In this study, a new parameter, S phase cell percentage (S fraction) normalized BrdU (SFN-BrdU) incorporation rate, was introduced to detect S arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, SFN-BrdU incorporation rate can be used to detecting S arrest.
Bromodeoxyuridine ; pharmacokinetics ; Cell Proliferation ; DNA Damage ; Epithelial Cells ; cytology ; Humans ; S Phase ; S Phase Cell Cycle Checkpoints

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

PURPOSE: Retinonic acid (RA) has been reported to induce differentiation and growth inhibition in various head and neck squamous cancer cell (HNSCC) lines. We hypothesized that this growth inhi bition might be explained by RA-induced apoptosis on cell cycle arrest mechanism. Therefore, we studied the degree of RA-induced apoptosis with variable RA concentration and exposure duration. MATERIAL AND METHODS: The flow cytometric evaluation of apoptosis degree and cell cycles were carried out with 7-amino actinomycin D (7AAD) and propium iodide (PI) respectively, with var ious RA exposure durations (2, 3, 6 day) and concentrations (conrol, 10 6, 10 7, 10 8, 10 9, 10 10 mole). Two different HNSCC lines (1483, SqCC/Y1) were used and the experiment was repeated twice. RESULTS: The maximal fraction of apoptosis in 1483 and SqCC/Y1 cell lines were observed at same concentration and exposure duration (1483: 6th day & 10 6, mole, and SqCC/Y1: 6th day & 10 6 mole). In our experimental model, RA did not induce specific cell cycle arrest in these HNSCC lines. However we observed S phase fraction increase in SqCC/Y1 cell line after RA treatment. CONCLUSION: We suppossed that in HNSCC lines, RA-induced cell growth inhibition could be explained by not only RA-induced apoptosis but also cell cycle arrest. Futher, in vitro study has been carried out to elucidate the RA-iduced cell growth inhibition mechanism in our laboratory.
Apoptosis ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line* ; Dactinomycin ; Head* ; Models, Theoretical ; Neck* ; S Phase ; Tretinoin

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

OBJECTIVE: To determine whether Indole-3-carbinol (I3C) can enhance the inhibitory effect of genistein on a human uterine leiomyoma cells. METHODS: Five uterine leiomyoma tissues were obtained from hysterectomies conducted on the benign diseases and cultured primarily. MTS reduction assay was carried out to determine the viability of human uterine leiomyoma cells. Cell cycle analysis for I3C and genistein treated human uterine leiomyoma cells was done by Fluorescent activated cell sorter (FACS) analysis. To detect the presence and expression of cell cycle related proteins was done by Western blot analysis. RESULTS: I3C and genistein induced growth inhibition in a dose dependent manner, treatment with 100 micro mol/L I3C and 100 micro mol/L genisten blocked 60% cell growth. FACS results showed that treatment with the I3C and genistein increased the percentage of cells in G2/M phase and decreased S phase. From Western blot analysis it revealed I3C and genistein induced the expression of p53, p21, and p27 increasing. Reduced expression of cyclin B1 and cyclin E were detected in treatment with I3C and genistein. The expression levels of these proteins correlate with G2/M cell cycle arrest. Activation of caspase pathway and fragmentation of PARP did not take place. CONCLUSIONS: These results demonstrate that I3C enhances genistein-mediated uterine leiomyoma cell growth inhibition through the cell cycle arrest at G2/M phase by decreasing the production of cyclin B1. Because of the synergistic effect of I3C and genistein, the potential exists for the therapeutic efficacy of each phytochemical when used in combination.
Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cyclin B1 ; Cyclin E ; Cyclins ; Genistein* ; Humans* ; Hysterectomy ; Leiomyoma* ; S Phase

