Abstract 3513: The underlying basis for divergent cell cycle kinetics following checkpoint activation by cisplatin and a platinum (IV) analog

Abstract

Abstract Cisplatin interacts with its macromolecular target to form DNA cross-links, and this DNA damage activates cell cycle checkpoints to induce first a transient S-phase cell cycle arrest, which is followed by a durable G2/M arrest. In contrast, DNA damage by the analog DAP [1R,2R-diaminocyclohexane-(trans-diacetato)-(dichloro)-Pt(IV)] induces primarily a G1-arrest, with no appreciable arrest in S-phase or at G2/M. We have examined the underlying basis for these differences between the platinum drugs in ovarian A2780 tumor cells, which are equally responsive to the two drugs, as seen in comparable upregulation of the p53/p21 pathway beginning at about 6 h after drug treatment at the 5 x IC50 level. The differences in cell cycle arrests in S and G2/M were consistent with the onset of cisplatin-mediated inhibition of CDK2/cyclin A in S-phase at about 12 h and of Cdc2/cyclin B in G2-phase at about 12-18 h that were not observed with DAP. Inhibitions of these CDKs by cisplatin were not mediated by p21, but were ascribed to activating phosphorylations of Chk1 (at Ser-345) and Chk2 (at Thr-68), which were first detected at 6 h after drug treatment and increased progressively thereafter for the next 24 h. Activations of Chk1 and Chk2 would be expected to downregulate Cdc25 phosphatase activity and, thereby, maintain these CDKs in their inhibitory phosphorylated form. For direct evidence, levels of phospho-CDKs were assessed by immunoblot analysis, and shown to increase by 12 h after exposure of cells to cisplatin. As would be anticipated from a lack of S- or G2/M-arrest, Chk1 and Chk2 were not activated by DAP. As further support for independent effects of cisplatin and DAP on Chk1 and Chk2, knockdown of Chk1 or Chk2 by siRNA attenuated activation of the p53/p21 pathway with cisplatin, but not with DAP. With respect to G1-phase CDKs, the effects of the two drugs were surprisingly similar. Thus, both cisplatin and DAP potently inhibited CDK4/cycin D1 and CDK2/cyclin E activities in a p21-dependent manner at ∼18 h after drug treatment. This strongly suggests that the differential effect of the two drugs in arresting cells in G1-phase must be due to the earlier effects of cisplatin in S- and G2/M-phase. That is, cisplatin activates all three checkpoints, but the sequential response, first in S-phase and then robustly at G2/M, effectively blocks cells from progressing further and accumulating in G1. The results not only provide a mechanistic understanding for differential cell cycle kinetics induced by the two platinum drugs, but also serve to demonstrate that inhibition of G1-phase CDK is a more accurate index of G1 checkpoint response than is cell cycle arrest. (Supported by NCI RO1 CA127263). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3513.