The Effects Of BaP On H2AX Phosphorylation And Nuclear Protein Expression

Polycyclic aromatic hydrocarbons (PAHs) are wide-spread environmentalpollutants which have been detected in surface waters, sediments, soil, plants, and bothrural and urban air. Automobile exhaust, domestic wood burning and industrial wastebyproducts are all sources of PAHs. Without an external stimulus, PAHs are extremelystable, making them a persistent environmental problem. Benzo[a]pyrene (BaP) is arepresentative of carcinogenic PAHs, and has been under intensive studies. BaP ismetabolized into BPDE, one of the strongest carcinogens. BaP can block signaltransductions,cell growth and differentiation, form adducts with DNA, induce DNAbreaks, as well as cause mutation in p53 gene. However, there were conflicting reportsabout the ability of BaP to induceγH2AX (the phospohorylated form of histoneH2AX), a marker for DNA double strand breaks (DSBs). In addition, it is not clearBaP-inducedγH2AX is cell cycle dependent, and the kinases involved inγH2AXformation are not known.In addition, previous studies usually only address one aspect of the possiblemechanisms for the carcinogenicity of BaP. However, it is clear that cancer is not theresult of a single or several genes, and carcinogenesis is a multi-factor, multi-stageprocess. Focusing on only a single gene is very difficult to elucidate the mechanismsfor cancer. The advent of high-throughput technologies, such as genomics andproteomics, provides a new angle for cancer study. By globally analyzing the changes of genes and proteins in a biological process, those involved can be identified,cellular response network can be constructed, key signal transduction pathway can beanalyzed, all of which could help understand the underlying molecular mechanism.Using such techniques, the cellular response to BaP treatment has been analyzed, and agroup of responsive genes/proteins has been identified. Nonetheless, as the maintarget for BaP is DNA, BaP-induced DNA damage would lead to DNA repair response,thus changes in gene expression, and eventually result in changes in protein expression.Since most of the DNA damage responsive proteins should reside in the nucleus, tostudy the effects of BaP on nuclear protein expression would provide betterinformation for the understanding of BaP-induced carcinogenesis.Therefore, in this study, we first investigated the effects of BaP treatment on cellsurvival and cell cycle, and whether BaP-induced DNA damage can lead to thephosphorylation of H2AX. In the first part of this dissertation, by using HeLa cells,we examined the ability of BaP to induce H2AX phosphorylation. The effects of BaPon HeLa cell survival was measured by MTT assay; cell cycle was analyzed by flowcytometry. The time- and dose-response of BaP to induceγH2AX, as well as thecorrelation betweenγH2AX and cell cycle was evaluated by Western-blot andimmunofluorescent microscopy. Furthermore, using specific gene-deficient cells andkinase inhibitors, we analyzed the kinases involved in BaP-induced H2AXphosphorylation.Our results showed that only at higher concentrations or longer incubation timesdid BaP affect cell survival. In contrast, BaP significantly affected cell cycle, causingarrest in the S and G2/M phase. BaP inducedγH2AX in a time- and dose-dependentmanner in HeLa cells, which also showed a cell cycle-dependent manner. BaP alsoinducedγH2AX in the different gene-deficient cell lines, and members of the PIKKsfamily, including ATM, ATR, and DNA-PK were involved in this process.In the second part of this dissertation, we used proteomic method analyzed the effects of BaP on nuclear protein expression in HeLa cells. HeLa cells were treatedwith 10μmol.L^-1 BaP for 6, 12, and 24 h. Nuclear protein was then extracted andprotein concentration was determined by Bradford method. After 2-dimensionalelectrophoresis, gel images were captured using a GS-800 scanner and analyzed byPDQuest 7.1 for differentially expressed protein spots. After in-gel digestion, proteinswere identified by liquid chromatography-tandem mass spectrometry.It was found that compared to control, BaP treatment induced expression changesin over 128 proteins, and 24 were successfully identified by mass spectrometry. Twoof them, Lamin A and Bub 3, were further examined by Western blot and Confocalmicroscopy, and thus validated the 2-dimensional electrophoresis results. Among theidentified proteins, several are involved in alternative splicing, suggesting that BaPtreatment could induce alternative splicing. Therefore, we further examined changesin the splicing variants of two genes, Fas and CD44, using RT-PCR, and the resultsshowed that indeed BaP caused changes in the soluble splicing variant of Fas.Taken together, results obtained from above study provided useful information forthe better understanding of the mechanisms for BaP-induced carcinogenesis, and thephenomenon of alternative splicing during DNA damage response is worth furtherstudy.