Abstract 3890: Regulation of mediator of DNA damage checkpoint 1 (MDC1) during mitosis

Abstract

Abstract Genetic instability is one of the hallmarks of human cancers. DNA double strand break (DSB), if improperly repaired, results in genetic instability and tumorigenesis. Previous research has established γH2AX as a bona fide marker of DSBs and the hierarchical foci assembly of DNA damage response (DDR) proteins at DSB sites is required for efficient repair. Recent findings from our group demonstrated that during either spontaneous or induced prolonged mitosis, cancer cells acquire DSBs which lead to further chromosomal abnormality [Dalton et al.(2007) Cancer Res. 67:11487]. Deciphering the mechanism of such DSB accumulation may provide new insights into the mechanism of genetic instability. We hypothesize that the increase of DSBs during prolonged mitosis is due to lack of efficient DDR. To this end, we focus on the protein Mediator of DNA damage Checkpoint 1 (MDC1), an important mediator in DSB repair. MDC1 directly binds to γH2AX and serves as a platform to accumulate/retain downstream DDR proteins at DSB sites and concomitantly initiates cell cycle arrest through its multiple interaction domains. We observed that during mitosis, many DDR proteins fail to colocalize with γH2AX in response to DSBs, while MDC1 exhibits a weakened interaction with γH2AX as shown by immunofluorescent microscopy. Inhibition of Cyclin-dependent kinase 1 (CDK1) activity, either by treatment with a specific CDK1 inhibitor RO-3306 or transient knockdown of CDK1 protein by siRNA, strengthened MDC1-γH2AX colocalization in colon cancer cells in mitosis. In vitro phosphorylation assay confirmed MDC1 as a direct substrate of CDK1. Moreover, a deletion mutant of MDC1 exhibits an increased colocalization with γH2AX compared to wild-type MDC1. Considering the published role of MDC1 as a positive regulator in metaphase-anaphase transition, we propose that phosphorylation of MDC1 by CDK1 promotes a partial disassembly of MDC1 from γH2AX foci during mitosis, in order for MDC1 to function as a modulator of mitotic progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2011-3890