Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis.

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Storm, P; Aits, S; Puthia, M K; Urbano, A; Northen, T; Powers, S; Bowen, B; Chao, Y; Reindl, W; Lee, D Y; Sullivan, N L; Zhang, J; Trulsson, M; Yang, H; Watson, J D; Svanborg, C

2011-12-01

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here, we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small-hairpin RNA (shRNA) inhibition, proteomic and metabolomic technology, we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, HAMLET sensitivity was modified by the glycolytic state of tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen, hexokinase 1 (HK1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 and hypoxia-inducible factor 1α modified HAMLET sensitivity. HK1 was shown to bind HAMLET in a protein array containing ∼8000 targets, and HK activity decreased within 15 min of HAMLET treatment, before morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 min. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene addiction or the Warburg effect.

Conserved features of cancer cells define their sensitivity of HAMLET-induced death; c-Myc and glycolysis

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Storm, Petter; Puthia, Manoj Kumar; Aits, Sonja; Urbano, Alexander; Northen, Trent; Powers, Scott; Bowen, Ben; Chao, Yinxia; Reindl, Wolfgang; Lee, Do Yup; Sullivan, Nancy Liu; Zhang, Jianping; Trulsson, Maria; Yang, Henry; Watson, James; Svanborg, Catharina

2014-01-01

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small hairpin RNA inhibition, proteomic and metabolomic technology we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted the sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, the HAMLET sensitivity was modified by the glycolytic state of the tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen Hexokinase 1, PFKFB1 and HIF1α modified HAMLET sensitivity. Hexokinase 1 was shown to bind HAMLET in a protein array containing approximately 8000 targets and Hexokinase activity decreased within 15 minutes of HAMLET treatment, prior to morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. The glycolytic machinery was modified and glycolysis was shifted towards the pentose phosphate pathway. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 minutes. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene-addiction or the Warburg effect. PMID:21643007

Targeting c-Myc: JQ1 as a promising option for c-Myc-amplified esophageal squamous cell carcinoma.

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Wang, Jingyuan; Liu, Zhentao; Wang, Ziqi; Wang, Shubin; Chen, Zuhua; Li, Zhongwu; Zhang, Mengqi; Zou, Jianling; Dong, Bin; Gao, Jing; Shen, Lin

2018-04-10

c-Myc amplification-induced cell cycle dysregulation is a common cause for esophageal squamous cell carcinoma (ESCC), but no approved targeted drug is available so far. The bromodomain inhibitor JQ1, which targets c-Myc, exerts anti-tumor activity in multiple cancers. However, the role of JQ1 in ESCC remains unknown. In this study, we reported that JQ1 had potent anti-proliferative effects on ESCC cells in both time- and dose-dependent manners by inducing cell cycle arrest at G1 phase, cell apoptosis, and the mesenchymal-epithelial transition. Follow-up studies revealed that both c-Myc/cyclin/Rb and PI3K/AKT signaling pathways were inactivated by JQ1, as indicated by the downregulation of c-Myc, cyclin A/E, and phosphorylated Rb, AKT and S6. Tumor suppression induced by JQ1 in c-Myc amplified or highly expressed xenografts was higher than that in xenografts with low expression, suggesting its potential role in prediction. In conclusion, targeting c-Myc by JQ1 could cause significant tumor suppression in ESCC both in vitro and in vivo. Also, c-Myc amplification or high expression might serve as a potential biomarker and provide a promising therapeutic option for ESCC. Copyright © 2018 Elsevier B.V. All rights reserved.

c-MYC expression sensitizes medulloblastoma cells to radio- and chemotherapy and has no impact on response in medulloblastoma patients

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Stearns Duncan

2011-02-01

Full Text Available Abstract Background To study whether and how c-MYC expression determines response to radio- and chemotherapy in childhood medulloblastoma (MB. Methods We used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio- and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium inner salt (MTS assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio- and chemotherapy as examined by neuroradiological imaging. Results In DAOY - and to a lesser extent in UW228 - cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation- and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio- (14 responders (CR, PR vs. 5 non-responders (SD, PD or chemotherapy (23 CR/PR vs. 20 SD/PD was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively. Conclusions c-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment.

EFFECT OF STENT ABSORBED c-myc ANTISENSE OLIGODEOXYNUCLEOTIDE ON SMOOTH MUSCLE CELLS APOPTOSIS IN RABBIT CAROTID ARTERY

Institute of Scientific and Technical Information of China (English)

张新霞; 崔长琮; 李江; 崔翰斌; 徐仓宝; 朱参战

2002-01-01

Objective To investigate the effect of gelatin coated Platinium-Iridium stent absorbed c-myc antisense oligodeoxynucleotide (ASODN) on smooth muscle cells apoptosis in a normal rabbit carotid arteries. Methods Gelatin coated Platinium-Iridium stents were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomly divided into control group and treated group receiving c-myc ASODN (n=16, respectively). On 7, 14, 30 and 90 days following the stenting procedure ,morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical method. Apoptotic smooth muscle cells was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Results At 7 and 14 days after stenting,there were no detectable apoptotic cells in both groups. The apoptotic cells occurred in the neointima 30 and 90 days after stenting, and the number of apoptotic cells at 30 days were less [4.50±1.29 vs 25.75±1.89 (number/0.1mm2)] than that at 90 days [13.50±1.91 vs 41.50±6.46 (number/0.1mm2)]. Meanwhile c-myc ASODN induced more apoptotic cells than the control group(P<0.0001). c-myc protein expression was weak positive or negative in treated group and positive in control group.Conclusion c-myc ASODN can induce smooth muscle cells apoptosis after stenting in normal rabbit carotid arteries,and it can be used to prevent in-stent restenosis.

Driver or passenger effects of augmented c-Myc and Cdc20 in gliomagenesis.

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Ji, Ping; Zhou, Xinhui; Liu, Qun; Fuller, Gregory N; Phillips, Lynette M; Zhang, Wei

2016-04-26

Cdc20 and c-Myc are commonly overexpressed in a broad spectrum of cancers, including glioblastoma (GBM). Despite this clear association, whether c-Myc and Cdc20 overexpression is a driver or passenger event in gliomagenesis remains unclear. Both c-Myc and Cdc20 induced the proliferation of primary glial progenitor cells. c-Myc also promoted the formation of soft agar anchorage-independent colonies. In the RCAS/Ntv-a glia-specific transgenic mouse model, c-Myc increased the GBM incidence from 19.1% to 47.4% by 12 weeks of age when combined with kRas and Akt3 in Ntv-a INK4a-ARF (also known as CDKN2A)-null mice. In contrast, Cdc20 decreased the GBM incidence from 19.1% to 9.1%. Moreover, cell differentiation was modulated by c-Myc in kRas/Akt3-induced GBM on the basis of Nestin/GFAP expression (glial progenitor cell differentiation), while Cdc20 had no effect on primary glial progenitor cell differentiation. We used glial progenitor cells from Ntv-a newborn mice to evaluate the role of c-Myc and Cdc20 in the proliferation and transformation of GBM in vitro and in vivo. We further determined whether c-Myc and Cdc20 have a driver or passenger role in GBM development using kRas/Akt3 signals in a RCAS/Ntv-a mouse model. These results suggest that the driver or passenger of oncogene signaling is dependent on cellular status. c-Myc is a driver when combined with kRas/Akt3 oncogenic signals in gliomagenesis, whereas Cdc20 overexpression is a passenger. Inhibition of cell differentiation of c-Myc may be a target for anti-glioma therapy.

The effect of Glut1 and c-myc on prognosis in esophageal squamous cell carcinoma of Kazakh and Han patients.

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Zhou, Ya-Xing; Zhou, Ke-Ming; Liu, Qian; Wang, Hui; Wang, Wen; Shi, Yi; Ma, Yu-Qing

2018-04-09

Glucose transporter type 1 (Glut1) plays a crucial role in cancer-specific metabolism. We explored the expression of Glut1 and c-myc, the relationship between them and the effect of Glut1, c-myc on prognosis in esophageal squamous cell carcinoma. Immunohistochemistry was used to examine the expression of Glut1 and c-myc. χ 2 test analyzes the relationship between c-myc, Glut1 and pathological parameters. Spearman correlation analyzes the relationship between c-myc and Glut1. Survival analysis was used to investigate the effect of Glut1 and c-myc on prognosis. Glut1 positivity was associated with tumor size (p C-myc positivity was associated with tumor location (p = 0.015), depth of invasion (p = 0.022) and lymph node metastasis (p = 0.035). There was a positive correlation between c-myc and Glut1 (r = 0.321). Patients with Glut1 c-myc co-expression had poorer prognosis. Inhibiting Glut1 c-myc co-expression may improve the prognosis of esophageal squamous cell carcinoma.

c-Myc Represses Transcription of Epstein-Barr Virus Latent Membrane Protein 1 Early after Primary B Cell Infection.

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Price, Alexander M; Messinger, Joshua E; Luftig, Micah A

2018-01-15

Recent evidence has shown that the Epstein-Barr virus (EBV) oncogene LMP1 is not expressed at high levels early after EBV infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased 50-fold between 7 days postinfection and the LCL state. Metabolic labeling of nascent transcribed mRNA indicated that this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to that in infected B cells early after infection. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B cell infection directly repress LMP1 transcription. IMPORTANCE EBV is a highly successful pathogen that latently infects more than 90% of adults worldwide and is also causally associated with a number of B cell malignancies. During the latent life cycle, EBV expresses a set of viral oncoproteins and noncoding RNAs with the potential to promote cancer. Critical among these is the viral latent membrane protein LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here we show that transcription of the LMP1 gene can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV oncogenes will allow us

The Max b-HLH-LZ can transduce into cells and inhibit c-Myc transcriptional activities.

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Martin Montagne

Full Text Available The inhibition of the functions of c-Myc (endogenous and oncogenic was recently shown to provide a spectacular therapeutic index in cancer mouse models, with complete tumor regression and minimal side-effects in normal tissues. This was achieved by the systemic and conditional expression of omomyc, the cDNA of a designed mutant of the b-HLH-LZ of c-Myc named Omomyc. The overall mode of action of Omomyc consists in the sequestration of Max and the concomitant competition of the Omomyc/Max complex with the endogenous c-Myc/Max heterodimer. This leads to the inhibition of the transactivation of Myc target genes involved in proliferation and metabolism. While this body of work has provided extraordinary insights to guide the future development of new cancer therapies that target c-Myc, Omomyc itself is not a therapeutic agent. In this context, we sought to exploit the use of a b-HLH-LZ to inhibit c-Myc in a cancer cell line in a more direct fashion. We demonstrate that the b-HLH-LZ domain of Max (Max* behaves as a bona fide protein transduction domain (PTD that can efficiently transduce across cellular membrane via through endocytosis and translocate to the nucleus. In addition, we show that the treatment of HeLa cells with Max* leads to a reduction of metabolism and proliferation rate. Accordingly, we observe a decrease of the population of HeLa cells in S phase, an accumulation in G1/G0 and the induction of apoptosis. In agreement with these phenotypic changes, we show by q-RT-PCR that the treatment of HeLa cells with Max* leads to the activation of the transcription c-Myc repressed genes as well as the repression of the expression of c-Myc activated genes. In addition to the novel discovery that the Max b-HLH-LZ is a PTD, our findings open up new avenues and strategies for the direct inhibition of c-Myc with b-HLH-LZ analogs.

c-Myc is essential to prevent endothelial pro-inflammatory senescent phenotype.

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Victoria Florea

Full Text Available The proto-oncogene c-Myc is vital for vascular development and promotes tumor angiogenesis, but the mechanisms by which it controls blood vessel growth remain unclear. In the present work we investigated the effects of c-Myc knockdown in endothelial cell functions essential for angiogenesis to define its role in the vasculature. We provide the first evidence that reduction in c-Myc expression in endothelial cells leads to a pro-inflammatory senescent phenotype, features typically observed during vascular aging and pathologies associated with endothelial dysfunction. c-Myc knockdown in human umbilical vein endothelial cells using lentivirus expressing specific anti-c-Myc shRNA reduced proliferation and tube formation. These functional defects were associated with morphological changes, increase in senescence-associated-β-galactosidase activity, upregulation of cell cycle inhibitors and accumulation of c-Myc-deficient cells in G1-phase, indicating that c-Myc knockdown in endothelial cells induces senescence. Gene expression analysis of c-Myc-deficient endothelial cells showed that senescent phenotype was accompanied by significant upregulation of growth factors, adhesion molecules, extracellular-matrix components and remodeling proteins, and a cluster of pro-inflammatory mediators, which include Angptl4, Cxcl12, Mdk, Tgfb2 and Tnfsf15. At the peak of expression of these cytokines, transcription factors known to be involved in growth control (E2f1, Id1 and Myb were downregulated, while those involved in inflammatory responses (RelB, Stat1, Stat2 and Stat4 were upregulated. Our results demonstrate a novel role for c-Myc in the prevention of vascular pro-inflammatory phenotype, supporting an important physiological function as a central regulator of inflammation and endothelial dysfunction.

Multiple fractions of gamma rays do not induce overexpression of c-myc or c-Ki-ras oncogenes in human cervical carcinoma cells

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Osmak, M.; Soric, J.; Matulic, M.

1993-01-01

Multiple fractions of gamma rays (0.5 Gy daily, 30 fractions) had previously been found to change the sensitivity of human cervical carcinoma HeLa cells to anticancer drugs. Preirradiated cells became resistant to cisplatin, methotrexate and vincristine but retained the same sensitivity to gamma rays and ultraviolet light. Some mechanisms involved in the resistance of preirradiated cells to cisplatin and vincristine were determined, i.e. the increased levels of metallothioneins and increased expression of plasma membrane P glycoprotein. As recent reports indicated that the resistance to cisplatin and ionizing radiation may involve the expression of oncogenes, the problem was studied whether multiple fractions of gamma rays can change the expression of c-myc and c-Ki-ras oncogenes in HeLa cells and whether there is a correlation between the expression of these oncogenes and the sensitivity of preirradiated cells to cisplatin and gamma rays. The expression of c-myc and c-Ki-ras oncogenes was examined using the DNA dot blot, the RNA dot blot and Northern blot analysis. The results show that preirradiation induced neither amplification nor elevated expression of c-myc and c-Ki-ras oncogenes. Furthermore, there is no correlation between the expression of c-myc and c-Ki-ras oncogenes and the acquired resistance to cisplatin. (author) 3 figs., 32 refs

Gamabufotalin triggers c-Myc degradation via induction of WWP2 in multiple myeloma cells.

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Yu, Zhenlong; Li, Tao; Wang, Chao; Deng, Sa; Zhang, Baojing; Huo, Xiaokui; Zhang, Bo; Wang, Xiaobo; Zhong, Yuping; Ma, Xiaochi

2016-03-29

Deciding appropriate therapy for multiple myeloma (MM) is challenging because of the occurrence of multiple chromosomal changes and the fatal nature of the disease. In the current study, gamabufotalin (GBT) was isolated from toad venom, and its tumor-specific cytotoxicity was investigated in human MM cells. We found GBT inhibited cell growth and induced apoptosis with the IC50 values <50 nM. Mechanistic studies using functional approaches identified GBT as an inhibitor of c-Myc. Further analysis showed that GBT especially evoked the ubiquitination and degradation of c-Myc protein, thereby globally repressing the expression of c-Myc target genes. GBT treatment inhibited ERK and AKT signals, while stimulating the activation of JNK cascade. An E3 ubiquitin-protein ligase, WWP2, was upregulated following JNK activation and played an important role in c-Myc ubiquitination and degradation through direct protein-protein interaction. The antitumor effect of GBT was validated in a xenograft mouse model and the suppression of MM-induced osteolysis was verified in a SCID-hu model in vivo. Taken together, our study identified the potential of GBT as a promising therapeutic agent in the treatment of MM.

Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter.

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Liu, Te; Cheng, Weiwei; Liu, Tianjin; Guo, Lihe; Huang, Qin; Jiang, Lizhen; Du, Xiling; Xu, Fuhui; Liu, Zhixue; Lai, Dongmei

2010-02-01

Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

Pre-clinical analysis of changes in intra-cellular biochemistry of glioblastoma multiforme (GBM) cells due to c-Myc silencing.

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Rajagopalan, Vishal; Vaidyanathan, Muthukumar; Janardhanam, Vanisree Arambakkam; Bradner, James E

2014-10-01

Glioblastoma Multiforme (GBM) is an aggressive form of brain Tumor that has few cures. In this study, we analyze the anti-proliferative effects of a new molecule JQ1 against GBMs induced in Wistar Rats. JQ1 is essentially a Myc inhibitor. c-Myc is also known for altering the biochemistry of a tumor cell. Therefore, the study is intended to analyze certain other oncogenes associated with c-Myc and also the change in cellular biochemistry upon c-Myc inhibition. The quantitative analysis of gene expression gave a co-expressive pattern for all the three genes involved namely; c-Myc, Bcl-2, and Akt. The cellular biochemistry analysis by transmission electron microscopy revealed high glycogen and lipid aggregation in Myc inhibited cells and excessive autophagy. The study demonstrates the role of c-Myc as a central metabolic regulator and Bcl-2 and Akt assisting in extending c-Myc half-life as well as in regulation of autophagy, so as to regulate cell survival on the whole. The study also demonstrates that transient treatment by JQ1 leads to aggressive development of tumor and therefore, accelerating death, emphasizing the importance of dosage fixation, and duration for clinical use in future.

1,25 dihydroxyvitamin D3 (1,25) regulation of c-myc mRNA in HL-60 leukemia cells

International Nuclear Information System (INIS)

Simpson, R.U.; Bresnick, E.H.; Begley, D.A.

1986-01-01

Recently, 1,25 was shown to induce differentiation and decrease c-myc levels in HL-60 cells. The authors have confirmed these observations by RNA dot blot analysis. Cells treated with 50 nM 1,25 for 4, 24 and 48 hr showed c-myc mRNA levels of 26, 17 and 15% of control respectively. β-Actin mRNA levels were not altered. To ascertain whether 1, 25 regulated c-myc transcriptionally, an HL-60 nuclear RNA runoff assay was developed. Assay of total nuclei transcriptional activity revealed that 50% of RNA elongation was α-amanitin (0.8 μg/ml) sensitive and was linear with nuclei concentration (0.1-1 x 10 7 nuclei). 1,25 (50 nM) treated (45-96 hr) cells had decreased (approx.40%) total transcription rate relative to control. Decreased total RNA synthesis occurred concomitant with NBT reducing activity. 32 P-RNA runoff transcripts from HL-60 nuclei were hybridized to excess (5 μg DNA was excess) Pst I linearized c-myc and β-actin clones (in pBR322) immobilized on nitrocellulose filter. 32 P-RNA input from 2 x 10 6 to 2 x 10 7 cpm yielded linear hybridization signal. Analysis of blot dot intensity revealed no difference in transcription of c-myc in nuclei from 1,25 dosed or control cells. (myc/actin ratios: 1,25 (50 nM, 72 hr) =1.1 +/- 0.3 and control (72 hr) = 1.0, N=3 or 2 or 3 dots ea). These preliminary data suggest 1,25 does not affect c-myc transcription in HL-60 nuclei and may regulate c-myc mRNA post-transcriptionally

Melatonin disturbs SUMOylation mediated crosstalk between c-Myc and Nestin via MT1 activation and promotes the sensitivity of Paclitaxel in brain cancer stem cells.

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Lee, Hyemin; Lee, Hyo-Jung; Jung, Ji Hoon; Shin, Eun Ah; Kim, Sung-Hoon

2018-04-14

Here the underlying antitumor mechanism of melatonin and its potency as a sensitizer of Paclitaxel was investigated in X02 cancer stem cells. Melatonin suppressed sphere formation and induced G2/M arrest in X02 cells expressing Nestin, CD133, CXCR4 and SOX-2 as biomarkers of stemness. Furthermore, melatonin reduced the expression of CDK2, CDK4, cyclin D1, cyclin E, and c-Myc and upregulated cyclin B1 in X02 cells. Notably, genes of c-Myc related mRNAs were differentially expressed in melatonin treated X02 cells by microarray analysis. Consistently, melatonin reduced the expression of c-Myc at mRNA and protein levels, which was blocked by MG132. Of note, overexpression of c-Myc increased the expression of Nestin, while overexpression of Nestin enhanced c-Myc through crosstalk despite different locations, nucleus and cytoplasm. Interestingly, melatonin attenuated small ubiquitin-related modifier-1 (SUMO-1) more than SUMO-2 or SUMO-3 and disturbed nuclear translocation of Nestin for direct binding to c-Myc by SUMOylation of SUMO-1 protein by immunofluorescence and immunoprecipitation. Also, melatonin reduced trimethylated histone H3K4me3 and H3K36me3 more than dimethylation in X02 cells by Western blotting and Chromatin immunoprecipitation assay. Notably, melatonin upregulated MT1, not MT2, in X02 cells and melatonin receptor inhibitor Luzindole blocked the ability of melatonin to decrease the expression of Nestin, p-c-Myc(S62) and c-Myc. Furthermore, melatonin promoted cytotoxicity, sub G1 accumulation and apoptotic body formation by Paclitaxcel in X02 cells. Taken together, these findings suggest that melatonin inhibits stemness via suppression of c-Myc, Nestin, and histone methylation via MT1 activation and promotes anticancer effect of Paclitaxcel in brain cancer stem cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Suppression of c-Myc induces apoptosis via an AMPK/mTOR-dependent pathway by 4-O-methyl-ascochlorin in leukemia cells.

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Shin, Jae-Moon; Jeong, Yun-Jeong; Cho, Hyun-Ji; Magae, Junji; Bae, Young-Seuk; Chang, Young-Chae

2016-05-01

4-O-Methyl-ascochlorin (MAC) is a methylated derivative of the prenyl-phenol antibiotic ascochlorin, which was isolated from an incomplete fungus, Ascochyta viciae. Although the effects of MAC on apoptosis have been reported, the underlying mechanisms remain unknown. Here, we show that MAC promoted apoptotic cell death and downregulated c-Myc expression in K562 human leukemia cells. The effect of MAC on apoptosis was similar to that of 10058-F4 (a c-Myc inhibitor) or c-Myc siRNA, suggesting that the downregulation of c-Myc expression plays a role in the apoptotic effect of MAC. Further investigation showed that MAC downregulated c-Myc by inhibiting protein synthesis. MAC promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its target proteins, including p70S6 K and 4E-BP-1. Treatment of cells with AICAR (an AMPK activator), rapamycin (an mTOR inhibitor), or mTOR siRNA downregulated c-Myc expression and induced apoptosis to a similar extent to that of MAC. These results suggest that the effect of MAC on apoptosis induction in human leukemia cells is mediated by the suppression of c-Myc protein synthesis via an AMPK/mTOR-dependent mechanism.

The c-Myc target glycoprotein1balpha links cytokinesis failure to oncogenic signal transduction pathways in cultured human cells.

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Qian Wu

2010-05-01

Full Text Available An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbalpha, a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbalpha in a p53-deficient background. GpIbalpha was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbalpha.

Increased radiation-induced transformation in C3H/10T1/2 cells after transfer of an exogenous c-myc gene

International Nuclear Information System (INIS)

Sorrentino, V.; Drozdoff, V.; Zeitz, L.; Fleissner, E.

1987-01-01

C3H/10T 1/2 cells were infected with a retroviral vector expressing a mouse c-myc oncogene and a drug-selection marker. The resulting cells, morphologically indistinguishable from C3H/10T l/1, displayed a greatly enhanced sensitivity to neoplastic transformation by ionizing radiation or by a chemical carcinogen. Constitutive expression of myc therefore appears to synergize with an initial carcinogenic event, providing a function analogous to a subsequent event that apparently is required for the neoplastic transformation of these cells. This cell system should prove useful in exploring early stages in radiation-induced transformation

Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

International Nuclear Information System (INIS)

Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

1987-01-01

After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43 0 C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37 0 C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 μg per 10 9 cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

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Pan, Christopher C.; Bloodworth, Jeffrey C. [Division of Pharmacology, Columbus, OH 43210 (United States); Mythreye, Karthikeyan [Duke University, Department of Medicine, Durham, NC 27708 (United States); Lee, Nam Y., E-mail: lee.5064@osu.edu [Division of Pharmacology, Columbus, OH 43210 (United States); Davis Heart and Lung Research Institute, Columbus, OH 43210 (United States)

2012-08-03

Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.

A proteomic study of cMyc improvement of CHO culture

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Dunn Michael J

2010-03-01

Full Text Available Abstract Background The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype. Results Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS. Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin, protein biosysnthesis (eIF6 and energy metabolism (ATP synthetase, and a decreased regulation of all proteins, indentified, involved in matrix and cell to cell adhesion. Conclusion These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

Nm23-M2/NDP kinase B induces endogenous c-myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death

International Nuclear Information System (INIS)

Arnaud-Dabernat, Sandrine; Masse, Karine; Smani, Moneim; Peuchant, Evelyne; Landry, Marc; Bourbon, Pierre-Marie; Le Floch, Renaud; Daniel, Jean-Yves; Larou, Monique

2004-01-01

The nm23 gene family encodes nucleoside diphosphate kinases (NDPKs) which supply the cell with (d)NTPs. The human NDPKB, also known as the PuF protein, binds the c-myc promoter and transactivates the c-myc protooncogene. We have now studied the effects of mouse NDPKA and NDPKB overexpression on endogenous c-myc transactivation in the mouse BAF3 and the rat PC12 cell lines. c-myc transcripts were found to be up-regulated by NDPKB only in the BAF3 line. This suggests that c-myc transcriptional control via NDPKB depends on the presence of cell-specific co-factors. Unexpectedly, NDPKB also induced NDPKA expression. This new effect was found in both cell lines, suggesting that NDPKB-dependent nm23-M1 gene transactivation requires cis and/or trans elements different from those involved in c-myc transactivation. Moreover, the BAF3 cell proliferation capacities were found to be independent of NDPKA or B cell contents. Interestingly, cell death induced by c-myc overexpression or H 2 O 2 exposure was decreased in nm23-transfected compared to control BAF3 cells. These data collectively suggest that NDPKs might improve cell survival by a mechanism coupling DNA repair and transcriptional regulation of genes involved in DNA damage response

SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

International Nuclear Information System (INIS)

Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

2012-01-01

Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc

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Lu Liu

2016-01-01

Full Text Available Introduction. Xeroderma pigmentosum group C (XPC, essential component of multisubunit stem cell coactivator complex (SCC, functions as the critical factor modulating pluripotency and genome integrity through interaction with Oct-4/Sox2. However, its specific role in regulating pluripotency and multilineage differentiation of human dental pulp cells (DPCs remains unknown. Methods. To elucidate the functional role XPC played in pluripotency and multilineage differentiation of DPCs, expressions of XPC in DPCs with long-term culture were examined by real-time PCR and western blot. DPCs were transfected with lentiviral-mediated human XPC gene; then transfection rate was investigated by real-time PCR and western blot. Cell cycle, apoptosis, proliferation, senescence, multilineage differentiation, and expression of Oct-4/Sox2/c-Myc in transfected DPCs were examined. Results. XPC, Oct-4, Sox2, and c-Myc were downregulated at P7 compared with P3 in DPCs with long-term culture. XPC genes were upregulated in DPCs at P2 after transfection and maintained high expression level at P3 and P7. Cell proliferation, PI value, and telomerase activity were enhanced, whereas apoptosis was suppressed in transfected DPCs. Oct-4/Sox2/c-Myc were significantly upregulated, and multilineage differentiation in DPCs with XPC overexpression was enhanced after transfection. Conclusions. XPC plays an essential role in the modulation of pluripotency and multilineage differentiation of DPCs through regulation of Oct-4/Sox2/c-Myc.

XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc.

Science.gov (United States)

Liu, Lu; Peng, Zhengjun; Xu, Zhezhen; Wei, Xi

2016-01-01

Introduction. Xeroderma pigmentosum group C (XPC), essential component of multisubunit stem cell coactivator complex (SCC), functions as the critical factor modulating pluripotency and genome integrity through interaction with Oct-4/Sox2. However, its specific role in regulating pluripotency and multilineage differentiation of human dental pulp cells (DPCs) remains unknown. Methods. To elucidate the functional role XPC played in pluripotency and multilineage differentiation of DPCs, expressions of XPC in DPCs with long-term culture were examined by real-time PCR and western blot. DPCs were transfected with lentiviral-mediated human XPC gene; then transfection rate was investigated by real-time PCR and western blot. Cell cycle, apoptosis, proliferation, senescence, multilineage differentiation, and expression of Oct-4/Sox2/c-Myc in transfected DPCs were examined. Results. XPC, Oct-4, Sox2, and c-Myc were downregulated at P7 compared with P3 in DPCs with long-term culture. XPC genes were upregulated in DPCs at P2 after transfection and maintained high expression level at P3 and P7. Cell proliferation, PI value, and telomerase activity were enhanced, whereas apoptosis was suppressed in transfected DPCs. Oct-4/Sox2/c-Myc were significantly upregulated, and multilineage differentiation in DPCs with XPC overexpression was enhanced after transfection. Conclusions. XPC plays an essential role in the modulation of pluripotency and multilineage differentiation of DPCs through regulation of Oct-4/Sox2/c-Myc.

Insertion of the LINE-1 element in the C-MYC gene and immunoreactivity of C-MYC, p53, p21 and p27 proteins in different morphological patterns of the canine TVT

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C.R.O. Lima

2016-06-01

Full Text Available ABSTRACT The canine transmissible venereal tumor (TVT affects the external genitalia of dogs by the natural transplant of viable tumor cells. Thus, this research aimed to diagnose and characterize TVT morphological patterns, identify the insertion of the LINE-1 element in C-MYC gene, by means of the polymerase chain reaction (PCR, and evaluate the immunohistochemical expression of C-MYC, p53, p21 and p27 proteins. The relationship between C-MYC and p53 proteins and their interference on the expression of p21 and p27 were also studied. For that, 20 samples of naturally occurring TVT were used, subjected to cytopathological, histopathological and immunohistochemical analysis, and to molecular diagnosis of neoplasia. The increased tissue expression and the correlation among C-MYC, p53, p21 and p27 proteins indicate reduction and/or loss of their functionality in the TVT microenvironment, with consequent apoptotic suppression, maintenance of cell growth and progression of neoplasia.

Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer

International Nuclear Information System (INIS)

Zhi, Xiuyi; Giroux-Leprieur, Etienne; Wislez, Marie; Hu, Mu; Zhang, Yi; Shi, Huaiyin; Du, Kaiqi; Wang, Lei

2015-01-01

Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studies indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis. - Highlights: • hPAF1C expression is a prognostic biomarker for early stage non-small cell lung cancer. • The expression of hPAF1C was positively correlated with c-MYC in tumor samples of patients and in several NSCLC cell lines. • hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription.

Glutathione Depletion Induced by c-Myc Downregulation Triggers Apoptosis on Treatment with Alkylating Agents1

Science.gov (United States)

Biroccio, Annamaria; Benassi, Barbara; Fiorentino, Francesco; Zupi, Gabriella

2004-01-01

Abstract Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by l-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis. PMID:15153331

c-Myc Represses Tumor-Suppressive microRNAs, let-7a, miR-16 and miR-29b, and Induces Cyclin D2-Mediated Cell Proliferation in Ewing's Sarcoma Cell Line.

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Masanori Kawano

Full Text Available Myc oncogenic transcription factor is known to inhibit tumor suppressive microRNAs (miRNAs, resulting in greater expression of their target protein related to cell cycle, invasion or anti-apoptotic factors in human cancer cells. To explore possible oncogenic factors in Ewing's sarcoma (ES, we conducted microarray-based approach to profile the changes in the expression of miRNAs and its downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs. Three miRNAs, let-7a, miR-16 and miR-29b were significantly down-regulated, whereas c-Myc and cyclin D2 (CCND2 were significantly up-regulated in all tested ES cells compared with hMSCs. To verify that let-7a, miR-16 and miR-29b were the targets of c-Myc in ES cell lines, we transfected siRNA against c-Myc and confirmed the coordinate up-regulation of let-7a, miR-16 and miR-29b through the repression of c-Myc. The ES cells transfected with c-Myc-siRNA and let-7a, miR-16 and miR-29b exhibited the inhibition of the cell cycle progression. The increased expression of let-7a, miR-16 and miR-29b resulted in the reduction of CCND2 protein expression. We also demonstrated that c-Myc-siRNA treatment of ES cells was associated with the decreased expression of CCND2 as a down-stream of three miRNAs. Furthermore, the introduction of let-7a, miR-16 and miR-29b in ES cells could inhibit the c-Myc-mediated up-regulation of CCND2 resulted in the prevention of cell cycle progression. In addition, the transfection of let-7a, miR-16 and miR-29b in ES cells suppressed tumor growth ex vivo treatment. These findings suggests that the up-regulation of c-Myc inhibited the expression of let-7a, miR-16 and miR-29b subsequently induced CCND2 expression in ES cells. The present study might identify a novel oncogenic axis that c-Myc regulates the expression of CCND2 via let-7a, miR-16 and miR-29b, leading to the development new therapeutic targets for ES.

Effects of c-myc oncogene modulation on differentiation of human small cell lung carcinoma cell lines

NARCIS (Netherlands)

Van Waardenburg, RCAM; Meijer, C; Pinto-Sietsma, SJ; De Vries, EGE; Timens, W; Mulder, NM

1998-01-01

Amplification and over-expression of oncogenes of the myc family are related to the prognosis of certain solid tumors such as small cell lung cancer (SCLC). For SCLC, c-myc is the oncogene most consistently found to correlate with the end stage behaviour of the tumour, in particular with survival

Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

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Pavan Kumar, Y; Saha, Puja; Saha, Dhurjhoti; Bessi, Irene; Schwalbe, Harald; Chowdhury, Shantanu; Dash, Jyotirmayee

2016-03-02

The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immortalization of human neural stem cells with the c-myc mutant T58A.

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Lidia De Filippis

Full Text Available Human neural stem cells (hNSC represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC. T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%. Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in

Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation

International Nuclear Information System (INIS)

Yoshida, Go J.; Saya, Hideyuki

2014-01-01

Highlights: •CD44 variant8–10 and c-Myc are inversely expressed in gastric cancer cells. •Redox-stress enhances c-Myc expression via canonical Wnt signal. •CD44v, but not CD44 standard, suppresses redox stress-induced Wnt activation. •CD44v expression promotes both transcription and proteasome degradation of c-Myc. •Inversed expression pattern between CD44v and c-Myc is often recognized in vivo. -- Abstract: Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 high / Fbw7 high / c-Myc low and proliferative cancer stem-like cells with CD44v8-10 high / Fbw7 low / c-Myc high

Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation

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Yoshida, Go J., E-mail: medical21go@yahoo.co.jp; Saya, Hideyuki

2014-01-10

Highlights: •CD44 variant8–10 and c-Myc are inversely expressed in gastric cancer cells. •Redox-stress enhances c-Myc expression via canonical Wnt signal. •CD44v, but not CD44 standard, suppresses redox stress-induced Wnt activation. •CD44v expression promotes both transcription and proteasome degradation of c-Myc. •Inversed expression pattern between CD44v and c-Myc is often recognized in vivo. -- Abstract: Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup high}/ c-Myc {sup low} and proliferative cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup low}/ c-Myc {sup high}.

Interrelationship between chromosome 8 aneuploidy, C-MYC amplification and increased expression in individuals from northern Brazil with gastric adenocarcinoma

Science.gov (United States)

Calcagno, Danielle Queiroz; Leal, Mariana Ferreira; Seabra, Aline Damaceno; Khayat, André Salim; Chen, Elizabeth Suchi; Demachki, Samia; Assumpção, Paulo Pimentel; Faria, Mario Henrique Girão; Rabenhorst, Silvia Helena Barem; Ferreira, Márcia Valéria Pitombeira; Smith, Marília de Arruda Cardoso; Burbano, Rommel Rodríguez

2006-01-01

AIM: To investigate chromosome 8 numerical aberrations, C-MYC oncogene alterations and its expression in gastric cancer and to correlate these findings with histopathological characteristics of gastric tumors. METHODS: Specimens were collected surgically from seven patients with gastric adenocarcinomas. Immunostaining for C-MYC and dual-color fluorescence in situ hybridization (FISH) for C-MYC gene and chromosome 8 centromere were performed. RESULTS: All the cases showed chromosome 8 aneuploidy and C-MYC amplification, in both the diffuse and intestinal histopathological types of Lauren. No significant difference (P < 0.05) was observed between the level of chromosome 8 ploidy and the site, stage or histological type of the adenocarcinomas. C-MYC high amplification, like homogeneously stained regions (HSRs) and double minutes (DMs), was observed only in the intestinal-type. Structural rearrangement of C-MYC, like translocation, was observed only in the diffuse type. Regarding C-MYC gene, a significant difference (P < 0.05) was observed between the two histological types. The C-MYC protein was expressed in all the studied cases. In the intestinal-type the C-MYC immunoreactivity was localized only in the nucleus and in the diffuse type in the nucleus and cytoplasm. CONCLUSION: Distinct patterns of alterations between intestinal and diffuse types of gastric tumors support the hypothesis that these types follow different genetic pathways. PMID:17036397

Aspirin therapy reduces the ability of platelets to promote colon and pancreatic cancer cell proliferation: Implications for the oncoprotein c-MYC

Science.gov (United States)

Sylman, Joanna L.; Ngo, Anh T. P.; Pang, Jiaqing; Sears, Rosalie C.; Williams, Craig D.; McCarty, Owen J. T.

2017-01-01

Aspirin, an anti-inflammatory and antithrombotic drug, has become the focus of intense research as a potential anticancer agent owing to its ability to reduce tumor proliferation in vitro and to prevent tumorigenesis in patients. Studies have found an anticancer effect of aspirin when used in low, antiplatelet doses. However, the mechanisms through which low-dose aspirin works are poorly understood. In this study, we aimed to determine the effect of aspirin on the cross talk between platelets and cancer cells. For our study, we used two colon cancer cell lines isolated from the same donor but characterized by different metastatic potential, SW480 (nonmetastatic) and SW620 (metastatic) cancer cells, and a pancreatic cancer cell line, PANC-1 (nonmetastatic). We found that SW480 and PANC-1 cancer cell proliferation was potentiated by human platelets in a manner dependent on the upregulation and activation of the oncoprotein c-MYC. The ability of platelets to upregulate c-MYC and cancer cell proliferation was reversed by an antiplatelet concentration of aspirin. In conclusion, we show for the first time that inhibition of platelets by aspirin can affect their ability to induce cancer cell proliferation through the modulation of the c-MYC oncoprotein. PMID:27903583

Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

Science.gov (United States)

Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

2017-06-01

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

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Hong, Kyung-Soo [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Park, Jun-Ik [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Dao, Trong Tuan; Oh, Won Keun [BK21 Project Team, College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Kang, Chi-Dug, E-mail: kcdshbw@pusan.ac.kr [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Kim, Sun-Hee, E-mail: ksh7738@pusan.ac.kr [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

2012-03-01

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

International Nuclear Information System (INIS)

Hong, Kyung-Soo; Park, Jun-Ik; Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won; Dao, Trong Tuan; Oh, Won Keun; Kang, Chi-Dug; Kim, Sun-Hee

2012-01-01

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

Aspirin regulation of c-myc and cyclinD1 proteins to overcome tamoxifen resistance in estrogen receptor-positive breast cancer cells.

