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*

OBJECTIVETo investigate the expression of long non-coding RNA-HOTAIR in prostate cancer cells and its effects on the growth and metastasis of the cells.

METHODSUsing quantitative reverse-transcription PCR (qRT-PCR), we determined the relative expression of HOTAIR in the normal human prostate epithelial cell line RWPE-I and prostate cancer cell lines PC-3 and DU145. We detected the effects of HOTAIR on the cell cycle and invasiveness of prostate cancer cells by RNA interference, flow cytometry, and Transwell mitration assay.

RESULTSThe expressions of HOTAIR in the PC3 and DU145 cells were increased 3.2 and 5.7 times, respectively, as compared with that in the normal RWPE-1 cells. After si-HOTAIR interference, the prostate cancer cells were arrested in the G2 phase and downregulated in the G1 phase. The invasive ability of the prostate cancer cells was evidently inhibited, with the inhibition rates of 32% and 44% of the PC3 cells and 43% and 34% of the DU145 cells for si-HOTAIR1 and si-HOTAIR2, respectively.

CONCLUSIONIncRNA HOTAIR is highly expressed in prostate cancer, which is associated with the growth and invasiveness of prostate cancer cells. HOTAIR is potentially a novel marker for the diagnosis and prognosis of prostate cancer.

Cell Cycle ; Cell Cycle Checkpoints ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; G1 Phase ; G2 Phase ; Humans ; Male ; Neoplasm Invasiveness ; Prognosis ; Prostatic Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Long Noncoding ; metabolism ; RNA, Untranslated ; metabolism

Human papilloma virus 16 E6 and E7 oncoproteins are well known to change cell functions, especially through p53 and pRb expression, so we studied their effects on molecular mechanisms and on the cell death associated with hypoxia and ionizing radiation. These treatments both caused cell death and increased p53 protein expression in HepG2 cells. This increased p53 expression by gamma-irradiation under hypoxia induced G1 cell cycle arrest and led to apoptosis even though HepG2 cells have a relatively reduced ability to induce p21 and pRb expression levels. Ablation of p53 expression by the HPV 16 E6 gene induced E2F-1 expression, which plays a role in cellular survival, especially under hypoxia or gamma-irradiation. The steady-state level of p53 action produced by HPV 16 E7 did not induce apoptotic cell death or the production of the apoptotic regulators, the bcl-2 family and caspase-3, so it did not appear to participate in apoptotic signaling in response to hypoxia and ionizing radiation. Thus, the HPV 16 E7 oncoprotein did not increase the rate of cell death induced by p53, although p53 might play a role in apoptosis in HepG2 cells.
Anoxia* ; Apoptosis* ; Caspase 3 ; Cell Death ; G1 Phase Cell Cycle Checkpoints ; Hep G2 Cells* ; Human papillomavirus 16 ; Humans* ; Oncogene Proteins ; Papilloma* ; Radiation, Ionizing

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

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

PURPOSE: Isocryptotanshinone (ICTS) is a natural bioactive product that is isolated from the roots of the widely used medical herb Salvia miltiorrhiza. However, few reports exist on the mechanisms underlying the therapeutic effects of ICTS. Here, we report that ICTS has anticancer activity and describe the mechanism underlying this effect. METHODS: The antiproliferative effect of ICTS was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and clonogenic assays. The effect of ICTS on the cell cycle was measured using flow cytometry. Apoptosis was determined by Hoechst 33342 staining, DNA fragmentation assays, and Western blotting for apoptotic proteins. Finally, the effect of ICTS on mitogen-activated protein kinases (MAPKs) was determined by Western blotting. RESULTS: ICTS significantly inhibited proliferation of MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 human liver cancer cells, and A549 human lung cancer cells in vitro. Among the tested cell lines, MCF-7 cells showed the highest sensitivity to ICTS. ICTS significantly inhibited colony formation by MCF-7 cells. Furthermore, exposure of MCF-7 cells to ICTS induced cell cycle arrest at the G1 phase and decreased mitochondrial membrane potential. Hoechst 33342 staining and Western blot analysis for apoptotic proteins suggested that ICTS induced apoptosis in MCF-7 cells. In addition, ICTS activated MAPK signaling in MCF-7 cells by inducing time- and concentration-dependent phosphorylation of JNK, ERK, and p38 MAPK. CONCLUSION: Our results suggest that ICTS inhibited MCF-7 cell proliferation by inducing apoptosis and activating MAPK signaling pathways.
Apoptosis* ; Blotting, Western ; Breast Neoplasms* ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line ; DNA Fragmentation ; Flow Cytometry ; G1 Phase ; Hep G2 Cells ; Humans ; Liver Neoplasms ; Lung Neoplasms ; MCF-7 Cells* ; Membrane Potential, Mitochondrial ; Mitogen-Activated Protein Kinases ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Salvia miltiorrhiza

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. METHODS: Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. RESULTS: The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. CONCLUSIONS: These results suggest that P53 plays a key role in the radiotherapy response of HCC.
Apoptosis/*radiation effects ; Blotting, Western ; Carcinoma, Hepatocellular/metabolism/pathology/radiotherapy ; Cell Line, Tumor ; Cell Survival/drug effects ; G1 Phase Cell Cycle Checkpoints/radiation effects ; *Gamma Rays ; Glutathione/metabolism ; Hep G2 Cells ; Humans ; Iodine Radioisotopes/chemistry/pharmacology/therapeutic use ; Liver Neoplasms/metabolism/pathology/radiotherapy ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Tumor Suppressor Protein p53/*metabolism

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

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

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*