ANALYSIS OF MALIGNANT TRANS FORMATION CHARACTERISTICS OF RAT ESOPHAGEAL EPITHELIUM USING FLOW CYTOMETRY
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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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Aim:To investigate the effects of reservatrol(RES) on the cell cycle,apoptosis and expression of PDCD5 mRNA in renal cell carcinoma 786-0 cells.Methods:786-0 cells in the experimental group were cultured with the medium containing 25 μmol/L RES for 24,48 and 72 h,and the cells in the blank control group were cultured with the medium without RES.The cell cycle phase distribution and the cell apoptosis of 786-0 cells were analyzed by using flow cytometry;and the expression of PDCD5 mRNA was analyzed by using in situ hybridization.Results:786-0 cells in the experimental group at G1 phase and G2 phase ratio decreased significantly,and the proportion of S phase significantly increased after RES treatment for 24 h.The mRNA level of PDCD5 expression and the number of apoptotic cells in the experimental group were higher than the blank control group at 24,48 and 72 h.Conclusion:RES could upregulate the expression of PDCD5 mRNA in 786-0 cells,which induces cell apoptosis by arresting cells at S phase.
Cytometry
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Objective To investigate the effects of MS-275 on the proliferation of oral squamous cell carcinoma(Tca-8113) and promotion of apoptosis. Methods Oral squamous cell carcinoma was intervened with 0,0.5,1,2,4,and 8μmol/L MS-275,and the morphologic changes of Tca-8113 cells were observed by the inverted phase contrastmicroscope.The cell proliferative activity was measured by MTT assay,the cell apoptotic rates were detected by flow cytometry through Annexin-V-FITC/PI staining,and cell cycles were analyzed by flow cytometry. Results ① Under the inverted phase contrast microscope,no changes of cell morphology could be observed in the control,with cells polygonal,adherent and active.However,the changes could be observed in groups treated with MS-275 at 2,4μmol/L for 48h.The cells became smaller and round,and shrunk cells increased.② A time-and dose-dependent inhibition was detected in Tca-8113 cells that were treated with different concentrations of MS-275 for 24,48 and 72h,with difference of proliferative inhibition between the control and the treated groups(P0.05).③ Cell apoptosis rate was 41.28% and 53.71% respectively with 2 and 4μmol/L MS-275 treatment for 48h,and cell cycles arrest in G0/G1,with statistical difference. Conclusion MS-275 inhibits the growth of oral squamous cell carcinoma,time-and dose-dependent,and promotes cell apoptosis and blocks cell cycle.
Cytometry
MTT assay
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OBJECTIVE:To observe the effects of cell apoptosis , MDR1 protein expression and cell cycle on human bladder cancer cell BIU-87 treated by arsenic trioxide (As 2O 3).METHODS: The cell growth inhibitory rates of BIU-87 cell were tested by MTT assay with various concentration As 2O 3 and various time. Fas, bcl-2 , MDR1 protein expression and cell cycle changes were detected by flow cytometry after 72 hours treated with various concentration As 2O 3.RESULTS: As 2O 3 effectively inhibited the growth of BIU-87 cell, which was positive correlation with concentration of As 2O 3 and reaction time,P0.05. Fas expression was positive correlation with the increasing concentration and bcl-2 expression was negative so,P0.05. It was negative correlation with the increasing concentration between Fas and bcl-2 expression,P0.05. MDR1 protein expression treated with 1 μmol/L As 2O 3 was higher than control, but it treated with 2 μmol/L and 5 μmol/L was lower than control,P0.05.Cell cycle was arrested in G 0/G 1 phase with the increasing As 2O 3 concentration.CONCLUSIONS: As 2O 3 could significantly inhibit the growth of bladder cancer cells. Inducing cell apoptosis and arrested cell cycle were probably its important action mechanism.
Arsenic Trioxide
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Objective To study the effects of 9-cis retinoic acid(9-cisRA) on cell cycle and expressions of CYCLIND1 and CDK4 in thyroid squamous cell carcinoma cells(SW579).Methods Cells were treated with different doses of 9-cisRA,the proliferation of the cells was observed with MTT and the cell cycle was measured by flow cytometry;the CDK4 and CYCLIND1 mRNA expression levels were determined by RT-PCR;CDK4 and CYCLIND1 protein expression were determined by Western blot.Results After cells were treated with 9-cisRA the proliferation index decreased markedly with a dose-dependent manner.Flow cytometry analysis showed the percent of G1 phase cells was significantly higher than that of the control groups and the percent of S phase cells was lower than that of the control groups.The cell cycle was arrested at G1 stage;The mRNA and protein expression of the CDK4 was not changed;the mRNA and protein expression of CYCLIND1 was descreased dramatically(P0.01).Conclusion 9-cisRA inhibits the proliferation of SW579 cell in a dose-dependent manner,It is related with down-regulation of CYCLIND1 and arresting SW579 cell at G1 phase.
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Objective To study the effects of chlorophyllin on proliferation and cell cycle in vitro in human bronchial epithelial cell line (16HBE) transformed by trans-benzo(a)pyrene-trans-7,8- dihydrodiol-9,10-epoxide (trans-BPDE).Methods MTT method and flow cytometry were used to investigate alteration of the growth curve and cell cycle among untreated control cells,malignant transformed cells induced by trans-BPDE and the anti-transformed cells treated with chlorophyllin and to analyse effects of chloropyllin on the cells.Results Compared with that of control 16HBE,the vitality of malignant transformed cells was higher and it was inhibited significantly in the anti-transformed cells treated with 100μmol/L chloropyllin.However it was uninhibited in the cells treated with only chloropyllin of the same concentration.The proportion of G_0/G_1 cells decreased significantly after being tansformed by trans-BPDE while the proportion increased after being anti-transformed by chlorophyllin.Meanwhile,no change was observed in G_0/G_1 phase of cells treated with the same concentration of chlorophyllin.Conclusion A certain concentration of chlorophyllin may have significant anti-profileration activity in human bronchial epithelial cell line 16HBE.It interfere cell cycle course by enhancing the G_0/G_1 phase proportion in malignant transformed cell and result in change of cell profileration and differentiation.
