Ataxia telangiectasia-mutated (ATM) is a serine/threonine protein kinase that has critical functions in the cellular responses to DNA damage, including cell cycle checkpoint activation and DNA repair. Since ataxia telangiectasia individuals, who have homozygous mutations in ATM, are exquisitely radiosensitive there is considerable interest in inhibiting the kinase activity of ATM to increase the efficacy of targeted radiotherapy. In this dissertation work, I sought to understand the cellular responses to radiation when ATM kinase activity is transiently inhibited using the small molecule ATM kinase inhibitor KU55933. During my PhD, our laboratory has shown that transient ATM kinase inhibition one hour post-irradiation results in radiosensitization, increased chromosome aberrations and abrogation of sister chromatid exchange. I contributed to these findings by showing that the cellular radiosensitization seen in H460 cells with kinase-inhibited ATM was identical to that seen when ATM protein was disrupted using siRNA prior to the insult. In addition, I demonstrated that 15 minutes of ATM kinase activity post-irradiation is sufficient to trigger the G2/M cell cycle checkpoint, and that subsequent transient inhibition of ATM with KU55933 does not affect recovery from this checkpoint. To gain a more global view of the functional consequences of kinase-inhibited ATM following irradiation, I utilized a SILAC-based tandem mass spectrometry approach, combined with a subcellular fractionation protocol, to determine ATM kinase-dependent spatial proteome dynamics in response to radiation-induced DNA damage. Analysis of the chromatin-associated proteome revealed that the retention of 53BP1 at chromatin is decreased when the kinase activity of ATM is inhibited following ionizing radiation (IR). Using fluorescence recovery after photobleaching in live cells, I determined that the stability of IR-induced GFP-53BP1 foci is decreased when the kinase activity of ATM is inhibited following IR. These results provide a roadmap for understanding ATM kinase-dependent spatial protein dynamics in response to DNA damage.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08032011-145452 |
| Date | 04 August 2011 |
| Creators | Choi, Serah |
| Contributors | Thomas Conrads, PhD, Christopher J. Bakkenist, PhD, Richard A. Steinman, MD, PhD, Bennett Van Houten, PhD, Jian Yu, PhD |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08032011-145452/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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