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Cell Cycle Analysis (cell + cycle_analysis)
Selected AbstractsDoes nicotine influence cytokine profile and subsequent cell cycling/apoptotic responses in inflammatory bowel disease?INFLAMMATORY BOWEL DISEASES, Issue 11 2008Marian C. Aldhous PhD Abstract Background: Smoking differentially influences susceptibility to the inflammatory bowel diseases (IBDs) Crohn's disease (CD) and ulcerative colitis (UC). We investigated the effects of nicotine on cytokine, cell cycle, and apoptotic responses in peripheral blood mononuclear cells (PBMCs) from IBD patients and healthy controls (HCs). Methods: PBMCs from IBD patients and HC were stimulated with lipopolysaccharide (LPS; 1 ,g/mL) or phytohemagglutinin (PHA, 5 or 0.5 ,g/mL), ± nicotine (1, 10, 100 ,g/mL). Cytokines (IL1,, IL2, IL10, IL12/IL23p40, TGF,, TNF,) were measured in supernatants at 24 hours. After 72 hours cells were analyzed by flow cytometry for cell cycle and apoptosis. Statistical modeling was used to identify interactions between cytokines and cell cycle / apoptosis and minimize confounding effects. Results: Stimulation by LPS and PHA (5 ,g/mL) increased IL12/IL23p40 production from CD and UC versus HC (P < 0.05); PHA (0.5 ,g/mL) increased IL1, in UC and decreased TGF, from CD and UC (P < 0.01). In all groups, nicotine reduced LPS- and PHA (0.5 ,g/mL)-stimulated production of IL1,, IL10, TGF,, and TNF, (P < 0.001). Cell cycle analysis showed that PHA, but not LPS, induced proliferation and decreased G0/G1 resting cells in CD and UC versus HC (P < 0.001). Nicotine decreased PHA-stimulated S-phase proliferation and increased G0/G1 resting cells (P < 0.01). Modeling showed independent associations between IL12/IL23p40 and apoptosis (P = 0.01), IL1, and resting cells (P = 0.006), TNF, and proliferating cells (P < 0.001). Disease activity and smoking habit had no effect. Conclusions: Dysregulated cytokine profiles in UC and CD are associated with specific alterations in cell cycle responses; these effects may be modified by nicotine, and potentially by anticytokine therapies. (Inflamm Bowel Dis 2008) [source] S100A6 (calcyclin) deficiency induces senescence-like changes in cell cycle, morphology and functional characteristics of mouse NIH 3T3 fibroblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010omnicki, ukasz P. S Abstract S100A6 (calcyclin) is a calcium binding protein with two EF-hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non-transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by ,-galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function. J. Cell. Biochem. 109: 576,584, 2010. © 2009 Wiley-Liss, Inc. [source] Effects of histone deacetylase inhibitors on p55CDC/Cdc20 expression in HT29 cell lineJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2006Giuseppe Iacomino Abstract In a previous work, taking advantage of the gene-array screening technology, we analysed the effects of histone deacetylase (HDAC) inhibitor sodium butyrate (NaBt), on gene transcription in HT29 human adenocarcinoma cell line. In this study, we focused our attention on p55CDC/Cdc20 gene, whose expression was dramatically reduced by NaBt treatment. Mammalian p55CDC/Cdc20 interacts with the anaphase promoting complex/cyclosome (APC/C), and is involved in regulating anaphase onset and late mitotic events. Using NaBt and trichostatin A (TSA), a member of the HDAC inhibitor family, we showed that both HDAC inhibitors totally downregulated p55CDC/Cdc20 transcription and expression. Cell cycle analysis demonstrated that NaBt arrested HT29 cells in G0/G1 phase, while TSA caused a double block in G0/G1 and G2/M phases. Moreover, p55CDC/Cdc20 showed maximal expression in S and G2/M phases of HT29 cell division cycle. Based on this evidence, and by means of specific cell cycle modulators, such as nocodazole and hydroxyurea, we demonstrated that both TSA and NaBt were responsible for loss of p55CDC/Cdc20 expression, but with different mechanisms of action. Taken together, these results suggest that targeting molecules involved in spindle mitotic checkpoint, such as p55CDC/Cdc20, might account for the high cytotoxicity of HDAC inhibitors versus malignant cells. J. Cell. Biochem. 99: 1122,1131, 2006. © 2006 Wiley-Liss, Inc. [source] Hydrogen sulfide inhibits cell proliferation and induces cell cycle arrest via an elevated p21Cip1 level in Ca9-22 cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2008H. Takeuchi Background and Objective:, Volatile sulfur compounds such as hydrogen sulfide (H2S) and methyl mercaptan (CH3SH) are the main causes of oral malodor. However, the physiological functions of H2S have not been investigated in oral tissues. The aim of this study was to evaluate the effect of H2S on cell proliferation and the cell cycle in oral epithelial-like cells. Material and Methods:, Ca9-22 cells were used in this study. Cells were cultured in 5% CO2/95% air with (5 or 10 ng/mL) or without H2S. DNA synthesis was measured using a 5-bromo-2-deoxyuridine enzyme-linked immunosorbent assay. The cell cycle was analyzed using a flow cytometer. The expressions of phosphorylated retinoblastoma protein (Rb), p21Cip1 and p27Kip1 were evaluated by western blotting. Results:, Exposure to 5 and 10 ng/mL of H2S significantly decreased DNA synthesis (p < 0.05). Cell cycle analysis also showed that exposure to both concentrations of H2S significantly increased the proportion of cells in G1 phase (p < 0.001) and significantly decreased the proportion of cells in S phase (p < 0.01). Western