BACKGROUND: Flow cytometric measurement of DNA can reveal G0/G1, S, G2/M phases of cell cycle, and BrdU labeling can determine the percentage of cells in active DNA synthesis. A monoclonal antibody (MoAb), Ki-67, recognizes a protein that is present only in the nucleus of cycling cells but absent in resting cells. We analyzed whether the resting and the proliferating fraction could be differentiated by double staining with Ki-67 MoAb and propidium iodide (PI), and observed the effects of GM-CSF on cell cycle in acute myelogenous leukemia (AML) cells by Ki-67 MoAb. METHODS: Blast cells were prepared from 9 AML patients. The cells were incubated for 48 hours with or without GM-CSF. Cells were stained with BrdU/PI and Ki-67/PI. Cell cycle was analyzed by flow cytometry. RESULTS: The average fraction of G0/G1, S, and G2/M phases was 84.6%, 10.9%, and 4.5 % by BrdU/PI and 87.8%, 8.6%, and 3.7% by Ki-67/PI, respectively. Ki-67/PI staining dis-criminated between G0 and G1 phases and the average was 71.5% and 16.3%, respectively. In cells incubated with GM-CSF, BrdU/ PI method showed that the average S phase fraction (SPF) significantly increased from 10.9 to 16.2% (P=0.01) and the fraction of G0/G1 phase decreased from 84.6% to 78.4% (P= .02). Ki-67/PI method showed that the median SPF significantly increased from 8.6% to 13.7% (P=0.05) and G0 fraction decreased from 71.5% to 58.1% (P=0.02) but G1 fraction increased from 16.3% to 22.3% (P=0.01). CONCLUSION: Cell cycle analysis by Ki-67 MoAb and PI in AML is rapid and simple. It is especially useful to determine the growth fraction and G0 fraction compared to BrdU/PI staining.
Bromodeoxyuridine ; Cell Cycle* ; DNA ; Flow Cytometry ; G0 Phase ; G1 Phase ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Ki-67 Antigen ; Leukemia, Myeloid, Acute* ; Propidium ; S Phase

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

The stem/progenitor cell has long been regarded as a central cell type in development, homeostasis, and regeneration, largely owing to its robust self-renewal and multilineage differentiation abilities. The balance between self-renewal and stem/progenitor cell differentiation requires the coordinated regulation of cell cycle progression and cell fate determination. Extensive studies have demonstrated that cell cycle states determine cell fates, because cells in different cell cycle states are characterized by distinct molecular features and functional outputs. Recent advances in high-resolution epigenome profiling, single-cell transcriptomics, and cell cycle reporter systems have provided novel insights into the cell cycle regulation of cell fate determination. Here, we review recent advances in cell cycle-dependent cell fate determination and functional heterogeneity, and the application of cell cycle manipulation for cell fate conversion. These findings will provide insight into our understanding of cell cycle regulation of cell fate determination in this field, and may facilitate its potential application in translational medicine.
Animals ; Cell Cycle ; Cell Physiological Phenomena ; Epigenomics ; G1 Phase ; G2 Phase ; Humans ; Translational Medical Research