PURPOSE: Several studies have proven that EGCG, the primary green tea catechin, and glucosamine-6-phosphate (PGlc) reduce triglyceride contents in 3T3-L1 adipocytes. The objective of this study is to evaluate the combination effect of EGCG and PGlc on decline of accumulated fat in differentiated 3T3-L1 adipocytes. METHODS: EGCG and PGlc were administered for 6 day for differentiation of 3T3-L1 adipocytes. Cell viability was measured using the CCK assay kit. In addition, TG accumulation in culture 3T3-L1 adipocytes was investigated by Oil Red O staining. We examined the expression level of several genes and proteins associated with adipogenesis and lipolysis using real-time RT-PCR and Western blot analysis. A flow cytometer Calibar was used to assess the effect of EGCG and PGluco on cell-cycle progression of differentiating 3T3-L1 cells. RESULTS: Intracelluar lipid accumulation was significantly decreased by combination treatment with EGCG 60 microM and PGlc 200 microg/m compared with control and EGCG treatment alone. In addition, use of combination treatment resulted in directly decreased expression of PPARgamma, C/EBPalpha, and SREBP1. In addition, it inhibited adipocyte differentiation and adipogenesis through downstream regulation of adipogenic target genes such as FAS, ACSL1, and LPL, and the inhibitory action of EGCG and PGlc was found to inhibit the mitotic clonal expansion (MCE) process as evidenced by impaired cell cycle entry into S phase and the S to G2/M phase transition of confluent cells and levels of cell cycle regulating proteins such as cyclin A and CDK2. CONCLUSION: Combination treatment of EGCG and PGlc inhibit-ed adipocyte differentiation through decreased expression of genes related to adipogenesis and adipogenic and cell cycle arrest in early stage of adipocyte differentiation.
3T3-L1 Cells ; Adipocytes* ; Adipogenesis* ; Blotting, Western ; Catechin ; Cell Cycle Checkpoints* ; Cell Cycle* ; Cell Survival ; Cyclin A ; Lipolysis ; Phase Transition ; PPAR gamma ; S Phase ; Tea ; Triglycerides

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

OBJECTIVE: Isoliquritizenin (ISL) is a chalcone flavonoid, present in licorice, shallot and bean sprouts, has cancer preventing properties and often used in chinese medicine. In this study, ISL to determine its effect on cell proliferation and cell cycle progression in human cervical cancer cells were evaluated. METHODS: Cell viability assay was carried out to determine the viability of human cervical cancer cells. We tested the several experimental methods for verification and functional identification, including MTT assay, FACS analysis, DNA fragmentation assay, and Western blot analysis for ISL treated human cervical cancer cells (HeLa). RESULTS: ISL, induced growth inhibition in a dose dependent manner, treatment with 50 microM/L ISL blocked 50% cell growth. FACS results showed that there was no change in the S phase, but on the other hand ISL increased the percentage of cells in G1 phase. DNA fragmentation assay by ELISA was done to find the rate of apoptosis. Apoptosis took place but in a reduced manner. From Western blot analysis, it revealed ISL induced the expression of p21(Cip1/Waf1) and p27(kip1) but not mediated by p53. Caspase pathway was revealed and cleavage of PARP took place. CONCLUSION: ISL, a chalcone flavonoid, inhibited cell proliferation and induced cell cycle arrest at sub G1 by enhancing the production of p21(Cip1/Waf1) and p27(kip1). These results indicate that ISL will be a promising agent for use in chemopreventive or therapeutic against human cervical cancer cells.
Apoptosis* ; Asian Continental Ancestry Group ; Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cell Survival ; Chalcone ; DNA Fragmentation ; Enzyme-Linked Immunosorbent Assay ; G1 Phase ; Glycyrrhiza ; Hand ; Humans* ; S Phase ; Shallots ; Uterine Cervical Neoplasms*