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Cheng, Ran; Liu, Ya-Jing; Cui, Jun-Wei; Yang, Man; Liu, Xiao-Ling; Li, Peng; Wang, Zhan; Zhu, Li-Zhang; Lu, Si-Yi; Zou, Li; Wu, Xiao-Qin; Li, Yu-Xia; Zhou, You; Fang, Zheng-Yu; Wei, Wei

2017-05-02

Tamoxifen is still the most commonly used endocrine therapy drug for estrogen receptor (ER)-positive breast cancer patients and has an excellent outcome, but tamoxifen resistance remains a great impediment to successful treatment. Recent studies have prompted an anti-tumor effect of aspirin. Here, we demonstrated that aspirin not only inhibits the growth of ER-positive breast cancer cell line MCF-7, especially when combined with tamoxifen, but also has a potential function to overcome tamoxifen resistance in MCF-7/TAM. Aspirin combined with tamoxifen can down regulate cyclinD1 and block cell cycle in G0/G1 phase. Besides, tamoxifen alone represses c-myc, progesterone receptor (PR) and cyclinD1 in MCF-7 cell line but not in MCF-7/TAM, while aspirin combined with tamoxifen can inhibit the expression of these proteins in the resistant cell line. When knocking down c-myc in MCF-7/TAM, cells become more sensitive to tamoxifen, cell cycle is blocked as well, indicating that aspirin can regulate c-myc and cyclinD1 proteins to overcome tamoxifen resistance. Our study discovered a novel role of aspirin based on its anti-tumor effect, and put forward some kinds of possible mechanisms of tamoxifen resistance in ER-positive breast cancer cells, providing a new strategy for the treatment of ER-positive breast carcinoma.

Transcriptional regulation of BRD7 expression by Sp1 and c-Myc

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Li Shufang

2008-12-01

Full Text Available Abstract Background Bromodomain is an evolutionally conserved domain that is found in proteins strongly implicated in signal-dependent transcriptional regulation. Genetic alterations of bromodomain genes contributed to the development of many human cancers and other disorders. BRD7 is a recently identified bromodomain gene. It plays a critical role in cellular growth, cell cycle progression, and signal-dependent gene expression. Previous studies showed that BRD7 gene exhibited much higher-level of mRNA expression in normal nasopharyngeal epithelia than in nasopharyngeal carcinoma (NPC biopsies and cell lines. However, little is known about its transcriptional regulation. In this study, we explored the transcriptional regulation of BRD7 gene. Method Potential binding sites of transcription factors within the promoter region of BRD7 gene were predicted with MatInspector Professional http://genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl. Mutation construct methods and luciferase assays were performed to define the minimal promoter of BRD7 gene. RT-PCR and western blot assays were used to detect the endogenous expression of transcription factor Sp1, c-Myc and E2F6 in all cell lines used in this study. Electrophoretic mobility shift assays (EMSA and Chromatin immunoprecipitation (ChIP were used to detect the direct transcription factors that are responsible for the promoter activity of BRD7 gene. DNA vector-based siRNA technology and cell transfection methods were employed to establish clone pools that stably expresses SiRNA against c-Myc expression in nasopharyngeal carcinoma 5-8F cells. Real-time PCR was used to detect mRNA expression of BRD7 gene in 5-8F/Si-c-Myc cells. Results We defined the minimal promoter of BRD7 gene in a 55-bp region (from -266 to -212bp, and identified that its promoter activity is inversely related to c-Myc expression. Sp1 binds to the Sp1/Myc-Max overlapping site of BRD7 minimal promoter, and slightly positively

Postnatal liver growth and regeneration are independent of c-myc in a mouse model of conditional hepatic c-myc deletion

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Sanders Jennifer A

2012-03-01

Full Text Available Abstract Background The transcription factor c-myc regulates genes involved in hepatocyte growth, proliferation, metabolism, and differentiation. It has also been assigned roles in liver development and regeneration. In previous studies, we made the unexpected observation that c-Myc protein levels were similar in proliferating fetal liver and quiescent adult liver with c-Myc displaying nucleolar localization in the latter. In order to investigate the functional role of c-Myc in adult liver, we have developed a hepatocyte-specific c-myc knockout mouse, c-mycfl/fl;Alb-Cre. Results Liver weight to body weight ratios were similar in control and c-myc deficient mice. Liver architecture was unaffected. Conditional c-myc deletion did not result in compensatory induction of other myc family members or in c-Myc's binding partner Max. Floxed c-myc did have a negative effect on Alb-Cre expression at 4 weeks of age. To explore this relationship further, we used the Rosa26 reporter line to assay Cre activity in the c-myc floxed mice. No significant difference in Alb-Cre activity was found between control and c-mycfl/fl mice. c-myc deficient mice were studied in a nonproliferative model of liver growth, fasting for 48 hr followed by a 24 hr refeeding period. Fasting resulted in a decrease in liver mass and liver protein, both of which recovered upon 24 h of refeeding in the c-mycfl/fl;Alb-Cre animals. There was also no effect of reducing c-myc on recovery of liver mass following 2/3 partial hepatectomy. Conclusions c-Myc appears to be dispensable for normal liver growth during the postnatal period, restoration of liver mass following partial hepatectomy and recovery from fasting.

Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

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Gao, Feng-Hou; Liu, Feng; Zhao, Ying-Zheng; Fang, Yong; Chen, Fang-Yuan; Wu, Ying-Li; Hu, Xiao-Hui; Li, Wei; Liu, Hua; Zhang, Yan-Jie; Guo, Zhu-Ying; Xu, Mang-Hua; Wang, Shi-Ting; Jiang, Bin

2010-01-01

Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment

Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

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Zhao Ying-Zheng

2010-11-01

Full Text Available Abstract Background Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Methods Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Results Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4 hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Conclusion Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

Shikonin regulates C-MYC and GLUT1 expression through the MST1-YAP1-TEAD1 axis

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Vališ, Karel, E-mail: karel.valis@biomed.cas.cz [Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, v.v.i., The Czech Academy of Sciences, Prague (Czech Republic); Faculty of Science, Charles University, Prague (Czech Republic); Talacko, Pavel; Grobárová, Valéria [Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, v.v.i., The Czech Academy of Sciences, Prague (Czech Republic); Faculty of Science, Charles University, Prague (Czech Republic); Černý, Jan [Faculty of Science, Charles University, Prague (Czech Republic); Novák, Petr, E-mail: pnovak@biomed.cas.cz [Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, v.v.i., The Czech Academy of Sciences, Prague (Czech Republic); Faculty of Science, Charles University, Prague (Czech Republic)

2016-12-10

The general mechanism underlying the tumor suppressor activity of the Hippo signaling pathway remains unclear. In this study, we explore the molecular mechanisms connecting the Hippo signaling pathway with glucose metabolism. We have found that two key regulators of glycolysis, C-MYC and GLUT1, are targets of the Hippo signaling pathway in human leukemia cells. Our results revealed that activation of MST1 by the natural compound shikonin inhibited the expression of GLUT1 and C-MYC. Furthermore, RNAi experiments confirmed the regulation of GLUT1 and C-MYC expression via the MST1-YAP1-TEAD1 axis. Surprisingly, YAP1 was found to positively regulate C-MYC mRNA levels in complex with TEAD1, while it negatively regulates C-MYC levels in cooperation with MST1. Hence, YAP1 serves as a rheostat for C-MYC, which is regulated by MST1. In addition, depletion of MST1 stimulates lactate production, whereas the specific depletion of TEAD1 has an opposite effect. The inhibition of lactate production and cellular proliferation induced by shikonin also depends on the Hippo pathway activity. Finally, a bioinformatic analysis revealed conserved TEAD-binding motifs in the C-MYC and GLUT1 promoters providing another molecular data supporting our observations. In summary, regulation of glucose metabolism could serve as a new tumor suppressor mechanism orchestrated by the Hippo signaling pathway. - Highlights: • Shikonin inhibits C-MYC and GLUT1 expression in MST1 and YAP1 dependent manner. • YAP1-TEAD1 interaction activates C-MYC and GLUT1 expression. • MST1 in cooperation with YAP1 inhibits C-MYC and GLUT1 expression. • MST1-YAP1-TEAD1 axis regulates lactate production by leukemic cells. • MST1 and YAP1 proteins block proliferation of leukemic cells.

Apoptosis and cell proliferation in the development of gastric carcinomas: associations with c-myc and p53 protein expression.

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Ishii, Hideaki H; Gobé, Glenda C; Pan, Wenshen; Yoneyama, Juichi; Ebihara, Yoshiro

2002-09-01

Patients with gastric carcinomas have a poor prognosis and low survival rates. The aim of the present paper was to characterize cellular and molecular properties to provide insight into aspects of tumor progression in early compared with advanced gastric cancers. One hundred and nine graded gastric carcinomas (early or advanced stage, undifferentiated or differentiated type) with paired non-cancer tissue were studied to define the correlation between apoptosis (morphology, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling), cell proliferation (Ki-67 expression, morphology) and expression and localization of two proteins frequently having altered expression in cancers, namely p53 and c-myc. Overall, apoptosis was lower in early stage, differentiated and undifferentiated gastric carcinomas compared with advanced-stage cancers. Cell proliferation was comparatively high in all stages. There was a high level of p53 positivity in all stages. Only the early- and advanced-stage undifferentiated cancers that were p53 positive had a significantly higher level of apoptosis (P cancers that had either c-myc or p53-positivity. The results indicate that low apoptosis and high cell proliferation combine to drive gastric cancer development. The molecular controls for high cell proliferation of the early stage undifferentiated gastric cancers involve overexpression of both p53 and c-myc. Overexpression of p53 may also control cancer development in that its expression is associated with higher levels of apoptosis in early and late-stage undifferentiated, cancers. Copyright 2002 Blackwell Publishing Asia Pty Ltd

The long non-coding RNA GAS5 cooperates with the eukaryotic translation initiation factor 4E to regulate c-Myc translation.

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Guangzhen Hu

Full Text Available Long noncoding RNAs (lncRNAs are important regulators of transcription; however, their involvement in protein translation is not well known. Here we explored whether the lncRNA GAS5 is associated with translation initiation machinery and regulates translation. GAS5 was enriched with eukaryotic translation initiation factor-4E (eIF4E in an RNA-immunoprecipitation assay using lymphoma cell lines. We identified two RNA binding motifs within eIF4E protein and the deletion of each motif inhibited the binding of GAS5 with eIF4E. To confirm the role of GAS5 in translation regulation, GAS5 siRNA and in vitro transcribed GAS5 RNA were used to knock down or overexpress GAS5, respectively. GAS5 siRNA had no effect on global protein translation but did specifically increase c-Myc protein level without an effect on c-Myc mRNA. The mechanism of this increase in c-Myc protein was enhanced association of c-Myc mRNA with the polysome without any effect on protein stability. In contrast, overexpression of in vitro transcribed GAS5 RNA suppressed c-Myc protein without affecting c-Myc mRNA. Interestingly, GAS5 was found to be bound with c-Myc mRNA, suggesting that GAS5 regulates c-Myc translation through lncRNA-mRNA interaction. Our findings have uncovered a role of GAS5 lncRNA in translation regulation through its interactions with eIF4E and c-Myc mRNA.

AP-2α Inhibits c-MYC Induced Oxidative Stress and Apoptosis in HaCaT Human Keratinocytes

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Lei Yu

2009-01-01

AP-2 may have a direct effect on the c-myc gene. Chromatin immunoprecipitation assays demonstrated that AP-2 proteins bound to a cluster of AP-2 binding sites located within a 2 kb upstream regulatory region of c-myc These results suggest that the negative regulation of AP-2 on c-MYC activity was achieved through binding of AP-2 protein to the c-myc gene. The effects of AP-2 on c-MYC induced ROS accumulation and apoptosis in epidermal keratinocytes are likely to play an important role in cell growth, differentiation and carcinogenesis of the skin.

Nac1 promotes self-renewal of embryonic stem cells through direct transcriptional regulation of c-Myc.

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Ruan, Yan; He, Jianrong; Wu, Wei; He, Ping; Tian, Yanping; Xiao, Lan; Liu, Gaoke; Wang, Jiali; Cheng, Yuda; Zhang, Shuo; Yang, Yi; Xiong, Jiaxiang; Zhao, Ke; Wan, Ying; Huang, He; Zhang, Junlei; Jian, Rui

2017-07-18

The pluripotency transcriptional network in embryonic stem cells (ESCs) is composed of distinct functional units including the core and Myc units. It is hoped that dissection of the cellular functions and interconnections of network factors will aid our understanding of ESC and cancer biology. Proteomic and genomic approaches have identified Nac1 as a member of the core pluripotency network. However, previous studies have predominantly focused on the role of Nac1 in psychomotor stimulant response and cancer pathogenesis. In this study, we report that Nac1 is a self-renewal promoting factor, but is not required for maintaining pluripotency of ESCs. Loss of function of Nac1 in ESCs results in a reduced proliferation rate and an enhanced differentiation propensity. Nac1 overexpression promotes ESC proliferation and delays ESC differentiation in the absence of leukemia inhibitory factor (LIF). Furthermore, we demonstrated that Nac1 directly binds to the c-Myc promoter and regulates c-Myc transcription. The study also revealed that the function of Nac1 in promoting ESC self-renewal appears to be partially mediated by c-Myc. These findings establish a functional link between the core and c-Myc-centered networks and provide new insights into mechanisms of stemness regulation in ESCs and cancer.

Aberrant immunoglobulin and c-myc gene rearrangements in patients with nonmalignant monoclonal cryoglobulinemia

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Perl, A.; Wang, N.; Williams, J.M.; Hunt, M.J.; Rosenfeld, S.I.; Condemi, J.J.; Packman, C.H.; Abraham, G.N.

1987-01-01

The status of the immunoglobulin (Ig) genes was investigated in patients with idiopathic nonmalignant monoclonal IgG cryoglobulinemia (NCG). In NCG, monoclonal antibodies are synthesized at an accelerated rate by nonmalignant B lymphocytes. In order to determine whether this high production rate is related to a clonal B cell expansion, the rearrangement of the Ig genes was investigated by Southern blot analysis of genomic, 32 P-labelled, DNA extracted from the peripheral blood lymphocytes of four NCG patients. In three of four (VI, BR, and CH) clonal expansion of B cells was detected using probes specific for the genes. BamHI digestion of DNA from VI and BR produced three rearranged fragments which cohybridized with two of the probes. This finding suggested the presence of additional nonsecretory B cell clones and/or disruption of the gene segments spanned by and detected with the probes. In addition, the possibility of aberrant gene rearrangements was supported by noting the alteration of the c-myc gene locus in genomic DNA from peripheral blood leukocytes of VI and CH. Northern blot analysis of RNA isolated from peripheral blood B cells of VI and CH demonstrated aberrant transcripts of the c-myc gene, showing an active role of the altered c-myc locus. Detection of c-myc rearrangement in NCG patients clearly shows that this event may not be a final step in malignant B cell transformation

In vivo distribution of c-myc antisense oligodeoxynucleotides local delivered by gelatin-coated platinmn-iridium stents in rabbits and its effect on apoptosis

Institute of Scientific and Technical Information of China (English)

张新霞; 崔长琮; 许香广; 胡雪松; 方卫华; 邝碧娟

2004-01-01

Background Post-stenting restenosis is a significant clinical problem, involving vascular smooth muscle cells(VSMCs) proliferation and apoptosis. It is reported that c-myc antisense oligodeoxynucleotides (ASODNs) local delivered by catheter can inhibit VSMCs proliferation. This study was designed to assess tissue distribution of c-myc ASODN local delivered using gelatin-coated platinum-iridium (Pt-Ir) stents, and its effect on apoptosis of VSMCs. Methods Gelatin-coated Pt-Ir stents that had absorbed caroboxyfluorescein-5-succimidyl ester (FAM) labeled c-myc ASODNs (550 μg per stent) were implanted into the right carotid arteries of 6 rabbits. Tissue samples were obtained at 45 minutes, 2 hours, and 6 hours. Tissue distribution of c- myc ASODNs was assessed by fluorescence microscopy. In addition, 32 rabbits were randomly divided into two groups. Rabbits in the control group (n=16) were implanted with gelatin-coated Pt-Ir stents, and those in the treatment group (n=16) were implanted with gelatin-coated stents that had absorbed c-myc ASODNs. 7, 14, 30, or 90 days (n=4, respectively, for each group) after the stenting procedure, the stented segments were harvested, and histopathological examinations were performed to calculate neointimal area and mean neointimal thickness. The expression of c-myc was assessed using in situ hybridization (ISH) and immunohistochemical methods. Apoptotic VSMCs were detected using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM). Results According to fluorescence microscopic results, FAM-labeled c-myc ASODNs were concentrated in the target vessel media at the 45 minutes time point, and then dispersed to the adventitia. Morphometric analysis showed that neointimal area and mean neointimal thickness increased continuously up to 90 days after stent implantation, but that total neointimal area and mean neointimal thickness were less in the treatment group than in the

Mechanism of estrogen activation of c-myc oncogene expression.

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Dubik, D; Shiu, R P

1992-08-01

The estrogen receptor complex is a known trans-acting factor that regulates transcription of specific genes through an interaction with a specific estrogen-responsive cis-acting element (ERE). In previous studies we have shown that in estrogen-responsive human breast cancer cells estrogen rapidly activates c-myc expression. This activated expression occurs through enhanced transcription and does not require the synthesis of new protein intermediates; therefore, an ERE is present in the human c-myc gene regulatory region. To localize the ERE, constructs containing varying lengths of the c-myc 5'-flanking region ranging from -2327 to +25 (relative to the P1 promoter) placed adjacent to the chloramphenicol acetyl transferase reporter gene (CAT) were prepared. They were used in transient transfection studies in MCF-7 and HeLa cells co-transfected with an estrogen receptor expression vector. These studies reveal that all constructs containing the P2 promoter region exhibited estrogen-regulated CAT expression and that a 116-bp region upstream and encompassing the P2 TATA box is necessary for this activity. Analysis of this 116-bp region failed to identify a cis-acting element with sequences resembling the consensus ERE; however, co-transfection studies with mutant estrogen receptor expression vectors showed that the DNA-binding domain of the receptor is essential for estrogen-regulated CAT gene expression. We have also observed that anti-estrogen receptor complexes can weakly trans-activate from this 116-bp region but fail to do so from the ERE-containing ApoVLDLII-CAT construct. To explain these results we propose a new mechanism of estrogen trans-activation in the c-myc gene promoter.

Astroglial c-Myc overexpression predisposes mice to primary malignant gliomas

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Jensen, Niels Aagaard; Pedersen, Karen-Marie; Lihme, Frederikke

2003-01-01

Malignant astrocytomas are common human primary brain tumors that result from neoplastic transformation of astroglia or their progenitors. Here we show that deregulation of the c-Myc pathway in developing astroglia predisposes mice to malignant astrocytomas within 2-3 weeks of age. The genetically...... engineered murine (GEM) gliomas harbor a molecular signature resembling that of human primary glioblastoma multiforme, including up-regulation of epidermal growth factor receptor and Mdm2. The GEM gliomas seem to originate in an abnormal population of glial fibrillary acidic protein-expressing cells...... the neoplastic process, presumably by inducing the sustained growth of early astroglial cells. This is in contrast to most other transgenic studies in which c-Myc overexpression requires co-operating transgenes for rapid tumor induction....

c-Myc Enhances Sonic Hedgehog-Induced Medulloblastoma Formation from Nestin-Expressing Neural Progenitors in Mice

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Ganesh Rao

2003-05-01

Full Text Available Medulloblastomas are malignant brain tumors that arise in the cerebella of children. The presumed cellsof-origin are undifferentiated precursors of granule neurons that occupy the external granule layer (EGL of the developing cerebellum. The overexpression of proteins that normally stimulate proliferation of neural progenitor cells may initiate medulloblastoma formation. Two known mitogens for neural progenitors are the c-Myc oncoprotein and Sonic hedgehog (Shh, a crucial determinant of embryonic pattern formation in the central nervous system. We modeled the ability of c-Myc and Shh to induce medulloblastoma in mice using the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell type-specific manner. We targeted the expression of Shh and c-Myc to nestin-expressing neural progenitor cells by injecting replication-competent ALV splice acceptor (RCAS vectors into the cerebella of newborn mice. Following injection with RCAS-Shh alone, 3/32 (9% mice developed medulloblastomas and 5/32 showed multifocal hyperproliferation of the EGL, possibly a precursor stage of medulloblastoma. Following injection with RCAS-Shh plus RCAS-Myc, 9/39 (23% mice developed medulloblastomas. We conclude that nestin-expressing neural progenitors, present in the cerebellum at birth, can act as the cells-of-origin for medulloblastoma, and that c-Myc cooperates with Shh to enhance tumorigenicity.

Expression of p27 and c-Myc by immunohistochemistry in breast ductal cancers in African American women.

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Khan, Farhan; Ricks-Santi, Luisel J; Zafar, Rabia; Kanaan, Yasmine; Naab, Tammey

2018-06-01

Proteins p27 and c-Myc are both key players in the cell cycle. While p27, a tumor suppressor, inhibits progression from G1 to S phase, c-Myc, a proto-oncogene, plays a key role in cell cycle regulation and apoptosis. The objective of our study was to determine the association between expression of c-Myc and the loss of p27 by immunohistochemistry (IHC) in the four major subtypes of breast cancer (BC) (Luminal A, Luminal B, HER2, and Triple Negative) and with other clinicopathological factors in a population of 202 African-American (AA) women. Tissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 AA women. Five micrometer sections were stained with a mouse monoclonal antibody against p27 and a rabbit monoclonal antibody against c-Myc. The sections were evaluated for intensity of nuclear reactivity (1-3) and percentage of reactive cells; an H-score was derived from the product of these measurements. Loss of p27 expression and c-Myc overexpression showed statistical significance with ER negative (p c-Myc expression/p27 loss and luminal A/B and Her2 overexpressing subtypes. In our study, a statistically significant association between c-Myc expression and p27 loss and the triple negative breast cancers (TNBC) was found in AA women. A recent study found that constitutive c-Myc expression is associated with inactivation of the axin 1 tumor suppressor gene. p27 inhibits cyclin dependent kinase2/cyclin A/E complex formation. Axin 1 and CDK inhibitors may represent possible therapeutic targets for TNBC. Copyright © 2018 Elsevier Inc. All rights reserved.

Integrin α6Bβ4 inhibits colon cancer cell proliferation and c-Myc activity

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Dydensborg, Anders Bondo; Teller, Inga C; Groulx, Jean-François; Basora, Nuria; Paré, Fréderic; Herring, Elizabeth; Gauthier, Rémy; Jean, Dominique; Beaulieu, Jean-François

2009-01-01

Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin β4 subunit is up-regulated in primary colon cancer. Its partner, the integrin α6 subunit, exists as two different mRNA splice variants, α6A and α6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these α6 splice variants is still lacking. In this work, we first analyzed the expression of integrin α6A and α6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of α6A and α6B on the regulation of cell proliferation in a colon cancer cell line. Using variant-specific antibodies, we observed that α6A and α6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express α6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed α6B. A relative decrease of α6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the α6A/α6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the α6A/α6B balance in favor of α6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc. The findings that the α6Bβ4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its α6Aβ4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this α6Bβ4 integrin. Taken together, these findings point out the importance of integrin

Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

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Wang, Zhaojing [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Xu, Yonghong [Institute of Ophthalmological Research, Department of Ophthalmology, Renmin Hospital of Wuhan University, 430060 Wuhan (China); Meng, Xiangning [School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Watari, Fumio [Department of Biomedical, Dental Materials and Engineering, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586 (Japan); Liu, Hudan, E-mail: hudanliu@hust.edu.cn [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Chen, Xiao, E-mail: mornsmile@yahoo.com [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China)

2015-01-01

Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

Science.gov (United States)

Phesse, T J; Myant, K B; Cole, A M; Ridgway, R A; Pearson, H; Muncan, V; van den Brink, G R; Vousden, K H; Sears, R; Vassilev, L T; Clarke, A R; Sansom, O J

2014-06-01

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility

Directory of Open Access Journals (Sweden)

Nunes Virginia

2008-01-01

Full Text Available Abstract Background Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. Results This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC. Conclusion This study proposes that variation at putative 8q24 cis-regulator(s of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.

SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation

International Nuclear Information System (INIS)

Xie, Yuexia; Tu, Wenzhi; Zhang, Jianghong; He, Mingyuan; Ye, Shuang; Dong, Chen; Shao, Chunlin

2015-01-01

Highlights: • γ-Irradiation induced bystander effects between hepatoma cells and hepatocyte cells. • SirT1 played a protective role in regulating this bystander effect. • SirT1 contributed to the protective effects via elimination the accumulation of ROS. • The activity of c-Myc is critical for maintaining the protective role of SirT1. - Abstract: Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved

SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation

Energy Technology Data Exchange (ETDEWEB)

Xie, Yuexia [Institute of Radiation Medicine, Fudan University, Shanghai (China); Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Tu, Wenzhi; Zhang, Jianghong; He, Mingyuan; Ye, Shuang; Dong, Chen [Institute of Radiation Medicine, Fudan University, Shanghai (China); Shao, Chunlin, E-mail: clshao@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, Shanghai (China)

2015-02-15

Highlights: • γ-Irradiation induced bystander effects between hepatoma cells and hepatocyte cells. • SirT1 played a protective role in regulating this bystander effect. • SirT1 contributed to the protective effects via elimination the accumulation of ROS. • The activity of c-Myc is critical for maintaining the protective role of SirT1. - Abstract: Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved.

Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients.

Science.gov (United States)

Attallah, A M; El-Far, M; Abdelrazek, M A; Omran, M M; Attallah, A A; Elkhouly, A A; Elkenawy, H M; Farid, K

2017-10-01

Hepatocellular carcinoma (HCC) is a multistage process resulting from various genetic changes. We aimed to determine nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 expression and to evaluate their importance in HCC diagnosis. One hundred and twenty chronic hepatitis C (CHC) patients (60 non-HCC CHC patients and 60 HCC patients who had a single small (c-Myc and p53 were identified in liver tissues and serum samples using immunostaining, western blot and ELISA. Immunohistochemical detection of c-Myc and p53 with monospecific antibodies revealed intense and diffuse cytoplasmic staining patterns. Accumulated mutant proteins, released from tumour cells into the extracellular serum, were detected at 62 KDa, for c-Myc, and 53 KDa, for p53, using western blotting. In contrast to alpha feto-protein, there was a significant increase (p c-Myc (86.7% vs. 6.7%) and p53 (78.3% vs. 8.3%) in the malignant vs. non-malignant patients. The parallel combination of c-Myc and p53 reach the absolute sensitivity (100%), for more accurate and reliable HCC detection (specificity was 87%). c-Myc and p53 are potential HCC diagnostic biomarkers, and convenient combinations of them could improve diagnostic accuracy of HCC.

Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse

International Nuclear Information System (INIS)

Skvortsova, Ira; Skvortsov, Sergej; Raju, Uma; Stasyk, Taras; Riesterer, Oliver; Schottdorf, Eva-Maria; Popper, Bela-Andre; Schiestl, Bernhard; Eichberger, Paul; Debbage, Paul; Neher, Andreas; Bonn, Guenther K.; Huber, Lukas A.; Milas, Luka; Lukas, Peter

2010-01-01

Purpose: Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. Methods and materials: Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8 mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. Results: In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. Conclusions: The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E

c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks.

Science.gov (United States)

Barfeld, Stefan J; Urbanucci, Alfonso; Itkonen, Harri M; Fazli, Ladan; Hicks, Jessica L; Thiede, Bernd; Rennie, Paul S; Yegnasubramanian, Srinivasan; DeMarzo, Angelo M; Mills, Ian G

2017-04-01

Prostate cancer (PCa) is the most common non-cutaneous cancer in men. The androgen receptor (AR), a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen), and Glycine N-Methyltransferase (GNMT), in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks

Directory of Open Access Journals (Sweden)

Stefan J. Barfeld

2017-04-01

Full Text Available Prostate cancer (PCa is the most common non-cutaneous cancer in men. The androgen receptor (AR, a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen, and Glycine N-Methyltransferase (GNMT, in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa.

DNA repair in the c-myc proto-oncogene locus: Possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice

International Nuclear Information System (INIS)

Beecham, E.J.; Mushinski, J.F.; Shacter, E.; Potter, M.; Bohr, V.A.

1991-01-01

This report describes an unexpected difference in the efficiency of removal of UV-induced DNA damage in the c-myc locus in splenic B lymphoblasts from two inbred strains of mice. In cells from plasmacytoma-resistant DBA/2N mice, 35% of UV-induced damage in the regulatory and 5' flank of c-myc is removed by 12 h. However, in cells from plasmacytoma-susceptible BALB/cAn mice, damage is not removed from this region. In the protein-encoding region and 3' flank of c-myc as well as in two dihydrofolate reductase gene fragments, UV damage is repaired with similar efficiency in B lymphoblasts from both strains of mice. Furthermore, in the protein-encoding portion and 3' flank of c-myc, damage is selectively removed from only the transcribed strand. No repair is detected in the nontranscribed strand. In contrast, DNA repair in the 5' flank of c-myc is not strand specific; in DNA from DBA/2N cells, UV damage is rapidly removed from both the transcribed and nontranscribed strands. In BALB/cAn cells no repair was detected in either strand in the 5'flank, consistent with the results with double-stranded, nick-translated probes to this region of c-myc. In addition to the repair studies, we have detected post-UV-damage formation: in most of the genes studied, we find that additional T4 endonuclease-sensitive sites are formed in the DNA 2 h after irradiation. Our findings provide new insights into the details of gene-specific and strand-specific DNA repair and suggest that there may be close links between DNA repair and B-cell neoplastic development

Somatostatin reduces 3H-thymidine incorporation and c-myc, but not thyroglobulin ribonucleic acid levels in human thyroid follicular cells in vitro

International Nuclear Information System (INIS)

degli Uberti, E.C.; Hanau, S.; Rossi, R.; Piva, R.; Margutti, A.; Trasforini, G.; Pansini, G.; del Senno, L.

1991-01-01

The action of somatostatin (SRIH) on 3 H-thymidine (thy) incorporation and on c-myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10 - 7 M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH-stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent

Evaluation of Molecular Inhibitors of the c-Myc Oncoprotein

National Research Council Canada - National Science Library

Prochownik, Edward V

2005-01-01

.... All of these functions require that C-Myc physically associate with another protein. Max. Example of diseases in which c-Myc deregulation occurs include breast cancer (approx. 30% of cases), colon cancer (>85...

High levels of stable p53 protein and the expression of c-myc in cultured human epithelial tissue after cobalt-60 irradiation

International Nuclear Information System (INIS)

Mothersill, C.; Seymour, C.B.; Harney, J.; Hennessy, T.P.

1994-01-01

When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells was noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in open-quotes edgeclose quotes cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed. 33 refs., 2 figs., 3 tabs

Effects on micronuclei formation of 60-Hz electromagnetic field exposure with ionizing radiation, hydrogen peroxide, or c-Myc overexpression.

Science.gov (United States)

Jin, Yeung Bae; Kang, Ga-Young; Lee, Jae Seon; Choi, Jong-Il; Lee, Ju-Woon; Hong, Seung-Cheol; Myung, Sung Ho; Lee, Yun-Sil

2012-04-01

Epidemiological studies have demonstrated a possible correlation between exposure to extremely low-frequency magnetic fields (ELF-MF) and cancer. However, this correlation has yet to be definitively confirmed by epidemiological studies. The principal objective of this study was to assess the effects of 60 Hz magnetic fields in a normal cell line system, and particularly in combination with various external factors, via micronucleus (MN) assays. Mouse embryonic fibroblast NIH3T3 cells and human lung fibroblast WI-38 cells were exposed for 4 h to a 60 Hz, 1 mT uniform magnetic field with or without ionizing radiation (IR, 2 Gy), H(2)O(2) (100 μM) and cellular myelocytomatosis oncogene (c-Myc) activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic effects were observed when ELF-MF was combined with IR, H(2)O(2), and c-Myc activation. Our results demonstrate that ELF-MF did not enhance MN frequency by IR, H(2)O(2) and c-Myc activation.

c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.

Science.gov (United States)

Musso, Loana; Mazzini, Stefania; Rossini, Anna; Castagnoli, Lorenzo; Scaglioni, Leonardo; Artali, Roberto; Di Nicola, Massimo; Zunino, Franco; Dallavalle, Sabrina

2018-03-01

Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins

International Nuclear Information System (INIS)

Zullo, J.; Stiles, C.D.; Garcea, R.L.

1987-01-01

The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP 1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP 1 protein stimulated [ 3 H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for the cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins

Inhibition of c-Myc by 10058-F4 induces growth arrest and chemosensitivity in pancreatic ductal adenocarcinoma.

Science.gov (United States)

Zhang, Meng; Fan, Hai-Yan; Li, Sheng-Chao

2015-07-01

Pancreatic ductal adenocarcinoma (PDAC) is a formidable medical challenge due to its malignancies and the absence of effective treatment. c-Myc, as an important transcription factor, plays crucial roles in cell cycle progression, apoptosis and cellular transformation. The c-Myc inhibitor, 10058-F4, has been reported act as a tumor suppressor in several different tumors. In current study, the tumor-suppressive roles of 10058-F4 was observed in human pancreatic cancer cells in vitro as demonstrated by decreased cell viability, cell cycle arrest at the G1/S transition and increased caspase3/7 activity. And tumor responses to gemcitabine were also significantly enhanced by 10058-F4 in PANC-1 and SW1990 cells. In a subcutaneous xenograft model, however, 10058-F4 showed no significant influence on pancreatic tumorigenesis. When combined with gemcitabine, tumorigenesis was drastically attenuated compared with gemcitabine group or 10058-F4 group; this synergistic effect was accompanied with decreased PCNA-positive cells and reduced TUNEL-positive cells in the combined treated group. Subsequent studies revealed that decreased glycolysis may be involved in the inhibitory effect of 10058-F4 on PDAC. Taken together, this study demonstrates the roles of 10058-F4 in PDAC and provides evidence that 10058-F4 in combination with gemcitabine showed significant clinical benefit over the usage of gemcitabine alone. Copyright © 2015. Published by Elsevier Masson SAS.

Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation

DEFF Research Database (Denmark)

Mathiasen, D.P.; Egebjerg, C.; Andersen, S.H.

2012-01-01

are essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene...... that counteracts protein phosphatase 2A-mediated dephosphorylation of c-Myc. Here we show that JNK2 regulates Cip2a transcription via ATF2. ATF2 and c-Myc cooperate to activate the transcription of ATF3. Remarkably, not only ectopic JNK2, but also ectopic ATF2, CIP2A, c-Myc and ATF3 are sufficient to rescue...... the defective ras transformation of JNK2-deficient cells. Thus, these data identify the key signal converging point of JNK2 and ERK pathways and underline the central role of CIP2A in ras transformation.Oncogene advance online publication, 27 June 2011; doi:10.1038/onc.2011.230....

Expression of c-myc and c-fos and binding sites for estradiol and progesterone in human pituitary tumors.

Science.gov (United States)

Machiavelli, G A; Rivolta, C M; Artese, R; Basso, A; Burdman, J A

1998-12-01

We studied the concentration of mRNA from the oncogenes c-myc and c-fos in human pituitary adenomas by Northern blot hybridization (35 somatotrophinomas, 9 prolactinomas, 21 nonsecreting and 3 adrenocorticotrophinomas). The concentration of estrogens and progesterone receptors was also investigated. The levels of c-myc and c-fos mRNA was higher in nonsecreting tumors which were generally the largest and had a higher percentage of recurrence after surgery than the other groups. High concentration of estrogen receptors was observed in tumors derived from cells which are normally the target of this hormone, mainly prolactinomas. They were also present in somatotrophic and nonsecreting adenomas, related to the presence of prolactin or gonadotrophin cells in these tumors. The presence of estrogen receptors indicates that the tumor cells maintain their differentiation and a good prognosis as is the case for prolactinomas. We did not find any relationship between estrogen receptors and the concentration of c-myc and c-fos oncogenes. Larger adenomas (mainly nonsecreting) had higher levels of c-myc and c-fos mRNA than the other tumors and they had an important percentage of recurrence after surgery. It is clear that tumor size is related to the outcome after surgery and that nonsecreting adenomas are usually large because of the late diagnosis. However two large somatotrophinomas with extrasellar expansion also had overexpression of both oncogenes and both relapsed after surgery.

Anti-proliferative activity of the quassinoid NBT-272 in childhood medulloblastoma cells

Directory of Open Access Journals (Sweden)

Helson Lawrence

2007-01-01

Full Text Available Abstract Background With current treatment strategies, nearly half of all medulloblastoma (MB patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to correlate with anaplasia and unfavorable prognosis. In neuroblastoma – an embryonal tumor with biological similarities to MB – the quassinoid NBT-272 has been demonstrated to inhibit cellular proliferation and to down-regulate c-MYC protein expression. Methods To study MB cell responses to NBT-272 and their dependence on the level of c-MYC expression, DAOY (wild-type, empty vector transfected or c-MYC transfected, D341 (c-MYC amplification and D425 (c-MYC amplification human MB cells were used. The cells were treated with different concentrations of NBT-272 and the impact on cell proliferation, apoptosis and c-MYC expression was analyzed. Results NBT-272 treatment resulted in a dose-dependent inhibition of cellular proliferation (IC50 in the range of 1.7 – 9.6 ng/ml and in a dose-dependent increase in apoptotic cell death in all human MB cell lines tested. Treatment with NBT-272 resulted in up to 90% down-regulation of c-MYC protein, as demonstrated by Western blot analysis, and in a significant inhibition of c-MYC binding activity. Anti-proliferative effects were slightly more prominent in D341 and D425 human MB cells with c-MYC amplification and slightly more pronounced in c-MYC over-expressing DAOY cells compared to DAOY wild-type cells. Moreover, treatment of synchronized cells by NBT-272 induced a marked cell arrest at the G1/S boundary. Conclusion In human MB cells, NBT-272 treatment inhibits cellular proliferation at nanomolar concentrations, blocks cell cycle progression, induces apoptosis, and down-regulates the expression of the oncogene c-MYC. Thus, NBT-272 may represent a novel drug candidate to inhibit

[Survival of patients with primary central nervous system diffuse large B-cell lymphoma: impact of gene aberrations and protein overexpression of bcl-2 and C-MYC, and selection of chemotherapy regimens].

