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DNA glycosylases as modulators of chemotherapeutic response

Svilar, David (2012) DNA glycosylases as modulators of chemotherapeutic response. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Median survival is less than two years due to several factors, including challenges in surgical removal and chemotherapy resistance, underlining the need for more effective therapeutic options. To identify genes that contribute to chemotherapy resistance, we conducted a synthetic lethal screen in a chemotherapy-resistant GBM derived cell line (T98G) with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. This screen for TMZ-sensitizing genes indicated that a subset of genes that were over-expressed in GBM cells increased the cell’s sensitivity to TMZ when knocked down.
An ubiquitin ligase, UBE3B, and a DNA glycosylase, UNG, were among the TMZ-sensitizing genes identified using the siRNA library. We demonstrate that UBE3B and UNG are sensitizing genes in the screen validation studies using unique siRNA and shRNA sequences. Although UNG is one of four human DNA glycosylases that remove uracil lesions, UNG was the only uracil removing glycosylase to sensitize GBM cells in the validation studies. Notably, analysis of archived transcription datasets revealed that over-expression of UNG was correlated with poor outcomes in glioma patients.
In order to uncover functional groupings of TMZ-sensitizing proteins, we conducted in situ pathway analysis of gene candidates for synthetic lethal functions from our screen. This analysis discovered statistically significant enrichment of ontogeny clusters related to base excision repair (BER), response to DNA damage, cellular proliferation and protein modification. Interestingly, this pathway topography overlapped with TMZ-sensitizing genes identified from similar experiments in yeast and bacteria.
In order to facilitate rapid in vitro identification of lesion-specific repair activity in cancer cells, we developed a novel fluorescent assay that extends the state of the art. The molecular beacon assay measures real-time DNA repair rates of specific DNA lesions by defined DNA repair proteins. These studies reveal that GBM up-regulates several TMZ-sensitizing genes that correlate with poor patient survival and inhibiting these genes may increase TMZ cytotoxicity in a tumor specific manner. These TMZ-sensitizing genes are not only potential targets for adjuvant therapy, but also represent potential biomarkers to predict TMZ response.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Svilar, Davidsvilar.david@medstudent.pitt.eduDAS98
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorSobol, Robertrws9@pitt.eduRWS9
Committee ChairSteinman, Richardsteinman@pitt.eduSTEINMAN
Committee MemberPagano, Patrickpagano+@pitt.eduPAGANO
Committee MemberOkada,
Committee MemberBakkenist, Christophercjb38@pitt.eduCJB38
Date: 19 April 2012
Date Type: Publication
Defense Date: 16 March 2012
Approval Date: 19 April 2012
Submission Date: 3 April 2012
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 231
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: DNA Repair, Temozolomide, Alkylation Response, Chemotherapy Resistance, Molecular Beacon
Date Deposited: 02 Jul 2013 20:04
Last Modified: 15 Nov 2016 13:57


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