Abstract 568: Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma.

Abstract

Abstract Cell cycle checkpoints prevent cells from dividing with damaged DNA by causing cell cycle arrest or apoptosis. DNA damage-induced cell cycle arrest resulting from treatment with the topoisomerase I inhibitor SN38 can be abrogated specifically in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). However, some p53 wild-type tumor cell lines have also been shown to be sensitive to checkpoint abrogation, suggesting that these cells have other defects in the p53 pathway that can be exploited for therapeutic purposes. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers. Thus, one possible explanation for the sensitivity of p53 wildtype tumors to checkpoint abrogation is a defect in oligomerization. In the current study, we investigate the DNA damage response and UCN-01 sensitivity of three p53 wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, and IMR32. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. All three cell lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25-100 nM UCN-01 for 6 and 24 hours. The SK-N-SH and IMR32 showed only slight sensitivity to the highest concentration of UCN-01 treatment as evidenced by partial abrogation of S and G2 arrest. The SH-SY5Y, however, abrogated S arrest after 6 hours at the lowest concentration of UCN-01 and abrogated G2 arrest after 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possess levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The IMR32 and SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation p53 wildtype neuroblastomas, although more recent studies refute these reports. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53, while in IMR32, p53 was evenly distributed between the nucleus and cytoplasm. The results of this study suggest that oligomerization status alone is insufficient as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor. Citation Format: Robert Lipski, Aime A. Levesque. Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2013-568