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*

The cell cycle is composed of G1, S, G2 and M phase. The transitions between different phases are regulated at checkpoint such as Start(restriction), S phase and mitotic checkpoint. These checkpoints are regulated by specific cyclins and Cdks(cyclin-dependent kinases). Especially, Start checkpoint in late G1 is though to be very important in control of cell cycle. In this study, it was shown various CDKN2(p16ink4A) alteration, including deletions, mutations, down regulations, and performed differential expression of p53, Cdk4, PCNA and pRb in stomach cancer tissues. 1. The frequency of CDKN2 mutations was not observed in the 19 primary stomach cancer tissues. In contrast to the mutations of CDKN2, mRNA levels was showed by Northern blot analysis that expression of CDKN2 was absent or decreased in 10 of the 19(53%) primary stomach adenocarcinoma. Western blot analysis was performed to determine the differential expression of p53, Cdk4, PCNA related to Start checkpoint. Overexpression of p53 was shown 38%, Cdk4 was expressed in all each specimens, and expression of PCNA was not shown. 2. As the other method to determine the differential expression of p53, Cdk4, PCNA and pRb, immunohistochemical analyses were performed on each 14 formalin-fixed and paraffin embedded tumor tissues of stomach adenocarcinoma. p53 overexpression was showed to clear nuclear staining only in tumor cells not in nonneoplastic cells. In staining for cdk4, the tumor was considered to be cdk4 positive if there was nuclear staining in tumor cells, regardless of cytoplasmic staining. PCNA staining for carcinoma tissues showed more intense nuclear staining in tumor cells than in nonneoplastic cells. pRb overexpression was show in tumor cells. Significant differences were observed in the expression of the proteins among the cancers from different anatomic site. Overexpression of adenocarcinomas had high rate of p53(57.1%) and pRb(71.4% ), and low late of cdk4(7.1% ) and PCNA(14.3% ), As these results, deletion of CDKN2 gene in human stomach cancer was not observed but mRNA expression was down regulated in restriction checkpoint, G1 phase. Inactivation of the CDKN2 gene due to hypermethylation may play an important role in development of cancer. And one of the abnormalities in p53, Cdk4, PCNA or pRb function occurs very common in various cancers, especially oral adenocarcinoma, osteosarcoma and squemous cell carcinoma, suggest that components in restriction checkpoint also play an critical role in the carcinogenesis and progression of cancers.
Adenocarcinoma ; Blotting, Northern ; Blotting, Western ; Carcinogenesis* ; Cell Cycle ; Cell Division ; Cyclins ; Cytoplasm ; G1 Phase Cell Cycle Checkpoints ; Genes, p16 ; Humans ; M Phase Cell Cycle Checkpoints ; Osteosarcoma ; Paraffin ; Proliferating Cell Nuclear Antigen ; RNA, Messenger ; S Phase ; Social Control, Formal ; Stomach ; Stomach Neoplasms

The cell cycle is the set of events that is responsible for the duplication of the cells. Recent studies indicate that cell cycle regulatory proteins, mainly the cyclins and cyclin-related genes, can be critical targets during oncogenesis. The genes and gene products normally control specific events in the cell cycles, particularly during the late G1 and early S phase and G2/M phase. A large body of date implicates cyclins in oncogenesis. The first evidence came from human cyclin A in oncogenesis. Cyclin A is expressed from the late G1 phase through the M-phase of the cell cycle. Cyclin A is known as positive regulator of cell cycle and participates in the tumorigenesis. Overexpression of cyclin A has been reported in several cancers. Ki-67 is a nuclear protein expressed during the cell cycle except in Go. The labeling index of Ki-67 in the tumor cell nuclei has been used as a good prognostic factor. In this study, we compared labeling index of cyclin A and Ki-67 to assess the feasibility between them with 30 cases of cervical intraepithelial neoplasia(CIN) and 20 cases of invasive squamous cell carcinoma(SCC)by immunohistochemistry. The results were as follow; 1. Cyclin A expressed in normal parabasal cells and their labeling index was 0.8+/-0.4%, while in CIN and invasive SCC 65.5+/-9.4% and 86.5+/-12.3% respectively. Ki-67 expressed in normal parabasal cells as 1.3+/-0.7% while in CIN and invasive SCC as 77.8+/-12.9% and 92.2+/-17.6% respectively. 2. In CIN, the expression of cyclin A increased according to the grades of the CIN as 32.5+/-5.7%, 75.8+/-9.0%, and 83.2+/-13.4% in CIN I, II and III respectively. The expression of the Ki-67 also increased according to the grades of the CIN as 51.8+/-9.8%, 87.9+/-11.3%, and 93.6+/-17.5% respectively in CIN I, II and III. 3. There was no differences of cyclin A and Ki-67 expressions according to the histologic types of invasive SCC. Above results suggests that the cyclin A labeling index could be used as a marker of tumor progression in the uterine cervical carcinoma as Ki-67.
Carcinogenesis ; Cell Cycle ; Cell Cycle Proteins ; Cell Nucleus ; Cyclin A* ; Cyclins* ; G1 Phase ; Humans ; Immunohistochemistry ; Nuclear Proteins ; S Phase