Science.gov (United States)

Yin, W J; Zhu, X; Yang, H Y; Sun, W Y; Wu, M J

2018-01-08

Objective: To investigate the impact of clinicopathological features, gene rearrangements and protein expression of bcl-6, bcl-2, C-MYC and chemotherapy regime on the prognosis of patients with primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL). Methods: Thirty-three cases of PCNS-DLBCL diagnosed from January 2006 to December 2016 at Zhejiang Cancer Hospital were collected. The expression of CD10, bcl-6, bcl-2, MUM1 and MYC were detected by immunohistochemical staining (IHC). The presence of EB virus was detected by in situ hybridization(EBER). Copy number variation (ICN) and translocation status of bcl-6, bcl-2 and C-MYC genes were detected by fluorescence in situ hybridization (FISH). The relationship between the above indexes and the prognosis was analyzed by univariate, bivariate survival analysis and multiple Cox hazard regression analysis. Results: The study included 33 patients of PCNS-DLBCL, without evidence of primary or secondary immunodeficient disease. Male to female ratio was 1.36∶1.00, and the average age was 56 years. Twenty cases had single lesion while 13 had multiple lesions. Deep brain involvement was seen in 12 cases. All patients underwent partial or total tumor resection. Five patients received whole brain post-surgery radiotherapy, nine patients received high-dose methotrexate (HD-MTX) based chemotherapy, and 12 patients received whole-brain radiotherapy combined with HD-MTX based chemotherapy. Severn patients received no further treatment and rituximab was used in 8 patients. According to the Hans model, 27 cases were classified as non-GCB subtypes (81.8%). Bcl-2 was positive in 25 cases (75.8%, 25/33) and highly expressed in 8 (24.2%). MYC was positive in 12 cases (36.4%) and double expression of bcl-2 and MYC was seen in 6 cases. EBER positive rate was 10.0%(3/30), all of which had multiple lesions. Two bcl-6 gene translocations and 3 amplifications were found in 28 patients. Two translocations, 3 ICN or with both

Cytotoxic effect of γ-sitosterol from Kejibeling (Strobilanthes crispus and its mechanism of action towards c-myc gene expression and apoptotic pathway

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Susi Endrini

2015-01-01

Full Text Available Background: This study aimed to analyze the cytotoxicity effect of γ-sitosterol isolated from “Kejibeling” (Strobilanthes crispus, a medicinal plant, on several cancer cell lines. The mechanisms of the effects were studied through the expression of cancer-caused gene, c-myc and apoptotic pathways.Methods: This in vitro study was done using human colon cancer cell lines (Caco-2, liver cancer cell lines (HepG2, hormone-dependent breast cancer cell lines (MCF-7 and the normal liver cell lines (Chang Liver. The cytotoxic effect was measured through MTT assay and the potential cytotoxic value was calculated by determining the toxic concentration which may kill up to 50% of the total cell used (IC50. Meanwhile, the cytotoxic mechanism was studied by determining the effect of adding γ-sitosterol to the c-myc gene expression by reverse transciptase-polymerase chain reaction (RT-PCR. The effect of γ-sitosterol through apoptotic pathway was studied by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay.Results: γ-sitosterol was cytotoxic against Caco-2, HepG2, and MCF-7 with IC50-values of 8.3, 21.8, and 28.8 μg/mL, respectively. There were no IC50-values obtained from this compound against Chang Liver cell line. This compound induced apotosis on Caco-2 and HepG2 cell lines and suppressed the c-myc genes expression in both cells.Conclusion: γ-sitosterol was cytotoxic against colon and liver cancer cell lines and the effect was mediated by down-regulation of c-myc expression and induction of the apoptotic pathways.

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

International Nuclear Information System (INIS)

Nikolova, Ekaterina; Mitev, Vanio; Zhelev, Nikolai; Deroanne, Christophe F.; Poumay, Yves

2007-01-01

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity

Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30{sup II} accessory protein and the induction of oncogenic cellular transformation by p30{sup II}/c-MYC

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Romeo, Megan M.; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C.; He, Jeffrey; Harrod, Carolyn K.; Barnett, Braden [Laboratory of Molecular Virology, Department of Biological Sciences, and The Dedman College Center for Drug Discovery, Design, and Delivery, Southern Methodist University, Dallas, TX 75275-0376 (United States); Ratner, Lee [Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Lairmore, Michael D. [University of California-Davis, School of Veterinary Medicine, One Shields Avenue, Davis, CA 95618 (United States); Martinez, Ernest [Department of Biochemistry, University of California, Riverside, CA 92521 (United States); Lüscher, Bernhard [Institute of Biochemistry, Klinikum, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen (Germany); Robson, Craig N. [Northern Institute for Cancer Research, Newcastle University, The Medical School, Newcastle upon Tyne, NE2 4HH (United Kingdom); Henriksson, Marie [Department of Microbiology, Cell and Tumor Biology, Karolinska Institutet, Stockholm (Sweden); Harrod, Robert, E-mail: rharrod@smu.edu [Laboratory of Molecular Virology, Department of Biological Sciences, and The Dedman College Center for Drug Discovery, Design, and Delivery, Southern Methodist University, Dallas, TX 75275-0376 (United States)

2015-02-15

The human T-cell leukemia retrovirus type-1 (HTLV-1) p30{sup II} protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30{sup II} interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30{sup II} and c-MYC remain to be completely understood. Herein we demonstrate that p30{sup II} induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30{sup II} in c-myc{sup −/−} HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30{sup II} is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30{sup II} inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30{sup II}/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30{sup II}/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30{sup II}/c-MYC. • The HTLV-1 p30{sup II} protein induces lysine-acetylation of c-MYC. • p30{sup II} is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30{sup II} inhibits apoptosis in c-MYC-expressing proliferating cells.

Distinct Histopathologic and Molecular Alterations in Inflammatory Bowel Disease-Associated Intestinal Adenocarcinoma: c-MYC Amplification is Common and Associated with Mucinous/Signet Ring Cell Differentiation.

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Hartman, Douglas J; Binion, David G; Regueiro, Miguel D; Miller, Caitlyn; Herbst, Cameron; Pai, Reetesh K

2018-05-17

Chronic idiopathic inflammatory bowel disease (IBD) is a significant risk factor for the development of intestinal adenocarcinoma. The underlying molecular alterations in IBD-associated intestinal adenocarcinoma remain largely unknown. We compared the clinicopathologic and molecular features of 35 patients with 47 IBD-associated intestinal adenocarcinomas with a consecutive series of 451 patients with sporadic colorectal carcinoma identified at our institution and published data on sporadic colorectal carcinoma. c-MYC amplification was the most frequent molecular alteration identified in 33% of IBD-associated intestinal adenocarcinoma that is a significantly higher frequency than in sporadic colorectal carcinoma (8%) (P = 0.0001). Compared to sporadic colorectal carcinoma, IBD-associated intestinal adenocarcinomas more frequently demonstrated mucinous differentiation (60% vs 25%, P < 0.001) and signet ring cell differentiation (28% vs 4%, P < 0.001). Mucinous and signet ring cell differentiation were significantly associated with the presence of c-MYC amplification (both with P < 0.05). HER2 positivity (11%), KRAS exon 2 or 3 mutation (10%), and IDH1 mutation (7%) were less commonly observed in IBD-associated intestinal adenocarcinoma. There was an association between poor survival and HER2 status with 3 of 4 patients having HER2-positive adenocarcinoma dead of disease at last clinical follow-up; however, no statistically significant survival effect was identified for any of the molecular alterations identified. We demonstrate that IBD-associated intestinal adenocarcinomas have a high frequency of c-MYC amplification that is associated with mucinous and signet ring cell differentiation. Many of the identified molecular alterations have potential therapeutic relevance, including HER2 amplification, IDH1 mutation, and low frequency KRAS mutation.

c-Myc-Dependent Cell Competition in Human Cancer Cells.

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Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

2017-07-01

Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma.

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Yu, X; Zhen, Y; Yang, H; Wang, H; Zhou, Y; Wang, E; Marincola, F M; Mai, C; Chen, Y; Wei, H; Song, Y; Lyu, X; Ye, Y; Cai, L; Wu, Q; Zhao, M; Hua, S; Fu, Q; Zhang, Y; Yao, K; Liu, Z; Li, X; Fang, W

2013-05-16

Connective tissue growth factor (CTGF) has different roles in different types of cancer. However, the involvement and molecular basis of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC) have almost never been reported. In this study, we observed that downregulated CTGF expression was significantly associated with NPC progression and poor prognosis. Knockdown of CTGF markedly elevated the ability of cell proliferation in vivo and in vitro. Subsequently, we discovered that the reduction of CTGF increased the expression of miR-18b, an oncomir-promoting cell proliferation. Further, we discovered that attenuated CTGF-mediated upregulation of miR-18b was dependent on the increased binding of transcription factors Jun proto-oncogene (C-Jun) and v-Myc myelocytomatosis viral oncogene homolog (C-Myc) to miR-18b promoter region via phosphoinositide 3-kinase (PI3K)/AKT pathway. Finally, we further found that miR-18b directly suppressed the expression of CTGF in NPC. In clinical fresh specimens, miR-18b was widely overexpressed and inversely correlated with CTGF expression in NPC. Our studies are the first to demonstrate that reduced CTGF as an unfavorable prognosis factor mediates the activation of miR-18b, an oncomir directly suppresses CTGF expression, by PI3K/AKT/C-Jun and C-Myc and promotes cell growth of NPC.

c-myc Amplification Is Frequent in Esophageal Adenocarcinoma and Correlated with the Upregulation of VEGF-A Expression

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Burkhard H.A. von Rahden

2006-09-01

Full Text Available BACKGROUND: Deregulation of c-myc plays a major role in the carcinogenesis of human malignancies. We investigated the amplification of the c-myc gene in a surgical series of Barrett cancers. METHODS: Primary resected esophageal (Barrett adenocarcinomas (n = 84 were investigated for c-myc amplification using chromogene in situ hybridization. Tumor samples were assembled in a tissue microarray. c-myc gene dosage was correlated with clinicopathologic parameters, including the survival and gene expression of cyclooxygenases (COX-1 and COX-2 and proangiogenic growth factors (VEGF-A and VEGF-C. RESULTS: The majority (70 of 84; 83.3% exhibited amplification of the c-myc gene. There were low-level amplifications in 63 (75.0% cases and high-level amplifications in 7 (8.3% cases. No amplification was found in 14 (16.7% cases. Tumors without c-myc amplification had lower VEGF-A, VEGF-C, and COX-2 expression levels than tumors with low-level and high-level c-myc amplification (statistically significant for VEGF-A; P = .0348. c-myc amplification was not correlated with clinicopathological parameters or survival. Only diffuse and mixed-type tumors, according to Lauren classification, exhibited c-myc amplifications more frequently (P = .0466. CONCLUSIONS: Amplifications of the c-myc gene are frequent in Barrett cancer. c-myc may be involved in the regulation of angiogenesis.

Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells

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Guo-Dan Liu

2017-04-01

Full Text Available AIM: To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs. METHODS: A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT and the nucleus localization signal (NLS polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3 Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. RESULTS: These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. CONCLUSION: The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies.

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Deng, Changchun; Lipstein, Mark R; Scotto, Luigi; Jirau Serrano, Xavier O; Mangone, Michael A; Li, Shirong; Vendome, Jeremie; Hao, Yun; Xu, Xiaoming; Deng, Shi-Xian; Realubit, Ronald B; Tatonetti, Nicholas P; Karan, Charles; Lentzsch, Suzanne; Fruman, David A; Honig, Barry; Landry, Donald W; O'Connor, Owen A

2017-01-05

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc-dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. © 2017 by The American Society of Hematology.

MYCT1-TV, A Novel MYCT1 Transcript, Is Regulated by c-Myc and May Participate in Laryngeal Carcinogenesis

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Fu, Shuang; Guo, Yan; Chen, Hong; Xu, Zhen-Ming; Qiu, Guang-Bin; Zhong, Ming; Sun, Kai-Lai; Fu, Wei-Neng

2011-01-01

Background MYCT1, a putative target of c-Myc, is a novel candidate tumor suppressor gene cloned from laryngeal squamous cell carcinoma (LSCC). Its transcriptional regulation and biological effects on LSCC have not been clarified. Methodology/Principal Findings Using RACE assay, we cloned a 1106 bp transcript named Myc target 1 transcript variant 1 (MYCT1-TV) and confirmed its transcriptional start site was located at 140 bp upstream of the ATG start codon of MYCT1-TV. Luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed c-Myc could regulate the promoter activity of MYCT1-TV by specifically binding to the E-box elements within −886 to −655 bp region. These results were further verified by site-directed mutagenesis and RNA interference (RNAi) assays. MYCT1-TV and MYCT1 expressed lower in LSCC than those in paired adjacent normal laryngeal tissues, and overexpression of MYCT1-TV and MYCT1 could inhibit cell proliferation and invasion and promote apoptosis in LSCC cells. Conclusions/Significance Our data indicate that MYCT1-TV, a novel MYCT1 transcript, is regulated by c-Myc and down-regulation of MYCT1-TV/MYCT1 could contribute to LSCC development and function. PMID:21998677

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo

NARCIS (Netherlands)

Phesse, T. J.; Myant, K. B.; Cole, A. M.; Ridgway, R. A.; Pearson, H.; Muncan, V.; van den Brink, G. R.; Vousden, K. H.; Sears, R.; Vassilev, L. T.; Clarke, A. R.; Sansom, O. J.

2014-01-01

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC

Troglitazone inhibits cell growth and induces apoptosis of B-cell acute lymphoblastic leukemia cells with t(14;18).

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Takenokuchi, M; Saigo, K; Nakamachi, Y; Kawano, S; Hashimoto, M; Fujioka, T; Koizumi, T; Tatsumi, E; Kumagai, S

2006-01-01

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear receptor superfamily, has been detected in several human leukemia cells. Recent studies reported that PPARgamma ligands inhibit cell proliferation and induce apoptosis in both normal and malignant B-lineage cells. We investigated the expression of PPARgamma and the effects of PPARgamma ligands on UTree-O2, Bay91 and 380, three B-cell acute lymphoblastic leukemia (B-ALL) cell lines with t(14;18), which show a poor prognosis, accompanying c-myc abnormality. Western blot analysis identified expression of PPARgamma protein and real-time PCR that of PPARgamma mRNA on the three cell lines. Troglitazone (TGZ), a synthetic PPARgamma ligand, inhibited cell growth in these cell lines in a dose-dependent manner, which was associated with G(1) cell cycle arrest and apoptosis. We also found this effect PPARgamma independent since PPARgamma antagonists failed to reverse this effect. We assessed the expression of c-myc, an apoptosis-regulatory gene, since c-myc abnormality was detected in most B-ALL cells with t(14;18). TGZ was found to dose-dependently downregulate the expression of c-myc mRNA and c-myc protein in the three cell lines. These results suggest that TGZ inhibits cell growth via induction of G(1) cell cycle arrest and apoptosis in these cell lines and that TGZ-induced apoptosis, at least in part, may be related to the downregulation of c-myc expression. Moreover, the downregulation of c-myc expression by TGZ may depend on a PPARgamma-independent mechanism. Further studies indicate that PPARgamma ligands may serve as a therapeutic agent in B-ALL with t(14;18).

The 5T mouse multiple myeloma model: Absence of c-myc oncogene rearrangement in early transplant generations

NARCIS (Netherlands)

Radl, J.; Punt, Y.A.; Enden-Vieveen, M.H.M. van den; Bentvelzen, P.A.J.; Bakkus, M.H.C.; Akker T., W. van den; Benner, R.

1990-01-01

Consistent chromosomal translocations involving the c-myc cellular oncogene and one of the three immunoglobulin loci are typical for human Burkitt's lymphoma, induced mouse plasmacytoma (MPC) and spontaneously arising rat immunocytoma (RIC). Another plasma cell malignancy, multiple myeloma (MM),

Low expression of c-Myc protein predicts poor outcomes in patients with hepatocellular carcinoma after resection.

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Ji, Fei; Zhang, Zhi-Heng; Zhang, Yi; Shen, Shun-Li; Cao, Qing-Hua; Zhang, Long-Juan; Li, Shao-Qiang; Peng, Bao-Gang; Liang, Li-Jian; Hua, Yun-Peng

2018-04-24

Embryonic Liver Fodrin (ELF) is an adaptor protein of transforming growth factor (TGF-β) signaling cascade. Disruption of ELF results in mislocalization of Smad3 and Smad4, leading to compromised TGF-β signaling. c-Myc is an important oncogenic transcription factor, and the disruption of TGF-β signaling promotes c-Myc-induced hepatocellular carcinoma (HCC) carcinogenesis. However, the prognostic significance of c-Myc in HCC is less understood METHODS: The expression of c-Myc protein and mRNA were measured by immunohistochemistry (IHC) and qRT- PCR, respectively. IHC was performed to detect TGF-β1 and ELF expression in HCC tissues. Their relationship with clinicopathological factors and overall survival (OS) and disease free survival (DFS) were examined. The expression of c-Myc protein and mRNA in HCC tissues were significantly higher in HCC area than those in normal liver tissues. However, the expression were low compared with those adjacent to HCC area. c-Myc protein was independently predictive of DFS and OS, and it was negatively correlated with tumor size (P = 0.031), tumor number (P = 0.038), and recurrence (P = 0.001). Low c-Myc expression was associated with short-term recurrence and poor prognosis. The predictive value of c-Myc combined with TGF-β1 or/and ELF was higher than that of any other single marker. Low c-Myc, high TGF-β1 or/and low ELF expression was associated with the worst DFS and OS. Low expression of c-Myc protein predicts poor outcomes in patients with HCC with hepatectomy. The combination of the expression of c-Myc, TGF-β1, and ELF can be used to accurately predict outcomes of patients with HCC.

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

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Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

1990-01-01

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

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Han, Yu; Zhong, Cuiping [Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China); Hong, Liu [First Division of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China); Wang, Ye; Qiao, Li [Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China); Qiu, Jianhua, E-mail: qiujh@fmmu.edu.cn [Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China)

2009-12-18

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

International Nuclear Information System (INIS)

Han, Yu; Zhong, Cuiping; Hong, Liu; Wang, Ye; Qiao, Li; Qiu, Jianhua

2009-01-01

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.

Transformation of follicular lymphoma to plasmablastic lymphoma with c-myc gene rearrangement.

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Ouansafi, Ihsane; He, Bing; Fraser, Cory; Nie, Kui; Mathew, Susan; Bhanji, Rumina; Hoda, Rana; Arabadjief, Melissa; Knowles, Daniel; Cerutti, Andrea; Orazi, Attilio; Tam, Wayne

2010-12-01

Follicular lymphoma (FL) is an indolent lymphoma that transforms to high-grade lymphoma, mostly diffuse large B-cell lymphoma, in about a third of patients. We present the first report of a case of FL that transformed to plasmablastic lymphoma (PBL). Clonal transformation of the FL to PBL was evidenced by identical IGH/BCL2 gene rearrangements and VDJ gene usage in rearranged IGH genes. IGH/ BCL2 translocation was retained in the PBL, which also acquired c-myc gene rearrangement. Genealogic analysis based on somatic hypermutation of the rearranged IGH genes of both FL and PBL suggests that transformation of the FL to PBL occurred most likely by divergent evolution from a common progenitor cell rather than direct evolution from the FL clone. Our study of this unusual case expands the histologic spectrum of FL transformation and increases our understanding of the pathogenetic mechanisms of transformation of indolent lymphomas to aggressive lymphomas.

Absence of DNA damage after 60-Hz electromagnetic field exposure combined with ionizing radiation, hydrogen peroxide, or c-Myc overexpression.

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Jin, Yeung Bae; Choi, Seo-Hyun; Lee, Jae Seon; Kim, Jae-Kyung; Lee, Ju-Woon; Hong, Seung-Cheol; Myung, Sung Ho; Lee, Yun-Sil

2014-03-01

The principal objective of this study was to assess the DNA damage in a normal cell line system after exposure to 60 Hz of extremely low frequency magnetic field (ELF-MF) and particularly in combination with various external factors, via comet assays. NIH3T3 mouse fibroblast cells, WI-38 human lung fibroblast cells, L132 human lung epithelial cells, and MCF10A human mammary gland epithelial cells were exposed for 4 or 16 h to a 60-Hz, 1 mT uniform magnetic field in the presence or absence of ionizing radiation (IR, 1 Gy), H(2)O(2) (50 μM), or c-Myc oncogenic activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic or additive effects were observed after 4 or 16 h of pre-exposure to 1 mT ELF-MF or simultaneous exposure to ELF-MF combined with IR, H(2)O(2), or c-Myc activation.

Perfusion of veins at arterial pressure increases the expression of KLF5 and cell cycle genes in smooth muscle cells

International Nuclear Information System (INIS)

Amirak, Emre; Zakkar, Mustafa; Evans, Paul C.; Kemp, Paul R.

2010-01-01

Vascular smooth muscle cell (VSMC) proliferation remains a major cause of veno-arterial graft failure. We hypothesised that exposure of venous SMCs to arterial pressure would increase KLF5 expression and that of cell cycle genes. Porcine jugular veins were perfused at arterial or venous pressure in the absence of growth factors. The KLF5, c-myc, cyclin-D and cyclin-E expression were elevated within 24 h of perfusion at arterial pressure but not at venous pressure. Arterial pressure also reduced the decline in SM-myosin heavy chain expression. These data suggest a role for KLF5 in initiating venous SMCs proliferation in response to arterial pressure.

Histopathological and molecular prognostic markers in medulloblastoma: c-myc, N-myc, TrkC, and anaplasia.

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Eberhart, Charles G; Kratz, John; Wang, Yunyue; Summers, Krista; Stearns, Duncan; Cohen, Kenneth; Dang, Chi V; Burger, Peter C

2004-05-01

Several molecular and histopathological prognostic markers have been proposed for the therapeutic stratification of medulloblastoma patients. Amplification of the c-myc oncogene, elevated levels of c-myc mRNA, or tumor anaplasia have been associated with worse clinical outcomes. In contrast, high TrkC mRNA expression generally presages longer survival. The goal of this study was to evaluate the prognostic value of c-myc, N-myc and TrkC expression in medulloblastomas and compare them to histopathological classification. We used in situ hybridization to measure expression of these molecular markers. c-myc mRNA was detected in 18 of 59 (31%) cases, and was significantly associated with shorter patient survival times on both univariate and multivariate analyses (p = 0.04). The presence of c-myc mRNA was also significantly associated with tumor anaplasia. While survival rates were higher for patients with low N-myc or high TrkC expression, these differences were not statistically significant. The group of patients with either moderate or severely anaplastic tumors showed only a trend towards shorter survival (p = 0.11). However, severe anaplasia alone was significantly prognostic (p = 0.002). Given the prognostic import of c-myc, we investigated 2 potential mechanisms by which its expression might be regulated: Wnt signaling and Mxi-1 mutation. Nuclear translocation of beta-catenin, a marker of Wnt pathway activation, was more common in medulloblastomas with high c-myc than in tumors overall, but the difference was not statistically significant. No Mxi-1 mutations were detected in the 22 cases examined. The association we describe between c-myc expression, tumor anaplasia, and worse clinical outcomes provides further evidence for the importance of this oncogene in medulloblastoma pathobiology.

Effect of JTV1 gene on the proliferation and apoptosis of K562 cells and its mechanism

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Yan WU

2011-05-01

Full Text Available Objective To investigate the effect of tumor-suppressing gene JTV1 on proliferation and apoptosis of leukemic K562 cells,and the changes in apoptosis factors Bcl-2,C-myc and Bax genes.Methods The recombinate vector pcDNA3.1-JTV1,and the empty vector pcDNA3.1 were transfected into K562 cells as control.The cell proliferation of K562 cells was evaluated by colony formation assay;the cell cycle and apoptosis rate were assessed by flow cytometry(FCM;the mRNA levels of apoptosis related genes Bax,Bcl-2 and C-myc were determined by RT-PCR;the protein levels of Bax,Bcl-2 and C-myc were assayed by Western Blotting.Results The colony formation assay showed that the proliferation of K562 cells decreased when the expression of JTV1 gene was up-regulated.FCM assay showed that the G phase cells in pcDNA3.1-JTV1 positive transfection group increased compared with that of the control group and the pcDNA3.1 empty vector transfected group,and the differences were statistically significant(P < 0.05.Compared with the control group and the empty vector group,the mRNA transcription level and the protein translation level of Bax gene increased significantly,and the mRNA transcription level and the protein translation level of Bcl-2 and C-myc gene were reduced significantly(P < 0.05.Conclusions The expressions of Bcl-2 and C-myc gene are inhibited when the gene JTV1 is up-regulated,leading to an increase in Bax gene expression,inhibition of K562 cell proliferation,and promotion of tumor cells apoptosis.Over expression of JTV1 gene can inhibit the proliferation of K562 cells and promote cell apoptosis by inhibiting Bcl-2 and C-myc expression and up-regulating that of Bax.

Overexpression and amplification of the c-myc gene in mouse tumors induced by chemical and radiations

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Niwa, Ohtsura; Enoki, Yoshitaka; Yokoro, Kenjiro

1989-03-01

We examined expression of the c-myc gene by the dot blot hybridization of total cellular RNA from mouse primary tumors induced by chemicals and radiations. Expression of the c-myc gene was found to be elevated in 69 cases among 177 independently induced tumors of 12 different types. DNA from tumors overexpressing the myc gene was analyzed by Southern blotting. No case of rearrangement was detected. However, amplification of the c-myc gene was found in 7 cases of primary sarcomas. These included 4 cases out of 24 methylcholanthrene-induced sarcomas and 3 cases out of 7 /alpha/-tocopherol-induced sacromas. We also analyzed 8 cases of sarcomas induced by radiations, but could not find changes in the gene structure of the c-myc gene. Thus, our data indicate tumor type specificity and agent specificity of c-myc gene amplification. (author).

AMPK promotes survival of c-Myc-positive melanoma cells by suppressing oxidative stress.

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Kfoury, Alain; Armaro, Marzia; Collodet, Caterina; Sordet-Dessimoz, Jessica; Giner, Maria Pilar; Christen, Stefan; Moco, Sofia; Leleu, Marion; de Leval, Laurence; Koch, Ute; Trumpp, Andreas; Sakamoto, Kei; Beermann, Friedrich; Radtke, Freddy

2018-03-01

Although c-Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras Q61K INK4a -/- mouse melanoma model to show that c-Myc is essential for tumor initiation, maintenance, and metastasis. c-Myc-expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c-Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c-Myc-positive melanoma cells activated and became dependent on the metabolic energy sensor AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c-Myc-expressing melanoma cells, while AMPK activation protected against cell death of c-Myc-depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early-stage human melanoma samples revealed an inverse correlation between C-MYC and patient survival, suggesting that C-MYC expression levels could serve as a prognostic marker for early-stage disease. © 2018 The Authors.

Expression Analysis of p16, c-Myc, and mSin3A in Non-small Cell Lung Cancer by Computer Aided Scoring and Analysis (CASA).

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Salmaninejad, Arash; Estiar, Mehrdad Asghari; Gill, Rajbir K; Shih, Joanna H; Hewitt, Stephen; Jeon, Hyo-Sung; Fukuoka, Junya; Shilo, Konstantin; Shakoori, Abbas; Jen, Jin

2015-01-01

Immunohistochemical analysis (IHC) of tissue microarray (TMA) slides enables large sets of tissue samples to be analyzed simultaneously on a single slide. However, manual evaluation of small cores on a TMA slide is time consuming and error prone. We describe a computer aided scoring and analysis (CASA) method to allow facile and reliable scoring of IHC staining using TMA containing 300 non-small cell lung cancer (NSCLC) cases. In the two previous published papers utilizing our TMA slides of lung cancer we examined 18 proteins involved in the chromatin machinery. We developed our study using more proteins of the chromatin complex and several transcription factors that facilitate the chromatin machinery. Then, a total of 78 antibodies were evaluated by CASA to derive a normalized intensity value that correlated with the overall staining status of the targeting protein. The intensity values for TMA cores were then examined for association to clinical variables and predictive significance individually and with other factors. RESULTs: Using our TMA, the intensity of several protein pairs were significantly correlated with an increased risk of death in NSCLC. These included c-Myc with p16, mSin3A with p16 and c-Myc with mSinA. Predictive values of these pairs remained significant when evaluated based on standard IHC scores. Our results demonstrate the usefulness of CASA as a valuable tool for systematic assessment of TMA slides to identify potential predictive biomarkers using a large set of primary human tissues.

Cdx1 and c-Myc foster the initiation of transdifferentiation of the normal esophageal squamous epithelium toward Barrett's esophagus.

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Douglas B Stairs

Full Text Available Barrett's esophagus is a premalignant condition whereby the normal stratified squamous esophageal epithelium undergoes a transdifferentiation program resulting in a simple columnar epithelium reminiscent of the small intestine. These changes are typically associated with the stratified squamous epithelium chronically exposed to acid and bile salts as a result of gastroesophageal reflux disease (GERD. Despite this well-defined epidemiologic association between acid reflux and Barrett's esophagus, the genetic changes that induce this transdifferentiation process in esophageal keratinocytes have remained undefined.To begin to identify the genetic changes responsible for transdifferentiaiton in Barrett's esophagus, we performed a microarray analysis of normal esophageal, Barrett's esophagus and small intestinal biopsy specimens to identify candidate signaling pathways and transcription factors that may be involved. Through this screen we identified the Cdx1 homeodomain transcription factor and the c-myc pathway as possible candidates. Cdx1 and c-myc were then tested for their ability to induce transdifferentiation in immortalized human esophageal keratinocytes using organotypic culturing methods. Analyses of these cultures reveal that c-myc and cdx1 cooperate to induce mucin production and changes in keratin expression that are observed in the epithelium of Barrett's esophagus.These data demonstrate the ability of Cdx1 and c-myc to initiate the earliest stages of transdifferentiation of esophageal keratinocytes toward a cell fate characteristic of Barrett's esophagus.

Evolutionarily conserved regions of the human c-myc protein can be uncoupled from transforming activity

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Sarid, J.; Halazonetis, T.D.; Murphy, W.; Leder, P.

1987-01-01

The myc family of oncogenes contains coding sequences that have been preserved in different species for over 400 million years. This conservation (which implies functional selection) is broadly represented throughout the C-terminal portion of the human c-myc protein but is largely restricted to three cluster of amino acid sequences in the N-terminal region. The authors have examined the role that the latter three regions of the c-myc protein might play in the transforming function of the c-myc gene. Several mutations, deletions and frameshifts, were introduced into the c-myc gene, and these mutant genes were tested for their ability to collaborate with the EJ-ras oncogene to transform rat embryo fibroblasts. Complete elimination of the first two N-terminal conserved segments abolished transforming activity. In contrast, genes altered in a portion of the second or the entire third conserved segment retained their transforming activity. Thus, the latter two segments are not required for the transformation process, suggesting that they serve another function related only to the normal expression of the c-myc gene

cAMP-Dependent Protein Kinase A (PKA)-Mediated c-Myc Degradation Is Dependent on the Relative Proportion of PKA-I and PKA-II Isozymes.

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Liu, Qingyuan; Nguyen, Eric; Døskeland, Stein; Ségal-Bendirdjian, Évelyne

2015-09-01

The transcription factor c-Myc regulates numerous target genes that are important for multiple cellular processes such as cell growth and differentiation. It is commonly deregulated in leukemia. Acute promyelocytic leukemia (APL) is characterized by a blockade of granulocytic differentiation at the promyelocyte stage. Despite the great success of all-trans retinoic acid (ATRA)-based therapy, which results in a clinical remission by inducing promyelocyte maturation, a significant number of patients relapse due to the development of ATRA resistance. A significant role has been ascribed to the cAMP/cAMP-dependent protein kinase A (PKA) signaling pathway in retinoid treatment since PKA activation is able to restore differentiation in some ATRA-resistant cells and eradicate leukemia-initiating cells in vivo. In this study, using NB4 APL cell variants resistant to ATRA-induced differentiation, we reveal distinct functional roles of the two PKA isozymes, PKA type I (PKA-I) and PKA-type II (PKA-II), on the steady-state level of c-Myc protein, providing a likely mechanism by which cAMP-elevating agents can restore differentiation in ATRA maturation-resistant APL cells. Therefore, both the inhibition of c-Myc activity and the PKA-I/PKA-II ratio should be taken into account if cAMP-based therapy is considered in the clinical management of APL. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Mitochondrial structure, function and dynamics are temporally controlled by c-Myc.

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J Anthony Graves

Full Text Available Although the c-Myc (Myc oncoprotein controls mitochondrial biogenesis and multiple enzymes involved in oxidative phosphorylation (OXPHOS, the coordination of these events and the mechanistic underpinnings of their regulation remain largely unexplored. We show here that re-expression of Myc in myc-/- fibroblasts is accompanied by a gradual accumulation of mitochondrial biomass and by increases in membrane polarization and mitochondrial fusion. A correction of OXPHOS deficiency is also seen, although structural abnormalities in electron transport chain complexes (ETC are not entirely normalized. Conversely, the down-regulation of Myc leads to a gradual decrease in mitochondrial mass and a more rapid loss of fusion and membrane potential. Increases in the levels of proteins specifically involved in mitochondrial fission and fusion support the idea that Myc affects mitochondrial mass by influencing both of these processes, albeit favoring the latter. The ETC defects that persist following Myc restoration may represent metabolic adaptations, as mitochondrial function is re-directed away from producing ATP to providing a source of metabolic precursors demanded by the transformed cell.

Induction of apoptosis by 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via modulation of MAPKs (p38 and c-Jun N-terminal kinase) and c-Myc in HL-60 human leukemia cells.

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Park, Eun-Jung; Kiselev, Evgeny; Conda-Sheridan, Martin; Cushman, Mark; Pezzuto, John M

2012-03-23

Recently, we reported that 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (AM6-36), sharing structural similarity with naturally occurring isoquinolines, induced activities mediated by retinoid X receptor (RXR) response element accompanied by antiproliferative effects on breast cancer cells. To further characterize the biologic potential of AM6-36, we currently report studies conducted with HL-60 human leukemia cells. AM6-36 significantly inhibited cellular proliferation in a dose- and time-dependent manner with an IC(50) value of 86 nM. When evaluated at low test concentrations (≤0.25 μM), AM6-36 induced arrest in the G2/M phase of the cell cycle. At higher concentrations (1 and 2 μM), the response shifted to apoptosis, which was consistent with the effect of AM6-36 on other apoptotic signatures including an increase of apoptotic annexin V(+) 7-AAD(-) cells, loss of mitochondrial membrane potential, induction of poly(ADP-ribose) polymerase cleavage, and activation of several caspases. These apoptotic effects are potentially due to up-regulation of p38 MAPK and JNK phosphorylation and down-regulation of c-Myc oncogene expression. Taken together, AM6-36 might serve as an effective anticancer agent by inducing G2/M cell cycle arrest and apoptosis through the activation of MAPKs and inhibition of c-Myc.

Vitamin K2 and cotylenin A synergistically induce monocytic differentiation and growth arrest along with the suppression of c-MYC expression and induction of cyclin G2 expression in human leukemia HL-60 cells.

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Maniwa, Yasuhisa; Kasukabe, Takashi; Kumakura, Shunichi

2015-08-01

Although all-trans retinoic acid (ATRA) is a standard and effective drug used for differentiation therapy in acute promyelocytic leukemia, ATRA-resistant leukemia cells ultimately emerge during this treatment. Therefore, the development of new drugs or effective combination therapy is urgently needed. We demonstrate that the combined treatment of vitamin K2 and cotylenin A synergistically induced monocytic differentiation in HL-60 cells. This combined treatment also synergistically induced NBT-reducing activity and non-specific esterase-positive cells as well as morphological changes to monocyte/macrophage-like cells. Vitamin K2 and cotylenin A cooperatively inhibited the proliferation of HL-60 cells in short-term and long-term cultures. This treatment also induced growth arrest at the G1 phase. Although 5 µg/ml cotylenin A or 5 µM vitamin K2 alone reduced c-MYC gene expression in HL-60 cells to approximately 45% or 80% that of control cells, respectively, the combined treatment almost completely suppressed c-MYC gene expression. We also demonstrated that the combined treatment of vitamin K2 and cotylenin A synergistically induced the expression of cyclin G2, which had a positive effect on the promotion and maintenance of cell cycle arrest. These results suggest that the combination of vitamin K2 and cotylenin A has therapeutic value in the treatment of acute myeloid leukemia.

Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

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Khodadad Khodadadi

2012-01-01

Full Text Available Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months and characterized. The equine iPS (EiPS cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression of OCT4, SOX2 NANOG, and STAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers both in vitro by embryoid bodies (EBs formation and in vivo by teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.

The Dysregulation of Polyamine Metabolism in Colorectal Cancer Is Associated with Overexpression of c-Myc and C/EBPβ rather than Enterotoxigenic Bacteroides fragilis Infection

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Anastasiya V. Snezhkina

2016-01-01

Full Text Available Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC. Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF. Bacterial enterotoxin activates spermine oxidase (SMO, which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPβ (CREBP, and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPβ may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPβ rather than ETBF infection.

Bcl-2 and N-Myc Coexpression Increases IGF-IR and Features of Malignant Growth in Neuroblastoma Cell Lines

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Rama Jasty

2001-01-01

Full Text Available The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB2, Bcl2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR.

Myeloblastic leukemia cells conditionally blocked by myc-estrogen receptor chimeric transgenes for terminal differentiation coupled to growth arrest and apoptosis.

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Selvakumaran, M; Liebermann, D; Hoffman-Liebermann, B

1993-05-01

Conditional mutants of the myeloblastic leukemic M1 cell line, expressing the chimeric mycer transgene, have been established. It is shown that M1 mycer cells, like M1, undergo terminal differentiation coupled to growth arrest and programmed cell death (apoptosis) after treatment with the physiologic differentiation inducer interleukin-6. However, when beta-estradiol is included in the culture medium, M1 mycer cells respond to differentiation inducers like M1 myc cell lines, where the differentiation program is blocked at an intermediate stage. By manipulating the function of the mycer transgene product, it is shown that there is a 10-hour window during myeloid differentiation, from 30 to 40 hours after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, where activation of c-myc has no apparent effect on mature macrophages. M1 mycer cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal myelopoiesis and in leukemogenesis, also providing a strategy to clone c-myc target genes.

Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.

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Leonetti, C; Biroccio, A; Benassi, B; Stringaro, A; Stoppacciaro, A; Semple, S C; Zupi, G

2001-06-01

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

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Fabiana Salm

Full Text Available Medulloblastoma (MB is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

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Ono, Junya; Shime, Hiroaki; Takaki, Hiromi; Takashima, Ken; Funami, Kenji; Yoshida, Sumito; Takeda, Yohei; Matsumoto, Misako; Kasahara, Masanori; Seya, Tsukasa

2017-10-17

Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b + and CD8α + cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.

Influence of x-rays and UV-light on the presence of oncogene proteins in spleen cells of leukemic mice

International Nuclear Information System (INIS)

Popovic Hadzija, M.; Poljak Blazi, M.

1996-01-01

Proto-oncogenes are involved in growth, defferentiation and proliferation of normal cells, and in process of neoplastic transformation. In genome of normal cells, exist also tumor-suppressor genes, which contribute to cancer when they are inactivated. Those genes are target for carcinogenesis provoked by radiation. However, species specific genetic factors are important in determing which, if any, gene will be transformed by radiation. It is possible to presume that oncogenes are involved in the development of radioresistant phenotype of ML. Because of that, we examined the presence of c-myc protein in ML cells during the growth of ML and after the irradiation of these cells. Also, we examined the presence of tumor-suppressor protein p53, because inactivation or loss of p53 gene is in connection with transformation of cells. ML is strain specific for RFM mice. Spleen cells were tested 9 (nonterminal phase NTP) or 12 days (terminal phase TP) after inoculation of ML. Cells from NTP were also irradiate with x-rays or UV-light. C-myc protein expresse 74.98% spleen cells of healthy RFM mice. Wild type of p53 protein was detected in 60% of these cells, but mp53 was found in only 5.3% of cells. These results could be explained by the role of c-myc and p53 proteins in regulation of biologic processes. A few spleen cells of NTP expressed c-myc (15%) and mp53 (9.6%) proteins. But, in the same phase higher expressions of wp53 protein (30.5%) was found. On the other hand, the number of c-myc positive cells in TP of leukemia explanation lies in connection of c-myc protein and process of programmed cell death (apoptosis). During growth of ML the number of mp53 positive cells increased (to 47.8%), but wp53 positive cells decreased (to13.4%9). Both types of irradiation provoked strong activation of cellular c-myc gene in ML cells of NTP. We found about 95% c-myc positive cells after x-rays and 93% after UV-light

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

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Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Disruption of MEK/ERK/c-Myc signaling radiosensitizes prostate cancer cells in vitro and in vivo.

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Ciccarelli, Carmela; Di Rocco, Agnese; Gravina, Giovanni Luca; Mauro, Annunziata; Festuccia, Claudio; Del Fattore, Andrea; Berardinelli, Paolo; De Felice, Francesca; Musio, Daniela; Bouché, Marina; Tombolini, Vincenzo; Zani, Bianca Maria; Marampon, Francesco

2018-06-29

Prostate cancer (PCa) cell radioresistance causes the failure of radiation therapy (RT) in localized or locally advanced disease. The aberrant accumulation of c-Myc oncoprotein, known to promote PCa onset and progression, may be due to the control of gene transcription and/or MEK/ERK-regulated protein stabilization. Here, we investigated the role of MEK/ERK signaling in PCa. LnCAP, 22Rv1, DU145, and PC3 PCa cell lines were used in in vitro and in vivo experiments. U0126, trametinib MEK/ERK inhibitors, and c-Myc shRNAs were used. Radiation was delivered using an x-6 MV photon linear accelerator. U0126 in vivo activity alone or in combination with irradiation was determined in murine xenografts. Inhibition of MEK/ERK signaling down-regulated c-Myc protein in PCa cell lines to varying extents by affecting expression of RNA and protein, which in turn determined radiosensitization in in vitro and in vivo xenograft models of PCa cells. The crucial role played by c-Myc in the MEK/ERK pathways was demonstrated in 22Rv1 cells by the silencing of c-Myc by means of short hairpin mRNA, which yielded effects resembling the targeting of MEK/ERK signaling. The clinically approved compound trametinib used in vitro yielded the same effects as U0126 on growth and C-Myc expression. Notably, U0126 and trametinib induced a drastic down-regulation of BMX, which is known to prevent apoptosis in cancer cells. The results of our study suggest that signal transduction-based therapy can, by disrupting the MEK/ERK/c-Myc axis, reduce human PCa radioresistance caused by increased c-Myc expression in vivo and in vitro and restores apoptosis signals.

[Relationship between the expression of beta-cat, cyclin D1 and c-myc and the occurance and biological behavior of pancreatic cancer].

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Li, Yu-jun; Ji, Xiang-rui

2003-06-01

To study the relationship between the abnormal expression of beta-catenin (beta-cat) and the high expressions of cyclin D1 and c-myc and the occurance, proliferation, infiltration, metastasis and prognosis of pancreatic cancer, and to provide rational basis for the clinical diagnosis and treatment. Immunohistochemical PicTure trade mark was used to examine the expressions of beta-cat, cyclin D1 and c-myc in 47 cases of the cancerous tissue of pancreas, 12 cases of the pancreatic intraepithelial neoplasia and 10 cases of normal tissue of pancreas, respectively. Pancreatic cancer proliferation cell nuclear antigen (PCNA) was also tested as the index of the extent of proliferation of the pancreatic cancer. beta-cat was expressed normally in the 10 cases of the normal pancreatic tissue, while cyclin D1 and c-myc were negative. The expression rates of beta-cat, cyclin D1 and c-myc in the tissues of the pancreatic intraepithelial neoplasia and the pancreatic cancer had no significant difference [6/12 and 68.1% (32/47), 6/12 and 74.5% (35/47), 5/12 and 70.2% (33/47) respectively;P values were all more than 0.05]. The abnormal expression rate of beta-cat was significantly correlated to the metastasis of the pancreatic cancer and the one-year survival rate (both P 0.05). The expression rate of cyclin D1 was correlated with the proliferation of the pancreatic cancer and the extent of differentiation (both P 0.05). The expression rate of c-myc was not correlated with the size, the extent of proliferation, infiltration, metastasis, or one-year survival rate (both P > 0.05), but closely with the proliferation activity of the cancerous tissue of pancreas (P < 0.05). The abnormal expression of beta-cat and the high expressions of cyclin D1 and c-myc had a parallel relationship with the pancreatic intraepithelial neoplasia and pancreatic cancer (both P < 0.05, gamma = 1.000, 0.845, 0.437, 0.452). The abnormal expression of beta-cat activates cyclin D1 and c-myc, and results in the

Lanatoside C inhibits cell proliferation and induces apoptosis through attenuating Wnt/β-catenin/c-Myc signaling pathway in human gastric cancer cell.

Science.gov (United States)

Hu, Yudong; Yu, Kaikai; Wang, Gang; Zhang, Depeng; Shi, Chaoji; Ding, Yunhe; Hong, Duo; Zhang, Dan; He, Huiqiong; Sun, Lei; Zheng, Jun-Nian; Sun, Shuyang; Qian, Feng

2018-04-01

Gastric cancer is the third common cause of cancer mortality in the world with poor prognosis and high recurrence due to lack of effective medicines. Our studies revealed that lanatoside C, a FDA-approved cardiac glycoside, had an anti-proliferation effect on different human cancer cell lines (MKN-45; SGC-7901; HN4; MCF-7; HepG2) and gastric cell lines MKN-45 and SGC-7901 were the most sensitive cell lines to lanatoside C. MKN-45 cells treated with lanatoside C showed cell cycle arrest at G2/M phase and inhibition of cell migration. Meanwhile, upregulation of cleaved caspase-9 and cleaved PARP and downregulation of Bcl-xl were accompanied with the loss of mitochondrial membrane potential (MMP) and induction of intracellular reactive oxygen species (ROS). Lanatoside C inhibited Wnt/β-catenin signaling with downregulation of c-Myc, while overexpression of c-Myc reversed the anti-tumor effect of lanatoside C, confirming that c-Myc is a key drug target of lanatoside C. Furthermore, we discovered that lanatoside C prompted c-Myc degradation in proteasome-ubiquitin pathway with attenuating the binding of USP28 to c-Myc. These findings indicate that lanatoside C targeted c-Myc ubiquitination to inhibit MKN-45 proliferation and support the potential value of lanatoside C as a chemotherapeutic candidate. Copyright © 2018 Elsevier Inc. All rights reserved.

c-Myc oncogene expression in selected odontogenic cysts and tumors: An immunohistochemical study

Science.gov (United States)

Moosvi, Zama; Rekha, K

2013-01-01

Aim: To investigate the role of c-Myc oncogene in selected odontogenic cysts and tumors. Materials and Methods: Ten cases each of ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst, and radicular cyst were selected and primary monoclonal mouse anti-human c-Myc antibody was used in a dilution of 1: 50. Statistical Analysis was performed using Mann Whitney U test. Results: 80% positivity was observed in ameloblastoma, AOT and OKC; 50% positivity in radicular cyst and 20% positivity in dentigerous cyst. Comparison of c-Myc expression between ameloblastoma and AOT did not reveal significant results. Similarly, no statistical significance was observed when results of OKC were compared with ameloblastoma and AOT. In contrast, significant differences were seen on comparison of dentigerous cyst with ameloblastoma and AOT and radicular cyst with AOT. Conclusion: From the above data we conclude that (1) Ameloblastoma and AOT have similar proliferative potential and their biologic behavior cannot possibly be attributed to it. (2) OKC has an intrinsic growth potential which is absent in other cysts and reinforces its classification as keratocystic odontogenic tumor. PMID:23798830

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

Energy Technology Data Exchange (ETDEWEB)

Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Noritake, Hidenao [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kimura, Wataru; Wu, Yi-Xin [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kobayashi, Yoshimasa [Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Uezato, Tadayoshi [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Miura, Naoyuki, E-mail: nmiura@hama-med.ac.jp [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan)

2012-01-06

Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

Immunodetection of rasP21 and c-myc oncogenes in oral mucosal swab preparation from clove cigarette smokers

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Silvi Kintawati

2008-12-01

Full Text Available Background: Smoking is the biggest factor for oral cavity malignancy. Some carcinogens found in cigar will stimulate epithel cell in oral cavity and cause mechanism disturbance on tissue resistance and produce abnormal genes (oncogenes. Oncogenes ras and myc are found on malignant tumor in oral cavity which are associated with smoking. Purpose: This research is to find the expression of oncogenes rasP21 and c-myc in oral mucosa epithelial of smoker with immunocytochemistry reaction. Methods: An oral mucosal swab was performed to 30 smokers categorized as light, moderate, and chain, and 10 non smokers which was followed by immunocytochemistry reaction using antibody towards oncogene rasP21 and c-myc is reacted to identify the influence of smoking towards malignant tumor in oral cavity. The result is statistically analyzed using Kruskal-Wallis test. Result: Based on the observation result of oncogene rasP21reaction, it shows that there is significant difference between non smoker group and light smoker, compared to moderate and chain smoker group (p < 0.01. On the other side, the observation result of oncogene c-myc indicates that there is no significant difference between the group of non smokers and the group of light, moderate, and chain smokers (p > 0.05. Conclusion: The higher the possibility of oral cavity malignancy and that the antibody for rasP21 oncogene can be used as a marker for early detection of oral cavity malignancy caused by smoking.

Cooverexpression of EpCAM and c-myc genes in malignant breast

Indian Academy of Sciences (India)

oncogene, affects progression, treatment, and diagnosis of many adenocarcinomas. C-myc has been shown to be a downstream target of EpCAM and is also one of the most important proto-oncogenes routinely overexpressed in breast cancer.

Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

DEFF Research Database (Denmark)

Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

1993-01-01

A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc......-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G2+M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had...

Targeting C-Myc Promoter: Helquats As Novel G-Quadruplex Stabilizing Ligands

Czech Academy of Sciences Publication Activity Database

Kužmová, Erika; Kozák, Jaroslav; Komárková, Veronika; Pytlík, R.; Teplý, Filip; Hájek, Miroslav

2014-01-01

Roč. 124, č. 21 (2014) ISSN 0006-4971. [Annual Meeting of the American Society of Hematology /56./. 06.12.2014-09.12.2014, San Francisco] Institutional support: RVO:61388963 Keywords : helquats * C-Myc * leukemia Subject RIV: CE - Biochemistry

Acquired Tumor Cell Radiation Resistance at the Treatment Site Is Mediated Through Radiation-Orchestrated Intercellular Communication

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Aravindan, Natarajan, E-mail: naravind@ouhsc.edu [Department of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (United States); Aravindan, Sheeja; Pandian, Vijayabaskar; Khan, Faizan H.; Ramraj, Satish Kumar; Natt, Praveen [Department of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (United States); Natarajan, Mohan [Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas (United States)

2014-03-01

Purpose: Radiation resistance induced in cancer cells that survive after radiation therapy (RT) could be associated with increased radiation protection, limiting the therapeutic benefit of radiation. Herein we investigated the sequential mechanistic molecular orchestration involved in radiation-induced radiation protection in tumor cells. Results: Radiation, both in the low-dose irradiation (LDIR) range (10, 50, or 100 cGy) or at a higher, challenge dose IR (CDIR), 4 Gy, induced dose-dependent and sustained NFκB-DNA binding activity. However, a robust and consistent increase was seen in CDIR-induced NFκB activity, decreased DNA fragmentation, apoptosis, and cytotoxicity and attenuation of CDIR-inhibited clonal expansion when the cells were primed with LDIR prior to challenge dose. Furthermore, NFκB manipulation studies with small interfering RNA (siRNA) silencing or p50/p65 overexpression unveiled the influence of LDIR-activated NFκB in regulating CDIR-induced DNA fragmentation and apoptosis. LDIR significantly increased the transactivation/translation of the radiation-responsive factors tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), cMYC, and SOD2. Coculture experiments exhibit LDIR-influenced radiation protection and increases in cellular expression, secretion, and activation of radiation-responsive molecules in bystander cells. Individual gene-silencing approach with siRNAs coupled with coculture studies showed the influence of LDIR-modulated TNF-α, IL-1α, cMYC, and SOD2 in induced radiation protection in bystander cells. NFκB inhibition/overexpression studies coupled with coculture experiments demonstrated that TNF-α, IL-1α, cMYC, and SOD2 are selectively regulated by LDIR-induced NFκB. Conclusions: Together, these data strongly suggest that scattered LDIR-induced NFκB-dependent TNF-α, IL-1α, cMYC, and SOD2 mediate radiation protection to the subsequent challenge dose in tumor cells.

Function of oval cells in hepatocellular carcinoma in rats.

Science.gov (United States)

Fang, Chi-Hua; Gong, Jia-Qing; Zhang, Wei

2004-09-01

To study oval cells' pathological characteristics and relationship with the occurrence of hepatocellular carcinoma (HCC); to observe the form and structural characteristics of oval cells; to explore the expression characteristics of C-kit, PCNA mRNA and c-myc gene during the occurrence and development of HCC and the effect of ulinastatin (UTI) on C-kit and PCNA expression. One hundred and twenty-five SD rats fed on 3,3'-diaminobenzidine (DAB) to construct HCC models were divided into control group, cancer-inducing group and UTI intervention group. In each group, rat liver samples were collected at weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 respectively to study pathological distribution characteristics of oval cells in the process of carcinogenesis under optical microscope. Oval cells were separated by the methods of improved density gradient centrifugation and their structural characteristics were observed under optical microscope and electronic microscope respectively; the oval cells expressing C-kit and PCNA in the collected samples were observed by the methods of immunohistochemistry and image analysis and the expression of c-myc mRNA was also detected by reverse transcription polymerase chain reaction (RT-PCR). Oval cells proliferated firstly in the portal area then gradually migrated into hepatic parenchyma in the inducing group and intervention group. The oval cells distributed inside and outside the carcinoma nodes. The oval cells presented the characteristics of undifferentiated cells: a high ratio of nucleolus and cellular plasm and obvious nucleoli, rare organelle in plasm. Only a few mitochondria and endoplasmic reticulum and some villus-like apophysis on surface of cells could be seen. Cells stained with C-kit and PCNA antibody were mainly oval cells distributed in the portal area. The expression of c-myc mRNA increased with the progression of HCC. However, in the intervention group, UTI could retard its increase. Oval cells work throughout

USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

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Zhenghong Lin

2013-12-01

Full Text Available The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

Profil Ekspresi mRNA Gen Murine Double Minute2, Kruppel-Like Factor4, dan c-Myc pada Fibrosarkoma

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- Humaryanto

2017-02-01

Full Text Available Abstrak Fibrosarkoma hanya terjadi 1–3% dari seluruh keganasan jaringan lunak. Hingga saat ini etiologi fibrosarkoma belum diketahui dengan pasti. Beberapa faktor dapat menjadi penyebab patogenesis fibrosarkoma antara lain radiasi, terpapar zat kimia tertentu, serta infeksi human herpes virus 8 (HHV8 dan Epstein-Barr virus (EBV. Penelitian terkini menunjukkan bahwa banyak sarkoma terkait dengan mutasi genetik. Penelitian ini bertujuan melihat profil ekspresi mRNA gen Krüppel-like Factor4, Murine Double Minute2, dan c-Myc pada fibrosarkoma menggunakan teknik real time PCR kuantitatif (quantitative real time PCR, qRT-PCR. Analisis data menggunakan metode kuantititatif relatif 2-ΔΔCt. Penelitian ini menggunakan 10 sampel kasus fibrosarkoma yang ditemukan di Kota Jambi dari tahun 2011–2015. Hasil ΔCt (+SD MDM2, KLF-4, dan c-Myc disusun dari nilai yang terkecil hingga tertinggi adalah 1,85±2,14; 2,06±3,86; 2,9±2,66 secara berurutan. Dibanding dengan level ekspresi dengan GAPDH sebagai housekeeping gene, gen MDM2 dan KLF-4 relatif menurun dua kali lipat, sedangkan gen c-Myc relatif menurun lebih dari tiga kali lipat. Simpulan, penelitian ini menunjukkan bahwa pada kasus fibrosarkoma, gen c-Myc disupresi lebih kuat dibanding dengan gen MDM2 dan KLF-4. Abstract Fibrosarcoma is a rare soft tissue sarcoma, reported only 1–3% of all soft tissue sarcomas. Like any other soft-tissue sarcomas the definitive caused has not yet understood. Recognized causes include exposure to ionizing radiation, various physical and chemical factors, infection with human herpes virus (HHV8 and Epstein-Barr virus (EBV. Current research indicates many sarcomas are associated with genetic mutations. In this study, we investigated profile of mRNA gene expression KLF4, MDM2, and c-Myc of RNA in fibrosarcoma cases. The genes expression was examined using quantitative real time PCR (qRT-PCR and we analyzed the relative gene expression using the 2-ΔΔCt method. Ten

Localisation of lung cancer by a radiolabelled monoclonal antibody against the c-myc oncogene product

Energy Technology Data Exchange (ETDEWEB)

Chan, S Y.T.; Evan, G I; Ritson, A; Watson, J; Wraight, P; Sikora, K

1986-11-01

A set of mouse monoclonal antibodies against the c-myc oncogene product, a 62,000 dalton nuclear binding protein involved in cell cycle control, has been constructed by immunisation with synthetic peptide fragments. One such antibody, CT14, was radiolabelled with /sup 131/I and administered to 20 patients with different malignant diseases. Good tumour localisation was observed in 12 out of 14 patients with primary bronchial carcinoma but not in patients with pulmonary metastases from primary tumours elsewhere. Successfully localised tumours were all 3 cm or more in diameter. Monoclonal antibodies against oncogene products may provide novel selective tools for the diagnosis and therapy of cancer.

Detecção imunoistoquímica das oncoproteínas p21ras, c-myc E p53 no carcinoma hepatocelular e no tecido hepático não-neoplásico Immunohistochemical detection of p21ras, c-myc and p53 oncoproteins in hepatocellular carcinoma and in non-neoplastic liver tissue

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Vera Lucia Nunes Pannain

2004-12-01

Full Text Available RACIONAL: A hepatocarcinogênese é um processo no qual as alterações genéticas e epigenéticas são bem conhecidas em modelos animais, mas carece de estudos no homem. OBJETIVOS: Analisar a freqüência das oncoproteínas p21ras, c-myc e p53 no carcinoma hepatocelular e no fígado não-neoplásico. Verificar ainda a associação destas oncoproteínas com os padrões e graus histológicos, assim como com as infecções pelos vírus das hepatites B e C. MÉTODOS: Foi analisada por método imunoistoquímico a detecção das oncoproteínas p21ras, c-myc e p53 em 47 casos de carcinoma hepatocelular e no tecido não-neoplásico circunjacente ao tumor (40 casos. RESULTADOS: As oncoproteínas p21ras, c-myc e p53 foram detectadas, respectivamente, em 44,7%, 53,2% e 36,2% dos casos de carcinoma hepatocelular. A imunorreatividade do p21ras e c-myc mostrou uma associação significativa. Contudo, não houve associação significativa entre a detecção do p21ras, c-myc e p53 com os diferentes graus e padrões histológicos, nem tampouco com as infecções pelos vírus das hepatites B e C. A mesma associação significativa entre o p21ras e c-myc foi encontrada no tecido não-neoplásico dos casos de cirrose em relação aos que não apresentaram cirrose, enquanto que o p53 foi negativo em todos os casos. CONCLUSÕES: A imunorreatividade das oncoproteínas p21ras, c-myc e p53 corrobora evidências prévias de sua detecção no carcinoma hepatocelular, o que sugere poder haver participação destas proteínas na hepatocarcinogênese humana. A significativa associação entre as proteínas p21ras, c-myc e p53 no carcinoma hepatocelular e na cirrose pode apontar uma interação entre as mesmas, sobretudo na hepatocarcinogênese pela via da cirrose.BACKGROUND: Genetic and epigenetic alterations have been described in animal hepatocarcinogenesis models but need to be studied in human being. AIMS: To assess the immunoreactivity of p21ras, c-myc and p53

Controlled and localized delivery of c-myc AS-ODN to cells by 3-aminopropyl-trimethoxylsilane modified SBA-15 mesoporous silica

Science.gov (United States)

Zhang, Juan; Chen, Minmin; Zhao, Xiqiu; Zhang, Min; Mao, Jinxiang; Cao, Xichuan; Zhang, Zhuoqi

2018-01-01

SBA-15 mesoporous silicate was synthesized and functionalized with 3-aminopropyl organic groups through a post-synthesis method. The materials were characterized consecutively by powder X-ray diffraction (XRD), N2 adsorption/desorption analysis and solid-state magic-angle spinning 29Si nuclear magnetic resonance (MAS NMR). Human c-myc anti-sense oligodeoxyneucleotide (AS-ODN) was selected as a model molecule to be loaded onto the surface of bare and functionalized SBA-15 via different loading conditions. It has been found that the amount of AS-ODN incorporated into the porous matrix is strongly dependent on the surface properties, pH of the loading solvent and AS-ODN concentration. The release behaviour of AS-ODN from modified SBA-15 materials was also investigated and depended on conditions chosen. Cellular uptake of the eluted AS-ODN into Hela cells was observed by fluorescent microscopy. The materials showed excellent cytocompatibility. The AS-ODN keeps full transfection and expression activities indicating its structural integrity. The functionalized SBA-15 is an excellent prospect as a biomedical material candidate for the future.

Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

Science.gov (United States)

Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

1997-07-18

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde

Directory of Open Access Journals (Sweden)

Lee Jae-Won

2011-04-01

Full Text Available Abstract Background Tumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL. Results PC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB, a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer. Conclusion This study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and

Genomic amplification patterns of human telomerase RNA gene and C-MYC in liquid-based cytological specimens used for the detection of high-grade cervical intraepithelial neoplasia

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Chen Shaomin

2012-04-01

Full Text Available Abstract Background The amplification of oncogenes initiated by high-risk human papillomavirus (HPV infection is an early event in cervical carcinogenesis and can be used for cervical lesion diagnosis. We measured the genomic amplification rates and the patterns of human telomerase RNA gene (TERC and C-MYC in the liquid-based cytological specimens to evaluate the diagnostic characteristics for the detection of high-grade cervical lesions. Methods Two hundred and forty-three residual cytological specimens were obtained from outpatients aged 25 to 64 years at Qilu Hospital, Shandong University. The specimens were evaluated by fluorescence in situ hybridization (FISH using chromosome probes to TERC (3q26 and C-MYC (8q24. All of the patients underwent colposcopic examination and histological evaluation. A Chi-square test was used for categorical data analysis. Results In the normal, cervical intraepithelial neoplasia grade 1 (CIN1, grade 2 (CIN2, grade 3 (CIN3 and squamous cervical cancer (SCC cases, the TERC positive rates were 9.2%, 17.2%, 76.2%, 100.0% and 100.0%, respectively; the C-MYC positive rates were 20.7%, 31.0%, 71.4%, 81.8% and 100.0%, respectively. The TERC and C-MYC positive rates were higher in the CIN2+ (CIN2, CIN3 and SCC cases than in the normal and CIN1 cases (p p p > 0.05. Conclusions The TERC test is highly sensitive and is therefore suitable for cervical cancer screening. The C-MYC test is not suitable for cancer screening because of its lower sensitivity. The amplification patterns of TERC become more diverse and complex as the severity of cervical diseases increases, whereas for C-MYC, the amplification patterns are similar between the normal/CIN1 and CIN2+ groups. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1308004512669913.

Increased expression of bHLH transcription factor E2A (TCF3) in prostate cancer promotes proliferation and confers resistance to doxorubicin induced apoptosis

International Nuclear Information System (INIS)

Patel, Divya; Chaudhary, Jaideep

2012-01-01

Highlights: ► E2A, considered as a tumor suppressor is highly expressed in prostate cancer. ► Silencing of E2A attenuates cell proliferation and promotes apoptosis. ► E2A regulates c-myc, Id1, Id3 and CDKN1A expression. ► Loss of E2A promotes doxorubicin dependent apoptosis in prostate cancer cells. ► Results suggest that E2A acts as a tumor promoter at least in prostate cancer. -- Abstract: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH), transcription factor. E2A regulates transcription of target genes by homo- or heterodimerization with cell specific bHLH proteins. In general, E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Given the diverse biological pathways regulated/influenced by E2A little is known about its expression in cancer. In this study we investigated the expression of E2A in prostate cancer. Unexpectedly, E2A immuno-histochemistry demonstrated increased E2A expression in prostate cancer as compared to normal prostate. Silencing of E2A in prostate cancer cells DU145 and PC3 led to a significant reduction in proliferation due to G1 arrest that was in part mediated by increased CDKN1A(p21) and decreased Id1, Id3 and c-myc. E2A silencing in prostate cancer cell lines also resulted in increased apoptosis due to increased mitochondrial permeability and caspase 3/7 activation. Moreover, silencing of E2A increased sensitivity to doxorubicin induced apoptosis. Based on our results, we propose that E2A could be an upstream regulator of Id1 and c-Myc which are highly expressed in prostate cancer. These results for the first time demonstrate that E2A could in fact acts as a tumor promoter at least in prostate cancer.

TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein

NARCIS (Netherlands)

Hateboer, G.; Timmers, H.T.M.; Rustgi, A.K.; Billaud, Marc; Veer, L.J. Van 't; Bernards, R.A.

1993-01-01

Using a protein binding assay, we show that the amino-teminal 204 amino acids of the c-Myc protein interact di y with a key component of the basal p tdon factor TFID, the TATA box-binding protein (TBP). Essentialy the same region of the c-Myc protein alo binds the product of the retinoblatoma

The human T-cell leukemia virus type-1 p30II protein activates p53 and induces the TIGAR and suppresses oncogene-induced oxidative stress during viral carcinogenesis.

Science.gov (United States)

Romeo, Megan; Hutchison, Tetiana; Malu, Aditi; White, Averi; Kim, Janice; Gardner, Rachel; Smith, Katie; Nelson, Katherine; Bergeson, Rachel; McKee, Ryan; Harrod, Carolyn; Ratner, Lee; Lüscher, Bernhard; Martinez, Ernest; Harrod, Robert

2018-05-01

In normal cells, aberrant oncogene expression leads to the accumulation of cytotoxic metabolites, including reactive oxygen species (ROS), which can cause oxidative DNA-damage and apoptosis as an intrinsic barrier against neoplastic disease. The c-Myc oncoprotein is overexpressed in many lymphoid cancers due to c-myc gene amplification and/or 8q24 chromosomal translocations. Intriguingly, p53 is a downstream target of c-Myc and hematological malignancies, such as adult T-cell leukemia/lymphoma (ATL), frequently contain wildtype p53 and c-Myc overexpression. We therefore hypothesized that p53-regulated pro-survival signals may thwart the cell's metabolic anticancer defenses to support oncogene-activation in lymphoid cancers. Here we show that the Tp53-induced glycolysis and apoptosis regulator (TIGAR) promotes c-myc oncogene-activation by the human T-cell leukemia virus type-1 (HTLV-1) latency-maintenance factor p30 II , associated with c-Myc deregulation in ATL clinical isolates. TIGAR prevents the intracellular accumulation of c-Myc-induced ROS and inhibits oncogene-induced cellular senescence in ATL, acute lymphoblastic leukemia, and multiple myeloma cells with elevated c-Myc expression. Our results allude to a pivotal role for p53-regulated antioxidant signals as mediators of c-Myc oncogenic functions in viral and non-viral lymphoid tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in ApcMin/+ mice

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Brudvik Kristoffer W

2011-12-01

Full Text Available Abstract Background The adenomatous polyposis coli (APC protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E2 (PGE2 - PI-3 kinase pathways. Recent reports show that PGE2-induced phosphorylation of β-catenin by protein kinase A (PKA increases nuclear translocation indicating two mechanisms of action of PGE2 on β-catenin homeostasis. Findings Treatment of ApcMin/+ mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116 revealed that Rp-8-Br-cAMPS blocked PGE2-induced β-catenin phosphorylation and c-Myc upregulation. Conclusion Based on our findings we suggest that PGE2 act through PKA to promote β-catenin nuclear translocation and tumor development in ApcMin/+ mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

Profiling and bioinformatic analysis of circular RNA expression regulated by c-Myc.

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Gou, Qiheng; Wu, Ke; Zhou, Jian-Kang; Xie, Yuxin; Liu, Lunxu; Peng, Yong

2017-09-22

The c-Myc transcription factor is involved in cell proliferation, cell cycle and apoptosis by activating or repressing transcription of multiple genes. Circular RNAs (circRNAs) are widely expressed non-coding RNAs participating in the regulation of gene expression. Using a high-throughput microarray assay, we showed that Myc regulates the expression of certain circRNAs. A total of 309 up- and 252 down-regulated circRNAs were identified. Among them, randomly selected 8 circRNAs were confirmed by real-time PCR. Subsequently, Myc-binding sites were found to generally exist in the promoter regions of differentially expressed circRNAs. Based on miRNA sponge mechanism, we constructed circRNAs/miRNAs network regulated by Myc, suggesting that circRNAs may widely regulate protein expression through miRNA sponge mechanism. Lastly, we took advantage of Gene Ontology and KEGG analyses to point out that Myc-regulated circRNAs could impact cell proliferation through affecting Ras signaling pathway and pathways in cancer. Our study for the first time demonstrated that Myc transcription factor regulates the expression of circRNAs, adding a novel component of the Myc tumorigenic program and opening a window to investigate the function of certain circRNAs in tumorigenesis.

NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III1

International Nuclear Information System (INIS)

Dexheimer, Thomas S.; Carey, Steven S.; Zuohe, Song; Gokhale, Vijay M.; Hu, Xiaohui; Murata, Lauren B.; Maes, Estelle M.; Weichsel, Andrzej; Sun, Daekyu; Meuillet, Emmanuelle J.; Montfort, William R.; Hurley, Laurence H.

2009-01-01

The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III 1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III 1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III 1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III 1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg 88 to Ala 88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III 1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.

A positive feedback regulation of ISL-1 in DLBCL but not in pancreatic β-cells

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Zhang, Qiao, E-mail: zhangqiao200824@126.com [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Yang, Zhe, E-mail: zheyang@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Wang, Weiping, E-mail: wwp@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Guo, Ting, E-mail: luckyguoting@bjmu.edu.cn [Department of Gastrointestinal Translation Research, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital, 52 Fucheng Road, 100142 Beijing (China); Jia, Zhuqing, E-mail: zhuqingjia@126.com [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Ma, Kangtao, E-mail: makangtao11@126.com [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Zhou, Chunyan, E-mail: chunyanzhou@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China)

2014-07-04

Highlights: • ISL-1 is highly expressed in human pancreatic β-cells and DLBCL. • ISL-1 accelerates the tumorigenesis of DLBCL in vivo. • c-Myc positively regulates ISL-1 expression in DLBCL but not in pancreatic β-cells. • ISL-1 and c-Myc forms an ISL-1/c-Myc transcriptional complex only in DLBCL. • Positive feedback regulation of ISL-1 does not exist in normal pancreatic β-cell. - Abstract: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, has been reported to play essential roles in promoting adult pancreatic β-cells proliferation. Recent studies indicate that ISL-1 may also involve in the occurrence of a variety of tumors. However, whether ISL-1 has any functional effect on tumorigenesis, and what are the differences on ISL-1 function in distinct conditions, are completely unknown. In this study, we found that ISL-1 was highly expressed in human pancreatic β-cells, as well as in diffuse large B cell lymphoma (DLBCL), but to a much less extent in other normal tissues or tumor specimens. Further study revealed that ISL-1 promoted the proliferation of pancreatic β-cells and DLBCL cells, and also accelerated the tumorigenesis of DLBCL in vivo. We also found that ISL-1 could activate c-Myc transcription not only in pancreatic β-cells but also in DLBCL cells. However, a cell-specific feedback regulation was detectable only in DLBCL cells. This auto-regulatory loop was established by the interaction of ISL-1 and c-Myc to form an ISL-1/c-Myc transcriptional complex, and synergistically to promote ISL-1 transcription through binding on the ISL-1 promoter. Taken together, our results demonstrate a positive feedback regulation of ISL-1 in DLBCL but not in pancreatic β-cells, which might result in the functional diversities of ISL-1 in different physiological and pathological processes.

A positive feedback regulation of ISL-1 in DLBCL but not in pancreatic β-cells

International Nuclear Information System (INIS)

Zhang, Qiao; Yang, Zhe; Wang, Weiping; Guo, Ting; Jia, Zhuqing; Ma, Kangtao; Zhou, Chunyan

2014-01-01

Highlights: • ISL-1 is highly expressed in human pancreatic β-cells and DLBCL. • ISL-1 accelerates the tumorigenesis of DLBCL in vivo. • c-Myc positively regulates ISL-1 expression in DLBCL but not in pancreatic β-cells. • ISL-1 and c-Myc forms an ISL-1/c-Myc transcriptional complex only in DLBCL. • Positive feedback regulation of ISL-1 does not exist in normal pancreatic β-cell. - Abstract: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, has been reported to play essential roles in promoting adult pancreatic β-cells proliferation. Recent studies indicate that ISL-1 may also involve in the occurrence of a variety of tumors. However, whether ISL-1 has any functional effect on tumorigenesis, and what are the differences on ISL-1 function in distinct conditions, are completely unknown. In this study, we found that ISL-1 was highly expressed in human pancreatic β-cells, as well as in diffuse large B cell lymphoma (DLBCL), but to a much less extent in other normal tissues or tumor specimens. Further study revealed that ISL-1 promoted the proliferation of pancreatic β-cells and DLBCL cells, and also accelerated the tumorigenesis of DLBCL in vivo. We also found that ISL-1 could activate c-Myc transcription not only in pancreatic β-cells but also in DLBCL cells. However, a cell-specific feedback regulation was detectable only in DLBCL cells. This auto-regulatory loop was established by the interaction of ISL-1 and c-Myc to form an ISL-1/c-Myc transcriptional complex, and synergistically to promote ISL-1 transcription through binding on the ISL-1 promoter. Taken together, our results demonstrate a positive feedback regulation of ISL-1 in DLBCL but not in pancreatic β-cells, which might result in the functional diversities of ISL-1 in different physiological and pathological processes

TNFα-mediated loss of β-catenin/E-cadherin association and subsequent increase in cell migration is partially restored by NKX3.1 expression in prostate cells.

Directory of Open Access Journals (Sweden)

Bilge Debelec-Butuner

Full Text Available Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered β-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of β-catenin following increased phosphorylation of Akt(S473 and GSK3β(S9. Consistently, we observed that subsequent increase in β-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the β-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt(S473 phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt(S308 priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA, high-grade prostatic intraepithelial neoplasia (H-PIN and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.

Technetium-99m labeled antisense probes uptake in vascular smooth muscle cells

International Nuclear Information System (INIS)

Zhang, Y.X.; Qin, G.M.; An, R.; Cao, G.X.; Cao, W.; Gao, Z.R.

2002-01-01

In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. The atherosclerotic plaques contained 3-4 fold more c-myc mRNA than those in the normal aortic arteries, while increased Bax and Bak coupled with lack/paucity of Bcl-2 and Bcl-xL are associated with SMC apoptosis in advanced lesions. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. Cell uptake studies: 99m Tc- MAG 3 -DNA radioactivity incorporation into porcine coronary smooth muscle cells in the log and plateau phases, respectively, was determined after different times of incubation at 37. The influence of extracellular 99m Tc- MAG 3 -DNA concentration on SMC uptake was also analyzed. [Results] Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The MAG 3 -DNA was labeled with 99m Tc at room temperature and neutral pH, with a mean labeling efficiency of 80.11%(s.d=2.96%,n=4). The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. After labeling, the stability of the DNA in saline or serum was retained as determined by reverse-phase Sep-Pak C18 chromatography analysis, except a shift at 30 min in serum incubation that suggesting a short time serum protein binding. 99m Tc-MAG 3 -c-myc uptake plateaued at 60 min and was directly proportional to the

Study on relationship between apoptosis-related genes and radiosensitivity of esophageal squamous cell carcinoma

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Li Huixiang; Wang Yaohe; Shi Yonggang; Gao Dongling; Zhang Yunhan

2000-01-01

Objective: To observing the relationship between apoptosis-related genes bcl-2,c-myc, p53 and the radiosensitivity of esophageal squamous cell carcinoma. Methods: The expression levels of bcl-2, c-myc and p53 genes in 57 biopsy samples from patients of esophageal squamous cell carcinoma were detected with the LSAB immunohistochemistry method. All the patients were treated with radiotherapy. The radiotherapeutic effect in these patients was observed and the relation between gene expression and radiosensitivity was analyzed. Results: Compared with the bcl-2-negative group, the radiosensitivity of bcl-2-positive one was lower(P<0.01). The radiosensitivity of p53-positive group was slightly lower than that of the p53-negative one (P<0.05). The c-myc protein expression was not related to radiosensitivity. Conclusion: Detection and comprehensive analysis of bcl-2, c-myc and p53 protein expressions are useful in forecasting the radiotherapeutic effect on squamous cell carcinoma of esophagus

Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line

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Vaskivuo, Liisa; Rysae, Jaana; Koivuperae, Johanna; Myllynen, Paeivi; Vaskivuo, Tommi; Chvalova, Katerina; Serpi, Raisa; Savolainen, Eeva-Riitta; Puistola, Ulla; Vaehaekangas, Kirsi

2006-01-01

p14 ARF tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14 ARF is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14 ARF to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14 ARF protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14 ARF protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14 ARF can respond to DNA damage without oncogene activation in cell lines without functional p53

RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

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Yu, W; Sanders, B G; Kline, K

1997-01-01

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately.