blotting showed that Rb phosphorylation was reduced and p21Cip1 was enhanced by exposure to H2S. Conclusion:, The results indicated that H2S inhibits cell proliferation and induces cell cycle arrest via the expression of p21Cip1 in Ca9-22 cells. [source] Inhibition of Aurora Kinase A enhances chemosensitivity of medulloblastoma cell lines,PEDIATRIC BLOOD & CANCER, Issue 1 2010Ayman El-Sheikh MD Abstract Background Medulloblastoma comprises approximately 20% of all primary pediatric brain tumors. Despite recent advances, the survival rate for high-risk patients and the morbidity associated with these treatments remains suboptimal. To improve outcomes and decrease morbidity, more targeted therapy is required. One possible target is the Aurora Kinase family. The objective of this study was to evaluate the impact of Aurora Kinase A inhibition in medulloblastoma cell lines. Procedure Cell proliferation was measured using an MTS assay after adding an Aurora Kinase inhibitor (C1368) at different concentrations. Cell cycle analysis was carried out by Flow Cytometry using propidium iodide (PI). RNAi experiments were performed using siRNA oligonucleotides. Luciferase experiments were carried out using the Cignal Finder 10 Pathway Reporter Arrays. Results Inhibition of Aurora Kinase A induces cell death in medulloblastoma cells and lowers the IC50 of other chemotherapeutic agents (etoposide and cisplatin) used in medulloblastoma treatment. Cell arrest at G2/M phase was significantly increased in medulloblastoma cell lines treated with C1368 Sigma at IC30 or transfected siRNA. Inhibition of Aurora Kinase A resulted in decreased activity of pro-proliferative signaling pathways including Wnt, Myc, and RB as measured by luciferase reporter assays. Conclusions These data indicate that inhibition of Aurora Kinase A inhibits cell growth in medulloblastoma through inhibition of pro-proliferative signaling pathways Wnt, Myc, and RB. Additionally, combining Aurora Kinase A inhibition with other chemotherapeutic agents significantly lowers their IC50, which make it a promising small molecule target for medulloblastoma therapy. Pediatr Blood Cancer 2010;55:35,41. © 2010 Wiley-Liss, Inc. [source] Pharmacological doses of dietary curcumin increase colon epithelial cell proliferation in vivo in ratsPHYTOTHERAPY RESEARCH, Issue 10 2007Sylvia Jeewon Kim Abstract Although curcumin has preventive actions in animal models of colon cancer, whether the mechanism of action is through anti-proliferation in normal environment is not clearly understood. Here, we studied the effects of chemopreventive doses of curcumin on the proliferation rate of colon epithelial cells (CEC), using a recently developed stable isotope , mass spectrometric method for measuring DNA synthesis rate. Adult male F344 rats were given diets containing 0, 2 and 4% curcumin for 5 weeks. 4% 2H2O was given in drinking water to label DNA, after a priming bolus, for 4 days prior to sacrifice. The isotopic enrichment of the deoxyribose moiety of deoxyadenosine from DNA was measured by gas chromatography , mass spectrometry. Cell cycle analysis was performed after propidium iodide staining of CECs. Curcumin administration did not reduce but instead resulted in dose-dependent increases in CEC proliferation rate (p < 0.05) for 2% and 4% curcumin vs 0%). The length of the colon crypts and the fraction of cells in S-phase were also increased in the 2% and 4% curcumin groups (p < 0.05). Thus, pharmacological doses of curcumin increase CEC proliferation rate and pool size in normal rats. Reduction of CEC proliferation therefore cannot explain the proposed chemopreventive actions of curcumin in colon cancer. Copyright © 2007 John Wiley & Sons, Ltd. [source] Rosiglitazone Inhibits Cell Proliferation by Inducing G1 Cell Cycle Arrest and Apoptosis in ADPKD Cyst-Lining Epithelia CellsBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2010Yawei Liu Many drugs inhibiting cell proliferation have been proved to be effective in slowing the disease progression in ADPKD. Recent evidence has suggested that peroxisome proliferator-activated receptor , (PPAR,) ligands have anti-neoplasm effects through inhibiting cell growth and inducing cell apoptosis in various cancer cells. In the present study, we examined the expression of PPAR, in human ADPKD kidney tissues and cyst-lining epithelial cell line, and found that the expression of PPAR, was greater in ADPKD kidney tissues and cyst-lining epithelial cell line than in normal kidney tissues and human kidney cortex (HKC) cell line. Rosiglitazone inhibited significantly proliferation of cyst-lining epithelial cells in a concentration- and time-dependent manner. These effects were diminished by GW9662, a specific PPAR, antagonist. Cell cycle analysis showed a G0/G1 arrest in human ADPKD cyst-lining epithelial cells with rosiglitazone treatment. Analysis of cell cycle regulatory proteins revealed that rosiglitazone decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, cyclin D2 and Cdk4 but increased the levels of p21 and p27 in a dose-dependent manner. Rosiglitazone also induced apoptosis in cyst-lining epithelial cells, which was correlated with increased bax expression and decreased bcl-2 expression. These results suggest PPAR, agonist might serve as a promising drug for the treatment of ADPKD. [source] No influence of magnetic fields on cell cycle progression using conditions relevant for patients during MRIBIOELECTROMAGNETICS, Issue 4 2003Ilka B. Schiffer Abstract The purpose of this study was to examine whether