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

BACKGROUND: Renin is secreted from the juxtaglomerular (JG) cells in response to a wide variety of extracellular stimuli. To study the underlying mechanism of regulation of renin secretion at molecular level, pure JG cell lines (As 4.1) cloned from renal JG tumor was used. In this study, to explore the feasibility of As 4.1 cells as an in vitro model for renin secretion, the changes of renin secretion from As 4.1 in culture during cell cycle were characterized. METHODS: To address this issue, As 4.1s were synchronized in G0, G1, S, G2, early M and late M phase during experiment. RESULTS: The rate of renin secretion was above 1 ng AI/well/hr in G0, G2/M and early mitotic phase and 0.5 ng AI/well/hr in G1, G1/S, S and late mitotic phase. ML-7 (6x10(-5) M), an inhibitor of MLCK which is known to stimulate renin secretion, increased the rate of renin secretion much greater in G1, G1/S, S and late M phase than the other phases; in particular, in early mitotic phase it had no stimulation. On the other hand, the rate of renin secretion was not influenced through out cell cycles by calyculin A, an inhibitor of type 1 protein phosphatase. Forskolin, an activator of adenlyate cyclase resulting in an elevation of intracellular cyclic AMP, stimulated renin secretion only in S phase in a concentration dependent manner. CONCLUSION: The present study demonstrated that As 4.1 cells in culture secrete active renin in much the similar manner to JG cells in situ but its rate varies during each phase of the cell cycle. Thus As 4.1 cells can be utilized as an in vitro model for renin secretion. But, changes in the rate of renin secretion and the secretory responses to stimulators or inhibitors during cell cycle must be considered in conducting experiments to elucidate the cellular and molecular mechanism of the renin secretion.
Cell Cycle* ; Cell Division ; Cell Line ; Clone Cells ; Colforsin ; Cyclic AMP ; Hand ; Renin* ; S Phase

BACKGROUNDBreast cancer is one of the most common malignancies in women and is highly resistant to chemotherapy. Due to its high tumour selectivity, 3-bromopyruvic acid (3-BrPA), a well-known inhibitor of energy metabolism has been proposed as a specific anticancer agent. The present study determined the effect of 3-BrPA on proliferation, cell cycle and apoptosis in the human breast cancer MCF-7 cell line and other antitumour mechanisms.

METHODSMCF-7 cells were treated with various concentrations of 3-BrPA for 1 - 4 days, and cell growth was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Marked morphological changes in MCF-7 cells after treatment with 3-BrPA were observed using transmission electron microscopy. The distributions of the cell cycle and apoptosis were analyzed by flow cytometry. Immunohistochemistry was used to indicate the changes in the expression of Bcl-2, c-Myc, and mutant p53.

RESULTS3-BrPA (25 microg/ml) significantly inhibited the proliferation of MCF-7 cells in a time-dependent manner. The MCF-7 cells exposed to 3-BrPA showed the typical morphological characteristics of apoptosis, including karyopycnosis, nuclear condensation and oversize cytoplasmic particles. In addition, flow cytometric assay also showed more apoptotic cells after 3-BrPA stimulation. The cells at the G0 and G1 phases were dramatically decreased while cells at the S and G2/M phases were increased in response to 3-BrPA treatment after 48 hours. Furthermore, 3-BrPA stimulation decreased the expressions of Bcl-2, c-Myc and mutant p53, which were strongly associated with the programmed cell death signal transduction pathway.

CONCLUSION3-BrPA inhibits proliferation, induces S phase and G2/M phase arrest, and promotes apoptosis in MCF-7 cells, which processes might be mediated by the downregulation of the expressions of Bcl-2, c-Myc and mutant p53.

Antineoplastic Agents ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Flow Cytometry ; G2 Phase ; drug effects ; Humans ; Immunohistochemistry ; Molecular Structure ; Pyruvates ; chemistry ; pharmacology ; S Phase ; drug effects