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Shi, Yan; Shu, Bin; Yang, Ronghua; Xu, Yingbin; Xing, Bangrong; Liu, Jian; Chen, Lei; Qi, Shaohai; Liu, Xusheng; Wang, Peng; Tang, Jinming; Xie, Julin

2015-06-16

Wnt and Notch signaling pathways are critically involved in relative cell fate decisions within the development of cutaneous tissues. Moreover, several studies identified the above two pathways as having a significant role during wound healing. However, their biological effects during cutaneous tissues repair are unclear. We employed a self-controlled model (Sprague-Dawley rats with full-thickness skin wounds) to observe the action and effect of Wnt/β-catenin and Notch signalings in vivo. The quality of wound repair relevant to the gain/loss-of-function Wnt/β-catenin and Notch activation was estimated by hematoxylin-and-eosin and Masson staining. Immunofluorescence analysis and Western blot analysis were used to elucidate the underlying mechanism of the regulation of Wnt and Notch signaling pathways in wound healing. Meanwhile, epidermal stem cells (ESCs) were cultured in keratinocyte serum-free medium with Jaggedl or in DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl) to investigate whether the interruption of Notch signaling contributes to the expression of Wnt/β-catenin signaling. The results showed that in vivo the gain-of-function Wnt/β-catenin and Notch activation extended the ability to promote wound closure. We further determined that activation or inhibition of Wnt signaling and Notch signaling can affect the proliferation of ESCs, the differentiation and migration of keratinocytes, and follicle regeneration by targeting c-Myc and Hes1, which ultimately lead to enhanced or delayed wound healing. Furthermore, Western blot analysis suggested that the two pathways might interact in vivo and in vitro. These results suggest that Wnt and Notch signalings play important roles in cutaneous repair by targeting c-Myc and Hes1 separately. What's more, interaction between the above two pathways might act as a vital role in regulation of wound healing.

The use of computerized video time lapse to study cell death in rat embryo cells transfected with c-ha-ras or c-myc

International Nuclear Information System (INIS)

Forrester, H.B.; Vidair, C.A.; Dewey, W.C.; Ling, C.C.

1998-01-01

Full text: Individual rat embryo fibroblasts that had been transfected with the c-myc (REC:myc) or c-Ha ras (REC:ras) oncogene were followed after irradiation using a computer video time lapse (CVTL) system in order to quantify the lethal events that resulted in loss of clonogenic survival after irradiation. By followed the cells for 2 to 3 generations before irradiation we were able to determine where they were in the cell cycle at the time of irradiation for cell cycle analysis. After irradiation, the individual cells and their progeny were followed in multiple fields for 5-6 days Then, pedigrees for individual irradiated cells were determined by noting the times of divisions fusions, and cell death. After X-irradiation, the clonogenic survival values for these two cell lines are similar. However, by using computerized video time lapse (CVTL) to follow individual cells we found that the loss of clonogenic survival was due to two different processes, cell death and a senescent-like process. The loss of clonogenic survival of x-irradiated (9.5 and 4 Gy) REC:myc cells was attributed almost entirely to the cells dying by apoptosis (∼99 and 90%). In contrast, approximately 60% of the x-irradiated (9.5 Gy) non-clonogenic REC:ras cells died by apoptosis (with a very small amount of necrosis), and the other 40% underwent a senescent-type process in which some of the cells and their progeny stopped dividing but remained as viable cells throughout 140 hours of observation. Both processes usually occurred after the cells had divided and continued to occur in the cells' progeny for up to five divisions after irradiation. The mode of cell death in the progeny of a non-clonogenic cell can be determined only by using CVTL and can not be determined by conventional clonogenic survival experiments. Also, only by following the individual cells and their progeny can the true amount of apoptosis be determined. The cumulative percentage of apoptosis scored in whole populations

Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells.

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Wang, Jiandong; Ma, Xiaoli; Jones, Hannah M; Chan, Leo Li-Ying; Song, Fang; Zhang, Weiyuan; Bae-Jump, Victoria L; Zhou, Chunxiao

2014-08-21

Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. Our objective was to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian carcinoma cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using MTT assay, colony formation, flow cytometry and Annexin V FITC assays, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. The response to 10058-F4 was independent the level of c-Myc protein over-expression in primary cultures of ovarian carcinoma. These novel findings suggest that the growth of ovarian cancer cells is dependent upon c-MYC activity and that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer.

Complex Biological Systems Analysis of Cell Cycling Models in Carcinogenesis: I. The essential roles of modifications in the c-Myc, TP53/p53, p27 and hTERT modules in Cancer Initiation and Progression

CERN Document Server

Prisecaru, V I

2004-01-01

A new approach to the integration of results from a modular, complex biological systems analysis of nonlinear dynamics in cell cycling network transformations that are leading to carcinogenesis is proposed. Carcinogenesis is a complex process that involves dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments that form numerous inter-related pathways referred to as networks. One such network module contains the cell cyclins whose functions are essential to cell cycling and division. Cyclins are proteins that also link to several critical pro-apoptotic and other cell cycling/division components, such as: c-Myc, p27, the tumor suppressor gene TP53 and its product-- the p53 protein with key roles in controlling DNA repair, inducing apoptosis and activating p21 (which can depress cell cyclins if activated), mdm2(with its biosynthesis activated by p53 and also, in its turn, inhibiting p53), p21, the Thomsen-Friedenreich antigen(T- antigen),Rb,Bax, Ba...

Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

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2014-11-01

increased by hydroxyurea, ATR inhibition, deregulated c-Myc expression and by PARPi treatment of BRCA1 deficient cells. This work was recently published...Genome Stability." 6: May 27, 2013-Collaborative Research Center 655 from Cells to Tissues seminar series at the Max-Planck-Institute in Dresden, Germany ...Eisenach, Germany -“Genome Stability during DNA Replication” 8: May 3, 2013- Chemical and Systems Biology Department Seminar Series at Stanford

E-cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor-κB in AGS cells.

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Park, Song Yi; Shin, Jee-Hye; Kee, Sun-Ho

2017-09-01

β-Catenin is a central player in Wnt signaling, and activation of Wnt signaling is associated with cancer development. E-cadherin in complex with β-catenin mediates cell-cell adhesion, which suppresses β-catenin-dependent Wnt signaling. Recently, a tumor-suppressive role for E-cadherin has been reconsidered, as re-expression of E-cadherin was reported to enhance the metastatic potential of malignant tumors. To explore the role of E-cadherin, we established an E-cadherin-expressing cell line, EC96, from AGS cells that featured undetectable E-cadherin expression and a high level of Wnt signaling. In EC96 cells, E-cadherin re-expression enhanced cell proliferation, although Wnt signaling activity was reduced. Subsequent analysis revealed that nuclear factor-κB (NF-κB) activation and consequent c-myc expression might be involved in E-cadherin expression-mediated cell proliferation. To facilitate rapid proliferation, EC96 cells enhance glucose uptake and produce ATP using both mitochondria oxidative phosphorylation and glycolysis, whereas AGS cells use these mechanisms less efficiently. These events appeared to be mediated by NF-κB activation. Therefore, E-cadherin re-expression and subsequent induction of NF-κB signaling likely enhance energy production and cell proliferation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Methylselenol, a selenium metabolite, plays common and different roles in cancerous colon HCT116 cell and noncancerous NCM460 colon cell proliferation.

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Zeng, Huawei; Briske-Anderson, Mary; Wu, Min; Moyer, Mary P

2012-01-01

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenol's stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenol's anticancer action.

Synergistic in-vitro effects of combining an antiglycolytic, 3-bromopyruvate, and a bromodomain-4 inhibitor on U937 myeloid leukemia cells.

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Kapp, Nicolette; Stander, Xiao X; Stander, Barend A

2018-06-01

This project investigated the in-vitro effects of a glycolytic inhibitor, 3-bromopyruvate (3-BrP), in combination with and a new in silico-designed inhibitor of the bromodomain-4 (BRD-4) protein, ITH-47, on the U937 acute myeloid leukemia cell line. 3-BrP is an agent that targets the altered metabolism of cancer cells by interfering with glucose metabolism in the glycolytic pathway. ITH-47 is an acetyl-lysine inhibitor that displaces bromdomain 4 proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of this bromodomain and extraterminal (BET) BRD protein, thereby preventing transcription of cancer-associated genes and further cell growth. Cell growth studies determined the IC50 after 48 h exposure for 3-BrP and ITH-47 to be 6 and 2 μmol/l, respectively. When combined, 2.4 and 1 μmol/l of 3-BrP and ITH-47, respectively, inhibited 50% of the cell population, yielding a synergistic combination index of 0.9. Subsequent mechanistic studies showed that the IC50 concentrations of ITH-47 and 3-BrP and the combination increased observable apoptotic bodies and cell shrinkage in U937 cells treated for 48 h. Cell cycle analysis showed an increase in the sub-G1 fraction in all treated cells, suggesting that cell death was increased in the treated samples. Annexin-V-FITC apoptosis analysis showed a statistically significant increase in the number of cells in early and late apoptosis, indicating that cell death occurred through apoptosis and not necrosis. Only U937 cells exposed to ITH-47 showed a decrease in mitochondrial membrane potential compared with the vehicle control. Reactive oxygen species production was decreased in all treated samples. ITH-47-exposed cells showed a decrease in c-Myc, Bcl-2, and p53 gene expressions. 3-BrP-treated cells showed an increase in c-myc and p53 gene expressions. The combination of ITH-47 and 3-BrP lead to downregulation of c-myc and Bcl-2 genes. ITH-47 exposure conditions yielded a marked decrease

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

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Q. Sun

2012-10-01

Full Text Available The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3 can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

International Nuclear Information System (INIS)

Sun, Q.; Xiong, J.; Lu, J.; Xu, S.; Li, Y.; Zhong, X.P.; Gao, G.K.; Liu, H.Q.

2012-01-01

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

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Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

2012-06-22

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

A novel form of the RelA nuclear factor kappaB subunit is induced by and forms a complex with the proto-oncogene c-Myc.

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Chapman, Neil R; Webster, Gill A; Gillespie, Peter J; Wilson, Brian J; Crouch, Dorothy H; Perkins, Neil D

2002-01-01

Members of both Myc and nuclear factor kappaB (NF-kappaB) families of transcription factors are found overexpressed or inappropriately activated in many forms of human cancer. Furthermore, NF-kappaB can induce c-Myc gene expression, suggesting that the activities of these factors are functionally linked. We have discovered that both c-Myc and v-Myc can induce a previously undescribed, truncated form of the RelA(p65) NF-kappaB subunit, RelA(p37). RelA(p37) encodes the N-terminal DNA binding and dimerization domain of RelA(p65) and would be expected to function as a trans-dominant negative inhibitor of NF-kappaB. Surprisingly, we found that RelA(p37) no longer binds to kappaB elements. This result is explained, however, by the observation that RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc. These results demonstrate a previously unknown functional and physical interaction between RelA and c-Myc with many significant implications for our understanding of the role that both proteins play in the molecular events underlying tumourigenesis. PMID:12027803

Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay.

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Abruzzese, Maria Pia; Bilotta, Maria Teresa; Fionda, Cinzia; Zingoni, Alessandra; Soriani, Alessandra; Vulpis, Elisabetta; Borrelli, Cristiana; Zitti, Beatrice; Petrucci, Maria Teresa; Ricciardi, Maria Rosaria; Molfetta, Rosa; Paolini, Rossella; Santoni, Angela; Cippitelli, Marco

2016-12-01

Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells. Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138 + MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays. Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi

Combinatorial treatment of DNA and chromatin-modifying drugs cause cell death in human and canine osteosarcoma cell lines.

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Venugopal Thayanithy

Full Text Available Downregulation of microRNAs (miRNAs at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat and the DNA methylation inhibitor Zebularine (Zeb, with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

[Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma].

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Ren, K; Qian, D; Wang, Y W; Pang, Q S; Zhang, W C; Yuan, Z Y; Wang, P

2018-01-23

, knockdown NS1-BP in KYSE510 cells induced c-Myc expression, increased cell proliferation and repressed apoptosis. Conclusions: NS1-BP is an independent favorable prognostic factor in ESCC. It inhibits cell proliferation and enhances cell apoptosis via repressing c-Myc. Targeting NS1-BP may be a new therapeutic strategy for ESCC patients.

Genetic dissimilarity between primary colorectal carcinomas and their lymph node metastases: ploidy, p53, bcl-2, and c-myc expression--a pilot study.

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Zalata, Khaled Refaat; Elshal, Mohamed Farouk; Foda, Abd AlRahman Mohammad; Shoma, Ashraf

2015-08-01

The current paradigm of metastasis proposes that rare cells within primary tumors acquire metastatic capability via sequential mutations, suggesting that metastases are genetically dissimilar from their primary tumors. This study investigated the changes in the level of expression of a well-defined panel of cell proliferation, differentiation, and apoptosis markers between the primary colorectal cancer (CRC) and the corresponding synchronous lymph node (LN) metastasis from the same patients. DNA flow cytometry and immunostaining of p53, bcl-2, and c-myc were carried out on 36 cases of CRC radical resection specimens with their corresponding LN metastases. There was very low probability that the histological patterns of primary tumors and LN metastases are independent (p < 0.001). Metastatic tumors were significantly more diffusely positive for p53 than the primary tumors (p < 0.001). Conversely, primary tumors were significantly more diffusely positive for c-myc than metastatic tumors (p = 0.011). No significant difference was found between the LNs and the primary tumors in bcl-2 positivity (p = 0.538) and DNA aneuploidy (p = 0.35), with a tendency towards negative bcl-2 and less aneuploidy in LN metastases than primary tumors. In conclusion, LN metastatic colorectal carcinomas have a tendency of being less differentiated, with a higher incidence of diffuse p53 staining, lower incidence of bcl-2 staining, and less aneuploidy in comparison to their primary counterparts suggesting a more aggressive biological behavior, which could indicate the necessity for more aggressive adjuvant therapy.

Phorbol esters induce interleukin 2 mRNA in sensitive but not in resistant EL4 cells

International Nuclear Information System (INIS)

Harrison, J.R.; Lynch, K.R.; Sando, J.J.

1986-01-01

Phorbol ester (PE) sensitive EL4 cells are growth-inhibited and produce interleukin 2 (IL2) when treated with PE. Resistant EL4 cells lack both responses. To determine whether the defect in resistant cells occurs pre or post-transcriptionally, an assay for IL2 mRNA was developed using a synthetic oligonucleotide to mouse IL2 as a probe. Total RNA (15 μg) from cells +/- PE was electrophoresed, blotted onto a cationic nylon membrane, and probed with radiolabeled oligomer. This probe hybridized to a 1.1 kb band in RNA from PE-treated sensitive cells. This RNA was detectable within 3h of PE administration, was clearly visible by 6h, and peaked by 9 to 12h. No bands hybridizing with the IL2 probe were detected in RNA isolated from unstimulated cells or from resistant EL4 cells at any time following PE stimulation. Since levels of the protooncogene c-myc have been shown to decrease in a number of cell lines during differentiation and growth inhibition, total RNA from EL4 cells was probed with a nick-translated plasmid containing the protein coding region of the c-myc gene. In PE sensitive cells, levels of c-myc RNA are markedly reduced by 3h. In a pilot experiment with resistant cells, c-myc levels appeared to remain constant. These results demonstrate that PE induced IL2 mRNA in PE sensitive but not resistant EL4 cells. Sensitive and resistant EL4 cell lines provide a useful model for the investigation of the regulation of gene expression by PE

MYC is a metastasis gene for non-small-cell lung cancer.

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Ulf R Rapp

Full Text Available BACKGROUND: Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. METHODOLOGY/PRINCIPAL FINDINGS: Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. CONCLUSIONS/SIGNIFICANCE: Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development.

Overexpression of c-myc and loss of heterozigosity on 2p, 3p, 5q, 17p and 18q in sporadic colorectal carcinoma Sobreexpresión de c-myc y pérdida de heterozigosidad en 2p, 3p, 5q, 17p y 18q en carcinoma colorrectal esporádico

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A. Sánchez-Pernaute

2005-03-01

Full Text Available Aim: the aim of the present study is to evaluate the prognostic influence of loss of heterozygosity on 2p, 3p, 5q, 17p and 18q, and c-myc overexpression on surgically treated sporadic colorectal carcinoma. Methods: tumor and non-tumor tissue samples from 153 patients were analyzed. Fifty-one percent of patients were male, and mean age in the series was 67 years. Tumors were located in the proximal colon in 37 cases, in the distal bowel in 37, and in the rectum in 79 patients. c-myc overexpression was studied by means of Northern blot analysis, and loss of heterozigosity through microsatellite analysis. Results: c-myc overexpression was detected in 25% of cases, and loss of heterozygosity in at least one of the studied regions in 48%. There was no association between clinical and pathologic features, and genetic alterations. The disease-free interval was significantly shorter for patients with both genetic alterations; the presence of both events was an independent prognostic factor for poor outcome in the multivariate analysis (RR: 4.34, p Objetivo: el objetivo del presente trabajo es evaluar la importancia pronóstica de la pérdida de heterozigosidad en las regiones 2p, 3p, 5q, 17p y 18q y de la sobreexpresión del gen c-myc en el carcinoma colorrectal esporádico, mediante el estudio de la supervivencia libre de enfermedad tras cirugía potencialmente curativa. Métodos: se han analizado muestras tumorales y no tumorales de mucosa colónica de 153 pacientes. El 51% de los pacientes eran varones y la edad media de la serie fue 67 años. Los tumores fueron proximales en 37 casos, distales en 37 y localizados en recto en 79. Se analizó la sobreexpresión del RNA de c-myc por Northern blot, y la presencia de pérdida de heterozigosidad en las diferentes regiones consideradas por análisis de microsatélites. Resultados: se detectó sobreexpresión de c-myc en el 25% de los casos, y pérdida de heterozigosidad en alguna de las regiones estudiadas

The adaptive immune system promotes initiation of prostate carcinogenesis in a human c-Myc transgenic mouse model.

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Melis, Monique H M; Nevedomskaya, Ekaterina; van Burgsteden, Johan; Cioni, Bianca; van Zeeburg, Hester J T; Song, Ji-Ying; Zevenhoven, John; Hawinkels, Lukas J A C; de Visser, Karin E; Bergman, Andries M

2017-11-07

Increasing evidence from epidemiological and pathological studies suggests a role of the immune system in the initiation and progression of multiple cancers, including prostate cancer. Reports on the contribution of the adaptive immune system are contradictive, since both suppression and acceleration of disease development have been reported. This study addresses the functional role of lymphocytes in prostate cancer development using a genetically engineered mouse model (GEMM) of human c-Myc driven prostate cancer (Hi-Myc mice) combined with B and T cell deficiency (RAG1 -/- mice). From a pre-cancerous stage on, Hi-Myc mice showed higher accumulation of immune cells in their prostates then wild-type mice, of which macrophages were the most abundant. The onset of invasive adenocarcinoma was delayed in Hi-MycRAG1 -/- compared to Hi-Myc mice and associated with decreased infiltration of leukocytes into the prostate. In addition, lower levels of the cytokines CXCL2, CCL5 and TGF-β1 were detected in Hi-MycRAG1 -/- compared to Hi-Myc mouse prostates. These results from a GEMM of prostate cancer provide new insights into the promoting role of the adaptive immune system in prostate cancer development. Our findings indicate that the endogenous adaptive immune system does not protect against de novo prostate carcinogenesis in Hi-Myc transgenic mice, but rather accelerates the formation of invasive adenocarcinomas. This may have implications for the development of novel treatment strategies.

Promoter trans-activation of protooncogenes c-fos and c-myc, but not c-Ha-ras, by products of adenovirus early region 1A

International Nuclear Information System (INIS)

Sassone-Corsi, P.; Borrelli, E.

1987-01-01

The E1A (early region 1A) oncogene products of adenovirus type 2 trans-activate the other early viral transcription units, as well as some cellular promoters. Using a short-term cotransfection assay in murine NIH 3T3 fibroblasts, we show that c-fos and c-myc promoter activities are stimulated by the E1A proteins, whereas c-Ha-ras transcription is not affected. The product of E1A 13S mRNA is responsible for the trans-activation, whereas the 12S mRNA product has no effect. Analysis of the c-fos promoter sequences required for the E1A stimulation shows that responsive sequences are located between positions -402 and -240 upstream of the transcription initiation site. This same region also contains the c-fos serum-responsive element. Furthermore, transcription of the endogenous c-fos gene in HeLa cells is increased after E1A transfection

Modulation of RIZ gene expression is associated to estradiol control of MCF-7 breast cancer cell proliferation

International Nuclear Information System (INIS)

Gazzerro, Patrizia; Abbondanza, Ciro; D'Arcangelo, Andrea; Rossi, Mariangela; Medici, Nicola; Moncharmont, Bruno; Puca, Giovanni Alfredo

2006-01-01

The retinoblastoma protein-interacting zinc-finger (RIZ) gene, a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N-terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI-BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumor suppressor. Estradiol treatment of MCF-7 cells produced a selective decrease of RIZ1 transcript and an increase of total RIZ mRNA. Experiments of chromatin immunoprecipitation indicated that RIZ2 protein expression was controlled by estrogen receptor and RIZ1 had a direct repressor function on c-myc gene expression. To investigate the role of RIZ gene products as regulators of the proliferation/differentiation transition, we analyzed the effects of forced suppression of RIZ1 induced in MCF-7 cells by siRNA of the PR domain-containing form. Silencing of RIZ1 expression stimulated cell proliferation, similar to the effect of estradiol on these cells, associated with a transient increase of c-myc expression

Overexpression of c-myc is associated with adverse clinical features and worse overall survival in multiple myeloma

DEFF Research Database (Denmark)

Szabo, Agoston Gyula; Gang, Anne Ortved; Pedersen, Mette Ølgod

2016-01-01

The role of c-myc in multiple myeloma (MM) is controversial. We conducted a retrospective study of 117 patients with MM diagnosed between 2004 and 2010 at Herlev Hospital. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) were performed on tissue microarrays (TMAs) made from...

Pifithrin-μ, an inhibitor of heat-shock protein 70, can increase the antitumor effects of hyperthermia against human prostate cancer cells.

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Kazumasa Sekihara

Full Text Available Hyperthermia (HT improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145 were treated with HT (43°C for 2 h. All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21(WAF1/Cip, indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.

Angiotensin II reduces cardiac AdipoR1 expression through AT1 receptor/ROS/ERK1/2/c-Myc pathway.

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Li Li

Full Text Available Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2 mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1 receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.

Periplocin from Cortex periplocae inhibits cell growth and down-regulates survivin and c-myc expression in colon cancer in vitro and in vivo via beta-catenin/TCF signaling.

Science.gov (United States)

Zhao, Lianmei; Shan, Baoen; Du, Yanyan; Wang, Mingxia; Liu, Lihua; Ren, Feng-Zhi

2010-08-01

Cancer of the colon and rectum is the third most commonly diagnosed cancer and accounts for approximately 10% of all cancer-related deaths. Although surgical resection or radiotherapy are potentially curative for localized disease, advanced colon cancer is currently associated with poor prognosis. Therefore, the development of a new and effective chemotherapeutic agent is required to target critical pathways to induce responsiveness of colon cancer cells to death signals. Dysregulation of the beta-catenin/TCF pathway plays a central role in early activities of colorectal carcinogenesis. In this study, human colon cancer SW480 cells were used to investigate the effect of CPP (periplocin from Cortex periplocae) on the modulation of the beta-catenin/TCF signaling pathway. Our research results showed that CPP caused a dose- and time-dependent inhibition of cell growth as assessed by MTT assay and an induction in apoptosis as measured by flow cytometry and transmission electron microscopy. Furthermore, the CPP- treated cells were characterized by a decreased expression of beta-catenin protein in the total cell lysates and cytosolic and nuclear extracts. This expression alleviates the binding activity of T-cell factor (Tcf) complexes to its specific DNA-binding sites. Thus, the protein expression of the downstream elements survivin and c-myc was down-regulated. To determine the precise inhibitory mechanisms involved, further in-depth in vivo studies of CPP are warranted. In conclusion, our data suggest that CPP wields a multi-prong strategy to target the beta-catenin/Tcf signaling pathway, leading to the induction of apoptosis and inhibition of growth of colon cancer cells in vitro and in vivo. Therefore, CPP may become a potential agent against colon cancer.

A mechanistic study of cigarette smoke and cyclooxygenase-2 on proliferation of gastric cancer cells

International Nuclear Information System (INIS)

Shin, Vivian Y.; Liu, Edgar S.L.; Ye, Yi-Ne; Koo, Marcel W.L.; Chu, K.-M.; Cho, C.-H.

2004-01-01

Cigarette smoke has been shown to cause gastric cancer. Overexpression of cyclooxygenase-2 (COX-2) is a common characteristic in gastric malignancy. The present study aimed to explore the correlation between cigarette smoke and COX-2 in the promotion of tumorigenesis in human gastric cancer cells (AGS). We further studied the action of COX-2 on other proto-oncogenes on gastric tumor growth. Results showed that chloroform extract (CE) and ethanol extract (EE) from cigarette smoke dose-dependently stimulated gastric cancer cell proliferation, which was accompanied with an activation of ornithine decarboxylase (ODC) activity, COX-2, and c-myc expressions. Both antisense of c-myc and α-difluoromethylornithine (DFMO, specific ODC inhibitor) inhibited cell proliferation without affecting COX-2 expression in response to cigarette smoke extracts (CSE). However, selective COX-2 inhibitor (SC-236) not only blocked the proliferative activity but also the ODC activity and c-myc protein expression by CSE in gastric cancer cells. Further, supplementation of exogenous prostaglandin (PG) E 2 reversed all the inhibitory actions of SC-236. Our results underline the importance of COX-2 in the cancer-promoting effect of CSE and its modulation on its downstream growth-related genes, such as c-myc and ODC in cancer cell proliferation. These results reveal that CSE-induced gastric carcinogenesis is via the COX-2/c-myc/ODC and PGE 2 -dependent pathway. Hence, selective COX-2 inhibitor could be an effective therapeutic agent for gastric cancer in smokers

Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis

International Nuclear Information System (INIS)

Hubbard, Kyle; Catalano, Jennifer; Puri, Raj K; Gnatt, Averell

2008-01-01

A common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture. RNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines. Knockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death. Although transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery

Cell cycle control by the thyroid hormone in neuroblastoma cells

International Nuclear Information System (INIS)

Garcia-Silva, Susana; Perez-Juste, German; Aranda, Ana

2002-01-01

The thyroid hormone (T3) blocks proliferation and induces differentiation of neuroblastoma N2a-β cells that overexpress the β1 isoform of the T3 receptor. An element in the region responsible for premature termination of transcription mediates a rapid repression of c-myc gene expression by T3. The hormone also causes a decrease of cyclin D1 gene transcription, and is able to antagonize the activation of the cyclin D1 promoter by Ras. In addition, a strong and sustained increase of the levels of the cyclin kinase inhibitor (CKI) p27 Kip1 are found in T3-treated cells. The increased levels of p27 Kip1 lead to a marked inhibition of the kinase activity of the cyclin-CDK2 complexes. As a consequence of these changes, retinoblastoma proteins are hypophosphorylated in T3-treated N2a-β cells, and progression through the restriction point in the cell cycle is blocked

Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1 in laryngeal squamous cell carcinoma

International Nuclear Information System (INIS)

Yang, Min; Li, Wei; Liu, Yi-Ying; Fu, Shuang; Qiu, Guang-Bin; Sun, Kai-Lai; Fu, Wei-Neng

2012-01-01

MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene. MYCT1 was cloned from laryngeal squamous cell cancer (LSCC) and has been found to be down-regulated in LSCC; however, the regulatory details have not been fully elucidated. Here, we sought to investigate the methylation status of the CpG islands of MYCT1 and mRNA levels by bisulfite-specific PCR (BSP) based on sequencing restriction enzyme digestion, reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR). The function of specific sites in the proximal promoter of MYCT1 in LSCC was measured by transient transfection, luciferase assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). The results suggested hypermethylation of 12 CpG sites of the promoter in both laryngeal cancer tissues and the laryngeal cancer line Hep-2 cell. The hypermethylation of the site CGCG (−695 to −692), which has been identified as the c-Myc binding site, was identified in laryngeal cancer tissues (59/73) compared to paired mucosa (13/73); in addition, statistical analysis revealed that the methylation status of this site significantly correlated with cancer cell differentiation(p < 0.01). The mRNA level of MYCT1 increased in Hep-2 cells treated with 5-aza-C (p < 0.01). The luciferase activity from mutant transfectants pGL3-MYCT1m (−852/+12, mut-695-C > A, mut-693-C > G) was significantly reduced compared with the wild type pGL3-MYCT1 (−852/+12), while the luciferase activity from wild transfectants pGL3-MYCT1 (−852/+12) rose after 5-aza treatment in Hep-2 cells. Finally, EMSA and ChIP confirmed that the methylation of the CGCG (−695 to −692) site prevented c-Myc from binding of the site and demethylation treatment of the 5′ flanking region of MYCT1 by 5-aza induced the increased occupation of the core promoter by c-Myc (p < 0.01). In summary, this study concluded that hypermethylation contributed to the transcriptional down

M2 macrophages activate WNT signaling pathway in epithelial cells: relevance in ulcerative colitis.

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Jesús Cosín-Roger

Full Text Available Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in

Control of amino acid transport coordinates metabolic reprogramming in T-cell malignancy.

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Grzes, K M; Swamy, M; Hukelmann, J L; Emslie, E; Sinclair, L V; Cantrell, D A

2017-12-01

This study explores the regulation and importance of System L amino acid transport in a murine model of T-cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN -/- T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T-cell malignancies, highlighting how a single amino acid transporter can have a key role.

Induction of Programmed Cell Death by Parvovirus H-1 in U937 Cells: Connection with the Tumor Necrosis Factor Alpha Signalling Pathway

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Rayet, Béatrice; Lopez-Guerrero, José-Antonio; Rommelaere, Jean; Dinsart, Christiane

1998-01-01

The human promonocytic cell line U937 undergoes apoptosis upon treatment with tumor necrosis factor alpha (TNF-α). This cell line has previously been shown to be very sensitive to the lytic effect of the autonomous parvovirus H-1. Parvovirus infection leads to the activation of the CPP32 ICE-like cysteine protease which cleaves the enzyme poly(ADP-ribose)polymerase and induces morphologic changes that are characteristic of apoptosis in a way that is similar to TNF-α treatment. This effect is also observed when the U937 cells are infected with a recombinant H-1 virus which expresses the nonstructural (NS) proteins but in which the capsid genes are replaced by a reporter gene, indicating that the induction of apoptosis can be assigned to the cytotoxic nonstructural proteins in this cell system. The c-Myc protein, which is overexpressed in U937 cells, is rapidly downregulated during infection, in keeping with a possible role of this product in mediating the apoptotic cell death induced by H-1 virus infection. Interestingly, four clones (designated RU) derived from the U937 cell line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642–1653, 1997) failed to decrease c-Myc expression upon treatment with differentiation agents and also resisted the induction of cell death after TNF-α treatment. Our data suggest that the RU clones have developed defense strategies against apoptosis, either by their failure to downregulate c-Myc and/or by activating antiapoptotic factors. PMID:9765434

Dental pulp stem cells differentiation reveals new insights in Oct4A dynamics.

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Federico Ferro

Full Text Available Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC. A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A. Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.

The total flavonoids of Clerodendrum bungei suppress A549 cells proliferation, migration, and invasion by impacting Wnt/β-Catenin signaling

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Na Yu

2017-01-01

Full Text Available Objectives: The objective of this study is to evaluate the effect of the total flavonoids of Clerodendrum bungei (TFCB on the proliferation, invasion, and metastasis of A549 lung cancer cells through the Wnt signaling pathway. Materials and Methods: A549 cells were transfected with a β-catenin overexpression plasmid and the empty vector pcDNA3.1. The A549 cells were divided into six groups: normal A549 cell group, normal A549 cells with TFCB group, vector control group, vector with TFCB group, β-catenin overexpression group, and β-catenin with TFCB group. We used the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay to detect cell proliferation, a scratch test was used to observe cell migration, and a transwell experiment was employed to evaluate cell invasion. Proteins related to the Wnt pathway were detected with Western blot analysis, including β-catenin, GSK-3 β, P-GSK-3 β, c-Myc, and CyclinD1. Results: The proliferation, invasion, and metastasis of A549 cells were significantly enhanced after being transfected with the β-catenin overexpression plasmid (P < 0.05 or 0.01, accompanied by increased expression of β-catenin, C-Myc, CyclinD1 and reduced expression of Gsk-3 β and P-GSK-3 β. Treatment of cells with TFCB resulted in inhibition of cell proliferation, migration, and invasion; downregulated expression of β-catenin, C-Myc, and CyclinD1; and upregulated expression of GSK-3 β and P-GSK-3 β, especially in the β-catenin overexpression group. Conclusion: TFCB has the potential to inhibit the Wnt/β-catenin pathway by prohibiting the overexpression of β-catenin and regulating its downstream factors.

Myc Decoy Oligodeoxynucleotide Inhibits Growth and Modulates Differentiation of Mouse Embryonic Stem Cells as a Model of Cancer Stem Cells.

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Johari, Behrooz; Ebrahimi-Rad, Mina; Maghsood, Faezeh; Lotfinia, Majid; Saltanatpouri, Zohreh; Teimoori-Toolabi, Ladan; Sharifzadeh, Zahra; Karimipoor, Morteza; Kadivar, Mehdi

2017-01-01

Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation.

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Song, Juhyun; Oh, Yumi; Kim, Jong Youl; Cho, Kyoung Joo; Lee, Jong Eun

2016-11-01

Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induced autophagy in a bovine kidney cell line

International Nuclear Information System (INIS)

Fiorito, Filomena; Ciarcia, Roberto; Granato, Giovanna Elvira; Marfe, Gabriella; Iovane, Valentina; Florio, Salvatore; De Martino, Luisa; Pagnini, Ugo

2011-01-01

Highlights: ► TCDD induces cell proliferation in MDBK cells. ► TCDD provokes morphological cell death in MDBK cells. ► TCDD induces autophagy in MDBK cells. ► TCDD induces a down-regulation of telomerase activity, bTERT, c-Myc in MDBK cells. ► TCDD induces an up-regulation of p53 and p21 protein levels in MDBK cells. -- Abstract: The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK) cells where no signs of apoptosis were observed, but herein, analysis of MDBK cell morphology, in a large number of exposed cells, revealed some alterations, as expanded cytoplasm, an increase of intercellular spaces and many pyknotic nuclei. Hence, the aim of the current study was to elucidate the influences of dioxin on cell proliferation and cell death. We found that dioxin increased proliferation, as well as, activated cell death with autophagy, as we detected by increased amount of LC3-II, an autophagosome marker. Furthermore, formation of acidic vesicular organelles was observed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. These results were accompanied by down-regulation of telomerase activity, bTERT and c-Myc. Key tumor-suppressor protein p53 and expression of cell cycle inhibitor p21Waf1/Cip1 were activated after TCDD exposure. These changes occurred with activation of ATM phosphorylation in the presence of a decrease in Mdm2 protein levels. Taken together, these results support the idea that TCDD in MDBK cells, may exert its action, in part, by enhancing cell proliferation, but also by modulating the incidence of induced cell death with autophagy.

The cucurbitacins D, E, and I from Ecballium elaterium (L. upregulate the LC3 gene and induce cell-cycle arrest in human gastric cancer cell line AGS

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Naser Jafargholizadeh

2018-03-01

Full Text Available Objective(s: Cucurbitacins exhibit a range of anti-cancer functions. We investigated the effects of cucurbitacins D, E, and I purified from Ecballium elaterium (L. A. Rich fruits on some apoptotic and autophagy genes in human gastric cancer cell line AGS. Materials and Methods: Using quantitative reverse transcription PCR (qRT-PCR, the expression of LC3, VEGF, BAX, caspase-3, and c-MYC genes were quantified in AGS cells 24 hr after treatment with cucurbitacins D, E, and I at concentrations 0.3, 0.1 and 0.5 μg/ml, respectively. Cell cycle and death were analyzed by flowcytometry. Results: Purified cucurbitacins induced sub-G1 cell-cycle arrest and cell death in AGS cells and upregulated LC3mRNA effectively, but showed a very low effect on BAX, caspase-3, and c-MYC mRNA levels. Also after treatment with cucurbitacin I at concentration 0.5 μg/ml, VEGF mRNA levels were increased about 4.4 times. Pairwise comparison of the effect of cucurbitacins D, E, and I on LC3 mRNA expression showed that the cucurbitacin I effect is 1.3 and 1.1 times that of cucurbitacins E and D, respectively; cucurbitacin D effect is 1.2 times that of cucurbitacin E (P-value

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis.

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Okada, Morihiro; Miller, Thomas C; Wen, Luan; Shi, Yun-Bo

2017-05-11

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc-Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad-Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development.

Replication confers β cell immaturity.

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Puri, Sapna; Roy, Nilotpal; Russ, Holger A; Leonhardt, Laura; French, Esra K; Roy, Ritu; Bengtsson, Henrik; Scott, Donald K; Stewart, Andrew F; Hebrok, Matthias

2018-02-02

Pancreatic β cells are highly specialized to regulate systemic glucose levels by secreting insulin. In adults, increase in β-cell mass is limited due to brakes on cell replication. In contrast, proliferation is robust in neonatal β cells that are functionally immature as defined by a lower set point for glucose-stimulated insulin secretion. Here we show that β-cell proliferation and immaturity are linked by tuning expression of physiologically relevant, non-oncogenic levels of c-Myc. Adult β cells induced to replicate adopt gene expression and metabolic profiles resembling those of immature neonatal β that proliferate readily. We directly demonstrate that priming insulin-producing cells to enter the cell cycle promotes a functionally immature phenotype. We suggest that there exists a balance between mature functionality and the ability to expand, as the phenotypic state of the β cell reverts to a less functional one in response to proliferative cues.

