Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

Directory of Open Access Journals (Sweden)

Tozeren Aydin

2006-11-01

Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

International Nuclear Information System (INIS)

Kapp, L.N.; Painter, R.B.

1989-01-01

SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G 2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

Fungal invasion of normally non-phagocytic host cells.

Directory of Open Access Journals (Sweden)

Scott G Filler

2006-12-01

Full Text Available Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

DNA-repair, cell killing and normal tissue damage

International Nuclear Information System (INIS)

Dahm-Daphi, J.; Dikomey, E.; Brammer, I.

1998-01-01

Background: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. Material and methods: Review. Results: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. Conclusion: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly. (orig.) [de

Mast cell distribution in normal adult skin

NARCIS (Netherlands)

A.S. Janssens (Artiena Soe); R. Heide (Rogier); J.C. den Hollander (Jan); P.G.M. Mulder (P. G M); B. Tank (Bhupendra); A.P. Oranje (Arnold)

2005-01-01

markdownabstract__AIMS:__ To investigate mast cell distribution in normal adult skin to provide a reference range for comparison with mastocytosis. __METHODS:__ Mast cells (MCs) were counted in uninvolved skin adjacent to basal cell carcinomas and other dermatological disorders in adults.

Cell line name recognition in support of the identification of synthetic lethality in cancer from text

Science.gov (United States)

Kaewphan, Suwisa; Van Landeghem, Sofie; Ohta, Tomoko; Van de Peer, Yves; Ginter, Filip; Pyysalo, Sampo

2016-01-01

Motivation: The recognition and normalization of cell line names in text is an important task in biomedical text mining research, facilitating for instance the identification of synthetically lethal genes from the literature. While several tools have previously been developed to address cell line recognition, it is unclear whether available systems can perform sufficiently well in realistic and broad-coverage applications such as extracting synthetically lethal genes from the cancer literature. In this study, we revisit the cell line name recognition task, evaluating both available systems and newly introduced methods on various resources to obtain a reliable tagger not tied to any specific subdomain. In support of this task, we introduce two text collections manually annotated for cell line names: the broad-coverage corpus Gellus and CLL, a focused target domain corpus. Results: We find that the best performance is achieved using NERsuite, a machine learning system based on Conditional Random Fields, trained on the Gellus corpus and supported with a dictionary of cell line names. The system achieves an F-score of 88.46% on the test set of Gellus and 85.98% on the independently annotated CLL corpus. It was further applied at large scale to 24 302 102 unannotated articles, resulting in the identification of 5 181 342 cell line mentions, normalized to 11 755 unique cell line database identifiers. Availability and implementation: The manually annotated datasets, the cell line dictionary, derived corpora, NERsuite models and the results of the large-scale run on unannotated texts are available under open licenses at http://turkunlp.github.io/Cell-line-recognition/. Contact: sukaew@utu.fi PMID:26428294

Mitogen-stimulated phospholipid synthesis in normal and immune-deficient human B cells

International Nuclear Information System (INIS)

Chien, M.M.; Yokoyama, W.M.; Ashman, R.F.

1986-01-01

Eight patients with common variable panhypogammaglobulinemia were shown in the in vitro Ig biosynthesis assay to have defective B cell responses to pokeweed mitogen (PWM). Phospholipid synthesis was assessed in the B cell plus monocyte fraction (MB) and irradiated T cells (T*) of patients and paired normal controls. Cell populations were studied separately and in the four possible combinations (1:1), with and without PWM, to reveal the effect of cell interactions. At 16 to 20 hr the mean stimulation index (SI) +/- standard error for MB cells alone was 1.01 +/- 0.02 for eight patients and 0.99 +/- 0.02 for the paired normals; the T* cell SI was 1.25 +/- 0.04 for patients and 1.28 +/- 0.05 for normals. Combinations of normal MB cells with normal T* cells showed significantly higher SI when compared with the combinations of normal MB cells with patient T* cells (p less than 0.005). However, the combination of patient MB cells with patient T* cells and the combination of patient MB cells with normal T* cells were not significantly different in SI (0.05 less than p less than 0.1). Isolation of patient and normal B cells, T* cells, and monocytes after the choline pulse showed that patient B cells gave a higher SI with normal T* help than with patient T* help. Of greatest interest is the finding that patient B cells that were defective in PWM-stimulated Ig production nevertheless showed a phospholipid synthesis response to PWM in the normal range, suggesting that the maturation defect in these B cells occurs later than the phospholipid synthesis acceleration step, or on a different pathway

Cell survival of human tumor cells compared with normal fibroblasts following 60Co gamma irradiation

International Nuclear Information System (INIS)

Lloyd, E.L.; Henning, C.B.; Reynolds, S.D.; Holmblad, G.L.; Trier, J.E.

1982-01-01

Three tumor cell lines, two of which were shown to be HeLa cells, were irradiated with 60 Co gamma irradiation, together with two cell cultures of normal human diploid fibroblasts. Cell survival was studied in three different experiments over a dose range of 2 to 14 gray. All the tumor cell lines showed a very wide shoulder in the dose response curves in contrast to the extremely narrow shoulder of the normal fibroblasts. In addition, the D/sub o/ values for the tumor cell lines were somewhat greater. These two characteristics of the dose response curves resulted in up to 2 orders of magnitude less sensitivity for cell inactivation of HeLa cells when compared with normal cells at high doses (10 gray). Because of these large differences, the extrapolation of results from the irradiation of HeLa cells concerning the mechanisms of normal cell killing should be interpreted with great caution

Glycosaminoglycan-sac formation in vitro. Interactions between normal and malignant cells

OpenAIRE

Logothetou-Rella, H.

1994-01-01

The interaction of monolayer normal human or normal rat cells with suspension Walker rat tumor cells was demonstrated cytologically, during a cocultivation period of thirty days. At ten days, Walker rat tumor cells were interiorized in the cytoplasm of the normal monolayer host cells. At twenty days, degeneration of the interiorized tumor cells followed by mucification led to glycosaminoglycan-sac formation. At thirty days, tumor nodules and protease (a,- c...

Identification of astrocytoma associated genes including cell surface markers

International Nuclear Information System (INIS)

Boon, Kathy; Edwards, Jennifer B; Eberhart, Charles G; Riggins, Gregory J

2004-01-01

Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide. We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [1]. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie [2], and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors. A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions. This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes

Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field.

LENUS (Irish Health Repository)

Shields, L

2014-10-31

Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells are not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci\\/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

Directory of Open Access Journals (Sweden)

Magnusson Magnus K

2010-07-01

Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

The rarity of ALDH(+) cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients.

Science.gov (United States)

Hoang, Van T; Buss, Eike C; Wang, Wenwen; Hoffmann, Isabel; Raffel, Simon; Zepeda-Moreno, Abraham; Baran, Natalia; Wuchter, Patrick; Eckstein, Volker; Trumpp, Andreas; Jauch, Anna; Ho, Anthony D; Lutz, Christoph

2015-08-01

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH(+) cells; cells (≥1.9%; ALDH-numerous AML). In patients with ALDH-rare AML, normal HSC could be separated by their CD34(+) ALDH(+) phenotype, whereas LSC were exclusively detected among CD34(+) ALDH(-) cells. For patients with ALDH-numerous AML, the CD34(+) ALDH(+) subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH(+) cells from ALDH-numerous AML were quiescent, refractory to ARA-C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long-term outcome were also characteristic for patients with ALDH-numerous AML providing an additional risk-stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible. © 2014 UICC.

Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

1995-12-01

Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

Plurihormonal cells of normal anterior pituitary: Facts and conclusions

OpenAIRE

Mitrofanova, Lubov B.; Konovalov, Petr V.; Krylova, Julia S.; Polyakova, Victoria O.; Kvetnoy, Igor M.

2017-01-01

Introduction plurihormonality of pituitary adenomas is an ability of adenoma cells to produce more than one hormone. After the immunohistochemical analysis had become a routine part of the morphological study, a great number of adenomas appeared to be multihormonal in actual practice. We hypothesize that the same cells of a normal pituitary gland releases several hormones simultaneously. Objective To analyse a possible co-expression of hormones by the cells of the normal anterior pituitary of...

Identification of Cell Surface Targets through Meta-analysis of Microarray Data

Directory of Open Access Journals (Sweden)

Henry Haeberle

2012-07-01

Full Text Available High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

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Yukiko Kiniwa

Full Text Available Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+ T helper (Th cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1 as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+ T cell-mediated immunotherapy in melanoma.

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

Science.gov (United States)

Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

2015-01-01

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

The Glycome of Normal and Malignant Plasma Cells

Science.gov (United States)

Hose, Dirk; Andrulis, Mindaugas; Moreaux, Jèrôme; Hielscher, Thomas; Willhauck-Fleckenstein, Martina; Merling, Anette; Bertsch, Uta; Jauch, Anna; Goldschmidt, Hartmut; Klein, Bernard; Schwartz-Albiez, Reinhard

2013-01-01

The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10) and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i) malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii) be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii) Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14), t(4;14), hyperdiploidy, 1q21-gain and deletion of 13q14. iv) A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v) As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma. PMID:24386263

The glycome of normal and malignant plasma cells.

Directory of Open Access Journals (Sweden)

Thomas M Moehler

Full Text Available The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10 and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14, t(4;14, hyperdiploidy, 1q21-gain and deletion of 13q14. iv A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma.

Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.

Science.gov (United States)

Huang, Emina H; Hynes, Mark J; Zhang, Tao; Ginestier, Christophe; Dontu, Gabriela; Appelman, Henry; Fields, Jeremy Z; Wicha, Max S; Boman, Bruce M

2009-04-15

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development.

Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

International Nuclear Information System (INIS)

Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

1988-01-01

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes

Response of cultured normal human mammary epithelial cells to X rays

International Nuclear Information System (INIS)

Yang, T.C.; Stampfer, M.R.; Smith, H.S.

1983-01-01

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D 0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique

Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngeneic mice

International Nuclear Information System (INIS)

Brecher, G.; Ansell, J.D.; Micklem, H.S.; Tjio, J.H.; Cronkite, E.P.

1982-01-01

The widely held view that transfused bone marrow cells will not proliferate in normal mice, not exposed to irradiation or other forms of bone marrow ablation, was reinvestigated. Forty million bone marrow cells from male donors were given to female recipients on each of 5 consecutive days, 5 to 10 times the number customarily used in the past. When the recipients were examined 2-13 weeks after the last transfusion, donor cells were found to average 16-25% of total marrow cells. Similar percentages of donor cells were found when variants of the enzyme phosphoglycerate kinase determined electrophoretically were used for identification of donor and recipient cells. Evidence is presented that the proportion of donor cells is compatible with a nonlinear dependence on the number of cells transfused over the range tested - i.e., 20-200 million bone marrow cells injected intravenously. Special proliferative sites thus do not appear to be required

Chronic Lymphocytic Leukemia B-Cell Normal Cellular Counterpart: Clues From a Functional Perspective.

Science.gov (United States)

Darwiche, Walaa; Gubler, Brigitte; Marolleau, Jean-Pierre; Ghamlouch, Hussein

2018-01-01

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of small mature-looking CD19+ CD23+ CD5+ B-cells that accumulate in the blood, bone marrow, and lymphoid organs. To date, no consensus has been reached concerning the normal cellular counterpart of CLL B-cells and several B-cell types have been proposed. CLL B-cells have remarkable phenotypic and gene expression profile homogeneity. In recent years, the molecular and cellular biology of CLL has been enriched by seminal insights that are leading to a better understanding of the natural history of the disease. Immunophenotypic and molecular approaches (including immunoglobulin heavy-chain variable gene mutational status, transcriptional and epigenetic profiling) comparing the normal B-cell subset and CLL B-cells provide some new insights into the normal cellular counterpart. Functional characteristics (including activation requirements and propensity for plasma cell differentiation) of CLL B-cells have now been investigated for 50 years. B-cell subsets differ substantially in terms of their functional features. Analysis of shared functional characteristics may reveal similarities between normal B-cell subsets and CLL B-cells, allowing speculative assignment of a normal cellular counterpart for CLL B-cells. In this review, we summarize current data regarding peripheral B-cell differentiation and human B-cell subsets and suggest possibilities for a normal cellular counterpart based on the functional characteristics of CLL B-cells. However, a definitive normal cellular counterpart cannot be attributed on the basis of the available data. We discuss the functional characteristics required for a cell to be logically considered to be the normal counterpart of CLL B-cells.

LET effects on normal and radiosensitive cell lines

International Nuclear Information System (INIS)

Geard, C.R.; Travisano, M.

1986-01-01

Charged particles in the track segment mode were produced by the RARAF Van de Graaff accelerator and used to irradiate two CHO cell lines, a radiosensitive hypermutable line EM9 and its normal parent AA8. Asynchronous cells were irradiated attached to 6 micrometer thick Mylar with protons, deuterons and helium-3 particles at LETs ranging from 10 to 150 keV per micrometer. A 50 kVp x-ray tube integrated into the track segment facility provided a low LET comparison. Following irradiation cells were monitored for clonogenicity, and in a separate series of experiments frequencies of sister chromatid exchanges. Up to 9 experiments were carried out at each LET, with a total of 8 radiations of different LETs being compared. The optimally effective LET for cell survival was between 80 and 120 keV per micrometer, with the 150 keV per micrometer particles indicating energy wastage. The differential between the normal and radiosensitive cell lines was maintained at all LETs

Sub-cellular force microscopy in single normal and cancer cells

Energy Technology Data Exchange (ETDEWEB)

Babahosseini, H. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Carmichael, B. [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Strobl, J.S. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Mahmoodi, S.N., E-mail: nmahmoodi@eng.ua.edu [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Agah, M., E-mail: agah@vt.edu [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States)

2015-08-07

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

Sub-cellular force microscopy in single normal and cancer cells

International Nuclear Information System (INIS)

Babahosseini, H.; Carmichael, B.; Strobl, J.S.; Mahmoodi, S.N.; Agah, M.

2015-01-01

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain

Mast cell distribution in normal adult skin.

Science.gov (United States)

Janssens, A S; Heide, R; den Hollander, J C; Mulder, P G M; Tank, B; Oranje, A P

2005-03-01

To investigate mast cell distribution in normal adult skin to provide a reference range for comparison with mastocytosis. Mast cells (MCs) were counted in uninvolved skin adjacent to basal cell carcinomas and other dermatological disorders in adults. There was an uneven distribution of MCs in different body sites using the anti-tryptase monoclonal antibody technique. Numbers of MCs on the trunk, upper arm, and upper leg were similar, but were significantly different from those found on the lower leg and forearm. Two distinct groups were formed--proximal and distal. There were 77.0 MCs/mm2 at proximal body sites and 108.2 MCs/mm2 at distal sites. Adjusted for the adjacent diagnosis and age, this difference was consistent. The numbers of MCs in uninvolved skin adjacent to basal cell carcinomas and other dermatological disorders were not different from those in the control group. Differences in the numbers of MCs between the distal and the proximal body sites must be considered when MCs are counted for a reliable diagnosis of mastocytosis. A pilot study in patients with mastocytosis underlined the variation in the numbers of MCs in mastocytosis and normal skin, but showed a considerable overlap. The observed numbers of MCs in adults cannot be extrapolated to children. MC numbers varied significantly between proximal and distal body sites and these differences must be considered when MCs are counted for a reliable diagnosis of mastocytosis. There was a considerable overlap between the numbers of MCs in mastocytosis and normal skin.

Nuclear localization of the mitochondrial ncRNAs in normal and cancer cells.

Science.gov (United States)

Landerer, Eduardo; Villegas, Jaime; Burzio, Veronica A; Oliveira, Luciana; Villota, Claudio; Lopez, Constanza; Restovic, Franko; Martinez, Ronny; Castillo, Octavio; Burzio, Luis O

2011-08-01

We have previously shown a differential expression of a family of mitochondrial ncRNAs in normal and cancer cells. Normal proliferating cells and cancer cells express the sense mitochondrial ncRNA (SncmtRNA). In addition, while normal proliferating cells express two antisense mitochondrial ncRNAs (ASncmtRNAs-1 and -2), these transcripts seem to be universally down-regulated in cancer cells. In situ hybridization (ISH) of some normal and cancer tissues reveals nuclear localization of these transcripts suggesting that they are exported from mitochondria. FISH and confocal microscopy, in situ digestion with RNase previous to ISH and electron microscopy ISH was employed to confirm the extra-mitochondrial localization of the SncmtRNA and the ASncmtRNAs in normal proliferating and cancer cells of human and mouse. In normal human kidney and mouse testis the SncmtRNA and the ASncmtRNAs were found outside the organelle and especially localized in the nucleus associated to heterochromatin. In cancer cells, only the SncmtRNA was expressed and was found associated to heterochromatin and nucleoli. The ubiquitous localization of these mitochondrial transcripts in the nucleus suggests that they are new players in the mitochondrial-nuclear communication pathway or retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step on neoplastic transformation and cancer progression.

High content screening of defined chemical libraries using normal and glioma-derived neural stem cell lines.

Science.gov (United States)

Danovi, Davide; Folarin, Amos A; Baranowski, Bart; Pollard, Steven M

2012-01-01

Small molecules with potent biological effects on the fate of normal and cancer-derived stem cells represent both useful research tools and new drug leads for regenerative medicine and oncology. Long-term expansion of mouse and human neural stem cells is possible using adherent monolayer culture. These cultures represent a useful cellular resource to carry out image-based high content screening of small chemical libraries. Improvements in automated microscopy, desktop computational power, and freely available image processing tools, now means that such chemical screens are realistic to undertake in individual academic laboratories. Here we outline a cost effective and versatile time lapse imaging strategy suitable for chemical screening. Protocols are described for the handling and screening of human fetal Neural Stem (NS) cell lines and their malignant counterparts, Glioblastoma-derived neural stem cells (GNS). We focus on identification of cytostatic and cytotoxic "hits" and discuss future possibilities and challenges for extending this approach to assay lineage commitment and differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

Directory of Open Access Journals (Sweden)

Porceddu Enrico

2009-02-01

Full Text Available Abstract Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability

EXPRESSION OF CELLULAR ADHESION MOLECULES IN LANGERHANS CELL HISTIOCYTOSIS AND NORMAL LANGERHANS CELLS

NARCIS (Netherlands)

DEGRAAF, JH; TAMMINGA, RYJ; KAMPS, WA; TIMENS, W

1995-01-01

Langerhans cell histiocytosis (LCH) is characterized by lesions with an accumulation and/or proliferation of Langerhans cells (LCs). Little is known of the etiology and pathogenesis of LCH. Although the relation between the LCH cell and normal LCs is currently uncertain, the localizations of the LCH

Modulated Raman spectroscopy for enhanced identification of bladder tumor cells in urine samples.

Science.gov (United States)

Canetta, Elisabetta; Mazilu, Michael; De Luca, Anna Chiara; Carruthers, Antonia E; Dholakia, Kishan; Neilson, Sam; Sargeant, Harry; Briscoe, Tina; Herrington, C Simon; Riches, Andrew C

2011-03-01

Standard Raman spectroscopy (SRS) is a noninvasive technique that is used in the biomedical field to discriminate between normal and cancer cells. However, the presence of a strong fluorescence background detracts from the use of SRS in real-time clinical applications. Recently, we have reported a novel modulated Raman spectroscopy (MRS) technique to extract the Raman spectra from the background. In this paper, we present the first application of MRS to the identification of human urothelial cells (SV-HUC-1) and bladder cancer cells (MGH) in urine samples. These results are compared to those obtained by SRS. Classification using the principal component analysis clearly shows that MRS allows discrimination between Raman spectra of SV-HUC-1 and MGH cells with high sensitivity (98%) and specificity (95%). MRS is also used to distinguish between SV-HUC-1 and MGH cells after exposure to urine for up to 6 h. We observe a marked change in the MRS of SV-HUC-1 and MGH cells with time in urine, indicating that the conditions of sample collection will be important for the application of this methodology to clinical urine samples.

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

Science.gov (United States)

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

International Nuclear Information System (INIS)

Hasan, Yasmin; Schoenherr, Diane; Martinez, Alvaro A.; Wilson, George D.; Marples, Brian

2010-01-01

Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity was assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing γH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2μg/mL], Trinovin [10μg/mL], and Prostate Rx [50 μg/mL]). However, both Trinovin (10μg/mL) and Prostate Rx (6μg/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.

Comparison of the circadian variation in cell proliferation in normal and neoplastic colonic epithelial cells.

Science.gov (United States)

Kennedy, M F; Tutton, P J; Barkla, D H

1985-09-15

Circadian variations in cell proliferation in normal tissues have been recognised for many years but comparable phenomena in neoplastic tissues appear not to have been reported. Adenomas and carcinomas were induced in mouse colon by injection of dimethylhydrazine (DMH) and cell proliferation in these tumors was measured stathmokinetically. In normal intestine cell proliferation is fastest at night whereas in both adenomas and carcinomas it was found to be slower at night than in the middle of the day. Chemical sympathectomy was found to abolish the circadian variation in tumor cell proliferation.

CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

International Nuclear Information System (INIS)

Hwang, Chae Young; Lee, Seung-Min; Park, Sung Sup; Kwon, Ki-Sun

2012-01-01

Highlights: ► H 2 O 2 differently adjusted senescence and proliferation in normal and cancer cells. ► H 2 O 2 exposure transiently decreased PCNA levels in normal cells. ► H 2 O 2 exposure transiently increased CDK2 activity in cancer cells. ► p21 Cip1 is likely dispensable when H 2 O 2 induces senescence in normal cells. ► Suggestively, CDK2 and PCNA play critical roles in H 2 O 2 -induced cell fate decision. -- Abstract: Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H 2 O 2 decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H 2 O 2 increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H 2 O 2 -induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21 Cip1 /PCNA complex plays an important role as a regulator of cell fate decisions.

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

International Nuclear Information System (INIS)

Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

1985-01-01

Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and 14 C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of 14 C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

Science.gov (United States)

Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

2011-11-01

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

Discrimination Between Cervical Cancer Cells and Normal Cervical Cells Based on Longitudinal Elasticity Using Atomic Force Microscopy.

Science.gov (United States)

Zhao, Xueqin; Zhong, Yunxin; Ye, Ting; Wang, Dajing; Mao, Bingwei

2015-12-01

The mechanical properties of cells are considered promising biomarkers for the early diagnosis of cancer. Recently, atomic force microscopy (AFM)-based nanoindentation technology has been utilized for the examination of cell cortex mechanics in order to distinguish malignant cells from normal cells. However, few attempts to evaluate the biomechanical properties of cells have focused on the quantification of the non-homogeneous longitudinal elasticity of cellular structures. In the present study, we applied a variation of the method of Carl and Schillers to investigate the differences between longitudinal elasticity of human cervical squamous carcinoma cells (CaSki) and normal cervical epithelial cells (CRL2614) using AFM. The results reveal a three-layer heterogeneous structure in the probing volume of both cell types studied. CaSki cells exhibited a lower whole-cell stiffness and a softer nuclei zone compared to the normal counterpart cells. Moreover, a better differentiated cytoskeleton was found in the inner cytoplasm/nuclei zone of the normal CRL2614 cells, whereas a deeper cytoskeletal distribution was observed in the probing volume of the cancerous counterparts. The sensitive cortical panel of CaSki cells, with a modulus of 0.35~0.47 kPa, was located at 237~225 nm; in normal cells, the elasticity was 1.20~1.32 kPa at 113~128 nm. The present improved method may be validated using the conventional Hertz-Sneddon method, which is widely reported in the literature. In conclusion, our results enable the quantification of the heterogeneous longitudinal elasticity of cancer cells, in particular the correlation with the corresponding depth. Preliminary results indicate that our method may potentially be applied to improve the detection of cancerous cells and provide insights into the pathophysiology of the disease.

Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues.

Directory of Open Access Journals (Sweden)

Mehdi Mesri

Full Text Available Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.

Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells

NARCIS (Netherlands)

A.M. Sieuwerts (Anieta); J. Kraan (Jaco); J. Bolt (Joan); P. van der Spoel (Petra); F. Elstrodt (Fons); A.E.M. Schutte (Mieke); J.W.M. Martens (John); J.W. Gratama (Jan-Willem); S. Sleijfer (Stefan); J.A. Foekens (John)

2009-01-01

textabstractIdentification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all

Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells

International Nuclear Information System (INIS)

Escaffit, Fabrice; Pare, Frederic; Gauthier, Remy; Rivard, Nathalie; Boudreau, Francois; Beaulieu, Jean-Francois

2006-01-01

The homeobox gene Cdx2 is involved in the regulation of the expression of intestine specific markers such as sucrase-isomaltase and lactase-phlorizin hydrolase. Previous studies performed with immortalized or transformed intestinal cell lines have provided evidence that Cdx2 can promote morphological and functional differentiation in these experimental models. However, no data exist concerning the implication of this factor in normal human intestinal cell physiology. In the present work, we have investigated the role of Cdx2 in normal human intestinal epithelial crypt (HIEC) cells that lack this transcription factor. The establishment of HIEC cells expressing Cdx2 in an inducible manner shows that forced expression of Cdx2 significantly alters the proliferation of intestinal crypt cells and stimulates dipeptidylpeptidase IV expression but is not sufficient to trigger intestinal terminal differentiation. These observations suggest that Cdx2 requires additional factors to activate the enterocyte differentiation program in normal undifferentiated cells

Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

International Nuclear Information System (INIS)

Borek, C.

1982-01-01

In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

Distinguishing normal cells from cancer cells via lysosome-targetable pH biomarkers with benzo[a]phenoxazine skeleton

Energy Technology Data Exchange (ETDEWEB)

Zhan, Yan-Hua [College of Chemistry, Chemical Engineering and Material Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Soochow University, 199 Ren’Ai Road, Suzhou, 215123 (China); Li, Xiao-Jun [School of Radiation Medicine and Protection, Medicine College of Soochow University, Suzhou, 215123 (China); Sun, Ru, E-mail: sunru924@hotmail.com [College of Chemistry, Chemical Engineering and Material Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Soochow University, 199 Ren’Ai Road, Suzhou, 215123 (China); Xu, Yu-Jie [School of Radiation Medicine and Protection, Medicine College of Soochow University, Suzhou, 215123 (China); Ge, Jian-Feng, E-mail: ge_jianfeng@hotmail.com [College of Chemistry, Chemical Engineering and Material Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Soochow University, 199 Ren’Ai Road, Suzhou, 215123 (China); Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163 (China)

2016-08-24

In this paper, the design of a lysosome-targetable pH probe that has a fluorescent OFF (pH = 4) to ON (pH = 5–6) response is described to identify lysosomes in normal cells. The mechanism of photoinduced electron transfer with a fluorophore-based reaction (FBR-PET) was proposed. Benzo[a]phenoxazines with electro-donating aryl groups were selected, its (2,5-dimethoxyphenyl)imino-, (2-hydroxyphenyl)imino- and (2-hydroxy-5-methoxyphenyl)- imino-derivatives (probes 1a−c) were prepared and their optical responses towards pH were evaluated; their fluorescence pH titration experiments gave regularly changes with the increasing electro-donating abilities at the linked aryl groups, the (2-hydroxy-5-methoxyphenyl)iminobenzo[a]phenoxazine (probe 1c) exhibited a nearly OFF−ON response at 580–800 nm. All probes were reversible, and they showed excellent selectivity toward the proton over other competitive species. Fluorescence confocal images were performed with HeLa, KB cancer cells and V79 normal cells, probes 1a−c are all lysosome-targetable pH probes, and benzo[a]phenoxazine with (2-hydroxy-5-methoxyphenyl)imino-group (probe 1c) has potential applications in selective differentiation of normal cells from cancer cells. - Highlights: • pH probes for lysosome detection in normal cells. • Differentiation of normal cells from cancer cells by lysosome-biomarker. • The PET mechanism promoted by fluorophore based reactions (FBR-PET).

Distinguishing normal cells from cancer cells via lysosome-targetable pH biomarkers with benzo[a]phenoxazine skeleton

International Nuclear Information System (INIS)

Zhan, Yan-Hua; Li, Xiao-Jun; Sun, Ru; Xu, Yu-Jie; Ge, Jian-Feng

2016-01-01

In this paper, the design of a lysosome-targetable pH probe that has a fluorescent OFF (pH = 4) to ON (pH = 5–6) response is described to identify lysosomes in normal cells. The mechanism of photoinduced electron transfer with a fluorophore-based reaction (FBR-PET) was proposed. Benzo[a]phenoxazines with electro-donating aryl groups were selected, its (2,5-dimethoxyphenyl)imino-, (2-hydroxyphenyl)imino- and (2-hydroxy-5-methoxyphenyl)- imino-derivatives (probes 1a−c) were prepared and their optical responses towards pH were evaluated; their fluorescence pH titration experiments gave regularly changes with the increasing electro-donating abilities at the linked aryl groups, the (2-hydroxy-5-methoxyphenyl)iminobenzo[a]phenoxazine (probe 1c) exhibited a nearly OFF−ON response at 580–800 nm. All probes were reversible, and they showed excellent selectivity toward the proton over other competitive species. Fluorescence confocal images were performed with HeLa, KB cancer cells and V79 normal cells, probes 1a−c are all lysosome-targetable pH probes, and benzo[a]phenoxazine with (2-hydroxy-5-methoxyphenyl)imino-group (probe 1c) has potential applications in selective differentiation of normal cells from cancer cells. - Highlights: • pH probes for lysosome detection in normal cells. • Differentiation of normal cells from cancer cells by lysosome-biomarker. • The PET mechanism promoted by fluorophore based reactions (FBR-PET).

Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue

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Luo, Wei; Hu, Qiang; Wang, Dan; Deeb, Kristin K.; Ma, Yingyu; Morrison, Carl D.; Liu, Song; Johnson, Candace S.; Trump, Donald L.

2013-01-01

Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissues obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small

Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

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Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

2016-05-01

Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture

Quantitative identification of senescent cells in aging and disease.

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Biran, Anat; Zada, Lior; Abou Karam, Paula; Vadai, Ezra; Roitman, Lior; Ovadya, Yossi; Porat, Ziv; Krizhanovsky, Valery

2017-08-01

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single-cell basis. The method combines a senescence-associated beta-galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high-content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Dendritic cells and skin sensitization: Biological roles and uses in hazard identification

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Ryan, Cindy A.; Kimber, Ian; Basketter, David A.; Pallardy, Marc; Gildea, Lucy A.; Gerberick, G. Frank

2007-01-01

Recent advances have been made in our understanding of the roles played by cutaneous dendritic cells (DCs) in the induction of contact allergy. A number of associated changes in epidermal Langerhans cell phenotype and function required for effective skin sensitization are providing the foundations for the development of cellular assays (using DC and DC-like cells) for skin sensitization hazard identification. These alternative approaches to the identification and characterization of skin sensitizing chemicals were the focus of a Workshop entitled 'Dendritic Cells and Skin Sensitization: Biological Roles and Uses in Hazard Identification' that was given at the annual Society of Toxicology meeting held March 6-9, 2006 in San Diego, California. This paper reports information that was presented during the Workshop

Normalization of cell responses in cat striate cortex

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Heeger, D. J.

