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PRECLINICAL STUDIES ON ATM KINASE INHIBITORS AS ANTI-CANCER AGENTS

Choi, Serah (2011) PRECLINICAL STUDIES ON ATM KINASE INHIBITORS AS ANTI-CANCER AGENTS. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Choi, Serahchoi.serah@medstudent.pitt.educhoi.serah@medstudent.pitt.edu
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSteinman, Richard Asteinman@pitt.edusteinman@pitt.edu
Committee MemberVan Houten, Bennettvanhoutenb@upmc.eduvanhoutenb@upmc.edu
Committee MemberBakkenist, Christopher Jbakkenistcj@upmc.edubakkenistcj@upmc.edu
Committee MemberYu, Jianyuj2@upmc.eduyuj2@upmc.edu
Committee MemberConrads, Thomasconrads@whirc.orgconrads@whirc.org
Date: 4 August 2011
Date Type: Completion
Defense Date: 25 July 2011
Approval Date: 4 August 2011
Submission Date: 3 August 2011
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Pharmacology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: ATM; DNA damage; DNA repair
Other ID: http://etd.library.pitt.edu/ETD/available/etd-08032011-145452/, etd-08032011-145452
Date Deposited: 10 Nov 2011 19:56
Last Modified: 04 Aug 2016 05:15
URI: http://d-scholarship.pitt.edu/id/eprint/8872

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