3D view to tumor suppression: Lkb1, polarity and the arrest of oncogenic c-Myc.

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Partanen, Johanna I; Nieminen, Anni I; Klefstrom, Juha

2009-03-01

Machiavelli wrote, in his famous political treatise Il Principe, about disrupting organization by planting seeds of dissension or by eliminating necessary support elements. Tumor cells do exactly that by disrupting the organized architecture of epithelial cell layers during progression from contained benign tumor to full-blown invasive cancer. However, it is still unclear whether tumor cells primarily break free by activating oncogenes powerful enough to cause chaos or by eliminating tumor suppressor genes guarding the order of the epithelial organization. Studies in Drosophila have exposed genes that encode key regulators of the epithelial apicobasal polarity and which, upon inactivation, cause disorganization of the epithelial layers and promote unscheduled cell proliferation. These polarity regulator/tumor suppressor proteins, which include products of neoplastic tumor suppressor genes (nTSGs), are carefully positioned in polarized epithelial cells to maintain the order of epithelial structures and to impose a restraint on cell proliferation. In this review, we have explored the presence and prevalence of somatic mutations in the human counterparts of Drosophila polarity regulator/tumor suppressor genes across the human cancers. The screen points out LKB1, which is a causal genetic lesion in Peutz-Jeghers cancer syndrome, a gene mutated in certain sporadic cancers and a human homologue of the fly polarity gene par-4. We review the evidence linking Lkb1 protein to polarity regulation in the scope of our recent results suggesting a coupled role for Lkb1 as an architect of organized acinar structures and a suppressor of oncogenic c-Myc. We finally present models to explain how Lkb1-dependent formation of epithelial architecture is coupled to suppression of normal and oncogene-induced proliferation.

Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains

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Ye, Shoudong; Tan, Li; Yang, Rongqing; Fang, Bo; Qu, Su; Schulze, Eric N.; Song, Houyan; Ying, Qilong; Li, Ping

2012-01-01

Background Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools. PMID:22540008

Mechanisms for the proliferation of eosinophilic leukemia cells by FIP1L1-PDGFRα

International Nuclear Information System (INIS)

Ishihara, Kenji; Kitamura, Hajime; Hiraizumi, Kenji; Kaneko, Motoko; Takahashi, Aki; Zee, OkPyo; Seyama, Toshio; Hong, JangJa; Ohuchi, Kazuo; Hirasawa, Noriyasu

2008-01-01

The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor α (PDGFRα) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFRα. In this study, we analyzed the mechanism by which FIP1L1-PDGFRα induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFRα inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFRα induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils

Radiation related basic cancer research : research for radiation induced tumor cell killing

International Nuclear Information System (INIS)

Lee, Seung Hoon; Hong, Seok Il; Cho, Kyung Ja; Kim, Byung Gi; Lee, Kee Ho; Nam, Myung Jin

1999-04-01

The radioresistant clones was established from human U251 glioblastoma cell line through intermittently exposed to 3 Gy gamma-radiation for six months. Treatment of SNU-16 cells with various doses of radiation, TNF alpha and PMA resulted in a decrease in cell viability. The results prove that cell death of SNU16 is a apoptosis mediated by caspase-3. We have examined the expression of bcl-2 and c-myc in cervical cancer specimens and cervical intraepithelial neoplasia (CIN) to determine the role of coexpression of bcl-3 and c-myc during progression into cervical cancer. The frequent alterations in FHIT expression in many cervical carcinomas and their cell lines suggest that FHIT gene alterations are pla a role in cervical tumorigenesis. According to these correlation between the viability and apoptosis of RD cells, the proper range of the dosage for the investigation of differentiation potency in RD cells was assessed as 1 to 3Gy

Radiation related basic cancer research : research for radiation induced tumor cell killing

Energy Technology Data Exchange (ETDEWEB)

Lee, Seung Hoon; Hong, Seok Il; Cho, Kyung Ja; Kim, Byung Gi; Lee, Kee Ho; Nam, Myung Jin

1999-04-01

The radioresistant clones was established from human U251 glioblastoma cell line through intermittently exposed to 3 Gy gamma-radiation for six months. Treatment of SNU-16 cells with various doses of radiation, TNF alpha and PMA resulted in a decrease in cell viability. The results prove that cell death of SNU16 is a apoptosis mediated by caspase-3. We have examined the expression of bcl-2 and c-myc in cervical cancer specimens and cervical intraepithelial neoplasia (CIN) to determine the role of coexpression of bcl-3 and c-myc during progression into cervical cancer. The frequent alterations in FHIT expression in many cervical carcinomas and their cell lines suggest that FHIT gene alterations are pla a role in cervical tumorigenesis. According to these correlation between the viability and apoptosis of RD cells, the proper range of the dosage for the investigation of differentiation potency in RD cells was assessed as 1 to 3Gy.

Mechanisms for c-myc Induced Mouse Mammary Gland Carcinogenesis and for the Synergistic Role of TGF(alpha) in the Process

Science.gov (United States)

2001-07-01

1242 11-28. anti-tumor effects with microencapsulated c-myc antisense Panico L, D’Antonio A, Salvatore G, Mezza E, Tortora G, De oligonucleotide... enzymatic conversion of androgens to estrogens, since an estrogen receptor antagonist cannot block the lobular- alveolar induction by T, DHT

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

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Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

2016-01-01

This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

OpenAIRE

Paquette, Y; Doyon, L; Laperrière, A; Hanna, Z; Ball, J; Sekaly, R P; Jolicoeur, P

1992-01-01

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to...

Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway.

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Youlin, Kuang; Li, Zhang; Weiyang, He; Jian, Kang; Siming, Liang; Xin, Gou

2017-03-01

Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

Cytokine signalling in embryonic stem cells

DEFF Research Database (Denmark)

Kristensen, David Møbjerg; Kalisz, Mark; Nielsen, Jens Høiriis

2006-01-01

Cytokines play a central role in maintaining self-renewal in mouse embryonic stem (ES) cells through a member of the interleukin-6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK-STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric...... pathways seem to converge on c-myc as a common target to promote self-renewal. Whereas LIF does not seem to stimulate self-renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling...... via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem...

Shikonin regulates C-MYC and GLUT1 expression through the MST1-YAP1-TEAD1 axis

Czech Academy of Sciences Publication Activity Database

Vališ, Karel; Talacko, Pavel; Grobárová, Valeria; Černý, J.; Novák, Petr

2016-01-01

Roč. 349, č. 2 (2016), s. 273-281 ISSN 0014-4827 R&D Projects: GA ČR(CZ) GP14-21095P; GA ČR GA13-16565S; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Hippo * Glycolysis * C-MYC Subject RIV: EE - Microbiology, Virology Impact factor: 3.546, year: 2016

Concomitant targeting of multiple key transcription factors effectively disrupts cancer stem cells enriched in side population of human pancreatic cancer cells.

Directory of Open Access Journals (Sweden)

Xiyan Wang

Full Text Available A major challenge in the treatment of pancreatic ductal adenocarcinoma is the failure of chemotherapy, which is likely due to the presence of the cancer stem cells (CSCs.To identify side population (SP cells and characterize s-like properties in human pancreatic cancer cell lines (h-PCCLs and to exploit the efficacy of concomitant targeting of multiple key transcription factors governing the stemness of pancreatic CSCs in suppressing CSC-like phenotypes.Flow cytometry and Hoechst 33342 DNA-binding dye efflux assay were used to sort SP and non-SP (NSP cells from three h-PCCLs: PANC-1, SW1990, and BxPc-3. The self-renewal ability, invasiveness, migration and drug resistance of SP cells were evaluated. Expression of CSC marker genes was analyzed. Tumorigenicity was assessed using a xenograft model in nude mice. Effects of a complex decoy oligonucleotide (cdODN-SCO designed to simultaneously targeting Sox2, Oct4 and c-Myc were assessed.CSCs were enriched in the side proportion (SP cells contained in the h-PCCLs and they possessed aggressive growth, invasion, migration and drug-resistance properties, compared with NSP cells. SP cells overexpressed stem cell markers CD133 and ALDH1, pluripotency maintaining factors Nanog, Sox2 and Oct4, oncogenic transcription factor c-Myc, signaling molecule Notch1, and drug resistant gene ABCG2. Moreover, SP cells consistently demonstrated significantly greater tumorigenicity than NSP cells in xenograft model of nude mice. CdODN-SOC efficiently suppressed all CSC properties and phenotypes, and minimized the tumorigenic capability of the SP cells and the resistance to chemotherapy. By comparison, the negative control failed to do so.The findings indicate that targeting the key genes conferring the stemness of CSCs can efficiently eliminate CSC-like phenotypes, and thus may be considered a new approach for cancer therapy. Specifically, the present study establishes the combination of Sox2/Oct4/c-Myc targeting as a

Disturbance of Bcl-2, Bax, Caspase-3, Ki-67 and C-myc expression in acute and subchronic exposure to benzo(a)pyrene in cervix.

Science.gov (United States)

Gao, Meili; Li, Yongfei; Ji, Xiaoying; Xue, Xiaochang; Chen, Lan; Feng, Guodong; Zhang, Huqin; Wang, Huichun; Shah, Walayat; Hou, Zhanwu; Kong, Yu

2016-03-01

Epidemiological studies have demonstrated that cigarette smoking is an important cofactor or an independent risk factor for the development of cervical cancer. Benzo(a)pyrene (BaP) is one of the most potent tobacco smoke carcinogens in tobacco smoke. BaP induced DNA damage and over expression in p53 cervical tissue of mice as demonstrated in our previous study. Here we present the findings of exposure to BaP on the expression of Bcl-2, C-myc, Ki-67, Caspase-3 and Bax genes in mouse cervix. Acute intraperitoneal administration of BaP (12.5, 25, 50, 100mg/kg body weight) to ICR female mice induced a significant increase in Bcl-2, C-myc, Ki-67 mRNA and protein level till 72h except in Bcl-2 at 24h with 12.5, 25, 50mg/kg as well as at 48h with 12.5mg/kg body weight post treatment. A significant increase was also seen in Caspase-3 and Bax mRNA and protein level with peak level at 24h and gradual decrease till 72h, however, the expression of caspase-3 increased while that of Bax decreased with increasing dose of Bap after 24h. In sub chronic intraperitoneal and oral gavage administration of BaP (2.5, 5, 10mg/kg body weight), similar significant increase was observed for all the examined genes as compared to the control and vehicle groups, however the expression of Bax decreased in a dose dependent manner. The findings of this study will help in further understanding the molecular mechanism of BaP induced carcinogenesis of cervical cancer. Copyright © 2015 Elsevier GmbH. All rights reserved.

Study on the proliferation of human gastric cancer cell AGS by activation of EGFR in H2O2.

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Wang, Q; Shen, W; Tao, G-Q; Sun, J; Shi, L-P

2017-03-01

This study is to investigate the effect of low concentration hydrogen peroxide (H2O2) on the proliferation of gastric cancer AGS cell line in vitro and the mechanism. AGS cells were treated with different low concentrations of H2O2 (1, 0.1, 0.01, and 0.001 μm) for 48 hours. The effect of H2O2 concentration gradient on the activity of AGS cell activities was detected by methyl thiazolyl tetrazolium (MTT) method. The expression of the epidermal growth factor receptor (EGFR) and its downstream signaling pathway extracellular signal-regulated kinase (ERK) protein in H2O2 was detected by Western blot method; moreover, the effect of H2O2 on intracellular reactive oxygen species (ROS) in AGS cells was observed under the fluorescence microscope and quantitative analysis by flow cytometry. The effect of H2O2 on the level of c-myc mRNA in AGS cells was also detected by reverse transcription polymerase chain reaction (RT-PCR). MTT detection results showed that 1 μm and 0.1 μm H2O2 at 48h can effectively promote the proliferation of AGS cells (pH2O2 treatment of AGS cells, the EGFR protein levels and ERK protein phosphorylation levels increased significantly (pH2O2 increased the intracellular reactive oxygen species (ROS). RT-PCR results showed the levels of c-myc mRNA in AGS cells treated with a low concentration of H2O2 were significantly increased (pH2O2 can significantly promote the proliferation of AGS cells by activating EGFR/ERK signaling pathway.

Apoptotic potential and cell sensitivity to fractionated radiotherapy

International Nuclear Information System (INIS)

Rupnow, Brent A.; Murtha, Albert D.; Alarcon, Rodolfo M.; Giaccia, Amato J.; Knox, Susan J.

1997-01-01

Purpose/Objective: At present, the relationship between sensitivity to radiation-induced apoptosis and overall cellular radiosensitivity remains unclear. In particular, the relationship of apoptotic sensitivity to the survival of cells following fractionated irradiation has not been well studied. The purpose of the present study was to determine if increasing cell sensitivity to radiation-induced apoptosis would result in decreased clonogenic survival following single dose and fractionated irradiation in vitro. Materials and Methods: To address this, we chose a cell line (Rat-1MycER) in which the sensitivity to radiation-induced apoptosis could be altered by switching on or off the activity of a conditional c-Myc allele (c-MycER). The c-MycER construct expresses a full length c-Myc protein fused to a modified hormone binding domain of the estrogen receptor. Only in the presence of the estrogen analog 4-hydroxytamoxifen (4HT), does the conditional c-MycER become active. Apoptosis following irradiation in these cells (with and without c-MycER activation) was analyzed by flow cytometry to determine the percentage of cells undergoing apoptosis following various radiation doses and at different times after irradiation. Additionally, clonogenic survival analysis was performed following single radiation doses from 0 to 10 Gy and following five fractions of 2 or 4 Gy each. Survival of cells with and without c-MycER activation was compared. Furthermore, the effect of overexpressing the anti-apoptotic Bcl-2 gene on apoptosis induction and clonogenic survival of these cells was examined. Results: Rat-1MycER cells were strongly sensitized to radiation-induced apoptosis in a dose and time dependent manner when MycER was activated relative to cells treated without c-MycER activation. This c-Myc-mediated sensitivity to radiation-induced apoptosis was suppressed by overexpression of the anti-apoptotic protein Bcl-2. In addition to increasing apoptosis, activating c-MycER prior to

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma Irradiation

Science.gov (United States)

2014-12-01

the nucleolus fol- lowed by their transport into the cytoplasm (50). This process requires coordinated transcription catalyzed by all three RNA...these RPs, including L11, are released from the nucleolus or from intact ribosomes to suppress MDM2 (68). However, whether L11 suppresses c-Myc in...centrifugation. For isolation of the nucleolus fraction, the nuclear pellets were resuspended in buffer S1 containing 0.25 M sucrose and 10 mM MgCl2, layered over

Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

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Budd A Tucker

2011-04-01

Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

DEFF Research Database (Denmark)

Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

2010-01-01

Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

Mechanisms involved in growth inhibition induced by clofibrate in hepatoma cells

International Nuclear Information System (INIS)

Muzio, Giuliana; Maggiora, Marina; Trombetta, Antonella; Martinasso, Germana; Reffo, Patrizia; Colombatto, Sebastiano; Canuto, Rosa Angela

2003-01-01

Low concentrations of some peroxisome proliferators have been found to decrease apoptosis in rat liver cells, whereas higher but pharmacological concentrations have been found to inhibit cell proliferation or to induce apoptosis in human and rat hepatoma cells. The highly deviated JM2 rat hepatoma cell line was used to examine the mechanisms underlying the inhibitory effect on cell proliferation. Clofibrate chiefly inhibited cell proliferation in these cells. Parallel to the decrease in cell proliferation there was an increase of peroxisome proliferator activated receptor (PPAR) gamma and of protein phosphatase 2A, whose importance was confirmed, respectively, by using antisense oliginucleotides (AS-ODN) or okadaic acid. The increase of protein phosphatase 2A induced by PPARgamma caused a decrease of MAPK, an intracellular signaling transduction pathway, as shown by evaluation of Erk1,2 and c-myc. In light of these results, clofibrate, like conventional synthetic ligands of PPARgamma, may be regarded as a possible prototype anti-tumour drug

CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model

International Nuclear Information System (INIS)

Imayoshi, Natsuki; Yoshioka, Makoto; Chauhan, Jay; Nakata, Susumu; Toda, Yuki; Fletcher, Steven; Strovel, Jeffrey W.; Takata, Kazuyuki; Ashihara, Eishi

2017-01-01

Multiple myeloma (MM) is characterized by the clonal proliferation of neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC 50 value of 1.1 μM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250 inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM. - Highlights: • A novel bromodomain inhibitor CG13250 suppresses MM cell proliferation. • CG13250 decreases C-MYC expression, resulting in the induction of apoptosis. • CG13250 prolongs the survivals of MM-bearing mice.

Cellular and molecular studies on ataxia-telangiectasia lymphoblastoid cell lines

International Nuclear Information System (INIS)

Fiorilli, M.; Crescenzi, M.; Carbonari, M.; Russo, G.; Businco, L.; Aiuti, F.

1985-01-01

We have examined several AT-related lesions in lymphoblastoid cell lines (LCLs) derived from AT patients. Diminished sensitivity to gamma-irradiation was found in six of seven AT-LCLs. A seventh line, from a patient with apparently normal T-cell immunity, responded normally following radiation. Constitutive proteins from exponentially growing AT-LCLs were assessed by SDS-PAGE analysis and did not differ significantly from normals. IgM synthesis was also normal except for one AT-LCL that contained native IgM molecules of different sizes, corresponding to the presence of pentamers and oligomers. Analysis under reducing conditions showed normal-sized secretory mu-chains. Finally, we examined mRNAs corresponding to two oncogenes, c-myc and c-myb, in AT and normal LCLs and found marked overproduction of c-myc in one AT-LCL (i.e., ATL6). The latter findings suggest that AT cells might be prone to aberrantly express cellular oncogenes as a result of chromosomal instability and consequent transposition of oncogenes

Chicken Induced Pluripotent Stem Cells: Establishment and Characterization.

Science.gov (United States)

Fuet, Aurelie; Pain, Bertrand

2017-01-01

In mammals, the introduction of the OSKM (Oct4, Sox2, Klf4, and c-Myc) genes into somatic cells has allowed generating induced pluripotent stem (iPS) cells. So far, this process has been only clearly demonstrated in mammals. Here, using chicken as an avian model, we describe a set of protocols allowing the establishment, characterization, maintenance, differentiation, and injection of putative reprogrammed chicken Induced Pluripotent Stem (iPS) cells.

Melatonin promotes circadian rhythm-induced proliferation through Clock/histone deacetylase 3/c-Myc interaction in mouse adipose tissue.

Science.gov (United States)

Liu, Zhenjiang; Gan, Lu; Luo, Dan; Sun, Chao

2017-05-01

Melatonin is synthesized in the pineal gland and controls circadian rhythm of peripheral adipose tissue, resulting in changes in body weight. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms of circadian rhythm-mediated proliferation in adipose tissue is still limited. Here, we showed that melatonin (20 mg/kg/d) promoted circadian and proliferation processes in white adipose tissue. The circadian amplitudes of brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1, Pcircadian locomotor output cycles kaput (Clock, Pcircadian disruption and promoted adipocyte proliferation in chronic jet-lagged mice and obese mice. Thus, our study found that melatonin promoted adipocyte proliferation by forming a Clock/HDAC3/c-Myc complex and subsequently driving the circadian amplitudes of proliferation genes. Our data reveal a novel mechanism that links circadian rhythm to cell proliferation in adipose tissue. These findings also identify a new potential means for melatonin to prevent and treat sleep deprivation-caused obesity. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

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Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

2010-12-10

Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

International Nuclear Information System (INIS)

Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

2010-01-01

Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

Targeting human c-Myc promoter duplex DNA with actinomycin D by use of multi-way analysis of quantum-dot-mediated fluorescence resonance energy transfer

DEFF Research Database (Denmark)

Gholami, Somayeh; Kompany Zare, Mohsen

2013-01-01

Actinomycin D (Act D), an oncogenic c-Myc promoter binder, interferes with the action of RNA polymerase. There is great demand for high-throughput technology able to monitor the activity of DNA-binding drugs. To this end, binding of 7-aminoactinomycin D (7AAD) to the duplex c-Myc promoter...... pairs resulted in efficient energy transfer from drug to QD via fluorescence resonance energy transfer (FRET). Multi-way analysis of the three-way data array obtained from titration experiments was performed by use of restricted Tucker3 and hard trilinear decomposition (HTD). These techniques enable...... the important advantage over univariate classical methods of enabling us to investigate the source of variance in the fluorescence signal of the DNA-drug complex. It was established that hard trilinear decomposition analysis of FRET-measured data overcomes the problem of rank deficiency, enabling calculation...

NOTCH1 Is Aberrantly Activated in Chronic Lymphocytic Leukemia Hematopoietic Stem Cells

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Mauro Di Ianni

2018-04-01

Full Text Available To investigate chronic lymphocytic leukemia (CLL-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs. In NOTCH1-mutated CLL, we detected subclonal mutations in 57% CD34+/CD38− HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38− and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.

NOTCH1 Is Aberrantly Activated in Chronic Lymphocytic Leukemia Hematopoietic Stem Cells.

Science.gov (United States)

Di Ianni, Mauro; Baldoni, Stefano; Del Papa, Beatrice; Aureli, Patrizia; Dorillo, Erica; De Falco, Filomena; Albi, Elisa; Varasano, Emanuela; Di Tommaso, Ambra; Giancola, Raffaella; Accorsi, Patrizia; Rotta, Gianluca; Rompietti, Chiara; Silva Barcelos, Estevão Carlos; Campese, Antonio Francesco; Di Bartolomeo, Paolo; Screpanti, Isabella; Rosati, Emanuela; Falzetti, Franca; Sportoletti, Paolo

2018-01-01

To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1- mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.

Down-regulation of 5S rRNA by miR-150 and miR-383 enhances c-Myc-rpL11 interaction and inhibits proliferation of esophageal squamous carcinoma cells.

Science.gov (United States)

Wang, Xinyu; Ren, Yanli; Wang, Zhiqiong; Xiong, Xiangyu; Han, Sichong; Pan, Wenting; Chen, Hongwei; Zhou, Liqing; Zhou, Changchun; Yuan, Qipeng; Yang, Ming

2015-12-21

5S rRNA plays an important part in ribosome biology and is over-expression in multiple cancers. In this study, we found that 5S rRNA is a direct target of miR-150 and miR-383 in esophageal squamous cell carcinoma (ESCC). Overexpression of miR-150 and miR-383 inhibited ESCC cell proliferation in vitro and in vivo. Moreover, 5S rRNA silencing by miR-150 and miR-383 might intensify rpL11-c-Myc interaction, which attenuated role of c-Myc as an oncogenic transcriptional factor and dysregulation of multiple c-Myc target genes. Taken together, our results highlight the involvement of miRNAs in ribosomal regulation during tumorigenesis. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Lipopolysaccharide stimulates endogenous β-glucuronidase via PKC/NF-κB/c-myc signaling cascade: a possible factor in hepatolithiasis formation.

Science.gov (United States)

Yao, Dianbo; Dong, Qianze; Tian, Yu; Dai, Chaoliu; Wu, Shuodong

2017-11-29

Hepatolithiasis is commonly encountered in Southeastern and Eastern Asian countries, but the pathogenesis mechanism of stone formation is still not well understood. Now, the role of endogenous β-glucuronidase in pigment stones formation is being gradually recognized. In this study, the mechanism of increased expression and secretion of endogenous β-glucuronidase during hepatolithiasis formation was investigated. We assessed the endogenous β-glucuronidase, c-myc, p-p65, and p-PKC expression in liver specimens with hepatolithiasis by immunohistochemical staining, and found that compared with that in normal liver samples, the expression of endogenous β-glucuronidase, c-myc, p-p65, and p-PKC in liver specimens with hepatolithiasis significantly increased, and their expressions were positively correlated with each other. Lipopolysaccharide (LPS) induced increased expression of endogenous β-glucuronidase and c-myc in hepatocytes and intrahepatic biliary epithelial cells in a dose- and time-dependent manner, and endogenous β-glucuronidase secretion increased, correspondingly. C-myc siRNA transfection effectively inhibited the LPS-induced expression of endogenous β-glucuronidase. Furthermore, NF-κB inhibitor pyrrolidine dithiocarbamate or PKC inhibitor chelerythrine could effectively inhibit the LPS-induced expression of c-myc and endogenous β-glucuronidase, and the expression of p-p65 was also partly inhibited by chelerythrine. Our clinical observations and experimental data indicate that LPS could induce the increased expression and secretion of endogenous β-glucuronidase via a signaling cascade of PKC/NF-κB/c-myc in hepatocytes and intrahepatic biliary epithelial cells, and endogenous β-glucuronidase might play a possible role in the formation of hepatolithiasis.

Up-regulation of microRNA-1290 impairs cytokinesis and affects the reprogramming of colon cancer cells.

Science.gov (United States)

Wu, Jia; Ji, Xiaowei; Zhu, Linlin; Jiang, Qiaoli; Wen, Zhenzhen; Xu, Song; Shao, Wei; Cai, Jianting; Du, Qin; Zhu, Yongliang; Mao, Jianshan

2013-02-28

Abnormal cytokinesis increases the possibility of nuclear fusion in tumor cells. However, the role of microRNAs (miRNAs) in abnormal cytokinesis is unclear. Here, we found that miR-1290 was significantly up-regulated in clinical colon cancer tissues. Up-regulation of miR-1290 postponed cytokinesis and led to the formation of multinucleated cells. KIF13B was a target of miR-1290 that was involved in aberrant cytokinesis. Furthermore, enforced expression of miR-1290 activated the Wnt pathway and increased the reprogramming-related transcript factors c-Myc and Nanog. Our results suggest that up-regulation of miR-1290 in colon cancer cells impaired cytokinesis and affected reprogramming. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

Directory of Open Access Journals (Sweden)

Anna Frenzel

Full Text Available Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C. Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

Gene expression in skin tumors induced in hairless mice by chronic exposure to ultraviolet B irradiation

International Nuclear Information System (INIS)

Sato, Hiromi; Tanaka, Misao; Kobayashi, Shizuko; Suzuki, Junko S.; Ogiso, Manabu; Tohyama, Chiharu

1997-01-01

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumours induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m 2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumour appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumours, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m 2 ) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis of immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wide type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice. (author)

Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells

Directory of Open Access Journals (Sweden)

Liu Y

2016-06-01

Full Text Available Ying Liu,1,2,* Baocun Sun,1–3,* Tieju Liu,1,2,* Xiulan Zhao,1,2 Xudong Wang,3 Yanlei Li,1,2 Jie Meng,2 Qiang Gu,1,2 Fang Liu,1,2 Xueyi Dong,1,2 Peimei Liu,2 Ran Sun,2 Nan Zhao1 1Department of Pathology, General Hospital of Tianjin Medical University, 2Department of Pathology, Tianjin Medical University, 3Department of Pathology, Cancer Hospital of Tianjin Medical University, Tianjin, People’s Republic of China *These authors contributed equally to this work Abstract: Tumor cell vasculogenic mimicry (VM, a newly defined pattern of tumor blood supply, signifies the functional plasticity of aggressive cancer cells forming vascular networks. VM and cancer stem cells (CSCs have been shown to be associated with tumor growth, local invasion, and distant metastasis. In our previous study, CSCs in triple-negative breast cancer were potential to participate in VM formation. In this study, breast CSCs were isolated from the triple-negative breast cancer cell line MDA-MB-231 by using mammosphere culture. Western blotting and reverse transcription polymerase chain reaction showed that mammosphere cells displayed an increased expression of AURKA protein kinase and stem cell marker c-myc and sox2. The VM formation by mammosphere cells was inhibited by AURKA knockdown or the addition of AURKA inhibitor MLN8237. In the meantime, MLN8237 induced the increased E-cadherin and decreased c-myc, sox2, and β-catenin expressions. The function of AURKA in VM formation was further confirmed using a xenograft-murine model. The results suggested that AURKA protein kinase is involved in VM formation of CSCs and may become a new treatment target in suppressing VM and metastasis of breast cancer. Keywords: AURKA, cancer stem cells, vasculogenic mimicry, breast cancer

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

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Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

Directory of Open Access Journals (Sweden)

Syed Mohmad Shah

2015-11-01

Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

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Gonsalves, Wilson I.; Hitosugi, Taro; Ghosh, Toshi; Jevremovic, Dragan; Petterson, Xuan-Mai; Wellik, Linda; Kumar, Shaji K.; Nair, K. Sreekumaran

2018-01-01

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention. PMID:29321378

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

OpenAIRE

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

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors 1,2 . 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 linea...

The A-myb transcription factor in neoplastic and normal B cells.

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Golay, J; Facchinetti, V; Ying, G; Introna, M

1997-07-01

The myb family of transcription factors has been strongly implicated in the regulation of cell growth and differentiation in the haematopoietic system. The v-myb oncogene, carried by avian defective retroviruses, causes leukaemias in the chicken and transforms haematopoietic cells in vitro. Its normal cellular equivalent c-myb, has been shown to promote the proliferation and block the differentiation of haematopoietic cells in several experimental models and is required for fetal haematopoiesis. Two other members of the family have been cloned more recently, A-myb and B-myb, which show sequence homology with c-myb in several domains, of which the DNA binding domain as well as other regulatory domains. Both have been shown to be transcription factors. B-myb is also involved in the control of proliferation and differentiation, but, unlike c-myb, it is expressed in many cell types. The third member of the family, A-myb, shows the most restricted pattern of expression, suggesting a very specific role for this transcription factor. A-myb is expressed in a subpopulation of normal B lymphocytes activated in vivo and localised in the germinal center of peripheral lymphoid organs and is not detected at significant levels in all other mature or immature haematopoietic populations studied, including bone marrow cells, T lymphocytes, granulocytes, monocytes, either at rest or after in vitro activation. These studies indicate that A-myb plays a role during a narrow window of normal B cell differentiation. A-myb expression has also been studied in a wide range of neoplastic B cells, representing the whole spectrum of B cell differentiation. A-myb is strongly expressed in Burkitt's lymphomas (BL) and slg+ B-acute lymphoblastic leukaemias (B-ALL) and not in all other leukaemias/lymphomas tested, with the exception of a subset of CLL (about 25% of cases). It is intriguing that the A-myb genome has been localised relatively close to the c-myc gene on chromosome 8, suggesting that

4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and β in breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Ronghe, Amruta; Chatterjee, Anwesha [Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108 (United States); Singh, Bhupendra [Department of Genetics, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Dandawate, Prasad [ISTRA, Department of Chemistry, Abeda Inamdar Senior College, University of Pune (India); Abdalla, Fatma; Bhat, Nimee K. [Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108 (United States); Padhye, Subhash [ISTRA, Department of Chemistry, Abeda Inamdar Senior College, University of Pune (India); Bhat, Hari K., E-mail: bhath@umkc.edu [Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108 (United States)

2016-06-15

Breast cancer is a public health concern worldwide. Prolonged exposure to estrogens has been implicated in the development of breast neoplasms. Epidemiologic and experimental evidence suggest a chemopreventive role of phytoestrogens in breast cancers. Resveratrol, a naturally occurring phytoestrogen, has been shown to have potent anti-cancer properties. However, poor efficacy and bioavailability have prevented the use of resveratrol in clinics. In order to address these problems, we have synthesized a combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the proliferation of breast cancer cells. We have recently shown that 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), has better anti-cancer properties than resveratrol and any other resveratrol analog we have synthesized so far. The objective of this study was to investigate the regulation of estrogen receptors (ERs) α and β by TIMBD in breast cancer cell lines. We demonstrate that TIMBD significantly induces the mRNA and protein expression levels of ERβ and inhibits that of ERα. TIMBD inhibits mRNA and protein expression levels of oncogene c-Myc, and cell cycle protein cyclin D1, which are important regulators of cellular proliferation. TIMBD significantly induces protein expression levels of tumor suppressor genes p53 and p21 in MCF-7 cells. TIMBD inhibits c-Myc in an ERβ-dependent fashion in MCF-10 A and ERβ1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this analog. ERβ plays a partial role in inhibition of proliferation by TIMBD while ERα overexpression does not significantly affect TIMBD's inhibition. - Highlights: • Resveratrol analog TIMBD inhibits growth of breast cancer cells. • TIMBD induces protein expression levels of ERβ and inhibits that of ERα. • TIMBD inhibits c-Myc and cyclin D1, and induces p53 and p21. • TIMBD suppresses c-Myc in an ER-dependent fashion.

4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and β in breast cancer cells

International Nuclear Information System (INIS)

Ronghe, Amruta; Chatterjee, Anwesha; Singh, Bhupendra; Dandawate, Prasad; Abdalla, Fatma; Bhat, Nimee K.; Padhye, Subhash; Bhat, Hari K.

2016-01-01

Breast cancer is a public health concern worldwide. Prolonged exposure to estrogens has been implicated in the development of breast neoplasms. Epidemiologic and experimental evidence suggest a chemopreventive role of phytoestrogens in breast cancers. Resveratrol, a naturally occurring phytoestrogen, has been shown to have potent anti-cancer properties. However, poor efficacy and bioavailability have prevented the use of resveratrol in clinics. In order to address these problems, we have synthesized a combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the proliferation of breast cancer cells. We have recently shown that 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), has better anti-cancer properties than resveratrol and any other resveratrol analog we have synthesized so far. The objective of this study was to investigate the regulation of estrogen receptors (ERs) α and β by TIMBD in breast cancer cell lines. We demonstrate that TIMBD significantly induces the mRNA and protein expression levels of ERβ and inhibits that of ERα. TIMBD inhibits mRNA and protein expression levels of oncogene c-Myc, and cell cycle protein cyclin D1, which are important regulators of cellular proliferation. TIMBD significantly induces protein expression levels of tumor suppressor genes p53 and p21 in MCF-7 cells. TIMBD inhibits c-Myc in an ERβ-dependent fashion in MCF-10 A and ERβ1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this analog. ERβ plays a partial role in inhibition of proliferation by TIMBD while ERα overexpression does not significantly affect TIMBD's inhibition. - Highlights: • Resveratrol analog TIMBD inhibits growth of breast cancer cells. • TIMBD induces protein expression levels of ERβ and inhibits that of ERα. • TIMBD inhibits c-Myc and cyclin D1, and induces p53 and p21. • TIMBD suppresses c-Myc in an ER-dependent fashion.

Effects of short-hairpin RNA-inhibited {beta}-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Liang, Wenqing, E-mail: liangwenqing_1234@126.com [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Yang, Chengwei [Department of Spinal Surgery, Lanzhou General Hospital, Lanzhou Military Area Command, 333 Nanbinhe Road, Lanzhou 730050 (China); Qian, Yu [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Fu, Qiang, E-mail: chyygklwq@hotmail.com [Department of Orthopaedics, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433 (China)

2012-06-15

Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.

Clinical Significance of "Double-hit" and "Double-protein" expression in Primary Gastric B-cell Lymphomas.

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He, Miaoxia; Chen, Keting; Li, Suhong; Zhang, Shimin; Zheng, Jianming; Hu, Xiaoxia; Gao, Lei; Chen, Jie; Song, Xianmin; Zhang, Weiping; Wang, Jianmin; Yang, Jianmin

2016-01-01

Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. "Double-hit" and "double-protein" involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the "double-hit" and "double-protein" in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas. Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions. There was no definite "double-hit" case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the "double-protein" expression of MYC and BCL2/BCL6. All patients with "double-protein" gastric B-cell lymphomas had poor outcome compared with those without. More importantly, "MYC-BCL2-BCL6" negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better

Cytological and oncogene alterations in radiation-transformed Syrian hamster embryo cells

International Nuclear Information System (INIS)

Trutschler, K.; Hieber, L.; Kellerer, A.M.

1991-01-01

Syrian hamster embryo (SHE) cells were neoplastically transformed by different types of ionizing radiation (γ-rays, α-particles or carbon ions). Transformed and tumor cell lines (derived from nude mice tumors) were analysed for alterations of the oncogenes c-Ha-ras and c-myc, i.e. RFLPs, gene amplifications, activation by point mutation, gene expression, and for cytological changes. In addition, the chromosome number and the numbers of micronuclei per cell have been determined in a series of cell lines. (author)

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

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Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

2016-06-01

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Regulation of TFIIIB during F9 cell differentiation.

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Athineos, Dimitris; Marshall, Lynne; White, Robert J

2010-03-12

Differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE) provides a tractable model system for studying molecular events during early and inaccessible stages of murine development. PE formation is accompanied by extensive changes in gene expression both in vivo and in culture. One of the most dramatic is the ~10-fold decrease in transcriptional output by RNA polymerase (pol) III. This has been attributed to changes in activity of TFIIIB, a factor that is necessary and sufficient to recruit pol III to promoters. The goal of this study was to identify molecular changes that can account for the low activity of TFIIIB following F9 cell differentiation. Three essential subunits of TFIIIB decrease in abundance as F9 cells differentiate; these are Brf1 and Bdp1, which are pol III-specific, and TBP, which is also used by pols I and II. The decreased levels of Brf1 and Bdp1 proteins can be explained by reduced expression of the corresponding mRNAs. However, this is not the case for TBP, which is regulated post-transcriptionally. In proliferating cells, pol III transcription is stimulated by the proto-oncogene product c-Myc and the mitogen-activated protein kinase Erk, both of which bind to TFIIIB. However, c-Myc levels fall during differentiation and Erk becomes inactive through dephosphorylation. The diminished abundance of TFIIIB is therefore likely to be compounded by changes to these positive regulators that are required for its full activity. In addition, PE cells have elevated levels of the retinoblastoma protein RB, which is known to bind and repress TFIIIB. The low activity of TFIIIB in PE can be attributed to a combination of changes, any one of which could be sufficient to inhibit pol III transcription. Declining levels of essential TFIIIB subunits and of activators that are required for maximal TFIIIB activity are accompanied by an increase in a potent repressor of TFIIIB. These events provide fail-safe guarantees to ensure that pol III

Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1

International Nuclear Information System (INIS)

Wierstra, Inken; Alves, Juergen

2008-01-01

FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27

Human embryonic stem cells handbook

Directory of Open Access Journals (Sweden)

Carlo Alberto Redi

2013-03-01

Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

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Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

2014-01-01

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

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Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

[Molecular mechanism of cisplatin to enhance the ability of TRAIL in reversing multidrug resistance in gastric cancer cells].