1992-01-01

Simple cells in the striate cortex have been depicted as half-wave-rectified linear operators. Complex cells have been depicted as energy mechanisms, constructed from the squared sum of the outputs of quadrature pairs of linear operators. However, the linear/energy model falls short of a complete explanation of striate cell responses. In this paper, a modified version of the linear/energy model is presented in which striate cells mutually inhibit one another, effectively normalizing their responses with respect to stimulus contrast. This paper reviews experimental measurements of striate cell responses, and shows that the new model explains a significantly larger body of physiological data.

Redox biology in normal cells and cancer: restoring function of the redox/Fyn/c-Cbl pathway in cancer cells offers new approaches to cancer treatment.

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Noble, Mark; Mayer-Pröschel, Margot; Li, Zaibo; Dong, Tiefei; Cui, Wanchang; Pröschel, Christoph; Ambeskovic, Ibro; Dietrich, Joerg; Han, Ruolan; Yang, Yin Miranda; Folts, Christopher; Stripay, Jennifer; Chen, Hsing-Yu; Stevens, Brett M

2015-02-01

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/βpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries. Copyright © 2015. Published by Elsevier Inc.

Comparison of Gated Audiovisual Speech Identification in Elderly Hearing Aid Users and Elderly Normal-Hearing Individuals

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Lidestam, Björn; Rönnberg, Jerker

2016-01-01

The present study compared elderly hearing aid (EHA) users (n = 20) with elderly normal-hearing (ENH) listeners (n = 20) in terms of isolation points (IPs, the shortest time required for correct identification of a speech stimulus) and accuracy of audiovisual gated speech stimuli (consonants, words, and final words in highly and less predictable sentences) presented in silence. In addition, we compared the IPs of audiovisual speech stimuli from the present study with auditory ones extracted from a previous study, to determine the impact of the addition of visual cues. Both participant groups achieved ceiling levels in terms of accuracy in the audiovisual identification of gated speech stimuli; however, the EHA group needed longer IPs for the audiovisual identification of consonants and words. The benefit of adding visual cues to auditory speech stimuli was more evident in the EHA group, as audiovisual presentation significantly shortened the IPs for consonants, words, and final words in less predictable sentences; in the ENH group, audiovisual presentation only shortened the IPs for consonants and words. In conclusion, although the audiovisual benefit was greater for EHA group, this group had inferior performance compared with the ENH group in terms of IPs when supportive semantic context was lacking. Consequently, EHA users needed the initial part of the audiovisual speech signal to be longer than did their counterparts with normal hearing to reach the same level of accuracy in the absence of a semantic context. PMID:27317667

Radiosensitivity of normal human epidermal cells in culture

International Nuclear Information System (INIS)

Dover, R.; Potten, C.S.

1983-01-01

Using an in vitro culture system the authors have derived #betta#-radiation survival curves over a dose range 0-8 Gy for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells. (author)

Automatic identification and normalization of dosage forms in drug monographs

Science.gov (United States)

2012-01-01

Background Each day, millions of health consumers seek drug-related information on the Web. Despite some efforts in linking related resources, drug information is largely scattered in a wide variety of websites of different quality and credibility. Methods As a step toward providing users with integrated access to multiple trustworthy drug resources, we aim to develop a method capable of identifying drug's dosage form information in addition to drug name recognition. We developed rules and patterns for identifying dosage forms from different sections of full-text drug monographs, and subsequently normalized them to standardized RxNorm dosage forms. Results Our method represents a significant improvement compared with a baseline lookup approach, achieving overall macro-averaged Precision of 80%, Recall of 98%, and F-Measure of 85%. Conclusions We successfully developed an automatic approach for drug dosage form identification, which is critical for building links between different drug-related resources. PMID:22336431

Olfactory granule cell development in normal and hyperthyroid rats.

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Brunjes, P C; Schwark, H D; Greenough, W T

1982-10-01

Dendritic development was examined in olfactory bulbs of both normal 7-, 14-, 21- and 60-day-old rats and littermates treated on postnatal days 1-4 with 1 microgram/g body weight of L-thyroxine sodium. Tissue was processed via the Golgi-Cox technique and subjected to quantitative analyses of mitral and internal layer granule cell development. These populations of granule cells were selected because their pattern of late proliferation suggested potentially greater susceptibility to postnatal hormonal alterations. Although neonatal hyperthyroidism induces widespread acceleration of maturation, including precocious chemosensitivity, granule cell development was unaffected relative to littermate controls. Both normal and hyperthyroid groups exhibited an inverted U-shaped pattern of cellular development, with rapid dendritic dendritic growth and expansion occurring during the earliest ages tested, but with loss of processes and dendritic field size occurring after day 21.

Cell renewal of glomerular cell types in normal rats. An autoradiographic analysis

International Nuclear Information System (INIS)

Pabst, R.; Sterzel, R.B.

1983-01-01

Normal adult Sprague-Dawley rats received either a single or repetitive injection of the DNA precursor 3 H-thymidine ( 3 H-TdR). For autoradiography semi-thin sections were prepared 2 hr to 14 days after labeling. The majority of labeled cells noted in glomerular tufts were endothelial cells. Mesangial cells had a lower production rate. Podocytes revealed no evidence of proliferation. Bowman's capsule cells showed a higher labeling index than tuft cells at all times. Neither the urinary nor the vascular pole was found to be a proliferative zone for Bowman's capsule cells. The flash and repetitive labeling experiments demonstrated a constant rate of cell renewal of about 1% per day, resulting in a long life span for endothelial and mesangial cells as well as Bowman's capsule cells. These data provide a basis for cell kinetic studies in models of glomerular diseases

Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

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Clementina Sansone

Full Text Available Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD, 2-trans,4-trans-octadienal (OD and 2-trans,4-trans-heptadienal (HD on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1 and Fas Associated Death Domain (FADD leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP. The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

The use of HPLC-MS in T-cell epitope identification.

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Lemmel, Claudia; Stevanović, Stefan

2003-03-01

The hunt for T-cell epitopes is going on because hopes are set on such peptide sequences for diagnosis and vaccine development in the fight against infectious and tumor diseases. In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometers coupled to microcapillary liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we review the basics of mass spectrometric techniques and their on-line coupling to microcapillary liquid chromatography (microcap-LC). Furthermore, we introduce current strategies for the identification of new T-cell epitopes using microcapillary liquid chromatography-mass spectrometry (microcap-LC-MS).

Small copper fixed-point cells of the hybrid type to be used in place of normal larger cells

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Battuello, M.; Girard, F.; Florio, M.

2012-10-01

Two small cells for the realization of the fixed point of copper were constructed and investigated at INRIM. They are of the same hybrid design generally adopted for the eutectic high-temperature fixed-point cells, namely a structure with a sacrificial graphite sleeve and a layer of flexible carbon-carbon composite sheet (C/C sheet). Because of the largely different design with respect to the cells normally adopted for the construction of pure metal fixed points, they were compared and characterized with respect to the normal cells used at INRIM for the ITS-90 realization. Two different furnaces were used to compare hybrid and normal cells. One of the hybrid cells was also used in different configurations, i.e. without the C/C sheet and with two layers of sheet. The cells were compared with different operative conditions, i.e. temperature settings of the furnaces for inducing the freeze, and repeatability and reproducibility were investigated. Freezing temperature and shape of the plateaux obtained under the different conditions were analysed. As expected the duration of the plateaux obtained with the hybrid cells is considerably shorter than with the normal cell, but this does not affect the results in terms of freezing temperature. Measurements with the modified cell showed that the use of a double C/C sheet may improve both repeatability and reproducibility of the plateaux.

Small copper fixed-point cells of the hybrid type to be used in place of normal larger cells

International Nuclear Information System (INIS)

Battuello, M; Girard, F; Florio, M

2012-01-01

Two small cells for the realization of the fixed point of copper were constructed and investigated at INRIM. They are of the same hybrid design generally adopted for the eutectic high-temperature fixed-point cells, namely a structure with a sacrificial graphite sleeve and a layer of flexible carbon–carbon composite sheet (C/C sheet). Because of the largely different design with respect to the cells normally adopted for the construction of pure metal fixed points, they were compared and characterized with respect to the normal cells used at INRIM for the ITS-90 realization. Two different furnaces were used to compare hybrid and normal cells. One of the hybrid cells was also used in different configurations, i.e. without the C/C sheet and with two layers of sheet. The cells were compared with different operative conditions, i.e. temperature settings of the furnaces for inducing the freeze, and repeatability and reproducibility were investigated. Freezing temperature and shape of the plateaux obtained under the different conditions were analysed. As expected the duration of the plateaux obtained with the hybrid cells is considerably shorter than with the normal cell, but this does not affect the results in terms of freezing temperature. Measurements with the modified cell showed that the use of a double C/C sheet may improve both repeatability and reproducibility of the plateaux. (paper)

CDDO-Me protects normal lung and breast epithelial cells but not cancer cells from radiation.

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Mariam El-Ashmawy

Full Text Available Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs. In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF = 1.3, and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients.

Vesicles mimicking normal and cancer cell membranes exhibit differential responses to the cell-penetrating peptide Pep-1.

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Almarwani, Bashiyar; Phambu, Esther Nzuzi; Alexander, Christopher; Nguyen, Ha Aimee T; Phambu, Nsoki; Sunda-Meya, Anderson

2018-06-01

The cell-penetrating peptide (CPP) Pep-1 presents a great potential in drug delivery due to its intrinsic property to cross plasma membrane. However, its mechanism of entry into the cell remains unresolved. In this study, we compare the selectivity of Pep-1 towards vesicles mimicking normal and cancer cell membranes. The interaction was performed in a wide range of peptide-to-lipid molar ratios using infrared (IR), fluorescence, scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. At low peptide concentration, fluorescence experiments show that lipid-phosphatidylserine (PS) seems to enable Pep-1 translocation into cancer cell membrane as evidenced by the blue shift of its maximal emission wavelength. DSC data show that Pep-1 induces segregation of lipids. At high peptide concentration, IR data indicate that the interaction of Pep-1 is relatively stronger with normal cell membrane than with cancer cell membrane through the phosphate groups, while the interaction is weaker with normal cell membrane than with cancer cell membrane through the carbonyl groups. TGA and DSC data reveal that vesicles of normal cell membrane are thermally more stable than vesicles of cancer cell membrane. This suggests that the additional lipid PS included in cancer cell membrane has a destabilizing effect on the membrane structure. SEM images reveal that Pep-1 form superstructures including spherical particles and fibrils in the presence of both model membranes. PS seems to enhance peptide transport across cellular membranes. The biophysical techniques in this study provide valuable insights into the properties of CPPs in drug delivery systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

International Nuclear Information System (INIS)

Kheradmand, Arash; Dezfoulian, Omid; Alirezaei, Masoud; Rasoulian, Bahram

2012-01-01

Highlights: ► Spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. ► Numerous studies have documented the direct action of ghrelin in the modulation of apoptosis in different cell types. ► Ghrelin may be considered as a modulator of spermatogenesis in normal adult rats. ► Ghrelin may be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors. -- Abstract: Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n = 7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P 0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.

Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

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Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

2009-03-09

Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

RON kinase isoforms demonstrate variable cell motility in normal cells.

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Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

2016-09-01

Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

Identification of cell wall proteins in the flax (Linum usitatissimum) stem.

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Day, Arnaud; Fénart, Stéphane; Neutelings, Godfrey; Hawkins, Simon; Rolando, Christian; Tokarski, Caroline

2013-03-01

Sequential salt (CaCl2 , LiCl) extractions were used to obtain fractions enriched in cell wall proteins (CWPs) from the stem of 60-day-old flax (Linum usitatissimum) plants. High-resolution FT-ICR MS analysis and the use of recently published genomic data allowed the identification of 11 912 peptides corresponding to a total of 1418 different proteins. Subcellular localization using TargetP, Predotar, and WoLF PSORT led to the identification of 152 putative flax CWPs that were classified into nine different functional classes previously established for Arabidopsis thaliana. Examination of different functional classes revealed the presence of a number of proteins known to be involved in, or potentially involved in cell-wall metabolism in plants. The flax stem cell wall proteome was also compared with transcriptomic data previously obtained on comparable samples. This study represents a major contribution to the identification of CWPs in flax and will lead to a better understanding of cell wall biology in this species. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New castanospermine glycoside analogues inhibit breast cancer cell proliferation and induce apoptosis without affecting normal cells.

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Ghada Allan

Full Text Available sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231 cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4, cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21(Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.

Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

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Lin-Lin Liu

Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

Identification of a developmental gene expression signature, including HOX genes, for the normal human colonic crypt stem cell niche: overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis.

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Bhatlekar, Seema; Addya, Sankar; Salunek, Moreh; Orr, Christopher R; Surrey, Saul; McKenzie, Steven; Fields, Jeremy Z; Boman, Bruce M

2014-01-15

Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region--the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (∼18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, ∼30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.

Bioactive form of resveratrol in glioblastoma cells and its safety for normal brain cells

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Xiao-Hong Shu

2013-05-01

Full Text Available ABSTRACTBackground: Resveratrol, a plant polyphenol existing in grapes and many other natural foods, possesses a wide range of biological activities including cancer prevention. It has been recognized that resveratrol is intracellularly biotransformed to different metabolites, but no direct evidence has been available to ascertain its bioactive form because of the difficulty to maintain resveratrol unmetabolized in vivo or in vitro. It would be therefore worthwhile to elucidate the potential therapeutic implications of resveratrol metabolism using a reliable resveratrol-sensitive cancer cells.Objective: To identify the real biological form of trans-resveratrol and to evaluate the safety of the effective anticancer dose of resveratrol for the normal brain cells.Methods: The samples were prepared from the condition media and cell lysates of human glioblastoma U251 cells, and were purified by solid phase extraction (SPE. The samples were subjected to high performance liquid chromatography (HPLC and liquid chromatography/tandem mass spectrometry (LC/MS analysis. According to the metabolite(s, trans-resveratrol was biotransformed in vitro by the method described elsewhere, and the resulting solution was used to treat U251 cells. Meanwhile, the responses of U251 and primarily cultured rat normal brain cells (glial cells and neurons to 100μM trans-resveratrol were evaluated by multiple experimental methods.Results: The results revealed that resveratrol monosulfate was the major metabolite in U251 cells. About half fraction of resveratrol monosulfate was prepared in vitro and this trans-resveratrol and resveratrol monosulfate mixture showed little inhibitory effect on U251 cells. It is also found that rat primary brain cells (PBCs not only resist 100μM but also tolerate as high as 200μM resveratrol treatment.Conclusions: Our study thus demonstrated that trans-resveratrol was the bioactive form in glioblastoma cells and, therefore, the biotransforming

A Comparison of Exogenous Labels for the Histological Identification of Transplanted Neural Stem Cells

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Nicholls, Francesca J.; Liu, Jessie R.; Modo, Michel

2017-01-01

The interpretation of cell transplantation experiments is often dependent on the presence of an exogenous label for the identification of implanted cells. The exogenous labels Hoechst 33342, 5-bromo-2′-deoxyuridine (BrdU), PKH26, and Qtracker were compared for their labeling efficiency, cellular effects, and reliability to identify a human neural stem cell (hNSC) line implanted intracerebrally into the rat brain. Hoechst 33342 (2 mg/ml) exhibited a delayed cytotoxicity that killed all cells within 7 days. This label was hence not progressed to in vivo studies. PKH26 (5 μM), Qtracker (15 nM), and BrdU (0.2 μM) labeled 100% of the cell population at day 1, although BrdU labeling declined by day 7. BrdU and Qtracker exerted effects on proliferation and differentiation. PKH26 reduced viability and proliferation at day 1, but this normalized by day 7. In an in vitro coculture assay, all labels transferred to unlabeled cells. After transplantation, the reliability of exogenous labels was assessed against the gold standard of a human-specific nuclear antigen (HNA) antibody. BrdU, PKH26, and Qtracker resulted in a very small proportion (Exogenous labels can therefore be reliable to identify transplanted cells without exerting major cellular effects, but validation is required. The interpretation of cell transplantation experiments should be presented in the context of the label's limitations. PMID:27938486

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

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Botcheva K.; McCorkle S. R.; McCombie W. R.; Dunn J. J.; Anderson C. W.

2011-12-15

We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

The effects of environmental deuterium on normal and neoplastic cultured cell development

International Nuclear Information System (INIS)

Bild, W.; Schuller, T.; Zhihai, Qin; Blankenstein, T.; Nastasa, V.; Haulica, I.

2000-01-01

The powdered culture media (RPMI - 1640) were reconstituted either with normal distilled water (150 ppm deuterium) either with deuterium - depleted water (DDW) in various concentrations (30, 60, 90 ppm) and sterilized by filtration with 0.2 μm filters. The cell lines used were NIH (normal mouse fibroblasts), RAG (mouse renal carcinoma) and TS/A (mouse mammary adenocarcinoma). In auxiliary tests, BAIBC mouse splenocytes in direct culture were used, stimulated for growth with concanavalin A or LPS (bacterial lipopolysaccharide). The estimation of the growth was made using the MTT assay or direct counting with trypan blue exclusion. The following results were obtained: Deuterium - depleted water had a stimulating effect on cell growth, the most important stimulating action being from the 90 ppm deuterium-water. The growth curves show, in a first phase, a stimulation of the rapid -growing neoplastic cells, followed by a slower growth of the normal cells. Amiloride 100 mM blocking of the Na + /K + membrane pump did not affect the cell growth curves, while the lansoprazole 100 mM blocking of the K + /H + ATP-ase brought the growth curves at the level of those with normal water. This might show an eventual involvement of the K + /H + antiport in the stimulating effects of the DDW. (authors)

Study of nuclear proteins in normal and xeroderma pigmentosum lymphoblastoid cells

International Nuclear Information System (INIS)

Amari, N.M.B.

1985-01-01

Nuclear histone and nonhistone (NHP) proteins from normal human and xeroderma pigmentosum, complementation group A (XP-A) lymphoblastoid cells were compared both qualitatively, quantitatively and for binding affinity for DNA. Histones and four NHP fractions (NHP/sub 1-4/) were isolated from purified cell nuclei. Binding affinity to [ 3 H] melanoma DNA of histones and each NHP fraction was then determined using gradient dialysis followed by a filter assay. Histones and each NHP fraction were then sub-fractionated by polyacrylamide gel electrophoresis. Densitometric scans of the separation of these proteins on the gels were qualitatively, and quantitatively analyzed and compared between the two cell lines. No qualitative or quantitative differences were observed between histones from XP-A or normal cells

Identification of human embryonic progenitor cell targeting peptides using phage display.

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Paola A Bignone

Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

Natural killer activity and suppressor cells in irradiated mice repopulated with a mixture of cells from normal and 89Sr-treated donors

International Nuclear Information System (INIS)

Levy, E.M.; Kumar, V.; Bennett, M.

1981-01-01

Mice that have been injected with 89 Sr have fairly normal B and T cell function, but are abnormal in that they lack natural killer (NK) activity and other functions that require an intact bone marrow. These mice also have an increased potential for suppressor cell activity. We had previously shown that spleen cells from 89 Sr-treated mice could transfer low NK activity and increased suppressor cell function to lethally irradiated syngeneic recipients. To investigate the mechanisms involved in perpetuating these defects, groups of normal spleen or bone marrow cells. Recipients were assayed for their NK activity and suppressor cell function 5 to 14 wk later. it was found that the addition of normal cells in the donor inoculum resulted in normal NK activity. This indicates that low NK activity in 89 Sr-treated mice was not due to the presence of a suppressor cell that prevented NK cell generation. It was additionally found that low NK activity in recipient mice could be boosted by interferon inducers. This would indicate that NK activity in the recipients was not due to a lack of interferon-sensitive pre-NK cells. Suppressor cell function in recipient mice depended on the type and number of normal cells in the donor inoculum. Bone marrow cells were very efficient in overcoming the tendency to produce suppressor cells. It took approximately 20 times more normal spleen cells to produce the same results. The implications of these findings are discussed

CD4+ T Cell Activation and Vascular Normalization: Two Sides of the Same Coin?

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De Palma, Michele; Jain, Rakesh K

2017-05-16

Normalization of tumor blood vessels enhances the infiltration and functions of T cells. Tian et al. (2017) report that effector CD4 + T cells, in turn, support vascular normalization, highlighting intertwined roles for blood vessels and T cells in cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

A comparative study on the mast cells count in oral squamous cell carcinoma and normal oral mucosa

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Mahsa Dastpak

2015-03-01

Full Text Available Introduction: Oral squamous cell carcinoma (OSCC is one of the 10 most common malignant tumors and SCC accounts 94% of all oral malignancies. Mast cells are regarded as complex and multifunctional cells, playing a significant role in immunopathology . The aim of this study is to evaluate the number of mast cells in tissue sections of oral squamous cell carcinoma (OSCC in comparison with normal mucosa. Materials & Methods: Sixty paraffin-embedded specimens were obtained from the archives of the Department of Oral and Maxillofacial Pathology,dental school of Babol university of medical science (15 high grade,15 low grade and 30 Iritation Fibroma. Classification of OSCC cases was according to the BRODER`S malignancy grading system. Hematoxylin and Eosin-stained slides were re-evaluated before entering the samples in our study. Toluidine blue(1% staining was used to identify Mast cells in samples . We used SPSS software version 18 and one way ANOVA test for analyzing data. Results: The highest mast cell count was seen in normal tissue and it was higher in low grade OSCC in comparison with high grade, but the differences between groups weren’t statistically significant. The Mean count of mast cell between OSCC and normal oral mucosa was statistically significant different(p=0.019.We didn’t observe any statistically significant difference between Mast cell counts of control group and low grade OSCC . The same result was seen between high garde and low grade OSCC . The Mean mast cell count difference between male and female groups weren’t statistically significant. The Mean mast cell count difference between high grade OSCC and control group was significant (p<0.05. Conclusion: According to the results, the average amount of mast cells decreased in OSCC in comparison with normal oral mucosa . It does not seem that mast cells play an important role in tumor progression, although further study is needed. 

MUC-1-ESA+ progenitor cells in normal benign and malignant human breast epithelial cells

OpenAIRE

Lu, Xinquan; Li, Huixiang; Xu, Kejia; Nesland, Jahn M.; Suo, Zhenhe

2009-01-01

The existence of mammary epithelial stem/progenitor cells has been demonstrated in MUC-1-/ ESA+ subpopulations of breast epithelial cells. However, knowledge about the expression and localization in benign and malignant breast lesions is unknown. Using a double-staining immunohistochemistry method, we investigated MUC-1-/ESA+ cells in 10 normal breast tissues, 49 cases with fibrocystic disease, 40 fibroadenomas, 36 invasive ductal carcinomas and the breast cancer ce...

Amniotic Fluid Cells Proliferation in Normal and Down Syndrome Subjects

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Honcea Adina

2016-02-01

Full Text Available Down Syndrome/Trisomy 21 is the most common chromosomal anomaly, and it represents the most common congenital cause of infants’ intellectual disability. Subjects with this syndrome are affected by degenerative processes caused by accelerated aging or unknown ethyologies. In recent years, accumulating evidence revealed increased potential of amniotic fluid-derived stem cells to be used in regenerative therapy. Our aim was to assess differences in immunophenotype, cell morphology and proliferation of amniotic fluid cells from normal and Down Syndrome pregnancies using a quantitative cytometry approach. Results revealed the emergence of a population of small sized cells in Down Syndrome derived amniotic fluid cells that are readily visible upon microscopic inspection. Hence, the fluorescence–based quantitative image cytometry determinations showed a tendency of decrease in both cell and nuclei size in trisomy, with no significant modification in nuclei circularity, as measured following actin cytoskeleton and nuclei labeling. The propensity of Ki67 positive cells was found to be increased in Down Syndrome derived cells (48.92% as compared to normal specimens (28.68%. However, cells in S and G2/M cell cycle phases decreased from 32.91% to 4.49% in diseased cells. Further studies are devoted to understanding the molecular basis of the observed differences in the proliferation ability of Down Syndrome amniotic cells, in order to evaluate the potential therapeutic effect of amniotic fluid stem cells for tissue regeneration in subjects with trisomy and to find correlations between amniotic cells phenotype and patient prognosis.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

Science.gov (United States)

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Single cell analysis of normal and leukemic hematopoiesis.

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Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

2018-02-01

The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

Culture of normal human blood cells in a diffusion chamber system II. Lymphocyte and plasma cell kinetics

International Nuclear Information System (INIS)

Chikkappa, G.; Carsten, A.L.; Chanana, A.D.; Cronkite, E.P.

1979-01-01

Normal human blood leukocytes were cultured in Millipore diffusion chambers implanted into the peritoneal cavities of irradiated mice. The evaluation of survival and proliferation kinetics of cells in lymphyocytic series suggested that the lymphoid cells are formed from transition of small and/or large lymphocytes, and the lymphoblasts from the lymphoid cells. There was also evidence indicating that some of the cells in these two compartments are formed by proliferation. The evaluation of plasmacytic series suggested that the plasma cells are formed from plasmacytoid-lymphocytes by transition, and the latter from the transition of lymphocytes. In addition, relatively a small fraction of cells in these two compartments are formed by proliferation. mature plasma cells do not and immature plasma cells do proliferate. Estimation of magnitude of plasma cells formed in the cultures at day 18 indicated that at least one plasma cell is formed for every 6 normal human blood lymphocytes introduced into the culture

Stem Cell Therapy to Reduce Radiation-Induced Normal Tissue Damage

NARCIS (Netherlands)

Coppes, Rob P.; van der Goot, Annemieke; Lombaert, Isabelle M. A.

Normal tissue damage after radiotherapy is still a major problem in cancer treatment. Stem cell therapy may provide a means to reduce radiation-induced side effects and improve the quality of life of patients. This review discusses the current status in stem cell research with respect to their

Study on activity measurement of Nostoc flagelliforme cells based on color identification

Science.gov (United States)

Wang, Yizhong; Su, Jianyu; Liu, Tiegen; Kong, Fanzhi; Jia, Shiru

2008-12-01

In order to measure the activities of Nostoc flagelliforme cells, a new method based on color identification was proposed in this paper. N. flagelliforme cells were colored with fluoreseein diaeetate. Then, an image of colored N. flagelliforme cells was taken, and changed from RGB model to HIS model. Its histogram of hue H was calculated, which was used as the input of a designed BP network. The output of the BP network was the description of measured activity of N. flagelliforme cells. After training, the activity of N. flagelliforme cells was identified by the BP network according to the histogram of H of their colored image. Experiments were conducted with satisfied results to show the feasibility and usefulness of activity measurement of N. flagelliforme cells based on color identification.

Duchenne muscular dystrophy: normal ATP turnover in cultured cells

International Nuclear Information System (INIS)

Fox, I.H.; Bertorini, T.; Palmieri, G.M.A.; Shefner, R.

1986-01-01

This paper examines ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients and there was no difference in ATP synthesis or degradation. The ATP synthesis was measured by the incorporation of C 14-U-adenine into aTP and ADP. although there was a significant decrease in radioactively labelled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration of ADP metabolism

Identification of IFN-gamma-producing CD4+ T cells following PMA stimulation

DEFF Research Database (Denmark)

Kemp, K; Bruunsgaard, H

2001-01-01

Treatment of T cells with phorbol esters, such as phorbol myristate acetate (PMA), induces downregulation of CD4, making unambiguous identification of this subset difficult. In this study, the kinetics of intracellular expression of interferon-gamma (IFN-gamma) and downmodulation of surface CD4...... were measured in peripheral blood mononuclear cells (PBMC) after PMA stimulation. The number of IFN-gamma-producing cells increased within a 4-h period while the fluorescence intensity of the CD4(+) cell population decreased, and the two phenomena were correlated (n = 9; p = 0.01). Our data suggest...... that intracellular staining of CD4 together with cytokine staining will make identification of CD4(+) cells possible and facilitate the procedure of intracellular staining of cytokines....

GC-MS-Based Endometabolome Analysis Differentiates Prostate Cancer from Normal Prostate Cells

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Ana Rita Lima

2018-03-01

Full Text Available Prostate cancer (PCa is an important health problem worldwide. Diagnosis and management of PCa is very complex because the detection of serum prostate specific antigen (PSA has several drawbacks. Metabolomics brings promise for cancer biomarker discovery and for better understanding PCa biochemistry. In this study, a gas chromatography–mass spectrometry (GC-MS based metabolomic profiling of PCa cell lines was performed. The cell lines include 22RV1 and LNCaP from PCa with androgen receptor (AR expression, DU145 and PC3 (which lack AR expression, and one normal prostate cell line (PNT2. Regarding the metastatic potential, PC3 is from an adenocarcinoma grade IV with high metastatic potential, DU145 has a moderate metastatic potential, and LNCaP has a low metastatic potential. Using multivariate analysis, alterations in levels of several intracellular metabolites were detected, disclosing the capability of the endometabolome to discriminate all PCa cell lines from the normal prostate cell line. Discriminant metabolites included amino acids, fatty acids, steroids, and sugars. Six stood out for the separation of all the studied PCa cell lines from the normal prostate cell line: ethanolamine, lactic acid, β-Alanine, L-valine, L-leucine, and L-tyrosine.

Survival of human osteosarcoma cells and normal human fibroblasts following alpha particle irradiation

International Nuclear Information System (INIS)

Lloyd, E.L.; Gemmell, M.A.

1981-01-01

Cell survival of human osteosarcoma cells in culture following alpha particle irradiation is reported here for the first time. The osteosarcoma cell line (TE-85) is found to be less sensitive to inactivation by 5.6 MeV alpha particles (LET 86 keV/μm) than normal diploid human fibroblasts (NFS). Values for the mean lethal doses were estimated to be 103 rads for the TE-85 cells compared with 68 rads for the NFS cultures irradiated under identical conditions. It is postulated that the aneuploidy of the tumor cells with increased DNA chromosomal material may confer a selective advantage for the survival of tumor cells relative to normal cells with diploid chromosomes

Analysis of epothilone B-induced cell death in normal ovarian cells.

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Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

2013-12-01

We have investigated the mode of cell death induced by a new microtubule-stabilizing agent, epothilone B (EpoB, patupilone), and a clinically used medicine, paclitaxel (PTX), in normal ovarian cells. Using fluorescence microscopy, polyacrylamide gel electrophoresis preceding Western blot analysis, as well as spectrofluorimetric and colorimetric detection, we demonstrate that, compared to EpoB, PTX induced high time-dependent morphological and biochemical changes typical of apoptosis. Induction of apoptosis followed an early increase in p53 levels. Apoptosis reached its maximum at 24-48 h. At the same time, there was a significant increase in caspase-9 and -3 activity and PARP fragmentation, which suggests that an intrinsic path was involved. Apoptosis in MM14 cells was increased more by PTX than EpoB, and also induced more necrosis responsible for inflammation (1.4-fold) than EpoB. © 2013 International Federation for Cell Biology.

