The Study Of Malignant Transformation Of BEAS-2B Cells Induced By Coal Tar Pitch Smoke Extract

Coal tar pitch (CTP) is by-product made from coal duing the process of coking is mainly used in paint, rubber production. Epidemiological studies showed that there was a high incidence rate of lung cancer among the workors exposed to CTP defined as occupational tumor. In previous studies, single component of CTP was used to study the hazards and carcinogenic mechanisms of people exposed to coal tar pitch h, but as mixture there is rare reports about the carcinogenic mechanism of coal tar pitch.To study the malignant transformation effects of coal tar pitch smoke extract on BEAS-2B in vitro and establish malignant transformation of BEAS-2B model induced by coal tar pitch extract, then further investigate the genotoxicity of coal tar pitch smoke extract by single cell gel electrophoresis (SCGE). Using RealTime PCR and cellular immune staining method to analyze the differences in the gene and proteins expression profiles between the early transformation cells and normal BEAS-2B cells for revealing the carcinogenesis and mechanism of lung cancer in coal tar pitch smoke extracts.Methods1 Malignant transformation model of BEAS-2B induced by coal tar pitch smoke extract.BEAS-2B cells were reacted with coal tar pitch smoke, including a DMSO control group and a blank control grounp then with conventional culture. The characteristics of the cellular biology were observed and the malignant transformation of cells were identified through observing, anchorage independent growth and soon. Then the malignant transformation model of cells at different phases were established.2 The CIN of BEAS-2B induced by coal tar pitch smoke extracts.The chromosome aberration analyses showed that the CIN of BEAS-2B cells reacted by coal tar pitch smoke and the chromosome changes of BEAS-2B with cell transformation experiment. 3 The genotoxicity of coal tar pitch smoke extract and the function test of spindle checkpoint.Investigating the genotoxicity of coal tar pitch smoke extract by SCGE. Using RealTime PCR and cellular immune staining method to analyze the differences of the spindle checkpoint-related genes and proteins expression profiles between the different groups of 30th generation cells.4 Statistical analysisStatistical software SPSS 12.0 was used for data analysis.χ2 test; one-way ANOVA, LSD were used to analyze (α=0.05)Results1 Malignant transformation experimentThe 30th generation cell of experiment group showed malignant transformation phenotype:different sizes; loss of the original spindle appearance, most of the changes for the class circle shape; cytoplasm increased; nuclear atypia; compound layer growth.Flow cytometry showed that the proportion of G1 phase cells was more decreased and G2/M phase cells was more increased of the 30th generations cell in experiment group than those of the 30th generations cell in control group, and there were significantly statistical difference between these two groups (P<0.05) indicating that cells altered growth kineties. The experimental group cells were formed small cell colonies in soft agar in 20th colonies The anchorage independent growth was appeared in 30th generation, after three weeks, there was few number of cells can grow into large experimental group cells.2 Chromosome instabilityIn earlier period of malignant transformation, the cell included by coal tar pitch smoke extract had shown chromosome instability and had statistical difference with control group cells significantly (P<0.05), which mainly showed that the abnormal number and structure of chromosome.3 Test of the genetoxic of coal tar pitch smoke extract and the function of spindle checkpointIn the comet assay analyses in the BEAS-2B cells was included by coal tar pitch smoke extract, the results are positive. It means that coal tar pitch smoke extract may cause DNA damage. The cells of experiment group have longer tail length and had statistical difference with control group cells significantly (P<0.05). Coal tar pitch smoke extract has genetoxic for cell. The mRNA expression of spindle checkpoint-related genes were detected by RealTime PCR in the control group (P<0.05). Gene expression of Mad2, Bub1 in exposure group was lower than that of control group significantly. The protein expression of Mad2,Bub1 mainly located in the cytoplasm in coal tar pitch smoke extract cells, and the expression of Mad2, Bub1 were lower in the group of cells included by coal tar pitch smoke extract and had statistical difference with control group cells significantly (P<0.05)ConclusionCoal tar pitch smoke extract can induce malignant transformation of BEAS-2B cells in vitro.Coal tar pitch smoke extracts exposured can lead to chromosomal instability. DNA damage may be the initiating agent of the CIN and malignant transformation to the cell included with coal tar pitch smoke extract. The disfunction of spindle checkpoint may be an important agent of the CIN and malignant transformation to the cell included with coal tar pitch smoke extract.