene copy number in metastases was higher than that in primary tumor, RR=0.74 (95%CI: 0.53-1.02, P=0.06. There was no statistically significant difference of EGFR mutation frequency in primary tumors and metastases (P=0.31. The discordant rate in primary and metastases was 17.09% for EGFR mutation, 27.07% for EGFR amplification, 27.84% for EGFR protein expression and 25.91% for KRAS mutation. Conclusion The systematic analysis showed that the EGFR mutation status in primary lung cancer and corresponding metastases was more stable than KRAS gene. KRAS mutation in primary lung cancerous foci seems to better reflect systemically cancerous genetic characteristics of KRAS gene. Determination of KRAS gene status based merely on metastatic foci might lead to more resistant selections of EGFR tyrosine kinase inhibitor (TKI therapy. Combined detection of EGFR and KRAS mutation from primary NSCLC foci might serve as a better predictive biomarker for anti-EGFR targeted

The prognostic values of EGFR expression and KRAS mutation in patients with synchronous or metachronous metastatic colorectal cancer

International Nuclear Information System (INIS)

Huang, Ching-Wen; Wang, Jaw-Yuan; Tsai, Hsiang-Lin; Chen, Yi-Ting; Huang, Chun-Ming; Ma, Cheng-Jen; Lu, Chien-Yu; Kuo, Chao-Hung; Wu, Deng-Chyang; Chai, Chee-Yin

2013-01-01

The epidermal growth factor receptor (EGFR)/RAS/RAF/MEK/MAPK pathway is an important pathway in the carcinogenesis, invasion and metastasis of colorectal cancers (CRCs). We conducted a retrospective study to determine the prognostic values of EGFR expression and KRAS mutation in patients with metastatic CRC (mCRC) based on synchronous or metachronous status. From October 2002 to March 2012, 205 patients with mCRC were retrospectively analyzed; 98 were found to have metachronous mCRC while 107 were found to have synchronous mCRC. The EGFR expressions were determinate by IHC (immunohistochemistry) analysis and categorized 1+ (weak intensity), 2+ (moderate intensity), and 3+ (strong intensity). Genomic DNA was isolated from frozen primary CRC tissues and direct sequencing of KRAS was performed. The clinicopathological features of these mCRC patients were retrospectively investigated according to EGFR expression and KRAS mutation status. Moreover, we analyzed the prognostic values of EGFR expression and KRAS mutation among these patients. Of the 205 patients with mCRC, EGFR expression was analyzed in 167 patients, and positive EGFR expression was noted in 140 of those patients (83.8%). KRAS mutation was investigated in 205 patients and mutations were noted in 88 of those patients (42.9%). In patients with metachronous mCRC, positive EGFR expression was significantly correlated with well-and moderately-differentiated tumors (P = 0.028), poorer disease-free survival (DFS) (P < 0.001), and overall survival (OS) (P < 0.001). Furthermore, positive EGFR expression was a significant independent prognostic factor of DFS (P = 0.006, HR: 4.012, 95% CI: 1.130–8.445) and OS (P = 0.028, HR: 3.090, 95% CI: 1.477–10.900) in metachronous mCRC patients. KRAS mutation status was not significantly related to DFS and OS of patients with metachronous mCRC; likewise, KRAS mutation status was not significantly different in the progression-free survival (PFS) and OS of patients with

Clinical implementation of KRAS testing in metastatic colorectal carcinoma: the pathologist's perspective.

Science.gov (United States)

Ross, Jeffrey S

2012-10-01

Mutation status of the KRAS gene identifies a distinct disease subtype of metastatic colorectal carcinoma that does not respond to antibody therapeutics targeting the epidermal growth factor receptor. This is currently the only validated marker in metastatic colorectal carcinoma with a clear implication in treatment selection. KRAS testing is widely accepted in clinical practice to guide metastatic colorectal carcinoma therapeutic decisions, and there are many commercially available platforms to perform the test. To evaluate the critical role of pathologists in the full implementation of KRAS testing by optimizing tumor tissue collection and fixation procedures and by choosing testing technologies and reliable Clinical Laboratory Improvement Amendments of 1988-certified laboratories to perform the tests. Prospective clinical trials, retrospective studies, and quality assessment and survey reports were identified in the following databases: PubMed, American Society of Clinical Oncology Proceedings (American Society of Clinical Oncology Annual Meeting and Gastrointestinal Cancer Symposium) and European Society for Medical Oncology Proceedings (Annals of Oncology European Society for Medical Oncology Congress and Annals of Oncology World Congress on Gastrointestinal Cancers). More bona fide standards are needed to address the variety of available test methods, which have different performance characteristics including speed, sensitivity to detect rare mutations, and technical requirements. Refined standards addressing timing of KRAS testing, laboratory performance and accuracy, quality assurance and control, proper tissue collection, and appropriate result reporting would also be greatly beneficial. Pathologists should be aware that the amount of information they need to manage will increase, because future trends and technological advances will enhance the predictive power of diagnostic tests or the scope of the biomarker panels tested routinely across tumor types.

Naked Mole Rat Cells Have a Stable Epigenome that Resists iPSC Reprogramming

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Li Tan

2017-11-01

Full Text Available Naked mole rat (NMR is a valuable model for aging and cancer research due to its exceptional longevity and cancer resistance. We observed that the reprogramming efficiency of NMR fibroblasts in response to OSKM was drastically lower than that of mouse fibroblasts. Expression of SV40 LargeT antigen (LT dramatically improved reprogramming of NMR fibroblasts. Inactivation of Rb alone, but not p53, was sufficient to improve reprogramming efficiency, suggesting that NMR chromatin may be refractory to reprogramming. Analysis of the global histone landscape revealed that NMR had higher levels of repressive H3K27 methylation marks and lower levels of activating H3K27 acetylation marks than mouse. ATAC-seq revealed that in NMR, promoters of reprogramming genes were more closed than mouse promoters, while expression of LT led to massive opening of the NMR promoters. These results suggest that NMR displays a more stable epigenome that resists de-differentiation, contributing to the cancer resistance and longevity of this species.

A unique Oct4 interface is crucial for reprogramming to pluripotency

NARCIS (Netherlands)

Esch, Daniel; Vahokoski, Juha; Groves, Matthew R; Pogenberg, Vivian; Cojocaru, Vlad; Vom Bruch, Hermann; Han, Dong; Drexler, Hannes C A; Araúzo-Bravo, Marcos J; Ng, Calista K L; Jauch, Ralf; Wilmanns, Matthias; Schöler, Hans R

Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot

Nove interpretacije fluvialnih sedimentov na krasu = New interpretations of fluvial sediments from the Kras

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Andrej Mihevc

2007-01-01

Full Text Available Important unroofed caves with fluvial sediments from Divaški kras, Matarsko podoljePodgorski kras are presented. Extend of the phenomena and relation to the existingand karst surface and geomorphological meaning of them are described. Sedimentsthem were analysed and dated with different methods. The largest age of the sedimentfound in the unroofed cave excavated in Črnotiče quarry. In the cave wall fossil remainsstygobiont Marifugia cavatica were covered by 3.2-4.1 Ma old fluvial sediments.

Left Dorsal Premotor Cortex and Supramarginal Gyrus Complement Each Other during Rapid Action Reprogramming

DEFF Research Database (Denmark)

Hartwigsen, Gesa; Bestmann, Sven; Ward, Nick S

2012-01-01

The ability to discard a prepared action plan in favor of an alternative action is critical when facing sudden environmental changes. We tested whether the functional contribution of left supramarginal gyrus (SMG) during action reprogramming depends on the functional integrity of left dorsal...... premotor cortex (PMd). Adopting a dual-site repetitive transcranial magnetic stimulation (rTMS) strategy, we first transiently disrupted PMd with "off-line" 1 Hz rTMS and then applied focal "on-line" rTMS to SMG while human subjects performed a spatially precued reaction time (RT) task. Effective on-line r......TMS of SMG but not sham rTMS of SMG increased errors when subjects had to reprogram their action in response to an invalid precue regardless of the type of preceding off-line rTMS. This suggests that left SMG primarily contributes to the on-line updating of actions by suppressing invalidly prepared responses...

KRAS mutations and CDKN2A promoter methylation show an interactive adverse effect on survival and predict recurrence of rectal cancer.

Science.gov (United States)

Kohonen-Corish, Maija R J; Tseung, Jason; Chan, Charles; Currey, Nicola; Dent, Owen F; Clarke, Stephen; Bokey, Les; Chapuis, Pierre H

2014-06-15

Colonic and rectal cancers differ in their clinicopathologic features and treatment strategies. Molecular markers such as gene methylation, microsatellite instability and KRAS mutations, are becoming increasingly important in guiding treatment decisions in colorectal cancer. However, their association with clinicopathologic variables and utility in the management of rectal cancer is still poorly understood. We analyzed CDKN2A gene methylation, CpG island methylator phenotype (CIMP), microsatellite instability and KRAS/BRAF mutations in a cohort of 381 rectal cancers with extensive clinical follow-up data. BRAF mutations (2%), CIMP-high (4%) and microsatellite instability-high (2%) were rare, whereas KRAS mutations (39%), CDKN2A methylation (20%) and CIMP-low (25%) were more common. Only CDKN2A methylation and KRAS mutations showed an association with poor overall survival but these did not remain significant when analyzed with other clinicopathologic factors. In contrast, this prognostic effect was strengthened by the joint presence of CDKN2A methylation and KRAS mutations, which independently predicted recurrence of cancer and was associated with poor overall and cancer-specific survival. This study has identified a subgroup of more aggressive rectal cancers that may arise through the KRAS-p16 pathway. It has been previously shown that an interaction of p16 deficiency and oncogenic KRAS promotes carcinogenesis in the mouse and is characterized by loss of oncogene-induced senescence. These findings may provide avenues for the discovery of new treatments in rectal cancer. © 2013 UICC.

Direct lineage reprogramming of mouse fibroblasts to functional midbrain dopaminergic neuronal progenitors

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Han-Seop Kim

2014-01-01

Full Text Available The direct lineage reprogramming of somatic cells to other lineages by defined factors has led to innovative cell-fate-change approaches for providing patient-specific cells. Recent reports have demonstrated that four pluripotency factors (Oct4, Sox2, Klf4, and c-Myc are sufficient to directly reprogram fibroblasts to other specific cells, including induced neural stem cells (iNSCs. Here, we show that mouse fibroblasts can be directly reprogrammed into midbrain dopaminergic neuronal progenitors (DPs by temporal expression of the pluripotency factors and environment containing sonic hedgehog and fibroblast growth factor 8. Within thirteen days, self-renewing and functional induced DPs (iDPs were generated. Interestingly, the inhibition of both Jak and Gsk3β notably enhanced the iDP reprogramming efficiency. We confirmed the functionality of the iDPs by showing that the dopaminergic neurons generated from iDPs express midbrain markers, release dopamine, and show typical electrophysiological profiles. Our results demonstrate that the pluripotency factors-mediated direct reprogramming is an invaluable strategy for supplying functional and proliferating iDPs and may be useful for other neural progenitors required for disease modeling and cell therapies for neurodegenerative disorders.

Multifunctional imaging signature for V-KI-RAS2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in colorectal cancer.

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Miles, Kenneth A; Ganeshan, Balaji; Rodriguez-Justo, Manuel; Goh, Vicky J; Ziauddin, Zia; Engledow, Alec; Meagher, Marie; Endozo, Raymondo; Taylor, Stuart A; Halligan, Stephen; Ell, Peter J; Groves, Ashley M

2014-03-01

This study explores the potential for multifunctional imaging to provide a signature for V-KI-RAS2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutations in colorectal cancer. This prospective study approved by the institutional review board comprised 33 patients undergoing PET/CT before surgery for proven primary colorectal cancer. Tumor tissue was examined histologically for presence of the KRAS mutations and for expression of hypoxia-inducible factor-1 (HIF-1) and minichromosome maintenance protein 2 (mcm2). The following imaging parameters were derived for each tumor: (18)F-FDG uptake ((18)F-FDG maximum standardized uptake value [SUVmax]), CT texture (expressed as mean of positive pixels [MPP]), and blood flow measured by dynamic contrast-enhanced CT. A recursive decision tree was developed in which the imaging investigations were applied sequentially to identify tumors with KRAS mutations. Monte Carlo analysis provided mean values and 95% confidence intervals for sensitivity, specificity, and accuracy. The final decision tree comprised 4 decision nodes and 5 terminal nodes, 2 of which identified KRAS mutants. The true-positive rate, false-positive rate, and accuracy (95% confidence intervals) of the decision tree were 82.4% (63.9%-93.9%), 0% (0%-10.4%), and 90.1% (79.2%-96.0%), respectively. KRAS mutants with high (18)F-FDG SUVmax and low MPP showed greater frequency of HIF-1 expression (P = 0.032). KRAS mutants with low (18)F-FDG SUV(max), high MPP, and high blood flow expressed mcm2 (P = 0.036). Multifunctional imaging with PET/CT and recursive decision-tree analysis to combine measurements of tumor (18)F-FDG uptake, CT texture, and perfusion has the potential to identify imaging signatures for colorectal cancers with KRAS mutations exhibiting hypoxic or proliferative phenotypes.

Role of [{sup 18}F]FDG PET in prediction of KRAS and EGFR mutation status in patients with advanced non-small-cell lung cancer

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Caicedo, Carlos; Garcia-Velloso, Maria Jose; Vigil Diaz, Carmen; Richter Echevarria, Jose Angel [University of Navarra, Nuclear Medicine Department, University Clinic of Navarra, Pamplona (Spain); Lozano, Maria Dolores; Labiano, Tania [University of Navarra, Pathology Department, University Clinic of Navarra, Pamplona (Spain); Lopez-Picazo, Jose Maria; Gurpide, Alfonso; Perez Gracia, Jose Luis [University of Navarra, Oncology Department, University Clinic of Navarra, Pamplona (Spain); Zulueta, Javier [University of Navarra, Pulmonology Department, University Clinic of Navarra, Pamplona (Spain)

2014-11-15

The tumour molecular profile predicts the activity of epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). However, tissue availability and tumour heterogeneity limit its assessment. We evaluated whether [{sup 18}F]FDG PET might help predict KRAS and EFGR mutation status in NSCLC. Between January 2005 and October 2011, 340 NSCLC patients were tested for KRAS and EGFR mutation status. We identified patients with stage III and IV disease who had undergone [{sup 18}F]FDG PET/CT scanning for initial staging. SUVpeak, SUVmax and SUVmean of the single hottest tumour lesions were calculated, and their association with KRAS and EGFR mutation status was assessed. A receiver operator characteristic (ROC) curve analysis and a multivariate analysis (including SUVmean, gender, age and AJCC stage) were performed to identify the potential value of [{sup 18}F]FDG PET/CT for predicting KRAS mutation. From 102 patients staged using [{sup 18}F]FDG PET/CT, 28 (27 %) had KRAS mutation (KRAS+), 22 (22 %) had EGFR mutation (EGFR+) and 52 (51 %) had wild-type KRAS and EGFR profiles (WT). KRAS+ patients showed significantly higher [{sup 18}F]FDG uptake than EGFR+ and WT patients (SUVmean 9.5, 5.7 and 6.6, respectively; p < 0.001). No significant differences were observed in [{sup 18}F]FDG uptake between EGFR+ patients and WT patients. ROC curve analysis for KRAS mutation status discrimination yielded an area under the curve of 0.740 for SUVmean (p < 0.001). The multivariate analysis showed a sensitivity and specificity of 78.6 % and 62.2 %, respectively, and the AUC was 0.773. NSCLC patients with tumours harbouring KRAS mutations showed significantly higher [{sup 18}F]FDG uptake than WT patients, as assessed in terms of SUVpeak, SUVmax and SUVmean. A multivariate model based on age, gender, AJCC stage and SUVmean might be used as a predictive marker of KRAS mutation status in patients with stage III or IV NSCLC. (orig.)

Genetic analysis of tumorigenesis: XXXII. Localization of constitutionally amplified KRAS sequences to Chinese hamster chromosomes X and Y by in situ hybridization.

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Stenman, G; Anisowicz, A; Sager, R

1988-11-01

The KRAS gene is constitutionally amplified in the Chinese hamster. We have mapped the amplified sequences by in situ hybridization to two major sites on the X and Y chromosomes, Xq4 and Yp2. No autosomal site was detected despite a search under relaxed hybridization conditions. KRAS DNA is amplified about 50-fold compared to a human cell line known to have a diploid number of KRAS sequences, whereas mRNA expression is 5- to 10-fold lower than in normal human cells. While mRNA expression levels do not necessarily parallel gene copy number, the low expression level strongly suggests that the amplified sequences are transcriptionally silent. It is suggested that the amplified sequences arose from the original KRAS gene on chromosome 8 and that the KRAS sequences on the Y chromosome arose by X-Y recombination.

Developmental Programming of Adult Disease: Reprogramming by Melatonin?

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You-Lin Tain

2017-02-01

Full Text Available Adult-onset chronic non-communicable diseases (NCDs can originate from early life through so-called the “developmental origins of health and disease” (DOHaD or “developmental programming”. The DOHaD concept offers the “reprogramming” strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs.

The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

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Brix, Jacob; Zhou, Yan; Luo, Yonglun

2015-01-01

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation cap...

Phase II marker-driven trial of panitumumab and chemotherapy in KRAS wild-type biliary tract cancer

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Jensen, L H; Lindebjerg, J; Ploen, J

2012-01-01

BACKGROUND: Combination chemotherapy has proven beneficial in biliary tract cancer and further improvements may be achieved by individualizing treatment based on biomarkers and by adding biological agents. We report the effect of chemotherapy with panitumumab as first-line therapy for KRAS wild....... Combination chemotherapy with panitumumab in patients with KRAS wild-type tumors met the efficacy criteria for future testing in a randomized trial....

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

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Alvaro Muñoz-López

2016-10-01

Full Text Available Induced pluripotent stem cells (iPSCs are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

One-step isothermal detection of multiple KRAS mutations by forming SNP specific hairpins on a gold nanoshell.

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Chung, Chan Ho; Kim, Joong Hyun

2018-04-24

We developed a one-step isothermal method for typing multiple KRAS mutations using a designed set of primers to form a hairpin on a gold nanoshell upon being ligated by a SNP specific DNA ligase after binding of targets. As a result, we could detect as low as 20 attomoles of KRAS mutations within 1 h.

Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma.

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Jangchul Park

Full Text Available This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB-IV and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups' characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159, KRAS mutations (n = 55, or ALK rearrangements (n = 51. Among the three groups, we evaluated only patients with stage IIIB-IV lung adenocarcinoma who had EGFR mutations (n = 126, KRAS mutations (n = 35, or ALK rearrangements (n = 47. We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009. Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003. Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively. Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049. Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively. Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively. In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

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Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

2014-01-01

Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

The Current State of Nanoparticle-Induced Macrophage Polarization and Reprogramming Research

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Xiaoyuan Miao

2017-02-01

Full Text Available Macrophages are vital regulators of the host defense in organisms. In response to different local microenvironments, resting macrophages (M0 can be polarized into different phenotypes, pro-inflammatory (M1 or anti-inflammatory (M2, and perform different roles in different physiological or pathological conditions. Polarized macrophages can also be further reprogrammed by reversing their phenotype according to the changed milieu. Macrophage polarization and reprogramming play essential roles in maintaining the steady state of the immune system and are involved in the processes of many diseases. As foreign substances, nanoparticles (NPs mainly target macrophages after entering the body. NPs can perturb the polarization and reprogramming of macrophages, affect their immunological function and, therefore, affect the pathological process of disease. Optimally-designed NPs for the modulation of macrophage polarization and reprogramming might provide new solutions for treating diseases. Systematically investigating how NPs affect macrophage polarization is crucial for understanding the regulatory effects of NPs on immune cells in vivo. In this review, macrophage polarization by NPs is summarized and discussed.

Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

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Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

2016-01-01

Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA from Patients with Non-Small Cell Lung Cancer (NSCLC.

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James L Sherwood

Full Text Available Non-invasive mutation testing using circulating tumour DNA (ctDNA is an attractive premise. This could enable patients without available tumour sample to access more treatment options.Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits.2 hr incubation time and double plasma centrifugation (2000 x g reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA. Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT (Streck, after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield.This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

KRAS rs61764370 is associated with HER2-overexpressed and poorly-differentiated breast cancer in hormone replacement therapy users: a case control study

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Cerne, Jasmina-Ziva; Stegel, Vida; Gersak, Ksenija; Novakovic, Srdjan

2012-01-01

A single nucleotide polymorphism located in the 3'-untranslated region of the KRAS oncogene (KRAS variant; rs61764370) disrupts a let-7 miRNA binding and was recently reported to act as a genetic marker for increased risk of developing human cancers. We aimed to investigate an association of the KRAS variant with sporadic and familial breast cancer and breast tumor characteristics. Genotyping was accomplished in 530 sporadic postmenopausal breast cancer cases, 165 familial breast cancer cases (including N = 29, who test positive for BRCA1/2 mutations) and 270 postmenopausal control women using the flurogenic 5' nuclease assay. Information on hormone replacement therapy (HRT) use and tumor characteristics in sporadic breast cancer cases was ascertained from a postal questionnaire and pathology reports, respectively. Associations between the KRAS genotype and breast cancer or breast tumor characteristics were assessed using chi-square test and logistic regression models. No evidence of association was observed between the KRAS variant and risk of sporadic and familial breast cancer - either among BRCA carriers or non-BRCA carriers. The KRAS variant was statistically significantly more often associated with human epidermal growth factor receptor 2 (HER2) - positive tumors and tumors of higher histopathologic grade. However, both associations were detected only in HRT users. Our data do not support the hypothesis that the KRAS variant rs61764370 is implicated in the aetiology of sporadic or of familial breast cancer. In postmenopausal women using HRT, the KRAS variant might lead to HER2 overexpressed and poorly-differentiated breast tumors, both indicators of a worse prognosis

Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

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Quintana-Bustamante, Oscar; Segovia, Jose C

2016-01-01

Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

Statin use is not associated with improved progression free survival in cetuximab treated KRAS mutant metastatic colorectal cancer patients: results from the CAIRO2 study.

