The Effects Of P73 In Benzo(a)pyrene-induced Cells Death In MRC-5 And H1299 Cell Lines

Polycyclic Aromatic Hydrocarbons (PAHs) are widely distributed in the environment. Benzo(a)pyrene (BaP) is a environmental carcinogen as a representative member of the class of PAH. It is a byproduct of incomplete combustion or burning of organic compounds released by industrial sources, traffic pollution and household household cooking activities. Numerous epidemiological datas showed that BaP is associated with several tumous such as lung cancer and skin cancer in humans. Experimental datas indicated that the formed BaP-DNA adducts can result in cell cycle arrest and cell death including cell apoptosis and nocrosis, by interrupting the signal pathways of controlling cell growth and prolification.Apoptosis and nercrosis are two forms of cell death. Apoptosis or programmed cell death is a normal component of the development and health of multicellular organisms. Nercrosis refers to cells that are dying, which is a passive process. Cell fate was changed away from apoptosis and towards necrotic death after inhibiting cytokines of regulating apoptotic cell death. The findings suggest that p53-mediated sigling pathways were invoved in regulations of BaP-induced apoptosis and cell cycle arrest. However, the regulating mechanisms of p73 in BaP-induced cell death remains to be determined.p73, as a member of the p53 family, has been attracted much attention because it shares structural and functional similarities with p53. Over-expression of p73 can activate p53 down-modulated target genes involving in regulations of cell cycle and apoptosis in p53-deficient cells. However, there are differences in the functions and the molecular mechanisms for them. Whether p73 mediates BaP-induced cell death in p53-independent mechanisms remains to be determined.In the present study, two cell lines, including the human fetal lung fibroblasts (MRC-5) and human lung carcinoma cells (H1299) were treated with BaP at concentrations of 8, 16, 32, 64 and 128μM. The solvent control groups (final concentration of dimethyl sulfoxide (DMSO) <0.1% plus 3% S9 mixture) and blank control groups were established. At 24 h after the treatment, the cell viabilities were determined by MTT assay. The extent of DNA damage in two cell lines was determined by comet assay. Effects of BaP on the cell growth and cell death were determined using the flow cytometry (FCM). The transcriptional levels p73/p53 and their down- modulated genes involving in regulations of cell death and cell cycle arrest were detected by the real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). The major results are as follows:1. Cell viability assayMRC-5 cells and H1299 cells were treated with BaP at the indicated concentrations (8, 16, 32, 64 and 128μM) for 24 h. The decreased viabilities in the BaP-treated MRC-5 cells were observed except for 8μM BaP group, compared with the solevent control group (P<0.05); the viability was decreased by 30% (P<0.05) in 128μM Bap group. In H1299 cell line, the viabilities were decreased in a concentration-dependent manner in all BaP-treated groups; the viability was decreased by 40% in 32μM BaP group (P<0.05) compared with the solvent control group.2. Single cell gel electrophoresisMRC-5 cells and H1299 cells were treated with BaP at the indicated concentrations (8, 16, 32, 64 and 128μM) for 24 h. the Olive Tail Moment (TM) values were significantly increased in all BaP-treated MRC-5 cells compared with the solvent control group (P<0.05) with a peak value in 64μM BaP group. In H1299 cell line, the Oliver TM values were significantly increased in a concentration-dependent manner (P<0.01).3. Flow cytometryAfter treatment for 24 h, The FACS analysis was performed in both two cells. For MRC-5 cells, the cell cycle was mainly arrested in G1-phase when compared to control group (P<0.05). BaP induced cell necrosis in all treatment groups (P<0.01), but without the increase in the percentage of apoptosis cells. In BaP-treated H1299 cells, the cell cycle was mainly arrested in G2-phase comparing to control group. BaP induced cell necrosis in all treatment groups (P<0.01). Meanwhile, a marked increase in the percentage of necrotic cells in all treatment groups was observed that was greater than the increase in MRC-5 cells at the same concentrations (P<0.05). However, BaP- induced early apoptosis was only observed in 128μM BaP-treated H1299 cells (P<0.05).4. RT-qPCRMRC-5 cells were treated with BaP at the indicated concentrations (8, 16, 32, 64 and 128μM) for 24 h. ATM transcriptional levels were observed in BaP-treated cells with the exception of the 16μM BaPgroup (P<0.05). E2F1and Bcl-2 were observed in 16μM and128μM BaP-treated groups (P<0.05). The transcriptional levels of Puma were up-regulated in 16μM and128μM BaP-treated groups (P<0.05). The marked up-regulations in mRNA were observed for p53, p73 and p21 genes in all Bap-treated groups (P<0.05). But the transcriptional levels of 14-3-3σ, ATR, Bax, Noxa and caspase3 were not detected (P>0.05).H1299 cells were treated with BaP at the indicated concentrations (8, 16, 32, 64 and 128μM) for 24 h. With the exception of 32μM and 64μM BaP groups; the transcriptional levels of ATM and ATR were up-regulated (P<0.05). The transcriptional levels of E2F1, p73 and Puma were up-regulated in all BaP-treated groups (P<0.05). The transcriptional levels of Bcl-2 were up-regulated in the other BaP-treated groups, except for 64μM BaP group (P<0.05). However, the transcriptional levels of Gadd45, Bax, Noxa and caspase3 were not detected in all BaP-treated groups (P>0.05).The findings indicate that under the experimental conditions, BaP caused the decrease of cell variabilities and the obvious DNA damage in the two cell lines. The increased transcriptional level of p73 resulted from BaP-induced DNA damage may be involved in modulation of cell death in a p53-independent manner.