Analysis of molecular mechanism for modification of p53 tumor suppressor protein by anti-cancer drugs

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The aim of this research project is to obtain the information about the effects of anti-cancer drugs, especially vinca alkaloid, which play as a inhibitor of polymerization of microtubules, on the function of p53 superfamily. Head investigator, K. Arai found that β-tubulin isotypes transformed during establishment of drug-resistance for vinca alkaloid in mouse melanoma cell line, B16F10. Normally, expression of-tubulin expression was suppressed by p53 protein and decrease of DNA binding activity of Sp1 and p53 was induced by addition of vinca alkaloid to the culture medium. In 2006, to examine whether vinca alkaloid affect interaction between p53 superfamily and Sp1, in vitro translation system for p53 superfamily and Sp1 was established. Briefly, full length cDNAs corresponding to transactivating forms for p63, p73 and Sp1 in addition to wild-type and mutated p53 were amplified, then subcloned into pcDNA3.1 or pcDNA3.1/V5-His and used for in vitro transcription/translation. These enable to analyze interaction between p53 superfamily and Sp1 and their kinetics. In 2007, forced expression of p53 superfamily genes in p53-deficient mouse macrophage cell line was designed. Furthermore, mRNA distribution of p53 superfamily in the lung was examined by in situ hybridization and ribonuclease protection assay. As a result, mRNA corresponding to transactivating form of p73 was expressed in bronchial and type II alveolar epithelium. These findings indicated that p73 participate lung carcinogenesis.