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Lisanne L Krens

Full Text Available Statins may inhibit the expression of the mutant KRAS phenotype by preventing the prenylation and thus the activation of the KRAS protein. This study was aimed at retrospectively evaluating the effect of statin use on outcome in KRAS mutant metastatic colorectal cancer patients (mCRC treated with cetuximab. Treatment data were obtained from patients who were treated with capecitabine, oxaliplatin bevacizumab ± cetuximab in the phase III CAIRO2 study. A total of 529 patients were included in this study, of whom 78 patients were on statin therapy. In patients with a KRAS wild type tumor (n = 321 the median PFS was 10.3 vs. 11.4 months for non-users compared to statin users and in patients with a KRAS mutant tumor (n = 208 this was 7.6 vs. 6.2 months, respectively. The hazard ratio (HR for PFS for statin users was 1.12 (95% confidence interval 0.78-1.61 and was not influenced by treatment arm, KRAS mutation status or the KRAS*statin interaction. Statin use adjusted for covariates was not associated with increased PFS (HR = 1.01, 95% confidence interval 0.71-1.54. In patients with a KRAS wild type tumor the median OS for non-users compared to statin users was 22.4 vs. 19.8 months and in the KRAS mutant tumor group the OS was 18.1 vs. 14.5 months. OS was significantly shorter in statin users versus non-users (HR = 1.54; 95% confidence interval 1.06-2.22. However, statin use, adjusted for covariates was not associated with increased OS (HR = 1.41, 95% confidence interval 0.95-2.10. In conclusion, the use of statins at time of diagnosis was not associated with an improved PFS in KRAS mutant mCRC patients treated with chemotherapy and bevacizumab plus cetuximab.

A Fast, Sensitive and Accurate High Resolution Melting (HRM Technology-Based Assay to Screen for Common K-ras Mutations

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D. Kramer

2009-01-01

Full Text Available Background: Increasing evidence points to a negative correlation between K-ras mutations and patient’s response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed.

In vivo reprogramming for heart regeneration: A glance at efficiency, environmental impacts, challenges and future directions.

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Ebrahimi, Behnam

2017-07-01

Replacing dying or diseased cells of a tissue with new ones that are converted from patient's own cells is an attractive strategy in regenerative medicine. In vivo reprogramming is a novel strategy that can circumvent the hurdles of autologous/allogeneic cell injection therapies. Interestingly, studies have demonstrated that direct injection of cardiac transcription factors or specific miRNAs into the infarct border zone of murine hearts following myocardial infarction converts resident cardiac fibroblasts into functional cardiomyocytes. Moreover, in vivo cardiac reprogramming not only drives cardiac tissue regeneration, but also improves cardiac function and survival rate after myocardial infarction. Thanks to the influence of cardiac microenvironment and the same developmental origin, cardiac fibroblasts seem to be more amenable to reprogramming toward cardiomyocyte fate than other cell sources (e.g. skin fibroblasts). Thus, reprogramming of cardiac fibroblasts to functional induced cardiomyocytes in the cardiac environment holds great promises for induced regeneration and potential clinical purposes. Application of small molecules in future studies may represent a major advancement in this arena and pharmacological reprogramming would convey reprogramming technology to the translational medicine paradigm. This study reviews accomplishments in the field of in vitro and in vivo mouse cardiac reprogramming and then deals with strategies for the enhancement of the efficiency and quality of the process. Furthermore, it discusses challenges ahead and provides suggestions for future research. Human cardiac reprogramming is also addressed as a foundation for possible application of in vivo cardiac reprogramming for human heart regeneration in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electromagnetic fields mediate efficient cell reprogramming into a pluripotent state.

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Baek, Soonbong; Quan, Xiaoyuan; Kim, Soochan; Lengner, Christopher; Park, Jung-Keug; Kim, Jongpil

2014-10-28

Life on Earth is constantly exposed to natural electromagnetic fields (EMFs), and it is generally accepted that EMFs may exert a variety of effects on biological systems. Particularly, extremely low-frequency electromagnetic fields (EL-EMFs) affect biological processes such as cell development and differentiation; however, the fundamental mechanisms by which EMFs influence these processes remain unclear. Here we show that EMF exposure induces epigenetic changes that promote efficient somatic cell reprogramming to pluripotency. These epigenetic changes resulted from EMF-induced activation of the histone lysine methyltransferase Mll2. Remarkably, an EMF-free system that eliminates Earth's naturally occurring magnetic field abrogates these epigenetic changes, resulting in a failure to undergo reprogramming. Therefore, our results reveal that EMF directly regulates dynamic epigenetic changes through Mll2, providing an efficient tool for epigenetic reprogramming including the acquisition of pluripotency.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

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El-Heliebi, Amin; Hille, Claudia; Laxman, Navya; Svedlund, Jessica; Haudum, Christoph; Ercan, Erkan; Kroneis, Thomas; Chen, Shukun; Smolle, Maria; Rossmann, Christopher; Krzywkowski, Tomasz; Ahlford, Annika; Darai, Evangelia; von Amsberg, Gunhild; Alsdorf, Winfried; König, Frank; Löhr, Matthias; de Kruijff, Inge; Riethdorf, Sabine; Gorges, Tobias M; Pantel, Klaus; Bauernhofer, Thomas; Nilsson, Mats; Sedlmayr, Peter

2018-03-01

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices. © 2017 American Association for Clinical Chemistry.

Gender Differences in Global but Not Targeted Demethylation in iPSC Reprogramming

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Inês Milagre

2017-01-01

Full Text Available Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity.

Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

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Hu, Chenxia; Li, Lanjuan

2016-01-01

Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

Generating pluripotent stem cells: Differential epigenetic changes during cellular reprogramming

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Tobin, Stacey C.; Kim, Kitai

2012-01-01

Pluripotent stem cells hold enomous potential for therapuetic applications in tissue replacement therapy. Reprogramming somatic cells from a patient donor to generate pluripotent stem cells involves both ethical concerns inherent in the use of embryonic and oocyte-derived stem cells, as well as issues of histocompatibility. Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)—derived by ectopic expression of four reprogramming factors in donor somatic cells—are supe...

CT radiogenomic characterization of EGFR, K-RAS, and ALK mutations in non-small cell lung cancer

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Rizzo, Stefania; Rampinelli, Cristiano [European Institute of Oncology, Department of Radiology, Milan (Italy); Petrella, Francesco; Spaggiari, Lorenzo [European Institute of Oncology, Department of Thoracic Surgery, Milan (Italy); Buscarino, Valentina; De Maria, Federica [University of Milan, Department of Health Sciences, Milan (Italy); Raimondi, Sara [European Institute of Oncology, Department of Epidemiology and Biostatistics, Milan (Italy); Barberis, Massimo; Fumagalli, Caterina [European Institute of Oncology, Department of Pathology, Milan (Italy); Spitaleri, Gianluca; De Marinis, Filippo [European Institute of Oncology, Department of Thoracic Oncology, Milan (Italy); Bellomi, Massimo [European Institute of Oncology, Department of Radiology, Milan (Italy); University of Milan, Department of Health Sciences, Milan (Italy)

2016-01-15

To assess the association between CT features and EGFR, ALK, KRAS mutations in non-small cell lung cancer. Patients undergoing chest CT and testing for the above gene mutations were included. Qualitative evaluation of CTs included: lobe; lesion diameter; shape; margins; ground-glass opacity; density; cavitation; air bronchogram; pleural thickening; intratumoral necrosis; nodules in tumour lobe; nodules in non-tumour lobes; pleural retraction; location; calcifications; emphysema; fibrosis; pleural contact; pleural effusion. Statistical analysis was performed to assess association of features with each gene mutation. ROC curves for gene mutations were drawn; the corresponding area under the curve was calculated. P-values <0.05 were considered significant. Of 285 patients, 60/280 (21.43 %) were positive for EGFR mutation; 31/270 (11.48 %) for ALK rearrangement; 64/240 (26.67 %) for KRAS mutation. EGFR mutation was associated with air bronchogram, pleural retraction, females, non-smokers, small lesion size, and absence of fibrosis. ALK rearrangements were associated with age and pleural effusion. KRAS mutation was associated with round shape, nodules in non-tumour lobes, and smoking. This study disclosed associations between CT features and alterations of EGFR (air bronchogram, pleural retraction, small lesion size, absence of fibrosis), ALK (pleural effusion) and KRAS (round lesion shape, nodules in non-tumour lobes). (orig.)

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

Science.gov (United States)

Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

2016-04-01

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hi

Statin use is not associated with improved progression free survival in cetuximab treated KRAS mutant metastatic colorectal cancer patients: results from the CAIRO2 study

NARCIS (Netherlands)

Krens, Lisanne L.; Simkens, Lieke H. J.; Baas, Jara M.; Koomen, Els R.; Gelderblom, Hans; Punt, Cornelis J. A.; Guchelaar, Henk-Jan

2014-01-01

Statins may inhibit the expression of the mutant KRAS phenotype by preventing the prenylation and thus the activation of the KRAS protein. This study was aimed at retrospectively evaluating the effect of statin use on outcome in KRAS mutant metastatic colorectal cancer patients (mCRC) treated with

Braf, Kras and Helicobacter pylori epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer.

Science.gov (United States)

Sabry, Dina; Ahmed, Rasha; Abdalla, Sayed; Fathy, Wael; Eldemery, Ahmed; Elamir, Azza

2016-06-01

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.

KRAS rs61764370 is associated with HER2-overexpressed and poorly-differentiated breast cancer in hormone replacement therapy users: a case control study

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Cerne Jasmina-Ziva

2012-03-01

Full Text Available Abstract Background A single nucleotide polymorphism located in the 3'-untranslated region of the KRAS oncogene (KRAS variant; rs61764370 disrupts a let-7 miRNA binding and was recently reported to act as a genetic marker for increased risk of developing human cancers. We aimed to investigate an association of the KRAS variant with sporadic and familial breast cancer and breast tumor characteristics. Methods Genotyping was accomplished in 530 sporadic postmenopausal breast cancer cases, 165 familial breast cancer cases (including N = 29, who test positive for BRCA1/2 mutations and 270 postmenopausal control women using the flurogenic 5' nuclease assay. Information on hormone replacement therapy (HRT use and tumor characteristics in sporadic breast cancer cases was ascertained from a postal questionnaire and pathology reports, respectively. Associations between the KRAS genotype and breast cancer or breast tumor characteristics were assessed using chi-square test and logistic regression models. Results No evidence of association was observed between the KRAS variant and risk of sporadic and familial breast cancer - either among BRCA carriers or non-BRCA carriers. The KRAS variant was statistically significantly more often associated with human epidermal growth factor receptor 2 (HER2 - positive tumors and tumors of higher histopathologic grade. However, both associations were detected only in HRT users. Conclusion Our data do not support the hypothesis that the KRAS variant rs61764370 is implicated in the aetiology of sporadic or of familial breast cancer. In postmenopausal women using HRT, the KRAS variant might lead to HER2 overexpressed and poorly-differentiated breast tumors, both indicators of a worse prognosis.

Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming

DEFF Research Database (Denmark)

Østrup, Olga; Pedersen, Hanne Skovsgaard; Holm, Hanne M.

2015-01-01

When a cell is reprogrammed to a new phenotype, the nucleolus undergoes more or less dramatic modulations, which can be used as a marker for the occurrence of the reprogramming. This phenomenon is most pronounced when differentiated cells are reprogrammed to totipotency when they are submitted...... of the nucleolus are summarized in this developmental context, but also as they occur in assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer. Moreover, detailed protocols for monitoring the nucleolar changes by transmission electron microscopy and immunocytochemistry...

Review on comparative efficacy of bevacizumab, panitumumab and cetuximab antibody therapy with combination of FOLFOX-4 in KRAS-mutated colorectal cancer patients.

Science.gov (United States)

Pathak, Surajit; S, Sushmitha; Banerjee, Antara; Marotta, Francesco; Gopinath, Madhumala; Murugesan, Ramachandran; Zhang, Hong; B, Bhavani; Girigoswami, Agnishwar; Sollano, Jose; Sun, Xiao-Feng

2018-01-26

Colorectal cancer, fourth leading form of cancer worldwide and is increasing in alarming rate in the developing countries. Treating colorectal cancer has become a big challenge worldwide and several antibody therapies such as bevacizumab, panitumumab and cetuximab are being used with limited success. Moreover, mutation in KRAS gene which is linked with the colorectal cancer initiation and progression further interferes with the antibody therapies. Considering median progression free survival and overall survival in account, this review focuses to identify the most efficient antibody therapy in combination with chemotherapy (FOLFOX-4) in KRAS mutated colorectal cancer patients. The bevacizumab plus FOLFOX-4 therapy shows about 9.3 months and 8.7 months of progression free survival for KRAS wild and mutant type, respectively. The overall survival is about 34.8 months for wild type whereas for the mutant it is inconclusive for the same therapy. In comparison, panitumumab results in better progression-free survival which is about (9.6 months) and overall survival is about (23.9 months) for the wild type KRAS and the overall survival is about 15.5 months for the mutant KRAS . Cetuximab plus FOLFOX-4 therapy shows about 7.7 months and 5.5 months of progression-free survival for wild type KRAS and mutant type, respectively. Thus, panitumumab shows significant improvement in overall survival rate for wild type KRAS , validating as a cost effective therapeutic for colorectal cancer therapy. This review depicts that panitumumab along with FOLFOX-4 has a higher response in colorectal cancer patients than the either of the two monoclonal antibodies plus FOLFOX-4.

Increased NQO1 but Not c-MET and Survivin Expression in Non-Small Cell Lung Carcinoma with KRAS Mutations

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Ahmet Yilmaz

2014-09-01

Full Text Available Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor-targeted therapy has been used in the treatment of LC (lung cancer, mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(PH:quinone oxidoreductase, also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin in 108 patients with non-small cell lung carcinoma (NSCLC. NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1 oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2 selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3 since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.

Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

NARCIS (Netherlands)

Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

2016-01-01

Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated

Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

DEFF Research Database (Denmark)

Steffensen, Karina Dahl; Waldstrøm, Marianne; Pallisgård, Niels

2013-01-01

OBJECTIVE: The increasing number of negative trials for ovarian cancer treatment has prompted an evaluation of new biologic agents, which in combination with chemotherapy may improve survival. The aim of this study was to investigate the response rate in platinum-resistant, KRAS wild-type ovarian...... cancer patients treated with pegylated liposomal doxorubicin (PLD) supplemented with panitumumab. PATIENTS AND METHODS: Major eligibility criteria were relapsed ovarian/fallopian/peritoneal cancer patients with platinum-resistant disease, measurable disease by GCIG CA125 criteria and KRAS wild-type...

Genome-wide transcriptional reprogramming under drought stress

KAUST Repository

Chen, Hao; Xiong, Liming

2012-01-01

Soil water deficit is one of the major factors limiting plant productivity. Plants cope with this adverse environmental condition by coordinating the up- or downregulation of an array of stress responsive genes. Reprogramming the expression

Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming

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Derek K. Smith

2016-11-01

Full Text Available Pro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2, forskolin (F, and dorsomorphin (D can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals.

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

Science.gov (United States)

Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

2017-06-08

Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice via suppressing aldo-keto reductase family 1B10 (AKR1B10).

Science.gov (United States)

Li, Haonan; Yang, Allison L; Chung, Yeon Tae; Zhang, Wanying; Liao, Jie; Yang, Guang-Yu

2013-09-01

Sulindac has been identified as a competitive inhibitor of aldo-keto reductase 1B10 (AKR1B10), an enzyme that plays a key role in carcinogenesis. AKR1B10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl. There have been no studies though showing that the inhibition of PDAC by sulindac is via inhibition of AKR1B10, particularly the metabolism of farnesyl/geranylgeranyl and Kras protein prenylation. To determine the chemopreventive effects of sulindac on pancreatic carcinogenesis, 5-week-old LSL-Kras(G12D)-LSL-Trp53(R172H)-Pdx-1-Cre mice (Pan(kras/p53) mice) were fed an AIN93M diet with or without 200 p.p.m. sulindac (n = 20/group). Kaplan-Meier survival analysis showed that average animal survival in Pan(kras/p53) mice was 143.7 ± 8.8 days, and average survival with sulindac was increased to 168.0 ± 8.8 days (P < 0.005). Histopathological analyses revealed that 90% of mice developed PDAC, 10% with metastasis to the liver and lymph nodes. With sulindac, the incidence of PDAC was reduced to 56% (P < 0.01) and only one mouse had lymph node metastasis. Immunochemical analysis showed that sulindac significantly decreased Ki-67-labeled cell proliferation and markedly reduced the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Raf and mitogen-activated protein kinase kinase 1 and 2. In in vitro experiments with PDAC cells from Pan(kras/p53) mice, sulindac exhibited dose-dependent inhibition of AKR1B10 activity. By silencing AKR1B10 expression through small interfering RNA or by sulindac treatment, these in vitro models showed a reduction in Kras and human DNA-J homolog 2 protein prenylation, and downregulation of phosphorylated C-raf, ERK1/2 and MEK1/2 expression. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis by the inhibition of Kras protein prenylation by targeting AKR1B10.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

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Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

2016-01-01

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Eficacia de la terapia génica antisentido utilizando oligonucleótidos anti K-ras y antitelomerasa en cáncer colorrectal

OpenAIRE

Lledó, S.; Alfonso, R.; Aliño, S. F.

2005-01-01

Aim: to test the efficacy of anti-k-ras and antitelomerase oligonucleotides for disabling colorectal cancer cell growth. Material and methods: an established human colorectal cancer cell line (SW 480, ATTC®) was used. Oligodeoxiribonucleotides (ODNs) have a phosphorotioate modification to ensure intracellular intake. We used an antitelomerase ODN (Telp5) and two anti-k-ras ODNs (AS-KRAS and ISIS). AS-KRAS is designed to join the k-ras oncogene's exon 1. ISIS links to the terminal transcriptio...

First-Line Cetuximab Monotherapy in KRAS/NRAS/BRAF Mutation-Negative Colorectal Cancer Patients.

Science.gov (United States)

Moiseyenko, Vladimir M; Moiseyenko, Fedor V; Yanus, Grigoriy A; Kuligina, Ekatherina Sh; Sokolenko, Anna P; Bizin, Ilya V; Kudriavtsev, Alexey A; Aleksakhina, Svetlana N; Volkov, Nikita M; Chubenko, Vyacheslav A; Kozyreva, Kseniya S; Kramchaninov, Mikhail M; Zhuravlev, Alexandr S; Shelekhova, Kseniya V; Pashkov, Denis V; Ivantsov, Alexandr O; Venina, Aigul R; Sokolova, Tatyana N; Preobrazhenskaya, Elena V; Mitiushkina, Natalia V; Togo, Alexandr V; Iyevleva, Aglaya G; Imyanitov, Evgeny N

2018-06-01

Colorectal carcinomas (CRCs) are sensitive to treatment by anti-epidermal growth factor receptor (EGFR) antibodies only if they do not carry activating mutations in down-stream EGFR targets (KRAS/NRAS/BRAF). Most clinical trials for chemo-naive CRC patients involved combination of targeted agents and chemotherapy, while single-agent cetuximab or panitumumab studies included either heavily pretreated patients or subjects who were not selected on the basis of molecular tests. We hypothesized that anti-EGFR therapy would have significant efficacy in chemo-naive patients with KRAS/NRAS/BRAF mutation-negative CRC. Nineteen patients were prospectively included in the study. Two (11%) patients experienced partial response (PR) and 11 (58%) subjects showed stable disease (SD). Median time to progression approached 6.1 months (range 1.6-15.0 months). Cetuximab efficacy did not correlate with RNA expression of EGFR and insulin-like growth factor 2 (IGF2). Only one tumor carried PIK3CA mutation, and this CRC responded to cetuximab. Exome analysis of patients with progressive disease (PD) revealed 1 CRC with high-level microsatellite instability and 1 instance of HER2 oncogene amplification; 3 of 4 remaining patients with PD had allergic reactions to cetuximab, while none of the subjects with PR or SD had this complication. Comparison with 19 retrospective KRAS/NRAS/BRAF mutation-negative patients receiving first-line fluoropyrimidines revealed no advantages or disadvantages of cetuximab therapy. Cetuximab demonstrates only modest efficacy when given as a first-line monotherapy to KRAS/NRAS/BRAF mutation-negative CRC patients. It is of question, why meticulous patient selection, which was undertaken in the current study, did not result in the improvement of outcomes of single-agent cetuximab treatment.

Utilization of Cell-Transfer Technique for Molecular Testing on Hematoxylin-Eosin-Stained Sections: A Viable Option for Small Biopsies That Lack Tumor Tissues in Paraffin Block.