exposure to magnetic fields (MFs) relevant for magnetic resonance imaging (MRI) in clinical routine influences cell cycle progression in two tumor cell lines in vitro. HL60 and EA2 cells were exposed to four types of MFs: (i) static MF of 1.5 and 7.05 T, (ii) extremely low frequency magnetic gradient fields (ELFMGFs) with ±,10 mT/m and 100 Hz, as well as ±,100 mT/m and 100 Hz, (iii) pulsed high frequency MF in the radiofrequency (RF) range (63.6 MHz, 5.8 ,T), and (iv) a combination of (i,iii). Exposure periods ranged from 1 to 24 h. Cell cycle distribution (G0/G1, S, and G2/M phases) was analyzed by flow cytometry. Cell cycle analysis did not reveal differences between the exposed and the control cells. As expected, positive controls with irradiated (8 Gy) HL60 and EA2 cells showed a strong G2/M arrest. Using conditions that are relevant for patients during MRI, no influence of MFs on cell cycle progression was observed in these cell lines. Care was taken to control secondary parameters of influence, such as vibration by the MR scanner or temperature to avoid false positive results. Bioelectromagnetics 24:241-250, 2003. © 2003 Wiley-Liss, Inc. [source] Effects of Cloned Gene Dosage on the Response of Recombinant CHO Cells to Hyperosmotic Pressure in Regard to Cell Growth and Antibody ProductionBIOTECHNOLOGY PROGRESS, Issue 6 2001Joon Soo Ryu The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (,) and increased specific antibody productivity (qAb) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in qAb and less reduction in , at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and qAb, indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G1 -phase arrest, suggesting that the increase of cell population in G1 -phase may contribute in part to enhanced qAb at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in qAb at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage. [source] Cell cycle analysis and expression of cell cycle regulator genes in myeloma cells overexpressing cyclin D1BRITISH JOURNAL OF HAEMATOLOGY, Issue 3 2001Kenichiro Yata Among the recently discovered myeloma-specific gene alterations associated with chromosomal translocations, cyclin D1/PRAD1/Bcl-1 overexpression caused by t(11;14)(q13;q32) is considered to be the most frequent in myeloma patients and cell lines, and may be a prognostic factor clinically. To elucidate the cellular biological role of overexpressed cyclin D1 in myeloma cells, we examined the mRNA expression levels of cell cycle regulators including three cyclin Ds, cyclin-dependent kinase inhibitors (CDK-Is) and accelerators. Cyclin D1 overexpression was clearly demonstrated in the lines with abnormal 11q13 and associated with overexpression of S and G2 accelerator genes. The cyclin D1 -overexpressing lines tended to have a shortened G1 phase compared with the non-expressing lines. In addition, artificial silencing using antisense oligonucleotides for cyclin D1 suppressed the growth rate of some but not all cyclin D1 -overexpressing cells. These results indicate that overexpression of cyclin D1 caused by cytogenetic abnormalities may make cells progress through the cell cycle rapidly, but it seems that other factors such as cyclin D2 and translocation-related genes affect the cell cycle progression in myeloma cells. [source] Human ovarian carcinoma cells: Histone deacetylase inhibitors exhibit antiproliferative activity and potently induce apoptosisCANCER, Issue 12 2004Noriyuki Takai M.D., Ph.D. Abstract BACKGROUND Histone deacetylase inhibitors (HDACIs) can inhibit proliferation, stimulate apoptosis, and induce cell cycle arrest in malignant cells. METHODS The authors investigated the effects of four HDACIs on nine ovarian carcinoma cell lines in vitro and in vivo. Ovarian carcinoma cells were treated with a variety of HDACIs, and their effects on cell growth, the cell cycle, apoptosis, and related events were investigated. The ability of valproic acid (VPA) to inhibit the growth of ovarian tumors in immunodeficient mice was also assessed. RESULTS Clonogenic assays showed that all ovarian carcinoma cell lines were sensitive to the growth-inhibitory effects of the HDACIs. Cell cycle analysis indicated that their exposure to HDACIs decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases of the cell cycle. Terminal deoxynucleotidyltransferase-mediated uridine triphosphate end-labeling assays demonstrated that HDACIs induced apoptosis, which occurred in concert with alterations in the expression of genes related to apoptosis, cell growth, and malignant phenotype, including the activation of caspase-9 and caspase-3. Chromatin immunoprecipitation analysis revealed a notable increase in levels of acetylated histones associated with the p21 promoter after treatment with suberoylanilide bishydroxamine. In addition, in experiments involving nude mice, VPA significantly inhibited human ovarian tumor growth without toxic side effects. CONCLUSIONS The results of the current study suggest that HDACIs may be particularly effective in the treatment of ovarian tumors. Cancer 2004. © 2004 American Cancer Society. [source] Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferationCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2001Alison Colquhoun Abstract In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n -3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35,46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n -3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n -3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n -3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0,G1 phase cells and an increase in G2,M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n -3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA. Copyright © 2001 John Wiley & Sons, Ltd. [source] Ecology-based screen identifies new metabolites from a Cordyceps -colonizing fungus as cancer cell proliferation inhibitors and apoptosis inducersCELL PROLIFERATION, Issue 6 2009Y. Chen Objectives:, This study aims to identify new anti-cancer agents from Cordyceps -colonizing fungi, using an ecology-based approach. It also aims to explore their anti-cell proliferative mechanisms, and to evaluate their anti-tumour effects in vivo. Materials and methods:, Extracts from Cordyceps -colonizing fungi were tested on HeLa cells, and active extracts were separated to obtain anti-tumour metabolites; their structures were elucidated by mass and nuclear magnetic resonance spectroscopy. Cell cycle analysis was evaluated using flow cytometry. Tumour formation assays were performed using C57BL/6J mice. Results:, Based on ecological considerations, the selected extracts were subjected to initial anti-tumour screening. Bioassay-guided fractionation of the active extract afforded two new epipolythiodioxopiperazines, named gliocladicillins A (1) and B (2). (A) 1 and B (2) inhibited growth of HeLa, HepG2 and MCF-7 tumour cells. Further study demonstrated that both preparations arrested the cell cycle at G2/M phase in a dose-dependent manner, and induced apoptosis through up-regulation of expression of p53, p21, and cyclin B, and activation of caspases-8, -9 and -3. These data imply that gliocladicillins A (1) and B (2) induce tumour cell apoptosis through both extrinsic and intrinsic pathways. In addition, in vivo studies showed that they displayed significant inhibitory effects on cell population growth of melanoma B16 cells imlanted into immunodeficient mice. Conclusions:, Gliocladicillins A (1) and B (2) are effective anti-tumour agents in vitro and in vivo and should be further evaluated for their potential in clinical use. [source] Varying responses of human cells with discrepant p53 activity to ionizing radiation and heat shock exposureCELL PROLIFERATION, Issue 1 2007S. V. Tokalov Mutations of the p53 gene are observed at a high frequency in human tumours and are recognized in about half of all human cancers. Sensitivity to radiation, heat and anticancer agents has been observed in p53+/+ cells, but not in mutated or p53 -deficient cells. Moreover, enhancement of radiosensitivity by HS has been observed in wild-type p53 cells but not in p53 -deficient cells. The molecular mechanism of the differential cell response to HS or ionizing radiation is not yet understood. Materials and Methods: Differences in cellular response to radiation (200 kV X-ray, 1, 2, 5 Gy) and HS (39 °C, 41 °C and 43 °C for 30 min) on cell cycle progression of cultures of human p53 mutant cells were investigated by flow cytometry. In addition, the effects of stressors used on the expression of several heat shock genes (HSP27, HSP60, HSP70, HSC70, HSP75, HSP78, HSP90) were studied by reverse transcriptase,polymerase chain reaction. Results and Conclusions: Yet, with respect to HSP gene expression, different stressors produced similar effects. Combination of HS and radiation treatment significantly induced the transcription of the HSP70 gene above the level induced by each stressor alone. Cell cycle analysis, however, revealed striking differences in prolonged dynamics of cell division in response to each stressor. Thus, p53 status could be a useful indicator in predictive assays for hyperthermia cancer treatment in combination with radiation and/or chemotherapy. [source] DNA ploidy and cell cycle analyses in the bone marrow cells of patients with megaloblastic anemia using laser scanning cytometry,CYTOMETRY, Issue 2 2008Takayuki Tsujioka Abstract Background: Megaloblastic anemias are characterized by several hematopoietic cells with dysplastic nuclear morphology. The analyses of DNA ploidy and cell cycle of these cells are important to understand the property of such diseases. Methods: As laser scanning cytometry (LSC) is a useful tool to evaluate the morphology of the cells fixed on the slide glass together with the quantitative analysis of the fluorescence information of each cell by rapid scanning of the specimens, the authors examined the DNA ploidy and cell cycle of six cases with megaloblastic anemia using LSC. Results: Giant neutrophilic series such as giant metamyelocytes and giant band cells were found to have extraordinarily higher DNA ploidy, while hypersegmented neutrophils represented the normal diploid pattern like normal neutrophils. As to megaloblasts, cell cycle analysis showed that the proportion of the cells in S phase was increased as compared with the case of normal erythroblasts. Conclusions: The present study clearly demonstrates the abnormal aspects of the hematopoietic cells with megaloblastic anemia from the viewpoint of the DNA ploidy and cell cycle analyzed by the use of LSC. © 2007 Clinical Cytometry Society. [source] The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cellsLIVER INTERNATIONAL, Issue 1 2009Masaaki Arai Abstract Aims: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC). Methods: To isolate differentially expressed genes between HCC and adjacent non-cancerous liver tissues, cDNA microarray and quantitative reverse transcriptase polymerase chain reaction analyses were performed. Gene knockdown experiments in HepG2 cells were also