Cell Cycle Arrest by a Natural Product via G2/M Checkpoint
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AAb bs st tr ra ac ct t CKBM is a natural product that exhibits a novel anti-tumor activity through the induction of cell cycle arrest and apoptosis.We have investigated its effects on cell cycle regulation using a gastric cancer cell line, AGS.The effects of CKBM on cell proliferation, cell cycle regulation and apoptosis were analyzed using BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay and flow cytometric analysis, respectively.Specific cellular protein expressions were measured using Western blot analysis.Flow cytometric analysis indicated that CKBM induced G2/M cell cycle arrest and apoptosis, whereas differential protein expressions of p21, p53 and 14-3-3σ (stratifin) using Western blot analysis were enhanced.The differential expressions of p21, p53 and 14-3-3σ in AGS cancer cells after CKBM treatment may play critical roles in the G2/M cell cycle arrest that blocks cell proliferation and induces apoptosis. KKe ey y w wo or rd ds s 14-3-3σ (stratifin), G2/M arrest, cell proliferation, checkpoint proteinObjective to study the effect of SudanⅠ,Ⅲ,Ⅳ on the growth of SGC-7901.The change of DNA content is determined with Flow Cytometry,and the cell cycle is analyzed.Results show that the average content of DNA decreases in G_1 phase and increases in S and G_2 phase.The index of PI and SPF increase.It is indicated that SudanⅠ,Ⅲ,Ⅳ accelerate the cell cycle from G_1 phase to S and G_2 phase.It is also shown that the effects on SGC-7901 by Sudan Ⅲ in the dose of 50 μg·mL~(-1) and by SudanⅠ,Ⅳ in the dose of 10μg·mL~(-1) is very effective(P0.01).It is concluded that SudanⅠ,Ⅲ,Ⅳ affect SGC-7901 cell cycle to advance its differentiation and proliferation.
Cytometry
Proliferation index
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Objective To investigate the effect of miR-20b on cell proliferation and cell cycle in gastric cancer because of up-regulation of miR-20b in gastric cancer.Methods miR-20b mimics and its inhibitor were respectively transfected into MGC 803 gas-tric cancer cell and methyl thiazolyl tetrazolium （ MTT ） and fluorescence-activated cell sorting （ FACS ） were used to analyze cell growth and cell cycle.Western blot was used to explore the molecular basis of miR-20b.Results Compared with its control, cell growth was obvious elevated and the cell cycle transition was also increased from G 1 to S phase after miR-20b mimics transfection .After transfecting miR-20b inhibitor, cell growth was markedly decreased and cell cycle transition was also delayed from G 1 to S phase.Fur-thermore, miR-20b induced the expression of cyclin D1 （CCND1） and C-Myc, decreased the expressions of p21 and p15.Conclu-sions miR-20b was considered as a potential oncogene to modulate cell growth and cell cycle transition through regulating the expres -sion of cell cycle-related genes .
Key words:
微RNAs; MicroRNAs; Stomach neoplasms; Cell proliferation
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Abstract Measuring cell proliferation is important for studying the effects of various treatments on cultured primary cells or cell lines. Current proliferation analysis methods employ flow cytometry for fluorescence detection of CFSE-labeled cells. However, conventional flow cytometers require a considerable amount of cells per reading, which becomes an issue for kinetic measurements with rare cell population due to the lack of samples for flow cytometric analyses at multiple time points during proliferation period. Here we report the development of a novel cell proliferation kinetic detection method for low cell concentration samples using the new Cellometer Vision image-based cytometry system. Since the Cellometer system requires only 20 µl of sample, cell proliferation can be measured at multiple time points over the proliferation period, whereas typically, flow cytometry is only performed at the end of the proliferation period. To validate the detection method, B1 and B2 B cells were treated with a B cell mitogen and proliferation was measured on day 1, 3, 5, and 6. To demonstrate the capability, B1 B cells were treated with a panel of TLR agonists and proliferation was measured on day 2, 4, 6, and 7. Cellometer was able to obtain proliferation results on each day, which were comparable to flow cytometry. This novel method allows for kinetic measurements of the rare cell samples such as B1 B cell, which has the potential to revolutionize kinetic analysis of cell proliferation.
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Retinoblastoma protein
G1 phase
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Dysferlin
Immunofluorescence
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One of the most common uses of flow cytometry is to analyze the cell cycle of mammalian cells. Flow cytometry can measure the deoxyribonucleic acid (DNA) content of individual cells at a rate of several thousand cells per second and thus conveniently reveals the distribution of cells through the cell cycle. The DNA-content distribution of a typical exponentially growing cell population is composed of two peaks (cells in G1/G0 and G2/M phases) and a valley of cells in S phase (see Fig. 1). G2/M-phase cells have twice the amount of DNA as G1/G0-phase cells, and S-phase cells contain varying amounts of DNA between that found in G1 and G2 cells. Most flow-cytometric methods of cell cycle analysis cannot distinguish between G1 and G0 cells or G2 and M cells, so they are grouped together as G1/G0 and G2/M. However, there are flow-cytometric methods that can distinguish four or even all five cell cycle subpopulations: G0, G1, S, G2, and M (1–3). Furthermore, each subpopulation can be quantified (4). Obviously, flow cytometry with these unique features is irreplaceable for monitoring the cell cycle status and its regulation.
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Cell counting
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Abstract In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation‐specific marker (Ki‐67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co‐staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined. © 2015 by John Wiley & Sons, Inc.
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Proliferation Marker
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Plant-derived polyphenols are being tested as chemopreventive agents; some polyphenols arrest the cell cycle at G1 phase, whereas others inhibit cell cycle proliferation at G2/M phase. Therefore, polyphenols have been proposed to inhibit cell cycle progression at different phases via distinct mechanisms. Indeed, our previous studies showed that small structural differences in polyphenols cause large differences in their biological activities; however, the details of the structural properties causing G1 cell cycle arrest remain unknown. In this study, we prepared 27 polyphenols, including eight different scaffolds, to gain insight into the structural conditions that arrest the cell cycle at G1 phase in a quantitative structure–activity relationship study. We used cell cycle profiles to determine the biophores responsible for G1 cell cycle arrest and believe that the biophores identified in this study will help design polyphenols that cause G1 cell cycle arrest.
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Cell cycle arrest after different types of DNA damage can occur in either G1 phase or G2 phase of the cell cycle, involving the distinct mechanisms of p53/p21 Cip1/Waf1 induction, and phosphorylation of Cdc2, respectively. Treatment of asynchronously growing Swiss3T3 cells with the chemotherapeutic drug adriamycin induced a predominantly G2 cell cycle arrest. Here we investigate why Swiss3T3 cells were arrested in G2 phase and not in G1 phase after adriamycin‐induced damage. We show that adriamycin was capable of inducing a G1 cell cycle arrest, both during the G0‐G1 transition and during the G1 phase of the normal cell cycle. In G0 cells, adriamycin induced a prolonged cell cycle arrest. However, adriamycin caused only a transient cell cycle delay when added to cells at later time points during G0‐G1 transition or at the G1 phase of normal cell cycle. The G1 arrest correlated with the induction of p53 and p21 Cip1/Waf1 , and the exit from the arrest correlated with the decline of their expression. In contrast to the G1 arrest, adriamycin‐induced G2 arrest was relatively tight and correlated with the Thr‐14/Tyr‐15 phosphorylation of cyclin B‐Cdc2 complexes. The relative stringency of the G1 versus G2 cell cycle arrest may explain the predominance of G2 arrest after adriamycin treatment in mammalian cells.
G1 phase
Restriction point
Cyclin B1
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