Automated Photoreceptor Cell Identification on Nonconfocal Adaptive Optics Images Using Multiscale Circular Voting.

Science.gov (United States)

Liu, Jianfei; Jung, HaeWon; Dubra, Alfredo; Tam, Johnny

2017-09-01

Adaptive optics scanning light ophthalmoscopy (AOSLO) has enabled quantification of the photoreceptor mosaic in the living human eye using metrics such as cell density and average spacing. These rely on the identification of individual cells. Here, we demonstrate a novel approach for computer-aided identification of cone photoreceptors on nonconfocal split detection AOSLO images. Algorithms for identification of cone photoreceptors were developed, based on multiscale circular voting (MSCV) in combination with a priori knowledge that split detection images resemble Nomarski differential interference contrast images, in which dark and bright regions are present on the two sides of each cell. The proposed algorithm locates dark and bright region pairs, iteratively refining the identification across multiple scales. Identification accuracy was assessed in data from 10 subjects by comparing automated identifications with manual labeling, followed by computation of density and spacing metrics for comparison to histology and published data. There was good agreement between manual and automated cone identifications with overall recall, precision, and F1 score of 92.9%, 90.8%, and 91.8%, respectively. On average, computed density and spacing values using automated identification were within 10.7% and 11.2% of the expected histology values across eccentricities ranging from 0.5 to 6.2 mm. There was no statistically significant difference between MSCV-based and histology-based density measurements (P = 0.96, Kolmogorov-Smirnov 2-sample test). MSCV can accurately detect cone photoreceptors on split detection images across a range of eccentricities, enabling quick, objective estimation of photoreceptor mosaic metrics, which will be important for future clinical trials utilizing adaptive optics.

Evaluation of CD307a expression patterns during normal B-cell maturation and in B-cell malignancies by flow cytometry.

Science.gov (United States)

Auat, Mariangeles; Cardoso, Chandra Chiappin; Santos-Pirath, Iris Mattos; Rudolf-Oliveira, Renata Cristina Messores; Matiollo, Camila; Lange, Bárbara Gil; da Silva, Jessica Pires; Dametto, Gisele Cristina; Pirolli, Mayara Marin; Colombo, Maria Daniela Holthausen Perico; Santos-Silva, Maria Claudia

2018-02-24

Flow cytometric immunophenotyping is deemed a fundamental tool for the diagnosis of B-cell neoplasms. Currently, the investigation of novel immunophenotypic markers has gained importance, as they can assist in the precise subclassification of B-cell malignancies by flow cytometry. Therefore, the purpose of the present study was to evaluate the expression of CD307a during normal B-cell maturation and in B-cell malignancies as well as to investigate its potential role in the differential diagnosis of these entities. CD307a expression was assessed by flow cytometry in normal precursor and mature B cells and in 115 samples collected from patients diagnosed with precursor and mature B-cell neoplasms. CD307a expression was compared between neoplastic and normal B cells. B-acute lymphoblastic leukemia cases exhibited minimal expression of CD307a, displaying a similar expression pattern to that of normal B-cell precursors. Mantle cell lymphoma (MCL) cases showed the lowest levels of CD307a among mature B-cell neoplasms. CD307a expression was statistically lower in MCL cases than in chronic B lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL) cases. No statistical differences were observed between CD307a expression in neoplastic and normal plasma cells. These results indicate that the assessment of CD307a expression by flow cytometry could be helpful to distinguish CLL from MCL, and the latter from MZL. Although these results are not entirely conclusive, they provide a basis for further studies in a larger cohort of patients. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

Protection from intracellular oxidative stress by cytoglobin in normal and cancerous oesophageal cells.

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Fiona E McRonald

Full Text Available Cytoglobin is an intracellular globin of unknown function that is expressed mostly in cells of a myofibroblast lineage. Possible functions of cytoglobin include buffering of intracellular oxygen and detoxification of reactive oxygen species. Previous work in our laboratory has demonstrated that cytoglobin affords protection from oxidant-induced DNA damage when over expressed in vitro, but the importance of this in more physiologically relevant models of disease is unknown. Cytoglobin is a candidate for the tylosis with oesophageal cancer gene, and its expression is strongly down-regulated in non-cancerous oesophageal biopsies from patients with TOC compared with normal biopsies. Therefore, oesophageal cells provide an ideal experimental model to test our hypothesis that downregulation of cytoglobin expression sensitises cells to the damaging effects of reactive oxygen species, particularly oxidative DNA damage, and that this could potentially contribute to the TOC phenotype. In the current study, we tested this hypothesis by manipulating cytoglobin expression in both normal and oesophageal cancer cell lines, which have normal physiological and no expression of cytoglobin respectively. Our results show that, in agreement with previous findings, over expression of cytoglobin in cancer cell lines afforded protection from chemically-induced oxidative stress but this was only observed at non-physiological concentrations of cytoglobin. In addition, down regulation of cytoglobin in normal oesophageal cells had no effect on their sensitivity to oxidative stress as assessed by a number of end points. We therefore conclude that normal physiological concentrations of cytoglobin do not offer cytoprotection from reactive oxygen species, at least in the current experimental model.

Sub-cellular force microscopy in single normal and cancer cells.

Science.gov (United States)

Babahosseini, H; Carmichael, B; Strobl, J S; Mahmoodi, S N; Agah, M

2015-08-07

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. Copyright © 2015 Elsevier Inc. All rights reserved.

Patterns of proliferation and differentiation of irradiated haemopoietic stem cells cultured on normal 'stromal' cell colonies in vitro

International Nuclear Information System (INIS)

Mori, K.J.

1981-01-01

Experiments were designed to elucidate whether or not the irradiated bone marrow cells receive any stimulation for the self-replication and differentiation from normal 'stromal' cell colonies in the bone marrow cell culture in vitro. When irradiated or unirradiated bone marrow cells were overlaid on the normal adherent cell colonies, the proliferation of haemopoietic stem cells was supported, the degree of the stimulation depending on the starting cellular concentration. There was, however, no significant changes in the concentration of either CFUs or CFUc regardless of the dose of irradiation on the bone marrow cells overlaid. This was a great contrast to the dose-dependent decrease of CFUs or CFUc within the culture in which both the stem cells and stromal cells were simultaneously irradiated. These results suggest that the balance of self-replication and differentiation of the haemopoietic stem cells is affected only when haemopoietic microenvironment is perturbed. (author)

Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms

DEFF Research Database (Denmark)

Svingen, T; Jørgensen, Anne; Rajpert-De Meyts, E

2014-01-01

to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers......, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis...... and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further...

Expression of a family of noncoding mitochondrial RNAs distinguishes normal from cancer cells.

Science.gov (United States)

Burzio, Verónica A; Villota, Claudio; Villegas, Jaime; Landerer, Eduardo; Boccardo, Enrique; Villa, Luisa L; Martínez, Ronny; Lopez, Constanza; Gaete, Fancy; Toro, Viviana; Rodriguez, Ximena; Burzio, Luis O

2009-06-09

We reported the presence in human cells of a noncoding mitochondrial RNA that contains an inverted repeat (IR) of 815 nucleotides (nt) covalently linked to the 5' end of the mitochondrial 16S RNA (16S mtrRNA). The transcript contains a stem-loop structure and is expressed in human proliferating cells but not in resting cells. Here, we demonstrate that, in addition to this transcript, normal human proliferating cells in culture express 2 antisense mitochondrial transcripts. These transcripts also contain stem-loop structures but strikingly they are down-regulated in tumor cell lines and tumor cells present in 17 different tumor types. The differential expression of these transcripts distinguishes normal from tumor cells and might contribute a unique vision on cancer biology and diagnostics.

visnormsc: A Graphical User Interface to Normalize Single-cell RNA Sequencing Data.

Science.gov (United States)

Tang, Lijun; Zhou, Nan

2017-12-26

Single-cell RNA sequencing (RNA-seq) allows the analysis of gene expression with high resolution. The intrinsic defects of this promising technology imports technical noise into the single-cell RNA-seq data, increasing the difficulty of accurate downstream inference. Normalization is a crucial step in single-cell RNA-seq data pre-processing. SCnorm is an accurate and efficient method that can be used for this purpose. An R implementation of this method is currently available. On one hand, the R package possesses many excellent features from R. On the other hand, R programming ability is required, which prevents the biologists who lack the skills from learning to use it quickly. To make this method more user-friendly, we developed a graphical user interface, visnormsc, for normalization of single-cell RNA-seq data. It is implemented in Python and is freely available at https://github.com/solo7773/visnormsc . Although visnormsc is based on the existing method, it contributes to this field by offering a user-friendly alternative. The out-of-the-box and cross-platform features make visnormsc easy to learn and to use. It is expected to serve biologists by simplifying single-cell RNA-seq normalization.

Corneal endothelial cell density and morphology in normal Iranian eyes

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Fallah Mohammad

2006-03-01

Full Text Available Abstract Background We describe corneal endothelial cell density and morphology in normal Iranian eyes and compare endothelial cell characteristics in the Iranian population with data available in the literature for American and Indian populations. Methods Specular microscopy was performed in 525 eyes of normal Iranian people aged 20 to 85 years old. The studied parameters including mean endothelial cell density (MCD, mean cell area (MCA and coefficient of variation (CV in cell area were analyzed in all of the 525 eyes. Results MCD was 1961 ± 457 cell/mm2 and MCA was 537.0 ± 137.4 μm2. There was no statistically significant difference in MCD, MCA and CV between genders (Student t-test, P = 0.85, P = 0.97 and P = 0.15 respectively. There was a statistically significant decrease in MCD with age (P r = -0.64. The rate of cell loss was 0.6% per year. There was also a statistically significant increase in MCA (P r = 0.56 and CV (P r = 0.30 from 20 to 85 years of age. Conclusion The first normative data for the endothelium of Iranian eyes seems to confirm that there are no differences in MCD, MCA and CV between genders. Nevertheless, the values obtained in Iranian eyes seem to be different to those reported by the literature in Indian and American populations.

Studies by radioiodination of normal adult, fetal and leukemic cell membranes

Energy Technology Data Exchange (ETDEWEB)

Kannourakis, G; Cauchi, M N [Department of Pathology and Immunology, Monash Medical School, Melbourne, Australia

1978-01-01

A comparison was made between cord blood lymphocytes, normal adult lymphocytes and leukemic cells after membrane iodination with lactoperoxidase. A double-labeling technique using lactoperoxidase iodination with /sup 125/I and /sup 131/I followed by analysis on polyacrylamide gel electrophoresis revealed a number of membrane differences between leukemic, normal and fetal cells. There was a reduction in the 70,000 molecular weight component in cord blood cells compared to adult lymphocytes, and an increase in membrane peptides with molecular weights of 35,000, 20,000, 9,000 and 4,000. Although smaller molecular weight peptides were also present in chronic lymphatic leukemia as well as acute myeloid leukemia, these were shown to be distinct from fetal type membrane components.

Clinical and Pathologic Study of Feline Merkel Cell Carcinoma With Immunohistochemical Characterization of Normal and Neoplastic Merkel Cells.

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Dohata, A; Chambers, J K; Uchida, K; Nakazono, S; Kinoshita, Y; Nibe, K; Nakayama, H

2015-11-01

The authors herein describe the morphologic and immunohistochemical features of normal Merkel cells as well as the clinicopathologic findings of Merkel cell carcinoma in cats. Merkel cells were characterized as vacuolated clear cells and were individually located in the epidermal basal layer of all regions examined. Clusters of Merkel cells were often observed adjacent to the sinus hair of the face and carpus. Immunohistochemically, Merkel cells were positive for cytokeratin (CK) 20, CK18, p63, neuron-specific enolase, synaptophysin, and protein gene product 9.5. Merkel cell carcinoma was detected as a solitary cutaneous mass in 3 aged cats (13 to 16 years old). On cytology, large lymphocyte-like cells were observed in all cases. Histologic examinations of surgically resected tumors revealed nests of round cells separated by various amounts of a fibrous stroma. Tumor cells were commonly immunopositive for CK20, CK18, p63, neuron-specific enolase, and synaptophysin, representing the characteristics of normal Merkel cells. © The Author(s) 2015.

Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: combined application of atomic force microscopy and modulated Raman spectroscopy.

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Canetta, Elisabetta; Riches, Andrew; Borger, Eva; Herrington, Simon; Dholakia, Kishan; Adya, Ashok K

2014-05-01

Atomic force microscopy (AFM) and modulated Raman spectroscopy (MRS) were used to discriminate between living normal human urothelial cells (SV-HUC-1) and bladder tumour cells (MGH-U1) with high specificity and sensitivity. MGH-U1 cells were 1.5-fold smaller, 1.7-fold thicker and 1.4-fold rougher than normal SV-HUC-1 cells. The adhesion energy was 2.6-fold higher in the MGH-U1 cells compared to normal SV-HUC-1 cells, which possibly indicates that bladder tumour cells are more deformable than normal cells. The elastic modulus of MGH-U1 cells was 12-fold lower than SV-HUC-1 cells, suggesting a higher elasticity of the bladder cancer cell membranes. The biochemical fingerprints of cancer cells displayed a higher DNA and lipid content, probably due to an increase in the nuclear to cytoplasm ratio. Normal cells were characterized by higher protein contents. AFM studies revealed a decrease in the lateral dimensions and an increase in thickness of cancer cells compared to normal cells; these studies authenticate the observations from MRS. Nanostructural, nanomechanical and biochemical profiles of bladder cells provide qualitative and quantitative markers to differentiate between normal and cancerous cells at the single cellular level. AFM and MRS allow discrimination between adhesion energy, elasticity and Raman spectra of SV-HUC-1 and MGH-U1 cells with high specificity (83, 98 and 95%) and sensitivity (97, 93 and 98%). Such single-cell-level studies could have a pivotal impact on the development of AFM-Raman combined methodologies for cancer profiling and screening with translational significance. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Pivotal advance: CTLA-4+ T cells exhibit normal antiviral functions during acute viral infection.

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Raué, Hans-Peter; Slifka, Mark K

2007-05-01

Previous studies have shown that T cells, which are genetically deficient in CTLA-4/CD152 expression, will proliferate uncontrollably, resulting in lethal autoimmune disease. This and other evidence indicate that CTLA-4 plays a critical role in the negative regulation of effector T cell function. In contrast to expectations, BrdU incorporation experiments demonstrated that CTLA-4 expression was associated with normal or even enhanced in vivo proliferation of virus-specific CD4+ and CD8+ T cells following acute lymphocytic choriomeningitis virus or vaccinia virus infection. When compared with CTLA-4- T cells directly ex vivo, CTLA-4+ T cells also exhibited normal antiviral effector functions following stimulation with peptide-coated cells, virus-infected cells, plate-bound anti-CD3/anti-CTLA-4, or the cytokines IL-12 and IL-18. Together, this indicates that CTLA-4 does not directly inhibit antiviral T cell expansion or T cell effector functions, at least not under the normal physiological conditions associated with either of these two acute viral infections.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

Science.gov (United States)

McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

2011-04-01

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

RF Breakdown in Normal Conducting Single-cell Structures

CERN Document Server

Dolgashev, Valery A; Higo, Toshiyasu; Nantista, Christopher D; Tantawi, Sami G

2005-01-01

Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM01 mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials an...

Normal endometrial stromal cells regulate 17β-estradiol-induced epithelial-mesenchymal transition via slug and E-cadherin in endometrial adenocarcinoma cells in vitro.

Science.gov (United States)

Zhang, Hui; Li, Hongyan; Qi, Shasha; Liu, Zhao; Fu, Yibing; Li, Mingjiang; Zhao, Xingbo

2017-01-01

Stroma-tumor communication participates in the pathogenesis of endometrial carcinomas. In previous studies, we found that normal stromal cells inhibited the growth of endometrial carcinoma cells. Here, we investigated the role of normal stromal cells in the epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells and explored the possible mechanism implied. We found that conditioned medium (CM) by normal endometrial stromal cells (NSC) reduced cell growth and induced cell apoptosis in Ishikawa cells. CM by NSC inhibited 17β-estradiol-induced cell growth and apoptosis decrease in Ishikawa cells. Moreover, CM by NSC inhibited the migration and invasion, and 17β-estradiol-induced migration and invasion in Ishikawa cells. Meanwhile, CM by NSC decreased Slug expression and 17β-estradiol-induced Slug expression, increased E-cadherin expression and abolished 17β-estradiol-induced E-cadherin reduction in Ishikawa cells. In conclusion, normal stromal factors can inhibit 17β-estradiol-induced cell proliferation and apoptosis inhibition, and abolished 17β-estradiol-induced EMT in endometrial cancer cell via regulating E-cadherin and Slug expression.

Gamma-ray excision repair in normal and diseased human cells

International Nuclear Information System (INIS)

Cerutti, P.A.; Remsen, J.F.

1976-01-01

Radiation products of the 5,6-dihydroxy-dihydrothymine type (t') are efficiently removed from the DNA during postirradiation incubation of bacterial and mammalian cells. In this chapter we describe the t'-excision system contained in normal human cells, in human carcinoma HeLa S-3 cells, and in skin fibroblasts from xeroderma pigmentosum (XP) and Fanconi's anemia (FA) patients. The latter diseases are characterized among other symptoms by a genetically increased susceptibility for the development of cancer

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

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Kheradmand, Arash, E-mail: arashkheradmand@yahoo.com [Department of Clinical Sciences, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad (Iran, Islamic Republic of); Dezfoulian, Omid [Department of Pathobiology, School of Veterinary Medicine, Lorestan University, Khorram Abad (Iran, Islamic Republic of); Alirezaei, Masoud [Division of Biochemistry, School of Veterinary Medicine, Lorestan University, P.O. Box: 465, Khorram Abad (Iran, Islamic Republic of); Rasoulian, Bahram [Razi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Khorram Abad (Iran, Islamic Republic of)

2012-03-09

Highlights: Black-Right-Pointing-Pointer Spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. Black-Right-Pointing-Pointer Numerous studies have documented the direct action of ghrelin in the modulation of apoptosis in different cell types. Black-Right-Pointing-Pointer Ghrelin may be considered as a modulator of spermatogenesis in normal adult rats. Black-Right-Pointing-Pointer Ghrelin may be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors. -- Abstract: Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n = 7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P < 0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact

Cell adhesion-mediated radioresistance (CAM-RR). Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro

International Nuclear Information System (INIS)

Cordes, N.; Meineke, V.

2003-01-01

Background: Cell-extracellular matrix (ECM) contact is thought to have great impact on cellular mechanisms resulting in increased cell survival upon exposure to ionizing radiation. Several human tumor cell lines and normal human fibroblastic cell strains of different origin, all of them expressing the wide-spread and important integrin subunit β1, were irradiated, and clonogenic cell survival, β1-integrin cell surface expression, and adhesive functionality were investigated. Material and Methods: Human tumor cell lines A172 (glioblastoma), PATU8902 (pancreas carcinoma), SKMES1 (lung carcinoma), A549 (lung carcinoma), and IPC298 (melanoma) as well as normal human skin (HSF1) and lung fibroblasts (CCD32) and human keratinocytes (HaCaT) were irradiated with 0-8 Gy. Besides colony formation assays, β1-integrin cell surface expression by flow cytometry and adhesive functionality by adhesion assays were analyzed. Results: All cell lines showed improved clonogenic survival after irradiation in the presence of fibronectin as compared to plastic. Irradiated cells exhibited a significant, dose-dependent increase in β1-integrin cell surface expression following irradiation. As a parameter of the adhesive functionality of the β1-integrin, a radiation-dependent elevation of cell adhesion to fibronectin in comparison with adhesion to plastic was demonstrated. Conclusion: The in vitro cellular radiosensitivity is highly influenced by fibronectin according to the phenomenon of cell adhesion-mediated radioresistance. Additionally, our emerging data question the results of former and current in vitro cytotoxicity studies performed in the absence of an ECM. These findings might also be important for the understanding of malignant transformation, anchorage-independent cell growth, optimization of radiotherapeutic regimes and the prevention of normal tissue side effects on the basis of experimental radiobiological data. (orig.)

Generation of hiPSTZ16 (ISMMSi003-A cell line from normal human foreskin fibroblasts

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Marion Dejosez

2018-01-01

Full Text Available Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A. These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

Identification of Biomarkers for Esophageal Squamous Cell Carcinoma Using Feature Selection and Decision Tree Methods

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Chun-Wei Tung

2013-01-01

Full Text Available Esophageal squamous cell cancer (ESCC is one of the most common fatal human cancers. The identification of biomarkers for early detection could be a promising strategy to decrease mortality. Previous studies utilized microarray techniques to identify more than one hundred genes; however, it is desirable to identify a small set of biomarkers for clinical use. This study proposes a sequential forward feature selection algorithm to design decision tree models for discriminating ESCC from normal tissues. Two potential biomarkers of RUVBL1 and CNIH were identified and validated based on two public available microarray datasets. To test the discrimination ability of the two biomarkers, 17 pairs of expression profiles of ESCC and normal tissues from Taiwanese male patients were measured by using microarray techniques. The classification accuracies of the two biomarkers in all three datasets were higher than 90%. Interpretable decision tree models were constructed to analyze expression patterns of the two biomarkers. RUVBL1 was consistently overexpressed in all three datasets, although we found inconsistent CNIH expression possibly affected by the diverse major risk factors for ESCC across different areas.

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

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Ma, Hailun; Galvin, Teresa A; Glasner, Dustin R; Shaheduzzaman, Syed; Khan, Arifa S

2014-06-01

The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell

Identification of Contaminated Cells with Viruses, Bacteria, or Fungi by Fourier Transform Infrared Microspectroscopy

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V. Erukhimovitch

2013-01-01

Full Text Available Fourier transform infrared microspectroscopy (FTIR-M can detect small molecular changes in cells and therefore was previously applied for the identification of different biological samples. In the present study, FTIR spectroscopy was used for the identification and discrimination of Vero cells infected with herpes viruses or contaminated with bacteria or fungi in cell culture. Vero cells in culture were infected herpes simplex virus type 1 (HSV-1 or contaminated with E. coli bacteria or Candida albicans fungi and analyzed by FTIR microscopy at 24 h postinfection/contamination. Specific different spectral changes were observed according to the infecting or contaminating agent. For instance, both pure fungi and cell culture contaminated with this fungi showed specific peaks at 1030 cm−1 and at 1373 cm−1 regions, while pure E. coli and cell culture contaminated with this bacteria showed a specific and unique peak at 1657 cm−1. These results support the potential of developing FTIR microspectroscopy as a simple, reagent free method for identification and discrimination between different tissue infection or contamination with various pathogens.

Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

Science.gov (United States)

Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

2015-01-01

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

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Yukihiro Nishikawa

Full Text Available Withaferin A (WA, a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD. WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L. Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

DEFF Research Database (Denmark)

Roslind, A.; Balslev, E.; Kruse, H.

2008-01-01

. YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

Comparative evaluation of eosinophils in normal mucosa, dysplastic mucosa and oral squamous cell carcinoma with hematoxylin-eosin, Congo red, and EMR1 immunohistochemical staining techniques.

Science.gov (United States)

Kargahi, Neda; Razavi, Sayyed Mohammad; Deyhimi, Parviz; Homayouni, Solmaz

2015-01-01

Oral squamous cell carcinoma is the most common malignant lesion of the oral cavity, and it involves various molecular mechanisms. The development of oral squamous cell carcinoma is influenced by the host immune cells, such as eosinophils. The present study was conducted to compare the presence of eosinophils in normal mucosa, dysplastic mucosa, and oral squamous cell carcinoma by -hematoxylin- eosin staining, Congo red staining, and epidermal growth factor-like (EGF-like) module containing a mucin-like hormone receptor1 (EMR1) immunohistochemical marker. In this cross-sectional study, 60 paraffinized samples were selected, consisting of 20 normal mucosae, 20 dysplastic mucosae, and 20 squamous cell carcinoma samples. After confirmation of the diagnosis, the mean number of eosinophils was evaluated by hematoxylin-eosin, Congo red, and immunohystochemical staining techniques. The data were analyzed by SPSS-10 software using the Kruskal-Wallis and Friedman tests. The results showed that the number of eosinophils in dysplastic mucosa was significantly higher than the number in normal mucosa, and the number of eosinophils in squamous cell carcinoma was significantly higher than the number in dysplastic mucosa in all staining techniques (p<0.001). Moreover, the comparison of staining techniques showed a significantly higher number of eosinophils in EMR1immunohistochemicalmarker than were observed when Congo red and hematoxylin - eosin (H&E) staining techniques were used (p<0.001). It can be argued that eosinophil contributes to the identification of lesions that have a higher potential of malignant transformation. Moreover, eosinophil can be suggested as an indicator in the differentiation of oral lesions in cases with borderline diagnosis and in targeted molecular therapy.

RF Breakdown in Normal Conducting Single-Cell Structures

International Nuclear Information System (INIS)

Dolgashev, V.A.; Nantista, C.D.; Tantawi, S.G.; Higashi, Y.; Higo, T.

2006-01-01

Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM 01 mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials and preparation techniques with short turn-around time. Simple 2D geometry of the test structures simplifies modeling of the breakdown currents and their thermal effects

Androgen receptor signalling in peritubular myoid cells is essential for normal differentiation and function of adult Leydig cells

DEFF Research Database (Denmark)

Welsh, M.; Moffat, L.; Belling, Kirstine Christensen

2012-01-01

Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM-ARKO) did not affect LC number, but res......Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM-ARKO) did not affect LC number......’ subpopulation that had arrested development and only weakly expressed INSL3, luteinizing hormone receptor, and several steroidogenic enzymes. Furthermore, unlike ‘normal’ LCs in PTM-ARKOs, the ‘abnormal’ LCs did not involute as expected in response to exogenous testosterone. Differential function of these LC...... sub-populations is likely to mean that the ‘normal’ LCs work harder to compensate for the ‘abnormal’ LCs to maintain normal serum testosterone. These findings reveal new paracrine mechanisms underlying adult LC development, which can be further investigated using PTM-ARKOs....

HIV-Specific ADCC Improves After Antiretroviral Therapy and Correlates With Normalization of the NK Cell Phenotype

DEFF Research Database (Denmark)

Jensen, Sanne S; Hartling, Hans J; Tingstedt, Jeanette L

2015-01-01

analyzed. RESULTS: The ability of NK cells to mediate ADCC was significantly increased after only 6 months of HAART and was not explained by a normalization of NK cell subsets (CD56 CD16 and CD56 CD16 NK cells) but rather by normalization in the frequency of NK cells expressing CCR7 and CD27...

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin

DEFF Research Database (Denmark)

Damsgaard, T E; Olesen, A B; Sørensen, Flemming Brandt

1997-01-01

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results...... of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out...... yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found...

Cell biological effects of total body irradiation on growth and differentiation of acute myelogenous leukemia cells compared to normal bone marrow

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Greenberger, J S; Weichselbaum, R R; Botnick, L E; Sakakeeny, M; Moloney, W C

1979-01-01

Radiation therapy is used as total body treatment in preparation of the acute myelogenous leukemia (AML) patient for bone marrow transplantation. Many AML patients will have residual leukemia cells at the time of total body irradiation (TBI). In the present study, the effect of TBI on leukemic myeloid cells was compared to the effect on normal marrow granulocytic stem cells (CFUc) in vitro. Little difference from that of normal CFUc was found in the radiosensitivity of two mouse myeloid leukemia cell lines. The effect of TBI on growth of WEHI-3 or J774 cells in millipore diffusion chambers was stimulatory. These AML cell lines as well as others derived from Friend or Abelson virus infected in vitro long term mouse marrow cultures showed some morphologic differentiation by 7 days growth in diffusion chambers in irradiated heterologous rat hosts, but immature cells predominated by day 21. Thus, evidence in murine models of AML indicates that residual AML cells surviving chemotherapy will show no greater susceptibility to radiation killing compared to normal stem cells and will rapidly repopulate the irradiated host.

Comparison of radiosensitivity between tumor and normal tissue in terms of cell population kinetics

International Nuclear Information System (INIS)

Sugahara, Tsutomu; Utsumi, Hiroshi

1975-01-01

Puck and Marcus in 1956 established the in vitro colony formation of mammalian cells and demonstrated a dose-survival curve of mammalian cells well fitted to the target theory. Since then almost all of the work on the radiosensitivity of malignant and normal cells has been based on the reproductive integrity of cells. However, in the author's laboratory, a recent work was done on the effect of ionizing radiation on the differentiative trait, using clonal cell cultures developed by Coon (1966) in chick embryonic cartilage cells. This work demonstrated clearly that the differentiative trait is more radiosensitive than is reproduction. Based on this finding a new compartment model is proposed for a cell renewal system which demonstrates the difference between normal and malignant tissue. (author)

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

Science.gov (United States)

Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

2013-04-01

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013

Wnt/β-catenin Signaling in Normal and Cancer Stem Cells

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Kenneth C. Valkenburg

2011-04-01

Full Text Available The ability of Wnt ligands to initiate a signaling cascade that results in cytoplasmic stabilization of, and nuclear localization of, β-catenin underlies their ability to regulate progenitor cell differentiation. In this review, we will summarize the current knowledge of the mechanisms underlying Wnt/β-catenin signaling and how the pathway regulates normal differentiation of stem cells in the intestine, mammary gland, and prostate. We will also discuss how dysregulation of the pathway is associated with putative cancer stem cells and the potential therapeutic implications of regulating Wnt signaling.

Ubiquitous expression of MAKORIN-2 in normal and malignant hematopoietic cells and its growth promoting activity.

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King Yiu Lee

Full Text Available Makorin-2 (MKRN2 is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.

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

Stem cells: a model for screening, discovery and development of drugs.

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Kitambi, Satish Srinivas; Chandrasekar, Gayathri

2011-01-01

The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson's disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.

Comparative histochemical study of Bowen’s disease and actinic keratosis: preserved normal basal cells in Bowen’s disease

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H Ishida

2009-12-01

Full Text Available The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen’s disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen’s disease were completely negative. Compatible with this result, the basal cells in Bowen’s disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 ß E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14, which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen’s disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen’s disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 ß B4 (monoclonal antibody against cytokeratin 1, which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen’s disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen’s disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

International Nuclear Information System (INIS)

Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

2011-01-01

Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

Normal function of immunologic stem cells from aged mice

International Nuclear Information System (INIS)

Harrison, D.E.; Doubleday, J.W.

