Molecular Mechanism Of JWA Involved In DNA Damage And Repair After Benzo(a) Pyrene Exposure

Human health and diseases usually result in interactions between environmental factors and individual genetic susceptibility. An increasing number of environmental responsive genes are cloned and found to be related to human diseases. To investigate the interactions, we could elucidate the molecular mechanism of environmental-induced diseases. Benzo[a]pyrene, B(a)P, a prototypical member of this class of chemicals, has been extensively studied for its toxic effects in laboratory animals and human populations. B(a)P toxicity is often mediated by oxidative metabolism to reactive intermediates that interact with macromolecules leading to alterations in target cell structure and function. Benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE), a putative carcinogenic metabolite of B(a)P, is known to form DNA adducts with the N position of guanine. Metabolism of B(a)P can produce ROS in the form of O₂, OH, OH^-, H₂O₂, all of which are capable of incurring some form of injury whenpresent in high levels in the cell. If the damage couldn't be repaired promptly, these damaged DNA will result to mutation in the next replication, finally the accumulation of mutation will lead to cancer.In 1998, using the differential display high-throughput technique, Zhou et al cloned a novel environmental responsive gene from human bronchial epithelial (HBE) cells, named JWA, which is broadly distributed in most of examined eukaryotic cells and tissues. The intracellular distribution of the protein is very similar to that of cytoskeleton under immunofluorescence microscope. JWA might play an important role during cell mitosis. The gene expressions are modulated by some cell differentiation inducers, such as all trans retinoic acid (ATRA), 13-cis-retinoic acid and 12-O-tetradecanoyl-phorbol-13 acetate (TPA). Our recent studies showed that JWA is also a functional protein associated with cell differentiation and apoptosis, and might play an important role in development of acute promyelocytic leukemia (APL). JWA gene may be an important signal molecule that widely involves in various environmental stresses, such as heat stress, oxidative stress et al. In this study, we firstly establish the models of DNA damage of ccc-HPF-1 cells by treatment of the cells with B(a)P plus S9. Secondly we examined the expression pattern of JWA and hsp70 during the DNA damage and repair. Further more, we explored the possible mechanisms of JWA involving in DNA damage and repair.1. Establishing the models of DNA damage of ccc-HPF-1 cells by treatment of the cells with B(a)PThe models of DNA damage of ccc-FIPF-1 cells were established by treatment of the cells with B(a)P plus S9 at 10 to 100 μM for 3 hours. The DNA damage was detected by single cell gel electrophoresis assay (SCGE) or in other words, the comet assay. We found that with the dose exposed to B(a)P increasing, the DNA damagewas more and more serious after 3 hours, having a good dose-dependent relationship.2. JWA expression pattern during the DNA damage and repairThe immuno-blotting assay was used for detecting expressions of JWA and hsp70. JWA expression was actively modulated by B(a)P exposure. The expressions of both JWA and hsp70 were increased greatly at B(a)P treated cells and maintaining at over expressed levels during the restoring time. In addition, JWA expression pattern was very similar to that of hsp70. However, we failed to detect another heat shock protein 27 whenever exposed to B(a)P for 3h or restoring 24 hours. In order to confirm JWA involved in DNA damage and repair, pEGFP-Cl-asJT%( expression vector was reconstructed, and then was transfected into HeLa cells. As a result, the JWA knock down HeLa cells (KD-HeLa) have been stable screened by G418. The KD-HeLa cells and controlled HeLa cells were incubated for 3h with activated B(a)P at concentration of 50uM, then with 30, 60, 120, 180min and 24 hours recovery for DNA repairing. We found that the KD-HeLa cells were more damaged than the controlled HeLa cells after exposing to activated B(a)P 3 hours and the rate of repairing...