Science.gov (United States)

Wu, Howard H; Jovonovich, Stephen M; Randolph, Melissa; Post, Kristin M; Sen, Joyashree D; Curless, Kendra; Cheng, Liang

2016-12-01

- In some instances the standard method of doing molecular testing from formalin-fixed, paraffin-embedded block is not possible because of limited tissue. Tumor cell-enriched cell-transfer technique has been proven useful for performing immunocytochemistry and molecular testing on cytologic smears. - To establish the cell-transfer technique as a viable option for isolating tumor cells from hematoxylin-eosin (H&E)-stained slides. - Molecular testing was performed by using the cell-transfer technique on 97 archived H&E-stained slides from a variety of different tumors. Results were compared to the conventional method of molecular testing. - Polymerase chain reaction-based molecular testing via the cell-transfer technique was successfully performed on 82 of 97 samples (85%). This included 39 of 47 cases for EGFR, 10 of 11 cases for BRAF, and 33 of 39 cases for KRAS mutations. Eighty-one of 82 cell-transfer technique samples (99%) showed agreement with previous standard method results, including 4 mutations and 35 wild-type alleles for EGFR, 4 mutations and 6 wild-type alleles for BRAF, and 11 mutations and 21 wild-type alleles for KRAS. There was only 1 discrepancy: a cell-transfer technique with a false-negative >KRAS result (wild type versus G12C). - Molecular testing performed on H&E-stained sections via cell-transfer technique is useful when tissue from cell blocks and small surgical biopsy samples is exhausted and the only available material for testing is on H&E-stained slides.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

Science.gov (United States)

Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

2012-04-10

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

Generation of Induced Progenitor-like Cells from Mature Epithelial Cells Using Interrupted Reprogramming

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Li Guo

2017-12-01

Full Text Available Summary: A suitable source of progenitor cells is required to attenuate disease or affect cure. We present an “interrupted reprogramming” strategy to generate “induced progenitor-like (iPL cells” using carefully timed expression of induced pluripotent stem cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc; OSKM from non-proliferative Club cells. Interrupted reprogramming allowed controlled expansion yet preservation of lineage commitment. Under clonogenic conditions, iPL cells expanded and functioned as a bronchiolar progenitor-like population to generate mature Club cells, mucin-producing goblet cells, and cystic fibrosis transmembrane conductance regulator (CFTR-expressing ciliated epithelium. In vivo, iPL cells can repopulate CFTR-deficient epithelium. This interrupted reprogramming process could be metronomically applied to achieve controlled progenitor-like proliferation. By carefully controlling the duration of expression of OSKM, iPL cells do not become pluripotent, and they maintain their memory of origin and retain their ability to efficiently return to their original phenotype. A generic technique to produce highly specified populations may have significant implications for regenerative medicine. : In this article Waddell, Nagy, and colleagues present an “interrupted reprogramming” strategy to produce highly specified functional “induced progenitor-like cells” from mature quiescent cells. They propose that careful control of the duration of transient expression of iPSC reprogramming factors (OSKM allows controlled expansion yet preservation of parental lineage without traversing the pluripotent state. Keywords: generation of induced progenitor-like cells

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Ming-Wai Poon

Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

Science.gov (United States)

Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

Synergistic activity of vorinostat combined with gefitinib but not with sorafenib in mutant KRAS human non-small cell lung cancers and hepatocarcinoma.

Science.gov (United States)

Jeannot, Victor; Busser, Benoit; Vanwonterghem, Laetitia; Michallet, Sophie; Ferroudj, Sana; Cokol, Murat; Coll, Jean-Luc; Ozturk, Mehmet; Hurbin, Amandine

2016-01-01

Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (gefitinib) or a multi-targeted kinase inhibitor (sorafenib) in combination with a histone deacetylase inhibitor (vorinostat) on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT)-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G 2 /M cell cycle arrest without apoptosis. The sorafenib and vorinostat combination sustained the IGF-1R-, AKT-, and mitogen-activated protein kinase-dependent signaling pathways. These results showed that there was synergistic cytotoxicity when vorinostat was combined with gefitinib for both lung adenocarcinoma and hepatocarcinoma with mutant KRAS in vitro and in vivo but that the combination of vorinostat with sorafenib did not show any benefit. These findings highlight the important role of the IGF-1R/AKT pathway in the resistance to targeted therapies and support the use of histone deacetylase inhibitors in combination with EGFR-tyrosine kinase inhibitors, especially for

Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

DEFF Research Database (Denmark)

Dahl Steffensen, Karina; Waldstrøm, Marianne; Lund, Bente

, and head and neck cancer. No previous studies have evaluated the effect of panitumumab in OC based on KRAS mutation status. Methods: Eligibility criteria are confirmed stage I-IV primary epithelial ovarian/fallopian/peritoneal cancer patients with progression either during or within 6 months after end...... to a total of 33 patients. At present, 15 patients have been enrolled. The primary endpoint is to investigate the response rate in platinum-resistant, KRAS wild- type OC patients treated with PLD supplemented with panitumumab. Translational research is included as a secondary endpoint and tumor tissue...

External quality assessment for KRAS testing is needed: setup of a European program and report of the first joined regional quality assessment rounds

DEFF Research Database (Denmark)

Bellon, Ellen; Ligtenberg, Marjolijn J L; Tejpar, Sabine

2011-01-01

. In an effort to ensure optimal, uniform, and reliable community-based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer...... expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization....... prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use...

Joint Contribution of Left Dorsal Premotor Cortex and Supramarginal Gyrus to Rapid Action Reprogramming

DEFF Research Database (Denmark)

Hartwigsen, Gesa; Siebner, Hartwig R

2015-01-01

human subjects performed a spatially-precued reaction time task. RESULTS: Relative to sham rTMS, effective online perturbation of left PMd significantly impaired both the response speed and accuracy in trials that were invalidly pre-cued and required the subject to reprogram the prepared action......BACKGROUND: The rapid adaptation of actions to changes in the environment is crucial for survival. We previously demonstrated a joint contribution of left dorsal premotor cortex (PMd) and left supramarginal gyrus (SMG) to action reprogramming. However, we did not probe the contribution of PMd...... to the speed and accuracy of action reprogramming and how the functional relevance of PMd changes in the presence of a dysfunctional SMG. OBJECTIVE: This study further dissociated the unique contribution of left PMd and SMG to action reprogramming. Specifically, we tested whether the critical contribution...

A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

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Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

2015-07-01

It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

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Byungkuk Min

2016-05-01

Full Text Available Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.

Prognostic value of KRAS genotype in metastatic colorectal cancer (MCRC patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx according to extension of metastatic disease

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Bruera Gemma

2012-11-01

Full Text Available Abstract Background Bevacizumab (BEV plus triplet chemotherapy can increase efficacy of first-line treatment of metastatic colorectal cancer (MCRC, particularly integrated with secondary liver surgery in liver-limited (L-L patients. The prognostic value of the KRAS genotype in L-L and other or multiple metastatic (O/MM MCRC patients treated with the FIr-B/FOx regimen was retrospectively evaluated. Methods Tumoral and metastatic samples were screened for KRAS codon 12 and 13 and BRAF mutations by SNaPshot and/or direct sequencing. Fit MCRC patients 2, days 1, 2, 8, 9, 15, 16, 22 and 23; irinotecan (CPT-11 160 mg/m2 plus BEV 5 mg/kg, days 1, 15; oxaliplatin (OXP 80 mg/m2, days 8, 22; every 4 weeks. MCRC patients were classified as L-L and O/MM. Activity and efficacy were evaluated and compared using log-rank test. Results In all, 59 patients were evaluated: 31 KRAS wild-type (53%, 28 KRAS mutant (47%. At 21.5 months median follow-up, objective response rate (ORR, progression-free survival (PFS and overall survival (OS were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were not significantly different. PFS and OS were significantly different in L-L compared to O/MM evaluable patients. In KRAS wild-type patients, clinical outcome of 12 L-L compared to 18 O/MM was significantly different: PFS 21 versus 12 months and OS 47 versus 28 months, respectively. In KRAS mutant patients, the clinical outcome of 13 L-L compared to 14 O/MM was not significantly different: PFS 11 months equivalently and OS 39 versus 19 months, respectively. Conclusions The KRAS genotype wild-type and mutant does not significantly affect different clinical outcomes for MCRC patients treated with the first-line FIr-B/FOx intensive regimen. KRAS wild-type patients with L-L disease may achieve a significantly prolonged clinical outcome due to integration with secondary liver surgery, with respect to KRAS mutant patients.

Craniosynostosis and Noonan syndrome with KRAS mutations: Expanding the phenotype with a case report and review of the literature.

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Addissie, Yonit A; Kotecha, Udhaya; Hart, Rachel A; Martinez, Ariel F; Kruszka, Paul; Muenke, Maximilian

2015-11-01

Noonan syndrome (NS) is a multiple congenital anomaly syndrome caused by germline mutations in genes coding for components of the Ras-mitogen-activated protein kinase (RAS-MAPK) pathway. Features include short stature, characteristic facies, congenital heart anomalies, and developmental delay. While there is considerable clinical heterogeneity in NS, craniosynostosis is not a common feature of the condition. Here, we report on a 2 month-old girl with Noonan syndrome associated with a de novo mutation in KRAS (p.P34Q) and premature closure of the sagittal suture. We provide a review of the literature of germline KRAS mutations and find that approximately 10% of published cases have craniosynostosis. Our findings expand on the NS phenotype and suggest that germline mutations in the KRAS gene are causally involved in craniosynostosis, supporting the role of the RAS-MAPK pathway as a mediator of aberrant bone growth in cranial sutures. The inclusion of craniosynostosis as a possible phenotype in KRAS-associated Noonan Syndrome has implications in the differential diagnosis and surgical management of individuals with craniosynostosis. © 2015 Wiley Periodicals, Inc.

KRAS mutation detection in colorectal cancer by a commercially available gene chip array compares well with Sanger sequencing.

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French, Deborah; Smith, Andrew; Powers, Martin P; Wu, Alan H B

2011-08-17

Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer. KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers. Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension. This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist. Copyright © 2011 Elsevier B.V. All rights reserved.

Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

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Biswas, Dhruba; Jiang, Peng

2016-02-06

The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors

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Andreas Hermann

2016-01-01

Full Text Available Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC or two (OCT4, KLF4; hiPSC2F-NSC reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB or four reprogramming factors (hiPSC4F-FIB. After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.

Endometrial cancer and somatic G>T KRAS transversion in patients with constitutional MUTYH biallelic mutations.

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Tricarico, Rossella; Bet, Paola; Ciambotti, Benedetta; Di Gregorio, Carmela; Gatteschi, Beatrice; Gismondi, Viviana; Toschi, Benedetta; Tonelli, Francesco; Varesco, Liliana; Genuardi, Maurizio

2009-02-18

MUTYH-associated polyposis (MAP) is an autosomal recessive condition predisposing to colorectal cancer, caused by constitutional biallelic mutations in the base excision repair (BER) gene MUTYH. Colorectal tumours from MAP patients display an excess of somatic G>T mutations in the APC and KRAS genes due to defective BER function. To date, few extracolonic manifestations have been observed in MAP patients, and the clinical spectrum of this condition is not yet fully established. Recently, one patient with a diagnosis of endometrial cancer and biallelic MUTYH mutations has been described. We here report on two additional unrelated MAP patients with biallelic MUTYH germline mutations who developed endometrioid endometrial carcinoma. The endometrial tumours were evaluated for PTEN, PIK3CA, KRAS, BRAF and CTNNB1 mutations. A G>T transversion at codon 12 of the KRAS gene was observed in one tumour. A single 1bp frameshift deletion of PTEN was observed in the same sample. Overall, these findings suggest that endometrial carcinoma is a phenotypic manifestations of MAP and that inefficient repair of oxidative damage can be involved in its pathogenesis.

Long-term follow-up of patients with a clinically benign extrahepatic biliary stenosis and K-ras mutation in endobiliary brush cytology

NARCIS (Netherlands)

van Heek, N. Tjarda; Rauws, Erik A. J.; Caspers, Eric; Drillenburg, Paul; Gouma, Dirk J.; Offerhaus, G. Johan A.

2002-01-01

Background. K-ras mutations in endobiliary brush cytology are an early event in carcinogenesis and justify a suspicion of malignancy in patients with extrahepatic biliary stenosis. However, K-ras mutations have been detected in specimens obtained by brushing of clinically benign extrahepatic biliary

Analysis of the K-ras and p53 pathways in x-ray-induced lung tumors in the rat

Energy Technology Data Exchange (ETDEWEB)

Belinsky, S.A.; Middleton, S.K.; Hahn, F.F.; Nikula, K.J. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States); Picksley, S.M. [Medical Sciences Inst., Dundee (United Kingdom)

1996-04-01

The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to lmited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT {r_arrow} GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat. 49 refs., 5 figs.

Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations

International Nuclear Information System (INIS)

Kalari, Krishna R.; Rossell, David; Necela, Brian M.; Asmann, Yan W.; Nair, Asha

2012-01-01

KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.

System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato1[W][OA

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Etalo, Desalegn W.; Stulemeijer, Iris J.E.; Peter van Esse, H.; de Vos, Ric C.H.; Bouwmeester, Harro J.; Joosten, Matthieu H.A.J.

2013-01-01

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR. PMID:23719893

PENGARUH KEPEMIMPINAN TRANSFORMASIONAL DAN KEPUASAN KERJA TERHADAP PERILAKU KEWARGAAN ORGANISASI GURU SEKOLAH DASAR NEGERI KECAMATAN KRAS KABUPATEN KEDIRI

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Imam Gunawan

2016-11-01

Full Text Available The objective of this research is to found out the influence of transformational leadership and job satisfaction toward organizational citizenship behavior of the teacher in primary school Kras District of Kediri. This research used quantitative approach. The participants of the research are 159 teachers of 30 primary school Kras, District of Kediri. The data found using closed model questionnaire instrument. The data analyzed using path analyzing, with the help of SPSS PASW Statistics 18 program. The results of the research show that there is a significance influence between transformational leadership and job satisfaction toward organizational citizenship behavior of the teacher in primary school Kras, District of Kediri. The transformational leadership contributes 15.1% while the job satisfaction contribution is 20.4%. Other variables beyond this research contribute 64.5%.

New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status.

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Telechea-Fernández, Marcelino; Rodríguez-Fernández, Lucia; García, Concha; Zaragozá, Rosa; Viña, Juan; Cervantes, Andrés; García-Trevijano, Elena R

2018-02-06

Calpain-2 belongs to a family of pleiotropic Cys-proteases with modulatory rather than degradative functions. Calpain (CAPN) overexpression has been controversially correlated with poor prognosis in several cancer types, including colorectal carcinoma (CRC). However, the mechanisms of substrate-recognition, calpain-2 regulation/deregulation and specific functions in CRC remain elusive. Herein, calpain subcellular distribution was studied as a key event for substrate-recognition and consequently, for calpain-mediated function. We describe a new localization for calpain-2 in the nucleoli of CRC cells. Calpain-2 nucleolar distribution resulted dependent on its enzymatic activity and on the mutational status of KRAS. In KRASWT/- cells serum-starvation induced CAPN2 expression, nucleolar accumulation and increased binding to the rDNA-core promoter and intergenic spacer (IGS), concomitant with a reduction in pre-rRNA levels. Depletion of calpain-2 by specific siRNA prevented pre-rRNA down-regulation after serum removal. Conversely, ribosomal biogenesis proceeded in the absence of serum in unresponsive KRASG13D/- cells whose CAPN2 expression, nucleolar localization and rDNA-occupancy remained unchanged during the time-course of serum starvation. We propose here that nucleolar calpain-2 might be a KRAS-dependent sensor to repress ribosomal biogenesis in growth limiting conditions. Under constitutive activation of the pathway commonly found in CRC, calpain-2 is deregulated and tumor cells become insensitive to the extracellular microenvironment.

precise delta extraction scheme for reprogramming of wireless

African Journals Online (AJOL)

eobe

Keywords- reprogramming; operating system, wireless sensor network, Delta. 1. INTRODUCTION ... It entails the transmission of only modified modules that are then ... higher power consumption and slow system execution are drawbacks ...

KRASness and PIK3CAness in patients with advanced colorectal cancer: outcome after treatment with early-phase trials with targeted pathway inhibitors.

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Ignacio Garrido-Laguna

Full Text Available To evaluate clinicopathologic and molecular features of patients with metastatic colorectal cancer (mCRC and their outcomes in early-phase trials using pathway-targeting agents.We analyzed characteristics of 238 patients with mCRC referred to the phase 1 trials unit at MD Anderson Cancer Center. KRAS, PIK3CA and BRAF status were tested using PCR-based DNA sequencing.Fifty-one percent of patients harbored KRAS mutations; 15% had PIK3CA mutations. In the multivariate regression model for clinical characteristics KRAS mutations were associated with an increased incidence of lung and bone metastases and decreased incidence of adrenal metastases; PIK3CA mutations were marginally correlated with mucinous tumors (p = 0.05. In the univariate analysis, KRAS and PIK3CA mutations were strongly associated. Advanced Duke's stage (p<0.0001 and KRAS mutations (p = 0.01 were the only significant independent predictors of poor survival (Cox proportional hazards model. Patients with PIK3CA mutations had a trend toward shorter progression-free survival when treated with anti-EGFR therapies (p = 0.07. Eighteen of 78 assessable patients (23% treated with PI3K/Akt/mTOR axis inhibitors achieved stable disease [SD] ≥6 months or complete response/partial response (CR/PR, only one of whom were in the subgroup (N = 15 with PIK3CA mutations, perhaps because 10 of these 15 patients (67% had coexisting KRAS mutations. No SD ≥6 months/CR/PR was observed in the 10 patients treated with mitogen-activating protein kinase (MAPK pathway targeting drugs.KRAS and PIK3CA mutations frequently coexist in patients with colorectal cancer, and are associated with clinical characteristics and outcome. Overcoming resistance may require targeting both pathways.

New insights on the voltage dependence of the KCa3.1 channel block by internal TBA.

Science.gov (United States)

Banderali, Umberto; Klein, Hélène; Garneau, Line; Simoes, Manuel; Parent, Lucie; Sauvé, Rémy

2004-10-01

We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.

Comparison of clinical outcome after first-line platinum-based chemotherapy in different types of KRAS mutated advanced non-small-cell lung cancer.

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Mellema, Wouter W; Masen-Poos, Lucie; Smit, Egbert F; Hendriks, Lizza E L; Aerts, Joachim G; Termeer, Arien; Goosens, Martijn J; Smit, Hans J M; van den Heuvel, Michel M; van der Wekken, Anthonie J; Herder, Gerarda J M; Krouwels, Frans H; Stigt, Jos A; van den Borne, Ben E E M; Haitjema, Tjeerd J; Staal-Van den Brekel, Agnes J; van Heemst, Robbert C; Pouw, Ellen; Dingemans, Anne-Marie C

2015-11-01

As suggested by in-vitro data, we hypothesize that subtypes of KRAS mutated non-small cell lung cancer (NSCLC) respond differently to chemotherapy regimens. Patients with advanced NSCLC and known KRAS mutation, treated with first-line platinum-based chemotherapy, were retrieved from hospital databases. to investigate overall response rate (ORR), progression free survival (PFS) and overall survival (OS) between different types of platinum-based chemotherapy per type of KRAS mutation. 464 patients from 17 hospitals, treated between 2000 and 2013, were included. The majority of patients had stage IV disease (93%), had a history of smoking (98%) and known with an adenocarcinoma (91%). Most common types of KRAS mutation were G12C (46%), G12V (20%) and G12D (10%). Platinum was combined with pemetrexed (n=334), taxanes (n=68) or gemcitabine (n=62). Patients treated with taxanes had a significant improved ORR (50%) compared to pemetrexed (21%) or gemcitabine (25%; pchemotherapy had best ORR. Response to chemotherapy regimens was different in types of KRAS mutation. Especially patients with G12V had better response to taxane treatment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Nuclear reprogramming of somatic nucleus hybridized with embryonic stem cells by electrofusion.

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Tada, Masako; Tada, Takashi

2006-01-01

Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

Science.gov (United States)

Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

2017-03-01

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

Induced pluripotent stem cells reprogramming: Epigenetics and applications in the regenerative medicine

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Kátia Maria Sampaio Gomes

Full Text Available Summary Induced pluripotent stem cells (iPSCs are somatic cells reprogrammed into an embryonic-like pluripotent state by the expression of specific transcription factors. iPSC technology is expected to revolutionize regenerative medicine in the near future. Despite the fact that these cells have the capacity to self-renew, they present low efficiency of reprogramming. Recent studies have demonstrated that the previous somatic epigenetic signature is a limiting factor in iPSC performance. Indeed, the process of effective reprogramming involves a complete remodeling of the existing somatic epigenetic memory, followed by the establishment of a "new epigenetic signature" that complies with the new type of cell to be differentiated. Therefore, further investigations of epigenetic modifications associated with iPSC reprogramming are required in an attempt to improve their self-renew capacity and potency, as well as their application in regenerative medicine, with a new strategy to reduce the damage in degenerative diseases. Our review aimed to summarize the most recent findings on epigenetics and iPSC, focusing on DNA methylation, histone modifications and microRNAs, highlighting their potential in translating cell therapy into clinics.

CF3DODA-Me induces apoptosis, degrades Sp1, and blocks the transformation phase of the blebbishield emergency program.

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Taoka, Rikiya; Jinesh, Goodwin G; Xue, Wenrui; Safe, Stephen; Kamat, Ashish M

2017-05-01

Cancer stem cells are capable of undergoing cellular transformation after commencement of apoptosis through the blebbishield emergency program in a VEGF-VEGFR2-dependent manner. Development of therapeutics targeting the blebbishield emergency program would thus be important in cancer therapy. Specificity protein 1 (Sp1) orchestrates the transcription of both VEGF and VEGFR2; hence, Sp1 could act as a therapeutic target. Here, we demonstrate that CF 3 DODA-Me induced apoptosis, degraded Sp1, inhibited the expression of multiple drivers of the blebbishield emergency program such as VEGFR2, p70S6K, and N-Myc through activation of caspase-3, inhibited reactive oxygen species; and inhibited K-Ras activation to abolish transformation from blebbishields as well as transformation in soft agar. These findings confirm CF 3 DODA-Me as a potential therapeutic candidate that can induce apoptosis and block transformation from blebbishields.

(Re-)programming of subtype specific cardiomyocytes.