1975-01-01

Marrow or spleen grafts from aged donor mice produced antibody-forming cells as effectively as did grafts from younger controls in recipients tested 3 to 10 months after the transplantation. All recipients were lethally irradiated, and the T6 chromosome marker was used to demonstrate that they were populated by donor cell lines. Recipients of aged or younger control grafts gave similar responses when stimulated with varying doses of antigen and when tested at different times after the transplantation except in two cases. Recipients of aged spleen grafts gave significantly lower responses than younger controls for the first few weeks after the transplantation. If recipients had been thymectomized before lethal irradiation, aged cell lines (pooled marrow and spleen cells) gave only 37 percent of the responses of younger controls. Given sufficient time and intact young recipients, immunologic stem cell lines from old donors populated recipients with cells having normal immune responses. These results suggest that age-related immunologic defects are not intrinsically timed in the precursor cell lines that populate the immune system. (U.S.)

Chromosomal Aberrations in Normal and AT Cells Exposed to High Dose of Low Dose Rate Irradiation

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Kawata, T.; Shigematsu, N.; Kawaguchi, O.; Liu, C.; Furusawa, Y.; Hirayama, R.; George, K.; Cucinotta, F.

2011-01-01

Ataxia telangiectasia (A-T) is a human autosomally recessive syndrome characterized by cerebellar ataxia, telangiectases, immune dysfunction, and genomic instability, and high rate of cancer incidence. A-T cell lines are abnormally sensitive to agents that induce DNA double strand breaks, including ionizing radiation. The diverse clinical features in individuals affected by A-T and the complex cellular phenotypes are all linked to the functional inactivation of a single gene (AT mutated). It is well known that cells deficient in ATM show increased yields of both simple and complex chromosomal aberrations after high-dose-rate irradiation, but, less is known on how cells respond to low-dose-rate irradiation. It has been shown that AT cells contain a large number of unrejoined breaks after both low-dose-rate irradiation and high-dose-rate irradiation, however sensitivity for chromosomal aberrations at low-dose-rate are less often studied. To study how AT cells respond to low-dose-rate irradiation, we exposed confluent normal and AT fibroblast cells to up to 3 Gy of gamma-irradiation at a dose rate of 0.5 Gy/day and analyzed chromosomal aberrations in G0 using fusion PCC (Premature Chromosomal Condensation) technique. Giemsa staining showed that 1 Gy induces around 0.36 unrejoined fragments per cell in normal cells and around 1.35 fragments in AT cells, whereas 3Gy induces around 0.65 fragments in normal cells and around 3.3 fragments in AT cells. This result indicates that AT cells can rejoin breaks less effectively in G0 phase of the cell cycle? compared to normal cells. We also analyzed chromosomal exchanges in normal and AT cells after exposure to 3 Gy of low-dose-rate rays using a combination of G0 PCC and FISH techniques. Misrejoining was detected in the AT cells only? When cells irradiated with 3 Gy were subcultured and G2 chromosomal aberrations were analyzed using calyculin-A induced PCC technique, the yield of unrejoined breaks decreased in both normal and AT

A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

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Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antczak, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J; Guindani, Michele; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

2016-04-01

The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks

A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

Directory of Open Access Journals (Sweden)

Victor Trevino

2016-04-01

Full Text Available The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell

Geometrical nuclear diagnosis and total paths of cervical cell evolution from normality to cancer

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Javier Oswaldo Rodríguez Velásquez

2015-01-01

Full Text Available Background: The diagnosis of cervix cytology has problems of inter-observer reproducibility. Methodologies based on fractal geometry objectively differentiated normal, low-grade squamous intraepithelial lesion (L-SIL and high-grade squamous intraepithelial lesion (H-SIL states. Aims: The aim was to develop a mathematical-physical diagnosis and a theoretical generalization of the evolution paths of cervical cells from normal to carcinoma based on their occupation in the box-counting space. Subjects and Methods: Overlaying a grid of 8x8 pixels, the a number of squares occupying the nucleus surface and cytoplasm of 5 normal cells, 5 ASCUS, 5 L-SIL and 5 H-SIL were evaluated, as well as the ratio C/N, establishing differences between states. Sensitivity, specificity, negative likelihood ratio, and Kappa coefficient over the gold standard were calculated. Also was developed a generalization of all possible paths from normality to carcinoma. Results: The occupancy spaces of the nuclear surface allow differentiating normal L-SIL and H-SIL thus avoiding the indeterminacy of ASCUS cells. Compared to the Gold Standard, this method has sensitivity and specificity of 100%, negative likelihood ratio of 0, and Kappa coefficient of 1. 62,900 possible routes of evolution were determined between normal and H-SIL, states, based on the structural basis of the cells. Conclusions: it was obtained an objective and reproducible diagnostic methodology of the development of preneoplastic and neoplastic cervical cells for clinical application. Additionally were developed all possible paths of preneoplastic cellular alteration to carcinoma which facilitates the tracking of patients over time to clinical level, warning of alterations that lead to malignancy, based on the spatial occupation measurements of the nucleus in fractal space regardless of causes or risk factors.

Leptin and Adiponectin Modulate the Self-renewal of Normal Human Breast Epithelial Stem Cells.

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Esper, Raymond M; Dame, Michael; McClintock, Shannon; Holt, Peter R; Dannenberg, Andrew J; Wicha, Max S; Brenner, Dean E

2015-12-01

Multiple mechanisms are likely to account for the link between obesity and increased risk of postmenopausal breast cancer. Two adipokines, leptin and adiponectin, are of particular interest due to their opposing biologic functions and associations with breast cancer risk. In the current study, we investigated the effects of leptin and adiponectin on normal breast epithelial stem cells. Levels of leptin in human adipose explant-derived conditioned media positively correlated with the size of the normal breast stem cell pool. In contrast, an inverse relationship was found for adiponectin. Moreover, a strong linear relationship was observed between the leptin/adiponectin ratio in adipose conditioned media and breast stem cell self-renewal. Consistent with these findings, exogenous leptin stimulated whereas adiponectin suppressed breast stem cell self-renewal. In addition to local in-breast effects, circulating factors, including leptin and adiponectin, may contribute to the link between obesity and breast cancer. Increased levels of leptin and reduced amounts of adiponectin were found in serum from obese compared with age-matched lean postmenopausal women. Interestingly, serum from obese women increased stem cell self-renewal by 30% compared with only 7% for lean control serum. Taken together, these data suggest a plausible explanation for the obesity-driven increase in postmenopausal breast cancer risk. Leptin and adiponectin may function as both endocrine and paracrine/juxtacrine factors to modulate the size of the normal stem cell pool. Interventions that disrupt this axis and thereby normalize breast stem cell self-renewal could reduce the risk of breast cancer. ©2015 American Association for Cancer Research.

Identification and regulation of a molecular module for bleb-based cell motility

NARCIS (Netherlands)

Goudarzi, M.; Banisch, T.U.; Mobin, M.B.; Maghelli, N.; Tarbashevich, K.; Strate, I.; ter Berg, J.; Blaser, H.; Bandemer, S.; Paluch, E.; Bakkers, J.; Tolic-Norrelykke, I.M.; Raz, E.

2012-01-01

Single-cell migration is a key process in development, homeostasis, and disease. Nevertheless, the control over basic cellular mechanisms directing cells into motile behavior in vivo is largely unknown. Here, we report on the identification of a minimal set of parameters the regulation of which

Cell-Type-Specific Gene Programs of the Normal Human Nephron Define Kidney Cancer Subtypes.

Science.gov (United States)

Lindgren, David; Eriksson, Pontus; Krawczyk, Krzysztof; Nilsson, Helén; Hansson, Jennifer; Veerla, Srinivas; Sjölund, Jonas; Höglund, Mattias; Johansson, Martin E; Axelson, Håkan

2017-08-08

Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Responses of human normal osteoblast cells and osteoblast-like cell line, MG-63 cells, to pulse electromagnetic field (PEMF

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Suttatip Kamolmatyakul

2008-01-01

Full Text Available The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell/wells with 2 ml αMEM media supplemented with 10% FBS. The experimental plates were placed between a pair of Helmoltz coils powered by a pulse generator (PEMF, 50 Hz, 1.5 mV/cm in the upper compartment of a dual incubator (Forma. The control plates were placed in the lower compartment of the incubator without Helmotz coils. After three days, the cell proliferation was measured by the method modified from Mossman (J. Immunol Methods 1983; 65: 55-63. Other sets of plates were used for osteocalcin production assessment. Media from these sets were collected after 6 days and assessed for osteocalcin production using ELISA kits. The data were analyzed using a one-way analysis of variance (ANOVA. The results showed that MG-63 cells from the experimental group proliferated significantly more than those from the control group (20% increase, p<0.05. No significant difference in osteocalcin production was detected between the two groups. On the other hand, NHOC from the experimental group produced larger amount of osteocalcin (25% increase, p<0.05 and proliferated significantly more than those from the control group (100% increase, p<0.05. In conclusion, PEMF effect on osteoblasts might depend on their cell type of origin. For osteoblast-like cell line, MG-63 cells, PEMF increased proliferation rate but not osteocalcin production of the cells. However, PEMF stimulation effect on human normal osteoblast cells was most likely associated with enhancement of both osteocalcin production and cell proliferation.

The Fate of a Normal Human Cell Traversed by a Single Charged Particle

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Fournier, C.; Zahnreich, S.; Kraft, D.; Friedrich, T.; Voss, K.-O.; Durante, M.; Ritter, S.

2012-01-01

The long-term “fate” of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability. PMID:22966418

Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

International Nuclear Information System (INIS)

Miller, R.C.; Hiraoka, Toshio; Kopecky, K.J.; Nakamura, Nori; Jones, M.P.; Ito, Toshio; Clifton, K.H.

1986-09-01

Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and X-irradiated to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5 %, the average mean lethal dose D 0 was 97.6 cGy for HY cells, and 96.7 cGy and 94.3 cGy, respectively, for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average D 0 of 85.0 cGy and PC cells a significantly (p = .001) lower average D 0 of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (p = .82) or sex (p = .10). These results suggest that malignant thyroid cells may be especially radiosensitive. (author)

Accelerating parameter identification of proton exchange membrane fuel cell model with ranking-based differential evolution

International Nuclear Information System (INIS)

Gong, Wenyin; Cai, Zhihua

2013-01-01

Parameter identification of PEM (proton exchange membrane) fuel cell model is a very active area of research. Generally, it can be treated as a numerical optimization problem with complex nonlinear and multi-variable features. DE (differential evolution), which has been successfully used in various fields, is a simple yet efficient evolutionary algorithm for global numerical optimization. In this paper, with the objective of accelerating the process of parameter identification of PEM fuel cell models and reducing the necessary computational efforts, we firstly present a generic and simple ranking-based mutation operator for the DE algorithm. Then, the ranking-based mutation operator is incorporated into five highly-competitive DE variants to solve the PEM fuel cell model parameter identification problems. The main contributions of this work are the proposed ranking-based DE variants and their application to the parameter identification problems of PEM fuel cell models. Experiments have been conducted by using both the simulated voltage–current data and the data obtained from the literature to validate the performance of our approach. The results indicate that the ranking-based DE methods provide better results with respect to the solution quality, the convergence rate, and the success rate compared with their corresponding original DE methods. In addition, the voltage–current characteristics obtained by our approach are in good agreement with the original voltage–current curves in all cases. - Highlights: • A simple and generic ranking-based mutation operator is presented in this paper. • Several DE (differential evolution) variants are used to solve the parameter identification of PEMFC (proton exchange membrane fuel cells) model. • Results show that our method accelerates the process of parameter identification. • The V–I characteristics are in very good agreement with experimental data

Pluripotent stem cells with normal or reduced self renewal survive lethal irradiation

International Nuclear Information System (INIS)

Brecher, G.; Neben, S.; Yee, M.; Bullis, J.; Cronkite, E.P.

1988-01-01

Transfusion with 10,000 or 20,000 marrow cells resulted in 30+ days survival of 15%-50% of mice exposed to an Ld90 or LD100 or radiation. The use of congenic mice with alloenzyme markers permitted the identification of host and donor cells in the peripheral blood of transfused animals. Donor cells were present initially in all hosts. Between 55% and 92% of the animals became 100% host type by 12-24 weeks after transfusion in three separate experiments. To explore whether the temporary repopulation by donor cells was due to short-lived stem cells, the marrows of several primary hosts were transfused into secondary, lethally irradiated hosts. Some of the retransplanted primary donor and host cells persisted only temporarily. It is suggested that some of the donor stem cells in both the primary and secondary hosts had an intrinsically shortened life span

E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

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Satoshi Ohtsuka

Full Text Available Mouse epiblast stem cells (mEpiSCs are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse

International Nuclear Information System (INIS)

Morgan, J.E.; Hoffman, E.P.; Partridge, T.A.

1990-01-01

Dystrophin deficiency in skeletal muscle of the x-linked dystrophic (mdx) mouse can be partially remedied by implantation of normal muscle precursor cells (mpc). However, it is difficult to determine whether this biochemical rescue results in any improvement in the structure or function of the treated muscle, because the vigorous regeneration of mdx muscle more than compensates for the degeneration. By using x-ray irradiation to prevent mpc proliferation, it is possible to study loss of mdx muscle fibers without the complicating effect of simultaneous fiber regeneration. Thus, improvements in fiber survival resulting from any potential therapy can be detected easily. Here, we have implanted normal mpc, obtained from newborn mice, into such preirradiated mdx muscles, finding that it is far more extensively permeated and replaced by implanted mpc than is nonirradiated mdx muscle; this is evident both from analysis of glucose-6-phosphate isomerase isoenzyme markers and from immunoblots and immunostaining of dystrophin in the treated muscles. Incorporation of normal mpc markedly reduces the loss of muscle fibers and the deterioration of muscle structure which otherwise occurs in irradiated mdx muscles. Surprisingly, the regenerated fibers are largely peripherally nucleated, whereas regenerated mouse skeletal muscle fibers are normally centrally nucleated. We attribute this regeneration of apparently normal muscle to the tendency of newborn mouse mpc to recapitulate their neonatal ontogeny, even when grafted into 3-wk-old degenerating muscle

Comparison of radiosensitivities of human autologous normal and neoplastic thyroid epithelial cells

International Nuclear Information System (INIS)

Miller, R.C.; Kopecky, K.J.; Hiraoka, T.; Ezaki, H.; Clifton, K.H.

1986-01-01

Studies were conducted to examine differences between the radiosensitivities of normal and neoplastic epithelial cells of the human thyroid. Freshly excised thyroid tissues from the tumours of eight patients with papillary carcinoma (PC) and five with follicular adenoma (FA) were cultured in vitro separately from normal thyroid tissue obtained from the surgical margins of the same patients. Plating efficiency of unirradiated control tissue was lower, on average for tumour tissue compared with normal tissue. Radiosensitivity, measured by the 37% inactivation dose D 0 , was greater for carcinoma tissue than for normal tissue in seven out of eight PC cases. Adenomatous tissue was less radiosensitive than normal tissue in four out of five FA cases. This is the first report comparing the radiosensitivity of autologous normal and abnormal epithelial tissue from the human thyroid. (author)

Use of two predictive algorithms of the world wide web for the identification of tumor-reactive T-cell epitopes.

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Lu, J; Celis, E

2000-09-15

Tumor cells can be effectively recognized and eliminated by CTLs. One approach for the development of CTL-based cancer immunotherapy for solid tumors requires the use of the appropriate immunogenic peptide epitopes that are derived from defined tumor-associated antigens. Because CTL peptide epitopes are restricted to specific MHC alleles, to design immune therapies for the general population it is necessary to identify epitopes for the most commonly found human MHC alleles. The identification of such epitopes has been based on MHC-peptide-binding assays that are costly and labor-intensive. We report here the use of two computer-based prediction algorithms, which are readily available in the public domain (Internet), to identify HL4-B7-restricted CTL epitopes for carcinoembryonic antigen (CEA). These algorithms identified three candidate peptides that we studied for their capacity to induce CTL responses in vitro using lymphocytes from HLA-B7+ normal blood donors. The results show that one of these peptides, CEA9(632) (IPQQHTQVL) was efficient in the induction of primary CTL responses when dendritic cells were used as antigen-presenting cells. These CTLs were efficient in killing tumor cells that express HLA-B7 and produce CEA. The identification of this HLA-B7-restricted CTL epitope will be useful for the design of ethnically unbiased, widely applicable immunotherapies for common solid epithelial tumors expressing CEA. Moreover, our strategy of identifying MHC class I-restricted CTL epitopes without the need of peptide/HLA-binding assays provides a convenient and cost-saving alternative approach to previous methods.

Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

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Takahashi Takashi

2007-04-01

Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

Shape-dependent regulation of proliferation in normal and malignant human cells and its alteration by interferon

International Nuclear Information System (INIS)

Kulesh, D.A.; Greene, J.J.

1986-01-01

The relationship between cell morphology, proliferation, and contact inhibition was studied in normal and malignant human cells which varied in their sensitivity to contact inhibition. Their ability to proliferate was examined under conditions where the cells were constrained into different shapes by plating onto plastic surfaces coated with poly(2-hydroxyethyl methacrylate). Poly(2-hydroxyethyl methacrylate) can precisely vary the shape of cells without toxicity. Cell proliferation was quantitated by cell counts and labeling indices were determined by autoradiography. The normal JHU-1 foreskin fibroblasts and IMR-90 lung fibroblasts exhibited contact-inhibited growth with a saturation density of 2.9 X 10(5) and 2.0 X 10(5) cells/cm2, respectively. These cells also exhibited stringent dependency on cell shape with a mitotic index of less than 3% at poly(2-hydroxyethyl methacrylate) concentrations at which the cells were rounded versus a labeling index of 75-90% when the cells were flat. The malignant bladder carcinoma line RT-4 exhibited partial contact-inhibited growth. Its dependency on cell shape was less stringent than that of normal cells with a mitotic index of 37-40% when rounded and 79% when flat. The malignant fibrosarcoma line, HT1080, was not contact inhibited and was entirely shape independent with a mitotic index of 70-90% regardless of cell shape. Treatment of HT1080 cells with low concentration of human fibroblast interferon (less than 40 units/ml) restored shape-dependent proliferation while having little effect on normal cells. Subantiproliferative doses of interferon were also shown to restore contact-inhibited proliferation control to malignant cells previously lacking it

Radiation-induced chromosome aberrations and cell killing in normal human fibroblasts and ataxia telangiectasia fibroblasts

International Nuclear Information System (INIS)

Kawata, T.; Saito, M.; Uno, T.; Ito, H.; Shigematsu, N.

2003-01-01

Full text: When cells are held in a non-dividing state (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating. A change of survival depending on post irradiation condition is known to be repair of potentially lethal damage (RPLD). The effects of confluent holding recovery (24-h incubation following irradiation) on chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) were examined. A chemical-induced premature chromosome condensation (PCC) technique with fluorescent in situ hybridization (FISH) was applied to study chromosome aberrations in G2 and M-phase. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. Especially complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. Confluent holding can effectively reduce chromosome aberrations, especially complex type exchanges in normal cells

Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation

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Elena Grossini

2016-09-01

Full Text Available We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article “Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions” (Grossini et al., in press [1].

The rate of DNA synthesis in normal human and ataxia telangiectasia cells after exposure to X-irradiation

International Nuclear Information System (INIS)

Wit, J. de; Bootsma, D.; Jaspers, N.G.J.; Rijksverdedigingsorganisatie TNO, Rijswijk

1981-01-01

The rate of DNA synthesis was studied in normal cell strains and in strains from patients suffering from the inherited disorder ataxia telangiectasia (AT). After exposure to relatively low doses of oxic X-rays (0- 4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an excision-deficient and an excision-proficient strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray-sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad. These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis. (orig.)

Involvement of ERK-Nrf-2 signaling in ionizing radiation induced cell death in normal and tumor cells.

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Raghavendra S Patwardhan

Full Text Available Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.

Label-free identification of intestinal metaplasia in the stomach using multiphoton microscopy

International Nuclear Information System (INIS)

Wu, G; Wei, J; Zheng, Z; Ye, J; Zeng, S

2014-01-01

The early diagnosis of intestinal metaplasia (IM) in the stomach together with effective therapeutic interventions is crucial to reducing the mortality-rates of the patients associated with gastric cancer. However, it is challenging during conventional white-light endoscopy, and histological analysis remains the ‘gold standard’ for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), for the identification of IM in the stomach. It was found that multiphoton imaging provides cellular and subcellular details to the identification of IM from normal gastric tissues. In particular, there is significant difference in the population density of goblet cells between normal and IM gastric tissues, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early gastric lesions. To our knowledge, this is the first demonstration of the potential of MPM for the identification of IM. (letters)

Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome

DEFF Research Database (Denmark)

Pedersen, Martin Bjerregård; Hamilton-Dutoit, Stephen Jacques; Bendix, Knud

2014-01-01

Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome.......Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome....

PKH26 staining defines distinct subsets of normal human colon epithelial cells at different maturation stages.

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Anna Pastò

Full Text Available BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos population survived in culture and formed spheroids; this population included subsets with slow (PKH(high and rapid (PKH(low replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high cells; by cytofluorimetric analysis, Msi-1(+/Lgr5(+ cells were only found within PKH(high cells, whereas Msi-1(+/Lgr5(- cells were also observed in the PKH(low population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1 was highly enriched in Msi-1(+/Lgr5(+ cells. While CK20 expression was mainly found in PKH(low and PKH(neg cells, a small PKH(high subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high/Lgr5(+/Msi-1(+/CK20(-, PKH(high/Lgr5(-/Msi-1(+/CK20(+, PKH(low/Lgr5(-/Msi-1(+/Ck20(-, and PKH(low/Lgr5(-/Msi-1(-/CK20(+ cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any

Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

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Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

2015-08-27

Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

Toxic effect of C60 fullerene-doxorubicin complex towards tumor and normal cells in vitro

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Prylutska S. V.

2014-09-01

Full Text Available Creation of new nanostructures possessing high antitumor activity is an important problem of modern biotechnology. Aim. To evaluate cytotoxicity of created complex of pristine C60 fullerene with the anthracycline antibiotic doxorubicin (Dox, as well as of free C60 fullerene and Dox, towards different cell types – tumor, normal immunocompetent and hepatocytes. Methods. Measurement of size distribution for particles in C60 + Dox mixture was performed by a dynamic light scattering (DLS technique. Toxic effect of C60 + Dox complex in vitro towards tumor and normal cells was studied using the MTT assay. Results. DLS experiment demonstrated that the main fraction of the particles in C60 + Dox mixture had a diameter in the range of about 132 nm. The toxic effect of C60 + Dox complex towards normal (lymphocytes, macrophages, hepatocytes and tumor (Ehrlich ascites carcinoma, leukemia L1210, Lewis lung carcinoma cells was decreased by ~10–16 % and ~7–9 %, accordingly, compared with the same effect of free Dox. Conclusions. The created C60 + Dox composite may be considered as a new pharmacological agent that kills effectively tumor cells in vitro and simultaneously prevents a toxic effect of the free form of Dox on normal cells.

Biogenic amines as regulators of the proliferative activity of normal and neoplastic intestinal epithelial cells (review).

Science.gov (United States)

Tutton, P J; Barkla, D H

1987-01-01

The role of extracellular amines such as noradrenaline and serotonin and their interaction with cyclic nucleotides and intracellular polyamines in the regulation of intestinal epithelial cell proliferation is reviewed with particular reference to the differences between normal and neoplastic cells. In respect to the normal epithelium of the small intestine there is a strong case to support the notion that cell proliferation is controlled by, amongst other things, sympathetic nerves. In colonic carcinomas, antagonists for certain serotonin receptors, for histamine H2 receptors and for dopamine D2 receptors inhibit both cell division and tumour growth. Because of the reproducible variations between tumour lines in the response to these antagonists, this inhibition appears to be due to a direct effect on the tumour cells rather than an indirect effect via the tumour host or stroma. This conclusion is supported by the cytocidal effects of toxic congeners of serotonin on the tumour cells. The most salient difference between the amine responses of normal and neoplastic cells relates to the issue of amine uptake. Proliferation of crypt cells is promoted by amine uptake inhibitors, presumably because they block amine re-uptake by the amine secreting cells--sympathetic neurones and enteroendocrine cells. However, tumour cell proliferation is strongly inhibited by amine uptake inhibitors, suggesting that neoplastic cells can, and need to take up the amine before being stimulated by it. Recent revelations in the field of oncogenes also support an important association between amines, cyclic nucleotides and cell division. The ras oncogenes code for a protein that is a member of a family of molecules which relay information from extracellular regulators, such as biogenic amines, to the intracellular regulators, including cyclic nucleotides. Evidence is presented suggesting that enteroendocrine cells, enterocytes, carcinoid tumour cells and adenocarcinoma cells all have the same

Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes.

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Francesca Faggioli

Full Text Available Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability.

Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

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Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

2016-02-01

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

International Nuclear Information System (INIS)

Beretta, L.; Boutterin, M.C.; Sobel, A.

1988-01-01

We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and [ 32 P] phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins

Identification of Suitable Endogenous Normalizers for qRT- PCR Analysis of Plasma microRNA Expression in Essential Hypertension

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Solayman, Mohamed Hassan M.; Langaee, Taimour; Patel, Archanakumari; El-Wakeel, Lamia; El-Hamamsy, Manal; Badary, Osama; Johnson, Julie A.

2016-01-01

Circulating microRNAs (miRNAs) are promising biomarkers for many diseases. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a gold standard for miRNA expression profiling that requires proper data normalization. Since there is no universal normalizer, it is recommended to evaluate normalizers under every experimental condition. This study describes the identification of suitable endogenous normalizer(s) (ENs) for plasma miRNA expression in essential hypertension. Expression levels of 5 candidate ENs and 2 plasma quality markers were determined by qRT-PCR in plasma samples from 18 hypertensive patients and 10 healthy controls. NormFinder, GeNorm, and DataAssist software programs were used to select the best EN(s). Expression levels of the 5 candidate ENs were also analyzed in urine samples from hypertensive patients and compared to the plasma samples of the hypertensive patients. Among the analyzed candidates, hsa-miR-92a-3p was identified as the best EN, and hsa-miR-21-5p and hsa-miR-16-5p as next best. Moreover, hsa-miR-92a-3p showed the most consistent expression between plasma and urine In conclusion, this study showed that hsa-miR-92a-3p, hsa-miR-21-5p, and hsa-miR-16-5p may be used as normalizers for plasma miRNA expression data in essential hypertension studies. PMID:26798072

Characterization of exosomes derived from ovarian cancer cells and normal ovarian epithelial cells by nanoparticle tracking analysis.

Science.gov (United States)

Zhang, Wei; Peng, Peng; Kuang, Yun; Yang, Jiaxin; Cao, Dongyan; You, Yan; Shen, Keng

2016-03-01

Cellular exosomes are involved in many disease processes and have the potential to be used for diagnosis and treatment. In this study, we compared the characteristics of exosomes derived from human ovarian epithelial cells (HOSEPiC) and three epithelial ovarian cancer cell lines (OVCAR3, IGROV1, and ES-2) to investigate the differences between exosomes originating from normal and malignant cells. Two established colloid-chemical methodologies, electron microscopy (EM) and dynamic light scattering (DLS), and a relatively new method, nanoparticle tracking analysis (NTA), were used to measure the size and size distribution of exosomes. The concentration and epithelial cellular adhesion molecule (EpCAM) expression of exosomes were measured by NTA. Quantum dots were conjugated with anti-EpCAM to label exosomes, and the labeled exosomes were detected by NTA in fluorescent mode. The normal-cell-derived exosomes were significantly larger than those derived from malignant cells, and exosomes were successfully labeled using anti-EpCAM-conjugated quantum dots. Exosomes from different cell lines may vary in size, and exosomes might be considered as potential diagnosis biomarkers. NTA can be considered a useful, efficient, and objective method for the study of different exosomes and their unique properties in ovarian cancer.

DNA strand breaking and rejoining in response to ultraviolet light in normal human and xeroderma pigmentosum cells

International Nuclear Information System (INIS)

Dingman, C.W.; Kakunaga, T.

1976-01-01

A description is given of a reproducible technique for measuring DNA strand breaking and rejoining in cells after treatment with U.V.-light. Results obtained with normal human cells, xeroderma pigmentosum cells (XP, complementation group A) and XP variant cells suggested that all three of these cell-types can carry out single-strand incision with equal rapidity. However, the breaks so induced appeared to be only slowly rejoined in the XP variant cells and rejoined not at all in XP complementation group A cells. Furthermore, parental strand rejoining was inhibited by caffeine in XP variant cells but not in normal cells. (author)

Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents

DEFF Research Database (Denmark)

Lottrup, Grete; Belling, Kirstine González-Izarzugaza; Leffers, Henrik

2017-01-01

the normally clustered and hyperplastic ALCs.WHAT IS KNOWN ALREADY: LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance...... of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of > 15 LCs in a cross...... with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC....

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells

Science.gov (United States)

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells

International Nuclear Information System (INIS)

Fuhrmann, A; Staunton, J R; Banyai, N; Davies, P C W; Ros, R; Nandakumar, V

2011-01-01

The mechanical stiffness of individual cells is important in tissue homeostasis, cell growth, division and motility, and the epithelial–mesenchymal transition in the initiation of cancer. In this work, a normal squamous cell line (EPC2) and metaplastic (CP-A) as well as dysplastic (CP-D) Barrett's Esophagus columnar cell lines are studied as a model of pre-neoplastic progression in the human esophagus. We used the combination of an atomic force microscope (AFM) with a scanning confocal fluorescence lifetime imaging microscope to study the mechanical properties of single adherent cells. Sixty four force indentation curves were taken over the nucleus of each cell in an 8 × 8 grid pattern. Analyzing the force indentation curves, indentation depth-dependent Young's moduli were found for all cell lines. Stiffness tomograms demonstrate distinct differences between the mechanical properties of the studied cell lines. Comparing the stiffness for indentation forces of 1 nN, most probable Young's moduli were calculated to 4.7 kPa for EPC2 (n = 18 cells), 3.1 kPa for CP-A (n = 10) and 2.6 kPa for CP-D (n = 19). We also tested the influence of nuclei and nucleoli staining organic dyes on the mechanical properties of the cells. For stained EPC2 cells (n = 5), significant stiffening was found (9.9 kPa), while CP-A cells (n = 5) showed no clear trend (2.9 kPa) and a slight softening was observed (2.1 kPa) in the case of CP-D cells (n = 16). Some force–indentation curves show non-monotonic discontinuities with segments of negative slope, resembling a sawtooth pattern. We found the incidence of these 'breakthrough events' to be highest in the dysplastic CP-D cells, intermediate in the metaplastic CP-A cells and lowest in the normal EPC2 cells. This observation suggests that the microscopic explanation for the increased compliance of cancerous and pre-cancerous cells may lie in their susceptibility to 'crumble and yield' rather than their

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

NARCIS (Netherlands)

Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

2008-01-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

Directory of Open Access Journals (Sweden)

Xiuchun Ge

2010-07-01

Full Text Available Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

Science.gov (United States)

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

Science.gov (United States)

Pivonello, Rosario; Waaijers, Marlijn; Kros, Johan M; Pivonello, Claudia; de Angelis, Cristina; Cozzolino, Alessia; Colao, Annamaria; Lamberts, Steven W J; Hofland, Leo J

2017-08-01

The dopamine D 2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D 2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D 2 receptor. D 2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D 2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D 2 receptors. D 2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

Science.gov (United States)

Salmond, G P; Lutkenhaus, J F; Donachie, W D

1980-01-01

We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes.

