The Effect Of Human Tumor Suppressor P53 On Apoptosis In Yeast

Objective:To investigate the effect of human tumor suppressor p53 on apoptosis by heterogenously expressing p53 in yeast.Methods: The wild type gene of human tumor suppressor p53(TP53)was amplified by using cDNA as PCR template that was reversely transcribed from total RNA extracted from the MCF-7 breast cancer cell line(wild-type p53).The TP53 was subcloned into the galactose-induced yeast expression plasmid pRS426Gal1/10.Mutations in p53(R248W?G245S?R273H and R249S)were constructed using site-directed mutagenesis.Both alleles of wild-type and mutated TP53 were confirmed by DNA sequencing and were expressed in yeast.The growth and apoptotic phenotypes of yeast cells carrying wild-type and mutated TP53 were investigated.The expressions of p53 were assayed using Western blot at different time points of induction.The apoptosis was detected by using Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and Annexin V-FITC/PI assay.Results:The inhibition of yeast growth was observed after induction of wild type p53 expression at 3rd hour.p53 was detected at 9th of induction.The growth inhibition was more apparent with the induction times and the yeast cells presented typical phenotypes of apoptosis.However,expressions of mutated p53(R248W?G245S?R273H and R249S)did not significantly modified the apoptosis and growth of yeast cells.Conclusion: Human tumor suppressor p53 may be heterogenously expressed in yeast,which may indicate the p53-dependent apoptosis according to the growth inhibition of the yeast cells.