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Abstract: Environmental carcinogen benzo(a)pyrene (BaP) can damage DNA by forming bulky adducts that are degraded further to DNA strand breaks, thus contributing to induce DNA damage checkpoint response. Claspin is a critical checkpoint protein in response to multiple forms of genotoxic stress including UV, IR and hydroxyurea (HU). In the present study we have investigated the role of human Claspin in the DNA damage checkpoint elicited by BaP in 16HBE cells. We observed that Claspin levels are increased in a time-dependent manner in response to S-phase arrest induced by BaP. In addition, the levels of phosphorylation of Chk1 on S345 were increased, but the levels of Cdc25A were decreased after treatment with BaP. Inhibition of Claspin expression (siRNA) attenuated the effect of BaP on S-phase arrest and abrogated the activation of Chk1 and degradation of Cdc25A in response to BaP. Taken together, these data imply that Claspin plays an important role in S-phase checkpoint induced by BaP, and it participates in the activation of Chk1 and Cdc25A in this checkpoint pathway. [Copyright &y& Elsevier]

Abstract: Our previous results have indicated that Cdc25A is involved in benzo(a)pyrene (BaP)-induced S-phase checkpoint in 16HBE cells and A549 cells. In this paper, we reported the changes of the downstream molecular pathway of Cdc25A and the effects of over-expression and suppression of Cdc25A on BaP-induced S-phase checkpoint. In the S-phase checkpoint induced by BaP the reduction of Cdc25A contributes to cyclin A inhibition. Over-expression of Cdc25A abrogated BaP-induced S-phase arrest in 16HBE cells and concomitantly the expression levels of Cdk2 and cyclin A were not obviously changed by BaP when compared with the control. Cdc25A down-regulation by RNA interference (RNAi) prolonged the S-phase arrest induced by BaP and decreased clearly the expression levels of cyclin A and cyclin E. Therefore, our results further demonstrated that Cdc25A was an effector in Chk1–Cdc25A–cyclin A/Cdk2 pathway of S-phase checkpoint elicited by the carcinogen BaP in 16HBE cells. [Copyright &y& Elsevier]

Abstract: Environmental carcinogen benzo(a)pyrene (BaP) generates electrophilic products BaP diolepoxide (BPDE) that react covalently with genomic DNA. Cells that acquire BaP/BPDE-induced DNA damage undergo S-phase arrest in a p53-independent manner. However, the role of Cdc25A in the BaP/BPDE-induced checkpoint is not clear. In the present study, we investigated the change of checkpoint kinase 1 (Chk1) and Cdc25A in S-phase arrest elicited by BaP. The results indicated that BaP (10μM, with S9 mixture) treatment induced S-phase arrest in both human lung carcinoma A549 cells and human bronchial epithelial cells line 16HBE cells, increasing the proportions of cells in S-phase 19.0% and 21.1%, respectively, at 12h after treatment, compared with DMSO control (p <0.01). Then, the S-phase arrest was weakened after 24h. The level of phorsphorylated Chk1 obviously increased and Cdc25A protein level decreased in both two cell lines after treatment with BaP. The results of RT-PCR indicate Cdc25A mRNA in both A549 cells and 16HBE cells was not changed after BaP treatment 12h, and 24h. The treatment of the proteasome inhibitor MG132 greatly increased Cdc25A protein in abundance. Over all, our results indicated Chk1–Cdc25A checkpoint pathway is involved in BaP-induced S-phase arrest. Moreover, transcription of Cdc25A did not change in BaP induced S-phase arrest, the decrease of Cdc25A level was due to increased degradation through the ubiqutin–proteasome pathway. [Copyright &y& Elsevier]