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Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

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Ling Wang

2017-12-01

Full Text Available The development of human induced pluripotent stem cells (iPSCs holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4. Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

Experiences from treatment-predictive KRAS testing; high mutation frequency in rectal cancers from females and concurrent mutations in the same tumor

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Jönsson, Mats; Ekstrand, Anna; Edekling, Thomas

2009-01-01

. METHODS: We used a real-time PCR based method to determine KRAS mutations in 136 colorectal cancers with mutations identified in 53 (39%) tumors. RESULTS: KRAS mutations were significantly more often found in rectal cancer (21/38, 55%) than in colon cancer (32/98, 33%) (P = 0.02). This finding...... was explained by marked differences mutation rates in female patients who showed mutations in 33% of the colon cancers and in 67% of the rectal cancers (P = 0.01). Concurrent KRAS mutations were identified in three tumors; two colorectal cancers harbored Gly12Asp/Gly13Asp and Gly12Cys/Gly13Asp and a third tumor...... carried Gly12Cys/Gly12Asp in an adenomatous component and additionally acquired Gly12Val in the invasive component. CONCLUSION: The demonstration of a particularly high KRAS mutation frequency among female rectal cancer patients suggests that this subset is the least likely to respond to anti...

SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

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Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

2014-01-01

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

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Kinoshita, Taisuke [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kosaka, Takeo [Department of Urology, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Takubo, Keiyo [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Hotta, Akitsu [Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto (Japan); Ellis, James [Ontario Human iPS Cell Facility, Molecular Genetics, University of Toronto, Developmental and Stem Cell Biology, SickKids, Toronto, Canada MG1L7 (Canada); Suda, Toshio, E-mail: sudato@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan)

2011-04-08

Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

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Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

2011-01-01

Highlights: → iPS cells were induced with a fluorescence monitoring system. → ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. → iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. → ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

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Hisataka Ogawa

Full Text Available Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

Mesenchymal to Epithelial Transition Mediated by CDH1 Promotes Spontaneous Reprogramming of Male Germline Stem Cells to Pluripotency

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Junhui An

2017-02-01

Full Text Available Cultured spermatogonial stem cells (GSCs can spontaneously form pluripotent cells in certain culture conditions. However, GSC reprogramming is a rare event that is largely unexplained. We show GSCs have high expression of mesenchymal to epithelial transition (MET suppressors resulting in a developmental barrier inhibiting GSC reprogramming. Either increasing OCT4 or repressing transforming growth factor β (TGF-β signaling promotes GSC reprogramming by upregulating CDH1 and boosting MET. Reducing ZEB1 also enhances GSC reprogramming through its direct effect on CDH1. RNA sequencing shows that rare GSCs, identified as CDH1+ after trypsin digestion, are epithelial-like cells. CDH1+ GSCs exhibit enhanced reprogramming and become more prevalent during the course of reprogramming. Our results provide a mechanistic explanation for the spontaneous emergence of pluripotent cells from GSC cultures; namely, rare GSCs upregulate CDH1 and initiate MET, processes normally kept in check by ZEB1 and TGF-β signaling, thereby ensuring germ cells are protected from aberrant acquisition of pluripotency.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

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Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

Comparison of HER2 gene amplification and KRAS alteration in eyelid sebaceous carcinomas with that in other eyelid tumors.

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Kwon, Mi Jung; Shin, Hyung Sik; Nam, Eun Sook; Cho, Seong Jin; Lee, Min Joung; Lee, Samuel; Park, Hye-Rim

2015-05-01

Eyelid sebaceous carcinoma (SC) represents a highly aggressive malignancy. Despite the poor prognosis, genetic alterations as potential molecular targets are not available. KRAS mutation and HER2 gene amplification may be candidates related to their genetic alterations. We examined the HER2 and KRAS alteration status in eyelid SCs and compared it with that in other eyelid tumors. The controversial topics of the human papillomavirus (HPV) and p16 expression were also investigated. HER2 amplification was determined by silver in situ hybridization, while immunohistochemistry was performed to study protein expressions in 14 SCs and controls, including 23 other eyelid malignancies and 14 benign tumors. Peptide nucleic acid-mediated PCR clamping and direct sequencing were used to detect KRAS mutations. HER2 protein overexpression was observed in 85.7% (12/14) of the SCs, of which two-thirds showed HER2 gene amplification. HER2 protein overexpression and HER2 amplification were found more frequently in eyelid SCs than in other eyelid tumors. All SCs harbored wild type KRAS genes. No HPV infections were identified in the SCs. Nevertheless, p16 overexpression was found in 71.4% (10/14) of SCs, irrespective of the status of HPV infection. Furthermore, p16 overexpression in eyelid SCs was also significantly higher than that in other eyelid tumors. HER2 protein overexpression, HER2 gene amplifications, and wild type KRAS genes are common in eyelid SCs. HER2 gene amplification may represent potential therapeutic targets for the treatment of eyelid SCs. Copyright © 2014 Elsevier GmbH. All rights reserved.

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

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Schmitt Fernando

2008-09-01

Full Text Available Abstract Background BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC. In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP, MLH1 methylation and microsatellite instability (MSI. We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Methods Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. Results KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112, but statistically different from what is found in microsatellite stable (MSS tumours (P = 0.0191. Conclusion Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

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Velho, Sérgia; Moutinho, Cátia; Cirnes, Luís; Albuquerque, Cristina; Hamelin, Richard; Schmitt, Fernando; Carneiro, Fátima; Oliveira, Carla; Seruca, Raquel

2008-01-01

BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191). Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated

Influence of K-ras status and anti-tumour treatments on complications due to colorectal self-expandable metallic stents: a retrospective multicentre study.

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Fuccio, Lorenzo; Correale, Loredana; Arezzo, Alberto; Repici, Alessandro; Manes, Gianpiero; Trovato, Cristina; Mangiavillano, Benedetto; Manno, Mauro; Cortelezzi, Claudio Camillo; Dinelli, Marco; Cennamo, Vincenzo; de Bellis, Mario

2014-06-01

This study aimed to explore the relationship between K-ras status, anti-tumour treatments, and the complications of colorectal self-expandable metallic stenting in colorectal cancer. This is a retrospective, multicentre study of 91 patients with obstructive advanced colorectal cancer palliated with enteral stents between 2007 and 2011. K-ras wild-type tumours were diagnosed in 44 patients (48.4%); 82 (90.1%) received chemotherapy and 45 (49.4%) had additional biological therapy (34 bevacizumab, 11 cetuximab). Twenty-one (23.1%) experienced stent-related complications: 11 (52.4%) occurred in the K-ras mutant group (P=0.9). K-ras wild-type patients were not less likely to develop adverse events than K-ras mutant patients (OR, 0.99; 95% CI: 0.4-2.7). Overall mean time to complication was 167.6 days (range 4-720 days), with no difference between the two groups (141 vs. 197 days; P=0.5). Chemotherapy did not influence the risk of complications (OR, 0.56; 95% CI: 0.14-2.9), and there was no evidence that patients treated with chemotherapy and cetuximab were more likely to experience stent-related complications than patients treated with chemotherapy alone, or untreated (OR, 1.2; 95% CI: 0.2-5.9). Although perforation rates were higher with bevacizumab-based treatment (11.8% vs. 7%), this result was not statistically significant (P=0.69). K-ras mutation status, chemotherapy, and biological treatments should not influence colorectal stent-related complication rates. Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming

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Jun Chen

2016-02-01

Full Text Available The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies.

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

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Li, Min; Riddle, Suzette; Zhang, Hui; D'Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J; Hansen, Kirk C; Moldvan, Radu; McKeon, B Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Caánovas, Angela; Medrano, Juan F; Thomas, Milton G; Iloska, Dijana; Plecitá-Hlavatá, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A; El Kasmi, Karim C; Zhang, QingHong; Stenmark, Kurt R

2016-10-11

Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). RNA sequencing, quantitative polymerase chain reaction, 13 C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U- 13 C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD + ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD + ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly

Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

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Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

2015-01-01

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.

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Loboda, Andrey; Nebozhyn, Michael; Klinghoffer, Rich; Frazier, Jason; Chastain, Michael; Arthur, William; Roberts, Brian; Zhang, Theresa; Chenard, Melissa; Haines, Brian; Andersen, Jannik; Nagashima, Kumiko; Paweletz, Cloud; Lynch, Bethany; Feldman, Igor; Dai, Hongyue; Huang, Pearl; Watters, James

2010-06-30

Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

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Paweletz Cloud

2010-06-01

Full Text Available Abstract Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90% sensitivity but relatively low (50% specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical

Direct Cardiac Reprogramming: Advances in Cardiac Regeneration

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Olivia Chen

2015-01-01

Full Text Available Heart disease is one of the lead causes of death worldwide. Many forms of heart disease, including myocardial infarction and pressure-loading cardiomyopathies, result in irreversible cardiomyocyte death. Activated fibroblasts respond to cardiac injury by forming scar tissue, but ultimately this response fails to restore cardiac function. Unfortunately, the human heart has little regenerative ability and long-term outcomes following acute coronary events often include chronic and end-stage heart failure. Building upon years of research aimed at restoring functional cardiomyocytes, recent advances have been made in the direct reprogramming of fibroblasts toward a cardiomyocyte cell fate both in vitro and in vivo. Several experiments show functional improvements in mouse models of myocardial infarction following in situ generation of cardiomyocyte-like cells from endogenous fibroblasts. Though many of these studies are in an early stage, this nascent technology holds promise for future applications in regenerative medicine. In this review, we discuss the history, progress, methods, challenges, and future directions of direct cardiac reprogramming.

Efficient generation of fully reprogrammed human iPS cells via polycistronic retroviral vector and a new cocktail of chemical compounds.

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Zhonghui Zhang

Full Text Available Direct reprogramming of human somatic cells into induced pluripotent stem (iPS cells by defined transcription factors (TFs provides great potential for regenerative medicine and biomedical research. This procedure has many challenges, including low reprogramming efficiency, many partially reprogrammed colonies, somatic coding mutations in the genome, etc. Here, we describe a simple approach for generating fully reprogrammed human iPS cells by using a single polycistronic retroviral vector expressing four human TFs in a single open reading frame (ORF, combined with a cocktail containing three small molecules (Sodium butyrate, SB431542, and PD0325901. Our results demonstrate that human iPS cells generated by this approach express human ES cells markers and exhibit pluripotency demonstrated by their abilities to differentiate into the three germ layers in vitro and in vivo. Notably, this approach not only provides a much faster reprogramming process but also significantly diminishes partially reprogrammed iPS cell colonies, thus facilitating efficient isolation of desired fully reprogrammed iPS cell colonies.

Fluctuating levels of reprogramming factor expression in cultured ...

African Journals Online (AJOL)

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pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the ... could serve as disease models and aid in the discovery of drugs and genes; furthermore, this approach to gene-.

Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.

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David J Kahler

Full Text Available Current methods to derive induced pluripotent stem cell (iPSC lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS to isolate single cells expressing the cell surface marker signature CD13(NEGSSEA4(POSTra-1-60(POS on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral or non- integrating (Sendai virus reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.

Temperature-dependent thermo-elastic properties of s-, p- and d-block liquid metals

International Nuclear Information System (INIS)

Singh, R.N.; Arafin, S.; George, A.K.

2007-01-01

Thermodynamic relations involving experimentally known physical quantities are used to determine the ratio of specific heats, γ, for s-block (Na, K, Rb, Cs, Mg), d-block (Fe, Co, Ni, Cu, Ag, Au, Zn, Cd, Hg,) and p-block (Al, Ga, In, Tl, Sn, Pb, Sb, Bi, Se, Te) liquid metals. γ is further utilized to determine reliable values for the isothermal compressibility (κ T ) and the specific heat at constant volume (C V ) for these liquid metals near and above the melting temperatures. These data are of considerable significance for various thermo-physical analysis of liquid metals. We have also evaluated the Grueneisen function and its temperature dependence to establish the correlation between the thermal expansion and the elastic response to thermally induced stress. The values of acoustic impedance for liquid metals are evaluated and its physical significance is discussed for the transmission of elastic waves across metallic interfaces

Syndecan-2 promotes perineural invasion and cooperates with K-ras to induce an invasive pancreatic cancer cell phenotype

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De Oliveira Tiago

2012-04-01

Full Text Available Abstract Background We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC cells. Methods Syndecan-2 (SDC-2 expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling. Results SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and - further downstream - phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered. Conclusion SDC-2 is a novel (perineural invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.

Preliminary results of structural profiling of the Kras edge and Istria (Kozina – Srmin Motorway, Sečovlje

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2002-06-01

Full Text Available On the section Kozina-Srmin the new motorway Ljubljana-Koper is crossing the Kras edge, which in geologic terminology is refered to as the Kras thrust edge. In the widest sense it comprises the area between the edge of the Trieste-Komen plateau and the Savudrija ridge, creating the boundary between the Adriatic foreland and the External Dinarides. Detailed geologic mapping of the motorway section Kozina-Srmin slowed that the Kras thrust edge is not a monophase tectonic structure, but has been formed through several different deformation phases from the Eocene until today. Besides smaller onesthese phases include responses of three significant events; the Dinaric nappe thrusting, displacements along the strice-slipe faults with NW-SE trending and underthrusting of the Istria toward the NE. The latter event destroyed the primary of the SW boundary ofthe External Dinarides between Southern Alps and the Velebit arc.

Cellular reprogramming through mitogen-activated protein kinases

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Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Elixir of Life: Thwarting Aging With Regenerative Reprogramming.

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Beyret, Ergin; Martinez Redondo, Paloma; Platero Luengo, Aida; Izpisua Belmonte, Juan Carlos

2018-01-05

All living beings undergo systemic physiological decline after ontogeny, characterized as aging. Modern medicine has increased the life expectancy, yet this has created an aged society that has more predisposition to degenerative disorders. Therefore, novel interventions that aim to extend the healthspan in parallel to the life span are needed. Regeneration ability of living beings maintains their biological integrity and thus is the major leverage against aging. However, mammalian regeneration capacity is low and further declines during aging. Therefore, modalities that reinforce regeneration can antagonize aging. Recent advances in the field of regenerative medicine have shown that aging is not an irreversible process. Conversion of somatic cells to embryonic-like pluripotent cells demonstrated that the differentiated state and age of a cell is not fixed. Identification of the pluripotency-inducing factors subsequently ignited the idea that cellular features can be reprogrammed by defined factors that specify the desired outcome. The last decade consequently has witnessed a plethora of studies that modify cellular features including the hallmarks of aging in addition to cellular function and identity in a variety of cell types in vitro. Recently, some of these reprogramming strategies have been directly used in animal models in pursuit of rejuvenation and cell replacement. Here, we review these in vivo reprogramming efforts and discuss their potential use to extend the longevity by complementing or augmenting the regenerative capacity. © 2017 American Heart Association, Inc.

Detection of TET2 , KRAS and CBL variants by Next Generation ...

African Journals Online (AJOL)

Aim: In this study, we aimed to find possible genetic markers for molecular analysis in childhood AML by screening hot-spot exons of TET2, KRAS, and CBL using Next Generation Sequencing (NGS) analysis. In addition, association between found variants and mutations of Januse Kinase-2 (JAK2) and Fms Related ...

Rationale and Methodology of Reprogramming for Generation of Induced Pluripotent Stem Cells and Induced Neural Progenitor Cells

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Zuojun Tian

2016-04-01

Full Text Available Great progress has been made regarding the capabilities to modify somatic cell fate ever since the technology for generation of induced pluripotent stem cells (iPSCs was discovered in 2006. Later, induced neural progenitor cells (iNPCs were generated from mouse and human cells, bypassing some of the concerns and risks of using iPSCs in neuroscience applications. To overcome the limitation of viral vector induced reprogramming, bioactive small molecules (SM have been explored to enhance the efficiency of reprogramming or even replace transcription factors (TFs, making the reprogrammed cells more amenable to clinical application. The chemical induced reprogramming process is a simple process from a technical perspective, but the choice of SM at each step is vital during the procedure. The mechanisms underlying cell transdifferentiation are still poorly understood, although, several experimental data and insights have indicated the rationale of cell reprogramming. The process begins with the forced expression of specific TFs or activation/inhibition of cell signaling pathways by bioactive chemicals in defined culture condition, which initiates the further reactivation of endogenous gene program and an optimal stoichiometric expression of the endogenous pluri- or multi-potency genes, and finally leads to the birth of reprogrammed cells such as iPSCs and iNPCs. In this review, we first outline the rationale and discuss the methodology of iPSCs and iNPCs in a stepwise manner; and then we also discuss the chemical-based reprogramming of iPSCs and iNPCs.

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

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Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

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Zhai, Yingying; Chen, Xi; Yu, Dehai [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Tao [Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Cui, Jiuwei; Wang, Guanjun [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Hu, Ji-Fan, E-mail: jifan@stanford.edu [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Wei, E-mail: jdyylw@163.com [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China)

2015-09-10

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells

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Marta Trevisan

2017-01-01

Full Text Available Induced pluripotent stem cells (iPSCs are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

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Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

The importance of KRAS mutations and EGF61A>G polymorphism to the effect of cetuximab and irinotecan in metastatic colorectal cancer

DEFF Research Database (Denmark)

Spindler, Karen-Lise Garm; Pallisgaard, N; Rasmussen, Anders Aamann

2009-01-01

-line cetuximab-irinotecan. Blood samples were analysed for SNPs. KRAS analysis was carried out by sequencing analysis and quantitative PCR (DxS kit) in primary tumour and distant metastases. RESULTS: There was a clear correlation between KRAS status in primary tumours and metastasis. The DxS kit presented...

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

DEFF Research Database (Denmark)

Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno

2016-01-01

, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE...

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Co-Repressor C-terminal Binding Protein-1

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Li, Min; Riddle, Suzette; Zhang, Hui; D’Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J.; Hansen, Kirk C.; Moldvan, Radu; McKeon, B. Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Cánovas, Angela; Medrano, Juan F.; Thomas, Milton G.; Iloska, Dijana; Plecita-Hlavata, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A.; El Kasmi, Karim C.; Zhang, Qinghong; Stenmark, Kurt R.

2016-01-01

Background Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells including fibroblasts in pulmonary hypertension (PH). Herein, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and pro-inflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional co-repressor C-terminal binding protein 1 (CtBP1). Methods RNA-Sequencing, qPCR, 13C-NMR, fluorescence-lifetime imaging, mass spectrometry-based metabolomics and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure NADH/NAD+ ratio in bovine and human adventitial fibroblasts, and mouse lung tissues. Immunohistochemistry was utilized to assess CtBP1 expression in the whole lung tissues. CtBP1 siRNA and the pharmacologic inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were utilized to abrogate CtBP1 activity in cells and hypoxic mice. Results We found adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with IPAH (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with IPAH and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB, or genetically blocking CtBP1 using siRNA, upregulated the cyclin-dependent genes (p15 and p21) and pro-apoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. ChIP analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated

KRAS polymorphisms are associated with survival of CRC in Chinese population.

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Dai, Qiong; Wei, Hui Lian; Huang, Juan; Zhou, Tie Jun; Chai, Li; Yang, Zhi-Hui

2016-04-01

rs12245, rs12587, rs9266, rs1137282, rs61764370, and rs712 of KRAS oncogene are characterized in the 3'UTR. The study highlights the important role of these polymorphisms playing in the susceptibility, oxaliplatin-based chemotherapy sensitivity, progression, and prognosis of CRC. Improved multiplex ligation detection reaction (iMLDR) technique is used for genotyping. An unconditional logistic regression model was used to estimate the association of certain polymorphism and CRC risk. The Kaplan-Meier method, log-rank test, and Cox regression model were used to evaluate the effects of polymorphisms on survival analysis. Results demonstrated that TT genotype and T allele of rs712 were associated with the increased risk of CRC; the patients with GG genotype and G allele of rs61764370 had a shorter survival and a higher risk of relapse or metastasis of CRC. Our studies supported the conclusions that rs61764370 and rs712 polymorphisms of the KRAS are functional and it may play an important role in the development of CRC and oxaliplatin-based chemotherapy efficiency and prognosis of CRC.

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

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Orue, Andrea; Chavez, Valery; Strasberg-Rieber, Mary; Rieber, Manuel

2016-11-18

The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA. In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine. This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

CtIP-Specific Roles during Cell Reprogramming Have Long-Term Consequences in the Survival and Fitness of Induced Pluripotent Stem Cells

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Daniel Gómez-Cabello

2017-02-01

Full Text Available Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs. All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.

Reserve stem cells: Differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract.

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Mills, Jason C; Sansom, Owen J

2015-07-14

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, postmitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the long-term maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like the stomach and intestine, reprogramming may allow mature cells to serve as reserve ("quiescent") stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, postmitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferation in the stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. Copyright © 2015, American Association for the Advancement of Science.

In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.

Science.gov (United States)

Li, Hong-Tu; Jiang, Fang-Xu; Shi, Ping; Zhang, Tao; Liu, Xiao-Yu; Lin, Xue-Wen; San, Zhong-Yan; Pang, Xi-Ning

2017-02-01

Islet transplantation provides curative treatments to patients with type 1 diabetes, but donor shortage restricts the broad use of this therapy. Thus, generation of alternative transplantable cell sources is intensively investigated worldwide. We previously showed that bone marrow-derived mesenchymal stem cells (bmMSCs) can be reprogrammed to pancreatic-like cells through simultaneously forced suppression of Rest/Nrsf (repressor element-1 silencing transcription factor/neuronal restrictive silencing factor) and Shh (sonic hedgehog) and activation of Pdx1 (pancreas and duodenal transcription factor 1). We here aimed to reprogram bmMSCs further along the developmental pathway towards the islet lineages by improving our previous strategy and by overexpression of Ngn3 (neurogenin 3) and NeuroD1 (neurogenic differentiation 1), critical regulators of the development of endocrine pancreas. We showed that compared to the previous protocol, the overexpression of only Pdx1 and Ngn3 reprogrammed bmMSCs into cells with more characteristics of islet endocrine lineages verified with bioinformatic analyses of our RNA-Seq datasets. These analyses indicated 2325 differentially expressed genes including those involved in the pancreas and islet development. We validated with qRT-PCR analysis selective genes identified from the RNA-Seq datasets. Thus, we reprogrammed bmMSCs into islet endocrine-like cells and advanced the endeavor to generate surrogate functional insulin-secreting cells.

Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State.

Science.gov (United States)

Li, Xiang; Liu, Defang; Ma, Yantao; Du, Xiaomin; Jing, Junzhan; Wang, Lipeng; Xie, Bingqing; Sun, Da; Sun, Shaoqiang; Jin, Xueqin; Zhang, Xu; Zhao, Ting; Guan, Jingyang; Yi, Zexuan; Lai, Weifeng; Zheng, Ping; Huang, Zhuo; Chang, Yanzhong; Chai, Zhen; Xu, Jun; Deng, Hongkui

2017-08-03

Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state. Copyright © 2017 Elsevier Inc. All rights reserved.

A gene expression predictor of response to EGFR-targeted therapy stratifies progression-free survival to cetuximab in KRAS wild-type metastatic colorectal cancer

Directory of Open Access Journals (Sweden)

Black Esther P

2009-05-01

Full Text Available Abstract Background The anti-EGFR monoclonal antibody cetuximab is used in metastatic colorectal cancer (CRC, and predicting responsive patients garners great interest, due to the high cost of therapy. Mutations in the KRAS gene occur in ~40% of CRC and are a negative predictor of response to cetuximab. However, many KRAS-wildtype patients do not benefit from cetuximab. We previously published a gene expression predictor of sensitivity to erlotinib, an EGFR inhibitor. The purpose of this study was to determine if this predictor could identify KRAS-wildtype CRC patients who will benefit from cetuximab therapy. Methods Microarray data from 80 metastatic CRC patients subsequently treated with cetuximab were extracted from the study by Khambata-Ford et al. The study included KRAS status, response, and PFS for each patient. The gene expression data were scaled and analyzed using our predictive model. An improved predictive model of response was identified by removing features in the 180-gene predictor that introduced noise. Results Forty-three of eighty patients were identified as harboring wildtype-KRAS. When the model was applied to these patients, the predicted-sensitive group had significantly longer PFS than the predicted-resistant group (median 88 days vs. 56 days; mean 117 days vs. 63 days, respectively, p = 0.008. Kaplan-Meier curves were also significantly improved in the predicted-sensitive group (p = 0.0059, HR = 0.4109. The model was simplified to 26 of the original 180 genes and this further improved stratification of PFS (median 147 days vs. 56.5 days in the predicted sensitive and resistant groups, respectively, p Conclusion Our model of sensitivity to EGFR inhibition stratified PFS following cetuximab in KRAS-wildtype CRC patients. This study represents the first true external validation of a molecular predictor of response to cetuximab in KRAS-WT metastatic CRC. Our model may hold clinical utility for identifying patients responsive

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

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Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

2000-05-01

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

Elucidating the Metabolic Plasticity of Cancer: Mitochondrial Reprogramming and Hybrid Metabolic States

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Dongya Jia

2018-03-01

Full Text Available Aerobic glycolysis, also referred to as the Warburg effect, has been regarded as the dominant metabolic phenotype in cancer cells for a long time. More recently, it has been shown that mitochondria in most tumors are not defective in their ability to carry out oxidative phosphorylation (OXPHOS. Instead, in highly aggressive cancer cells, mitochondrial energy pathways are reprogrammed to meet the challenges of high energy demand, better utilization of available fuels and macromolecular synthesis for rapid cell division and migration. Mitochondrial energy reprogramming is also involved in the regulation of oncogenic pathways via mitochondria-to-nucleus retrograde signaling and post-translational modification of oncoproteins. In addition, neoplastic mitochondria can engage in crosstalk with the tumor microenvironment. For example, signals from cancer-associated fibroblasts can drive tumor mitochondria to utilize OXPHOS, a process known as the reverse Warburg effect. Emerging evidence shows that cancer cells can acquire a hybrid glycolysis/OXPHOS phenotype in which both glycolysis and OXPHOS can be utilized for energy production and biomass synthesis. The hybrid glycolysis/OXPHOS phenotype facilitates metabolic plasticity of cancer cells and may be specifically associated with metastasis and therapy-resistance. Moreover, cancer cells can switch their metabolism phenotypes in response to external stimuli for better survival. Taking into account the metabolic heterogeneity and plasticity of cancer cells, therapies targeting cancer metabolic dependency in principle can be made more effective.

Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells

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Ras Trokovic

2015-07-01

Full Text Available Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time.

SOX2 Reprograms Resident Astrocytes into Neural Progenitors in the Adult Brain

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Wenze Niu

2015-05-01

Full Text Available Glial cells can be in vivo reprogrammed into functional neurons in the adult CNS; however, the process by which this reprogramming occurs is unclear. Here, we show that a distinct cellular sequence is involved in SOX2-driven in situ conversion of adult astrocytes to neurons. This includes ASCL1+ neural progenitors and DCX+ adult neuroblasts (iANBs as intermediates. Importantly, ASCL1 is required, but not sufficient, for the robust generation of iANBs in the adult striatum. These progenitor-derived iANBs predominantly give rise to calretinin+ interneurons when supplied with neurotrophic factors or the small-molecule valproic acid. Patch-clamp recordings from the induced neurons reveal subtype heterogeneity, though all are functionally mature, fire repetitive action potentials, and receive synaptic inputs. Together, these results show that SOX2-mediated in vivo reprogramming of astrocytes to neurons passes through proliferative intermediate progenitors, which may be exploited for regenerative medicine.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

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Kate E. Hawkins

2016-03-01

Full Text Available The potential of induced pluripotent stem cells (iPSCs in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.

Excitation block in a nerve fibre model owing to potassium-dependent changes in myelin resistance.

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Brazhe, A R; Maksimov, G V; Mosekilde, E; Sosnovtseva, O V

2011-02-06

The myelinated nerve fibre is formed by an axon and Schwann cells or oligodendrocytes that sheath the axon by winding around it in tight myelin layers. Repetitive stimulation of a fibre is known to result in accumulation of extracellular potassium ions, especially between the axon and the myelin. Uptake of potassium leads to Schwann cell swelling and myelin restructuring that impacts the electrical properties of the myelin. In order to further understand the dynamic interaction that takes place between the myelin and the axon, we have modelled submyelin potassium accumulation and related changes in myelin resistance during prolonged high-frequency stimulation. We predict that potassium-mediated decrease in myelin resistance leads to a functional excitation block with various patterns of altered spike trains. The patterns are found to depend on stimulation frequency and amplitude and to range from no block (less than 100 Hz) to a complete block (greater than 500 Hz). The transitional patterns include intermittent periodic block with interleaved spiking and non-spiking intervals of different relative duration as well as an unstable regime with chaotic switching between the spiking and non-spiking states. Intermittent conduction blocks are accompanied by oscillations of extracellular potassium. The mechanism of conductance block based on myelin restructuring complements the already known and modelled block via hyperpolarization mediated by the axonal sodium pump and potassium depolarization.

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

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Erben, Philipp, E-mail: philipp.erben@medma.uni-heidelberg.de [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Stroebel, Philipp [Pathologisches Institut, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Horisberger, Karoline [Chirurgische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Kaehler, Georg; Kienle, Peter; Post, Stefan [Chirurgische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Wenz, Frederik [Klinik fuer Strahlentherapie und Radioonkologie, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Hochhaus, Andreas [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Klinik fuer Innere Medizin II, Abteilung Haematologie/Onkologie, Universitaetsklinikum Jena, Jena (Germany); Hofheinz, Ralf-Dieter [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany)

2011-11-15

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan-Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

International Nuclear Information System (INIS)

Erben, Philipp; Ströbel, Philipp; Horisberger, Karoline; Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin; Kähler, Georg; Kienle, Peter; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas; Hofheinz, Ralf-Dieter

2011-01-01

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan–Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

Combinatorial Modulation of Signaling Pathways Reveals Cell-Type-Specific Requirements for Highly Efficient and Synchronous iPSC Reprogramming

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Simon E. Vidal

2014-10-01

Full Text Available The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs. To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor β (TGF-β together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-β inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-β/mitogen-activated protein (MAP kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA.

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Ersin Akinci

Full Text Available The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.

Exploratory biomarker analysis for treatment response in KRAS wild type metastatic colorectal cancer patients who received cetuximab plus irinotecan

International Nuclear Information System (INIS)

Kim, Seung Tae; Ahn, Tae Jin; Lee, Eunjin; Do, In-Gu; Lee, Su Jin; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Kim, Suk Hyeong; Lee, Jeeyun; Kim, Hee Cheol

2015-01-01

More than half of the patients selected based on KRAS mutation status fail to respond to the treatment with cetuximab in metastatic colorectal cancer (mCRC). We designed a study to identify additional biomarkers that could act as indicators for cetuximab treatment in mCRC. We investigated 58 tumor samples from wild type KRAS CRC patients treated with cetuximab plus irinotecan (CI). We conducted the genotyping for mutations in either BRAF or PIK3CA and profiled comprehensively the expression of 522 kinase genes. BRAF mutation was detected in 5.1 % (3/58) of patients. All 50 patients showed wild type PIK3CA. Gene expression patterns that categorized patients with or without the disease control to CI were compared by supervised classification analysis. PSKH1, TLK2 and PHKG2 were overexpressed significantly in patients with the disease control to IC. The higher expression value of PSKH1 (r = 0.462, p < 0.001) and TLK2 (r = 0.361, p = 0.005) had the significant correlation to prolonged PFS. The result of this work demonstrated that expression nature of kinase genes such as PSKH1, TLK2 and PHKG2 may be informative to predict the efficacy of CI in wild type KRAS CRC. Mutations in either BRAF or PIK3CA were rare subsets in wild type KRAS CRC

CpG island methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations with male sex and KRAS mutations.

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Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Loda, Massimo; Fuchs, Charles S

2006-11-01

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation seems to be a distinct epigenotype of colorectal cancer. However, no study has comprehensively examined features of colorectal cancer with less extensive promoter methylation (designated as "CIMP-low"). Using real-time polymerase chain reaction (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1] in 840 relatively unbiased, population-based colorectal cancer samples, obtained from two large prospective cohort studies. CIMP-low (defined as 1/5 to 3/5 methylated promoters) colorectal cancers were significantly more common among men (38 versus 30% in women, P = 0.01) and among KRAS-mutated tumors (44 versus 30% in KRAS/BRAF wild-type tumors, P = 0.0003; 19% in BRAF-mutated tumors, P CIMP-low tumors (47%) than in CIMP-high tumors (with > or =4/5 methylated promoters, 12%, P CIMP-0 tumors (with 0/5 methylated promoters, 37%, P = 0.007). The associations of CIMP-low tumors with male sex and KRAS mutations still existed after tumors were stratified by microsatellite instability status. In conclusion, CIMP-low colorectal cancer is associated with male sex and KRAS mutations. The hypothesis that CIMP-low tumors are different from CIMP-high and CIMP-0 tumors needs to be tested further.

PPARα inhibition modulates multiple reprogrammed metabolic pathways in kidney cancer and attenuates tumor growth.

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Abu Aboud, Omran; Donohoe, Dallas; Bultman, Scott; Fitch, Mark; Riiff, Tim; Hellerstein, Marc; Weiss, Robert H

2015-06-01

Kidney cancer [renal cell carcinoma (RCC)] is the sixth-most-common cancer in the United States, and its incidence is increasing. The current progression-free survival for patients with advanced RCC rarely extends beyond 1-2 yr due to the development of therapeutic resistance. We previously identified peroxisome proliferator-activating receptor-α (PPARα) as a potential therapeutic target for this disease and showed that a specific PPARα antagonist, GW6471, induced apoptosis and cell cycle arrest at G0/G1 in RCC cell lines associated with attenuation of cell cycle regulatory proteins. We now extend that work and show that PPARα inhibition attenuates components of RCC metabolic reprogramming, capitalizing on the Warburg effect. The specific PPARα inhibitor GW6471, as well as a siRNA specific to PPARα, attenuates the enhanced fatty acid oxidation and oxidative phosphorylation associated with glycolysis inhibition, and PPARα antagonism also blocks the enhanced glycolysis that has been observed in RCC cells; this effect did not occur in normal human kidney epithelial cells. Such cell type-specific inhibition of glycolysis corresponds with changes in protein levels of the oncogene c-Myc and has promising clinical implications. Furthermore, we show that treatment with GW6471 results in RCC tumor growth attenuation in a xenograft mouse model, with minimal obvious toxicity, a finding associated with the expected on-target effects on c-Myc. These studies demonstrate that several pivotal cancer-relevant metabolic pathways are inhibited by PPARα antagonism. Our data support the concept that targeting PPARα, with or without concurrent inhibition of glycolysis, is a potential novel and effective therapeutic approach for RCC that targets metabolic reprogramming in this tumor.

A stochastic model of epigenetic dynamics in somatic cell reprogramming

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Max eFloettmann

2012-06-01

Full Text Available Somatic cell reprogramming has dramatically changed stem cell research inrecent years. The high pace of new findings in the field and an ever increasingamount of data from new high throughput techniques make it challengingto isolate core principles of the process. In order to analyze suchmechanisms, we developed an abstract mechanistic model of a subset of theknown regulatory processes during cell differentiation and production of inducedpluripotent stem cells. This probabilistic Boolean network describesthe interplay between gene expression, chromatin modifications and DNAmethylation. The model incorporates recent findings in epigenetics and reproducesexperimentally observed reprogramming efficiencies and changes inmethylation and chromatin remodeling. It enables us to investigate in detail,how the temporal progression of the process is regulated. It also explicitlyincludes the transduction of factors using viral vectors and their silencing inreprogrammed cells, since this is still a standard procedure in somatic cellreprogramming. Based on the model we calculate an epigenetic landscape.Simulation results show good reproduction of experimental observations duringreprogramming, despite the simple stucture of the model. An extensiveanalysis and introduced variations hint towards possible optimizations of theprocess, that could push the technique closer to clinical applications. Fasterchanges in DNA methylation increase the speed of reprogramming at theexpense of efficiency, while accelerated chromatin modifications moderatelyimprove efficiency.

No clinical utility of KRAS variant rs61764370 for ovarian or breast cancer

NARCIS (Netherlands)

A. Hollestelle (Antoinette); F.H. Van Der Baan (Frederieke H.); A. Berchuck (Andrew); S.E. Johnatty (Sharon); K.K.H. Aben (Katja); B.A. Agnarsson (Bjarni); K. Aittomäki (Kristiina); E. Alducci (Elisa); I.L. Andrulis (Irene); H. Anton-Culver (Hoda); N.N. Antonenkova (Natalia); A.C. Antoniou (Antonis C.); C. Apicella (Carmel); V. Arndt (Volker); N. Arnold (Norbert); B.K. Arun (Banu); B. Arver (Brita Wasteson); A. Ashworth (Alan); L. Baglietto (Laura); R. Balleine (Rosemary); E.V. Bandera (Elisa); D. Barrowdale (Daniel); Y.T. Bean (Yukie); L. Beckmann (Lars); M.W. Beckmann (Matthias); J. Benítez (Javier); A. Berger (Andreas); R. Berger (Raanan); B. Beuselinck (B.); M. Bisogna (Maria); L. Bjorge (Line); C. Blomqvist (Carl); N.V. Bogdanova (Natalia); A. Bojesen (Anders); S.E. Bojesen (Stig); M.K. Bolla (Manjeet); B. Bonnani (Bernardo); J.S. Brand (Judith S.); H. Brauch (Hiltrud); H. Brenner (Hermann); L.A. Brinton (Louise); A. Brooks-Wilson (Angela); F. Bruinsma (Fiona); J. Brunet (Joan); T. Brüning (Thomas); A. Budzilowska (Agnieszka); C.H. Bunker (Clareann H.); B. Burwinkel (Barbara); R. Butzow (Ralf); S.S. Buys (Saundra S.); M.A. Caligo (Maria); I. Campbell (Ian); J. Carter (Jonathan); J. Chang-Claude (Jenny); S.J. Chanock (Stephen J.); K.B.M. Claes (Kathleen B.M.); J.M. Collée (Margriet); L.S. Cook (Linda S.); F.J. Couch (Fergus); A. Cox (Angela); D.W. Cramer (Daniel); S.S. Cross (Simon); J.M. Cunningham (Julie); C. Cybulski (Cezary); K. Czene (Kamila); F. Damiola (Francesca); A. Dansonka-Mieszkowska (Agnieszka); H. Darabi (Hatef); M. de La Hoya (Miguel); A. DeFazio (Anna); J. Dennis (Joe); P. Devilee (Peter); E. Dicks (Ed); O. Díez (Orland); J.A. Doherty (Jennifer A.); S.M. Domchek (Susan); C.M. Dorfling (Cecilia); T. Dörk (Thilo); I. dos Santos Silva (Isabel); A. Du Bois (Andreas); M. Dumont (Martine); A.M. Dunning (Alison); M. Duran (Mercedes); D.F. Easton (Douglas F.); D. Eccles (Diana); R. Edwards (Robert); H. Ehrencrona (Hans); B. Ejlertsen (Bent); A.B. Ekici (Arif); S.D. Ellis (Steve); C. Engel (Christoph); M. Eriksson (Mikael); P.A. Fasching (Peter); L. Feliubadaló (L.); J.D. Figueroa (Jonine); D. Flesch-Janys (Dieter); O. Fletcher (Olivia); A. Fontaine (Annette); S. Fortuzzi (S.); F. Fostira (Florentia); B.L. Fridley (Brooke); M.O.W. Friebel (Mark ); E. Friedman (Eitan); G. Friel (Grace); D. Frost (Debra); J. Garber (Judy); M. García-Closas (Montserrat); S.A. Gayther (Simon); A. Gentry-Maharaj (Aleksandra); A-M. Gerdes (Anne-Marie); G.G. Giles (Graham); R. Glasspool (Rosalind); G. Glendon (Gord); A.K. Godwin (Andrew K.); M.T. Goodman (Marc T.); M. Gore (Martin); M.H. Greene (Mark H.); M. Grip (Mervi); J. Gronwald (Jacek); D. Gschwantler-Kaulich (Daphne); P. Guénel (Pascal); S.R. Guzman (Starr R.); L. Haeberle (Lothar); C.A. Haiman (Christopher A.); P. Hall (Per); S.L. Halverson (Sandra L.); U. Hamann (Ute); T.V.O. Hansen (Thomas); P. Harter (Philipp); J.M. Hartikainen (J.); S. Healey (Sue); R. Hein (Rebecca); P.U. Heitz; B.E. Henderson (Brian); J. Herzog (Josef); M.A. T Hildebrandt (Michelle A.); C.K. Høgdall (Claus); E. Høgdall (Estrid); F.B.L. Hogervorst (Frans); J.L. Hopper (John); K. Humphreys (Keith); T. Huzarski (Tomasz); E.N. Imyanitov (Evgeny N.); C. Isaacs (Claudine); A. Jakubowska (Anna); R. Janavicius (Ramunas); K. Jaworska (Katarzyna); A. Jensen (Allan); U.B. Jensen; N. Johnson (Nichola); A. Jukkola-Vuorinen (Arja); M. Kabisch (Maria); B.Y. Karlan (Beth Y.); V. Kataja (Vesa); N. Kauff (Noah); L.E. Kelemen (Linda); M. Kerin (Michael); L.A.L.M. Kiemeney (Bart); M. Kjaer (Michael); J.A. Knight (Julia); J.P. Knol-Bout (Jacoba P.); I. Konstantopoulou (I.); V-M. Kosma (Veli-Matti); C. Krakstad (Camilla); V. Kristensen (Vessela); K.B. Kuchenbaecker (Karoline); J. Kupryjanczyk (Jolanta); Y. Laitman (Yael); D. Lambrechts (Diether); S. Lambrechts (Sandrina); M.C. Larson (Melissa); A. Lasa (Adriana); P. Laurent-Puig (Pierre); C. Lazaro (Conxi); N. Le (Nhu); L. Le Marchand (Loic); A. Leminen (Arto); K.J. Lester (Kathryn); D.A. Levine (Douglas); J. Li (Jingmei); D. Liang (Dong); A. Lindblom (Annika); N.M. Lindor (Noralane); J. Lissowska (Jolanta); J. Long (Jirong); K.H. Lu (Karen); J. Lubinski (Jan); L. Lundvall (Lene); G. Lurie (Galina); P.L. Mai (Phuong); A. Mannermaa (Arto); S. Margolin (Sara); F. Mariette (F.); F. Marme (Federick); J.W.M. Martens (John); L.F. Massuger (Leon); C. Maugard; S. Mazoyer (Sylvie); L. McGuffog (Lesley); W.P. McGuire; C.A. McLean (Catriona Ann); I. McNeish (Iain); A. Meindl (Alfons); F. Menegaux (Florence); P. Menéndez (Primitiva); J. Menkiszak (Janusz); U. Menon (Usha); A.R. Mensenkamp (Arjen); N. Miller (Nicola); R.L. Milne (Roger); F. Modugno (Francesmary); M. Montagna (Marco); K.B. Moysich (Kirsten B.); H. Mul̈ler (Heiko); A.-M. Mulligan (Anna-Marie); T.A. Muranen (Taru); S.A. Narod (Steven A.); K.L. Nathanson (Katherine); R.B. Ness (Roberta B.); S.L. Neuhausen (Susan); H. Nevanlinna (Heli); P. Neven (Patrick); F. Nielsen (Finn); S.F. Nielsen (Sune); B.G. Nordestgaard (Børge); R. Nussbaum (Robert); K. Odunsi (Kunle); K. Offit (Kenneth); E. Olah; O.I. Olopade (Olufunmilayo I.); J.E. Olson (Janet); S.H. Olson (Sara); J.C. Oosterwijk (Jan); I. Orlow (Irene); N. Orr (Nick); S. Orsulic (Sandra); A. Osorio (Ana); L. Ottini (Laura); J. Paul (James); C.L. Pearce (Celeste); I.S. Pedersen (Inge Sokilde); B. Peissel (Bernard); T. Pejovic (Tanja); L.M. Pelttari (Liisa); J. Perkins (Jo); J. Permuth-Wey (Jenny); P. Peterlongo (Paolo); J. Peto (Julian); C. Phelan (Catherine); K.-A. Phillips (Kelly-Anne); M. Piedmonte (Marion); M.C. Pike (Malcolm C.); R. Platte (Radka); J. Plisiecka-Halasa (Joanna); E.M. Poole (Elizabeth); B. Poppe (Bruce); K. Pykäs (Katri); P. Radice (Paolo); S.J. Ramus (Susan); R. Rebbeck (Timothy); M.W.R. Reed (Malcolm W.R.); G. Rennert (Gad); H. Risch (Harvey); M. Robson (Mark); G. Rodriguez (Gustavo); A. Romero (Atocha); M.A. Rossing (Mary Anne); J.H. Rothstein (Joseph H.); A. Rudolph (Anja); I.B. Runnebaum (Ingo); R. Salani (Ritu); H.B. Salvesen (Helga); E.J. Sawyer (Elinor); J.M. Schildkraut (Joellen); M.K. Schmidt (Marjanka); R.K. Schmutzler (Rita); A. Schneeweiss (Andreas); M. Schoemaker (Minouk); A. Schrauder (André); F.R. Schumacher (Fredrick); I. Schwaab (Ira); G. Scuvera (Giulietta); T.A. Sellers (Thomas A.); G. Severi (Gianluca); C.M. Seynaeve (Caroline); M. Shah (Mitul); M. Shrubsole (Martha); N. Siddiqui (Nadeem); W. Sieh (Weiva); J. Simard (Jacques); C.F. Singer (Christian); O. Sinilnikova (Olga); D. Smeets (Dominiek); C. Sohn (Christof); M. Soller (Maria); H. Song (Honglin); P. Soucy (Penny); M.C. Southey (Melissa); C. Stegmaier (Christa); D. Stoppa-Lyonnet (Dominique); L. Sucheston (Lara); A.J. Swerdlow (Anthony ); I.L. Tangen (Ingvild L.); M.-K. Tea; P.J. Teixeira; K.L. Terry (Kathryn); M.B. Terry (Mary Beth); M. Thomassen (Mads); P.J. Thompson (Pamela J.); L. Tihomirova (Laima); M. Tischkowitz (Marc); A.E. Toland (Amanda); R.A.E.M. Tollenaar (Rob); I. Tomlinson (Ian); D. Torres (Diana); T. Truong (Thérèse); H. Tsimiklis (Helen); N. Tung (Nadine); S. Tworoger (Shelley); J.P. Tyrer (Jonathan); C. Vachon (Celine); L.J. van 't Veer (Laura); A.M. van Altena (Anne); C.J. van Asperen (Christi); D. Van Den Berg (David); A.M.W. van den Ouweland (Ans); H.C. van Doorn (Helena); E. Van Nieuwenhuysen (Els); E.J. van Rensburg (Elizabeth); I. Vergote (Ignace); S. Verhoef; R.A. Vierkant (Robert); J. Vijai (Joseph); A.F. Vitonis (Allison); A. von Wachenfeldt (Anna); C.S. Walsh (Christine); Q. Wang (Qing); S. Wang-Gohrke (Shan); B. Wapenschmidt (Barbara); M. Weischer (Maren); J.N. Weitzel (Jeffrey); C. Weltens (Caroline); N. Wentzensen (N.); A.S. Whittemore (Alice S.); L.R. Wilkens (Lynne R.); R. Winqvist (Robert); A.H. Wu (Anna); X. Wu (Xifeng); H.P. Yang (Hannah P.); D. Zaffaroni (Daniela); M.P. Zamora (Pilar); W. Zheng (Wei); A. Ziogas (Argyrios); G. Chenevix-Trench (Georgia); P.D.P. Pharoah (Paul); M.A. Rookus (Matti); M.J. Hooning (Maartje); E.L. Goode (Ellen L.); Breast Cancer Family Register; EMBRACE; GENICA Network; HEBON; SWE-BRCA