performed using small interfering RNAs (siRNAs). Proteins were detected by immunostaining, and cell proliferation was analysed using the MTT/WST-8 assay. Apoptosis and cell cycle analyses were performed using flow cytometry. Results: After an intensive screening for differentially expressed genes in HCC tissues, we isolated 23 upregulated genes in these lesions. Among these, we focused on the replication factor C4 (RFC4) gene. The expression of endogenous RFC4 proteins in HepG2 cells was found to be significantly reduced by RFC4 -specific siRNA. This inhibition of RFC4 expression correlated with a decrease in cellular proliferation, increased levels of apoptosis and a sensitizing of the cells to the DNA-damaging chemotherapeutic agents, doxorubicin and camptothecin. Conclusion: The replication factor C4 gene may be a novel target for developing cancer therapeutics, which can enhance the antitumour activity of chemotherapeutic agents that induce DNA damage. [source] Monodemethylated polymethoxyflavones from sweet orange (Citrus sinensis) peel Inhibit growth of human lung cancer cells by apoptosisMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 3 2009Hang Xiao Abstract Polymethoxyflavones (PMFs) are almost exclusively found in the Citrus genus, particularly in the peels of sweet orange (Citrus sinensis L. Osbeck) and mandarin (C. reticulate Blanco). We studied the effects of two major PMFs, namely, nobiletin and 3,5,6,7,8,3,,4,-heptamethoxyflavone (HMF), and two major monodemethylated PMFs, namely 5-hydroxy-3,7,8,3,,4,-pentamethoxyflavone (5HPMF), and 5-hydroxy-3,6,7,8,3,,4,-hexamethoxyflavone (5HHMF), on the growth of human lung cancer H1299, H441, and H460 cells. Monodemethylated PMFs were much more potent in growth inhibition of lung cancer cells than their permethoxylated counterpart PMFs. In H1299 cells, cell cycle analyses further revealed that monodemethylated PMFs caused significant increase in sub-G0/G1 phase, suggesting possible role of apoptosis in the growth inhibition observed, whereas the permethoxylated counterpart PMFs did not affect cell cycle distribution at same concentrations tested. These results strongly suggested that the phenolic group is essential for the growth inhibitory activity of monodemethylated PMFs. Further studies in H1299 cells demonstrated that monodemethylated PMFs downregulated oncogenic proteins, such as iNOS, COX-2, Mcl-1, and K-ras, as well as induced apoptosis evidenced by activation of caspase-3 and cleavage of PARP. Our results provide rationale to develop orange peel extract enriched with monodemethylated PMFs into value-added nutraceutical products for cancer prevention. [source] Cell Cycle Arrest and Apoptosis Induction by an Anticancer Chalcone EpoxideARCHIV DER PHARMAZIE, Issue 8 2010Haiyong Han Abstract Safe and effective chemotherapeutic agents for the treatment of pancreatic cancer remain elusive. We found that chalcone epoxides (1,3-diaryl-2,3-epoxypropanones) inhibited growth in two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2. Three compounds were active, with GI50 values of 5.6 to 15.8,µM. Compound 4a, 1,3-bis-(3,4,5-trimethoxyphenyl)-2,3-epoxypropanone, had an average GI50 of 14.1,µM in the NCI 60-cell-line panel. To investigate the mode of action, cell cycle analyses of BxPC-3 cells were carried out. Treatment of cells with 50,µM 4a resulted in dramatic accumulation at G2/M (61% after 12,h for 4avs. 15% for untreated cells). The cells rapidly entered apoptosis. After 12,h, 26% of cells treated with 50,µM 4a had entered apoptosis vs. 4% for cells treated with 100,µM etoposide and 2% for untreated cells. Compound 4a interfered with paclitaxel enhancement of tubulin polymerization, suggesting microtubules as the site of action. Reaction of thiol nucleophiles with 4a under basic conditions resulted in epoxide ring-opening and retroaldol fragmentation, yielding alkylated thiol. MALDI mass spectrometry showed that retroaldol reaction occurred upon treatment of ,-tubulin with 4a. The site of alkylation was identified as Cys354. Chalcone epoxides warrant further study as potential agents for treatment of cancer. [source] DNA ploidy and cell cycle analyses in the bone marrow cells of patients with megaloblastic anemia using laser scanning cytometry,CYTOMETRY, Issue 2 2008Takayuki Tsujioka Abstract Background: Megaloblastic anemias are characterized by several hematopoietic cells with dysplastic nuclear morphology. The analyses of DNA ploidy and cell cycle of these cells are important to understand the property of such diseases. Methods: As laser scanning cytometry (LSC) is a useful tool to evaluate the morphology of the cells fixed on the slide glass together with the quantitative analysis of the fluorescence information of each cell by rapid scanning of the specimens, the authors examined the DNA ploidy and cell cycle of six cases with megaloblastic anemia using LSC. Results: Giant neutrophilic series such as giant metamyelocytes and giant band cells were found to have extraordinarily higher DNA ploidy, while hypersegmented neutrophils represented the normal diploid pattern like normal neutrophils. As to megaloblasts, cell cycle analysis showed that the proportion of the cells in S phase was increased as compared with the case of normal erythroblasts. Conclusions: The present study clearly demonstrates the abnormal aspects of the hematopoietic cells with megaloblastic anemia from the viewpoint of the DNA ploidy and cell cycle analyzed by the use of LSC. © 2007 Clinical Cytometry Society. [source] Viability study of HL60 