Chlorophyllin
Malignant Transformation
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Objective To investigate the effects of 2-(3carboxy-1-oxoprogy1)amino-2-deoxy-D-glucose(COPADG) on the proliferation and cell cycle of human esophageal cancer cells Eca-109 and TE-1.Methods In vitro experiments,MTT colorimetric assay was performed to determine the growth inhibitory rates of Eca-109 and TE-1 cells treated by COPADG.Flow cytometry was applied to observe the change of cell proliferation cycle and apoptosis index.Cell morphological change was observed by transmission electron microscopy.Results COPADG inhibited effectively the growth of esophageal cancer cells Eca-109 and TE-1 in a time-and dose-dependent manner(P0.01).In Eca-109 and TE-1 cells treated by COPADG of different concentrations for 48 h,flow cytometry analysis showed that the percentage of G_(0)/G_(1)phase was higher and the percentage of S phase was lower than that in untreated cells(P0.01).A marked apoptosis peak was formed in Eca109 cells,and the typical apoptotic morphological change was observed by transmission electron microscopy.Conclusion COPADG can inhibit effectively the proliferation of esophageal cancer cells Eca-109 and TE-1,and it can change the distribution of cell cycle and induce apoptosis of tumor cells at certain concentrations.
Proliferation index
Cytometry
MTT assay
Growth inhibition
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Objective To study the effect of β-elemene on the proliferation and cell cycle of hepatic carcinoma cells.Methods Hepatic carcinoma 7402 cells were cultured in vitro,and treated with β-elemene for 48 h;MTT assay was used to evaluate the proliferation of 7402 cells;Cell cycle was analyzed by using flow cytometry with PI staining.Results After treated with β-elemene,the proliferation of 7402 cells was apparently inhibited in a dose-dependent manner;FCM assays showed that cells in S and G2 phases were decreased,while increased in G1 phase,which indicated that G1 phase arrest happened.Conclusion β-elemene can directly influence the proliferation and cell cycle of hepatic carcinoma cells,arrest cells entering S phase from G1 phase.
Elemene
MTT assay
Cytometry
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Objective To investigate the effect of all-trans retinoic acid( ATRA) on the cell proliferation and differentiation of human ovarian cancer SKOV3 cells. Methods In the experimental group,human ovarian cancer cell line SKOV3 was treated with 1x10- 6mol / L ATRA; and in the control group,the SKOV3 cells were not treated with ATRA. The inhibitory rate of cell proliferation was calculated with MTT assay,the cell cycle was analyzed by flow cytometry,the expression level of N-myc downstream-regulated gene 1( NDRG1) was determined by immunohistochemistry method and the cellular morphological changes were observed by inverted microscope. Results Compared with the control group,both the inhibitory rate of cell proliferation and the expression level of NDRG1 were higher in the experimental group( all P 0. 05). In the experimental group,along with the extension of treated time,the number of cells in the G1/ G0phases was increased and was decreased in the S phase; meanwhile,the inverted microscope showed the cells were in poor growth,and had slowing proliferation and few disintegration. Conclusion ATRA could inhibit the proliferation of ovarian cancer SKOV3 cells but also induce the differentiation.
Cell counting
MTT assay
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Objective To study the role of NR2F2 in murine pre-osteoblast proliferation.Methods Quantitative real-time PCR was used to measure the mRNA level,MTS assay was used to assess the cell proliferation rate,flow cytometry was used to figure out the cell cycle distribution,and ELISA-Brdu was used to check the rate of DNA synthesis.Results When murine pre-osteoblast MC3T3-E1 cell proliferation was accelerated,the expression level of NR2F2 remarkably increased to 4.57±0.30(P0.01) fold compared with that of the control.Over-expression of NR2F2 accelerated MC3T3-E1 cell proliferation(P0.01),and the proportion of cells in S phase markedly increased to 2 fold compared with that of the control,and the proportion of cells in G2/M phase also went up(P0.05).More Brdu was incorporated into NR2F2 over-expressing cells,indicating the increase of DNA replication rate(P0.01).Conclusion NR2F2 accelerates murine pre-osteoblast MC3T3-E1 proliferation through increasing the proportion of cells in S phase.
Proliferation Marker
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The growth rate of a tumor is dependent on both cell proliferation and cell loss. We have established subpopulations of human colon cancer cells with different in vivo growth rates by selecting the cells according to their adhesiveness to laminin-1 in vitro.Laminin-1-adhesion selected colon cancer cells were injected into the cecal wall of nude mice. The tumors were examined 30 days later. Cell proliferation was assessed by proliferating cell nuclear antigen (PCNA) index and apoptotic cells were labeled by digoxigenin-11-dUTP using terminal deoxynucleotidyl transferase.The laminin-1-adherent cells, which formed larger tumors in vivo, showed increased proliferative activity and reduced apoptosis in comparison with the laminin-1-nonadherent cells.Laminin-1 may enhance the malignant behavior of colon cancer cells by accelerating proliferation as well as by decreasing cell loss.
Terminal deoxynucleotidyl transferase
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