2016-01-01

textabstractObjective Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3′ UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies,

PCR-based assays versus direct sequencing for evaluating the effect of KRAS status on anti-EGFR treatment response in colorectal cancer patients: a systematic review and meta-analysis.

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Lianfeng Shan

Full Text Available The survival rate of colorectal cancer (CRC patients carrying wild-type KRAS is significantly increased by combining anti-EGFR monoclonal antibody (mAb with standard chemotherapy. However, conflicting data exist in both the wild-type KRAS and mutant KRAS groups, which strongly challenge CRC anti-EGFR treatment. Here we conducted a meta-analysis in an effort to provide more reliable information regarding anti-EGFR treatment in CRC patients.We searched full reports of randomized clinical trials using Medline, the American Society of Clinical Oncology (ASCO, and the European Society for Medical Oncology (ESMO. Two investigators independently screened the published literature according to our inclusive and exclusive criteria and the relative data were extracted. We used Review Manager 5.2 software to analyze the data.The addition of anti-EGFR mAb to standard chemotherapy significantly improved both progression-free survival (PFS and median overall survival (mOS in the wild-type KRAS group; hazard ratios (HRs for PFS and mOS were 0.70 [95% confidence interval (CI, 0.58-0.84] and 0.83 [95% CI, 0.75-0.91], respectively. In sub-analyses of the wild-type KRAS group, when PCR-based assays are employed, PFS and mOS notably increase: the HRs were 0.74 [95% CI, 0.62-0.88] and 0.87 [95% CI, 0.78-0.96], respectively. In sub-analyses of the mutant KRAS group, neither PCR-based assays nor direct sequencing enhance PFS or mOS.Our data suggest that PCR-based assays with high sensitivity and specificity allow accurate identification of patients with wild-type KRAS and thus increase PFS and mOS. Furthermore, such assays liberate patients with mutant KRAS from unnecessary drug side effects, and provide them an opportunity to receive appropriate treatment. Thus, establishing a precise standard reference test will substantially optimize CRC-targeted therapies.

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs.

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Pareja, Kristeen; Chu, Elaine; Dodyk, Katrina; Richter, Kristofer; Miller, Alan

2013-01-01

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

KRAS mutation testing of tumours in adults with metastatic colorectal cancer: a systematic review and cost-effectiveness analysis.

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Westwood, Marie; van Asselt, Thea; Ramaekers, Bram; Whiting, Penny; Joore, Manuela; Armstrong, Nigel; Noake, Caro; Ross, Janine; Severens, Johan; Kleijnen, Jos

2014-10-01

Bowel cancer is the third most common cancer in the UK. Most bowel cancers are initially treated with surgery, but around 17% spread to the liver. When this happens, sometimes the liver tumour can be treated surgically, or chemotherapy may be used to shrink the tumour to make surgery possible. Kirsten rat sarcoma viral oncogene (KRAS) mutations make some tumours less responsive to treatment with biological therapies such as cetuximab. There are a variety of tests available to detect these mutations. These vary in the specific mutations that they detect, the amount of mutation they detect, the amount of tumour cells needed, the time to give a result, the error rate and cost. To compare the performance and cost-effectiveness of KRAS mutation tests in differentiating adults with metastatic colorectal cancer whose metastases are confined to the liver and are unresectable and who may benefit from first-line treatment with cetuximab in combination with standard chemotherapy from those who should receive standard chemotherapy alone. Thirteen databases, including MEDLINE and EMBASE, research registers and conference proceedings were searched to January 2013. Additional data were obtained from an online survey of laboratories participating in the UK National External Quality Assurance Scheme pilot for KRAS mutation testing. A systematic review of the evidence was carried out using standard methods. Randomised controlled trials were assessed for quality using the Cochrane risk of bias tool. Diagnostic accuracy studies were assessed using the QUADAS-2 tool. There were insufficient data for meta-analysis. For accuracy studies we calculated sensitivity and specificity together with 95% confidence intervals (CIs). Survival data were summarised as hazard ratios and tumour response data were summarised as relative risks, with 95% CIs. The health economic analysis considered the long-term costs and quality-adjusted life-years associated with different tests followed by treatment

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

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Cantone, Irene; Fisher, Amanda G

2017-11-05

X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Novel molecular targets for kRAS downregulation: promoter G-quadruplexes

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2016-11-01

proteins studied. 6. Products: • Publications, conference papers , and presentations o Journal Publications • Morgan, RK; Batra, H; Gaerig, VC; Hockings, J... papers , and presentations • Batra, H; Brooks, TA. Binding and function of regulatory proteins to the kRAS promoter: a role in pancreatic cancer. 6th...development due to difficulties with delivery and excessive albumin binding, and antisoma’s G-rich phosphodiester oligonucleotide AS1411, a DNA aptamer with

Antisense gene therapy using anti-k-ras and antitelomerase oligonucleotides in colorectal cancer Eficacia de la terapia génica antisentido utilizando oligonucleótidos anti K-ras y antitelomerasa en cáncer colorrectal

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S. Lledó

2005-07-01

Full Text Available Aim: to test the efficacy of anti-k-ras and antitelomerase oligonucleotides for disabling colorectal cancer cell growth. Material and methods: an established human colorectal cancer cell line (SW 480, ATTC® was used. Oligodeoxiribonucleotides (ODNs have a phosphorotioate modification to ensure intracellular intake. We used an antitelomerase ODN (Telp5 and two anti-k-ras ODNs (AS-KRAS and ISIS. AS-KRAS is designed to join the k-ras oncogene's exon 1. ISIS links to the terminal transcription unit 5' of k-ras. Telp5 joins the template region of the hTR telomerase subunit. ODNs have been tested in different concentrations (1, 5, 10, 20 micromolar. Cell viability has been tested at 48 and 72 hours. Statistical analysis and graphic design were made with the statistical package "Analyzing Data with GraphPad Prism-1999", GraphPad Sofware Inc., San Diego CA©. We used the Student's t test for statistical analysis. Results: the lowest dose (1 µM was not effective. Using the highest dose (20 mM for 48 hours of combined AS-KRAS and Telp5 cell viability decreased to 99.67%. The rest of results varied depending on ODN type, dose, and exposure time. Conclusions: tested antisense ODNs stop colorectal cancer cell growth, and a combination of anti-telomerase and anti-k-ras is the most useful treatment. Efficacy is best with a higher dose and longer treatment period.Objetivo: evaluar la eficacia de oligonucleótidos anti k-ras y antitelomerasa para detener el crecimiento tumoral en el cáncer colorrectal. Material y métodos: se ha empleado una línea celular establecida de cáncer colorrectal humano (SW 480, ATTC®. Los oligodesoxirribonucleótidos (ODN utilizados en el presente trabajo presentan modificación fosforotioato con el fin de mejorar su estabilidad en presencia de fluidos biológicos. Hemos utilizado un ODN antitelomerasa (Telp5, y dos ODN anti k-ras (AS-KRAS e ISIS. AS-KRAS actúa en el exón 1 e ISIS actúa a nivel de la unidad terminal de

The ultrasound-guided retrolaminar block: volume-dependent injectate distribution

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Damjanovska M

2018-02-01

Full Text Available Marija Damjanovska,1 Tatjana Stopar Pintaric,1,2 Erika Cvetko,2 Kamen Vlassakov3 1Clinical Department of Anesthesiology and Intensive Therapy, University Medical Centre Ljubljana, Ljubljana, Slovenia; 2Institute of Anatomy, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 3Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Purpose: The ultrasound-guided retrolaminar block is one of the newer and simpler alternatives to the traditional, often technically challenging, paravertebral (PV block. Its feasibility, safety, and efficacy have already been clinically demonstrated in patients with multiple rib fractures using higher volumes of local anesthetic, when compared with the traditional approach. The primary aim of this observational anatomical study was to assess the spread of local anesthetic from the retrolaminar injection point to the PV space and its volume dependence. Second, we assessed the incidence of epidural and contralateral PV spread in the both groups.Methods: Ten fresh porcine cadavers were randomized into 2 groups (n=5 each to receive ultrasound-guided retrolaminar injections at Th4-Th5 level with either 10 mL (low-volume group or 30 mL (high-volume group of 2% lidocaine and methylene blue mixture. After the procedure, the cadavers were dissected and frozen. Cross-section cuts (~1 cm thick were performed to evaluate the injectate spread.Results: In the high-volume group, injectate spread from the retrolaminar to the PV space was observed in all specimens (5 out of 5; 100%, while in the low-volume group, no apparent spread to the PV space was found (0 out of 5; 0%. No epidural or contralateral PV spread was observed in any of the specimens.Conclusion: Following ultrasound-guided retrolaminar injections in fresh porcine cadavers, injectate spread from the retrolaminar tissue plane to the PV space is strongly volume dependent

Animal products and K-ras codon 12 and 13 mutations in colon carcinomas

NARCIS (Netherlands)

Kampman, E.; Voskuil, D.W.; Kraats, A.A. van; Balder, H.F.; Muijen, G.N.P. van; Goldbohm, R.A.; Veer, P. van 't

2000-01-01

K-ras gene mutations (codons 12 and 13) were determined by PCR-based mutant allele-specific amplification (MASA) in tumour tissue of 185 colon cancer patients: 36% harboured mutations, of which 82% were located in codon 12. High intakes of animal protein, calcium and poultry were differently

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

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Warren, Luigi; Manos, Philip D; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj K; Smith, Zachary D; Meissner, Alexander; Daley, George Q; Brack, Andrew S; Collins, James J; Cowan, Chad; Schlaeger, Thorsten M; Rossi, Derrick J

2010-11-05

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. Copyright © 2010 Elsevier Inc. All rights reserved.

Direct Reprogramming of Human Bone Marrow Stromal Cells into Functional Renal Cells Using Cell-free Extracts

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Evangelia Papadimou

2015-04-01

Full Text Available The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs, also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

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Sirirat Seekhuntod

Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

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Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

Presence of activating KRAS mutations correlates significantly with expression of tumour suppressor genes DCN and TPM1 in colorectal cancer

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Rems Miran

2009-08-01

Full Text Available Abstract Background Despite identification of the major genes and pathways involved in the development of colorectal cancer (CRC, it has become obvious that several steps in these pathways might be bypassed by other as yet unknown genetic events that lead towards CRC. Therefore we wanted to improve our understanding of the genetic mechanisms of CRC development. Methods We used microarrays to identify novel genes involved in the development of CRC. Real time PCR was used for mRNA expression as well as to search for chromosomal abnormalities within candidate genes. The correlation between the expression obtained by real time PCR and the presence of the KRAS mutation was investigated. Results We detected significant previously undescribed underexpression in CRC for genes SLC26A3, TPM1 and DCN, with a suggested tumour suppressor role. We also describe the correlation between TPM1 and DCN expression and the presence of KRAS mutations in CRC. When searching for chromosomal abnormalities, we found deletion of the TPM1 gene in one case of CRC, but no deletions of DCN and SLC26A3 were found. Conclusion Our study provides further evidence of decreased mRNA expression of three important tumour suppressor genes in cases of CRC, thus implicating them in the development of this type of cancer. Moreover, we found underexpression of the TPM1 gene in a case of CRCs without KRAS mutations, showing that TPM1 might serve as an alternative path of development of CRC. This downregulation could in some cases be mediated by deletion of the TPM1 gene. On the other hand, the correlation of DCN underexpression with the presence of KRAS mutations suggests that DCN expression is affected by the presence of activating KRAS mutations, lowering the amount of the important tumour suppressor protein decorin.

Reprogrammed Functional Brown Adipocytes Ameliorate Insulin Resistance and Dyslipidemia in Diet-Induced Obesity and Type 2 Diabetes

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Tsunao Kishida

2015-10-01

Full Text Available Brown adipocytes (BAs play important roles in body temperature regulation, energy balance, and carbohydrate and lipid metabolism. Activities of BAs are remarkably diminished in obese and diabetic patients, providing possibilities of transplanting functional BAs resulting in therapeutic benefit. Here, we show generation of functional BAs by cellular reprogramming procedures. Transduction of the PRDM16 gene into iPSC-derived embryoid bodies induced BA phenotypes (iBAs. Moreover, normal human fibroblasts were directly converted into BAs (dBAs by C/EBP-β and C-MYC gene transduction. Approximately 90% of the fibroblasts were successfully converted within 12 days. The dBAs were highly active in mitochondrial biogenesis and oxidative metabolism. Mouse dBAs were induced by Prdm16, C/ebp-β, and L-myc genes, and after transplantation, they significantly reduced diet-induced obesity and insulin resistance in an UCP1-dependent manner. Thus, highly functional BAs can be generated by cellular reprogramming, suggesting a promising tailor-made cell therapy against metabolic disorders including type 2 diabetes mellitus.

Reprogrammed Functional Brown Adipocytes Ameliorate Insulin Resistance and Dyslipidemia in Diet-Induced Obesity and Type 2 Diabetes.

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Kishida, Tsunao; Ejima, Akika; Yamamoto, Kenta; Tanaka, Seiji; Yamamoto, Toshiro; Mazda, Osam

2015-10-13

Brown adipocytes (BAs) play important roles in body temperature regulation, energy balance, and carbohydrate and lipid metabolism. Activities of BAs are remarkably diminished in obese and diabetic patients, providing possibilities of transplanting functional BAs resulting in therapeutic benefit. Here, we show generation of functional BAs by cellular reprogramming procedures. Transduction of the PRDM16 gene into iPSC-derived embryoid bodies induced BA phenotypes (iBAs). Moreover, normal human fibroblasts were directly converted into BAs (dBAs) by C/EBP-β and C-MYC gene transduction. Approximately 90% of the fibroblasts were successfully converted within 12 days. The dBAs were highly active in mitochondrial biogenesis and oxidative metabolism. Mouse dBAs were induced by Prdm16, C/ebp-β, and L-myc genes, and after transplantation, they significantly reduced diet-induced obesity and insulin resistance in an UCP1-dependent manner. Thus, highly functional BAs can be generated by cellular reprogramming, suggesting a promising tailor-made cell therapy against metabolic disorders including type 2 diabetes mellitus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

Science.gov (United States)

Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

2016-02-01

K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. © 2016 Federation of European Biochemical Societies.

Lineage Reprogramming of Astroglial Cells from Different Origins into Distinct Neuronal Subtypes

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Malek Chouchane

2017-07-01

Full Text Available Astroglial cells isolated from the rodent postnatal cerebral cortex are particularly susceptible to lineage reprogramming into neurons. However, it remains unknown whether other astroglial populations retain the same potential. Likewise, little is known about the fate of induced neurons (iNs in vivo. In this study we addressed these questions using two different astroglial populations isolated from the postnatal brain reprogrammed either with Neurogenin-2 (Neurog2 or Achaete scute homolog-1 (Ascl1. We show that cerebellum (CerebAstro and cerebral cortex astroglia (CtxAstro generates iNs with distinctive neurochemical and morphological properties. Both astroglial populations contribute iNs to the olfactory bulb following transplantation in the postnatal and adult mouse subventricular zone. However, only CtxAstro transfected with Neurog2 differentiate into pyramidal-like iNs after transplantation in the postnatal cerebral cortex. Altogether, our data indicate that the origin of the astroglial population and transcription factors used for reprogramming, as well as the region of integration, affect the fate of iNs.

Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

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Edwards Jeremy

2003-07-01

Full Text Available Abstract Background The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH, in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. Results Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. Conclusions In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence.

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Marta Winiecka-Klimek

Full Text Available Tumorigenic potential of induced pluripotent stem cells (iPSCs infiltrating population of induced neural stem cells (iNSCs generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc obtained with different methods-direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like or SOX2 and c-MYC (SMiNSc-like and induced pluripotent stem cells differentiation to ebiNSc-in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU incorporation and senescence-associated beta-galactosidase (SA-β-gal assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or

NANOG priming before full reprogramming may generate germ cell tumours

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I Grad

2011-11-01

Full Text Available Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Reprogramming mediated radio-resistance of 3D-grown cancer cells

International Nuclear Information System (INIS)

Xue Gang; Ren Zhenxin; Chen Yaxiong; Zhu Jiayun; Du Yarong; Pan Dong; Li Xiaoman; Hu Burong; Grabham, Peter W.

2015-01-01

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. (author)

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

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Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

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Bose, Bipasha; Shenoy P, Sudheer

2016-02-01

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Embelin suppresses growth of human pancreatic cancer xenografts, and pancreatic cancer cells isolated from KrasG12D mice by inhibiting Akt and Sonic hedgehog pathways.

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Minzhao Huang

Full Text Available Pancreatic cancer is a deadly disease, and therefore effective treatment and/or prevention strategies are urgently needed. The objectives of this study were to examine the molecular mechanisms by which embelin inhibited human pancreatic cancer cell growth in vitro, and xenografts in Balb C nude mice, and pancreatic cancer cell growth isolated from KrasG12D transgenic mice. XTT assays were performed to measure cell viability. AsPC-1 cells were injected subcutaneously into Balb c nude mice and treated with embelin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of Akt, and Sonic Hedgehog (Shh and their target gene products were measured by the immunohistochemistry, and Western blot analysis. The effects of embelin on pancreatic cancer cells isolated from 10-months old KrasG12D mice were also examined. Embelin inhibited cell viability in pancreatic cancer AsPC-1, PANC-1, MIA PaCa-2 and Hs 766T cell lines, and these inhibitory effects were blocked either by constitutively active Akt or Shh protein. Embelin-treated mice showed significant inhibition in tumor growth which was associated with reduced expression of markers of cell proliferation (Ki67, PCNA and Bcl-2 and cell cycle (cyclin D1, CDK2, and CDK6, and induction of apoptosis (activation of caspase-3 and cleavage of PARP, and increased expression of Bax. In addition, embelin inhibited the expression of markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8, and metastasis (MMP-2 and MMP-9 in tumor tissues. Antitumor activity of embelin was associated with inhibition of Akt and Shh pathways in xenografts, and pancreatic cancer cells isolated from KrasG12D mice. Furthermore, embelin also inhibited epithelial-to-mesenchymal transition (EMT by up-regulating E-cadherin and inhibiting the expression of Snail, Slug, and ZEB1. These data suggest that embelin can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt and

Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

OpenAIRE

Warlich, Eva; Kuehle, Johannes; Cantz, Tobias; Brugman, Martijn H; Maetzig, Tobias; Galla, Melanie; Filipczyk, Adam A; Halle, Stephan; Klump, Hannes; Schöler, Hans R; Baum, Christopher; Schroeder, Timm; Schambach, Axel

2011-01-01

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iP...