Science.gov (United States)

Tripathi, S K; Farman, M; Nandi, S; Mondal, S; Gupta, Psp; Kumar, V Girish

2016-07-01

The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were

Cerebral blood flow and red cell delivery in normal subjects and in multiple sclerosis

International Nuclear Information System (INIS)

Swank, R.L.; Roth, J.G.; Woody, D.C. Jr.

1983-01-01

Regional cerebral blood flow (rCBF) was determined in 77 normal females and 53 normal males of different ages and in 26 men and 45 women with multiple sclerosis by the inhalation of radioactive Xe133 method. In the normal subjects the CBF was relatively high in the teens and fell, at first rapidly and then slowly in both sexes with age. During adult life the flow in females was significantly higher than in males. The delivery of packed red cells (RCD) was determined by multiplying the CBF by the percentage concentration of red cells (HCT). The RCD for both sexes was nearly the same. In the patients with multiple sclerosis there occurred a progressive generalized decrease in CBF and in RCD with age which was significantly greater than observed in normal subjects. The rate of decrease in CBF and RCD correlated directly with the rate of progress of the disease

ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

Science.gov (United States)

Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

2016-05-01

ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells. © 2016 Federation of European Biochemical Societies.

Comparison of radiosensitivity between human hematopoietic cell lines derived from patients with Down's syndrome and from normal persons

International Nuclear Information System (INIS)

Huang, C.C.; Banerjee, A.; Tan, J.C.; Hou, Y.

1977-01-01

Seven hematopoietic cell lines, four derived from the peripheral blood of patients with Down's syndrome (DS) and three from normal persons, were irradiated with 100, 150, 300, and 500 rads from a 60 Co source and harvested for cell count and chromosome aberration studies every 12 hours for 72 hours post irradiation. Cell growth inhibition and an increase in chromosome aberration were observed in all the cell lines at each dose level and time interval. No significant difference was observed in the effects between DS and normal cell lines. The most common types of aberrations in the 12-hour samples were chromosome and/or chromatid breaks. In the later samples, chromatid exchanges were predominant. The results of the variance analyses on the induced chromosome aberrations in six lines (three DS and three normal lines) showed radiation dosage to be the largest component of total variance, following postirradiation duration and cell lines. The samples harvested 24 and 36 hours post irradiation generally showed greater effects than the samples of other harvest durations. The cell line variance could only be attributed to the differences among and between individual cell lines rather than the difference between DS and normal cell lines

Detection of genomic instability in normal human bronchial epithelial cells exposed to 238Pu

International Nuclear Information System (INIS)

Kennedy, C.H.; Fukushima, N.H.; Neft, R.E.; Lechner, J.F.

1994-01-01

Alpha particle-emitting radon daughters constitute a risk for development of lung cancer in humans. The development of this disease involves multiple genetic alterations. These changes and the time course they follow are not yet defined despite numerous in vitro endeavors to transform human lung cells with various physical or chemical agents. However, genomic instability, characterized both by structural and numerical chromosomal aberrations and by elevated rates of point mutations, is a common feature of tumor cells. Further, both types of genomic instability have been reported in the noncancerous progeny of normal murine hemopoietic cells exposed in vitro to α-particles. The purpose of this investigation was to determine if genomic instability is also a prominent feature of normal human bronchial epithelial cells exposed to α-particle irradiation from the decay of inhaled radon daughters

Simultaneous identification of a contaminant source and hydraulic conductivity via the restart normal-score ensemble Kalman filter

Science.gov (United States)

Xu, Teng; Gómez-Hernández, J. Jaime

2018-02-01

Detecting where and when a contaminant entered an aquifer from observations downgradient of the source is a difficult task; this identification becomes more challenging when the uncertainty about the spatial distribution of hydraulic conductivity is accounted for. In this paper, we have implemented an application of the restart normal-score ensemble Kalman filter (NS-EnKF) for the simultaneous identification of a contaminant source and the spatially variable hydraulic conductivity in an aquifer. The method is capable of providing estimates of the spatial location, initial release time, the duration of the release and the mass load of a point-contamination event, plus the spatial distribution of hydraulic conductivity together with an assessment of the estimation uncertainty of all the parameters. The method has been applied in synthetic aquifers exhibiting both Gaussian and non-Gaussian patterns. The identification is made possible by assimilating in time both piezometric head and concentration observations from an array of observation wells. The method is demonstrated in three different synthetic scenarios that combine hydraulic conductivities with unimodal and bimodal histograms, and releases in high and low conductivity zones. The results prove that the specific implementation of the EnKF is capable of recovering the source parameters with some uncertainty and of recovering the main patterns of heterogeneity of the hydraulic conductivity fields by assimilating a sufficient number of state variable observations. The proposed approach is an important step towards contaminant source identification in real aquifers, which may have logconductivity spatial distributions with either Gaussian or non-Gaussian features, yet, it is still far from practical applications since the transport parameters, the external sinks and sources and the initial and boundary conditions are assumed known.

Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate

International Nuclear Information System (INIS)

Cleaver, J.E.

1982-01-01

Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex

Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin

DEFF Research Database (Denmark)

Kamstrup, M.; Faurschou, A.; Gniadecki, R.

2008-01-01

In this review we focus on epidermal stem cells in the normal regeneration of the skin as well as in wounded and psoriatic skin. Furthermore, we discuss current data supporting the idea of cancer stem cells in the pathogenesis of skin carcinoma and malignant melanoma. Epidermal stem cells present...... or transit amplifying cells constitute a primary pathogenetic factor in the epidermal hyperproliferation seen in psoriasis. In cutaneous malignancies mounting evidence supports a stem cell origin in skin carcinoma and malignant melanoma and a possible existence of cancer stem cells Udgivelsesdato: 2008/5...

Damaging and protective bystander cross-talk between human lung cancer and normal cells after proton microbeam irradiation

International Nuclear Information System (INIS)

Desai, Sejal; Kobayashi, Alisa; Konishi, Teruaki; Oikawa, Masakazu; Pandey, Badri N.

2014-01-01

Graphical abstract: - Highlights: • Proton-microbeam irradiated A549 cells send damaging signals to bystander A549 cells. • Irradiated A549–A549 bystander response is through gap junctional communication. • Bystander WI38 cells exert protective signalling in irradiated A549 cells. • Rescue of irradiated A549 cells by WI38 cells is independent of gap junctions. - Abstract: Most of the studies of radiation-induced bystander effects (RIBE) have been focused on understanding the radiobiological changes observed in bystander cells in response to the signals from irradiated cells in a normal cell population with implications to radiation risk assessment. However, reports on RIBE with relevance to cancer radiotherapy especially investigating the bidirectional and criss-cross bystander communications between cancer and normal cells are limited. Hence, in present study employing co-culture approach, we have investigated the bystander cross-talk between lung cancer (A549) and normal (WI38) cells after proton-microbeam irradiation using γ-H2AX foci fluorescence as a measure of DNA double-strand breaks (DSBs). We observed that in A549–A549 co-cultures, irradiated A549 cells exert damaging effects in bystander A549 cells, which were found to be mediated through gap junctional intercellular communication (GJIC). However, in A549–WI38 co-cultures, irradiated A549 did not affect bystander WI38 cells. Rather, bystander WI38 cells induced inverse protective signalling (rescue effect) in irradiated A549 cells, which was independent of GJIC. On the other hand, in response to irradiated WI38 cells neither of the bystander cells (A549 or WI38) showed significant increase in γ-H2AX foci. The observed bystander signalling between tumour and normal cells may have potential implications in therapeutic outcome of cancer radiotherapy

Damaging and protective bystander cross-talk between human lung cancer and normal cells after proton microbeam irradiation

Energy Technology Data Exchange (ETDEWEB)

Desai, Sejal [Radiation Signalling and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kobayashi, Alisa; Konishi, Teruaki; Oikawa, Masakazu [Radiation System and Engineering Section, Department of Technical Support and Development, Research, Development and Support Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Pandey, Badri N., E-mail: badrinarain@yahoo.co.in [Radiation Signalling and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)

2014-05-15

Graphical abstract: - Highlights: • Proton-microbeam irradiated A549 cells send damaging signals to bystander A549 cells. • Irradiated A549–A549 bystander response is through gap junctional communication. • Bystander WI38 cells exert protective signalling in irradiated A549 cells. • Rescue of irradiated A549 cells by WI38 cells is independent of gap junctions. - Abstract: Most of the studies of radiation-induced bystander effects (RIBE) have been focused on understanding the radiobiological changes observed in bystander cells in response to the signals from irradiated cells in a normal cell population with implications to radiation risk assessment. However, reports on RIBE with relevance to cancer radiotherapy especially investigating the bidirectional and criss-cross bystander communications between cancer and normal cells are limited. Hence, in present study employing co-culture approach, we have investigated the bystander cross-talk between lung cancer (A549) and normal (WI38) cells after proton-microbeam irradiation using γ-H2AX foci fluorescence as a measure of DNA double-strand breaks (DSBs). We observed that in A549–A549 co-cultures, irradiated A549 cells exert damaging effects in bystander A549 cells, which were found to be mediated through gap junctional intercellular communication (GJIC). However, in A549–WI38 co-cultures, irradiated A549 did not affect bystander WI38 cells. Rather, bystander WI38 cells induced inverse protective signalling (rescue effect) in irradiated A549 cells, which was independent of GJIC. On the other hand, in response to irradiated WI38 cells neither of the bystander cells (A549 or WI38) showed significant increase in γ-H2AX foci. The observed bystander signalling between tumour and normal cells may have potential implications in therapeutic outcome of cancer radiotherapy.

Comparative analysis of gene expression in normal and cancer human prostate cell lines

Directory of Open Access Journals (Sweden)

E. E. Rosenberg

2014-04-01

Full Text Available Prostate cancer is one of the main causes of mortality in men with malignant tumors. The urgent problem was a search for biomarkers of prostate cancer, which would allow distinguishing between aggressive metastatic and latent tumors. The aim of this work was to search for differentially expressed genes in normal epithelial cells PNT2 and prostate cancer cell lines LNCaP, DU145 and PC3, produced from tumors with different aggressiveness and metas­tatic ability. Such genes might be used to create a panel of prognostic markers for aggressiveness and metastasis. Relative gene expression of 65 cancer-related genes was determined by the quantitative polymerase chain reaction (Q-PCR. Expression of 29 genes was changed in LNCaP cells, 20 genes in DU145 and 16 genes in PC3 cell lines, compared with normal line PNT2. The obtained data make it possible to conclude that the epithelial-mesenchymal cell transition took place, which involved the loss of epithelial markers, reduced cell adhesion and increased migration. We have also found few differentially expressed genes among 3 prostate cancer cell lines. We have found that genes, involved in cell adhesion (CDH1, invasiveness and metastasis (IL8, CXCL2 and cell cycle control (P16, CCNE1 underwent most changes. These genes might be used for diagnosis and prognosis of invasive metastatic prostate tumors.

Sequence of activation of template biosyntheses in normal and transformed human cells after synchronization with a double thimidine block

International Nuclear Information System (INIS)

Alekseev, S.B.; Boikov, P.Ya.; Ebralidze, L.K.; Stepanova, L.G.

1986-01-01

The sequences of synthesis of DNA, RNA, and various groups of proteins in normal and transformed human fibroblasts was studied in the first mitotic cycle synchronization of the cells by a double thymidine block. Two peculiarities of the synthesis of acid-soluble histone and acid-insoluble proteins in the normal and transformed cells, were detected: (1) in normal fibroblasts the synthesis of the two groups of proteins is a minimum before DNA replication, and the greatest activity is achieved in the G 2 phase; in transformed cells protein synthesis is a maximum after the removal of the thymine block, while in the G 2 phase it is decreased; (2) in normal fibroblasts the synthesis of acid-insoluble proteins is a maximum before the maximum synthesis of DNA, and that of acid-soluble proteins is a maximum after the maximum of DNA synthesis. The opposite picture is observed in transformed cells. RNA synthesis in normal and transformed cells is activated at the end of the G 2 phase. In normal cells the synthesis of proteins is coupled with the activation of RNA synthesis, while in transformed cells protein synthesis is evidently transferred to the following mitotic cycle. Especially pronounced differences were detected in the expression of certain LMG proteins. Thus, in transformed cells the regulation of the coupling of the template syntheses is modified

White blood cells identification system based on convolutional deep neural learning networks.

Science.gov (United States)

Shahin, A I; Guo, Yanhui; Amin, K M; Sharawi, Amr A

2017-11-16

White blood cells (WBCs) differential counting yields valued information about human health and disease. The current developed automated cell morphology equipments perform differential count which is based on blood smear image analysis. Previous identification systems for WBCs consist of successive dependent stages; pre-processing, segmentation, feature extraction, feature selection, and classification. There is a real need to employ deep learning methodologies so that the performance of previous WBCs identification systems can be increased. Classifying small limited datasets through deep learning systems is a major challenge and should be investigated. In this paper, we propose a novel identification system for WBCs based on deep convolutional neural networks. Two methodologies based on transfer learning are followed: transfer learning based on deep activation features and fine-tuning of existed deep networks. Deep acrivation featues are extracted from several pre-trained networks and employed in a traditional identification system. Moreover, a novel end-to-end convolutional deep architecture called "WBCsNet" is proposed and built from scratch. Finally, a limited balanced WBCs dataset classification is performed through the WBCsNet as a pre-trained network. During our experiments, three different public WBCs datasets (2551 images) have been used which contain 5 healthy WBCs types. The overall system accuracy achieved by the proposed WBCsNet is (96.1%) which is more than different transfer learning approaches or even the previous traditional identification system. We also present features visualization for the WBCsNet activation which reflects higher response than the pre-trained activated one. a novel WBCs identification system based on deep learning theory is proposed and a high performance WBCsNet can be employed as a pre-trained network. Copyright © 2017. Published by Elsevier B.V.

DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas.

OpenAIRE

Erickson, L C; Bradley, M O; Ducore, J M; Ewig, R A; Kohn, K W

1980-01-01

Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was ...

Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

Science.gov (United States)

Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

2015-09-29

Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.

Directory of Open Access Journals (Sweden)

Rebecca Williams

Full Text Available BACKGROUND: Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC, are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage. METHODS AND FINDINGS: Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect. CONCLUSIONS: In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell

Marked differences in immunocytological localization of [3H]estradiol-binding protein in rat pancreatic acinar tumor cells compared to normal acinar cells

International Nuclear Information System (INIS)

Beaudoin, A.R.; Grondin, G.; St Jean, P.; Pettengill, O.; Longnecker, D.S.; Grossman, A.

1991-01-01

[ 3 H]Estradiol can bind to a specific protein in normal rat pancreatic acinar cells. Electron microscopic immunocytochemical analysis has shown this protein to be localized primarily in the rough endoplasmic reticulum and mitochondria. Rat exocrine pancreatic tumor cell lines, whether grown in tissue culture (AR42J) or as a tumor mass after sc injection into rats (DSL-2), lacked detectable amounts of this [ 3 H]estradiol-binding protein (EBP), as determined by the dextran-coated charcoal assay. Furthermore, primary exocrine pancreatic neoplasms induced with the carcinogen azaserine contained little or no detectable [ 3 H]estradiol-binding activity. However, electron immunocytochemical studies of transformed cells indicated the presence of material that cross-reacted with antibodies prepared against the [ 3 H]EBP. The immunopositive reaction in transformed cells was localized almost exclusively in lipid granules. Such lipid organelles in normal acinar cells, although present less frequently than in transformed cells, have never been observed to contain EBP-like immunopositive material. Presumably, the aberrant localization of EBP in these acinar tumor cells results in loss of function of this protein, which in normal pancreatic acinar cells appears to exert a modulating influence on zymogen granule formation and the process of secretion

Normal telomere lengths in naive and memory CD4+ T cells in HIV type 1 infection: a mathematical interpretation

NARCIS (Netherlands)

Wolthers, K. C.; Noest, A. J.; Otto, S. A.; Miedema, F.; de Boer, R. J.

1999-01-01

To study CD4+ T cell productivity during HIV-1 infection, CD4+ T cell telomere lengths were measured. Cross-sectional and longitudinal analysis of HIV-1-infected individuals with CD4+ T cells counts >300 cells/mm3 showed normal average telomeric restriction fragment (TRF) length and normal

Normal telomere lengths in naive and memory CD4 T cells in HIV type 1 infection : a mathematical interpretation

NARCIS (Netherlands)

Wolthers, K.C.; Noest, A.J.; Otto, S.A.; Miedema, F.; Boer, R.J. de

1999-01-01

To study CD4+ T cell productivity during HIV-1 infection, CD4+ T cell telomere lengths were measured. Cross-sectional and longitudinal analysis of HIV-1-infected individuals with CD4+ T cells counts >300 cells/mm3 showed normal average telomeric restriction fragment (TRF) length and normal

Mechanism of suppression of normal hemopoietic activity by lymphokine-activated killer cells and their products

International Nuclear Information System (INIS)

Gibson, F.M.; Malkovska, V.; Myint, A.A.; Meager, A.; Gordon-Smith, E.C.

1991-01-01

Interleukin 2 (IL-2)-activated lymphocytes (lymphokine-activated killer [LAK] cells) have been shown to inhibit the formation of autologous human granulocyte-macrophage hemopoietic progenitors (granulocyte-macrophage colony-forming units, CFU-GM) in vitro. Effects of LAK cells on these progenitors may include a number of different mechanisms. LAK cells are potent cytotoxic lymphocytes capable of lysing certain normal autologous cells. They also produce cytokines known to inhibit hemopoiesis (interferon gamma [IFN-gamma] and tumor necrosis factor alpha [TNF-alpha]) or enhance it (granulocyte-macrophage colony-stimulating factor, GM-CSF). In the authors' current study they analyzed the mechanism of suppression of autologous CFU-GM by LAK cells. Their results suggest that LAK cells are not directly cytotoxic to normal CFU-GM. They show that it is possible to abolish the hemopoiesis-inhibiting activity of LAK cells without abrogating their cytotoxicity against tumor cell lines using inhibitors of DNA synthesis, namely hydroxyurea or irradiation

Stem cells: a model for screening, discovery and development of drugs

Directory of Open Access Journals (Sweden)

Kitambi SS

2011-09-01

Full Text Available Satish Srinivas Kitambi1, Gayathri Chandrasekar21Department of Medical Biochemistry and Biophysics; 2Department of Biosciences, Karolinska Institutet, Stockholm, SwedenAbstract: The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson's disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.Keywords: therapeutics, stem cells, cancer stem cells, screening models, drug development, high throughput screening

Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine

International Nuclear Information System (INIS)

Lobo, Nazleen C.; Gedye, Craig; Apostoli, Anthony J.; Brown, Kevin R.; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J.; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A. S.; Ailles, Laurie

2016-01-01

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability

Investigating the Role of Working Memory in Speech-in-noise Identification for Listeners with Normal Hearing.

Science.gov (United States)

Füllgrabe, Christian; Rosen, Stuart

2016-01-01

With the advent of cognitive hearing science, increased attention has been given to individual differences in cognitive functioning and their explanatory power in accounting for inter-listener variability in understanding speech in noise (SiN). The psychological construct that has received most interest is working memory (WM), representing the ability to simultaneously store and process information. Common lore and theoretical models assume that WM-based processes subtend speech processing in adverse perceptual conditions, such as those associated with hearing loss or background noise. Empirical evidence confirms the association between WM capacity (WMC) and SiN identification in older hearing-impaired listeners. To assess whether WMC also plays a role when listeners without hearing loss process speech in acoustically adverse conditions, we surveyed published and unpublished studies in which the Reading-Span test (a widely used measure of WMC) was administered in conjunction with a measure of SiN identification. The survey revealed little or no evidence for an association between WMC and SiN performance. We also analysed new data from 132 normal-hearing participants sampled from across the adult lifespan (18-91 years), for a relationship between Reading-Span scores and identification of matrix sentences in noise. Performance on both tasks declined with age, and correlated weakly even after controlling for the effects of age and audibility (r = 0.39, p ≤ 0.001, one-tailed). However, separate analyses for different age groups revealed that the correlation was only significant for middle-aged and older groups but not for the young (< 40 years) participants.

Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

Science.gov (United States)

2014-01-01

Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

Competition between clonal plasma cells and normal cells for potentially overlapping bone marrow niches is associated with a progressively altered cellular distribution in MGUS vs myeloma.

Science.gov (United States)

Paiva, B; Pérez-Andrés, M; Vídriales, M-B; Almeida, J; de las Heras, N; Mateos, M-V; López-Corral, L; Gutiérrez, N C; Blanco, J; Oriol, A; Hernández, M T; de Arriba, F; de Coca, A G; Terol, M-J; de la Rubia, J; González, Y; Martín, A; Sureda, A; Schmidt-Hieber, M; Schmitz, A; Johnsen, H E; Lahuerta, J-J; Bladé, J; San-Miguel, J F; Orfao, A

2011-04-01

Disappearance of normal bone marrow (BM) plasma cells (PC) predicts malignant transformation of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) into symptomatic multiple myeloma (MM). The homing, behavior and survival of normal PC, but also CD34(+) hematopoietic stem cells (HSC), B-cell precursors, and clonal PC largely depends on their interaction with stromal cell-derived factor-1 (SDF-1) expressing, potentially overlapping BM stromal cell niches. Here, we investigate the distribution, phenotypic characteristics and competitive migration capacity of these cell populations in patients with MGUS, SMM and MM vs healthy adults (HA) aged >60 years. Our results show that BM and peripheral blood (PB) clonal PC progressively increase from MGUS to MM, the latter showing a slightly more immature immunophenotype. Of note, such increased number of clonal PC is associated with progressive depletion of normal PC, B-cell precursors and CD34(+) HSC in the BM, also with a parallel increase in PB. In an ex vivo model, normal PC, B-cell precursors and CD34(+) HSC from MGUS and SMM, but not MM patients, were able to abrogate the migration of clonal PC into serial concentrations of SDF-1. Overall, our results show that progressive competition and replacement of normal BM cells by clonal PC is associated with more advanced disease in patients with MGUS, SMM and MM.

Effect of radiation dosage changes on the cell viability and the apoptosis induction on normal and tumorigenic cells

International Nuclear Information System (INIS)

Park, In Woo; Choi, Soon Chul; Lee, Sam Sun; Heo, Min Suk

1999-01-01

The study was aimed to detect the differences in the cell viability and the apoptosis induction after irradiation on normal and tumorigenic cells. The study, that was generated for two human normal cells (RHEK, HGF-1) and two human tumor cells (KB, HT-1080), was tested using MTT assay at 1 day and 3 day after irradiation and TUNEL assay under confocal laser scanning microscope at 1 day after irradiation. Single irradiation of 0.5, 1, 2, 4, and 8 Gy were applied to the cells. The two fractions of 1, 2, 4, and 8 Gy were separated with a 4 hour time interval. The irradiation was done with 5.38 Gy/min dose rate using Cs-137 irradiator at room temperature. 1. In 3-day group, the cell viability of HGF-1 cell was significantly decreased at 2, 4 and 8 Gy irradiation, the cell viability of KB cell was significantly decreased at 8 Gy irradiation and the cell viability of HT-1080 cell was significantly decreased at 4 and 8 Gy irradiation. 2. There was significant difference between RHEK and KB cell line in the cell viability of 3-day group at 8 Gy irradiation. There was significant difference between RHEK and HGF-1 cell line in the cell viability of 3-day group at 4 and 8 Gy irradiation. 3. There was a significantly decreased cell viability in 3-day group than those in 1-day group at 2, 4 and 8 Gy on HGF-1 cell, at 4 and 8 Gy on HT-1080 cell, at 8 Gy on KB cell.4. We could detect DNA fragmented cells only on KB cell. Number of apoptotic cells of KB cell was significantly increased at 4 and 8 Gy irradiation. However, there was no correlation between cell viability and apoptosis.5. On all 4 cell lines, there were no differences between single and split irradiation method in cell viability and apoptosis.

Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

Directory of Open Access Journals (Sweden)

Ann-Marie Baker

2014-08-01

Full Text Available Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+. Furthermore, we show that, in adenomatous crypts (APC−/−, there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.

Aging and insulin signaling differentially control normal and tumorous germline stem cells.

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Kao, Shih-Han; Tseng, Chen-Yuan; Wan, Chih-Ling; Su, Yu-Han; Hsieh, Chang-Che; Pi, Haiwei; Hsu, Hwei-Jan

2015-02-01

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Nonphotochemical Hole-Burning Imaging Studies of in vitro Carcinoma and Normal Cells Utilizing a Mitochondrial Specific Dye

Energy Technology Data Exchange (ETDEWEB)

Walsh, Richard Joseph [Iowa State Univ., Ames, IA (United States)

2002-01-01

Low temperature Nonphotochemical Hole Burning (NPHB) Spectroscopy of the dye rhodamine 800 (MF680) was applied for the purpose of discerning differences between cultured normal and carcinoma ovarian surface epithelial (OSE) cells. Both the cell lines were developed and characterized at the Mayo Clinic (Rochester, MN), with the normal cell line having been transfected with a strain of temperature sensitive Simian Virus 40 Large T Antigen (SV40) for the purpose of extending the life of the cell culture without inducing permanent changes in the characteristics of the cell line. The cationic lipophilic fluorophore rhodamine 800 preferentially locates in in situ mitochondria due to the high lipid composition of mitochondria and the generation of a large negative membrane potential (relative to the cellular cytoplasm) for oxidative phosphorylation. Results presented for NPHB of MF680 located in the cells show significant differences between the two cell lines. The results are interpreted on the basis of the NPHB mechanism and characteristic interactions between the host (cellular mitochondrial) and the guest (MF680) in the burning of spectral holes, thus providing an image of the cellular ultrastructure. Hole growth kinetics (HGK) were found to differ markedly between the two cell lines, with the carcinoma cell line burning at a faster average rate for the same exposure fluence. Theoretical fits to the data suggest a lower degree of structural heterogeneity in the carcinoma cell line relative to the normal cell line. Measurement of changes in the permanent dipole moment (fΔμ) were accomplished by measurement of changes in hole width in response to the application of an external electric field (the Stark effect), and found that Δμ values for the carcinoma line were 1.5x greater than those of the SV40 antigen-free normal analogs. These findings are interpreted in terms of effects from the mitochondrial membrane potential. Results for HGK on the scale of single cells is

Nonphotochemical Hole-Burning Imaging Studies of In Vitro Carcinoma and Normal Cells Utilizing a Mitochondrial Specific Dye

Energy Technology Data Exchange (ETDEWEB)

Walsh, Richard Joseph [Iowa State Univ., Ames, IA (United States)

2002-01-01

Low temperature Nonphotochemical Hole Burning (NPHB) Spectroscopy of the dye rhodamine 800 (MF680) was applied for the purpose of discerning differences between cultured normal and carcinoma ovarian surface epithelial (OSE) cells. Both the cell lines were developed and characterized at the Mayo Clinic (Rochester, MN), with the normal cell line having been transfected with a strain of temperature sensitive Simian Virus 40 Large T Antigen (SV40) for the purpose of extending the life of the cell culture without inducing permanent changes in the characteristics of the cell line. The cationic lipophilic fluorophore rhodamine 800 preferentially locates in in situ mitochondria due to the high lipid composition of mitochondria and the generation of a large negative membrane potential (relative to the cellular cytoplasm) for oxidative phosphorylation. Results presented for NPHB of MF680 located in the cells show significant differences between the two cell lines. The results are interpreted on the basis of the NPHB mechanism and characteristic interactions between the host (cellular mitochondrial) and the guest (MF680) in the burning of spectral holes, thus providing an image of the cellular ultrastructure. Hole growth kinetics (HGK) were found to differ markedly between the two cell lines, with the carcinoma cell line burning at a faster average rate for the same exposure fluence. Theoretical fits to the data suggest a lower degree of structural heterogeneity in the carcinoma cell line relative to the normal cell line. Measurement of changes in the permanent dipole moment (fΔμ)were accomplished by measurement of changes in hole width in response to the application of an external electric field (the Stark effect), and found that Δμ values for the carcinoma line were 1.5x greater than those of the SV40 antigen-free normal analogs. These findings are interpreted in terms of effects from the mitochondrial membrane potential. Results for HGK on the scale of single cells is

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

Directory of Open Access Journals (Sweden)

Ruth Merkle

2016-08-01

Full Text Available Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC, is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO. However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR. The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.

Science.gov (United States)

van Rhenen, Anna; van Dongen, Guus A M S; Kelder, Angèle; Rombouts, Elwin J; Feller, Nicole; Moshaver, Bijan; Stigter-van Walsum, Marijke; Zweegman, Sonja; Ossenkoppele, Gert J; Jan Schuurhuis, Gerrit

2007-10-01

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

Identification of murine T-cell epitopes in Ebola virus nucleoprotein

International Nuclear Information System (INIS)

Simmons, Graham; Lee, Anee; Rennekamp, Andrew J.; Fan Xin; Bates, Paul; Shen Hao

2004-01-01

CD8 T cells play an important role in controlling Ebola infection and in mediating vaccine-induced protective immunity, yet little is known about antigenic targets in Ebola that are recognized by CD8 T cells. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding Ebola nucleoprotein (NP). CD8 T-cell responses were mapped to a H-2 d -restricted epitope (NP279-288) and two H-2 b -restricted epitopes (NP44-52 and NP288-296). The identification of these epitopes will facilitate studies of immune correlates of protection and the evaluation of vaccine strategies in murine models of Ebola infection

Identification of surface species by vibrational normal mode analysis. A DFT study

Science.gov (United States)

Zhao, Zhi-Jian; Genest, Alexander; Rösch, Notker

2017-10-01

Infrared spectroscopy is an important experimental tool for identifying molecular species adsorbed on a metal surface that can be used in situ. Often vibrational modes in such IR spectra of surface species are assigned and identified by comparison with vibrational spectra of related (molecular) compounds of known structure, e. g., an organometallic cluster analogue. To check the validity of this strategy, we carried out a computational study where we compared the normal modes of three C2Hx species (x = 3, 4) in two types of systems, as adsorbates on the Pt(111) surface and as ligands in an organometallic cluster compound. The results of our DFT calculations reproduce the experimental observed frequencies with deviations of at most 50 cm-1. However, the frequencies of the C2Hx species in both types of systems have to be interpreted with due caution if the coordination mode is unknown. The comparative identification strategy works satisfactorily when the coordination mode of the molecular species (ethylidyne) is similar on the surface and in the metal cluster. However, large shifts are encountered when the molecular species (vinyl) exhibits different coordination modes on both types of substrates.

