BACKGROUND: Type I collagen is a major component of extracellular cellular matrix in tissue. It is synthesized by human skin fibroblasts. However, synthesis of type I collagen is markedly-decreased in senescent fibroblasts. In the cell cycle, the hallmark of senescent fibroblasts is a permanent G1 phase arrest. However it is largely unknown whether the expression of type I collagen protein is decreased by the G1 phase arrest in human skin fibroblasts after UVB irradiation, serum starvation, or mimosine, an inducer of the G1 phase arrest treatment. OBJECTIVE: The purpose of this study was to investigate the expression of type I collagen protein in G1 phase- arrested human skin fibroblasts after UVB irradiation, serum starvation, and mimosine treatment. METHODS: To induce G1 phase arrest in the cell cycle, human skin fibroblasts were irradiated by UVB (100, 200, 300 mJ/cm(2)), subjected to serum starvation for 5 days, or mimosine treatment (50, 100, 200 uM). The expressions of type I collagen protein were analyzed by Western blot analysis. RESULTS: The G1 phase of cell populations were increased by a dose or time-dependent manner with UVB irradiation, serum starvation, and mimosine-treated human skin fibroblasts. The expression of type I collagen protein was markedly-decreased by UVB, serum starvation, and mimosine treatment. CONCLUSION: The expression of type I collagen protein in human skin fibroblasts is decreased by UVB irradiation, serum starvation, and mimosine treatment through induction of G1 phase cell cycle arrest.
Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Collagen Type I* ; Down-Regulation* ; Fibroblasts* ; G1 Phase ; G1 Phase Cell Cycle Checkpoints* ; Gene Expression* ; Humans* ; Mimosine ; Skin* ; Starvation

OBJECTIVE: We investigated the effects on the cell cycle and p53 expression by the treatment of various concentrations of staurosporine to elucidate the molecular mechanism of staurosporine induced cell cycle arrest in MCF-7 cell line. METHODS: Various concentrations of staurosporine were treated in MCF-7 cells cultured with RPMI 1640 media. The harvested cells were fixed and permeabilized with 1% paraformaldehyde and absolute methanol. Then the cells were stained indirectly with anti-p53 primary antibody and FITC conjugated goat anti-mouse(GAM)-IgG secondary antibody. Sequentially DNA were stained with 0.1% RNase and PI solution. These stained cells were analyzed by the standard FACScan flow cytometer. The obtained results were analyzed further with WinList 3.0, and ModiFit LT software program. RESULTS: MCF-7 cells were arrested mostly in G1 phase of cell cycle at 5-10 nM of staurosporine, however, the cells were arrested in G2 phase at 20-100 nM of staurosporine. The expressions of p53 protein were higher in the MCF-7 cells treated with both concentrations of 10 nM and 100 nM of staurosporine compaired with the control cells. This suggests that the p53 may be involved in the mechanism of G1 and G2M arrest of cell cycle in MCF-7 cell. CONCLUSIONS: The points of arrest in cell cycle differred depending on the concentrations of staurosporine and these cell cycle arrests at G0G1 and G2M pahse were related with p53 protein expression. It suggested that these results could be extended to study for staurosporine to be usefull as a potential anti-tumor agent.
Cell Cycle Checkpoints ; Cell Cycle* ; DNA ; Fluorescein-5-isothiocyanate ; G1 Phase ; G2 Phase ; Goats ; MCF-7 Cells* ; Methanol ; Ribonucleases ; Staurosporine*

OBJECTIVE: We compared the cell responsiveness of activated lymphocytes to rapamycin, which blocks the G1/S transition, between patients with Alzheimer's disease (AD) and normal controls to assess the early phase control defect in cell cycle. METHODS: Blood samples of 26 patients with AD and 28 normal controls were collected to separate peripheral lymphocytes. We measured the proportion of each cell cycle phase in activated lymphocytes using flow cytometry and evaluated the responsiveness of these lymphocytes to rapamycin. RESULTS: The patients with AD were older than the normal controls (AD 74.03+/-7.90 yr vs. control 68.28+/-6.21 yr, p=0.004). The proportion of G1 phase cells in the AD group was significantly lower than that in the control group (70.29+/-6.32% vs. 76.03+/-9.05%, p=0.01), and the proportion of S phase cells in the AD group was higher than that in control group (12.45+/-6.09% vs. 6.03+/-5.11%, p=0.001). Activated lymphocytes in patients with AD were not arrested in the G1 phase and they progressed to the late phase of the cell cycle despite rapamycin treatment, in contrast to those of normal subjects. CONCLUSION: The patients with AD probably have a control defect of early phase cell cycle in peripheral lymphocytes that may be associated with the underlying pathology of neuronal death.
Alzheimer Disease ; Cell Cycle ; Cell Cycle Checkpoints ; Flow Cytometry ; G1 Phase ; Humans ; Lymphocytes ; Neurons ; S Phase ; Sirolimus

