ANALYSIS OF MALIGNANT TRANS FORMATION CHARACTERISTICS OF RAT ESOPHAGEAL EPITHELIUM USING FLOW CYTOMETRY
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Abstract:
The DNA content of the epithelium cells and the characteristics of cell proliferation cycle of various phase of malignant transformation were analysed by flow-cytometry. Comparison has been made amony three cell lines withdifferent proliferation capacityf33the normal esophgeal epithelium(NRE),the anaplasticepithelium induced by NSEE(DRE) and RE25 -3 (malignant transformation cell from DRE) Without using carcinogen in vitro .The experimental results showed that, NRE cells and DRE :ells have similar DNA content, DI-1.0, while the DI of various generations of the kE25-3 cells incerased to 1.60--3.36. The analysis of the cell proliferation cycle showed that the percentage of S phase cells was 5.04% in NRE, 29.92% in DRE, while in R-E5-3 it increased froml0.62%to 45.85% much higher than NRE and DRE. When RE25-3 was transplanted to the nude mice (RE25/N) its S phase cell decreased markedly which impress that the proliferation capacity of the cell may be affected by the immunity of the host.Keywords:
Malignant Transformation
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Objective To study the effect of acrylonitrile(CAN)to cell growth,apoptosis,proliferation and related gene expression of rat normal glial ceUs(DI TNCI)and tumor glial cells(C6).Methods The concentration of CAN on DI TNCI and C6 were 25,50 and 75μg/mL For cell growth,prolifcration and apoptosis assay,the treated time was 24 hours.for microarray assay,the treated time wa84 and 24 houm.Results After treatment of DI TNCl cell with 25,50 and 75μ/ml CAN,the DNA synthesis index were decreased 93.1%.81.3%and 74.9%as compared to control respectively,the apoptosis index Was increased 118%.122%and 143%as compared to controls respectively.The DNA syntllesis and apoptosis indexes of C6 cell showed no change after treatment with CAN.The cell cycle and apoptosis pathway related genes,such as cyclin and p53,also showed changes after treatment with AC N.Conclusion CAN inhibited the cell proliferation of DI TNCl.induced the apoptosis of DI TNCl and had no effect on cell proliferation and apoptosis of C6 cells,and the related regulation gene expression changes further confirmed the results.
Key words:
Acrylonitrile; Cell proliferation; Apoptosis; Bats; Neurog]ia
Proliferation index
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AIM: To examine the effects of diethyldithiocarbamate (DDC) on the proliferation, redifferentiation, and apoptosis in human hepatoma cells. METHODS: Cell surface charge, biochemical changes, cell growth in soft agar, single cell electrophoresis, electron microscopy examination, and flow cytometry analysis were measured. RESULTS: After being treated with DDC 3 mmol/L the growth curve and mitotic index of human hepatoma cells decreased remarkably, and the cellular growth inhibitory rate amounted to 52.4 % . The indices related with cell malignancy were alleviated significantly, such as the cell surface charge decreased significantly, the electrophoresis rate dropped from 1.6 to 0.8 μm·s-1·V-1·cm-1, the average value of a-fetoprotein (a-FP) content decreased from 314 to 95μg/g (protein), and r-glutamyl-transpeptidase (γ-GT) activity from 0.9 to 0.14 U/g (protein). The cell differentiation index increased significantly, such as the average levels of tyrosine-a-ketoglutarate transaminase (TAT) activity increased from 11.6 to 36 μmol/g (protein), and the colonogenic potential decreased by 95.6 % . The apoptotic bodies, detached cells, and apoptotic morphological features appeared, and the treated cells' DNA was fragmented as observed by the comet assay. The flow cytometric results showed that a 42.9 % fractional DNA contentexisted in the treated cells. CONCLUSION: DDC can inhibit human hepatoma cells proliferation, and can induce redifferentiation as well as apoptosis.
Mitotic index
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Objective To investigate the effect and mechanism of narigenin on human leukemia K562 cell line.Methods K562 cell line cultured in vitro was exposed to different concentration of narigenin and cell proliferation manner was detected by MTT assay,and cell PCNA protein was detected by immunhis- tochemical staining.K562 cell morphology and apoptosis were observed by light microscope and electron mi- croscope,and flow cytometry was used to decide cell cycle and apoptosis rate.Expression of p53 and p21~(WAF1) mRNA and protein was detected by RT-PCR and Western blot technology respectively.Results Proliferation of K562 cell was significantly inhibited by nerigenin and the IC_(50) for 24 h,48 h and 72 h were 455μmol/L, 225μmol/L and 175μmol/L respectively.PCNA protein in treated groups was remarkably poorer than in con- trol group,cell growth-inhibition and morphologic change of apoptosis were observed under light microscope and electron microscope.Observation by flow cytometry indicates that K562 cells were blocked in G_0/G_1 phase of cell cycle in nerigenin group and the apoptosis rates were remarkably increased.Both RT-PCR and West- ern blot tests showed gradually higher expression of p21/WAF1 gene in treated group,whereas p53 gene ex- pression no detectable change between control group and nerigenin group.Conclusion Nerigenin has signifi- cant inhibitory effect on the growth of K562 cell in vitro;Cell cycle blockage and apoptosis induce maybe the important mechanisms underlied;and a p53-independent up regulation of p21/WAF1 may be one of an im- portant pathway during the process.