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

DEFF Research Database (Denmark)

Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

2006-01-01

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

Science.gov (United States)

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-11-27

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of the correlation between KRAS mutated allele frequency and pathologist tumorous nuclei percentage assessment in colorectal cancer suggests a role for zygosity status.

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Libbrecht, Louis; Baldin, Pamela; Dekairelle, Anne-France; Jouret-Mourin, Anne

2018-04-27

Evaluation of molecular tumour heterogeneity relies on the tumorous nuclei percentage (TNP) assessment by a pathologist, which has been criticised for being inaccurate and suffering from interobserver variability. Based on the 'Big Bang theory' which states that KRAS mutation in colorectal cancer is mostly homogeneous, we investigated this issue by performing a critical analysis of the correlation of the KRAS mutant allele fraction with the TNP in 99 colorectal tumour samples with a positive KRAS mutation status as determined by next-generation sequencing. Our results yield indirect evidence that the KRAS zygosity status influences the correlation between these parameters and we show that a well-trained pathologist is indeed capable of accurately assessing TNP. Our findings indicate that tumour zygosity, a feature which has largely been neglected until now, should be taken into account in future studies on (colorectal) molecular tumour heterogeneity. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Intrinsic and extrinsic molecular determinants or modulators for epigenetic remodeling and reprogramming of somatic cell-derived genome in mammalian nuclear-transferred oocytes and resultant embryos.

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Samiec, M; Skrzyszowska, M

2018-03-01

The efficiency of somatic cell cloning in mammals remains disappointingly low. Incomplete and aberrant reprogramming of epigenetic memory of somatic cell nuclei in preimplantation nuclear- transferred (NT) embryos is one of the most important factors that limit the cloning effectiveness. The extent of epigenetic genome-wide alterations, involving histone or DNA methylation and histone deacetylation, that are mediated by histone-lysine methyltransferases (HMTs) or DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be modulated/reversed via exogenous inhibitors of these enzymes throughout in vitro culture of nuclear donor cells, nuclear recipient oocytes and/or cloned embryos. The use of the artificial modifiers of epigenomically-conditioned gene expression leads to inhibition of both chromatin condensation and transcriptional silencing the genomic DNA of somatic cells that provide a source of nuclear donors for reconstruction of enucleated oocytes and generation of cloned embryos. The onset of chromatin decondensation and gene transcriptional activity is evoked both through specific/selective inactivating HMTs by BIX-01294 and through non-specific/non-selective blocking the activity of either DNMTs by 5-aza-2'-deoxycytidine, zebularine, S-adenosylhomocysteine or HDACs by trichostatin A, valproic acid, scriptaid, oxamflatin, sodium butyrate, m-carboxycinnamic acid bishydroxamide, panobinostat, abexinostat, quisinostat, dacinostat, belinostat and psammaplin A. Epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or cloned embryos may facilitate and accelerate the reprogrammability for gene expression of donor cell nuclei that have been transplanted into a host ooplasm and subsequently underwent dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during pre- and postimplantation development of NT embryos. Nevertheless, a comprehensive additional work is necessary to determine

Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming

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Kyung Tae Lim

2016-04-01

Full Text Available Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps. However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.

Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

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Sara E. Howden

2015-12-01

Full Text Available The derivation of genetically modified induced pluripotent stem (iPS cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

Bio markers and Anti-EGFR therapies for Krads wild-type tumors in metastatic colorectal cancer patients; Biomarcadores y terapeutica ANTI-EGFR en el cancer colorrectal metastasico en pacientes con K-Ras no mutado

Energy Technology Data Exchange (ETDEWEB)

Diaz Rubio Garcia, E

2009-07-01

The natural history of metastasis colorectal cancer has being clearly modified in terms of response rate, time to progression and overall survival, once the anti-EGFR monoclonal antibodies (cetuximab and panitumumab) have emerged in combination with the standard cytotoxic chemotherapy (FOLFOX and FOLFIRI). However, the benefit from cetuximab and panitumumab is only confined to KRAS-wild type (KRAS-wt) colorectal tumors, while KRAS mutated tumors do not respond to these drugs. The 65 % of colorectal tumors are KRAS-wt tumors, but efficacy of antiEGFR therapies is detected only in 60-70 % of these KRAS-wt tumors. Other biomarkers and molecular pathways must be involved in the response of the antiEGFR therapies for the KRAS-wt colorectal tumors, such as the EGFR ligands, the EGFR-phosphorilated levels, the number of EGFR copies, the status of the KRAS effected B-RAF and the alternative intracellular signaling pathways PIK3CA/PTEN/AKT and JAK/STAT. A battery of these biomarkers is needed to select the most sensitive patients to the antiEGFR therapies. This pattern may represent a novel favorable cost-effectiveness tool to develop tailored treatments. A review of these biomarkers and molecular pathways, involved in the antiEGFR therapies response, is performed. (Author) 68 refs.

Regenerating the human heart: direct reprogramming strategies and their current limitations.

Science.gov (United States)

Ghiroldi, Andrea; Piccoli, Marco; Ciconte, Giuseppe; Pappone, Carlo; Anastasia, Luigi

2017-10-27

Cardiovascular diseases are the leading cause of death in the Western world. Unfortunately, current therapies are often only palliative, consequently essentially making heart transplantation necessary for many patients. However, several novel therapeutic approaches in the past two decades have yielded quite encouraging results. The generation of induced pluripotent stem cells, through the forced expression of stem cell-specific transcription factors, has inspired the most promising strategies for heart regeneration by direct reprogramming of cardiac fibroblasts into functional cardiomyocytes. Initial attempts at this reprogramming were conducted using a similar approach to the one used with transcription factors, but during years, novel strategies have been tested, e.g., miRNAs, recombinant proteins and chemical molecules. Although preliminary results on animal models are promising, the low reprogramming efficiency, as well as the incomplete maturation of the cardiomyocytes, still represents important obstacles. This review covers direct transdifferentiation strategies that have been proposed and developed and illustrates the pros and cons of each approach. Indeed, as described in the manuscript, there are still many unanswered questions and drawbacks that require a better understanding of the basic signaling pathways and transcription factor networks before functional cells, suitable for cardiac regeneration and safe for the patients, can be generated and used for human therapies.

k-RAS mutations in non-small cell lung cancer patients treated with TKIs among smokers and non-smokers: a meta-analysis

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Ai-Gui Jiang

2016-06-01

Full Text Available Aim of the study : Recent studies have suggested that k-RAS mutations are related to the response to epidermal growth factor receptor (EGFR tyrosine-kinase inhibitions (TKIs in advanced non-small cell lung cancer (NSCLC treatment. The aim of this meta-analysis was to assess the relationship between smoking history and k-RAS mutations in NSCLC treated with TKIs. Material and methods : We searched MEDLINE and Web of Science up to 15 March 2014. The pooled relative risk (RR was estimated by using fixed effect model or random effect model, according to heterogeneity between studies. We also carried out power analyses. Results : We identified 12 studies with 1193 patients, including 196 patients (16.4% with k-RAS mutations. The pooled k-RAS mutations incidence was 22.8% (174/764 in patients with smoke expose vs. 5.4% (23/429 in those with no smoke exposure. The pooled RR was 2.991 (95% CI: 1.884–4.746; Z = 4.65, p = 0.000. No publication bias was found (Begg’s test: z = 1.09, p = 0.274 and Egger’s test: t = 1.38, p = 0.201. In subgroup analyses, the pooled RR was 3.336 (95% CI: 1.925–5.779; Z = 4.30, p = 0.000 in the Caucasian subgroup, while in the Asian subgroup the pooled RR was 2.093 (95% CI: 0.909–4.822; Z = 1.73, p = 0.083, but the sample size was underpowered (0.465. Conclusions : The current meta-analysis found that smoking was related to increased incidence of k-RAS mutations in non-small cell lung cancer treated with TKIs. This may be further evidence that smoking will lead to a worse prognosis in NSCLC patients treated with TKIs.

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene.

Science.gov (United States)

Mariani, Sara; Bertero, Luca; Osella-Abate, Simona; Di Bello, Cristiana; Francia di Celle, Paola; Coppola, Vittoria; Sapino, Anna; Cassoni, Paola; Marchiò, Caterina

2017-07-25

Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies. We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs. By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12). mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

International Nuclear Information System (INIS)

Wang Liu; Zheng Aihua; Yi Ling; Xu Chongren; Ding Mingxiao; Deng Hongkui

2004-01-01

Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation

Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B.

Science.gov (United States)

Lee, Chih-Yung Sean; Lu, Tu; Seydoux, Geraldine

2017-11-07

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin.

Genetic reprogramming of host cells by bacterial pathogens.

Science.gov (United States)

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

Using Naïve Bayesian Analysis to Determine Imaging Characteristics of KRAS Mutations in Metastatic Colon Cancer.

Science.gov (United States)

Pershad, Yash; Govindan, Siddharth; Hara, Amy K; Borad, Mitesh J; Bekaii-Saab, Tanios; Wallace, Alex; Albadawi, Hassan; Oklu, Rahmi

2017-09-02

Genotype, particularly Ras status, greatly affects prognosis and treatment of liver metastasis in colon cancer patients. This pilot aimed to apply word frequency analysis and a naive Bayes classifier on radiology reports to extract distinguishing imaging descriptors of wild-type colon cancer patients and those with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. In this institutional-review-board-approved study, we compiled a SNaPshot mutation analysis dataset from 457 colon adenocarcinoma patients. From this cohort of patients, we analyzed radiology reports of 299 patients (> 32,000 reports) who either were wild-type (147 patients) or had a KRAS (152 patients) mutation. Our algorithm determined word frequency within the wild-type and mutant radiology reports and used a naive Bayes classifier to determine the probability of a given word belonging to either group. The classifier determined that words with a greater than 50% chance of being in the KRAS mutation group and which had the highest absolute probability difference compared to the wild-type group included: "several", "innumerable", "confluent", and "numerous" ( p colon adenocarcinoma. Moreover, likely characteristic imaging traits of mutant tumors make probabilistic word analysis useful in identifying unique characteristics and disease course, with applications ranging from radiology and pathology reports to clinical notes.

Long-Term Survival with Regorafenib in KRAS-Mutated Metastatic Rectal Cancer

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Marie-Laure Amram

2017-11-01

Full Text Available Regorafenib, an oral multikinase inhibitor, was approved in September 2012 by the US Food and Drug Administration for the treatment of patients with metastatic colorectal cancer progressing on standard therapies. Here, we describe the clinical history of a 63-year-old male patient who was treated with regorafenib in the pivotal CORRECT trial. The patient was initially diagnosed in November 2008 with nonmetastatic KRAS-mutated (exon 2, codon 12 rectal cancer. He underwent successful surgery and was treated with 5 cycles of adjuvant chemotherapy. In 2010, lung metastases (KRAS-mutated were detected and the patient received 6 cycles of FOLFIRI plus bevacizumab. By January 2011, the metastases had progressed. The patient, who was asymptomatic with an Eastern Cooperative Oncology Group performance status of 0, was enrolled onto the CORRECT trial and received best supportive care plus regorafenib (160 mg once daily for 3 weeks of a 4-week cycle over a period of 2 years, during which time the disease remained stable and the patient remained asymptomatic. Grade 1 anemia and thrombocytopenia were the only treatment-emergent adverse events reported. After receiving 26 cycles of regorafenib, a majority of the lung lesions progressed, and third-line palliative 5-fluorouracil, leucovorin, and oxaliplatin chemotherapy was administered. The patient died in May 2016.

Dependence of present and future European heat waves and cold spells on the location of atmospheric blocking

Science.gov (United States)

Brunner, L.; Schaller, N.; Sillmann, J.; Steiner, A. K.

2017-12-01

Atmospheric blocking describes stationary anti-cyclones, which weaken or reverse the climatological flow at mid-latitudes. In the northern hemisphere one of the main blocking regions is located over the North Atlantic and Northern Europe. The link between blocking and European temperature extremes, such as heat waves and cold spells, strongly depends on several aspects like season, longitudinal location of the block, and location of the extremes (particularly Northern Europe versus Southern Europe). We use a 50-member ensemble of the Canadian CanESM2 model to investigate historical (1981-2010) and future (2070-2099) blocking cases and their relationship with European temperature extremes. For the historical period the model results are also compared to those from the ERA-Interim reanalysis. Atmospheric blocking is detected on a daily basis in different 30° longitude windows between 60°W and 60°E, using a standard geopotential height-based detection index. Temperature extremes are defined by the daily Heat/Cold Wave Magnitude Index (HWMId/CWMId). The role of cold advection is found particularly important in winter conditions leading to a more than threefold increase in cold wave occurrence during blocking between 60°W and 0°. During blocking over Northern Europe (0° to 60°E) a split relationship is found with cold wave occurrence being strongly increased in Southern Europe, while it is decreased in Northern Europe. Direct, radiative effects dominate in summer, therefore blocking westward of Europe has a weaker effect, while blocking over Northern Europe leads to an increase of heat waves by at least a factor three at the location of the block and a decrease in cold wave occurrence in almost all of Europe. Comparing the historical and future period we find the link between blocking and temperature extremes in Europe to be robust, even though blocking frequency and temperatures are changing.

K-Ras and β-catenin mutations cooperate with Fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder

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Imran Ahmad

2011-07-01

The human fibroblast growth factor receptor 3 (FGFR3 gene is frequently mutated in superficial urothelial cell carcinoma (UCC. To test the functional significance of FGFR3 activating mutations as a ‘driver’ of UCC, we targeted the expression of mutated Fgfr3 to the murine urothelium using Cre-loxP recombination driven by the uroplakin II promoter. The introduction of the Fgfr3 mutations resulted in no obvious effect on tumorigenesis up to 18 months of age. Furthermore, even when the Fgfr3 mutations were introduced together with K-Ras or β-catenin (Ctnnb1 activating mutations, no urothelial dysplasia or UCC was observed. Interestingly, however, owing to a sporadic ectopic Cre recombinase expression in the skin and lung of these mice, Fgfr3 mutation caused papilloma and promoted lung tumorigenesis in cooperation with K-Ras and β-catenin activation, respectively. These results indicate that activation of FGFR3 can cooperate with other mutations to drive tumorigenesis in a context-dependent manner, and support the hypothesis that activation of FGFR3 signaling contributes to human cancer.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

Science.gov (United States)

Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

2013-01-01

Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This

Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

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Chunli Zhao

Full Text Available A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers.

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Przybojewska, B; Jagiello, A; Jalmuzna, P

2000-08-01

Bladder cancer is one of the leading causes of cancer death in most developed countries. In this work, 19 bladder cancer specimens, along with their infiltrations of the urinary bladder wall from the same patients, were examined for the presence of H-RAS, K-RAS, and N-RAS activation using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The H-RAS activation was found in 15 (about 84%) of the 19 bladder cancers studied. The same results were obtained in the infiltrating urinary bladder wall samples. N-RAS gene mutations were observed in all cases (except 1) in which H-RAS gene mutations were detected. The results suggest a strong relationship between H-RAS and N-RAS gene activation in bladder cancer. Changes in the K-RAS gene in bladder cancers seem to be a rare event; this is in agreement with findings of other authors. We found activation of the gene in one specimen of bladder cancer and its infiltration of the urinary bladder wall in the same patient.

Detection of TET2, KRAS and CBL variants by Next Generation Sequencing and analysis of their correlation with JAK2 and FLT3 in childhood AML

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Dilara Fatma Akin

2016-04-01

Conclusion: We found novel mutations for TET2, KRAS, and CBL. The detected variants in this article seem to be the first screening results of genes studied by NGS in childhood AML patients. Our results also showed some degree of association between FLT3-ITD and TET2, KRAS, CBL mutations.

Developmental Programming of Renal Function and Re-Programming Approaches.

Science.gov (United States)

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin-angiotensin-aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early

The Role of Conserved Waters in Conformational Transitions of Q61H K-ras

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Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Gorfe, Alemayehu A.

2012-01-01

To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 µs. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance (d) between the Cα atoms of G60 at switch 2 and G10 at the P-loop and the N-Cα-C-O dihedral (ξ) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/ξ plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ in effector recognition. PMID:22359497

Endogenous n-3 Polyunsaturated Fatty Acids Delay Progression of Pancreatic Ductal Adenocarcinoma in Fat-1-p48Cre/+-LSL-KrasG12D/+ Mice

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Altaf Mohammed

2012-12-01

Full Text Available Preclinical studies suggest that diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs may be beneficial for prevention of pancreatic cancer. Nutritional intervention studies are often complex, and there is no clear evidence, without potential confounding factors, on whether conversion of n-6 PUFAs to n-3 PUFAs in pancreatic tissues would provide protection. Experiments were designed using n-3 fatty acid desaturase (Fat-1 transgenic mice, which can convert n-6 PUFA to n-3 FAs endogenously, to determine the impact of n-3 PUFAs on pancreatic intraepithelial neoplasms (PanINs and their progression to pancreatic ductal adenocarcinoma (PDAC. Six-weekold female p48Cre/+-LSL-KrasG12D/+ andcompoundFat-1-p48Cre/+-LSL-KrasG12D/+ mice were fed (AIN-76A diets containing 10% safflower oil for 35 weeks. Pancreata were evaluated histopathologically for PanINs and PDAC. Results showed a dramatic reduction in incidence of PDAC (84%; P 85%; P < .05–0.01 in pancreas of compound transgenic mice than in those of p48Cre/+-LSL-KrasG12D/+ mice. Molecular analysis of the pancreas showed a significant down-regulation of proliferating cell nuclear antigen, cyclooxygenase-2, 5-lipoxygenase (5-LOX, 5-LOX-activating protein, Bcl-2, and cyclin D1 expression levels in Fat-1-p48Cre/+-LSL-KrasG12D/+ mice compared to p48Cre/+-LSL-KrasG12D/+ mice. These data highlight the promise of dietary n-3 FAs for chemoprevention of pancreatic cancer in high-risk individuals.

Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2

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Jessica Alves de Medeiros Araújo

2018-06-01

Full Text Available Reprogramming of somatic cells into induced pluripotent stem cells (iPS or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs SRY (sex determining region Y-box 2 (Sox2, Mammalian achaete-scute homolog 1 (Ascl1, or Neurogenin 2 (Neurog2 is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC into induced neurons (iNs. Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2 may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.

Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

Science.gov (United States)

Cerdá-Esteban, Nuria; Spagnoli, Francesca M

2014-01-01

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family

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Eriona Hysolli

2016-07-01

Full Text Available Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs have been shown to be highly similar to embryonic stem cells (ESCs. However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.

Fat and K-ras mutations in sporadic colorectal cancer in The Netherlands Cohort Study

NARCIS (Netherlands)

Brink, M.; Weijenberg, M.P.; Goeij, A.F.P.M. de; Schouten, L.J.; Koedijk, F.D.H.; Roemen, G.M.J.M.; Lentjes, M.H.F.M.; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den

2004-01-01

Associations between dietary intake of various fats and specific K-ras mutations in colorectal cancer (CRC) were investigated within the framework of The Netherlands Cohort Study on diet and cancer (NLCS). After 7.3 years of follow-up and with exclusion of the first 2.3 years, 448 colon and 160

A systematic evaluation of integration free reprogramming methods for deriving clinically relevant patient specific induced pluripotent stem (iPS cells.

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Pollyanna A Goh

Full Text Available A systematic evaluation of three different methods for generating induced pluripotent stem (iPS cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2% and episomal plasmid (0.10% methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells.

The wild tapered block bootstrap

DEFF Research Database (Denmark)

Hounyo, Ulrich

In this paper, a new resampling procedure, called the wild tapered block bootstrap, is introduced as a means of calculating standard errors of estimators and constructing confidence regions for parameters based on dependent heterogeneous data. The method consists in tapering each overlapping block...... of the series first, the applying the standard wild bootstrap for independent and heteroscedastic distrbuted observations to overlapping tapered blocks in an appropriate way. Its perserves the favorable bias and mean squared error properties of the tapered block bootstrap, which is the state-of-the-art block......-order asymptotic validity of the tapered block bootstrap as well as the wild tapered block bootstrap approximation to the actual distribution of the sample mean is also established when data are assumed to satisfy a near epoch dependent condition. The consistency of the bootstrap variance estimator for the sample...

Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver? A molecular analysis of 101 liver lesions from 93 patients

NARCIS (Netherlands)

Hruban, R. H.; Sturm, P. D.; Slebos, R. J.; Wilentz, R. E.; Musler, A. R.; Yeo, C. J.; Sohn, T. A.; van Velthuysen, M. L.; Offerhaus, G. J.