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells

International Nuclear Information System (INIS)

Sutherland, B.M.; Oliver, R.; Fuselier, C.O.; Sutherland, J.C.

1976-01-01

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobutyl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis--syn thymine--thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts

System Identification of a Non-Uniformly Sampled Multi-Rate System in Aluminium Electrolysis Cells

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Håkon Viumdal

2014-07-01

Full Text Available Standard system identification algorithms are usually designed to generate mathematical models with equidistant sampling instants, that are equal for both input variables and output variables. Unfortunately, real industrial data sets are often disrupted by missing samples, variations of sampling rates in the different variables (also known as multi-rate systems, and intermittent measurements. In industries with varying events based maintenance or manual operational measures, intermittent measurements are performed leading to uneven sampling rates. Such is the case with aluminium smelters, where in addition the materials fed into the cell create even more irregularity in sampling. Both measurements and feeding are mostly manually controlled. A simplified simulation of the metal level in an aluminium electrolysis cell is performed based on mass balance considerations. System identification methods based on Prediction Error Methods (PEM such as Ordinary Least Squares (OLS, and the sub-space method combined Deterministic and Stochastic system identification and Realization (DSR, and its variants are applied to the model of a single electrolysis cell as found in the aluminium smelters. Aliasing phenomena due to large sampling intervals can be crucial in avoiding unsuitable models, but with knowledge about the system dynamics, it is easier to optimize the sampling performance, and hence achieve successful models. The results based on the simulation studies of molten aluminium height in the cells using the various algorithms give results which tally well with the synthetic data sets used. System identification on a smaller data set from a real plant is also implemented in this work. Finally, some concrete suggestions are made for using these models in the smelters.

Characterization of adenoviral transduction profile in prostate cancer cells and normal prostate tissue.

Science.gov (United States)

Ai, Jianzhong; Tai, Phillip W L; Lu, Yi; Li, Jia; Ma, Hong; Su, Qin; Wei, Qiang; Li, Hong; Gao, Guangping

2017-09-01

Prostate diseases are common in males worldwide with high morbidity. Gene therapy is an attractive therapeutic strategy for prostate diseases, however, it is currently underdeveloped. As well known, adeno virus (Ad) is the most widely used gene therapy vector. The aims of this study are to explore transduction efficiency of Ad in prostate cancer cells and normal prostate tissue, thus further providing guidance for future prostate pathophysiological studies and therapeutic development of prostate diseases. We produced Ad expressing enhanced green fluorescence protein (EGFP), and characterized the transduction efficiency of Ad in both human and mouse prostate cancer cell lines in vitro, as well as prostate tumor xenograft, and wild-type mouse prostate tissue in vivo. Ad transduction efficiency was determined by EGFP fluorescence using microscopy and flow cytometry. Cell type-specific transduction was examined by immunofluorescence staining of cell markers. Our data showed that Ad efficiently transduced human and mouse prostate cancer cells in vitro in a dose dependent manner. Following intratumoral and intraprostate injection, Ad could efficiently transduce prostate tumor xenograft and the major prostatic cell types in vivo, respectively. Our findings suggest that Ad can efficiently transduce prostate tumor cells in vitro as well as xenograft and normal prostate tissue in vivo, and further indicate that Ad could be a potentially powerful toolbox for future gene therapy of prostate diseases. © 2017 Wiley Periodicals, Inc.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

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Kaseb, Hatem O. [Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261 (United States); Department of Clinical Pathology, National Cancer Institute (NCI), Cairo University, Cairo (Egypt); Fohrer-Ting, Helene [Department of Pathology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261 (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219 (United States); Lewis, Dale W. [Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261 (United States); Lagasse, Eric [Department of Pathology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261 (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219 (United States); Gollin, Susanne M., E-mail: gollin@pitt.edu [Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261 (United States); University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232 (United States)

2016-10-15

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance. - Highlights: • Spheroid enrichment selects cancer stem cells (CSC) from head & neck tumors (HNSCC). • Compared to normal epithelial cells, isolated CSC express increased SC/CSC markers. • Isolated CSC display enhanced radioresistance, clonogenicity and tumorigenicity. • HNSCC CSC express chromosomal instability. • CD44+/CD66− is a promising, consistent biomarker for HNSCC CSC.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

International Nuclear Information System (INIS)

Kaseb, Hatem O.; Fohrer-Ting, Helene; Lewis, Dale W.; Lagasse, Eric; Gollin, Susanne M.

2016-01-01

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance. - Highlights: • Spheroid enrichment selects cancer stem cells (CSC) from head & neck tumors (HNSCC). • Compared to normal epithelial cells, isolated CSC express increased SC/CSC markers. • Isolated CSC display enhanced radioresistance, clonogenicity and tumorigenicity. • HNSCC CSC express chromosomal instability. • CD44+/CD66− is a promising, consistent biomarker for HNSCC CSC.

Identification of nuclear τ isoforms in human neuroblastoma cells

International Nuclear Information System (INIS)

Loomis, P.A.; Howard, T.H.; Castleberry, R.P.; Binder, L.I.

1990-01-01

The τ proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, τ has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer's disease and of most aged individuals with Down syndrome (trisomy 21). The authors report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, they further demonstrated the existence of the entire τ molecule in the isolated nuclei of neuroblastoma cells. Nuclear τ proteins, like the τ proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that τ may function in processes not directly associated with microtubules and that highly insoluble complexes of τ may also play a role in normal cellular physiology

Effects of radiation from a radiofrequency identification (RFID) microchip on human cancer cells.

Science.gov (United States)

Lai, Henry C; Chan, Ho Wing; Singh, Narendra P

2016-01-01

Radiofrequency identification (RFID) microchips are used to remotely identify objects, e.g. an animal in which a chip is implanted. A passive RFID microchip absorbs energy from an external source and emits a radiofrequency identification signal which is then decoded by a detector. In the present study, we investigated the effect of the radiofrequency energy emitted by a RFID microchip on human cancer cells. Molt-4 leukemia, BT474 breast cancer, and HepG2 hepatic cancer cells were exposed in vitro to RFID microchip-emitted radiofrequency field for 1 h. Cells were counted before and after exposure. Effects of pretreatment with the spin-trap compound N-tert-butyl-alpha-phenylnitrone or the iron-chelator deferoxamine were also investigated. Results We found that the energy effectively killed/retarded the growth of the three different types of cancer cells, and the effect was blocked by the spin-trap compound or the iron-chelator, whereas an inactive microchip and energy from the external source had no significant effect on the cells. Conclusions Data of the present study suggest that radiofrequency field from the microchip affects cancer cells via the Fenton Reaction. Implantation of RFID microchips in tumors may provide a new method for cancer treatment.

Effects of D2O on biochemical parameters of normal cells and tumour cells

International Nuclear Information System (INIS)

Biesewig, G.

1975-01-01

The influence of high temperatures (Hyperthermia) on normal tissue and Ehrlich-Ascites tumour cells ('ATZ') was examined under several conditions with regard to the application of deuterium oxide as a stabilising factor. It was proven that the DNA-synthesis of normal tissue (liver, mouse) is not sensitive to temperature. This effect of hyperthermia only occurs when the tissue is damaged, e.g. by trypsinising. The influence of hyperthermia on several biochemical parameters and on morphological changes of the Ascites cells was examined. The findings show that deuterium oxide (D 2 O) is able to reduce both the thermal and the ureal denaturation of enzymes. Thus tests were carried out to find out if D 2 O also reduces toxic influence in complicated biological systems. The assumption of high D 2 O concentrations to prevent several reactions was confirmed. When the Ascites tumour cells in the H 2 O-buffer were exposed to the damaging influence of hyperthermia, the high degree of damage was seen with the decreasing DNA synthesis, reduced aerobic glycose capacity, a drop in the ATP values and breakdown of the permeability of the membrane. Deuterium oxide was able under high temperature (from appr. 44 0 C on) to reduce the degree of damage to DNA synthesis, while auto-effects (inhibition of synthesis) of D 2 O predominate in the lower region. Aerobic glycolysis was damaged in both cases to the same degree, however. In D 2 O after hyperthermia the ATP-level dropped faster than in H 2 O. D 2 O not only reduces the thermal denaturation of the Ascites tumour cells, but it also eliminates the toxic influence of the zytostaticum TRENIMONsup(R) (under 38 0 or 46 0 C incubation). (orig./AJ) [de

Lethality and the depression on DNA synthesis in UV-irradiated normal human and xeroderma pigmentosum cells

Energy Technology Data Exchange (ETDEWEB)

Shinohara, K. (Kobe Univ. (Japan). School of Medicine)

1983-12-01

Ultraviolet radiation suppresses the semiconservative DNA replication in mammalian cells. The rate of DNA synthesis is initially depressed and later recovers after low doses of UV radiation in human cells. Such a response is more sensitive to UV radiation in cells derived from patients with xeroderma pigmentosum (XP) than that in normal human cells. The relative rate of DNA synthesis is not always correlated with cell survival because, unlike cell survival, the dose-response curve of the relative rate of DNA synthesis shows the biphasic nature of the sensitivity. In the experiments reported herein, the total amount (not the rate) of DNA synthesized during a long interval of incubation which covers the period of inhibition and recovery (but not longer than one generation time) after irradiation with various doses of UV radiation was examined in normal human and XP cells, and was found to be well correlated with cell survival in all the cells tested.

Identification of normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections

International Nuclear Information System (INIS)

Zhou, Yi; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin; Chen, Zhifen; Guan, Guoxian; Kang, Deyong

2016-01-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a potential diagnostic tool is attractive. MPM can effectively provide information about morphological and biochemical changes in biological tissues at the molecular level. In this paper, we attempt to identify normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections (both in transverse and longitudinal sections). The results show that MPM can display different microstructure changes in the transverse and longitudinal sections of colorectal muscularis propria. MPM also can quantitatively describe the alteration of collagen content between normal and cancerous muscle layers. These are important pathological findings that MPM images can bring more detailed complementary information about tissue architecture and cell morphology through observing the transverse and longitudinal sections of colorectal muscularis propria. This work demonstrates that MPM can be better for identifying the microstructural characteristics of normal and cancerous human colorectal muscularis propria in different sections. (paper)

Identification of normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections

Science.gov (United States)

Zhou, Yi; Chen, Zhifen; Kang, Deyong; li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Guan, Guoxian; Chen, Jianxin

2016-01-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a potential diagnostic tool is attractive. MPM can effectively provide information about morphological and biochemical changes in biological tissues at the molecular level. In this paper, we attempt to identify normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections (both in transverse and longitudinal sections). The results show that MPM can display different microstructure changes in the transverse and longitudinal sections of colorectal muscularis propria. MPM also can quantitatively describe the alteration of collagen content between normal and cancerous muscle layers. These are important pathological findings that MPM images can bring more detailed complementary information about tissue architecture and cell morphology through observing the transverse and longitudinal sections of colorectal muscularis propria. This work demonstrates that MPM can be better for identifying the microstructural characteristics of normal and cancerous human colorectal muscularis propria in different sections.

Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

Directory of Open Access Journals (Sweden)

Yan Wusheng

2012-01-01

Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC, the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB, normal differentiated squamous epithelium (ND, and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets. Results As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA were verified to be dysregulated in the same pattern at both the mRNA and protein levels. Conclusions These data reveal insight into genes and

The Effects of Imatinib Mesylate on Cellular Viability, Platelet Derived Growth Factor and Stem Cell Factor in Mouse Testicular Normal Leydig Cells.

Science.gov (United States)

Kheradmand, Fatemeh; Hashemnia, Seyyed Mohammad Reza; Valizadeh, Nasim; Roshan-Milani, Shiva

2016-01-01

Growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells. Treatment of cancer with anti-cancer agents like imatinib mesylate may interfere with normal leydig cell activity, growth and fertility through failure in growth factors' production or their signaling pathways. The purpose of the study was to determine cellular viability and the levels of, platelet derived growth factor (PDGF) and stem cell factor (SCF) in normal mouse leydig cells exposed to imatinib, and addressing the effect of imatinib on fertility potential. The mouse TM3 leydig cells were treated with 0 (control), 2.5, 5, 10 and 20 μM imatinib for 2, 4 and 6 days. Each experiment was repeated three times (15 experiments in each day).The cellular viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, one-way ANOVA with Tukey's post hoc and Kruskal-Wallis test were performed. A p-value less than 0.05 was considered statistically significant. With increasing drug concentration, cellular viability decreased significantly (pcellular viability, PDGF and SCF levels. Imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability. The effect of imatinib on leydig cells is associated with PDGF stimulation. Of course future studies can be helpful in exploring the long term effects of this drug.

Identification of 4 ataxia telangiectasia cell lines hypersensitive to. gamma. -irradiation but not to hydrogen peroxide

Energy Technology Data Exchange (ETDEWEB)

Cantoni, O.; Sestili, P.; Santoro, M.P.; Tannoia, M.C.; Cattabeni, F. (Universita degli Studi di Urbino (Italy). Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologia Sperimentale); Novelli, G.; Dallapiccola, B. (Universit degli Studi di Urbino (Italy). Cattedra di Genetica); Fiorilli, M. (Universita di Roma ' La Sapienze' (Italy). Cattedra di Allergologia e Immunologia Clinica)

1989-09-01

The effct of hydrogen peroxide on the rate of semi-conservative DNA synthesis in ataxia telangiectasia (AT) and normal human lymphoblastoid cells was investigated. The rate of DNA synthesis in AT cells was not depressed to a lesser extent than in normal cells, as might have been expected since H{sub 2O2} is a radiomimetic agent. On the contrary, 4 AT cell lines displayed a higher sensitivity to the inhibitory effect of H{sub 2O2} on DNA synthesis than 2 normal cell lines. Comparable levels of cytotoxicity were detected in cell vaibility studies. Furthermore, neither the level of DNA breakage produced by H{sub 2O2}, nor the rate of repair of these lesions was signigicantly different in normal and AT cells. Together, these results indicate that the AT cell lines utilized in this study are not hypersensitive to the oxidant. It is suggested that H-2-O-2 may not induce lethality via the direct ation of the hydroxyl radical (OH). (Author). 20 refs.; 3 figs.; 1 tab.

Identification of 4 ataxia telangiectasia cell lines hypersensitive to γ-irradiation but not to hydrogen peroxide

International Nuclear Information System (INIS)

Cantoni, O.; Sestili, P.; Santoro, M.P.; Tannoia, M.C.; Cattabeni, F.; Novelli, G.; Dallapiccola, B.; Fiorilli, M.

1989-01-01

The effct of hydrogen peroxide on the rate of semi-conservative DNA synthesis in ataxia telangiectasia (AT) and normal human lymphoblastoid cells was investigated. The rate of DNA synthesis in AT cells was not depressed to a lesser extent than in normal cells, as might have been expected since H 2O2 is a radiomimetic agent. On the contrary, 4 AT cell lines displayed a higher sensitivity to the inhibitory effect of H 2O2 on DNA synthesis than 2 normal cell lines. Comparable levels of cytotoxicity were detected in cell vaibility studies. Furthermore, neither the level of DNA breakage produced by H 2O2 , nor the rate of repair of these lesions was signigicantly different in normal and AT cells. Together, these results indicate that the AT cell lines utilized in this study are not hypersensitive to the oxidant. It is suggested that H-2-O-2 may not induce lethality via the direct ation of the hydroxyl radical (OH). (Author). 20 refs.; 3 figs.; 1 tab

Expression of UV-irradiated adenovirus in normal and UV-sensitive Chinese hamster ovary cells

International Nuclear Information System (INIS)

Rainbow, A.J.

1985-01-01

The chinese hamster ovary (CHO) cell mutants UV-20, UV-24, and UV-41 are abnormally sensitive to UV and harbour various defects lin their ability to repair cellular DNA. This study has examined the expression of UV-irradiated AD2 in these cells. HCR of UV-irradiated Ad2, as measured by viral structural antigen (Vag) formation or progeny production, was found to be similar for the normal and the UV-sensitive CHO strains. UV-irradiation of Ad2 (1200 J/m/sup 2/) resulted in a delay of Vag expression of 18 hours in normal human fibroblasts, which is thought to reflect the time required for removal of UV-induced lesions from the DNA before viral DNA synthesis can proceed. However, a similar UV-irradiation of Ad2 did not result in a delay of Vag expression for infection of CHO cells, suggesting that UV-induced lesions in Ad2 DNA do not inhibit its replication in CHO cells. These results indicate a fundamental difference in the processing of UV-irradiated AD2-DNA in CHO as compared to human cells

Effect of capping agents on the cytotoxicity of silver nanoparticles in human normal and cancer skin cell lines

Energy Technology Data Exchange (ETDEWEB)

Netchareonsirisuk, Ponsawan [Chulalongkorn University, Program in Biotechnology, Faculty of Science (Thailand); Puthong, Songchan [Chulalongkorn University, Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering (Thailand); Dubas, Stephan [Chulalongkorn University, Petroleum and Petrochemical College (Thailand); Palaga, Tanapat [Chulalongkorn University, Department of Microbiology, Faculty of Science (Thailand); Komolpis, Kittinan, E-mail: kittinan.k@chula.ac.th [Chulalongkorn University, Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering (Thailand)

2016-11-15

Silver nanoparticles (AgNPs) are among the most widely used nanomaterials in medical and consumer products. However, safety in the uses of AgNPs is still controversial. The toxicity of AgNPs toward various cell types has been reported to depend on the surface properties of the nanoparticles. In this study, the effect of AgNPs with the average size of 5–15 nm on the viability of the CCD-986SK human normal skin fibroblast cell line and A375 human malignant melanoma cell line was evaluated. Comparative toxicity studies, based on MTT assay, were performed by using either sodium alginate or poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) as capping agent in the nanoparticle preparation. The cytotoxicity tests revealed that AgNO{sub 3} alone was highly toxic to both cell types while both alginate and PSSMA alone were not toxic. AgNPs capped with alginate were selectively toxic to the cancer cell line but not to the normal cell line while AgNPs capped with PSSMA were toxic to both cancer and normal cell lines. Judging from the 50 % inhibition concentration (IC{sub 50}), it was found that the cancer cell line was more sensitive to AgNPs than the normal cell line. Study on the mode of cell death by annexin V and propidium iodide staining revealed that AgNPs induced more apoptotic cell death (84–90 %) than necrosis (8–12 %) in the skin cancer cell line. These results suggest that the toxicity of AgNPs depended on the type of capping agent and the type of cell line.

Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

Science.gov (United States)

Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2011-06-01

Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

Local stem cell depletion model for normal tissue damage

International Nuclear Information System (INIS)

Yaes, R.J.; Keland, A.

1987-01-01

The hypothesis that radiation causes normal tissue damage by completely depleting local regions of tissue of viable stem cells leads to a simple mathematical model for such damage. In organs like skin and spinal cord where destruction of a small volume of tissue leads to a clinically apparent complication, the complication probability is expressed as a function of dose, volume and stem cell number by a simple triple negative exponential function analogous to the double exponential function of Munro and Gilbert for tumor control. The steep dose response curves for radiation myelitis that are obtained with our model are compared with the experimental data for radiation myelitis in laboratory rats. The model can be generalized to include other types or organs, high LET radiation, fractionated courses of radiation, and cases where an organ with a heterogeneous stem cell population receives an inhomogeneous dose of radiation. In principle it would thus be possible to determine the probability of tumor control and of damage to any organ within the radiation field if the dose distribution in three dimensional space within a patient is known

Cell Proliferation during Lymphopoiesis in the Thymus of Normal and Continuously Irradiated Mice

Energy Technology Data Exchange (ETDEWEB)

Fabrikant, J. I. [Department of Radiological Science, Johns Hopkins University, Baltimore, MD (United States)

1968-08-15

The patterns of lymphoid cell proliferation in the thymus and spleen in normal and continuously irradiated young C57BL mice have been examined with techniques of flash and repeated labelling with tritiated thymidine and high resolution autoradiography. Changes in percentage labelling indices and labelled mitoses data have provided information on sites and rates of lymphoid cell proliferation in the thymus cortex (reticular cells, large, medium and small lymphocytes) and the spleen white pulp (germinal centre cells, large, medium and small lymphocytes). Labelling rates were fastest in the more primitive cell forms; in both lymphoid organs, the stem-cell labelling - reticular cells and germinal centre cells - reached 100% rapidly, whereas this was not the case for the different lymphocyte populations, and thymic lymphopoiesis was more rapid than splenic lymphopoiesis. Mean cycle times for thymus lymphoid cells were {approx} 12.5 hours for reticular cells, {approx} 9.5 hours for large lymphocytes, and {approx} 10.0 hours for medium and small lymphocytes; in the spleen, representative cycle times were significantly longer. Small lymphocytes were replaced at a greater rate in the thymus than in the spleen. Under continuous {gamma}-irradiation (caesium-137) at 45 rad/day and 75 rad/day for 15 days, there was a progressive depopulation of all lymphoid cell classes, an increase in the relative proportion of the more primitive forms, and a marked decrease in the numbers of small lymphocytes in both tissues. In the thymus and in the spleen, there was an increase in proliferation rates in both stem-cell populations and in all lymphoid cell forms, a decrease in mean cell cycle times to shorter values and a possible reduction in the spread of cell cycle times. In irradiated tissues, there was little evidence for lymphoid cell emigration. Tentative patterns of lymphopoiesis in the normal thymus and spleen based on the autoradiographic data aredescribed and changes in the

Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization.

Science.gov (United States)

Kamada, Mizuna; Kumazaki, Tsutomu; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2012-06-01

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

The identification of specialized pacemaking cells in the anal sphincters.

Science.gov (United States)

Shafik, Ahmed; El Sibai, Olfat; Ahmed, Ismail

2006-07-01

Interstitial cells of Cajal (ICC) are claimed to generate the electrical activity in the colon and stomach. As the external (EAS) and internal (IAS) anal sphincters exhibit resting electrical activity, we hypothesized the presence of ICC in these sphincters. This hypothesis was investigated in the current study. Specimens from the EAS and IAS were taken from normal areas of the anorectum which had been surgically excised by abdominoperineal operation for rectal cancer of 28 patients (16 men, 12 women, mean age 42.2+/-4.8 years). The specimens were subjected to c-kit immunohistochemistry. Controls for the specificity of the antisera consisted of tissue incubation with normal rabbit serum substituted for the primary antiserum. Fusiform, c-kit positive, ICC-like cells were detected in the anal sphincters; they had dendritic processes. They were clearly distinguishable from the non-branching, c-kit negative smooth and striated muscle cells of the anal sphincters. The specimens contained also c-kit positive mast cells, but they had a rounded body with no dendritic processes. Immunoreactivity was absent in negative controls in which the primary antibody was omitted. We have identified, for the first time, cells in EAS and IAS with morphological and immunological phenotypes similar to ICCs of the gut. These cells appear to be responsible for initiating the slow waves recorded from the anal sphincters and for controlling their activity. A deficiency or absence of these cells may affect the anal motile activity. Studies are needed to explore the role of these cells in anal motility disorders.

Radiation effects in silicon and gallium arsenide solar cells using isotropic and normally incident radiation

Science.gov (United States)

Anspaugh, B. E.; Downing, R. G.

1984-01-01

Several types of silicon and gallium arsenide solar cells were irradiated with protons with energies between 50 keV and 10 MeV at both normal and isotropic incidence. Damage coefficients for maximum power relative to 10 MeV were derived for these cells for both cases of omni-directional and normal incidence. The damage coefficients for the silicon cells were found to be somewhat lower than those quoted in the Solar Cell Radiation Handbook. These values were used to compute omni-directional damage coefficients suitable for solar cells protected by coverglasses of practical thickness, which in turn were used to compute solar cell degradation in two proton-dominated orbits. In spite of the difference in the low energy proton damage coefficients, the difference between the handbook prediction and the prediction using the newly derived values was negligible. Damage coefficients for GaAs solar cells for short circuit current, open circuit voltage, and maximum power were also computed relative to 10 MeV protons. They were used to predict cell degradation in the same two orbits and in a 5600 nmi orbit. Results show the performance of the GaAs solar cells in these orbits to be superior to that of the Si cells.

Enhanced effect of geldanamycin nanocomposite against breast cancer cells growing in vitro and as xenograft with vanquished normal cell toxicity

Energy Technology Data Exchange (ETDEWEB)

Prabhu, Suma [Department of Radiation Biology and Toxicology, School of Life Sciences, Manipal University, Manipal 576 104, Karnataka (India); Ananthanarayanan, Preeta; Aziz, Sajida Kannangar [Department of Biotechnology, School of Life Sciences, Manipal University, Manipal 576 104, Karnataka (India); Rai, Sharada [Department of Pathology, Kasturba Medical College, Mangalore Campus, Manipal University, Mangalore 575 001, Karnataka (India); Mutalik, Srinivas [Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576 104, Karnataka (India); Sadashiva, Satish Rao Bola, E-mail: rao.satish@manipal.edu [Department of Radiation Biology and Toxicology, School of Life Sciences, Manipal University, Manipal 576 104, Karnataka (India)

2017-04-01

Despite enormous advances in remedies developed for breast cancer, an effective therapeutic strategy by targeting malignant cells with the least normal tissue toxicity is yet to be developed. Hsp90 is considered to be an important therapeutic target to inhibit cell proliferation. Geldanamycin (GDM), a potent inhibitor of Hsp90 was withdrawn from clinical trials due to its undesirable hepatotoxicity. We report a superparamagnetic iron oxide (SPION) based polymeric nanocomposite of GDM augmenting anticancer competence with decreased hepatic toxicity. The particle size of nanocomposite was ascertained to be 76 ± 10 nm with acceptable stability. A comparative dose dependent in vitro validation of cytotoxicity showed an enhanced cellular damage and necrosis in breast cancer (MCF-7) cell line at a low dose of 5.49 nM (in GDM nanocomposite) in contrast to 20 nM of pure GDM, while normal breast epithelial cells (MCF-10A) were least affected. Besides, in vivo study (in breast cancer xenografts) substantiated 2.7 fold delay in tumor progression mediated by redundancy in the downstream functions of p-Akt and MAPK-Erk leading to apoptosis with negligible hepatotoxicity. Pure GDM disrupted the function and morphology of liver with lesser therapeutic efficacy than the GDM nanocomposite. These findings deduce that GDM based polymeric magnetite nanocomposite play a vital role in efficacious therapy while vanquishing normal cells and hepatic toxicity and thereby promising it to be reinstated in clinics. - Highlights: • GDM nanocomposite shows selective cell kill of cancerous breast cells. • Nanocomposite delays the growth of tumor in comparison to pure GDM treatment. • GDM promotes apoptosis by down-regulation of p-Akt and MAPK-Erk. • GDM nanocomposite nullifies the hepatotoxicity generally exhibited by pure GDM.

Identification of melanoma cells: a method based in mean variance of signatures via spectral densities.

Science.gov (United States)

Guerra-Rosas, Esperanza; Álvarez-Borrego, Josué; Angulo-Molina, Aracely

2017-04-01

In this paper a new methodology to detect and differentiate melanoma cells from normal cells through 1D-signatures averaged variances calculated with a binary mask is presented. The sample images were obtained from histological sections of mice melanoma tumor of 4 [Formula: see text] in thickness and contrasted with normal cells. The results show that melanoma cells present a well-defined range of averaged variances values obtained from the signatures in the four conditions used.

Effects of recombinant human interleukin-8 (rhIL-8) on the bone marrow cells of normal BALB/c mice

International Nuclear Information System (INIS)

Liu Yulong; Zhou Jianying; Wang Guoquan; Dai Hong; Duan Yingying; Guo Xiaokui

2001-01-01

Objective: To observe the colony formation ability of recombinant human interleukin-8 (rhIL-8) on bone marrow cells (BMCs) of normal mice in vivo. Methods: By means of cells culture and flow cytometry (FCM), the colony-stimulating activity of rhIL-8 on BMCs of normal mice was studied. Results: The experimental studies in vivo demonstrated that rhIL-8 could not changed the counts of CFU-GM and distribution of cell cycle in BMCs. Conclusion: rhIL-8 has no colony-stimulating activity to BMCs of normal mice

Characteristics and function of bone marrow stromal adherent cells in normal and irradiated mice and guinea pigs

Energy Technology Data Exchange (ETDEWEB)

Changyu, Zheng; Ji, Liu; Xiaoying, Bi

1986-04-01

It has been shown from cytochemical and other characteristic studies of bone marrow stromal cells in CFU-F that there are seven types of stromal cells in the stromal adherent cell layer of normal and irradiated C/sub 57/ mice whereas there are only six types in guinea pigs. On the other hand, a radioresistant cell subtype appears in adherent layer after irradiation of both C/sub 57/ mice and guinea pig since the supernatant of cultured CFU-F of the normal and irradiated C/sub 57/ mice can stimulate production of CFU-Gm. It is justifiable that the bone marrow stromal adherent cells of the C/sub 57/ mice could produce CSF.

Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

International Nuclear Information System (INIS)

Lane, M.C.

1989-01-01

Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous β-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in 35 SO 4 -labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed

Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

Directory of Open Access Journals (Sweden)

Biddle Adrian

2010-04-01

Full Text Available Abstract Background Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Methods Cells isolated from fresh human head and neck carcinomas (n = 11, cell lines derived from head and neck, prostate and breast human carcinomas (n = 7, and from normal human oral mucosa (n = 5, were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin. Flow cytometry for CD44 and epithelial-specific antigen (ESA expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR. The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH and siRNA. Results In both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block

Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

International Nuclear Information System (INIS)

Harper, Lisa J; Costea, Daniela Elena; Gammon, Luke; Fazil, Bilal; Biddle, Adrian; Mackenzie, Ian C

2010-01-01

Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA. In both cancer biopsies and carcinoma cell lines a subset of CD44 high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44 high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44 high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance. These data

Androgen-Forming Stem Leydig cells: Identification, Function and Therapeutic Potential

Directory of Open Access Journals (Sweden)

Yunhui Zhang

2008-01-01

Full Text Available Leydig cells are the primary source of testosterone in the male, and differentiation of Leydig cells in the testes is one of the primary events in the development of the male body and fertility. Stem Leydig cells (SLCs exist in the testis throughout postnatal life, but a lack of cell surface markers previously hindered attempts to obtain purified SLC fractions. Once isolated, the properties of SLCs provide interesting clues for the ontogeny of these cells within the embryo. Moreover, the clinical potential of SLCs might be used to reverse age-related declines in testosterone levels in aging men, and stimulate reproductive function in hypogonadal males. This review focuses on the source, identification and outlook for therapeutic applications of SLCs. Separate pools of SLCs may give rise to fetal and adult generations of Leydig cell, which may account for their observed functional differences. These differences should in turn be taken into account when assessing the consequences of environmental pollutants such as the phthalate ester, diethylhexylphthalate (DEHP.

