Centrosome Abnormalities And Chromosomal Instability In BEAS-2B Cells Induced By Coal Tar Pitch Fume Extract

Coal tar pitch is mainly composed of polycyclic aromatic hydrocarbons and has been recognized as a human carcinogen, however, its carcinogenic mechanism remains unclear, which is needed to study and research further.chromosomal instability theory has drawn increasing attention in the field of tumor mechanism research. Chromosomal instability is a common feature of tumor cells. It is considered that chromosomal instability is related to the mechanism of tumor development. In addition to chromosomal instability, there are centrosome abnormalities that show an increase in the number of centrosome and an abnormal shape in many solid tumors. Centrosome abnormalities may be the main reason for chromosomal instability. The specific mechanism is still unclear, pending to further study. It has been found that many tumor suppressor genes and their products and some kinases, such as P53, BRCA1/2 and CDK2/CyclinE complex, are all involved in the regulation of centrosome replication. Inactivation or decreased expression of P53 and overexpression of CyclinE may result in centrosome amplification. Centrosome abnormalities can lead to chromosomal instability, which has a profound impact on the occurrence of cancer. ObjectiveThe study, which makes BEAS-2B cells (human bronchial epithelial cells) as a object of study and use coal tar pitch fume extract as an inducer, is to probe the changes of chromosomal karyotype and centrosome during the process of malignant transformation of cells as well as the molecular mechanisms of abnormal centrosome for provide possible theoretical basis to explore the mechanism of the pathogenesis of lung cancer caused by coal tar pitch. Methods1. The components of Coal tar pitch fume extract solution were analysis by Gas chromatography-mass spectrometry. There are three groups in this study, including coal tar pitch group (coal tar pitch fume extract solution), DMSO group (dimethylsulfoxide) and control group (saline).2. With coal tar pitch fume extract as an inducer of cells, the median lethal concentration of BEAS-2B cells was assessded by MTT assay. Cells were induced at 20% LC50,and then cultured conventionally to observe the changes of proliferation rate and morphology.3. Chromosomes of the 10th,20th and 30th generation cells were observed by the oil-immersion lens. Indirect immunofluorescence staining was used to observe changes of centrosome in induced BEAS-2B cells.4. Total RNA of the 10th,20th and 30th generation cells were extracted respectively to detect the gene expression of P53, P21 and CyclinE by real-time quantitative PCR. Protein expression of P53, P21 and CyclinE of the 10th,20th and 30th generation cells were semi-quantitatively detected by immunohistochemical staining.5. The data was analyzed by the statistical software SPSS 12.0 and indicated by mean and standard deviation. Test levelα=0.05.Results1. After analyzing coal tar pitch fume extracts by Gas chromatography-mass spectrometry, Polycyclic aromatic hydrocarbon compounds were the main ingredient, accounted for 87.91%. The median lethal concentration was 8.64 mg/L, and then the final concentration which was determined to induce cells was 2 mg/L.2. After induction, From 10 passages to 30 passages, the proportion of diploid karyotype of cells in coal tar pitch group was lower than that in normal group and DMSO group respectively, with a large number of aneuploid, indicating there was chromosomal instability at this passage.3. After induction:total ratio of abnormal centrosome of the 10th generation cells was 2.79%, however, there were no significant differences among the three groups. At 20 passages, the total ratio of abnormal centrosome (6.56%) increased, which mainly showed that the number of centrosome was abnormal (3.41%). The ratio of abnormal centrosome of the 30th generation cells increased further, showing number increase, volume augmentation and the number and size abnormalities simultaneously.4. There was a positive correlation between centrosome abnormalities and chromosomal instability(r=0.75, P=0.02), indicating that centrosome abnormalities was closely related to chromosomal instability.5. After induction, the gene and protein expression of P53, P21 and CyclinE of the 10th generation cells showed no significant differences among three groups. At 20 and 30 passages, the gene and protein expression of P53 and P21 reduced, while those of CyclinE increased significantly (P<0.05). There was a positive correlation between the protein expression of P53 and P21 and so was a negative correlation between the protein expression of CyclinE and P53 and P21 respectively. From 10 to 30 generations, the protein expression of P53 and P21 gradually decreased, while the protein expression of CyclinE increased.6. After induced, the analysis of correlation between the rate of centrosome abnormality and the expression of proteins P53, P21 and CyclinE in the 20th and 30th cells showed that there was a negative correlation beween the total rate of abnormal centrosome and P53 and P21 respectively (r=-0.57, P=0.01; r=-0.65, P=0.00), while there was a positive correlation between the total rate of abnormal centrosome and the expression of CyclinE (r=0.57, P=0.01).ConclusionThe induced cells regulate the replication of centrosome by P53/P21 pathway during malignant transformation of BEAS-2B cells induced by coal tar pitch extracts. Centrosome abnormal may play an important role in the formation of chromosome instability.