Abstract 2507: Defective Chk2 expression in cisplatin-resistant ovarian tumor cells impedes wild-type p53 function
Authors: Meraz Ismail M. , Khokhar Abdul R. , Siddik Zahid H. 
Published: February 2011

Abstract
The p53 tumor suppressor in its wild-type state correlates with sensitivity of tumor cells to cisplatin, and mutant p53 with resistance. However, resistance is also a significant impediment in tumors harboring wild-type p53. Identifying the mechanism preventing wild-type p53 from inducing cell death becomes vital in understanding resistance. Our studies in an isogenic sensitive-resistant pair of wild-type p53 ovarian tumor models demonstrate that unlike sensitive A2780 cells, the ∼25-fold resistant 2780CP variant cells do not upregulate p21 at 24 hr in response to cisplatin (1 or 5 µM). Since defects in wild-type p53 and chk2 are reported to be mutually exclusive and Chk2 defects are known to impact p53 function, we investigated chk2 expression in the model pair. By immunoblot analysis, A2780 cells robustly expressed Chk2, whereas levels were undetectable in resistant cells. Real-time PCR demonstrated an absence of Chk2 transcripts in resistant cells, and since chronic exposure to decitabine (1 or 5 µM for 5, 7 or 9 days) failed to upregulate Chk2, this suggested that loss of Chk2 expression in 2780CP cells is likely due to a non-epigenetic mechanism. In order to test whether loss of Chk2 indeed contributes to loss of p53 function and also loss of sensitivity to cisplatin, A2780 cells were exposed to Chk2 siRNA (100 nM for 5 hr), which reduced Chk2 levels by &gt;75%. Exposure of siRNA-treated cells to cisplatin (1.0 µM) followed by immunoblot analysis 24 hr later demonstrated an attenuated p21 expression. Similarly, cytotoxic assessment by the MTT assay provided evidence that Chk2-knockdown in A2780 cells increased resistance to cisplatin by about 2-fold. Since the level of cisplatin resistance in 2780CP cells and other similar models is much greater than those seen in resistant cells harboring mutant- or null-p53, we tested the concept that p53 knockdown by siRNA in Chk2-deficient 2780CP cells may actually sensitize these cells. Support for this was provided by a 2-fold decrease in the IC50 of cisplatin. In contrast, the IC50 of cisplatin increased by about 2-fold in p53-knockdown A2780 cells. If Chk2-deficiency contributes to cisplatin resistance in tumor cells harboring wild-type p53, then specific non-cross-resistant platinum analogs must be activating p53 and inducing cell killing in a Chk2-independent manner. This was confirmed by the demonstration that the platinum(IV) analog DAP increased levels of p21 in Chk2-knockdown A2780 cells without loss of cytotoxic potency, and that knockdown of p53 in either sensitive or resistant cells increased resistance to the analog by about 2- to 3-fold. These results demonstrate the relative significance of Chk2 in the mode of action of cisplatin and the analog DAP. (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 2507.