Fruit extract from a Sechium edule hybrid induce apoptosis in leukaemic cell lines but not in normal cells.

Science.gov (United States)

Aguiñiga-Sánchez, Itzen; Soto-Hernández, Marcos; Cadena-Iñiguez, Jorge; Ruíz-Posadas, Lucero del Mar; Cadena-Zamudio, Jorge David; González-Ugarte, Ana Karen; Steider, Benny Weiss; Santiago-Osorio, Edelmiro

2015-01-01

The antiproliferative potential of a crude extract from the chayote hybrid H-837-07-GISeM® and its potential for apoptosis induction were assessed in leukaemic cell lines and normal mouse bone marrow mononuclear cells (BM-MNCs). The extract strongly inhibited the proliferation of the P388, J774, and WEHI-3 cell lines (with an IC50 below 1.3 μg·mL(-1)), reduced cell viability, and induced apoptotic body production, phosphatidylserine translocation, and DNA fragmentation. However, the extract had no effect on BM-MNCs. We postulate that these properties make the extract a good candidate for an anti-tumour agent for clinical use.

Role of growth factors in the growth of normal and transformed cells

International Nuclear Information System (INIS)

Lokeshwar, V.B.

1989-01-01

Growth factors play an important role in the growth of normal cells. However, their untimely and/or excess production leads to neoplastic transformation. The role of growth factors in the growth of normal cells was studied by investigating the mechanism of transmodulation of the cell surface EGF receptor number by protamine. Protamine increased the EGF stimulated mitogenic response in Swiss mouse 3T3 cells and A431 cells by increasing the number of functionally active EGF receptors. Protamine also increased EGF receptor number in plasma membranes and solubilized membranes. This was evidenced by an increase in both 125 I-EGF-EGF-receptor complex and EGF stimulated phosphorylation of the EGF receptor. The solubilized EGF receptor was retained on a protamine-agarose gel indicating that protamine might increase EGF receptor number by directly activating cryptic EGF receptors in the plasma membranes. The role of growth factors in neoplastic transformation was studied by investigating the role of the oncogene v-sis in the growth of Simian sarcoma virus (SSV) transformed cells. The product of the oncogene v-sis is 94% homologous to the B chain of PDGF. This study found that (i) v-sis gene product is synthesized as a 32 kDa unglycosylated monomer which is glycosylated, dimerized and proteolytically processed into p36, p72, p68, p58, p44 and p27 mol. wt. species respectively. (ii) p36, p72, p68 and p58 are very likely formed in the endoplasmic reticulum and/or Golgi complex. A fraction of newly synthesized p72, p68 and p58 is degraded intracellularly at a fast rate. (iii) p44 is a secretory product which remains tightly associated with the cell surface. p44 is recaptured by the cells through interaction with cell surface PDGF receptors and degraded into p27. (iv) During long term cultures p44 is extracellularly cleaved into a 27 kDa product

Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

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Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

2018-06-30

Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions

Age-Related Change in Vestibular Ganglion Cell Populations in Individuals With Presbycusis and Normal Hearing.

Science.gov (United States)

Gluth, Michael B; Nelson, Erik G

2017-04-01

We sought to establish that the decline of vestibular ganglion cell counts uniquely correlates with spiral ganglion cell counts, cochlear hair cell counts, and hearing phenotype in individuals with presbycusis. The relationship between aging in the vestibular system and aging in the cochlea is a topic of ongoing investigation. Histopathologic age-related changes the vestibular system may mirror what is seen in the cochlea, but correlations with hearing phenotype and the impact of presbycusis are not well understood. Vestibular ganglion cells, spiral ganglion cells, and cochlear hair cells were counted in specimens from individuals with presbycusis and normal hearing. These were taken from within a large collection of processed human temporal bones. Correlations between histopathology and hearing phenotype were investigated. Vestibular ganglion cell counts were positively correlated with spiral ganglion cell counts and cochlear hair cell counts and were negatively correlated with hearing phenotype. There was no statistical evidence on linear regression to suggest that the relationship between age and cell populations differed significantly according to whether presbycusis was present or not. Superior vestibular ganglion cells were more negatively correlated with age than inferior ganglion cells. No difference in vestibular ganglion cells was noted based on sex. Vestibular ganglion cell counts progressively deteriorate with age, and this loss correlates closely with changes in the cochlea, as well as hearing phenotype. However, these correlations do not appear to be unique in individuals with presbycusis as compared with those with normal hearing.

Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells.

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Liu, Xunxian; Allen, Jeffrey D; Arnold, Julia T; Blackman, Marc R

2008-04-01

Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3beta and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

Selenoprotein P Inhibits Radiation-Induced Late Reactive Oxygen Species Accumulation and Normal Cell Injury

Energy Technology Data Exchange (ETDEWEB)

Eckers, Jaimee C.; Kalen, Amanda L.; Xiao, Wusheng; Sarsour, Ehab H.; Goswami, Prabhat C., E-mail: prabhat-goswami@uiowa.edu

2013-11-01

Purpose: Radiation is a common mode of cancer therapy whose outcome is often limited because of normal tissue toxicity. We have shown previously that the accumulation of radiation-induced late reactive oxygen species (ROS) precedes cell death, suggesting that metabolic oxidative stress could regulate cellular radiation response. The purpose of this study was to investigate whether selenoprotein P (SEPP1), a major supplier of selenium to tissues and an antioxidant, regulates late ROS accumulation and toxicity in irradiated normal human fibroblasts (NHFs). Methods and Materials: Flow cytometry analysis of cell viability, cell cycle phase distribution, and dihydroethidium oxidation, along with clonogenic assays, were used to measure oxidative stress and toxicity. Human antioxidant mechanisms array and quantitative real-time polymerase chain reaction assays were used to measure gene expression during late ROS accumulation in irradiated NHFs. Sodium selenite addition and SEPP1 overexpression were used to determine the causality of SEPP1 regulating late ROS accumulation and toxicity in irradiated NHFs. Results: Irradiated NHFs showed late ROS accumulation (4.5-fold increase from control; P<.05) that occurs after activation of the cell cycle checkpoint pathways and precedes cell death. The mRNA levels of CuZn- and Mn-superoxide dismutase, catalase, peroxiredoxin 3, and thioredoxin reductase 1 increased approximately 2- to 3-fold, whereas mRNA levels of cold shock domain containing E1 and SEPP1 increased more than 6-fold (P<.05). The addition of sodium selenite before the radiation treatment suppressed toxicity (45%; P<.05). SEPP1 overexpression suppressed radiation-induced late ROS accumulation (35%; P<.05) and protected NHFs from radiation-induced toxicity (58%; P<.05). Conclusion: SEPP1 mitigates radiation-induced late ROS accumulation and normal cell injury.

On the use of plate-type normal pressure cells in silos

DEFF Research Database (Denmark)

Ramirez, Alvaro; Nielsen, Jørgen; Ayuga, F.

2010-01-01

the interpretation of results. Once the cells have been delivered from the manufacturer to the researcher, they should be calibrated and validated with reference to the measurement of pressure from a granular material against a silo wall. Two related papers deal with a specific plate-type normal pressure cell...... for use in an installation of three full-scale steel silos with different hopper eccentricities (concentric, half-eccentric and full-eccentric) as part of a silo research project. It was found to be necessary to validate the performance of the cells when measuring pressures in the silos in order to arrive...... at a solid basis for the interpretation of the pressure measurements in the silo installation aforementioned. This paper presents calibration results from three investigated methods as well as results from a finite element analysis of the plate deflection of the pressure cell which were performed to evaluate...

Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects.

Science.gov (United States)

Somoza, R; Beutler, E

1983-10-01

Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.

Application of Artificial Bee Colony in Model Parameter Identification of Solar Cells

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Rongjie Wang

2015-07-01

Full Text Available The identification of values of solar cell parameters is of great interest for evaluating solar cell performances. The algorithm of an artificial bee colony was used to extract model parameters of solar cells from current-voltage characteristics. Firstly, the best-so-for mechanism was introduced to the original artificial bee colony. Then, a method was proposed to identify parameters for a single diode model and double diode model using this improved artificial bee colony. Experimental results clearly demonstrate the effectiveness of the proposed method and its superior performance compared to other competing methods.

Cytokine Release and Focal Adhesion Proteins in Normal Thyroid Cells Cultured on the Random Positioning Machine

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Elisabeth Warnke

2017-08-01

Full Text Available Background/Aims: Spaceflight impacts on the function of the thyroid gland in vivo. In vitro normal and malignant thyrocytes assemble in part to multicellular spheroids (MCS after exposure to the random positioning machine (RPM, while a number of cells remain adherent (AD. We aim to elucidate possible differences between AD and MCS cells compared to 1g-controls of normal human thyroid cells. Methods: Cells of the human follicular epithelial thyroid cell line Nthy-ori 3-1 were incubated for up to 72 h on the RPM. Afterwards, they were investigated by phase-contrast microscopy, quantitative real-time PCR and by determination of cytokines released in their supernatants. Results: A significant up-regulation of IL6, IL8 and CCL2 gene expression was found after a 4h RPM-exposure, when the whole population was still growing adherently. MCS and AD cells were detected after 24 h on the RPM. At this time, a significantly reduced gene expression in MCS compared to 1g-controls was visible for IL6, IL8, FN1, ITGB1, LAMA1, CCL2, and TLN1. After a 72 h RPM-exposure, IL-6, IL-8, and TIMP-1 secretion rates were increased significantly. Conclusion: Normal thyrocytes form MCS within 24 h. Cytokines seem to be involved in the initiation of MCS formation via focal adhesion proteins.

The Expression of Markers for Intratubular Germ Cell Neoplasia in Normal Infantile Testes

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Kolja Kvist

2018-06-01

Full Text Available BackgroundPositive immunohistochemical expression of testicular cancer markers is often reported beyond 12 months of age in cryptorchid testes, which is assumed to indicate delayed maturation of the fetal germ cells, or neoplastic changes. These findings allowed for questions as to the extent of positive reaction in normal testes. The aim of the study was to clarify the expression of these markers in a normal material up to 2 years.MethodsTesticular material from 69 boys aged 1–690 days, who died of causes with no association of testicular pathology. Histology sections were incubated with primary antibodies including anti-placental-like alkaline phosphatase (PLAP, anti-C-Kit, anti-D2–40, and anti-Oct3/4. The mean germ cell number per tubular transverse section (G/T was calculated based on the G/T of both testes of every boy.ResultsThe mean G/T declined through the 690 days. PLAP appeared stably expressed throughout the ages studied. The likelihood of a positive reaction for C-Kit waned with increasing age within the study period. Positive staining for D2–40 and Oct3/4 was demonstrated up to 6 and 9 months respectively.ConclusionUp to 1 or 2 years of age, normal infantile testes contain germ cells positive for the immunohistochemical markers commonly utilized to aid in the detection of testicular cancer. This finding supports the concept of germ cells undergoing a continuous maturational process in a heterogeneous fashion, and that this process is not complete by 2 years of age.

Automatic cell identification and visualization using digital holographic microscopy with head mounted augmented reality devices.

Science.gov (United States)

O'Connor, Timothy; Rawat, Siddharth; Markman, Adam; Javidi, Bahram

2018-03-01

We propose a compact imaging system that integrates an augmented reality head mounted device with digital holographic microscopy for automated cell identification and visualization. A shearing interferometer is used to produce holograms of biological cells, which are recorded using customized smart glasses containing an external camera. After image acquisition, segmentation is performed to isolate regions of interest containing biological cells in the field-of-view, followed by digital reconstruction of the cells, which is used to generate a three-dimensional (3D) pseudocolor optical path length profile. Morphological features are extracted from the cell's optical path length map, including mean optical path length, coefficient of variation, optical volume, projected area, projected area to optical volume ratio, cell skewness, and cell kurtosis. Classification is performed using the random forest classifier, support vector machines, and K-nearest neighbor, and the results are compared. Finally, the augmented reality device displays the cell's pseudocolor 3D rendering of its optical path length profile, extracted features, and the identified cell's type or class. The proposed system could allow a healthcare worker to quickly visualize cells using augmented reality smart glasses and extract the relevant information for rapid diagnosis. To the best of our knowledge, this is the first report on the integration of digital holographic microscopy with augmented reality devices for automated cell identification and visualization.

Human respiratory syncytial virus load normalized by cell quantification as predictor of acute respiratory tract infection.

Science.gov (United States)

Gómez-Novo, Miriam; Boga, José A; Álvarez-Argüelles, Marta E; Rojo-Alba, Susana; Fernández, Ana; Menéndez, María J; de Oña, María; Melón, Santiago

2018-05-01

Human respiratory syncytial virus (HRSV) is a common cause of respiratory infections. The main objective is to analyze the prediction ability of viral load of HRSV normalized by cell number in respiratory symptoms. A prospective, descriptive, and analytical study was performed. From 7307 respiratory samples processed between December 2014 to April 2016, 1019 HRSV-positive samples, were included in this study. Low respiratory tract infection was present in 729 patients (71.54%). Normalized HRSV load was calculated by quantification of HRSV genome and human β-globin gene and expressed as log10 copies/1000 cells. HRSV mean loads were 4.09 ± 2.08 and 4.82 ± 2.09 log10 copies/1000 cells in the 549 pharyngeal and 470 nasopharyngeal samples, respectively (P respiratory tract infection and 4.22 ± 2.28 log10 copies/1000 cells with upper respiratory tract infection or febrile syndrome (P < 0.05). A possible cut off value to predict LRTI evolution was tentatively established. Normalization of viral load by cell number in the samples is essential to ensure an optimal virological molecular diagnosis avoiding that the quality of samples affects the results. A high viral load can be a useful marker to predict disease progression. © 2018 Wiley Periodicals, Inc.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

Energy Technology Data Exchange (ETDEWEB)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Ansari, Nariman [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Esner, Milan; Bickle, Marc [Max Planck Institute of Molecular Cell Biology and Genetics, High-Throughput Technology Development Studio (TDS), Dresden (Germany); Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H. [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Parczyk, Karsten; Prechtl, Stefan [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Steigemann, Patrick, E-mail: Patrick.Steigemann@bayer.com [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany)

2014-04-15

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

International Nuclear Information System (INIS)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H.; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

2014-01-01

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

Localization of cells containing sedimented amyloplasts in the shoots of normal and lazy rice seedlings.

Science.gov (United States)

Abe, K; Takahashi, H; Suge, H

1994-12-01

We have examined the localization of the cells containing sedimented amyloplasts (putative statocytes) and its relation to the graviresponding sites in the shoots of normal and lazy rice seedlings. All graviresponsive organs of the shoots of normal rice seedlings, the mesocotyl, the coleoptile and the leaf-sheath base, were found to possess the statocytes. This is the first indication that mesocotyl senses gravity by its own cells in inducing gravitropic bending in rice seedlings. In lazy-Kamenoo, although the shoots lost their gravitropic response with the advance of age, sedimentation of amyloplasts itself might not be attributable to the agravitropic growth of the shoots, because, including those of the leaf-sheath bases that had lost their response to gravity, sedimented amyloplasts appeared to be identical to those of normal Kamenoo and of younger seedlings of lazy-Kamenoo whose gravitropism is still apparent.

Myeloperoxidase-positive cell infiltration of normal colorectal mucosa is related to body fatness and is predictive of adenoma occurrence.

Science.gov (United States)

Mariani, F; Boarino, V; Bertani, A; Merighi, A; Pedroni, M; Rossi, G; Mancini, S; Sena, P; Benatti, P; Roncucci, L

2017-06-01

Body fatness is a risk factor for colorectal cancer, and promotes an inflammatory environment. Indeed, inflammation in normal colorectal mucosa may be a factor linking body fatness to colorectal carcinogenesis. In this study, we evaluated myeloperoxidase (MPO)-positive cells infiltration of normal colorectal mucosa as a marker of cancer-promoting inflammation in overweight and obese subjects. One hundred and three subjects with normal colonoscopy entered the study. Waist circumference (WC) and body mass index (BMI) were measured, and MPO-positive cells on histological sections of biopsies of normal colorectal mucosa were counted under a light microscope. The occurrence of adenomas was then evaluated on follow-up colonoscopies. Mean MPO-positive cell count (±s.e.m.) was higher in subject with a WC equal or above the obesity cutoff values according to gender (2.63±0.20 vs 2.06±0.18, P=0.03), and in subjects with BMI equal or above 25 kg m - 2 (2.54±0.18 vs 1.97±0.20, P=0.03). A Cox proportional hazard model showed that mean MPO-positive cell count in normal colorectal mucosa was the only factor independently related to occurrence of adenomas in follow-up colonoscopies. Though preliminary, these results show that MPO-positive cell infiltration in normal colorectal mucosa is related with body fatness, as evaluated by WC and BMI, and it may be considered a useful and simple marker to estimate adenoma occurrence risk.

Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

Science.gov (United States)

Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

2017-02-28

Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

Merging Mixture Components for Cell Population Identification in Flow Cytometry

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Greg Finak

2009-01-01

Full Text Available We present a framework for the identification of cell subpopulations in flow cytometry data based on merging mixture components using the flowClust methodology. We show that the cluster merging algorithm under our framework improves model fit and provides a better estimate of the number of distinct cell subpopulations than either Gaussian mixture models or flowClust, especially for complicated flow cytometry data distributions. Our framework allows the automated selection of the number of distinct cell subpopulations and we are able to identify cases where the algorithm fails, thus making it suitable for application in a high throughput FCM analysis pipeline. Furthermore, we demonstrate a method for summarizing complex merged cell subpopulations in a simple manner that integrates with the existing flowClust framework and enables downstream data analysis. We demonstrate the performance of our framework on simulated and real FCM data. The software is available in the flowMerge package through the Bioconductor project.

Insulin resistance according to β-cell function in women with polycystic ovary syndrome and normal glucose tolerance.

Science.gov (United States)

Song, Do Kyeong; Hong, Young Sun; Sung, Yeon-Ah; Lee, Hyejin

2017-01-01

Polycystic ovary syndrome (PCOS) is associated with insulin resistance (IR) and compensatory hyperinsulinemia. IR is recognized as a major risk factor for the development of type 2 diabetes mellitus. However, few studies have investigated IR in women with PCOS and normal glucose tolerance. The objective of this study was to evaluate IR and β-cell function in women with PCOS and normal glucose tolerance. Additionally, we sought to evaluate the usefulness of oral glucose tolerance test (OGTT)-derived IR indices in lean women with PCOS. We recruited 100 women with PCOS and normal glucose tolerance and 100 age- and BMI-matched women as controls. IR and insulin secretory indices, including the homeostasis-model assessment (HOMA)-IR, HOMA-M120, HOMA-F and the Stumvoll index, were calculated from an OGTT. Increased β-cell function was defined as>75th percentile for the HOMA-F in control women. Women with PCOS had higher values for post-load 2-hour glucose, fasting insulin, post-load 2-hour insulin, HOMA-IR, HOMA-M120, HOMA-F and lower values for the Stumvoll index than the controls (all PsWomen with PCOS and increased β-cell function showed lower Stumvoll index values than the matched controls (Plean women with PCOS (all PsWomen with PCOS and normal glucose tolerance showed higher IR than controls matched for age, BMI, and β-cell function. β-cell function was increased in women with PCOS when compared to the matched controls, but not when the lean subjects were compared to the matched controls separately. Therefore, early evaluation of IR in women with PCOS and normal glucose tolerance may be needed.

Procalcitonin NH2-terminal cleavage peptide has no mitogenic effect on normal human osteoblast-like cells

International Nuclear Information System (INIS)

Hassager, C.; Bonde, S.K.; Anderson, M.A.; Rink, H.; Spelsberg, T.C.; Riggs, B.L.

1991-01-01

The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen. The authors have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 microM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6-10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 micrograms/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus they data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts

Effect of resveratrol and zinc on intracellular zinc status in normal human prostate epithelial cells

Science.gov (United States)

To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated with 6 levels of resveratrol (0, 0.5, 1, 2.5, 5 and 10 microM) and 4 levels of zinc [0, 4, 16, and 32 microM for zinc-deficient (ZD), zinc-normal (ZN), zinc-adequate (ZA), an...

Identification of progenitor cancer stem cell in lentigo maligna melanoma.

Science.gov (United States)

Bongiorno, M R; Doukaki, S; Malleo, F; Aricò, M

2008-07-01

The potential role of stem cells in neoplasia has aroused considerable interest over the past few years. A number of known biologic characteristics of melanomas support the theory that they may originate in a mutated stem cell. Melanocytic stem cell markers have been described recently. Moreover, the CD133 cells that show surface markers for CD34 are stem cells primitive. These stem cells are capable of differentiating into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. The identification of cancer stem/initiating cells with a crucial role in tumor formation may open up new pharmacologic perspectives. The purpose of this study is to detect the expression of CD133 and CD34, two putative markers of cancer stem cells in the lentigo maligna melanoma. Thirty cases of lentigo maligna melanoma were analyzed using indirect immunohistochemical staining. The vast majority of the samples analyzed showed the presence of rare cells, which were clearly positive for CD133 and CD34. Strong CD133 and CD34 staining was found in the outer root sheath of the mid-lower hair follicles, intermixed with atypical melanocytes extending along layers of the hair follicles. A number of these staminal cells were adjacent and intermixed with melanoma cells. This study supports the stem cell origin of this tumor and suggests that the precursor of the melanoma in question is a stem-like cell rather than the primitive melanoblast committed to be exclusively involved in melanocytic differentiation.

Data-driven identification of intensity normalization region based on longitudinal coherency of 18F-FDG metabolism in the healthy brain.

Science.gov (United States)

Zhang, Huiwei; Wu, Ping; Ziegler, Sibylle I; Guan, Yihui; Wang, Yuetao; Ge, Jingjie; Schwaiger, Markus; Huang, Sung-Cheng; Zuo, Chuantao; Förster, Stefan; Shi, Kuangyu

2017-02-01

In brain 18 F-FDG PET data intensity normalization is usually applied to control for unwanted factors confounding brain metabolism. However, it can be difficult to determine a proper intensity normalization region as a reference for the identification of abnormal metabolism in diseased brains. In neurodegenerative disorders, differentiating disease-related changes in brain metabolism from age-associated natural changes remains challenging. This study proposes a new data-driven method to identify proper intensity normalization regions in order to improve separation of age-associated natural changes from disease related changes in brain metabolism. 127 female and 128 male healthy subjects (age: 20 to 79) with brain 18 F-FDG PET/CT in the course of a whole body cancer screening were included. Brain PET images were processed using SPM8 and were parcellated into 116 anatomical regions according to the AAL template. It is assumed that normal brain 18 F-FDG metabolism has longitudinal coherency and this coherency leads to better model fitting. The coefficient of determination R 2 was proposed as the coherence coefficient, and the total coherence coefficient (overall fitting quality) was employed as an index to assess proper intensity normalization strategies on single subjects and age-cohort averaged data. Age-associated longitudinal changes of normal subjects were derived using the identified intensity normalization method correspondingly. In addition, 15 subjects with clinically diagnosed Parkinson's disease were assessed to evaluate the clinical potential of the proposed new method. Intensity normalizations by paracentral lobule and cerebellar tonsil, both regions derived from the new data-driven coherency method, showed significantly better coherence coefficients than other intensity normalization regions, and especially better than the most widely used global mean normalization. Intensity normalization by paracentral lobule was the most consistent method within both

Mesenchymal Stem and Progenitor Cells in Normal and Dysplastic Hematopoiesis—Masters of Survival and Clonality?

Science.gov (United States)

Pleyer, Lisa; Valent, Peter; Greil, Richard

2016-01-01

Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell disorders that have the capacity to progress to acute myeloid leukemia (AML). Accumulating evidence suggests that the altered bone marrow (BM) microenvironment in general, and in particular the components of the stem cell niche, including mesenchymal stem cells (MSCs) and their progeny, play a pivotal role in the evolution and propagation of MDS. We here present an overview of the role of MSCs in the pathogenesis of MDS, with emphasis on cellular interactions in the BM microenvironment and related stem cell niche concepts. MSCs have potent immunomodulatory capacities and communicate with diverse immune cells, but also interact with various other cellular components of the microenvironment as well as with normal and leukemic stem and progenitor cells. Moreover, compared to normal MSCs, MSCs in MDS and AML often exhibit altered gene expression profiles, an aberrant phenotype, and abnormal functional properties. These alterations supposedly contribute to the “reprogramming” of the stem cell niche into a disease-permissive microenvironment where an altered immune system, abnormal stem cell niche interactions, and an impaired growth control lead to disease progression. The current article also reviews molecular targets that play a role in such cellular interactions and possibilities to interfere with abnormal stem cell niche interactions by using specific targeted drugs. PMID:27355944

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

Science.gov (United States)

Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

2005-01-01

MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

Combined Treatment with Low Concentrations of Decitabine and SAHA Causes Cell Death in Leukemic Cell Lines but Not in Normal Peripheral Blood Lymphocytes

Directory of Open Access Journals (Sweden)

Barbora Brodská

2013-01-01

Full Text Available Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells and to preserve normal cells. Combination epigenetic therapy may further improve the effect of individual drugs. We investigated combined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat on different leukemic cell lines in comparison with peripheral blood lymphocytes. Large decrease of viability, as well as huge p21WAF1 induction, reactive oxygen species formation, and apoptotic features due to combined decitabine and SAHA action were detected in leukemic cell lines irrespective of their p53 status, while essentially no effect was observed in response to the combined drug action in normal peripheral blood lymphocytes of healthy donors. p53-dependent apoptotic pathway was demonstrated to participate in the wtp53 CML-T1 leukemic cell line response, while significant influence of reactive oxygen species on viability decrease has been detected in p53-null HL-60 cell line.

Identification and Investigation of Native Chromosomal Fragile Sites in the Avian Cell Line DT40

DEFF Research Database (Denmark)

Pentzold, Constanze

cell systems. With the identification and investigation of CFSs in avian DT40 cells, this study reveals the genome-­‐wide evolutionary conservation of CFSs beyond the mammalian lineage for the first time. It opens the way for speculations on the beneficial existence of CFSs throughout the animal...... kingdom....

Human Mesenchymal Stem Cell Treatment Normalizes Cortical Gene Expression after Traumatic Brain Injury.

Science.gov (United States)

Darkazalli, Ali; Vied, Cynthia; Badger, Crystal-Dawn; Levenson, Cathy W

2017-01-01

Traumatic brain injury (TBI) results in a progressive disease state with many adverse and long-term neurological consequences. Mesenchymal stem cells (MSCs) have emerged as a promising cytotherapy and have been previously shown to reduce secondary apoptosis and cognitive deficits associated with TBI. Consistent with the established literature, we observed that systemically administered human MSCs (hMSCs) accumulate with high specificity at the TBI lesion boundary zone known as the penumbra. Substantial work has been done to illuminate the mechanisms by which MSCs, and the bioactive molecules they secrete, exert their therapeutic effect. However, no such work has been published to examine the effect of MSC treatment on gene expression in the brain post-TBI. In the present study, we use high-throughput RNA sequencing (RNAseq) of cortical tissue from the TBI penumbra to assess the molecular effects of both TBI and subsequent treatment with intravenously delivered hMSCs. RNAseq revealed that expression of almost 7000 cortical genes in the penumbra were differentially regulated by TBI. Pathway analysis using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database revealed that TBI regulated a large number of genes belonging to pathways involved in metabolism, receptor-mediated cell signaling, neuronal plasticity, immune cell recruitment and infiltration, and neurodegenerative disease. Remarkably, hMSC treatment was found to normalize 49% of all genes disrupted by TBI, with notably robust normalization of specific pathways within the categories mentioned above, including neuroactive receptor-ligand interactions (57%), glycolysis and gluconeogenesis (81%), and Parkinson's disease (100%). These data provide evidence in support of the multi-mechanistic nature of stem cell therapy and suggest that hMSC treatment is capable of simultaneously normalizing a wide variety of important molecular pathways that are disrupted by brain injury.

The cytogenetic estimate of the radioprotective effect of antioxydant on normal and defected human cells

International Nuclear Information System (INIS)

Zvereva, S.V.; Mutovina, G.R.; Khandogina, E.K.; Marchenko, L.F.; Neudakhin, E.V.; Artamonov, R.G.; Akif'ev, A.P.

1993-01-01

In studying the radioprotective action of natural and synthesised antioxydants a decreased yield of chromosome aberrations with respect to those in untreated cells was noted in normal cells irradiated in phase G 1 whereas no radioprotective effect was found in cells irradiated in G 0 . The addition of antioxydants into the cell cultures from patients with Turner's syndrome did not change their radiosensitivity. No adaptive response was induced in lymphocytes from patients with Down's syndrome cultivated with vitamine E

Identification of mononuclear cells in human blood. II. Evaluation of morphological and immunological aspects of native and formaldehyde-fixed cell populations

NARCIS (Netherlands)

Schuit, H.R.E.; Hijmans, W.

1980-01-01

The presence of surface-associated immunoglobulins and Fc receptors on mononuclear cells from normal human blood waas investigated by the direct immunofluorescence technique combined with phase-contrast microscopy. Formaldehyde-fixed cells were compared to unfixed cells and to cells preincubated at

Effects of ultraviolet irradiation on the cell cycle in normal and UV-sensitive cell lines with reference to the nature of the defect in xeroderma pigmentosum variant

International Nuclear Information System (INIS)

Imray, P.; Mangan, T.; Saul, A.; Kidson, C.

1983-01-01

Analysis of the distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry has been utilized to investigate the effects of ultraviolet (UV) irradiation on cell-cycle progression in normal and UV-sensitive lymphoblastoid cell lines. In time-course studies only slight perturbation of DNA distribution was seen in normal cells, or UV-sensitive familial melanoma (FM) lines in the 48 h following irradiation. Xeroderma pigmentosum (XPA) excision-deficient cells showed a large increase in the proportion of cells in S phase 16-40 h post-irradiation. XP variant (XPV) cells were blocked in G 1 and S phases with the complete absence of cells with G 2 DNA content 16-28 h after irradiation. By 48 h post-irradiation the DNA distribution of XPA and XPV cells had returned to that of an unirradiated control. When colcemid was added to the cultures immediately after irradiation to prevent mitotic cells dividing and re-entering the cell cycle, progression through the first cycle after irradiation was followed. UV irradiation did not affect the rate of movement of cells out of G 1 into S phase in normal, FM or XPA cells. The proportion of cells in S phase was increased in UV-irradiated cultures in these cell types and the number of cells entering the G 2 +M compartment was reduced. (orig./AJ)

Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

Energy Technology Data Exchange (ETDEWEB)

Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J. [and others

1995-12-01

In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

International Nuclear Information System (INIS)

Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

1995-01-01

In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to α-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMVΒ vector; and (2) the antibiotic hygromycin-resistant transfected cells

Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.

