Alt Actives Dna Polymerase Overexpression Via Special Signaling Pathway Of Camp-pka-creb

Background and objective:It is well known that the main function of DNA polymeraseβ(DNA polβ) is repairing DNA damages in the base excision repair.Under normal conditions,DNA polβis expressed at a constant low level throughout the cell cycle.In our previous studies,the mutations and overexpression of DNA polβcould increase genetic instability and the mutationrate by interfering other DNA polymerases,furthermore induce the tumor.Altenuene(ALT) is one mainly metabolite of the carcinogenicity of fungi isolated from the grain in the area of high incidence of esophageal carcinomaour in Henan province which called Alternaria alternate.Whether it can damage DNA directly and increase the transcription of DNA polymerase-βgene is not very clearly. In the previous research of the signaling pathway,together with other observations, someone found that the coincidence of cAMP-PKA activation and the CREB phosphorylation after 60 min treatment indicated low concentration MNNG activate DNA polymerase-βexpression via cAMP-PKA-CREB pathway.This result was consistent with what has been supposed that cAMP-PKA pathway might play a role in cellular response to alkylating agents and mediate the induced polβexpression.So in our study,we summarized the transcription of DNA polymerase-βgene's regulation and effect through the cAMP signal pathway and some t ranscription factors influenced by the cAMP pathway,after ALT stimulating. Method:NIH/3T3 cells were cultured in the medium supplemented with ALT at different concentrations and MMS for positive control,then were observed in the level of cAMP by ¹²⁵I-cAMP RIA,the expression of DNApolβmRNA by RT-PCR,and the expression of the protein of active catalytic subunits of PKA,p-CREB and DNApolβby Western Blotting and immunocytochemistry.Result:1.The expression of DNApolβmRNA and protein was increased both in the experimental group and in the positive control group.(P＜0.05)2.After NIH/3T3 cells were exposed to different concentrations of ALT for 30min, the cellular level of cAMP was significantly increased in a concentration-dependent, and in positive control group the level of cAMP was increased the same(P＜0.05).3.The contents of protein of active catalytic subunits of PKA was increased both in the experimental group and in the positive control group.When H89 was added in the experimental group,the increasing level was decreased.(P＜0.05)4.The contents of protein of p-CREB was increased both in the experimental group and in the positive control group.When H89 was added in the experimental group,the increasing level was decreased.(P＜0.05)Conclusion:1.When NIH3T3 cells are exposed to ALT,the cellular level of cAMP and the expressions of p-CREB and PKA are both increased.2.These results are consistent with what has been supposed that cAMP-PKA pathway might play a role in cellular response to ALT agents and mediate the induced polβexpression.