Science.gov (United States)

Zhu, Xingchao; Zhang, Kaiguang; Wang, Qiaomin; Chen, Si; Gou, Yawen; Cui, Yufang; Li, Qin

2015-06-01

To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells. MTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis. After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells.

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Yanting Xue

Full Text Available Induced pluripotent stem cell (iPS cell holds great potential for applications in regenerative medicine, drug discovery, and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency, offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study, we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research, facilitating future applications of human iPS cells.

Dual roles for coactivator activator and its counterbalancing isoform coactivator modulator in human kidney cell tumorigenesis.

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Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y; Tsai, Ming-Jer; O'Malley, Bert W

2008-10-01

Coactivator activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein, we show that CoAA is a dual-function coregulator that inhibits G(1)-S transition in human kidney cells and suppresses anchorage-independent growth and xenograft tumor formation. Suppression occurs in part by down-regulating c-myc and its downstream effectors ccnd1 and skp2 and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, coactivator modulator (CoAM), antagonizes CoAA-induced G(1)-S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma compared with normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice isoform. This is, thus far, the only example of a nuclear receptor coregulator involved in suppression of kidney cancer and suggests potentially significant new roles for coregulators in renal cancer biology.

Dual roles for CoAA and its counterbalancing isoform CoAM in human kidney cell tumorigenesis

Science.gov (United States)

Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y.; Tsai, Ming-Jer; W. O’Malley, Bert

2008-01-01

Co-Activator Activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein we show that CoAA is a dual-function coregulator that inhibits G1/S transition in human kidney cells and suppresses anchorage independent growth and xenograft tumor formation. Suppression occurs in part by downregulating c-myc and its downstream effectors ccnd1 and skp2, and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene, c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, Coactivator Modulator (CoAM), antagonizes CoAA-induced G1/S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma as compared to normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice-isoform. This is so far the only example of a nuclear receptor coregulator involved in suppression of kidney cancer, and suggests potentially significant new roles for coregulators in renal cancer biology. PMID:18829545

Antileukemic Potential of Momordica charantia Seed Extracts on Human Myeloid Leukemic HL60 Cells

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Ramani Soundararajan

2012-01-01

Full Text Available Momordica charantia (bitter gourd has been used in the traditional system of medicine for the treatment of various diseases. Anticancer activity of M. charantia extracts has been demonstrated by numerous in vitro and in vivo studies. In the present study, we investigated the differentiation inducing potential of fractionated M. charantia seed extracts in human myeloid HL60 cells. We found that the HL60 cells treated with the fractionated seed extracts differentiated into granulocytic lineage as characterized by NBT staining, CD11b expression, and specific esterase activity. The differentiation inducing principle was found to be heat-stable, and organic in nature. The differentiation was accompanied by a downregulation of c-myc transcript, indicating the involvement of c-myc pathway, at least in part, in differentiation. Taken together these results indicate that fractionated extracts of M. charantia seeds possess differentiation inducing activity and therefore can be evaluated for their potential use in differentiation therapy for leukemia in combination with other inducers of differentiation.

Antileukemic Potential of Momordica charantia Seed Extracts on Human Myeloid Leukemic HL60 Cells

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Soundararajan, Ramani; Prabha, Punit; Rai, Umesh; Dixit, Aparna

2012-01-01

Momordica charantia (bitter gourd) has been used in the traditional system of medicine for the treatment of various diseases. Anticancer activity of M. charantia extracts has been demonstrated by numerous in vitro and in vivo studies. In the present study, we investigated the differentiation inducing potential of fractionated M. charantia seed extracts in human myeloid HL60 cells. We found that the HL60 cells treated with the fractionated seed extracts differentiated into granulocytic lineage as characterized by NBT staining, CD11b expression, and specific esterase activity. The differentiation inducing principle was found to be heat-stable, and organic in nature. The differentiation was accompanied by a downregulation of c-myc transcript, indicating the involvement of c-myc pathway, at least in part, in differentiation. Taken together these results indicate that fractionated extracts of M. charantia seeds possess differentiation inducing activity and therefore can be evaluated for their potential use in differentiation therapy for leukemia in combination with other inducers of differentiation. PMID:22654956

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

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Zhang, Yali; Guo, Zonglou; Xu, Lihong

2014-03-01

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

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Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2017-03-01

The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox ( Nanog ), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog ( c-Myc ), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc , were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

Generation of iPSC lines from primary human chorionic villi cells

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Björn Lichtner

2015-11-01

Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

Non-invasive bioluminescence imaging to monitor the immunological control of a plasmablastic lymphoma-like B cell neoplasia after hematopoietic cell transplantation.

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Martin Chopra

Full Text Available To promote cancer research and to develop innovative therapies, refined pre-clinical mouse tumor models that mimic the actual disease in humans are of dire need. A number of neoplasms along the B cell lineage are commonly initiated by a translocation recombining c-myc with the immunoglobulin heavy-chain gene locus. The translocation is modeled in the C.129S1-Igha(tm1(MycJanz/J mouse which has been previously engineered to express c-myc under the control of the endogenous IgH promoter. This transgenic mouse exhibits B cell hyperplasia and develops diverse B cell tumors. We have isolated tumor cells from the spleen of a C.129S1-Igha(tm1(MycJanz/J mouse that spontaneously developed a plasmablastic lymphoma-like disease. These cells were cultured, transduced to express eGFP and firefly luciferase, and gave rise to a highly aggressive, transplantable B cell lymphoma cell line, termed IM380. This model bears several advantages over other models as it is genetically induced and mimics the translocation that is detectable in a number of human B cell lymphomas. The growth of the tumor cells, their dissemination, and response to treatment within immunocompetent hosts can be imaged non-invasively in vivo due to their expression of firefly luciferase. IM380 cells are radioresistant in vivo and mice with established tumors can be allogeneically transplanted to analyze graft-versus-tumor effects of transplanted T cells. Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival. These traits make the IM380 model very valuable for the study of B cell lymphoma pathophysiology and for the development of innovative cancer therapies.

mRNA in exosomas as a liquid biopsy in non-Hodgkin Lymphoma: a multicentric study by the Spanish Lymphoma Oncology Group.

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Provencio, Mariano; Rodríguez, Marta; Cantos, Blanca; Sabín, Pilar; Quero, Cristina; García-Arroyo, Francisco R; Rueda, Antonio; Maximiano, Constanza; Rodríguez-Abreu, Delvys; Sánchez, Antonio; Silva, Javier; García, Vanesa

2017-08-01

To determine the feasibility of mRNAs ( C-MYC, BCL-XL, BCL-6, NF-κβ, PTEN and AKT ) in exosomes of plasma as a liquid biopsy method for monitoring and prognostic evolution in B-cell lymphomas. Exosomes were isolated from 98 patients with B-cell Lymphoma and 68 healthy controls. mRNAs were analyzed by quantitative PCR. An additional 31 post-treatment samples were also studied. In the general and follicular lymphoma series, the presence of AKT mRNA was associated with poor response to rituximab-based treatment. Patients with first relapse or disease progression showed a lower percentage of PTEN and BCL-XL mRNA. The presence of BCL-6 mRNA was associated with a high death rate. The absence of PTEN mRNA in the general series, and presence of C-MYC mRNA in follicular lymphomas, were associated with short progression-free survival. BCL-6 and C-MYC mRNA were independent prognostic variables of overall survival. C-MYC mRNA may provide prognostic information with respect to overall survival. BCL-XL mRNA and increase of BCL-6 mRNA in post-treatment samples could serve as molecular monitoring markers. This is the first large study to evaluate the prognostic and predictive values of pretreatment tumor-associated mRNA in exosomes. BCL-6 and C-MYC mRNA positivity in pretreatment samples were predictors of worse PFS compared to patients with mRNA negativity. C-MYC mRNA positivity was also a statistically significant predictor of inability to obtain complete response with first-line therapy.

Creatine Enhances Transdifferentiation of Bone Marrow Stromal Cell-Derived Neural Stem Cell Into GABAergic Neuron-Like Cells Characterized With Differential Gene Expression.

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Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Taheri, Taher; Hassoun, Hayder K

2017-04-01

Creatine was reported to induce bone marrow stromal cells (BMSC) into GABAergic neuron-like cells (GNLC). In a previous study, creatine was used as a single inducer for BMSC into GNLC with low yield. In this study, BMSC-derived neurospheres (NS) have been used in generating GABAergic phenotype. The BMSC were isolated from adult rats and used in generating neurospheres and used for producing neural stem cells (NSC). A combination of all-trans-retinoic acid (RA), the ciliary neurotrophic factor (CNTF), and creatine was used in order to improve the yield of GNLC. We also used other protocols for the transdifferentiation including RA alone; RA and creatine; RA and CNTF; and RA, CNTF, and creatine. The BMSC, NSC, and GNLC were characterized by specific markers. The activity of the GNLC was evaluated using FM1-43. The isolated BMSC expressed Oct4, fibronectin, and CD44. The NS were immunoreactive to nestin and SOX2, the NSC were immunoreactive to nestin, NF68 and NF160, while the GNLC were immunoreactive to GAD1/2, VGAT, GABA, and synaptophysin. Oct4 and c-MYC, pluripotency genes, were expressed in the BMSC, while SOX2 and c-MYC were expressed in the NSC. The activity of GNLC indicates that the synaptic vesicles were released upon stimulation. The conclusion is that the combination of RA, CNTF, and creatine induced differentiation of neurosphere-derived NSC into GNLC within 1 week. This protocol gives higher yield than the other protocols used in this study. The mechanism of induction was clearly associated with several differential pluripotent genes.

Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

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Kazantseva, Yuliya A.; Yarushkin, Andrei A. [Institute of Molecular Biology and Biophysics SB RAMS, Novosibirsk, Timakova str., 2, 630117 (Russian Federation); Pustylnyak, Vladimir O., E-mail: pustylnyak@ngs.ru [Institute of Molecular Biology and Biophysics SB RAMS, Novosibirsk, Timakova str., 2, 630117 (Russian Federation); Novosibirsk State University, Novosibirsk, Pirogova str., 2, 630090 (Russian Federation)

2013-09-01

Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p′-DDT (85%) and o,p′-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CAR and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. - Highlights: • DDT activated both CAR and ERα and their cell

Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

International Nuclear Information System (INIS)

Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

2013-01-01

Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p′-DDT (85%) and o,p′-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CAR and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. - Highlights: • DDT activated both CAR and ERα and their cell

[miR-182 promotes cell proliferation of cervical cancer cells by targeting adenomatous polyposis coli (APC) gene].

Science.gov (United States)

Li, Pei; Hu, Jing; Zhang, Ying; Li, Jianping; Dang, Yunzhi; Zhang, Rui; Wei, Lichun; Shi, Mei

2018-02-01

Objective To investigate the role and mechanism of microRNA-182 (miR-182) in the proliferation of cervical cancer cells. Methods With liposome-mediated transient transfection method, the level of miR-182 in HeLa and SiHa cells was increased or decreased. CCK-8 assay and colony formation assay were used to observe the effect of miR-182 on the proliferation of cervical cancer cells. Using bioinformatics predictions, real-time quantitative PCR, and dual luciferase reporter assay, we clarified the role of miR-182 in posttranscriptional regulation of adenomatous polyposis coli (APC) gene and its effect on the downstream molecules (c-Myc and cyclin D1) of Wnt singling pathway. Results Up-regulation of miR-182 significantly promoted the proliferation of cervical cancer cells, while down-regulation of miR-182 significantly inhibited the proliferation of cervical cancer cells. Over-expression of miR-182 inhibited the expression of APC gene in cervical cancer cells and the regulation of miR-182 affected the expression of canonical Wnt signaling pathway downstream molecules in cervical cancer cells. Conclusion The miR-182 stimulates canonical Wnt signaling pathway by targeting APC gene and enhances the proliferation of cervical cancer cells.

Thermo-Responsive Complexes of c-Myc Antisense Oligonucleotide with Block Copolymer of Poly(OEGMA) and Quaternized Poly(4-Vinylpyridine).

Science.gov (United States)

Topuzogullari, Murat; Elalmis, Yeliz Basaran; Isoglu, Sevil Dincer

2017-04-01

Solution behavior of thermo-responsive polymers and their complexes with biological macromolecules may be affected by environmental conditions, such as the concentration of macromolecular components, pH, ion concentration, etc. Therefore, a thermo-responsive polymer and its complexes should be characterized in detail to observe their responses against possible environments under physiological conditions before biological applications. To briefly indicate this important issue, thermo-responsive block copolymer of quaternized poly(4-vinylpyridine) and poly(oligoethyleneglycol methyl ether methacrylate) as a potential nonviral vector has been synthesized. Polyelectrolyte complexes of this copolymer with the antisense oligonucleotide of c-Myc oncogene are also thermo-responsive but, have lower LCST (lower critical solution temperature) values compared to individual copolymer. LCST values of complexes decrease with molar ratio of macromolecular components and presence of salt. Dilution of solutions also affects solution behavior of complexes and causes a significant decrease in size and an increase in LCST, which indicates possible effects of severe dilutions in the blood stream. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[HSP90 Inhibitor 17-AAG Inhibits Multiple Myeloma Cell Proliferation by Down-regulating Wnt/β-Catenin Signaling Pathway].

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Chen, Kan-Kan; He, Zheng-Mei; Ding, Bang-He; Chen, Yue; Zhang, Li-Juan; Yu, Liang; Gao, Jian

2016-02-01

To investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism. The multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively. The 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P AAG concentration, the more high of cell ratio in G1 phase (P AAG, the more long time of culture, the more high of cell ratio in G1 phase (P AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.

Generation of iPSC line epiHUVEC from human umbilical vein endothelial cells

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Peggy Matz

2015-11-01

Full Text Available Human umbilical vein endothelial cells (HUVECs were used to generate the iPSC line epiHUVEC employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHUVEC and the human embryonic stem cell line — H1 have a Pearson correlation of 0.899.

Inhibition of STAT3 Expression and Signaling in Resveratrol-Differentiated Medulloblastoma Cells

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Li-Jun Yu

2008-07-01

Full Text Available In this study, the potential influence of resveratrol (3,5,4′-trihydroxy-trans-stilbene in signal transducer and activator of transcription 3 (STAT3 signaling of medulloblastoma cells was evaluated by checking the status of STAT3 signaling and its downstream gene expression in two medulloblastoma cell lines (UW228-2 and UW228-3 with and without resveratrol treatment. The results revealed that resveratrol induced neuronal differentiation of medulloblastoma cells. Signal transducer and activator of transcription 3 expression and phosphorylation were detected in normally cultured UW228-2 and UW228-3 cells that were apparently attenuated after resveratrol treatment. The expression of STAT3 downstream genes, survivin, cyclin D1, Cox-2, and c-Myc, was suppressed but Bcl-2 was enhanced by resveratrol. Meanwhile, the production and secretion of leukemia inhibitory factor, a STAT3 activator, became active in resveratrol-treated cells. To further ascertain the significance of STAT3 signaling for medulloblastoma cells, AG490, a selective inhibitor of STAT3 phosphorylation, was used to treat UW228-3 cells. Phosphorylation of STAT3 was inhibited by AG490 accompanied with growth suppression, differentiation-like changes, and down-regulation of survivin, cyclin D1, Cox-2, and c-Myc. Our data thus suggest the importance of STAT3 signaling in maintenance and survival of medulloblastoma cells. This signaling may be the major target of resveratrol. Enhanced leukemia inhibitory factor and Bcl-2 expressions in resveratrol-treated cells might reflect a compensatory response to the loss of STAT3 function.

BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

International Nuclear Information System (INIS)

Goupille, Olivier; Penglong, Tipparat; Lefèvre, Carine; Granger, Marine; Kadri, Zahra; Fucharoen, Suthat; Maouche-Chrétien, Leila; Leboulch, Philippe; Chrétien, Stany

2012-01-01

Highlights: ► UT7 erythroleukemia cells are known to be refractory to differentiate. ► Brief JQ1 treatment initiates the first steps of erythroid differentiation program. ► Engaged UT7 cells then maturate in the presence of erythropoietin. ► Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

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Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Thalassemia Research Center and Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Thailand); Lefevre, Carine; Granger, Marine; Kadri, Zahra [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Fucharoen, Suthat [Thalassemia Research Center and Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Thailand); Maouche-Chretien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Genetics Division, Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chretien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France)

2012-12-07

Highlights: Black-Right-Pointing-Pointer UT7 erythroleukemia cells are known to be refractory to differentiate. Black-Right-Pointing-Pointer Brief JQ1 treatment initiates the first steps of erythroid differentiation program. Black-Right-Pointing-Pointer Engaged UT7 cells then maturate in the presence of erythropoietin. Black-Right-Pointing-Pointer Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

Molecular Pathogenesis of B-Cell Posttransplant Lymphoproliferative Disorder: What Do We Know So Far?

Directory of Open Access Journals (Sweden)

J. Morscio

2013-01-01

Full Text Available Posttransplant lymphoproliferative disorder (PTLD is a potentially fatal disease that arises in 2%–10% of solid organ and hematopoietic stem cell transplants and is most frequently of B-cell origin. This very heterogeneous disorder ranges from benign lymphoproliferations to malignant lymphomas, and despite the clear association with Epstein-Barr Virus (EBV infection, its etiology is still obscure. Although a number of risk factors have been identified (EBV serostatus, graft type, and immunosuppressive regimen, it is currently not possible to predict which transplant patient will eventually develop PTLD. Genetic studies have linked translocations (involving C-MYC, IGH, BCL-2, various copy number variations, DNA mutations (PIM1, PAX5, C-MYC, RhoH/TTF, and polymorphisms in both the host (IFN-gamma, IL-10, TGF-beta, HLA and the EBV genome to B-cell PTLD development. Furthermore, the tumor microenvironment seems to play an important role in the course of disease representing a local niche that can allow antitumor immune responses even in an immunocompromised host. Taken together, B-cell PTLD pathogenesis is very complex due to the interplay of many different (patient-dependent factors and requires thorough molecular analysis for the development of novel tailored therapies. This review aims at giving a global overview of the currently known parameters that contribute to the development of B-cell PTLD.

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

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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.

Generation of human iPSC line GRX-MCiPS4F-A2 from adult peripheral blood mononuclear cells (PBMCs with Spanish genetic background

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Sonia Cabrera

2015-09-01

Full Text Available We have generated iPSCs from peripheral blood mononuclear cells (PBMCs of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

Tussilagone suppresses colon cancer cell proliferation by promoting the degradation of β-catenin

International Nuclear Information System (INIS)

Li, Hua; Lee, Hwa Jin; Ahn, Yeon Hwa; Kwon, Hye Jin; Jang, Chang-Young; Kim, Woo-Young; Ryu, Jae-Ha

2014-01-01

Highlights: •Tussilagone (TSL) was purified from plant as an inhibitor of Wnt/β-catenin pathway. •TSL suppressed the β-catenin/T-cell factor transcriptional activity. •The proteasomal degradation of β-catenin was induced by TSL. •TSL suppressed the Wnt/β-catenin target genes, cyclin D1 and c-myc. •TSL inhibit the proliferation of colon cancer cells. -- Abstract: Abnormal activation of the Wnt/β-catenin signaling pathway frequently induces colon cancer progression. In the present study, we identified tussilagone (TSL), a compound isolated from the flower buds of Tussilago farfara, as an inhibitor on β-catenin dependent Wnt pathway. TSL suppressed β-catenin/T-cell factor transcriptional activity and down-regulated β-catenin level both in cytoplasm and nuclei of HEK293 reporter cells when they were stimulated by Wnt3a or activated by an inhibitor of glycogen synthase kinase-3β. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of β-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the β-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/β-catenin signaling pathway, and consequently inhibited the proliferation of colon cancer cells. Taken together, TSL might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer

Tussilagone suppresses colon cancer cell proliferation by promoting the degradation of β-catenin

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Li, Hua [College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Gu, Seoul 140-742 (Korea, Republic of); Lee, Hwa Jin [Department of Natural Medicine Resources, Semyung University, 65 Semyung-ro, Jecheon, Chungbuk 390-711 (Korea, Republic of); Ahn, Yeon Hwa; Kwon, Hye Jin; Jang, Chang-Young; Kim, Woo-Young [College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Gu, Seoul 140-742 (Korea, Republic of); Ryu, Jae-Ha, E-mail: ryuha@sookmyung.ac.kr [College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Gu, Seoul 140-742 (Korea, Republic of)

2014-01-03

Highlights: •Tussilagone (TSL) was purified from plant as an inhibitor of Wnt/β-catenin pathway. •TSL suppressed the β-catenin/T-cell factor transcriptional activity. •The proteasomal degradation of β-catenin was induced by TSL. •TSL suppressed the Wnt/β-catenin target genes, cyclin D1 and c-myc. •TSL inhibit the proliferation of colon cancer cells. -- Abstract: Abnormal activation of the Wnt/β-catenin signaling pathway frequently induces colon cancer progression. In the present study, we identified tussilagone (TSL), a compound isolated from the flower buds of Tussilago farfara, as an inhibitor on β-catenin dependent Wnt pathway. TSL suppressed β-catenin/T-cell factor transcriptional activity and down-regulated β-catenin level both in cytoplasm and nuclei of HEK293 reporter cells when they were stimulated by Wnt3a or activated by an inhibitor of glycogen synthase kinase-3β. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of β-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the β-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/β-catenin signaling pathway, and consequently inhibited the proliferation of colon cancer cells. Taken together, TSL might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer.

Methylselenol, a selenium metabolite, induces cell cycle arrest in G1 phase and apoptosis via the extracellular-regulated kinase 1/2 pathway and other cancer signaling genes.

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Zeng, Huawei; Wu, Min; Botnen, James H

2009-09-01

Methylselenol has been hypothesized to be a critical selenium (Se) metabolite for anticancer activity in vivo, and our previous study demonstrated that submicromolar methylselenol generated by incubating methionase with seleno-l-methionine inhibits the migration and invasive potential of HT1080 tumor cells. However, little is known about the association between cancer signal pathways and methylselenol's inhibition of tumor cell invasion. In this study, we demonstrated that methylselenol exposure inhibited cell growth and we used a cancer signal pathway-specific array containing 15 different signal transduction pathways involved in oncogenesis to study the effect of methylselenol on cellular signaling. Using real-time RT-PCR, we confirmed that cellular mRNA levels of cyclin-dependent kinase inhibitor 1C (CDKN1C), heme oxygenase 1, platelet/endothelial cell adhesion molecule, and PPARgamma genes were upregulated to 2.8- to 5.7-fold of the control. BCL2-related protein A1, hedgehog interacting protein, and p53 target zinc finger protein genes were downregulated to 26-52% of the control, because of methylselenol exposure. These genes are directly related to the regulation of cell cycle and apoptosis. Methylselenol increased apoptotic cells up to 3.4-fold of the control and inhibited the extracellular-regulated kinase 1/2 (ERK1/2) signaling and cellular myelocytomatosis oncogene (c-Myc) expression. Taken together, our studies identify 7 novel methylselenol responsive genes and demonstrate that methylselenol inhibits ERK1/2 pathway activation and c-Myc expression. The regulation of these genes is likely to play a key role in G1 cell cycle arrest and apoptosis, which may contribute to the inhibition of tumor cell invasion.

Gene expression profiling for nitric oxide prodrug JS-K to kill HL-60 myeloid leukemia cells.

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Liu, Jie; Malavya, Swati; Wang, Xueqian; Saavedra, Joseph E; Keefer, Larry K; Tokar, Erik; Qu, Wei; Waalkes, Michael P; Shami, Paul J

2009-07-01

The nitric oxide (NO) prodrug JS-K is shown to have anticancer activity. To profile the molecular events associated with the anticancer effects of JS-K, HL-60 leukemia cells were treated with JS-K and subjected to microarray and real-time RT-PCR analysis. JS-K induced concentration- and time-dependent gene expression changes in HL-60 cells corresponding to the cytolethality effects. The apoptotic genes (caspases, Bax, and TNF-alpha) were induced, and differentiation-related genes (CD14, ITGAM, and VIM) were increased. For acute phase protein genes, some were increased (TP53, JUN) while others were suppressed (c-myc, cyclin E). The expression of anti-angiogenesis genes THBS1 and CD36 and genes involved in tumor cell migration such as tissue inhibitors of metalloproteinases, were also increased by JS-K. Confocal analysis confirmed key gene changes at the protein levels. Thus, multiple molecular events are associated with JS-K effects in killing HL-60, which could be molecular targets for this novel anticancer NO prodrug.

Reprogramming human amniotic fluid stem cells to functional pluripotency by manipulation of culture conditions.

OpenAIRE

sprotocols

2015-01-01

Authors: Dafni Moschidou & Pascale V Guillot ### Abstract Pluripotent stem cells have potential applications in regenerative medicine, disease modelling and drug screening. Induced pluripotent stem (iPS) cells have first been generated from fibroblasts using retroviral insertion of OCT4A, SOX2, c-MYC and KLF4. Since then, a number of methods have been developed to avoid the random integration of ectopic factors in the genome and the low efficiency of the process. Those include alterna...

Investigation of miRNA Biology by Bioinformatic Tools and Impact of miRNAs in Colorectal Cancer: Regulatory Relationship of c-Myc and p53 with miRNAs

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Yaguang Xi

2007-01-01

Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that mediate gene expression at the posttranscriptional and translational levels and have been demonstrated to be involved in diverse biological functions. Mounting evidence in recent years has shown that miRNAs play key roles in tumorigenesis due to abnormal expression of and mutations in miRNAs. High throughput miRNA expression profiling of several major tumor types has identified miRNAs associated with clinical diagnosis and prognosis of cancer treatment. Previously our group has discovered a novel regulatory relationship between tumor suppressor gene p53 with miRNAs expression and a number of miRNA promoters contain putative p53 binding sites. In addition, others have reported that c-myc can mediate a large number of miRNAs expression. In this review, we will emphasize algorithms to identify mRNA targets of miRNAs and the roles of miRNAs in colorectal cancer. In particular, we will discuss a novel regulatory relationship of miRNAs with tumor suppressor p53 and c-myc. miRNAs are becoming promising novel targets and biomarkers for future cancer therapeutic development and clinical molecular diagnosis.

Aging increases cell-to-cell transcriptional variability upon immune stimulation.

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Martinez-Jimenez, Celia Pilar; Eling, Nils; Chen, Hung-Chang; Vallejos, Catalina A; Kolodziejczyk, Aleksandra A; Connor, Frances; Stojic, Lovorka; Rayner, Timothy F; Stubbington, Michael J T; Teichmann, Sarah A; de la Roche, Maike; Marioni, John C; Odom, Duncan T

2017-03-31

Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4 + T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues. Copyright © 2017, American Association for the Advancement of Science.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

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Claudia Merkl

Full Text Available Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4 into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

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Ambrosini, Grazia; Sawle, Ashley D; Musi, Elgilda; Schwartz, Gary K

2015-10-20

Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease.

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Hama, Taketsugu; Nakanishi, Koichi; Sato, Masashi; Mukaiyama, Hironobu; Togawa, Hiroko; Shima, Yuko; Miyajima, Masayasu; Nozu, Kandai; Nagao, Shizuko; Takahashi, Hisahide; Sako, Mayumi; Iijima, Kazumoto; Yoshikawa, Norishige; Suzuki, Hiroyuki

2017-12-01

Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/ cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention. Copyright © 2017 the American Physiological Society.

Lymphoblast-derived integration-free ISRM-CON9 iPS cell line from a 75 year old female

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Soraia Martins

2018-01-01

Full Text Available Human lymphoblast cells were used to generate integration-free induced pluripotent stem cells (iPSCs employing episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The derived iPSCs were defined as pluripotent based on (i expression of pluripotency-associated markers, (ii embryoid body-based differentiation into cell types representative of the three germ layers and (iii the similarity between the transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95.

Perturbation biology nominates upstream-downstream drug combinations in RAF inhibitor resistant melanoma cells.

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Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

2015-08-18

Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.

Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig.

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Liu, Ying; Ostrup, Olga; Li, Rong; Li, Juan; Vajta, Gábor; Kragh, Peter M; Schmidt, Mette; Purup, Stig; Hyttel, Poul; Klærke, Dan; Callesen, Henrik

2014-08-01

In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

International Nuclear Information System (INIS)

Singh, Alok R.; Peirce, Susan K.; Joshi, Shweta; Durden, Donald L.

2014-01-01

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN fl/fl mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

Energy Technology Data Exchange (ETDEWEB)

Singh, Alok R. [UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093 (United States); Peirce, Susan K. [Department of Pediatrics, Emory University School of Medicine, Atlanta, GA (United States); Joshi, Shweta [UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093 (United States); Durden, Donald L., E-mail: ddurden@ucsd.edu [UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093 (United States); Division of Pediatric Hematology-Oncology, UCSD Rady Children' s Hospital, La Jolla, CA (United States)

2014-09-10

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN{sup fl/fl} mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI

Generation of iPS cell lines from schizophrenia patients using a non-integrative method

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Jaroslaw Sochacki

2016-07-01

Full Text Available Skin biopsies were collected from three male patients (age 45, 44 and 44 with clinically diagnosed schizophrenia. The patients were diagnosed according to DSM-5 criteria by a trained psychiatrist. Dermal fibroblast cell lines were established and expanded for subsequent reprogramming procedures. Induced pluripotent stem (iPS cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, containing Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

Science.gov (United States)

Selmin, Ornella I; Fang, Changming; Lyon, Adam M; Doetschman, Tom C; Thompson, Patricia A; Martinez, Jesse D; Smith, Jeffrey W; Lance, Peter M; Romagnolo, Donato F

2016-02-01

The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells

CCL5 promotes proliferation of MCF-7 cells through mTOR-dependent mRNA translation

International Nuclear Information System (INIS)

Murooka, Thomas T.; Rahbar, Ramtin; Fish, Eleanor N.

2009-01-01

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase in high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.

Generation of transgene-free induced pluripotent stem cells with non-viral methods.

Science.gov (United States)

Wang, Tao; Zhao, Hua-shan; Zhang, Qiu-ling; Xu, Chang-lin; Liu, Chang-bai

2013-03-01

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development

DEFF Research Database (Denmark)

Hall, Vanessa Jane; Christensen, Josef; Gao, Yu

2009-01-01

  The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass. Copyright (c) 2009 Wiley-Liss, Inc.......  The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... (LIF, LIFR, GP130), FGF pathway (bFGF, FGFR1, FGFR2), BMP pathway (BMP4), and downstream-activated genes (STAT3, c-Myc, c-Fos, and SMAD4). We discovered two different expression profiles exist in the developing porcine embryo. The D6 porcine blastocyst (inner cell mass stage) is devoid...

Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

International Nuclear Information System (INIS)

Rodríguez-Rigueiro, Teresa; Valladares-Ayerbes, Manuel; Haz-Conde, Mar; Aparicio, Luis A; Figueroa, Angélica

2011-01-01

The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells. Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed. Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The

Cadmium modifies the cell cycle and apoptotic profiles of human breast cancer cells treated with 5-fluorouracil.

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Asara, Yolande; Marchal, Juan A; Carrasco, Esther; Boulaiz, Houria; Solinas, Giuliana; Bandiera, Pasquale; Garcia, Maria A; Farace, Cristiano; Montella, Andrea; Madeddu, Roberto

2013-08-12

Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd), which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU) is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd.

Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil

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Roberto Madeddu

2013-08-01

Full Text Available Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd, which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd.

Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil

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Asara, Yolande; Marchal, Juan A.; Carrasco, Esther; Boulaiz, Houria; Solinas, Giuliana; Bandiera, Pasquale; Garcia, Maria A.; Farace, Cristiano; Montella, Andrea; Madeddu, Roberto

2013-01-01

Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd), which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU) is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd. PMID:23941782

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.

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells.

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Kim, Dae Seong; Ko, Young Jong; Lee, Myoung Woo; Park, Hyun Jin; Park, Yoo Jin; Kim, Dong-Ik; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2016-11-01

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O 2 ) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-β-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.

Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms

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Leidgens, Verena; Seliger, Corinna; Jachnik, Birgit; Welz, Tobias; Leukel, Petra; Vollmann-Zwerenz, Arabel; Bogdahn, Ulrich; Kreutz, Marina; Grauer, Oliver M.; Hau, Peter

2015-01-01

Background Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with anti-tumorigenic effects in different tumor entities. For glioma, research has generally focused on diclofenac; however data on other NSAIDs, such as ibuprofen, is limited. Therefore, we performed a comprehensive investigation of the cellular, molecular, and metabolic effects of ibuprofen and diclofenac on human glioblastoma cells. Methods Glioma cell lines were treated with ibuprofen or diclofenac to investigate functional effects on proliferation and cell motility. Cell cycle, extracellular lactate levels, lactate dehydrogenase-A (LDH-A) expression and activity, as well as inhibition of the Signal Transducer and Activator of Transcription 3 (STAT-3) signaling pathway, were determined. Specific effects of diclofenac and ibuprofen on STAT-3 were investigated by comparing their effects with those of the specific STAT-3 inhibitor STATTIC. Results Ibuprofen treatment led to a stronger inhibition of cell growth and migration than treatment with diclofenac. Proliferation was affected by cell cycle arrest at different checkpoints by both agents. In addition, diclofenac, but not ibuprofen, decreased lactate levels in all concentrations used. Both decreased STAT-3 phosphorylation; however, diclofenac led to decreased c-myc expression and subsequent reduction in LDH-A activity, whereas treatment with ibuprofen in higher doses induced c-myc expression and less LDH-A alteration. Conclusions This study indicates that both ibuprofen and diclofenac strongly inhibit glioma cells, but the subsequent metabolic responses of both agents are distinct. We postulate that ibuprofen may inhibit tumor cells also by COX- and lactate-independent mechanisms after long-term treatment in physiological dosages, whereas diclofenac mainly acts by inhibition of STAT-3 signaling and downstream modulation of glycolysis. PMID:26485029

MYC activates stem-like cell potential in hepatocarcinoma by a p53-dependent mechanism

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Akita, Hirofumi; Marquardt, Jens U; Durkin, Marian E

2014-01-01

Activation of c-MYC is an oncogenic hallmark of many cancers including liver cancer, and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c-MYC activation for the b...

PET/CT imaging of c-Myc transgenic mice identifies the genotoxic N-nitroso-diethylamine as carcinogen in a short-term cancer bioassay.

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Katja Hueper

Full Text Available BACKGROUND: More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay. METHODOLOGY/PRINCIPAL FINDINGS: μCT and ¹⁸F-FDG μPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced μCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis. CONCLUSIONS/SIGNIFICANCE: The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgenic animal models may possibly enable short-term cancer bioassays to significantly improve hazard identification and follow-up examinations of different organs by non-invasive methods.

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

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Xu, Ning; Shen, Sylvie; Dolnikov, Alla

2017-04-01

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Relationship of Amplification and Expression of the C-MYC Gene with Survival among Gastric Cancer Patients.

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Khaleghian, Malihea; Shakoori, Abbas; Razavi, Amirnader Emami; Azimi, Cyrus

2015-01-01

During the past decades, the incidence and mortality rate of stomach cancer has demonstrated a great decrease in the world, but it is still one of the most common and fatal cancers especially among men worldwide, including Iran. The MYC proto-oncogene, which is located at 8q24.1, regulates 15% of genes and is activated in 20% of all human tumors. MYC amplification and overexpression of its protein product has been reported in 15-30% of gastric neoplasias. The aim of this investigation was to find the relative efficacy of CISH (chromogenic in situ hybridization) or IHC (immunohistochemistry) in diagnosis and prognosis of gastric cancer, as well as the relationship of amplification and expression of C-MYC gene with patient survival. In this cross-sectional study, 102 samples of gastric cancer were collected from patients who had undergone primary surgical resection at the Cancer Institute Hospital, Tehran University of Medical Sciences, from July 2009 to March 2014. All samples were randomly selected from those who were diagnosed with gastric adenocarcinomas. CISH and IHC methods were performed on all of them. Patients were classified into two groups. The first consisted of stage I and II cases, and the second of stage III and IV. Survival tests for both groups was carried out with referrnce to CISH test reults. Group II (stage III and IV) with CISH+ featured lower survival than those with CISH- (p=0.233), but group I (stage I and II) patients demonstrated no significant variation with CISH+ or CISH- (p=0.630). Kaplan-Meier for both groups was carried out with IHC test findings and showed similar results. This data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in men than women. Our data also showed that CISH+ patients (43%) were more frequent in comparison with IHC+ patients (14.7%). For planning treatment of gastric cancer patients, by focusing on expanding tumors, which is the greatest concern of the surgeons and

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

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Ricolo, Delia; Deligiannaki, Myrto; Casanova, Jordi; Araújo, Sofia J

2016-10-24

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Constitutive expression of pluripotency-associated genes in mesodermal progenitor cells (MPCs.

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Simone Pacini

Full Text Available BACKGROUND: We recently characterized a progenitor of mesodermal lineage (MPCs from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. METHODOLOGY/PRINCIPAL FINDINGS: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. CONCLUSIONS/SIGNIFICANCE: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Characterization of Cancer Stem Cells in Colon Adenocarcinoma Metastasis to the Liver

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Hugo N. Humphries

2018-01-01

Full Text Available BackgroundFifty percent of colorectal cancer (CRC patients develop liver metastasis. This study identified and characterized cancer stem cells (CSCs within colon adenocarcinoma metastasis to the liver (CAML.Methods3,3-Diaminobenzidine immunohistochemical (IHC staining was performed on nine CAML samples for embryonic stem cell (ESC markers OCT4, SOX2, NANOG, c-Myc, and KLF4. Immunofluorescence (IF IHC staining was performed to investigate coexpression of two markers. NanoString mRNA expression analysis and colorimetric in situ hybridization (CISH were performed on four snap-frozen CAML tissue samples for transcript expression of these ESC markers. Cells stained positively and negatively for each marker by IHC and CISH staining were counted and analyzed.Results3,3-Diaminobenzidine IHC staining, and NanoString and CISH mRNA analyses demonstrated the expression of OCT4, SOX2, NANOG, c-Myc, and KLF4 within in all nine CAML samples, except for SOX2 which was below detectable levels on NanoString mRNA analysis. IF IHC staining showed the presence of a SOX2+/NANOG+/KLF4+/c-Myc+/OCT− CSC subpopulation within the tumor nests, and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− CSC subpopulation and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4+ CSC subpopulation within the peritumoral stroma.ConclusionThe novel finding of three CSC subpopulations within CAML provides insights into the biology of CRC.

cMyc/miR-125b-5p signalling determines sensitivity to bortezomib in preclinical model of cutaneous T-cell lymphomas

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Manfè, Valentina; Biskup, Edyta; Willumsgaard, Ayalah

2013-01-01

Successful/effective cancer therapy in low grade lymphoma is often hampered by cell resistance to anti-neoplastic agents. The crucial mechanisms responsible for this phenomenon are poorly understood. Overcoming resistance of tumor cells to anticancer agents, such as proteasome inhibitors, could...... improve their clinical efficacy. Using cutaneous T-cell lymphoma (CTCL) as a model of the chemotherapy-resistant peripheral lymphoid malignancy, we demonstrated that resistance to proteasome inhibition involved a signaling between the oncogene cMyc and miR-125b-5p. Bortezomib repressed c...