cells in contact with commonly used microchip materialsELECTROPHORESIS, Issue 24 2006Floor Wolbers Abstract This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (Rap) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower Rap as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24,h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing. [source] BSc2118 is a novel proteasome inhibitor with activity against multiple myelomaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2010Jan Sterz Abstract Objectives:, The ubiquitin,proteasome system emerged as a new therapeutic target in cancer treatment. The purpose of this study was to elucidate the effects of the novel proteasome inhibitor BSc2118 on t(4;14) positive and negative multiple myeloma (MM) cells and normal peripheral blood mononuclear cells (PBMNC). Methods:, Human MM cell lines OPM-2, RPMI-8226, and U266 and primary MM cells from bone marrow aspirates were exposed to BSc2118. Cytotoxicity levels were evaluated using the MTT-test. BSc2118-induced apoptosis was analyzed by annexin-V assay. Further methods used included proteasomal activity determination, cell cycle analysis, western blot, and transcription factor assays. Results:, In OPM-2, RPMI-8226, U266 cell lines and primary MM cells, BSc2118 caused dose-dependent growth inhibitory effects. After 48 h, dose-dependent apoptosis occurred both in cell lines and primary myeloma cells irrespective of t(4;14). A significant G2-M cell cycle arrest occurred after 24 h. Furthermore, we observed a marked inhibition of intracellular proteasome activity, an increase in intracellular p21 levels, and an inhibition of NF-,B activation. The toxicity against PBMNC remained low, suggesting a broad therapeutic range of this agent. Conclusion:, Taken together, BSc2118 shows significant antimyeloma activity and may be considered as a promising agent in cancer drug development. [source] Pharmacological screening of bryophyte extracts that inhibit growth and induce abnormal phenotypes in human HeLa cancer cellsFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 4 2009Lucie Krzaczkowski Abstract Antitumor activities of substances from natural sources apart from vascular plants and micro-organisms have been poorly investigated. Here we report on a pharmacological screening of a bryophyte extract library using a phenotypic cell-based assay revealing microtubules, centrosomes and DNA. Among the 219 moss extracts tested, we identified 41 extracts acting on cell division with various combinations of significant effects on interphasic and mitotic cells. Seven extracts were further studied using a cell viability assay, cell cycle analysis and the phenotypic assay. Three distinct pharmacological patterns were identified including two unusual phenotypes. [source] Seliciclib (CYC202, R-roscovitine) enhances the antitumor effect of doxorubicin in vivo in a breast cancer xenograft modelINTERNATIONAL JOURNAL OF CANCER, Issue 2 2009Maria Virginia C.L. Appleyard Abstract We sought to determine whether seliciclib (CYC202, R-roscovitine) could increase the antitumor effects of doxorubicin, with no increase in toxicity, in an MCF7 breast cancer xenograft model. The efficacy of seliciclib combined with doxorubicin was compared with single agent doxorubicin or seliciclib administered to MCF7 cells and to nude mice bearing established MCF7 xenografts. Post-treatment cells and tumors were examined by cell cycle analysis, immunohistochemistry and real-time PCR. Seliciclib significantly enhanced the antitumor effect of doxorubicin without additional murine toxicity. MIB1 (ki67) immunohistochemistry demonstrated reduced proliferation with treatment. The levels of p21 and p27 increased after treatment with doxorubicin or seliciclib alone or in combination, compared to untreated controls. However, no changes in p53 protein (DO1, CM1), survivin or p53 phosphorylation (SER15) were observed in treated tumors compared with controls. In conclusion, the CDK inhibitor seliciclib (R-roscovitine) enhances the antitumor effect of doxorubicin in MCF7 tumors without increased toxicity with a mechanism that involves cell cycle arrest rather than apoptosis. © 2008 Wiley-Liss, Inc. [source] Relevance of a new rat model of osteoblastic metastases from prostate carcinoma for preclinical studies using zoledronic acidINTERNATIONAL JOURNAL OF CANCER, Issue 4 2008François Lamoureux Abstract Animal models that mimic osteoblastic metastases associated with prostate carcinoma are required to improve the therapeutic options in humans. A new model was then developed and characterized in immunocompetent rats. The bisphosphonate zoledronic acid (ZOL) was tested to validate this model as a therapeutic application. Rat AT6-1 prostate tumor cells were characterized in vitro at the transcriptional (bone and epithelial markers) and functional (induction of mineralized nodules) levels. The bone lesions induced after their direct injection into the femur bone marrow were characterized by radiography, microscanner and histology analyses. ZOL effects were studied in vivo on bone lesion development and in vitro on AT6-1 cell proliferation, apoptosis and cell cycle analysis. Apart from epithelial markers, AT6-1 cells express an osteoblast phenotype as they express osteoblastic markers and are able to induce mineralized nodule formation in vitro. A disorganization of the trabecular bone at the growth zone level was observed in vivo after intraosseous AT6-1 cell injection as well as cortical erosion. The tumor itself is associated with bone formation as