K562 cells
Growth inhibition
MTT assay
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Objective To analyze the influence of cyclooxygenase-2(COX-2)inhibitor NS-398on cell morphology,cell cycle,proliferation and apoptosis of hepatocelluar carcinoma cells SMMC-7721,and to investigate the mechanism of the effect of NS-398on the growth of tumor cells.Methods The SMMC-7721cells were treated with different doses of NS-398(0,25,50,75,100,and 150μmol·L-1)in various groups for 24,48and 72h,and at the same time normal control group was set up.The changes of cell morphology in various groups were observed,the inhibitory rates of proliferation of hepatocellular carcinoma cells were detected by MTT assay,and the changes of cell cycle and apoptotic rates were detected by flow cytometry.Results After treated with 25,50,75,100,and 150μmol·L-1 NS-398,the cell adhesion was decreased,and some of the cells were floating,and the medium was not clear.Compared with normal control group,the ratios of SMMC-7721cells in G0/G1phase were reduced,and the ratios of SMMC-7721cells in G2/M phase were increased in the other groups.No apoptotic peak was observed in normal control group.The apoptotic peak was observed in 50μmol·L-1 NS-398group after treated for 24h,and the level of apoptosis was increased significantly 72hafter treatment.The inhibitory rates of proliferation had a concentration-and time-dependant trend in various groups.Conclusion The COX-2inhibitor NS-398can repress the proliferation of hepatocellular carcinoma cells SMMC-7721and induce apoptosis;the process is time-and dose-dependant.
MTT assay
Cytometry
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Objective:To investigate the effects of bone morphogenetic protein 9 on the proliferation, cell cycle and apoptosis of breast cancer cell line MDA-MB-231.Methods:The expression of BMP9 in high-metastatic cell line MDA-MB-231 and low-metastatic cell line MCF-7 were detected by Semi-quantitative PCR respectively,MDA-MB-231 were transfected with AdBMP9(as experimental groups) and AdGFP(as control groups) respectively,Then the ability of cell proliferation was detected by MTT,cell cycle and apoptosis were detected by flow cytometry.Results:The BMP9 mRNA was not detected in human breast cancer cell line MDA-MB-231.After transfection with AdBMP9,the MDA-MB-231 cells showed an inhibited proliferation,On the fifth day,the growth rate of experimental groups(0.886 0±0.053 2)was significantly lower than that of control groups(1.224 0±0.103 1)(P0.05);The results of flow cytometry showed that,BMP9 could induce the cell cycle arrest in MDA-MB-231,On the second day and third day after transfection,the cell number of G2/M phase increased significantly,which were 3.2 fold and 2.4 fold that of control groups respectively;It's also found that after 72h treatment,the apoptosis rate of experimental groups(31.55%±8.26%) was significantly higher than that of control group(3.80%±0.46%)(P0.05).Conclusion:These data demonstrated that BMP9 could inhibit the proliferation of MDA-MB-231 by blocking cell cycle and inducing apoptosis.
G1 phase
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Objective: To investigate the effects of proliferation and apoptosis induced by ARG in human esophageal cancer-1 cells.Methods: EC-1 cells were treated with different concentration ARG.Proliferation of EC-1 cell inhibited by ARG were assessed by MTT.The cell cycle was measured using flow cytometry(FCM),and the level of proliferating cell nuclear antigen(PCNA) with immunohistochemistry(IHC).Apoptosis rate of EC-1 cell were assessed by flow cytomeu-y(FCM).the changes of apoptosis of EC-1 cells were detected by TUNEL.The expression alteration of protein of Bcl-2 and Bax genes were detected by immunocytochemistry technique.Results: ARG could inhibit the growth and significantly suppressed expression of PCNA of EC-1 cells in a dose/time dependent manner(P0.05).G0/G1 to S phase transition was blocked after treated with ARG,the percentage of G1 phase of the cell cycle was significantly increased,whereas the percentage of S phase was remarkably decreased.ARG could inhibit the growth of EC-1 cells in a dose/time dependent manner(P0.05).TUNEL showed the ratio of apoptotic rate of EC-1 cells increased(P0.05).Immunocytochemistry technique showed that the Bax protein increased gradually,while the Bcl-2 protein decreased gradually(P0.05).Conclusion: The ARG could inhibit the growth and expression of PCNA,and retarded cell cycle of EC-1.ARG can induce apoptosis of EC-1 cells,the pathway that ARG induce apoptosis of EC-1 cells may be through regulation of protein expression of Bcl-2 gene and Bax gene.
