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Objective: To investigate expression and methylation pattern of RASSF1A, MGMT, p16, APC, DAPK-1 genes during the malignant transformation of human bronchial epithelial cells induced by cigarette smoke.Methods: The immortalized human bronchial epithelial cells (BEAS-2B) in exponential growth phase were seeded onto Transwell membrane with 1×10~5 per well one day before exposure to cigarette smoke. Cells were directly exposed to cigarette smoke for 10min once per two days with oxygen concentration of 21% and temperature at 37℃. The optimal time and concentration for induction of malignant transformation of BEAS-2B cells were determined by CCK-8 assay. After exposure, cells were cultured for another 40 passages. The exposed cells were trypsinized into dishes for further growth. Biological characteristics between the control group and different passages (5, 10, 15, 20, 25, 30, 35 and 40) cells were compared for cell growth, cell morphology, cell cycle and apoptotic rate. The extend of malignant transformation was detected by serum resistance, and soft agar colony formation. The expression of p16, Survivin and bcl-2 were detected by RT-PCR. The methylation status in the promoter regions of the RASSF1A, MGMT, p16, DAPK-1, APC genes were observed by methylation-specific PCR, based on modified genome DNA by bisulphate. Then the mRNA level of RASSF1A and MGMT were detected by RT-PCR.Results: (1) After the cells were exposed to cigarette smoke, the cytostasis rate was related to both concentration and treatment time. When the concentration of cigarette smoke was 20% and exposure time was 10min once, the BEAS-2B cells could be malignantly transformed after 40 passages. A series of sequential steps emerged among transformed cells, including altered growth kinetics, heteromorphism, increases the proportion of nuclear mass, lost contact inhibition, and formation of multiple layers overlap. With the increase of passage, morphological changes and growth showed similar characteristics of cancer cells. The percentage of G1 phase cells in the cigarette smoke treatment group was significantly lower, the proliferation index was significantly increased, and the apoptotic rates were significantly lower in all the cell generations when compared with controls. (2) Human bronchial epithelial cells were malignantly transformed after exposure to cigarette smoke for 5 passages, with an increased capacity of antiserum growth, colony formation rate of 32.34% (P<0.05), and acquired ability of growth in soft agar. The mRNA level of bcl-2 gene did not change but level of p16 gene was highly expressed in both the control and the exposure group. The mRNA level of Survivin gene was not expressed in the control group, and highly expressed in the cigarette exposure group, with a dose-dependent manner. (3) Methylation specific PCR was used to analyze the DNA methylation of promoter region of RASSF1A, MGMT, p16, DAPK-1, and APC genes in the control group and passage 5, 10, 15, 20, 25, 30, 35, 40 cells. The RASSF1A and MGMT methylation was found in the fifth passage cells and stable expressed in the other passages of the exposure cells. The p16 and APC methylation was not changed in both the control and cigarette exposure group. On the other hand, the DAPK-1 methylation was found in either the control or cigarette exposed group. The mRNA of RASSF1A, MGMT genes were highly expressed in the control group and the expression levels of the cigarette exposure group were much lower in a dose-dependent manner.Conclusion: 1. A model of malignant transformation of human cells in vitro induced by cigarette smoke was established to obtain the optimal exposure concentration and time. 2. Multiple genes expression change during the malignant transformation by exposure to cigarette smoke. 3. Aberrant promoter methylation of RASSF1A, MGMT genes and the reduced gene expression may play an important role in malignant transformation of BEAS-2B cells induced by cigarette smoke in vitro.
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