Objective To explore the role of ubiquitin protein ligase RING2 in the changes of BPDE-DNA adducts and checkpoint kinase 1 (CHK1) in human bronchial epithelial cells (BEAS-2B) exposed to benzo (a) pyrene (BaP).
Methods BESA-2B cells were exposed to BaP at concentrations of 0, 1, 2, 4, 8, 16, and 32μmol/L for 24h, respectively; BESA-2B cells were exposed to 16 μmol/L BaP for 0, 1, 2, 4, 8, 12, and 24 h, respectively. The expressions of RING2 were inhibited by small in terfering RNA (siRNA) in BEAS-2B cells, then the BESA-2B cells and BESA-2B (siRNA-RING2) cells were exposed to 16μmol/L BaP for 24 h. BPDE-DNA adducts were evaluated by fluorescent immunohistochemistry. The levels of CHK1 and CHK1 S345-p were detected by Western blot.
Results Compared with the normal control group, the fluorescence intensities were significantly elevated both in the BESA-2B cells and the BESA-2B (siRNA-RING2) cells exposed to BaP (P < 0.01), indicating elevated levels of BPDE-DNA adducts, and the fluorescence intensity of BESA-2B (siRNA-RING2) cells were increased by 32% compared with the BESA-2B cells. According to results of Western blot, the relative expression levels of CHK1 and CHK1 S345-p in BESA-2B cells were significantly elevated along with increasing concentration and exposure time (P < 0.01); the levels of CHK1 and CHK1 S345-p in BESA-2B (siRNA-RING2) cells exposed to 16 μmol/L BaP for 24 h were significantly decreased compared with the normal control group (P < 0.01). The results of factorial analysis showed both transfection and BaP treatment had impacts on the expression levels of CHK1 and CHK1 S345-p, as well as interactions between these two factors (P < 0.01). Compared with the BESA-2B cells, the difference between levels of CHK1 and CHK1 S345-p in BESA-2B (siRNA-RING2) cells exposed or non-exposed to BaP decreased by 54% or 51%. The results of covariance analysis showed that the estimated means of the levels of CHK1 and CHK1 S345-p (0.77 and 0.89 respectively) in BESA-2B (siRNA-RING2) cells were significantly lower than those in BESA-2B cells (1.24 and 1.32 respectively) (P < 0.01) with control of BaP exposure.
Conclusion The DNA of siRNA-RING2 cells are much more sensitive to BaP exposure than that of normal cells, which in dicates that RING2 may be involved in DNA repair by affecting the expression of CHK1 and CHK1 S345-p.
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