BACKGROUND: Apoptosis is an important negative growth regulatory mechanism in tumors. In some malignancies, the apoptotic index(the percentage of apoptotic cells/bodies in the total number of tumor cells) may reflect the degree of carcinogeneity. In cells lacking functional p53, there is reduced susceptibility to apoptosis, thereby facilitating tumor growth. The bcl-2 gene product is a potent inhibitor of apoptosis and increases proliferation. The bcl-2/bax ratio is the critical determinant for the induction or inhibition of apoptosis. Proliferating cell nuclear antigen(PCNA) is present in nuclei throughout the cell cycle and is synthesized in the late G1 and S phases. Cyclin D1 is a major regulator of the G1 restriction point and may act as an oncogene; it is altered in several neoplasms. OBJECTIVE: Our purposes were to investigate the apoptotic index and the correlation between the apoptotic index and p53, bcl-2, bax, PCNA, and cyclin D1 in porokeratosis, actinic keratosis, and squamous cell carcinoma. METHODS: We investigated the apoptotic index by TUNEL and the expression of p53, bcl-2, bax, PCNA, and cyclin D1 by immunohistochemistry in 12 cases of porokeratosis, 18 cases of actinic keratosis, and 7 cases of squamous cell carcinomas. RESULTS: 1. The apoptotic index(%) in the epidermis central to the cornoid lamella was significantly higher than that of the peripheral epidermis in porokeratosis, 36.4+/-10.37 vs 24.4+/-8.76(p=0.004). 2. The apoptotic index(%) of actinic keratosis was significantly higher than that of porokeratosis, 37.4+/-7.73 vs 28.3+/-8.01(p=0.008). The apoptotic index(%) of squamous cell carcinoma was significantly higher than that of actinic keratosis, 45.1+/-6.18 vs 37.4+/-7.73(p=0.029). 3. p53 had significant positive correlation to the apoptotic index in porokeratosis and squamous cell carcinoma(p=0.002, 0.018). In actinic keratosis, the apoptotic index had significant positive correlation to cyclin D1(p=0.005). CONCLUSIONS: Actinic keratosis is more frequently evolved in malignant tumors than porokeratosis, which is supported by a significantly higher apoptotic index(%). Also the apoptotic index(%) of cutaneous malignant tumors was significantly higher than that of precancerous lesions. Apoptosis and p53, rather than proliferation, may provide the pathogenesis and progression into malignant tumors in porokeratosis. Apoptosis and cyclin D1 may provide the pathogenesis in actinic keratosis. In squamous cell carcinoma, p53-mediated apoptosis may be the key to pathogenesis in tumorigenesis and its proliferation.
Actins* ; Apoptosis* ; Carcinogenesis ; Carcinoma, Squamous Cell* ; Cell Cycle ; Cyclin D1* ; Cyclins* ; Epidermis ; G1 Phase Cell Cycle Checkpoints ; Genes, bcl-2 ; Immunohistochemistry ; In Situ Nick-End Labeling ; Keratosis, Actinic* ; Oncogenes ; Porokeratosis* ; Proliferating Cell Nuclear Antigen* ; S Phase

OBJECTIVETo investigate the effect of solanine on the growth of human prostate cancer cell xenograft in nude mice.

METHODSHuman prostate cancer Du145 cells were injected into the subcutaneous layers on the back of nude mice. After a week, the mice bearing subcutaneous tumor graft were randomly divided into solanine treatment group and saline control group for treatment for 3 weeks. The tumor grafts were then harvested to evaluate the inhibition rate. The mRNA and protein expressions of cell cycle-related genes in the tumors were detected by qRT-PCR and Western blotting, respectively, and tumor cell apoptosis was detected using TUNEL method.

RESULTSThe tumor growth rate in solanine-treated group was significantly slower than that in the control group (P<0.01). The mRNA and protein expressions of C-myc, cyclin D1, cyclin E1, CDK2, CDK4 and CDK6 were significantly inhibited by solanine. Solanine significantly up-regulated p21 mRNA and protein expression in the tumors and induced a higher apoptosis rate of the tumor cells than saline (P<0.01).

CONCLUSIONThe tumor-inhibition effect of solanine is probably mediated by regulating the expressions of genes related with G1/S cell cycle arrest and cell apoptosis.

Animals ; Apoptosis ; Cyclin-Dependent Kinases ; metabolism ; Cyclins ; metabolism ; G1 Phase Cell Cycle Checkpoints ; Humans ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; pathology ; Prostatic Neoplasms ; drug therapy ; pathology ; S Phase ; Solanine ; pharmacology