Construction Of Vectors Containing Hsp70 And P53 And The Colocalization Of Hsp70 And P53 In HepG2 Cells

HSPs are series important stress proteins that take important roles in protecting cells against damaging. It was found that HSPs were correlated with the occurrence and development of the tumors in many studies. High expressions of hsp70 were detected in many tumors. Hsp70 participates in the process of tumor development and it plays the role of anti-tumor cells apoptosis through combining with the production of oncogene and tumor suppressor gene. The development of tumor is a complex multi-step process involved multiple gene. Tumor suppressor genes may be an important protective mechanism against tumors. As long as they have a complete activity, they can be effectively inhibited the occurrence of tumor. Point mutations in the p53 gene were detected inï¹¥50% of the most common tumors. HSPs may form complex with various oncoproteins and participate in the process of cellular transformation. It was reported that the hsp70-p53 complex have been detected in breast carcinoma, cervical cancer, osteosarcoma. Hsp70 and mutational p53 have a certain degree of relevance. This is probably one of the reasons for extending the half-life of mp53. Hsp70 participate in cell cycle regulation and proliferation of tumor cells through impacting mp53. We have detected high expressions of hsp70 in HCC, and a part of hsp70 and p53 were colocalized in nuclei. We have already proved their combination by co-immunoprecipitation.We synthesized p53 mutants 249, 273 with site-directed mutagenesis and constructed the relative GFP expression vectors, transfected HepG2 cells and then detected the co-localization of p53 and hsp70. We have found p53 and hsp70 positive products were colocalized in nuclei of human HCC, while the endogenous hsp70 were colocalized in cytoplasm with 273H p53 in HepG2 cells. The different localization of p53 may be related to the protein structure to cover the C-terminal nuclear localization signal of p53 after the site-directed mutagenesis. As a kind of molecular chaperon, hsp70 has the different colocalization and different point mutational p53. Furthermore, hsp70 was colocalized in cytoplasm with 273H p53 in HepG2 cells. But they mainly localized in the nucleus in HCC, these results were contradictory. This contradictory phenomenon suggests that perhaps there are unclear mechanisms existed between the relationship of hsp70 and p53. To further study the impact of hsp70 on biological behavior of HepG2 cells and the relationship between hsp70 and p53, we constructed recombinant adenovirus vector containing heat shock protein 70 gene with Adeasy Vector System and lentivirus vector containing shhsp70. Furthermore, we have succeeded in packaging adenovirus containing heat shock protein 70 gene and constructed FG12-shhsp70 lentivirus vector. The construction of these tools lay a good basis for further study.