OBJECTIVE: To examine the effect of resveratrol on cell proliferation and cell cycle progression in the human uterine leiomyoma cells. METHODS: MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay was carried out to determine the viability of human uterine leiomyoma cells. Western blot analysis was done using anti pRB, anti-p21cip1/waf1, anti-p53, anti-cyclin E, anti CDK2 antibodies to detect the presence and expression of these proteins in treatment with resveratrol. DNA fragmentation assay was done to find the rate of apoptosis. Cell cycle analysis for resveratrol treated in human uterine leiomyoma cells was done by FACS (fluorescence-activated cell sorter) analysis. RESULTS: Resveratrol induced growth inhibition in a dose dependent manner, treatment with 100 ?M/L resveratrol blocked 30% cell growth. From Western blot analysis it revealed resveratrol induced the expression of p53 increasing. Caspase pathway was activated and cleavage of PARP was occurred. Apoptosis took place but in a reduced manner. FACS results showed that resveratrol increased the percentage of cells in sub G1 phase. CONCLUSION: Resveratrol, a dietry phytoalexin, inhibited cell proliferation and induced cell cycle arrest at sub G1 by enhancing the production of p53. These results indicate that resveratrol will be a promising agent chemopreventives or therapeutics against human uterine leiomyoma cells.
Antibodies ; Apoptosis ; Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; DNA Fragmentation ; G1 Phase ; Humans ; Leiomyoma*

Gingival fibroblasts are major cellular component of gingiva. However, the molecular mechanisms of senescence of human gingival fibroblasts are unknown. Human fibroblasts undergo replicative senescence in vitro after a limited number of population doublings. A reduced rate of proliferation is a prominent phenomenon observed in senescent fibroblasts. This phenomenon is happened with cell cycle arrest that was controled by cell cycle regulatory proteins. The purpose of present study was to investigate the effect of replicative senescence on cell cycle progression and to find out its molecular mechanisms in human gingival fibroblasts. Replicative senescence of gingival fibroblasts were induced by subsequent cultures that were repeated up to 18 passage. In the present study, I examined change of cell proliferation, cell activity, cell viability and cell cycle progression during the replicative process. Also, I examined expression of cell cycle regulatory proteins which was estimated by western blot analysis. Cell proliferation, cell activity and cell viability of gingival fibroblasts were notably decreased with increase of population doubling level(PDL). S phase was decreased and G1 phase was increased with increase of PDL. Western blot analysis showed that levels of p16, p21 and p53 of senescent gingival fibroblasts(PDL41, PDL58) were higher than young fibroblasts(PDL27) and cdk4 were lower than young fibroblasts(PDL27). In conclusion, these results suggest that proliferative function of human gingival fibroblasts may be decreased by replicative senescence and its molecular mechanisms may be activatied with p16, p21, p53 and pRB, and repressed wtih cdk4.
Aging ; Blotting, Western ; Cell Aging* ; Cell Cycle Checkpoints ; Cell Cycle Proteins ; Cell Cycle* ; Cell Proliferation ; Cell Survival ; Fibroblasts* ; G1 Phase ; Gingiva ; Humans* ; S Phase