revealed by SEM analysis and polarized light microscopy. ZOL prevents the development of such osteoblastic lesions, related to a direct inhibitory effect on tumor cell proliferation independent of caspase 3 activation, but associated with cell cycle arrest. A new rat model of osteoblastic bone metastases was validated in immunocompetent rats and used to show the relevance of using ZOL in such lesions, as this compound shows bifunctional effects on both bone remodelling and tumor cell proliferation. © 2007 Wiley-Liss, Inc. [source] The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-azacytidine in human bladder cancer therapyINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Jose A. Karam Abstract The long-term disease-free survival in patients with metastatic transitional cell carcinoma (TCC) is still considerably low. Novel chemotherapeutic agents are needed to decrease the morbidity and mortality of TCC. In this study, we have evaluated several epigenetic modifiers for their therapeutic application in bladder cancer. Both histone deacetylase inhibitors (FK228, TSA) and DNA hypomethylating agent (5-Azacytidine) were tested using in vitro assays such as cell viability, cell cycle analysis and western blot to determine their mechanisms of action. Drug combination experiments were also designed to study any additive or synergistic effects of these agents. In addition, two bladder cancer xenograft models (one subcutaneous and one orthotopic) were employed to assess the therapeutic efficacy of these agents in vivo. Three agents exhibited various growth inhibitory effects on 5 different TCC cell lines in a dose- and time-dependent manner. In addition to G2/M cell cycle arrest, FK228 is more potent in inducting apoptosis than the two other single agents, and combination of both FK228 and 5-Aza further enhances this effect. p21 induction is closely associated with FK228 or TSA but not 5-Aza, which is mediated via p53-independent pathway. Consistent with in vitro results, FK228 exhibited a significant in vivo growth inhibition of TCC tumor in both subcutaneous and orthotopic xenograft models. FK228 is a potent chemotherapeutic agent for TCC in vivo with minimal undesirable side effects. The elevated p21 level mediated via p53 independent pathway is a hallmark of FK228 mechanism of action. © 2007 Wiley-Liss, Inc. [source] Effects of adenoviral-mediated coexpression of bone morphogenetic protein-7 and insulin-like growth factor-1 on human periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2010L. Yang Yang L, Zhang Y, Dong R, Peng L, Liu X, Wang Y, Cheng X. Effects of adenoviral-mediated coexpression of bone morphogenetic protein-7 and insulin-like growth factor-1 on human periodontal ligament cells. J Periodont Res 2010; 45: 532,540. © 2010 John Wiley & Sons A/S Background and Objective:, Bone morphogenetic protein-7 (BMP-7) and insulin-like growth factor-1 (IGF-1) are important in periodontal reconstruction. However, their synergistic effect in periodontal regeneration by gene delivery has not been reported. In this study, gene delivery of these two growth factors to human periodontal ligament cells (hPDLCs) was examined for its effects on cell proliferation and differentiation. Material and Methods:, Recombinant adenoviruses containing both human BMP-7 and IGF-1 cDNA created by introducing the internal ribosome entry site (IRES) sequence were used to transfer the genes into hPDLCs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis were used to observe their effects on cell proliferation, while alkaline phosphatase activity measurement, RT-PCR and in vivo tests were conducted to investigate their effects on cell differentiation. Results:, The proliferation of hPDLCs transduced by adenoviruses coexpressing BMP-7 and IGF-1 was suppressed while their differentiation ability was enhanced. There was a synergism of BMP-7 and IGF-1 in up-regulating alkaline phosphatase activity and mRNA levels of collagen type I and Runx2. Implantation in vivo with scaffolds illustrated that the transduced cells exhibited osteogenic differentiation and formed bone-like structures. Conclusion:, The combined delivery of BMP-7 and IGF-1 genes using an IRES-based strategy synergistically enhanced differentiation of hPDLCs. It is suggested that this could be a new potential method in gene therapy for periodontal reconstruction. [source] Differential expression of periodontal ligament-specific markers and osteogenic differentiation in human papilloma virus 16-immortalized human gingival fibroblasts and periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2007S.-H. Pi Background and Objective:, Periodontal ligament cells and gingival fibroblasts are important in the remodeling of periodontal tissue, but human papilloma virus (HPV)16-immortalized cell lines derived from human periodontal ligament cells and gingival fibroblasts has not been characterized. The purpose of this study was to establish and differentially characterize the immortalized cell lines from gingival fibroblasts and periodontal ligament by HPV16 transfection. Material and Methods:, Cell growth, cell cycle analysis, western blot for cell cycle regulatory proteins and osteogenic differentiation markers, and reverse transcription,polymerase chain reaction for periodontal ligament-specific markers were performed. Results:, Both immortalized cell lines (immortalized gingival fibroblasts and immortalized periodontal ligament cells) grew faster than primary cultured gingival fibroblasts or periodontal ligament cells. Immortalized gingival