Science.gov (United States)

Ofran, Yanay; Schlessinger, Avner; Rost, Burkhard

2008-11-01

Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.

The toxicity of saffron (Crocus sativus L. and its constituents against normal and cancer cells

Directory of Open Access Journals (Sweden)

Alireza Milajerdi

2016-03-01

Conclusion: In conclusion, emerging evidence suggests that saffron extract and its crocin, crocetin and safranal have a selective toxicity effects against cancer cells and also may have cancer preventive functions. However, Saffron and its constituent's toxicity against normal cells is negligible and they are even non-toxic in oral administration.

Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

International Nuclear Information System (INIS)

Rosenberg, S.A.; Schwarz, S.; Anding, H.; Hyatt, C.; Williams, G.M.; Johns Hopkins Univ., Baltimore, Md.

1977-01-01

Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

Comparison of the suppressor cells found in the spleens of 89Sr-treated mice and in normal murine bone marrow

International Nuclear Information System (INIS)

Levy, E.M.; Corvese, J.S.; Bennett, M.

1981-01-01

Normal murine bone marrow cells and spleen cells of mice treated with 89 Sr both have suppressive activity. These nonspecific suppressor cells inhibit the ability of normal spleen cells to undergo antibody responses in vitro. After being precultured for 24 hr, these cells will also suppress antibody responses in vivo and the responses of normal spleen cells to T and B cell mitogens in vitro. These cells have previously been shown not to be mature T or B lymphocytes or macrophages. Velocity sedimentation and cell-size analysis indicated that both suppressor cells are large (approx. =206 μ 3 ). Mitomycin C treatment eliminated the ability of both suppressor cells to inhibit an in vitro antibody response. In contrast, this treatment did not reduce the ability of the cells to inhibit an in vitro antibody response. In contrast, this treatment did not reduce the ability of the cells to suppress a mitogenic response. Irradiation (1000 R) was also ineffective in eliminating the ability of either cell to suppress a mitogenic response. We conclude that the 2 suppressor cells are closely related if not identical, and we speculate that these cells may function in vivo to suppress immune reactivity in areas of intense hematopoiesis

Sustained beta-cell dysfunction but normalized islet mass in aged thrombospondin-1 deficient mice.

Directory of Open Access Journals (Sweden)

Carl Johan Drott

Full Text Available Pancreatic islet endothelial cells have in recent years been shown to support beta-cell mass and function by paracrine interactions. Recently, we identified an islets endothelial-specific glycoprotein, thrombospondin-1 (TSP-1, that showed to be of importance for islet angiogenesis and beta-cell function in young mice. The present study aimed to investigate long-term consequences for islet morphology and beta-cell function of TSP-1 deficiency. Islet and beta-cell mass were observed increased at 10-12 weeks of age in TSP-1 deficient mice, but were normalized before 16 weeks of age when compared to wild-type controls. Islet vascularity was normal in 10-12 and 16-week-old TSP-1 deficient animals, whereas islets of one-year-old animals lacking TSP-1 were hypervascular. Beta-cell dysfunction in TSP-1 deficient animals was present at similar magnitudes between 10-12 and 52 weeks of age, as evaluated by glucose tolerance tests. The insulin secretion capacity in vivo of islets in one-year-old TSP-1 deficient animals was only ∼15% of that in wild-type animals. Using a transplantation model, we reconstituted TSP-1 in adult TSP-deficient islets. In contrast to neonatal TSP-1 deficient islets that we previously reported to regain function after TSP-1 reconstitution, adult islets failed to recover. We conclude that TSP-1 deficiency in islets causes changing vascular and endocrine morphological alterations postnatally, but is coupled to a chronic beta-cell dysfunction. The beta-cell dysfunction induced by TSP-1 deficiency is irreversible if not substituted early in life.

DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas.

Science.gov (United States)

Erickson, L C; Bradley, M O; Ducore, J M; Ewig, R A; Kohn, K W

1980-01-01

Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was measured by alkaline elution in cells treated with four chloroethylnitrosoureas. Whereas VA-13 cells exhibited dose-dependent interstrand crosslinking, little or none was detected in IMR-90 cells. The IMR-90 cells, however, exhibited at least as much DNA-protein crosslinking as did VA-13 cells. The results can be interpreted in terms of a possible difference in DNA repair between the cell lines. PMID:6928639

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

Science.gov (United States)

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

DEFF Research Database (Denmark)

Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

2009-01-01

Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

Immune cells in the normal ovary and spontaneous ovarian tumors in the laying hen (Gallus domesticus) model of human ovarian cancer.

Science.gov (United States)

Bradaric, Michael J; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L; Yu, Yi; Abramowicz, Jacques S; Bahr, Janice M; Luborsky, Judith L

2013-01-01

Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia

DEFF Research Database (Denmark)

Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte

2013-01-01

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface...... a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA...

Nonlinear spectral imaging of human normal skin, basal cell carcinoma and squamous cell carcinoma based on two-photon excited fluorescence and second-harmonic generation

Science.gov (United States)

Xiong, S. Y.; Yang, J. G.; Zhuang, J.

2011-10-01

In this work, we use nonlinear spectral imaging based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) for analyzing the morphology of collagen and elastin and their biochemical variations in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and normal skin tissue. It was found in this work that there existed apparent differences among BCC, SCC and normal skin in terms of their thickness of the keratin and epithelial layers, their size of elastic fibers, as well as their distribution and spectral characteristics of collagen. These differences can potentially be used to distinguish BCC and SCC from normal skin, and to discriminate between BCC and SCC, as well as to evaluate treatment responses.

Comparison of Gated Audiovisual Speech Identification in Elderly Hearing Aid Users and Elderly Normal-Hearing Individuals: Effects of Adding Visual Cues to Auditory Speech Stimuli.

Science.gov (United States)

Moradi, Shahram; Lidestam, Björn; Rönnberg, Jerker

2016-06-17

The present study compared elderly hearing aid (EHA) users (n = 20) with elderly normal-hearing (ENH) listeners (n = 20) in terms of isolation points (IPs, the shortest time required for correct identification of a speech stimulus) and accuracy of audiovisual gated speech stimuli (consonants, words, and final words in highly and less predictable sentences) presented in silence. In addition, we compared the IPs of audiovisual speech stimuli from the present study with auditory ones extracted from a previous study, to determine the impact of the addition of visual cues. Both participant groups achieved ceiling levels in terms of accuracy in the audiovisual identification of gated speech stimuli; however, the EHA group needed longer IPs for the audiovisual identification of consonants and words. The benefit of adding visual cues to auditory speech stimuli was more evident in the EHA group, as audiovisual presentation significantly shortened the IPs for consonants, words, and final words in less predictable sentences; in the ENH group, audiovisual presentation only shortened the IPs for consonants and words. In conclusion, although the audiovisual benefit was greater for EHA group, this group had inferior performance compared with the ENH group in terms of IPs when supportive semantic context was lacking. Consequently, EHA users needed the initial part of the audiovisual speech signal to be longer than did their counterparts with normal hearing to reach the same level of accuracy in the absence of a semantic context. © The Author(s) 2016.

Detection of Apoptosis and Necrosis in Normal Human Lung Cells Using 1H NMR Spectroscopy

Science.gov (United States)

Shih, Chwen-Ming; Ko, Wun-Chang; Yang, Liang-Yo; Lin, Chien-Ju; Wu, Jui-Sheng; Lo, Tsui-Yun; Wang, Shwu-Huey; Chen, Chien-Tsu

2005-05-01

This study aimed to detect apoptosis and necrosis in MRC-5, a normal human lung cell line, by using noninvasive proton nuclear magnetic resonance (1H NMR). Live MRC-5 cells were processed first for 1H NMR spectroscopy; subsequently their types and the percentage of cell death were assessed on a flow cytometer. Cadmium (Cd) and mercury (Hg) induced apoptosis and necrosis in MRC-5 cells, respectively, as revealed by phosphatidylserine externalization on a flow cytometer. The spectral intensity ratio of methylene (CH2) resonance (at 1.3 ppm) to methyl (CH3) resonance (at 0.9 ppm) was directly proportional to the percentage of apoptosis and strongly and positively correlated with PI staining after Cd treatment (r2 = 0.9868, P In contrast, this ratio only increased slightly within 2-h Hg treatment, and longer Hg exposure failed to produce further increase. Following 2-h Hg exposure, the spectral intensity of choline resonance (at 3.2 ppm) was abolished, but this phenomenon was absent in Cd-induced apoptosis. These findings together demonstrate that 1H NMR is a novel tool with a quantitative potential to distinguish apoptosis from necrosis as early as the onset of cell death in normal human lung cells.

Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

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Ana Paula Santin Bertoni

2015-01-01

Full Text Available Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P<0.0001; 2.39 times, P=0.01; 1.58 times, P=0.0003; and 1.87 times, P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P<0.0001; 1.75 times, P=0.037; and 1.95 times, P<0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P=0.069. These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

Denotational Aspects of Untyped Normalization by Evaluation

DEFF Research Database (Denmark)

Filinski, Andrzej; Rohde, Henning Korsholm

2005-01-01

of soundness (the output term, if any, is in normal form and ß-equivalent to the input term); identification (ß-equivalent terms are mapped to the same result); and completeness (the function is defined for all terms that do have normal forms). We also show how the semantic construction enables a simple yet...... formal correctness proof for the normalization algorithm, expressed as a functional program in an ML-like, call-by-value language. Finally, we generalize the construction to produce an infinitary variant of normal forms, namely Böhm trees. We show that the three-part characterization of correctness...

High load of Merkel cell polyomavirus DNA detected in the normal skin of Japanese patients with Merkel cell carcinoma.

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Hashida, Yumiko; Nakajima, Kimiko; Nakajima, Hideki; Shiga, Takeo; Tanaka, Moe; Murakami, Masanao; Matsuzaki, Shigenobu; Naganuma, Seiji; Kuroda, Naoki; Seki, Yasutaka; Katano, Harutaka; Sano, Shigetoshi; Daibata, Masanori

2016-09-01

Although Merkel cell polyomavirus (MCPyV) has the potential to cause Merkel cell carcinoma (MCC), it is also found in the normal skin of healthy individuals. However, the mechanism for transformation of MCPyV to an oncogenic form is unknown. To investigate the levels of MCPyV infection in the normal skin patients with MCC compared with those in a control cohort. We studied a total of six Japanese patients with cutaneous MCC. Sun-exposed and sun-unexposed skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time polymerase chain reaction. At first, we found a patient with MCC carrying an extremely high load of MCPyV DNA in normal skin. This unique case prompted us to further explore the levels of MCPyV as skin microbiota in patients with MCC. We showed that MCPyV DNA levels were significantly higher in swabs obtained from normal skin samples of six patients with MCC compared with those from 30 age-matched healthy individuals and 19 patients with other cutaneous cancers. Whereas MCPyV strains obtained from the normal skin of patients with MCC had gene sequences without structural alterations, sequences of the tumor-derived strains showed truncating mutations or deletions. Although the number of patients with MCC studied was small, our findings suggest that MCC may occur with a background of high MCPyV load in the skin, and are expected to stimulate further studies on whether such skin virome levels could be one of predictive markers for the development of MCC. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein Biomarkers Associated With Growth And Synaptogenesis In a cell culture model of neuronal development

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Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens fo...

Heterogeneity in c-jun gene expression in normal and malignant cells exposed to either ionizing radiation or hydrogen peroxide

International Nuclear Information System (INIS)

Horio, M.; Collart, F.R.; Huberman, E.

1993-01-01

We investigated the role of reactive oxygen intermediates and protein kinase C (PKC) in induction of c-jun gene expression in human ML-2 leukemic cells and normal DET-551 fibroblasts by comparing the effects of either ionizing radiation or H 2 O 2 exposure in the presence or absence of appropriate inhibitors. In these cell types, the radiation and H 2 O 2 -mediated increase in c-jun mRNA levels could be prevented by pretreatment of the cells with N-acetylcysteine, an antioxidant, or H7, an inhibitor of PKC and cAMP-dependent protein kinase (PKA), but not by HA1004, an inhibitor of PKA. These results suggest a role for PKC and reactive oxygen intermediates in the induction of c-jun gene expression in both normal and tumor cells. We also investigated potential differences in radiation- or H 2 O 2 -induced c-jun gene expression in normal and tumor cells by examining steady-state c-jun mRNA levels in a number of human fibroblast, leukemia, melanoma, sarcoma, and carcinoma cell types. We observed heterogeneity in the steady-state level of c-jun mRNA in both the untreated normal and tumor cells and in such cells exposed to ionizing radiation or to H 2 O 2 . Exposure to radiation or to hydrogen peroxide produced a varied response which ranged from little or no induction to a more than two orders of magnitude increase in the steady-state level of the c-jun mRNA

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

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Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data

Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

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Pijanka, Jacek K; Stone, Nicholas; Rutter, Abigail V; Forsyth, Nicholas; Sockalingum, Ganesh D; Yang, Ying; Sulé-Suso, Josep

2013-09-07

Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

Libraries for spectrum identification: Method of normalized coordinates versus linear correlation

International Nuclear Information System (INIS)

Ferrero, A.; Lucena, P.; Herrera, R.G.; Dona, A.; Fernandez-Reyes, R.; Laserna, J.J.

2008-01-01

In this work it is proposed that an easy solution based directly on linear algebra in order to obtain the relation between a spectrum and a spectrum base. This solution is based on the algebraic determination of an unknown spectrum coordinates with respect to a spectral library base. The identification capacity comparison between this algebraic method and the linear correlation method has been shown using experimental spectra of polymers. Unlike the linear correlation (where the presence of impurities may decrease the discrimination capacity), this method allows to detect quantitatively the existence of a mixture of several substances in a sample and, consequently, to beer in mind impurities for improving the identification

Evaluation of the Cytotoxic Effects of CAM Therapies: An In Vitro Study in Normal Kidney Cell Lines

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Shagun Arora

2014-01-01

Full Text Available The purpose of this current study was to justify the incorporation of complementary and alternate medicine (CAM in current cancer treatments. The major drawback of anticancer drugs is their nonselective killing, which ultimately leads to attrition of normal cells. Keeping this as the foundation of our study, we made an effort to compare the cytotoxicity associated with a known chemotherapeutic drug 5-Fluorouracil (5-FU, with certain CAM therapies previously reported to have anticancer activity. The parameters chosen for the study were based on antiproliferative and cytotoxic effects on normal, kidney epithelial cells (NRK-52E. The MTT assay, colony formation assay, DNA fragmentation, and differential staining using AO/EB, following treatment with either 5-FU or CAM therapies, were performed. The CAM therapies under study were various extracts of wheatgrass, roots of Achyranthes aspera (AA, mushroom extracts (Pleurotus ostreatus, Macrolepiota procera, and Auricularia polytricha, and a homeopathic drug, Ruta graveolens (Ruta. The results showed that treatment of normal cells with the CAM therapies led to minimum cell damage in comparison to 5-FU. This evidence-based study will lead to greater acceptance of alternative therapies against cancer.

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

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Reddy, Jay P; Atkinson, Rachel L; Larson, Richard; Burks, Jared K; Smith, Daniel; Debeb, Bisrat G; Ruffell, Brian; Creighton, Chad J; Bambhroliya, Arvind; Reuben, James M; Van Laere, Steven J; Krishnamurthy, Savitri; Symmans, William F; Brewster, Abenaa M; Woodward, Wendy A

2018-06-01

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44 + CD49f + CD133/2 + mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44 + CD49f + CD133/2 + stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44 + CD49f + CD133/2 + cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

International Nuclear Information System (INIS)

McMahon, Laura W.; Zhang Pan; Sridharan, Deepa M.; Lefferts, Joel A.; Lambert, Muriel W.

2009-01-01

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.

Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis

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Sabo Edmond

2006-09-01

Full Text Available Abstract Background We have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5 in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We hypothesized that liver cell gene expression is highly sensitive to alterations in the amino acid microenvironment and that tumor cells may differ substantially in gene sets sensitive to amino acid availability. To assess the potential number and classes of hepatic genes sensitive to arginine availability at the RNA level and compare these between normal and tumor cells, we used an Affymetrix microarray approach, a paired in vitro model of normal rat hepatic cells and a tumorigenic derivative with triplicate independent replicates. Cells were exposed to arginine-deficient or control conditions for 18 hours in medium formulated to maintain differentiated function. Results Initial two-way analysis with a p-value of 0.05 identified 1419 genes in normal cells versus 2175 in tumor cells whose expression was altered in arginine-deficient conditions relative to controls, representing 9–14% of the rat genome. More stringent bioinformatic analysis with 9-way comparisons and a minimum of 2-fold variation narrowed this set to 56 arginine-responsive genes in normal liver cells and 162 in tumor cells. Approximately half the arginine-responsive genes in normal cells overlap with those in tumor cells. Of these, the majority was increased in expression and included multiple growth, survival, and stress-related genes. GADD45, TA1/LAT1, and caspases 11 and 12 were among this group. Previously known amino acid regulated genes were among the pool in both cell types. Available cDNA probes allowed independent validation of microarray data for multiple genes. Among genes downregulated under arginine-deficient conditions were multiple genes involved in cholesterol and fatty acid metabolism. Expression of low-density lipoprotein receptor was

Asymmetric cell division of stem cells in the lung and other systems

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Mohamed eBerika

2014-07-01

Full Text Available New insights have been added to identification, behavior and cellular properties of embryonic and tissue-specific stem cells over the last few years. The modes of stem cell division, asymmetric versus symmetric, are tightly regulated during development and regeneration. The proper choice of a stem cell to divide asymmetrically or symmetrically has great consequences for development and disease because inappropriate asymmetric division disrupts organ morphogenesis, whereas uncontrolled symmetric division induces tumorigenesis. Therefore, understanding the behavior of lung stem cells could identify innovative solutions for restoring normal morphogenesis and/or regeneration of different organs. In this concise review, we describe recent studies in our laboratory about the mode of division of lung epithelial stem cells. We also compare asymmetric cell division in the lung stem cells with other tissues in different organisms.

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells.

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Yim, Ji-Hye; Yun, Jung Mi; Kim, Ji Young; Nam, Seon Young; Kim, Cha Soon

2017-11-01

Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells. We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8 h after low-dose (0.05 Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting. Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8 h after low-dose radiation. Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.

Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

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Nandakumar, Vivek; Kelbauskas, Laimonas; Hernandez, Kathryn F; Lintecum, Kelly M; Senechal, Patti; Bussey, Kimberly J; Davies, Paul C W; Johnson, Roger H; Meldrum, Deirdre R

2012-01-01

Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria. We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure. We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At pfibrocystic from the metastatic cell populations. Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.

Identification of copy number variations and translocations in cancer cells from Hi-C data.

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Chakraborty, Abhijit; Ay, Ferhat

2017-10-18

Eukaryotic chromosomes adapt a complex and highly dynamic three-dimensional (3D) structure, which profoundly affects different cellular functions and outcomes including changes in epigenetic landscape and in gene expression. Making the scenario even more complex, cancer cells harbor chromosomal abnormalities (e.g., copy number variations (CNVs) and translocations) altering their genomes both at the sequence level and at the level of 3D organization. High-throughput chromosome conformation capture techniques (e.g., Hi-C), which are originally developed for decoding the 3D structure of the chromatin, provide a great opportunity to simultaneously identify the locations of genomic rearrangements and to investigate the 3D genome organization in cancer cells. Even though Hi-C data has been used for validating known rearrangements, computational methods that can distinguish rearrangement signals from the inherent biases of Hi-C data and from the actual 3D conformation of chromatin, and can precisely detect rearrangement locations de novo have been missing. In this work, we characterize how intra and inter-chromosomal Hi-C contacts are distributed for normal and rearranged chromosomes to devise a new set of algorithms (i) to identify genomic segments that correspond to CNV regions such as amplifications and deletions (HiCnv), (Nurtdinov et al.) to call inter-chromosomal translocations and their boundaries (HiCtrans) from Hi-C experiments, and (iii) to simulate Hi-C data from genomes with desired rearrangements and abnormalities (AveSim) in order to select optimal parameters for and to benchmark the accuracy of our methods. Our results on 10 different cancer cell lines with Hi-C data show that we identify a total number of 105 amplifications and 45 deletions together with 90 translocations, whereas we identify virtually no such events for two karyotypically normal cell lines. Our CNV predictions correlate very well with whole genome sequencing (WGS) data among chromosomes

Identification of Cell Cycle-Regulated Genes by Convolutional Neural Network.

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Liu, Chenglin; Cui, Peng; Huang, Tao

2017-01-01

The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype are analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight to the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Identification of Cell Surface Proteins as Potential Immunotherapy Targets in 12 Pediatric Cancers

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Orentas, Rimas J. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Yang, James J. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States); Wen, Xinyu; Wei, Jun S. [Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States); Mackall, Crystal L. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Khan, Javed, E-mail: rimas.orentas@nih.gov [Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States)

2012-12-17

Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

Attenuation of radiation-induced DNA damage due to paracrine interactions between normal human epithelial and stromal cells

International Nuclear Information System (INIS)

Saenko, V.A.; Nakazawa, Yu.; Rogounovitch, T.I.; Suzuki, K.; Mitsutake, N.; Matsuse, M.; Yamashita, S.

2007-01-01

Complete text of publication follows. Objective: Developmentally, every tissue accommodates different types of cells, such as epitheliocytes and stromal cells in parenchymal organs. To better understand the complexity of radiation response, it is necessary to evaluate possible cross-talk between different tissue components. This work was set out to investigate reciprocal influence of normal human epithelial cells and fibroblasts on the extent of radiation-induced DNA damage. Methods: Model cultures of primary human thyrocytes (PT), normal diploid fibroblasts (BJ), PT/BJ cell co-culture and conditioned medium transfer were used to examine DNA damage in terms of γ-H2AX foci number per cell or by Comet assay after exposure to different doses of γ-rays. Results: In co-cultures, the kinetics of γ-H2AX foci number change was dose-dependent and similar to that in individual PT and BJ cultures. The number of γ-H2AX foci in co-cultures was significantly lower (∼25%) in both types of cells comparing to individual cultures. Reciprocal conditioned medium transfer to individual counterpart cells prior to irradiation resulted in approximately 35% reduction in the number γ-H2AX foci at 1 Gy and lower doses in both PT and BJ demonstrating the role of paracrine soluble factors. Comet assay corroborated the results of γ-H2AX foci counting in conditioned medium transfer experiments. In contrast to medium conditioned on PT cells, conditioned medium collected from several human thyroid cancer cell lines failed to establish DNA-protected state in BJ fibroblasts. In its turn, medium conditioned on BJ cells did not change the extent of radiation-induced DNA damage in cancer cell lines tested. Conclusion: The results imply the existence of a network of soluble factor-mediated paracrine interactions between normal epithelial and stromal cells that could be a part of natural mechanism by which cells protect DNA from genotoxic stress.

ADAMTS-1 Is Found in the Nuclei of Normal and Tumoral Breast Cells.

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Suély V Silva

Full Text Available Proteins secreted in the extracellular matrix microenvironment (ECM by tumor cells are involved in cell adhesion, motility, intercellular communication and invasion. The tumor microenvironment is expansively modified and remodeled by proteases, resulting in important changes in both cell-cell and cell-ECM interactions and in the generation of new signals from the cell surface. Metalloproteinases belonging to the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs family have been implicated in tissue remodeling events observed in cancer development, growth and progression. Here we investigated the subcellular localization of ADAMTS-1 in normal-like (MCF10-A and tumoral (MCF7 and MDA-MB-231 human breast cells. ADAMTS-1 is a secreted protease found in the extracellular matrix. However, in this study we show for the first time that ADAMTS-1 is also present in the nuclei and nucleoli of the three mammary cell lines studied here. Our findings indicate that ADAMTS-1 has proteolytic functions in the nucleus through its interaction with aggrecan substrate.

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2.

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Hodson, Daniel J; Shaffer, Arthur L; Xiao, Wenming; Wright, George W; Schmitz, Roland; Phelan, James D; Yang, Yandan; Webster, Daniel E; Rui, Lixin; Kohlhammer, Holger; Nakagawa, Masao; Waldmann, Thomas A; Staudt, Louis M

2016-04-05

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.

In-vitro secretion of inhibin-like activity by Sertoli cells from normal and prenatally irradiated immature rats

International Nuclear Information System (INIS)

Ultee-van Gessel, A.M.; Leemborg, F.G.; Jong, F.H. de; Molen, H.J. van der

1986-01-01

The influence of in-vitro conditions on the production of inhibin by Sertoli cells from 21-day-old normal and prenatally irradiated rat testes was studied by measuring inhibin activity in culture media, using the suppression of the release of FSH from cultured rat pituitary cells. Sertoli cells secreted inhibin-like activity during at least 21 days of culture, and cells cultured at 37 0 C produced significantly more inhibin than those cultured at 32 0 C. The presence of fetal calf serum had no significant effect on inhibin production at 32 0 C, while at 37 0 C the production was decreased. The presence of ovine FSH stimulated inhibin secretion, while inhibin concentrations in Sertoli cell culture media were decreased after the addition of testosterone. Testosterone, added together with ovine FSH, suppressed inhibin secretion when compared with the levels found in the presence of FSH alone. The presence of spermatogenic cells decreased the release of inhibin. From these results it was concluded that both Sertoli cells isolated from normal immature rat testes and those from testes without spermatogenic cells can secrete inhibin-like activity in culture. A number of discrepancies with in-vivo observations was observed. (author)

[Study on detoxication of kansui radix on normal liver cells LO2 after stir-baking with vinegar].

Science.gov (United States)

Yan, Xiaojing; Zhang, Li; Li, Lin; Cao, Yudan; Li, Zhengjun; Tang, Yuping; Ding, Anwei

2012-06-01

To compare the toxicity on normal liver cells LO2 before and after Kansui Radix stir-baked with vinegar, and make a preliminary study on the mechanism of detoxication of Kansui Radix stir-baked with vinegar. The MTT method was adopted to detect the cell activity, with normal liver cells LO2 as the study object. The morphology of cells were observed, and the level or content of AST, ALT, LDH, SOD, Na+-K+-ATPase, Ca2+-Mg2+ -ATPase, GSH and MDA were determined in cell culture supernatant and splitting supernatant. Compared with the control group, Kansui can obviously inhibit the cell activity (P baked with vinegar can significantly decrease the cell proliferation inhibition and the trend of morphological variation, and obviously decrease the levels of ALT, AST, and LDH (P baking with rice vinegar can release the hepatotoxicity of Kansui Radix. Its possible mechanism was that Kansui Radix stir-baked with vinegar can decrease the influence of Kansui Radix on the permeability of liver cells LO2 membrane and oxidative damage, in order to provide basis for further exploration of the detoxication mechanism of Kansui Radix stir-baked with vinegar.

Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

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Manabu Watanabe

2014-09-01

Full Text Available Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line were used as a model. Single-cell capture was performed using laser capture microdissection (LCM with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells were subjected to whole genome amplification (WGA. For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100× were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100× were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

Insulin resistance according to β-cell function in women with polycystic ovary syndrome and normal glucose tolerance.

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Do Kyeong Song

Full Text Available Polycystic ovary syndrome (PCOS is associated with insulin resistance (IR and compensatory hyperinsulinemia. IR is recognized as a major risk factor for the development of type 2 diabetes mellitus. However, few studies have investigated IR in women with PCOS and normal glucose tolerance. The objective of this study was to evaluate IR and β-cell function in women with PCOS and normal glucose tolerance. Additionally, we sought to evaluate the usefulness of oral glucose tolerance test (OGTT-derived IR indices in lean women with PCOS.We recruited 100 women with PCOS and normal glucose tolerance and 100 age- and BMI-matched women as controls. IR and insulin secretory indices, including the homeostasis-model assessment (HOMA-IR, HOMA-M120, HOMA-F and the Stumvoll index, were calculated from an OGTT. Increased β-cell function was defined as>75th percentile for the HOMA-F in control women.Women with PCOS had higher values for post-load 2-hour glucose, fasting insulin, post-load 2-hour insulin, HOMA-IR, HOMA-M120, HOMA-F and lower values for the Stumvoll index than the controls (all Ps<0.05. Women with PCOS and increased β-cell function showed lower Stumvoll index values than the matched controls (P<0.05. The HOMA-F was significantly associated with the HOMA-M120 and Stumvoll index when adjusted for age and BMI in a multiple regression analysis (all Ps<0.05. The HOMA-M120 was positively correlated with triglycerides and free testosterone, and the Stumvoll index was negatively correlated with triglycerides and free testosterone in lean women with PCOS (all Ps<0.05.Women with PCOS and normal glucose tolerance showed higher IR than controls matched for age, BMI, and β-cell function. β-cell function was increased in women with PCOS when compared to the matched controls, but not when the lean subjects were compared to the matched controls separately. Therefore, early evaluation of IR in women with PCOS and normal glucose tolerance may be needed.

Normalized inverse characterization of sound absorbing rigid porous media.

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Zieliński, Tomasz G

2015-06-01

This paper presents a methodology for the inverse characterization of sound absorbing rigid porous media, based on standard measurements of the surface acoustic impedance of a porous sample. The model parameters need to be normalized to have a robust identification procedure which fits the model-predicted impedance curves with the measured ones. Such a normalization provides a substitute set of dimensionless (normalized) parameters unambiguously related to the original model parameters. Moreover, two scaling frequencies are introduced, however, they are not additional parameters and for different, yet reasonable, assumptions of their values, the identification procedure should eventually lead to the same solution. The proposed identification technique uses measured and computed impedance curves for a porous sample not only in the standard configuration, that is, set to the rigid termination piston in an impedance tube, but also with air gaps of known thicknesses between the sample and the piston. Therefore, all necessary analytical formulas for sound propagation in double-layered media are provided. The methodology is illustrated by one numerical test and by two examples based on the experimental measurements of the acoustic impedance and absorption of porous ceramic samples of different thicknesses and a sample of polyurethane foam.

HIV enteropathy: HAART reduces HIV-induced stem cell hyperproliferation and crypt hypertrophy to normal in jejunal mucosa.

Science.gov (United States)

Batman, Philip A; Kapembwa, Moses S; Belmonte, Liliana; Tudor, Gregory; Kotler, Donald P; Potten, Christopher S; Booth, Catherine; Cahn, Pedro; Griffin, George E