BACKGROUND/OBJECTIVES: Inonotus obliquus (I. obliquus, Chaga mushroom) has long been used as a folk medicine to treat cancer. In the present study, we examined whether or not ethanol extract of I. obliquus (EEIO) inhibits cell cycle progression in HT-29 human colon cancer cells, in addition to its mechanism of action. MATERIALS/METHODS: To examine the effects of Inonotus obliquus on the cell cycle progression and the molecular mechanism in colon cancer cells, HT-29 human colon cancer cells were cultured in the presence of 2.5 - 10 microg/mL of EEIO, and analyzed the cell cycle arrest by flow cytometry and the cell cycle controlling protein expression by Western blotting. RESULTS: Treatment cells with 2.5 - 10 microg/mL of EEIO reduced viable HT-29 cell numbers and DNA synthesis, increased the percentage of cells in G1 phase, decreased protein expression of CDK2, CDK4, and cyclin D1, increased expression of p21, p27, and p53, and inhibited phosphorylation of Rb and E2F1 expression. Among I. obliquus fractions, fraction 2 (fractionated by dichloromethane from EEIO) showed the same effect as EEIO treatment on cell proliferation and cell cycle-related protein levels. CONCLUSIONS: These results demonstrate that fraction 2 is the major fraction that induces G1 arrest and inhibits cell proliferation, suggesting I. obliquus could be used as a natural anti-cancer ingredient in the food and/or pharmaceutical industry.
Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Colonic Neoplasms* ; Cyclin D1 ; DNA ; Drug Industry ; Ethanol* ; Flow Cytometry ; G1 Phase ; G1 Phase Cell Cycle Checkpoints* ; HT29 Cells ; Humans ; Medicine, Traditional ; Methylene Chloride ; Phosphorylation

BACKGROUND: In spite of the clinical introduction of brachytherapy to reduce restenosis, the biologic responses of vascular smooth muscle cells(VSMCs) to radiation have not been well studied. We investigated the effects and mechanisms of gamma-irradiation on the cell cycle of VSMCs using primary cultures of rat aortic VSMCs and 137Cs as a radiation source. METHODS & RESULTS: The cell counts after irradiation with 0, 2, 8, 16 Gray (Gy) (n=, each) were 3.28, 2.34, 1.94 and 1.30 x 105/ml at 24h, and 5.10, 2.00, 1.80 and 1.20 x 105/ml at 48h, respectively. The proportions of cells in the G0/G1, S and G2/M phases, as measured by Fluorescence Activated Cell Sorter, were 61, 9 and 30% at 12 hours after 16Gy radiation (control 61, 34 and 5%), 65, 9 and 26% at 24 hours (control 70, 16 and 14%); and 67, 7 and 26% (control 78, 12 and 10%) at 48 hours, which demonstrated G1 and G2 arrest. By immunoblot analysis and kinase assay, gamma-irradiation with 8 or 16 Gy increased the expression of p21, universal cell cycle inhibitor, and decreased the expression and activity of CDK2, an important kinase during the later stages of G1/S progression, as well as the expression and activity of CDK1, which is important in the G2/M phase transition. In contrast, radiation did not affect the expression or activity of either CDK4 or CDK6. The cell-cycle inhibitors, p27 and p16 were not involved in the radiation-induced cell cycle arrest of VSMCs. CONCLUSION: Gamma-irradiation can effectively inhibit VSMC proliferation because it causes cell cycle arrest at both the G1 phase by enhancing P21 expression and suppressing CDK2, and at the G2/M phase by suppressing CDK1.
Animals ; Brachytherapy ; Cell Count ; Cell Cycle ; Cell Cycle Checkpoints ; Cyclin-Dependent Kinases* ; Fluorescence ; G1 Phase ; Muscle, Smooth, Vascular* ; Phase Transition ; Phosphotransferases ; Rats

PURPOSE: Cyclins are groups of proteins that play a role as a major regulator of the G1 restriction point promoting inactivation of the retinoblastoma protein. The cyclin D1 gene, CCND1, is amplified in approximately 20% of breast carcinomas and the protein is reportedly overexpressed in 60~80% of all cases. Cyclin D1 overexpression was strongly correlated to estrogen receptor positivity and better histologic grade in breast cancer. The aim of this study was to correlate cyclin D1 overexpression using a well characterized antibody with estrogen receptor status and other clincopathologic parameters. METHODS: From March 1989 to December 1994, 85 patients with primary breast carcinoma were the subject in this study. We analyzed cyclin D1 expression by immnohistochemical staining using cyclin D1 antibody, cells were considered positive according to distinct nuclear staining. The correlation between cyclin D1 expression was compared with important clinicopathologic parameters (tumor size, axillary lymph node status, p53 expression, c-erbB2 expression, histologic grade, estrogen receptor status). RESULTS: Cyclin D1 expression was detected in 37 cases (43.5%). Cyclin D1 expression was high in patients with tumors that expressed estrogen receptor (58.5% vs 26.5%, P=0.019). Cyclin D1 was mainly overexpressed in the histologic grade I and II (75.0%), as compared with 65.2% in cyclin D1 negative tumor, however there was no statistical significance (P=0.067). There were no significant correlation with tumor size, axillary lymph node status, p53 expression, or c-erbB2 expression (P>0.05). CONCLUSION: Cyclin D1 expression in estrogen receptor (ER) positive patients was significantly higher than that seen in ER negative patients. There was a negative correlation between cyclin D1 and tumor histologic grade, however it was not statistically significant. Tumor size, axillary lymph node status, p53 expression, and c-erbB2 expression were not correlated with cyclin D1.
Breast Neoplasms* ; Breast* ; Cyclin D1* ; Cyclins* ; Estrogens* ; G1 Phase Cell Cycle Checkpoints ; Genes, bcl-1 ; Humans ; Lymph Nodes ; Retinoblastoma Protein