fibroblasts and immortalized periodontal ligament cells overexpressed proteins p16 and p21, and exhibited degradation of proteins pRb and p53, which normally cause cell cycle arrest in G2/M-phase. Western blotting and reverse transcription,polymerase chain reaction for periodontal ligament-specific and osteogenic differentiation marker studies demonstrated that a cell line, designated IPDL, mimicked periodontal ligament gene expression for alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein, bone morphogenic protein-2, periostin, S-100A4 and PDLs17. Conclusion:, These results indicate that IPDL and immortalized gingival fibroblast cell lines consistently retain normal periodontal ligament and gingival fibroblast phenotypes, respectively, and periodontal ligament markers and osteogenic differentiation in IPDL are distinct from immortalized gingival fibroblast cells. [source] Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2003a Jantová Previous studies on the anticancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human tumour HeLa and murine leukemia L1210 cell lines. An attempt was also made to investigate the relationship between the cytotoxic activity of berberine and its molecular mechanism of action. Cytotoxicity was measured in-vitro using a primary biochemical screening according to Oyama and Eagle, and the growth inhibition assay. The in-vitro cytotoxic techniques were complemented by cell cycle analysis and determination of apoptotic DNA fragmentation in L1210 cells. Berberine acted cytotoxically on both tumour cell lines. The sensitivity of leukemia L1210 cells to the berberine was higher than that of HeLa cells. The IC100 was below 100 ,g mL,1 for HeLa cells and approached a 10 , mL,1 limit for the leukemia L1210 cells. For both cell lines the IC50 was found to be less than 4 ,g mL,1, a limit put forward by the National Cancer Institute (NCI) for classification of the compound as a potential anticancer drug. In L1210 cells treated with 10,50 , mL,1 berberine, G0/G1 cell cycle arrest was observed. Futhermore, a concentration-dependent decrease of cells in S phase and increase in G2/M phase was detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25,100 ,g mL,1 berberine-treated cells by monitoring the apoptotic DNA fragmentation (DNA ladder) using agarose gel electrophoresis. [source] Survivin mediates prostate cell protection by HIF-1, against zinc toxicityTHE PROSTATE, Issue 11 2010Young-Joo Yun Abstract BACKGROUND The prostate contains extremely high concentrations of zinc, but survives and grows without apparent injury. This begs the question as to how prostate cells avoid the toxic effects of zinc. In a previous study, the authors found that; HIF-1, is expressed concomitantly with the accumulation of zinc in the epithelial cells of normal rat prostates, the zinc ion stabilizes HIF-1, in prostate cells, and that HIF-1, protects prostate cells from zinc toxicity. In the present study, the authors addressed the mechanism responsible for the protective effect of HIF-1, in a high zinc environment. METHODS Immunofluorescent staining, immunoblotting, reverse transcription-polymerase chain reaction, reporter assay, and cell cycle analysis. RESULTS Survivin was induced by ZnCl2 in a HIF-1 dependent manner in both DU-145 and PNT2 prostate cells. Furthermore, HIF-1 induced survivin expression at the transcriptional level and the induction of survivin was abolished by HIF-1, knock-down. In addition, HIF-1-dependent survivin overexpression promoted prostrate cell survival and prevented cell arrest in the presence of high zinc concentrations, and si-survivin transfected cells under zinc rich conditions contained markedly higher levels of cleaved caspase-9 and PARP than si-con transfected cells. Finally, survivin expression patterns well matched rat prostate proliferation statuses. CONCLUSION Under zinc rich conditions, prostate epithelial cells HIF-1-dependently express survivin, which promotes prostate cell proliferation, and prevents apoptosis and cell cycle arrest. Accordingly, the HIF-1,-survivin pathway appears to facilitate prostate cell survival and growth in zinc rich environments, and this pathway could be a therapeutic target for the treatment of prostate hyperplasia. Prostate 70: 1179,1188, 2010. © 2010 Wiley-Liss, Inc. [source] Synthesis, in-vitro Cytotoxicity, and a Preliminary Structure-Activity Relationship Investigation of Pyrimido[4,5- c]quinolin-1(2H)-onesARCHIV DER PHARMAZIE, Issue 8 2010Kamel Metwally Abstract As part of our ongoing research effort to develop new antimitotic agents based on the recently reported pyrimido[4,5- c]quinoline-1(2H)-one ring skeleton, we were interested in identifying structural elements that contribute to the cytotoxicity of this class of compounds. The effect of several quinoline-ring substituents was examined and the new compounds were evaluated in vitro for cytotoxicity against three human cancer cell lines namely, lung fibrosarcoma HT-1080, colon adenocarcinoma HT-29, and breast carcinoma MDA-MB-231. Most of the compounds showed cytotoxic activity in the low micromolar and sub-micromolar range. Structure-activity relationship information revealed that a combination of electronic and steric factors may be involved. Flow cytometric cell cycle analysis performed on HT-1080 cells revealed that the most cytotoxic compounds 48, 50, 54, 59, and 63 inhibit the S-phase and arrest the cells in the G2/M phase of the cell cycle suggesting an antimitotic action of these compounds. [source] | |||||||||||||||||||||||||||||||