1997-01-01

It can be difficult to distinguish benign bile duct proliferations (BDPs) from well-differentiated metastatic peripancreatic adenocarcinomas on histological grounds alone. Most peripancreatic carcinomas harbor activating point mutations in codon 12 of the K-ras oncogene, suggesting that K-ras

An Algorithmic Information Calculus for Causal Discovery and Reprogramming Systems

KAUST Repository

Zenil, Hector

2017-09-08

We introduce a conceptual framework and an interventional calculus to steer and manipulate systems based on their intrinsic algorithmic probability using the universal principles of the theory of computability and algorithmic information. By applying sequences of controlled interventions to systems and networks, we estimate how changes in their algorithmic information content are reflected in positive/negative shifts towards and away from randomness. The strong connection between approximations to algorithmic complexity (the size of the shortest generating mechanism) and causality induces a sequence of perturbations ranking the network elements by the steering capabilities that each of them is capable of. This new dimension unmasks a separation between causal and non-causal components providing a suite of powerful parameter-free algorithms of wide applicability ranging from optimal dimension reduction, maximal randomness analysis and system control. We introduce methods for reprogramming systems that do not require the full knowledge or access to the system\\'s actual kinetic equations or any probability distributions. A causal interventional analysis of synthetic and regulatory biological networks reveals how the algorithmic reprogramming qualitatively reshapes the system\\'s dynamic landscape. For example, during cellular differentiation we find a decrease in the number of elements corresponding to a transition away from randomness and a combination of the system\\'s intrinsic properties and its intrinsic capabilities to be algorithmically reprogrammed can reconstruct an epigenetic landscape. The interventional calculus is broadly applicable to predictive causal inference of systems such as networks and of relevance to a variety of machine and causal learning techniques driving model-based approaches to better understanding and manipulate complex systems.

Cell reprogramming by 3D bioprinting of human fibroblasts in polyurethane hydrogel for fabrication of neural-like constructs.

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Ho, Lin; Hsu, Shan-Hui

2018-04-01

3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration. There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be

Role of the GRAS transcription factor ATA/RAM1 in the transcriptional reprogramming of arbuscular mycorrhiza in Petunia hybrida

OpenAIRE

Rich, Melanie K.; Courty, Pierre-Emmanuel; Roux, Christophe; Reinhardt, Didier

2017-01-01

Background Development of arbuscular mycorrhiza (AM) requires a fundamental reprogramming of root cells for symbiosis. This involves the induction of hundreds of genes in the host. A recently identified GRAS-type transcription factor in Petunia hybrida, ATA/RAM1, is required for the induction of host genes during AM, and for morphogenesis of the fungal endosymbiont. To better understand the role of RAM1 in symbiosis, we set out to identify all genes that depend on activation by RAM1 in mycorr...

K-ras mutations in gastric stump carcinomas and in carcinomas from the non-operated stomach

NARCIS (Netherlands)

van Rees, B. P.; Musler, A.; Caspers, E.; Drillenburg, P.; Craanen, M. E.; Polkowski, W.; Chibowski, D.; Offerhaus, G. J.

1999-01-01

Partial gastrectomy is a well-established pre-malignant condition. It is postulated that in the gastric stump an accelerated neoplastic process takes place, similar to that of (intestinal type) adenocarcinoma from the non-operated stomach. K-ras codon 12 mutation is one of the most frequent

Hydrologic connections and dynamics of water movement in the classical Karst (Kras) Aquifer: evidence from frequent chemical and stable isotope sampling

Science.gov (United States)

Doctor, Daniel H.

2008-01-01

A review of past research on the hydrogeology of the Classical Karst (Kras) region and new information obtained from a two- year study using environmental tracers are presented in this paper. The main problems addressed are 1) the sources of water to the Kras aquifer resurgence zone-including the famous Timavo springs-under changing flow regimes; 2) a quantification of the storage volumes of the karst massif corresponding to flow regimes defined by hydrograph recessions of the Timavo springs; and 3) changing dynamics between deep phreatic conduit flow and shallow phreatic and epiphreatic storage within the aquifer resurgence zone as determined through changes in chemical and isotopic composition at springs and wells. Particular focus was placed on addressing the long-standing question of the influence of the Soca River on the ground waters of the aquifer resurgence zone. The results indicate that the alluvial aquifer supplied by the sinking of the Soca River on the northwestern edge of the massif contributes approximately 75% of the mean annual outflow to the smaller springs of the aquifer resurgence zone, and as much as 53% to the mean annual outflow of the Timavo springs. As a whole, the Soca River is estimated to contribute 56% of the average outflow of the Kras aquifer resurgence. The proportions of Soca River water increase under drier conditions, and decrease under wetter conditions. Time series analysis of oxygen stable isotope records indicate that the transit time of Soca River water to the Timavo springs, Sardos spring, and well B-4 is on the order of 1-2 months, depending on hydrological conditions. The total baseflow storage of the Timavo springs is estimated to be 518 million m3, and represents 88.5% of the storage capacity estimated for all flow regimes of the springs. The ratio of baseflow storage volume to the average annual volume discharged at the Timavo springs is 0.54. The Reka River sinking in Slovenia supplies substantial allogenic recharge to

Circadian Reprogramming in the Liver Identifies Metabolic Pathways of Aging.

Science.gov (United States)

Sato, Shogo; Solanas, Guiomar; Peixoto, Francisca Oliveira; Bee, Leonardo; Symeonidi, Aikaterini; Schmidt, Mark S; Brenner, Charles; Masri, Selma; Benitah, Salvador Aznar; Sassone-Corsi, Paolo

2017-08-10

The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD + -related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia.

Science.gov (United States)

Hattori, Ayuna; Tsunoda, Makoto; Konuma, Takaaki; Kobayashi, Masayuki; Nagy, Tamas; Glushka, John; Tayyari, Fariba; McSkimming, Daniel; Kannan, Natarajan; Tojo, Arinobu; Edison, Arthur S; Ito, Takahiro

2017-05-25

Reprogrammed cellular metabolism is a common characteristic observed in various cancers. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Here we show that BCAT1, a cytosolic aminotransferase for branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukaemia (CML) in humans and in mouse models of CML. BCAT1 is upregulated during progression of CML and promotes BCAA production in leukaemia cells by aminating the branched-chain keto acids. Blocking BCAT1 gene expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML both in vitro and in vivo. Stable-isotope tracer experiments combined with nuclear magnetic resonance-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function through BCAA production in blast crisis CML cells. Importantly, BCAT1 expression not only is activated in human blast crisis CML and de novo acute myeloid leukaemia, but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, we identified Musashi2 (MSI2), an oncogenic RNA binding protein that is required for blast crisis CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukaemia. Taken together, this work reveals that altered BCAA metabolism activated through the MSI2-BCAT1 axis drives cancer progression in myeloid leukaemia.

Mucinous Cystic Neoplasms Lined by Abundant Mucinous Epithelium Frequently Involve KRAS Mutations and Malignant Progression.

Science.gov (United States)

Shibata, Hideki; Ohike, Nobuyuki; Norose, Tomoko; Isobe, Tomohide; Suzuki, Reika; Imai, Hideyuki; Shiokawa, Akira; Aoki, Takeshi; Murakami, Masahiko; Mizukami, Hiroki; Tanaka, Jun-Ichi; Takimoto, Masafumi

2017-12-01

Pancreatic and hepatic mucinous cyst neoplasms (MCNs) have a malignant potential, but indolent MCNs are not uncommon. The pathological and genetic characteristics of resected MCNs (n=15) categorized by the amount of mucin of the lining epithelium were investigated. MCNs were divided into two groups: (i) a rich (r)-MCN group (n=6), in which more than half of the epithelium was lined by abundant mucinous epithelium; and (ii) a poor (p)-MCN group (n=9), which consisted of the remaining cases. Three patients in the r-MCN group showed invasive carcinoma or high-grade dysplasia, whereas all patients in the p-MCN group showed low-grade dysplasia. Mutations of Kirsten rat sarcoma viral oncogene homolog (KRAS) were more frequent in the r-MCN group (83%) (p-MCN; 11%, p<0.05). Mucinous MCNs more frequently have KRAS mutations and higher risk of malignant progression. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A randomized study of KRAS-guided maintenance therapy with bevacizumab, erlotinib or metronomic capecitabine after first-line induction treatment of metastatic colorectal cancer

DEFF Research Database (Denmark)

Hagman, H; Frödin, J-E; Berglund, Å

2016-01-01

without progression were eligible for randomization to mt; KRAS wild-type (wt) patients were randomized to bev ± erlo (arms wt-BE, N = 36 versus wt-B, N = 35), KRAS mutated (mut) patients were randomized to bev or metronomic cap (arms mut-B, N = 34 versus mut-C, N = 33). Primary end point was progression...... to influence the outcome of treatment with erlotinib. Metronomic cap warrants further investigation in mt strategies, given our explorative results. CLINICALTRIALSGOV: NCT01229813....

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D-induced skin carcinogenesis in vivo.

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Pawel K Mazur

Full Text Available BACKGROUND: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D mice with ablation of Notch1 and/or Notch2. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, mice with activated Kras(G12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities. CONCLUSIONS/SIGNIFICANCE: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.

Glycometabolic reprogramming associated with the initiation of human dental pulp stem cell differentiation.

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Wang, Linyan; Cheng, Li; Wang, Huning; Pan, Hongying; Yang, Hui; Shao, Meiying; Hu, Tao

2016-03-01

Glycometabolism, particularly mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, plays a central role in cell life activities. Glycometabolism can be reprogrammed to maintain the stemness or to induce the differentiation of stem cells, thereby regulating tissue repair and regeneration. However, research on the glycometabolism of human dental pulp stem cells (hDPSCs) remains scarce. Here, we investigated the relationship between glycometabolic reprogramming and initiation of hDPSC differentiation. We found the differentiation of hDPSCs commenced on day 3 when cells were cultured in mineralized medium. When cell differentiation commenced, mitochondria became elongated with well-developed cristae, and the oxygen consumption rate of mitochondria was enhanced, manifested as an increase in basal respiration, mitochondrial ATP production, and maximal respiration. Interestingly, glycolytic enzyme activities, glycolysis capacity, and glycolysis reserve were also upregulated at this time to match the powerful bioenergetic demands. More importantly, hDPSCs derived from different donors or cultured in various oxygen environments showed similar glycometabolic changes when they began to differentiate. Thus, glycometabolic reprogramming accompanies initiation of hDPSC differentiation and could potentially play a role in the regulation of dental pulp repair. © 2015 International Federation for Cell Biology.

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

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Lüchtenborg, Margreet; Weijenberg, Matty P; Wark, Petra A; Saritas, A Merdan; Roemen, Guido MJM; Muijen, Goos NP van; Bruïne, Adriaan P de; Brandt, Piet A van den; Goeij, Anton FPM de

2005-01-01

The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

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de Bruïne Adriaan P

2005-12-01

Full Text Available Abstract Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1 and Ras (K-ras pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45 in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656 and 36% (235/656, respectively. Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656. Nine percent of all tumours (58/656 lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer

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Therkildsen, Christina; Bergmann, Troels K; Henrichsen-Schnack, Tine

2014-01-01

BACKGROUND: In metastatic colorectal cancer, mutation testing for KRAS exon 2 is widely implemented to select patients with wild-type tumors for treatment with the monocloncal anti-EGFR antibodies cetuximab and panitumumab. The added predictive value of additional biomarkers in the RAS-RAF-MAPK a......BACKGROUND: In metastatic colorectal cancer, mutation testing for KRAS exon 2 is widely implemented to select patients with wild-type tumors for treatment with the monocloncal anti-EGFR antibodies cetuximab and panitumumab. The added predictive value of additional biomarkers in the RAS...

Evaluating the potential of poly(beta-amino ester nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

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Bhise NS

2013-12-01

Full Text Available Nupura S Bhise,1,* Karl J Wahlin,2,* Donald J Zack,2–4 Jordan J Green1,21Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for Nanobiotechnology, 2Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 3Solomon H Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, and Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Institut de la Vision, Paris, France*These authors contributed equally to this workBackground: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs from human fibroblasts.Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.Results: 1-(3-aminopropyl-4-methylpiperazine end-terminated poly(1,4-butanediol diacrylate-co-4-amino-1-butanol polymer (B4S4E7 self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available

Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

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Burns, David; Blau, Helen M

2014-07-01

Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

A systemic evaluation of cardiac differentiation from mRNA reprogrammed human induced pluripotent stem cells.

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Ashish Mehta

Full Text Available Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by "foot-print free" reprogramming of somatic cells to induced pluripotent stem cells (iPSC. In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

Susceptibility of Human Oral Squamous Cell Carcinoma (OSCC H103 and H376 cell lines to Retroviral OSKM mediated reprogramming

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Nalini Devi Verusingam

2017-04-01

Full Text Available Although numbers of cancer cell lines have been shown to be successfully reprogrammed into induced pluripotent stem cells (iPSCs, reprogramming Oral Squamous Cell Carcinoma (OSCC to pluripotency in relation to its cancer cell type and the expression pattern of pluripotent genes under later passage remain unexplored. In our study, we reprogrammed and characterised H103 and H376 oral squamous carcinoma cells using retroviral OSKM mediated method. Reprogrammed cells were characterized for their embryonic stem cells (ESCs like morphology, pluripotent gene expression via quantitative real-time polymerase chain reaction (RT-qPCR, immunofluorescence staining, embryoid bodies (EB formation and directed differentiation capacity. Reprogrammed H103 (Rep-H103 exhibited similar ESCs morphologies with flatten cells and clear borders on feeder layer. Reprogrammed H376 (Rep-H376 did not show ESCs morphologies but grow with a disorganized morphology. Critical pluripotency genes Oct4, Sox2 and Nanog were expressed higher in Rep-H103 against the parental counterpart from passage 5 to passage 10. As for Rep-H376, Nanog expression against its parental counterpart showed a significant decrease at passage 5 and although increased in passage 10, the level of expression was similar to the parental cells. Rep-H103 exhibited pluripotent signals (Oct4, Sox2, Nanog and Tra-1-60 and could form EB with the presence of three germ layers markers. Rep-H103 displayed differentiation capacity into adipocytes and osteocytes. The OSCC cell line H103 which was able to be reprogrammed into an iPSC like state showed high expression of Oct4, Sox2 and Nanog at late passage and may provide a potential iPSC model to study multi-stage oncogenesis in OSCC.

Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation: indicators of tumor staging and metastasis in adenocarcinomatous sporadic colorectal cancer in Indian population.

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Rupal Sinha

Full Text Available Colorectal cancer (CRC development involves underlying modifications at genetic/epigenetic level. This study evaluated the role of Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation together/independently in sporadic CRC in Indian population and correlation with clinicopathological variables of the disease.One hundred and twenty four consecutive surgically resected tissues (62 tumor and equal number of normal adjacent controls of primary sporadic CRC were included and patient details including demographic characteristics, lifestyle/food or drinking habits, clinical and histopathological profiles were recorded. Polymerase chain reaction - Restriction fragment length polymorphism and direct sequencing for Kras gene mutation and Methylation Specific-PCR for RASSF1A, FHIT and MGMT genes was performed.Kras gene mutation at codon 12 & 13 and methylated RASSF1A, FHIT and MGMT gene was observed in 47%, 19%, 47%, 37% and 47% cases, respectively. Alcohol intake and smoking were significantly associated with presence of Kras mutation (codon 12 and MGMT methylation (p-value <0.049. Tumor stage and metastasis correlated with presence of mutant Kras codon 12 (p-values 0.018, 0.044 and methylated RASSF1A (p-values 0.034, 0.044, FHIT (p-values 0.001, 0.047 and MGMT (p-values 0.018, 0.044 genes. Combinatorial effect of gene mutation/methylation was also observed (p-value <0.025. Overall, tumor stage 3, moderately differentiated tumors, presence of lymphatic invasion and absence of metastasis was more frequently observed in tumors with mutated Kras and/or methylated RASSF1A, FHIT and MGMT genes.Synergistic interrelationship between these genes in sporadic CRC may be used as diagnostic/prognostic markers in assessing the overall pathological status of CRC.

Synergistic activity of vorinostat combined with gefitinib but not with sorafenib in mutant KRAS human non-small cell lung cancers and hepatocarcinoma

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Jeannot V

2016-11-01

Full Text Available Victor Jeannot,1,2 Benoit Busser,1–3 Laetitia Vanwonterghem,1,2 Sophie Michallet,1,2 Sana Ferroudj,1,2 Murat Cokol,4 Jean-Luc Coll,1,2 Mehmet Ozturk,1,2,5 Amandine Hurbin1,2 1INSERM U1209, Department Cancer Targets and Experimental Therapeutics, Grenoble, France; 2University Grenoble Alpes, Institute for Advanced Biosciences, Grenoble, France; 3Department of Biochemistry, Toxicology and Pharmacology, Grenoble University Hospital, Grenoble, France; 4Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; 5Faculty of Medicine, Dokuz Eyul University, Izmir Biomedicine and Genome Center, Izmir, Turkey Abstract: Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR-tyrosine kinase inhibitor (gefitinib or a multi-targeted kinase inhibitor (sorafenib in combination with a histone deacetylase inhibitor (vorinostat on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G2/M cell cycle arrest without

Antitumor activity in RAS-driven tumors by blocking AKT and MEK.

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Tolcher, Anthony W; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R; Patnaik, Amita; Baird, Richard D; Olmos, David; Garrett, Christopher R; Skolnik, Jeffrey M; Rubin, Eric H; Smith, Paul D; Huang, Pearl; Learoyd, Maria; Shannon, Keith A; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A; Yan, Li; de Bono, Johann S

2015-02-15

KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical antitumor activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). ©2014 American Association for Cancer Research.

Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

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Yinxiang Wang

2018-01-01

Full Text Available Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

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Doulatov, Sergei; Vo, Linda T.; Macari, Elizabeth R.; Wahlster, Lara; Kinney, Melissa A.; Taylor, Alison M.; Barragan, Jessica; Gupta, Manav; McGrath, Katherine; Lee, Hsiang-Ying; Humphries, Jessica M.; DeVine, Alex; Narla, Anupama; Alter, Blanche P.; Beggs, Alan H.; Agarwal, Suneet; Ebert, Benjamin L.; Gazda, Hanna T.; Lodish, Harvey F.; Sieff, Colin A.; Schlaeger, Thorsten M.; Zon, Leonard I.; Daley, George Q.

2017-01-01

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. PMID:28179501

Chemical strategies for pancreatic β cell differentiation, reprogramming, and regeneration.

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Ma, Xiaojie; Zhu, Saiyong

2017-04-01

Generation of unlimited functional pancreatic β cells is critical for the study of pancreatic biology and treatment of diabetes mellitus. Recent advances have suggested several promising directions, including directed differentiation of pancreatic β cells from pluripotent stem cells, reprogramming of pancreatic β cells from other types of somatic cells, and stimulated proliferation and enhanced functions of existing pancreatic β cells. Small molecules are useful in generating unlimited numbers of functional pancreatic cells in vitro and could be further developed as drugs to stimulate endogenous pancreatic regeneration. Here, we provide an updated summary of recent major achievements in pancreatic β cell differentiation, reprogramming, proliferation, and function. These studies will eventually lead to significant advances in the field of pancreatic biology and regeneration. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Analysis of Kras gene from induced pancreatic cancer rats administered with Momordicacharantia and Ocimumbasilicum leaf extracts

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J.B. Minari

2018-04-01

Full Text Available Objective: To analyze K-ras gene from induced pancreatic cancer rats administered with Momordicacharantia and Ocimumbasilicum leaf extracts. Methods: Twenty-five (25 adult rats weighing between 90–120 g were divided into 5 groups namely RA, RB, RC, NC and PC, each group had 5 rats. The PC which served as the control was fed with normal fish meal and water ad libitum; the NC which is the negative control received 20 mg/ml/week of Nitrosamines only while other groups received different concentrations of aqueous extract of both M. charantia and O. basilicum (200 mg, 100 mg, 50 mg and Nitrosamine. Qualitative phytochemical screening of the aqueous extract of both M. charantia and O. basilicum was carried out. The extraction of DNA was done using Jena Bioscience DNA preparation kit and the protocol was based on the spin column based genomic DNA purification from blood, animal and plant cells. Agarose gel electrophoresis was used to analyze the K-ras gene extracted from the pancreas tissues of experimental rats while hematoxylinand eosin staining was used for histological assay. Results: Phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins and glycosides in M. charantia while saponins, tannins and glycosides were discovered in O. basilicum. Significant reduction in the weight of rats treated with 200 mg of aqueous extracts of M. charantia and O. basilicum while rats that were dosed with nitrosamines only showed a slight increase in weight in the first three weeks when compared to the positive control. Histological studies revealed that there is both enlargement and reduction in the islet cell size, with one of the sections showing a normal islet cell size. While the agarose gel electrophoresis revealed that there may be possibility of prevention of damage to k-ras gene as a result of the effect of plants extract. Conclusion: This work has shown that the leaf extracts of both M. charantia and O. basilicum

Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors

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Zhao, Chen; Tao, Zui; Xue, Langyue; Zeng, Yuxiao [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China); Wang, Yi, E-mail: wangyieye@aliyun.com [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China); Xu, Haiwei, E-mail: haiweixu2001@163.com [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China); Yin, Zheng Qin, E-mail: qinzyin@aliyun.com [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China)

2017-03-01

In lower-order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Müller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Müller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors. - Highlights: • Lin28b reprograms Müller glia to retinal progenitors. • Let-7 micrRNAs are suppressed by Lin28b. • Transplantation of reprogrammed Müller glia restores retinal function.

Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors

International Nuclear Information System (INIS)

Zhao, Chen; Tao, Zui; Xue, Langyue; Zeng, Yuxiao; Wang, Yi; Xu, Haiwei; Yin, Zheng Qin

2017-01-01

In lower-order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Müller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Müller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors. - Highlights: • Lin28b reprograms Müller glia to retinal progenitors. • Let-7 micrRNAs are suppressed by Lin28b. • Transplantation of reprogrammed Müller glia restores retinal function.

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline.

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Calvopina, Joseph Hargan; Cook, Helene; Vincent, John J; Nee, Kevin; Clark, Amander T

2015-07-01

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis

Czech Academy of Sciences Publication Activity Database

Tomasetti, M.; Amati, M.; Santarelli, L.; Neužil, Jiří