PURPOSE: Cyclins are groups of proteins that play a role as a major regulator of the G1 restriction point promoting inactivation of the retinoblastoma protein. The cyclin D1 gene, CCND1, is amplified in approximately 20% of breast carcinomas and the protein is reportedly overexpressed in 60~80% of all cases. Cyclin D1 overexpression was strongly correlated to estrogen receptor positivity and better histologic grade in breast cancer. The aim of this study was to correlate cyclin D1 overexpression using a well characterized antibody with estrogen receptor status and other clincopathologic parameters. METHODS: From March 1989 to December 1994, 85 patients with primary breast carcinoma were the subject in this study. We analyzed cyclin D1 expression by immnohistochemical staining using cyclin D1 antibody, cells were considered positive according to distinct nuclear staining. The correlation between cyclin D1 expression was compared with important clinicopathologic parameters (tumor size, axillary lymph node status, p53 expression, c-erbB2 expression, histologic grade, estrogen receptor status). RESULTS: Cyclin D1 expression was detected in 37 cases (43.5%). Cyclin D1 expression was high in patients with tumors that expressed estrogen receptor (58.5% vs 26.5%, P=0.019). Cyclin D1 was mainly overexpressed in the histologic grade I and II (75.0%), as compared with 65.2% in cyclin D1 negative tumor, however there was no statistical significance (P=0.067). There were no significant correlation with tumor size, axillary lymph node status, p53 expression, or c-erbB2 expression (P>0.05). CONCLUSION: Cyclin D1 expression in estrogen receptor (ER) positive patients was significantly higher than that seen in ER negative patients. There was a negative correlation between cyclin D1 and tumor histologic grade, however it was not statistically significant. Tumor size, axillary lymph node status, p53 expression, and c-erbB2 expression were not correlated with cyclin D1.
Breast Neoplasms* ; Breast* ; Cyclin D1* ; Cyclins* ; Estrogens* ; G1 Phase Cell Cycle Checkpoints ; Genes, bcl-1 ; Humans ; Lymph Nodes ; Retinoblastoma Protein

OBJECTIVES AND BACKGROUND: In head and neck cancer including hypopharyngeal carcinoma, cisplatin and 5-fluorouracil usually have been used as neoadjuvant chemotherapeutic agents. We investigated the difference in the influences of cisplatin and 5-fluorouracil (5-FU) on the p53 protein expression and cell responses (cell cycle arrest and apoptosis) in the hypopharyngeal cell line (PHUH-12). METHOD: PNUH-12 with a mutant type p53 (one point mutation at the 78th base, C to G, in exon 7) was treated with cisplatin and 5-FU. Changes in the cell line were assessed by MTT assay, Western blotting (p53 and p21 protein), DNA fragmentation, PI stain, and DNA flow cytometry. RESULTS: The p53 protein expression was increased after the treatment with cisplatin and 5-FU. The expression of p21 protein was increased after the treatment with 5-FU, not cisplatin. With cisplatin, we observed apoptosis by DNA fragmentation and PI stain and the increased S phase on DNA flow cytometry. But, with 5-FU, we couldn't observe apoptosis by DNA fragmentation, PI, and flow cytometry and only the increased G1 phase on DNA flow cytometry. CONCLUSION: In hypopharyngeal cell line (PNUH-12), cisplatin induced p53 dependent apoptosis and 5-FU induced p53 and p21 dependent G0/G1 cell cycle arrest, but not apoptosis.
Apoptosis ; Blotting, Western ; Cell Cycle Checkpoints ; Cell Line ; Cisplatin* ; DNA ; DNA Fragmentation ; Exons ; Flow Cytometry ; Fluorouracil* ; G1 Phase ; Head and Neck Neoplasms ; Hypopharyngeal Neoplasms ; Point Mutation ; S Phase