Status of p53 phosphorylation and function in sensitive and resistant human cancer models exposed to platinum-based DNA damaging agents.

Objective: Resistance to chemotherapeutic drugs is a hallmark of many human cancers, which can occur independent of p53 gene status; however, the presence of wild-type p53 in chemorefractory tumors confers greater resistance to cisplatin, but such tumors do not display complete cross-resistance to the platinum analog (1R,2R-diaminocyclohexane)(trans-diacetato)(dichloro)platinumIV (DACH-Ac-Pt). In this article we examine DNA damage-induced phosphorylation of p53 and downstream p53-dependent transactivation events in cisplatin-sensitive and cisplatin-resistant human cancer cell lines possessing wild-type p53.

Methods: Western-blot analysis was utilized to study the effect of cisplatin and the analog on p53 phosphorylation and p53-dependent target genes.

Results: In response to CDDP and DACH-Ac-Pt, both CDDP-sensitive and CDDP-resistant models demonstrated time- and dose-dependent inductions of total p53 protein and an increase in Ser-15 phosphorylation, which was more pronounced with CDDP. Although phosphorylation of p53 at Ser-392 was also observed in CDDP-treated sensitive and resistant cells, it was weak or absent in response to DACH-Ac-Pt. Lack of Ser-392 phosphorylation by DACH-Ac-Pt, however, did not affect the induction of p21(WAF1/CIP1) or Mdm2. Similarly, inductions of p21(WAF1/CIP1) and Mdm2 were observed in sensitive cells exposed to cisplatin. In marked contrast, cisplatin-mediated induction of p21(WAF1/CIP1) was minimal or absent in resistant cells, but that of Mdm2 was unaffected. Wortmannin, a PI3-kinase (PI3-K) inhibitor, caused a dose-dependent inhibition of total p53 accumulation, Ser-15 phosphorylation and p21(WAF1/CIP1) transactivation in response to both CDDP and DACH-Ac-Pt, indicating that members of the PI3-K family are involved in phosphorylation of p53 and that transactivation of p21(WAF1/CIP1) is p53 dependent.

Conclusions: These studies demonstrate that cisplatin and DACH-Ac-Pt differentially phosphorylate p53 through independent DNA damage-induced pathways, and that the kinase-mediated phosphorylation of p53 at Ser-15 or Ser-392 is unaltered in resistance. Moreover, the phosphorylation status of Ser-392 on its own does not appear to correlate with p21(WAF1/CIP1) or Mdm2 induction in these studies; however, a lack of increase in p21(WAF1/CIP1) by cisplatin, but not DACH-Ac-Pt, provides a correlation with resistance and its circumvention, and implicates the role for cyclin-dependent kinase inhibitor in the differential cytotoxic effects of the two platinum agents against resistant cells.