Effect of surgical resection combined with transcatheter arterial chemoembolization on postoperative serum tumor marker levels and stem cell characteristics during tumor recurrence

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Sen Yang

2017-05-01

Full Text Available Objective: To study the effect of surgical resection combined with transcatheter arterial chemoembolization (TACE on postoperative serum tumor marker levels and stem cell characteristics during tumor recurrence. Methods: A total of 98 patients with liver cancer who received radical resection in our hospital between May 2013 and July 2015 were reviewed and divided into TACE group and control group according to whether they received TACE within two months after surgical resection. Serum levels of tumor markers were detected 4 weeks after operation; the tumor recurrence was followed up within 3 years after operation, and the expression of stem cell marker molecules and cell proliferation molecules in recurrent lesions were detected. Results: 4 weeks after radical hepatectomy, serum AFP, AFP-L3, GP73 and GPC3 levels in TACE group were significantly lower than those in control group; Nanog, CD133, EpCAM, PICK1, CyclinD1, C-myc and Survivin expression in surgically removed lesions of TACE group were not different from those of control group while Nanog, CD133, EpCAM, PICK1, CyclinD1, C-myc and Survivin expression in recurrent lesions were significantly lower than those of control group. Conclusion: Surgical resection combined with TACE can more effectively remove liver cancer lesions, reduce the tumor marker levels and inhibit the tumor stem cell characteristics and cell proliferation activity in recurrent lesions.

The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

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Khan, Omar M.; Carvalho, Joana; Spencer-Dene, Bradley; Mitter, Richard; Frith, David; Snijders, Ambrosius P.; Wood, Stephen A.

2018-01-01

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations. PMID:29346117

Increased cell surface metallopeptidase activity in cells undergoing UV-induced apoptosis

International Nuclear Information System (INIS)

Piva, T.J.; Davern, C.M.; Ellem, K.A.O.

1999-01-01

Full text: We have previously shown that UVC irradiation activated a range of cell surface peptidases (CSP) in HeLa cell monolayer cultures 20 h post-irradiation (1). In cells undergoing apoptosis there is an increase in CSP activity compared to control viable cells in cultures which have been treated by a wide range of agents including UV-irradiation (2). In order to further understand the mechanism involved in this process, we induced apoptosis in HeLa cells using 500 Jm -2 UVB. The separation of viable, apoptotic and necrotic cells of irradiated HeLa cell cultures was made by FACS analysis and sorting. The three populations were distinguished by their staining with PI and Hoechst 33342 dyes. CSP activity was measured using the P9 assay developed in this laboratory (1-3). The viable fraction of the irradiated cells had a higher level of CSP activity compared to unirradiated controls. The level of CSP activity in the apoptotic fraction was higher than that of the viable fraction, however that of the necrotic fraction was significantly lower. This finding agreed with that seen in UVC-irradiated (50 Jm -2 ) cultures (2). In order to elucidate the mechanism by which CSP activity was increased in UVB-irradiated cells undergoing apoptosis, the cultures were treated with the following agents: bestatin, aminopeptidase inhibitor, DEVD, caspase 3 inhibitor, and 3-aminobenzamide (3AB), PARP activation inhibitor. Bestatin and DEVD did not affect the level of CSP activity in the different cell subpopulations following UVB-irradiation. Treatment with 3AB abolished the increased CSP activity seen in the viable and apoptotic fraction following UVB-irradiation. All treated cells had the same morphology as observed under EM. The degree of phosphatidylserine eversion on the cell membrane was similar as were the cleavage profiles of PARP and actin. Only DEVD-treated cells had reduced caspase 3 activity which confirmed that the activation of CSP activity in apoptotic cells is

Fisetin inhibits the growth and migration in the A549 human lung cancer cell line via the ERK1/2 pathway.

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Wang, Junjian; Huang, Shaoxiang

2018-03-01

Lung cancer is the most prevalent malignant tumor type in the developed world and the discovery of novel anti-tumor drugs is a research hotspot. Fisetin, a naturally occurring flavonoid, has been reported to have anti-cancer effects in multiple tumor types. The present study found that fisetin inhibited the growth and migration of non-small cell lung cancer in vitro . MTT, wound-healing, cell-matrix adhesion and Transwell assays were performed and demonstrated that fisetin suppressed proliferation, migration, adhesion and invasion, respectively. Flow cytometric analysis indicated that fisetin induced apoptosis in the A549 cell line by decreasing the expression of c-myc, cyclin-D1, cyclooxygenase-2, B cell lymphoma-2, CXC chemokine receptor type 4, cluster of differentiation 44 and metalloproteinase-2/9, increasing the expression of cyclin dependent kinase inhibitor (CDKN) 1A/B, CDKN2D and E-cadherin and increasing the activity of caspase-3/9 via targeting the extracellular signal-regulated kinase signaling pathway. The results provided comprehensive evidence for the anti-tumor effects of fisetin in non-small cell lung cancer in vitro , which may provide a novel approach for clinical treatment.

Ouabain Increases Gap Junctional Communication in Epithelial Cells

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Arturo Ponce

2014-11-01

Full Text Available Background/Aims: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC. Methods: We employed two different approaches: 1 analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2 measurement of the electrical capacitance. Results: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. Conclusion: Ouabain 10 nM increases GJC in MDCK cells.

Perturbation biology nominates upstream–downstream drug combinations in RAF inhibitor resistant melanoma cells

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Korkut, Anil; Wang, Weiqing; Demir, Emek; Aksoy, Bülent Arman; Jing, Xiaohong; Molinelli, Evan J; Babur, Özgün; Bemis, Debra L; Onur Sumer, Selcuk; Solit, David B; Pratilas, Christine A; Sander, Chris

2015-01-01

Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs. DOI: http://dx.doi.org/10.7554/eLife.04640.001 PMID:26284497

The Downregulation of the Expression of CD147 by Tumor Suppressor REIC/Dkk-3, and Its Implication in Human Prostate Cancer Cell Growth Inhibition.

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Mori, Akihiro; Watanabe, Masami; Sadahira, Takuya; Kobayashi, Yasuyuki; Ariyoshi, Yuichi; Ueki, Hideo; Wada, Koichiro; Ochiai, Kazuhiko; Li, Shun-Ai; Nasu, Yasutomo

2017-04-01

The cluster of differentiation 147 (CD147), also known as EMMPRIN, is a key molecule that promotes cancer progression. We previously developed an adenoviral vector encoding a tumor suppressor REIC/Dkk-3 gene (Ad-REIC) for cancer gene therapy. The therapeutic effects are based on suppressing the growth of cancer cells, but, the underlying molecular mechanism has not been fully clarified. To elucidate this mechanism, we investigated the effects of Ad-REIC on the expression of CD147 in LNCaP prostate cancer cells. Western blotting revealed that the expression of CD147 was significantly suppressed by Ad-REIC. Ad-REIC also suppressed the cell growth of LNCaP cells. Since other researchers have demonstrated that phosphorylated mitogen-activated protein kinases (MAPKs) and c-Myc protein positively regulate the expression of CD147, we investigated the correlation between the CD147 level and the activation of MAPK and c-Myc expression. Unexpectedly, no positive correlation was observed between CD147 and its possible regulators, suggesting that another signaling pathway was involved in the downregulation of CD147. This is the first study to show the downregulation of CD147 by Ad-REIC in prostate cancer cells. At least some of the therapeutic effects of Ad-REIC may be due to the downregulation of the cancer-progression factor, CD147.

Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone).

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Isaka, Keiichi; Fujito, Atsuya; Sagawa, Yasukazu; Yudate, Tamaki; Nishi, Hirotaka; Ito, Hiroe; Takayama, Masaomi

2002-01-01

Induction of apoptosis is an attractive strategy in cancer therapy but it clinical practice is not yet sufficient in choriocarcinoma. The quinolinone derivative, vesnarinone, is a novel inotropic agent used for treating congestive heart failure and may also have a potential anticancer activity. It induces apoptosis and differentiation in some tumor cell lines. We examined the antitumor effect of vesnarinone in eight cell lines established from human choriocarcinoma and hydatidiform mole using MTT assay and also analyzed the nuclear fragmentation of tumor cells by DNA electrophoresis assay. Vesnarinone inhibited the proliferation of choriocarcinoma cell lines in a dose-dependent manner and induced DNA fragmentation in cells. However, the BM cell line prepared by subcultivation from hydatidiform mole showed no growth suppression or DNA fragmentation in response to vesnarinone. On the other hand, PCR-SSCP analysis and direct DNA sequencing have shown that a human choriocarcinoma cell line, SCH, has a mutant p53 gene at codon 249. When SCH cells were treated with vesnarinone cellular proliferation was significantly inhibited. Vesnarinone suppressed the proliferation of all choriocarcinoma cell lines and induced apoptosis, regardless of the existence of p53 mutation. In addition, it has been found by RT-PCR that expression of c-Myc mRNA is upregulated by treating choriocarcinoma cells with vesnarinone. The finding suggests that vesnarinone might induce expression of c-Myc gene in choriocarcinoma cells, the product of which may be associated with the inhibition of cell growth and induce apoptosis. These results suggest that vesnarinone is a useful reagent for the treatment of choriocarcinoma.

Systemic GLIPR1-ΔTM protein as a novel therapeutic approach for prostate cancer.

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Karantanos, Theodoros; Tanimoto, Ryuta; Edamura, Kohei; Hirayama, Takahiro; Yang, Guang; Golstov, Alexei A; Wang, Jianxiang; Kurosaka, Shinji; Park, Sanghee; Thompson, Timothy C

2014-04-15

GLIPR1 is a p53 target gene known to be downregulated in prostate cancer, and increased endogenous GLIPR1 expression has been associated with increased production of reactive oxygen species, increased apoptosis, decreased c-Myc protein levels and increased cell cycle arrest. Recently, we found that upregulation of GLIPR1 in prostate cancer cells increases mitotic catastrophe through interaction with heat shock cognate protein 70 (Hsc70) and downregulation of Aurora kinase A and TPX2. In this study, we evaluated the mechanisms of recombinant GLIPR1 protein (glioma pathogenesis-related protein 1-transmembrane domain deleted [GLIPR1-ΔTM]) uptake by prostate cancer cells and the efficacy of systemic GLIPR1-ΔTM administration in a prostate cancer xenograft mouse model. GLIPR1-ΔTM was selectively internalized by prostate cancer cells, leading to increased apoptosis through reactive oxygen species production and to decreased c-Myc protein levels. Interestingly, GLIPR1-ΔTM was internalized through clathrin-mediated endocytosis in association with Hsc70. Systemic administration of GLIPR1-ΔTM significantly inhibited VCaP xenograft growth. GLIPR1-ΔTM showed no evidence of toxicity following elimination from mouse models 8 hr after injection. Our results demonstrate that GLIPR1-ΔTM is selectively endocytosed by prostate cancer cells, leading to increased reactive oxygen species production and apoptosis, and that systemic GLIPR1-ΔTM significantly inhibits growth of VCaP xenografts without substantial toxicity. © 2013 UICC.

MYC gene delivery to adult mouse utricles stimulates proliferation of postmitotic supporting cells in vitro.

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Burns, Joseph C; Yoo, James J; Atala, Anthony; Jackson, John D

2012-01-01

The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A) triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ~60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore regenerative

MYC gene delivery to adult mouse utricles stimulates proliferation of postmitotic supporting cells in vitro.

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Joseph C Burns

Full Text Available The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ~60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore

SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

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Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

2017-06-20

Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

Generation of an integration-free induced pluripotent stem cell line (CSC-43 from a patient with sporadic Parkinson's disease

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Ana Marote

2018-03-01

Full Text Available An induced pluripotent stem cell (iPSC line was generated from a 36-year-old patient with sporadic Parkinson's disease (PD. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line (CSC-43 exhibits expression of common pluripotency markers, in vitro differentiation into three germ layers and normal karyotype. This iPSC line can be used to study the mechanisms underlying the development of PD.

CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.

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Rogers, Natasha M; Zhang, Zheng J; Wang, Jiao-Jing; Thomson, Angus W; Isenberg, Jeffrey S

2016-08-01

Defects in renal tubular epithelial cell repair contribute to renal ischemia reperfusion injury, cause acute kidney damage, and promote chronic renal disease. The matricellular protein thrombospondin-1 and its receptor CD47 are involved in experimental renal ischemia reperfusion injury, although the role of this interaction in renal recovery is unknown. We found upregulation of self-renewal genes (transcription factors Oct4, Sox2, Klf4 and cMyc) in the kidney of CD47(-/-) mice after ischemia reperfusion injury. Wild-type animals had minimal self-renewal gene expression, both before and after injury. Suggestive of cell autonomy, CD47(-/-) renal tubular epithelial cells were found to increase expression of the self-renewal genes. This correlated with enhanced proliferative capacity compared with cells from wild-type mice. Exogenous thrombospondin-1 inhibited self-renewal gene expression in renal tubular epithelial cells from wild-type but not CD47(-/-) mice, and this was associated with decreased proliferation. Treatment of renal tubular epithelial cells with a CD47 blocking antibody or CD47-targeting small interfering RNA increased expression of some self-renewal transcription factors and promoted cell proliferation. In a syngeneic kidney transplant model, treatment with a CD47 blocking antibody increased self-renewal transcription factor expression, decreased tissue damage, and improved renal function compared with that in control mice. Thus, thrombospondin-1 via CD47 inhibits renal tubular epithelial cell recovery after ischemia reperfusion injury through inhibition of proliferation/self-renewal. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Adipose-Derived Stem Cells Respond to Increased Osmolarities.

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Urška Potočar

Full Text Available Cell therapies present a feasible option for the treatment of degenerated cartilaginous and intervertebral disc (IVD tissues. Microenvironments of these tissues are specific and often differ from the microenvironment of cells that, could be potentially used for therapy, e.g. human adipose-derived stem cells (hASC. To ensure safe and efficient implantation of hASC, it is important to evaluate how microenvironmental conditions at the site of implantation affect the implanted cells. This study has demonstrated that cartilaginous tissue-specific osmolarities ranging from 400-600 mOsm/L affected hASC in a dose- and time-dependent fashion in comparison to 300 mOsm/L. Increased osmolarities resulted in transient (nuclear DNA and actin reorganisation and non-transient, long-term morphological changes (vesicle formation, increase in cell area, and culture morphology, as well as reduced proliferation in monolayer cultures. Increased osmolarities diminished acid proteoglycan production and compactness of chondrogenically induced pellet cultures, indicating decreased chondrogenic potential. Viability of hASC was strongly dependent on the type of culture, with hASC in monolayer culture being more tolerant to increased osmolarity compared to hASC in suspension, alginate-agarose hydrogel, and pellet cultures, thus emphasizing the importance of choosing relevant in vitro conditions according to the specifics of clinical application.

Increased hydrostatic pressure enhances motility of lung cancer cells.

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Kao, Yu-Chiu; Lee, Chau-Hwang; Kuo, Po-Ling

2014-01-01

Interstitial fluid pressures within most solid tumors are significantly higher than that in the surrounding normal tissues. Therefore, cancer cells must proliferate and migrate under the influence of elevated hydrostatic pressure while a tumor grows. In this study, we developed a pressurized cell culture device and investigated the influence of hydrostatic pressure on the migration speeds of lung cancer cells (CL1-5 and A549). The migration speeds of lung cancer cells were increased by 50-60% under a 20 mmHg hydrostatic pressure. We also observed that the expressions of aquaporin in CL1-5 and A549 cells were increased under the hydrostatic pressure. Our preliminary results indicate that increased hydrostatic pressure plays an important role in tumor metastasis.

Increasing the lifetime of fuel cell catalysts

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Latsuzbaia, R.

2015-01-01

In this thesis, I discuss a novel idea of fuel cell catalyst regeneration to increase lifetime of the PEM fuel cell electrode/catalyst operation and, therefore, reduce the catalyst costs. As many of the catalyst degradation mechanisms are difficult to avoid, the regeneration is alternative option to

Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention

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Heather S. Smallwood

2017-05-01

Full Text Available Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.

Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention

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Smallwood, Heather S.; Duan, Susu; Morfouace, Marie; Rezinciuc, Svetlana; Shulkin, Barry L.; Shelat, Anang; Zink, Erika E.; Milasta, Sandra; Bajracharya, Resha; Oluwaseum, Ajayi J.; Roussel, Martine F.; Green, Douglas R.; Pasa-Tolic, Ljiljana; Thomas, Paul G.

2017-05-01

Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

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Linxu Chen

Full Text Available RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

Generation of a human induced pluripotent stem cell line (CSC-42 from a patient with sporadic form of Parkinson's disease

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Ekaterina Savchenko

2018-03-01

Full Text Available Skin fibroblasts were collected from a 44-year-old patient with sporadic case of Parkinson's disease (PD. The non-integrating Sendai virus vector encoding OCT3/4, SOX2, c-MYC and KLF4 was used to reprogram fibroblasts into induced pluripotent stem cells (iPSCs. Generated iPSCs had normal karyotypes, expressed common stem cell markers, and were capable of differentiating into all three germ layers. Generated line could be used for PD modeling to understand the mechanisms that influence the disorder.

Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

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Taku Saito

Full Text Available Induced pluripotent stem cells (iPSC are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

Invasive Glioblastoma Cells Acquire Stemness and Increased Akt Activation

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Jennifer R. Molina

2010-06-01

Full Text Available Glioblastoma multiforme (GBM is the most frequent and most aggressive brain tumor in adults. The dismal prognosis is due to postsurgery recurrences arising from escaped invasive tumor cells. The signaling pathways activated in invasive cells are under investigation, and models are currently designed in search for therapeutic targets. We developed here an in vivo model of human invasive GBM in mouse brain from a GBM cell line with moderate tumorigenicity that allowed simultaneous primary tumor growth and dispersal of tumor cells in the brain parenchyma. This strategy allowed for the first time the isolation and characterization of matched sets of tumor mass (Core and invasive (Inv cells. Both cell populations, but more markedly Inv cells, acquired stem cell markers, neurosphere renewal ability, and resistance to rapamycin-induced apoptosis relative to parental cells. The comparative phenotypic analysis between Inv and Core cells showed significantly increased tumorigenicity in vivo and increased invasion with decreased proliferation in vitro for Inv cells. Examination of a large array of signaling pathways revealed extracellular signal-regulated kinase (Erk down-modulation and Akt activation in Inv cells and an opposite profile in Core cells. Akt activation correlated with the increased tumorigenicity, stemness, and invasiveness, whereas Erk activation correlated with the proliferation of the cells. These results underscore complementary roles of the Erk and Akt pathways for GBM proliferation and dispersal and raise important implications for a concurrent inhibitory therapy.

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.

Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

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Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

2015-07-14

Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Deconstructing transcriptional heterogeneity in pluripotent stem cells

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Shalek, Alex K.; Satija, Rahul; DaleyKeyser, AJay; Li, Hu; Zhang, Jin; Pardee, Keith; Gennert, David; Trombetta, John J.; Ferrante, Thomas C.; Regev, Aviv; Daley, George Q.; Collins, James J.

2014-01-01

SUMMARY Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates, but the regulatory circuits specifying these states and enabling transitions between them are not well understood. We set out to characterize transcriptional heterogeneity in PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signaling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signaling pathways and chromatin regulators. Strikingly, either removal of mature miRNAs or pharmacologic blockage of signaling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal, and a distinct chromatin state, an effect mediated by opposing miRNA families acting on the c-myc / Lin28 / let-7 axis. These data illuminate the nature of transcriptional heterogeneity in PSCs. PMID:25471879

Generation of urine iPS cell line from a patient with obsessive-compulsive disorder using a non-integrative method

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Jaroslaw Sochacki

2016-07-01

Full Text Available Urine sample was collected from a 29-year-old male patient with an early onset form of DSM-5 obsessive-compulsive disorder (OCD, comorbid body dysmorphic and excoriation (skin-picking disorders, and a positive family history for OCD. Urine cell line was established and expanded for the reprogramming procedure. Induced pluripotent stem (iPS cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Deregulation of epidermal stem cell niche contributes to pathogenesis of non-healing venous ulcers

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Nusbaum, Aron G.; Vukelic, Sasa; Krzyzanowska, Agata; Tomic-Canic, Marjana

2014-01-01

The epidermis is maintained by epidermal stem cells (ESC) that reside in distinct niches and contribute to homeostasis and wound closure. Keratinocytes at the non-healing edges of venous ulcers (VUs) are healing-incompetent, hyper-proliferative and non-migratory suggesting deregulation of ESCs. To date genes which regulate ESC niches have been studied in mice only. Utilizing microarray analysis of VU non-healing edges, we identified changes in expression of genes harboring regulation of ESCs and their fate. In a prospective clinical study of ten VUs, we confirmed suppression of the bone morphogenetic protein receptor and GATA binding protein3 as well as inhibitors of DNA-binding proteins 2 and 4. We also found decreased levels of phosphorylated glycogen synthase kinase 3, nuclear presence of ß-catenin and overexpression of its transcriptional target, c-myc indicating activation of the Wnt pathway. Additionally, we found down-regulation of leucine-rich repeats and immunoglobulin-like domains protein 1, a gene important for maintaining ESCs in a quiescent state, and absence of keratin 15, a marker of the basal stem cell compartment suggesting local depletion of ESCs. Our study shows that loss of genes important for regulation of ESCs and their fate along with activation of ß-catenin and c-myc in the VU may contribute to ESC deprivation and a hyper-proliferative, non-migratory, healing incapable wound edge. PMID:24635172

New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells

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Gottfried, Eva; Lang, Sven A.; Renner, Kathrin; Bosserhoff, Anja; Gronwald, Wolfram; Rehli, Michael; Einhell, Sabine; Gedig, Isabel; Singer, Katrin; Seilbeck, Anton; Mackensen, Andreas; Grauer, Oliver; Hau, Peter; Dettmer, Katja; Andreesen, Reinhard; Oefner, Peter J.; Kreutz, Marina

2013-01-01

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies. PMID:23874405

Generation of human induced pluripotent stem cells from a Bombay individual: Moving towards 'universal-donor' red blood cells

International Nuclear Information System (INIS)

Seifinejad, Ali; Taei, Adeleh; Totonchi, Mehdi; Vazirinasab, Hamed; Hassani, Seideh Nafiseh; Aghdami, Nasser; Shahbazi, Ebrahim; Yazdi, Reza Salman; Salekdeh, Ghasem Hosseini; Baharvand, Hossein

2010-01-01

Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, α-globulin, and γ-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.

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.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells

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Liang-Fang Zhu

2017-03-01

Full Text Available Objective: The present study aimed to establish an induced pluripotent stem cell (iPSC line from acute myelogenous leukemia (AML cells in vitro and identify their biological characteristics. Methods: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. Results: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. Conclusion: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Increased Circulating Anti-inflammatory Cells in Marathon-trained Runners.

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Rehm, K; Sunesara, I; Marshall, G D

2015-10-01

Exercise training can alter immune function. Marathon training has been associated with an increased susceptibility to infectious diseases and an increased activity of inflammatory-based diseases, but the precise mechanisms are unknown. The purpose of this study was to compare levels of circulating CD4+  T cell subsets in the periphery of marathon-trained runners and matched non-marathon controls. 19 recreational marathoners that were 4 weeks from running a marathon and 19 demographically-matched healthy control subjects had the percentage of CD4+ T cell subpopulations (T helper 1, T helper 2, T helper 1/T helper 2 ratio, regulatory T cells, CD4+ IL10+, and CD4+ TGFβ+ (Transforming Growth Factor-beta) measured by flow cytometry. Marathon-trained runners had significantly less T helper 1 and regulatory T cells and significantly more T helper 2, CD4+ IL10+, and TGFβ+ cells than the control subjects. The alterations in the percentage of T helper 1 and T helper 2 cells led to a significantly lower T helper 1/T helper 2 ratio in the marathon-trained runners. These data suggest that endurance-based training can increase the number of anti-inflammatory cells. This may be a potential mechanism for the increased incidence of both infectious and inflammatory diseases observed in endurance athletes. © Georg Thieme Verlag KG Stuttgart · New York.

Overexpression of SOX18 correlates with accelerated cell growth and poor prognosis in human pancreatic ductal adenocarcinoma

International Nuclear Information System (INIS)

Wang, Yazhou; Guo, Huahu; Zhang, Dafang; Yu, Xin; Leng, Xisheng; Li, Shu; Zhu, Weihua

2016-01-01

Transcription factor SOX18 has been proved to play a significant role in carcinogenesis. However, no investigation was performed about the expression of SOX18 in pancreatic ductal adenocarcinoma (PDAC). In our work, we found that the PDAC tissues had higher level of SOX18 mRNA and protein expression than matched non-tumor pancreatic tissues and high level of SOX18 protein indicated poor prognosis for PDAC patients. After knockdown of SOX18 gene in PANC-1 and SW1990 cell lines, which showed higher expression level of SOX18 among five PDAC cell lines, the abilities of proliferation, migration and invasion were inhibited and the tumor growth was suppressed in vivo. In addition, the flow cytometry results indicated that down-regulation of SOX18 induced G1/S phase arrest. Furthermore, we found that the expression of cyclin D1, c-myc and MMP-7, three tumorigenesis promoters, was inhabited with downregulation of SOX18. In conclusion, our study reveals that SOX18 plays a significant role in promoting the growth of PDAC, and might serve as a promising target for PDAC therapy. - Highlights: • Overexpression of SOX18 correlates with poor prognosis for pancreatic cancer. • SOX18 promotes pancreatic cancer cell growth in vitro and in vivo. • SOX18 promotes pancreatic cancer cell migration and invasion. • Knockdown of SOX18 induces G1/S phase arrest. • Knockdown of SOX18 induces decrease of cyclin D1, c-myc and MMP-7.

Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells

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Grepper Susan

2009-09-01

Full Text Available Abstract Background Hepatocellular carcinoma (HCC is an aggressive cancer, and is the third leading cause of cancer death worldwide. Standard therapy is ineffective partly because HCC is intrinsically resistant to conventional chemotherapy. Its poor prognosis and limited treatment options make it critical to develop novel and selective chemotherapeutic agents. Since the Wnt/β-catenin pathway is essential in HCC carcinogenesis, we studied the inhibition of Wnt-1-mediated signaling as a potential molecular target in HCC. Results We demonstrated that Wnt-1 is highly expressed in human hepatoma cell lines and a subgroup of human HCC tissues compared to paired adjacent non-tumor tissues. An anti-Wnt-1 antibody dose-dependently decreased viability and proliferation of Huh7 and Hep40 cells over-expressing Wnt-1 and harboring wild type β-catenin, but did not affect normal hepatocytes with undetectable Wnt-1 expression. Apoptosis was also observed in Huh7 and Hep40 cells after treatment with anti-Wnt-1 antibody. In these two cell lines, the anti-Wnt-1 antibody decreased β-catenin/Tcf4 transcriptional activities, which were associated with down-regulation of the endogenous β-catenin/Tcf4 target genes c-Myc, cyclin D1, and survivin. Intratumoral injection of anti-Wnt-1 antibody suppressed in vivo tumor growth in a Huh7 xenograft model, which was also associated with apoptosis and reduced c-Myc, cyclin D1, and survivin expressions. Conclusion Our results suggest that Wnt-1 is a survival factor for HCC cells, and that the blockade of Wnt-1-mediated signaling may offer a potential pathway-specific therapeutic strategy for the treatment of a subgroup of HCC that over-expresses Wnt-1.

Mechanism of Ursolic Acid-Mediated Inhibition of Proliferation in ...

African Journals Online (AJOL)

performed to measure the endogenous mRNA levels of ERK1, C-Jun, C-Myc, and Cyclin D1, and. Western blotting was used to determine the expressions of ERK1, C-Jun, C-Myc, and Cyclin ... Cells were harvested, and the total RNA was extracted according to BIOZOL reagent instruction manual. Total RNA was used for.

Neural stem cells achieve and maintain pluripotency without feeder cells.

Directory of Open Access Journals (Sweden)

Hyun Woo Choi

Full Text Available BACKGROUND: Differentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells. METHODOLOGY/PRINCIPAL FINDING: Here, we show that adult mouse neural stem cells (NSCs, which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20. Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog, the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions. CONCLUSION/SIGNIFICANCE: This study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as a feeder-free reprogramming system may help generate iPS cells of a clinical grade for tissue or organ regeneration.

Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

Directory of Open Access Journals (Sweden)

Hidetatsu Outani

Full Text Available The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4 and one chondrogenic factor (SOX9 can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon cells from human dermal fibroblast (HDF culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

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Stampfer, Martha R; Garbe, James C

2015-02-24

Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

Epidermal stem cells: location, potential and contribution to cancer.

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Ambler, C A; Määttä, A

2009-01-01

Epidermal stem cells have been classically characterized as slow-cycling, long-lived cells that reside in discrete niches in the skin. Gene expression studies of niche-resident cells have revealed a number of stem cell markers and regulators, including the Wnt/beta-catenin, Notch, p63, c-Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow-cycling stem cells and rapid-cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi-lineage epidermal progenitors can be isolated from highly proliferative, non-niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi-lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side-population tumour cells.

Increased radiation resistance in lithium-counterdoped silicon solar cells

Science.gov (United States)

Weinberg, I.; Swartz, C. K.; Mehta, S.

1984-01-01

Lithium-counterdoped n(+)p silicon solar cells are found to exhibit significantly increased radiation resistance to 1-MeV electron irradiation when compared to boron-doped n(+)p silicon solar cells. In addition to improved radiation resistance, considerable damage recovery by annealing is observed in the counterdoped cells at T less than or equal to 100 C. Deep level transient spectroscopy measurements are used to identify the defect whose removal results in the low-temperature aneal. It is suggested that the increased radiation resistance of the counterdoped cells is primarily due to interaction of the lithium with interstitial oxygen.

Clinical Application of Induced Pluripotent Stem Cells in Cardiovascular Medicine.

Science.gov (United States)

Chi, Hong-jie; Gao, Song; Yang, Xin-chun; Cai, Jun; Zhao, Wen-shu; Sun, Hao; Geng, Yong-Jian

2015-01-01

Induced pluripotent stem cells (iPSCs) are generated by reprogramming human somatic cells through the overexpression of four transcription factors: Oct4, Sox2, Klf4 and c-Myc. iPSCs are capable of indefinite self-renewal, and they can differentiate into almost any type of cell in the body. These cells therefore offer a highly valuable therapeutic strategy for tissue repair and regeneration. Recent experimental and preclinical research has revealed their potential for cardiovascular disease diagnosis, drug screening and cellular replacement therapy. Nevertheless, significant challenges remain in terms of the development and clinical application of human iPSCs. Here, we review current progress in research related to patient-specific iPSCs for ex vivo modeling of cardiovascular disorders and drug screening, and explore the potential of human iPSCs for use in the field of cardiovascular regenerative medicine. © 2015 S. Karger AG, Basel.

Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement.

Science.gov (United States)

Basolo, Fulvio; Giannini, Riccardo; Toniolo, Antonio; Casalone, Rosario; Nikiforova, Marina; Pacini, Furio; Elisei, Rossella; Miccoli, Paolo; Berti, Piero; Faviana, Pinuccia; Fiore, Lisa; Monaco, Carmen; Pierantoni, Giovanna Maria; Fedele, Monica; Nikiforov, Yuri E; Santoro, Massimo; Fusco, Alfredo

2002-02-10

A novel human thyroid papillary carcinoma cell line (FB-2) has been established and characterized. FB-2 cells harbor the RET/PTC1 chimeric oncogene in which the RET kinase domain is fused to the H4 gene. FB-2 cells neither formed colonies in semisolid media nor induced tumors after heterotransplant into severe combined immunodeficient mice. However, HMGI(Y), HMGI-C and c-myc genes, which are associated to thyroid cell transformation, were abundantly expressed in FB-2 cells but not in normal thyroid cells. FB-2 cells only partially retained the differentiated thyroid phenotype. In fact, the PAX-8 gene, which codes for a transcriptional factor required for thyroid cell differentiation, was expressed, while thyroglobulin, TSH-receptor and thyroperoxidase genes were not. Moreover, FB-2 cells produced high levels of interleukin (IL)-6 and IL-8. Copyright 2001 Wiley-Liss, Inc.

Polymerase chain reaction-based detection of myc transduction in feline leukemia virus-infected cats.

Science.gov (United States)

Sumi, Ryosuke; Miyake, Ariko; Endo, Taiji; Ohsato, Yoshiharu; Ngo, Minh Ha; Nishigaki, Kazuo

2018-04-01

Feline lymphomas are associated with the transduction and activation of cellular proto-oncogenes, such as c-myc, by feline leukemia virus (FeLV). We describe a polymerase chain reaction assay for detection of myc transduction usable in clinical diagnosis. The assay targets c-myc exons 2 and 3, which together result in a FeLV-specific fusion gene following c-myc transduction. When this assay was conducted on FeLV-infected feline tissues submitted for clinical diagnosis of tumors, myc transduction was detected in 14% of T-cell lymphoma/leukemias. This newly established system could become a useful diagnostic tool in veterinary medicine.

TSA increases C/EBP‑α expression by increasing its lysine acetylation in hepatic stellate cells.

Science.gov (United States)

Tao, Li-Li; Ding, Di; Yin, Wei-Hua; Peng, Ji-Ying; Hou, Chen-Jian; Liu, Xiu-Ping; Chen, Yao-Li

2017-11-01

CCAAT enhancer binding protein‑α (C/EBP‑α) is a transcription factor expressed only in certain tissues, including the liver. It has been previously demonstrated that C/EBP‑α may induce apoptosis in hepatic stellate cells (HSCs), raising the question of whether acetylation of C/EBP‑α is associated with HSCs, and the potential associated mechanism. A total of three histone deacetylase inhibitors (HDACIs), including trichostatin A (TSA), suberoylanilide hydroxamic acid and nicotinamide, were selected to determine whether acetylation affects C/EBP‑α expression. A Cell Counting Kit‑8 assay was used to determine the rate of proliferation inhibition following treatment with varying doses of the three HDACIs in HSC‑T6 and BRL‑3A cells. Western blot analysis was used to examine Caspase‑3, ‑8, ‑9, and ‑12 levels in HSC‑T6 cells treated with adenoviral‑C/EBP‑α and/or TSA. Following treatment with TSA, a combination of reverse transcription‑quantitative polymerase chain reaction and western blot analyses was used to determine the inherent C/EBP‑α mRNA and protein levels in HSC‑T6 cells at 0, 1, 2, 4, 8, 12, 24, 36 and 48 h. Nuclear and cytoplasmic proteins were extracted to examine C/EBP‑α distribution. Co‑immunoprecipitation analysis was used to examine the lysine acetylation of C/EBP‑α. It was observed that TSA inhibited the proliferation of HSC‑T6 cells to a greater extent compared with BRL‑3A cells, following treatment with the three HDACIs. TSA induced apoptosis in HSC‑T6 cells and enhanced the expression of C/EBP‑α. Following treatment of HSC‑T6 cells with TSA, inherent C/EBP‑α expression increased in a time‑dependent manner, and its lysine acetylation simultaneously increased. Therefore, the results of the present study suggested that TSA may increase C/EBP‑α expression by increasing its lysine acetylation in HSCs.

Endorsing cellular competitiveness in aberrant epithelium of oral submucous fibrosis progression: neighbourhood analysis of immunohistochemical attributes.

Science.gov (United States)

Anura, Anji; Kazi, Anees; Pal, Mousumi; Paul, Ranjan Rashmi; Sengupta, Sanghamitra; Chatterjee, Jyotirmoy

2018-04-23

Epithelial abnormality during the transformation of oral submucous fibrosis (OSF) into oral squamous cell carcinoma has been well studied and documented. However, the differential contribution of atrophy and hyperplasia for malignant potentiality of OSF is yet to be resolved. Existing diagnostic conjectures lack precise diagnostic attributes which may be effectively resolved by substantiation of specific molecular pathology signatures. Present study elucidates existence of cellular competitiveness in OSF conditions using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. The concept of field cancerization was contributory in finding correspondence among neighbouring cells of epithelial layers with reference to differential expression of cardinal cancer-related genes [c-Myc (oncogene), p53 (tumour suppressor), and HIF-1α (hypoxia regulator)] which are known to be important sensors in recognizing cellular competitive interface. Our analyses indicate that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighbouring cells which might be responsible to shape the present and future of the pre-malignant condition. Analytical findings indicated association of atrophic epithelium with stress-driven competitive environment having low c-Myc, high-p53, and stable HIF-1α (the looser cells) which undergo apoptosis. Whereas, the cells with high c-Myc + (winner cells) give rise to hyperplastic epithelium via possible mutation in p53. The epithelial dysplasia plausibly occurs due to clonal expansion of c-Myc and p53 positive supercompetitor cells. Present study proposes quantitative IHC along with neighbourhood analysis which might help us to dig deeper on to the interaction among epithelial cell population to provide a better understanding of field cancerization and malignant transformation of pre-malignancy.

Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells

DEFF Research Database (Denmark)

Hall, Vanessa Jane; Christensen, Marianne; Rasmussen, Mikkel Aabech

2012-01-01

Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression...... pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species....... transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1¿µg/mL doxycycline or 4¿µg/mL doxycycline. Our results revealed that endogenous...

Generation of human induced pluripotent stem cells from a Bombay individual: Moving towards 'universal-donor' red blood cells

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Seifinejad, Ali; Taei, Adeleh [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Totonchi, Mehdi; Vazirinasab, Hamed [Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Hassani, Seideh Nafiseh [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Aghdami, Nasser [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Department of Regenerative Biomedicine, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahbazi, Ebrahim [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Yazdi, Reza Salman [Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Salekdeh, Ghasem Hosseini, E-mail: Salekdeh@royaninstitute.org [Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj (Iran, Islamic Republic of); Baharvand, Hossein, E-mail: Baharvand@royaninstitute.org [Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, P.O. Box 19395-4644, ACECR, Tehran (Iran, Islamic Republic of); Department of Regenerative Biomedicine, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Developmental Biology, University of Science and Culture, ACECR, Tehran (Iran, Islamic Republic of)

2010-01-01

Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, {alpha}-globulin, and {gamma}-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.