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Objective To investigate the biological effects of arsenic trioxide(As2O3) on human tumor cells in vitro.Methods Human gastric cancer cells(HGC-27) and human hepatoma cells(HepG2) were treated with different concentrations of As2O3(1.25、2.50、5.00、10.00 and 20.00(mol/L).The apoptosis of these cells was detected at the indicated times(48,72 and 96 hours) and the morphology of apoptosis cells were observed by the fluorescent microscope after staining of HT33258 dye.The change of cell cycle was measured by flow cytometry.Results As2O3 significantly inhibited the proliferation of tumor cells in both dose-and time-dependent manners.After treatment of As2O3,the nuclear chromatin in apoptotic cells tendered to condensation and marginalization.The data of flow cytometry showed that when treated with As2O3,G0/G1 phase cells increased from 68.55% to 80.30% and G2/M phase cells decreased from 11.56% to 2.53%(P0.05).Conclusion As2O3 significantly inhibited the proliferation of tumor cells in both dose-and time-dependent manners.As2O3 arrested most of HGC-27 cells at G1/G0 phase.The mechanism may be associated with the inhibition of cellular growth and proliferation and promotion of apoptosis.
Arsenic Trioxide
Cytometry
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Objective To investigate the effects of ARG on PCNA expression and cell cycling in invitro cultured esophageal cancer-1 cells Methods EC-1 cells were treated with different concentration ARG.Proliferation of EC-1 cell inhibited by ARG were assessed by MTT.The cell cycle was measured using flow cytometry(FCM),and the level of proliferating cell nuclear antigen(PCNA)with immunohistochemistry(IHC).Results ARG could inhibit the growth and significantly suppressed expression of PCNA of EC-1 cells in a dose/time dependent manner(P0.05).G0/G1 to S phase transition was blocked after treated with ARG,the percentage of G1 phase of the cell cycle was significantly increased,whereas the percentage of S phase was remarkably decreased.Conclusion The findings suggested the ARG could inhibit the growth and expression of PCNA,and retarded cell cycle of EC-1.
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OBJECTIVE To investigate the effect of microRNA-155(miR-155) over-expression on malignant phenotype in Hep-2 cell line of laryngeal squamous cell carcinoma.METHODS miR-155 mimics
were transfected into Hep-2 cells by Lipofectamine RNAiMAX.miR-155 expression level was detected by qRT-PCR.Cell proliferation were evaluated by CCK8 assay and colony formation.Cell apoptosis were analyzed by flow cytometry and Hoechst 33342 propidium iodide staining.Cell migration was detected by wound healing assay.RESULTS After transfection of miR-155 mimics into Hep-2 cells,the data of qRTPCR showed that miR-155 expression level was significantly up-regulated.CCK-8 assay and colony formation revealed that growth or proliferation ability of miR-155 group was obviously promoted.Cell apoptosis or death was suppressed after over-expressed miR155 which was analyzed by flow cytometry and Hoechst 33342 propidium iodide staining.Remarkable enhancement of cell migration was observed in miR155 group which was detected by wound healing assay.CONCLUSION Over-expression of miR-155 could induce proliferation and migration of Hep-2 cell line,and inhibit apoptosis.It might play the function as an oncogene in the development of laryngeal squamous cell carcinoma.
Propidium iodide
Lipofectamine
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Using FX-4000 strain unit, the proliferation of human lung adenocarcinoma A549 cells and Squamous Carcinoma of Tongue Tca8113 Cells that underwent different mechanical strain were studied. Flow Cytometer was adopted to investigate the proliferation of A549 cells and Tca8113 cells. Experimental results indicated that cell proliferation index (PI) reduced significantly after A549 cells and Tca8113 cells were subjected to square wave with 10% elongation at frequency 0.5Hz for 4h, the PI had no distinct difference for sine wave-treated A549 cells and Tca8113 cells when compared with the control group. It was indicated that cyclic stretch could inhibit the proliferation of A549 cells and Tca8113 cells, the waveform play important roles in inhibiting proliferation.
Squamous carcinoma
Proliferation index
Strain (injury)
Elongation
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