Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Michael D Wyatt

Abstract

Cancer is a deadly manifestation of diseases in which abnormal cells undergo uncontrolled proliferation and can invade nearby tissues. Despite the recent advances in cancer drug discovery, traditional chemotherapeutics such as 5-fluorouracil (5-FU) remain one of the most widely used agents for management of cancer in clinic. Currently, 5-FU is approved in USA for therapy of various cancers including that of colorectal and breast. Pharmacological actions of 5-FU are mediated by intracellular conversion to its active metabolites that inhibits thymidylate synthase (TS), thus depriving cells of thymidylate and subsequently increasing the uracil (U) content. Alternatively, 5-FU metabolites can also cause DNA damage by direct incorporation into DNA or RNA. Incorporation of U and 5-FU activates the DNA glycosylase-initiated base excision repair (BER) in cells to remove the damaged base and replace it with a correct base (i.e. T). However, lack of thymidylate could lead to re-introduction of U and 5-FU leading to futile BER cycles. It is commonly hypothesized that accumulation of DNA strand breaks from futile cycles of BER commits the cells to apoptosis. However, the involvement of specific DNA glycosylases in the formation and resolution of strand breaks is uncertain.

In the work presented here, we utilized cell lines with short hairpin RNA (shRNA) mediated knockdown of either uracil-DNA glycosylase-2 (UNG2) or single-strand-selective monofunctional uracil-DNA glycosylase-1 (SMUG1), both of which have been implicated in the repair of U and 5-FU lesions. Interestingly, in spite of a decreased efficiency of UNG2 or SMUG1-depleted cells to remove 5-FU from a synthetic oligonucleotide, loss of UNG2 did not affect the cytotoxicity of 5-FU. However, loss of SMUG1 corresponded with >2-fold increase in sensitivity to 5-FU. Whereas the wild-type (WT) cells have the ability to repair the 5-FU-induced DNA damage, UNG2 as well as SMUG1-depleted cells show an increased formation of BER intermediates in the form of single-strand breaks and abasic sites. Interestingly, only the SMUG1-depleted cells show a significant increase in the lethal DNA double-strand breaks (DSBs) formed following 5-FU treatments. Overall, results from this study indicate that expression of SMUG1 is a key determinant to the downstream DNA damage signaling and cell death consequences of 5-FU. It suggests that SMUG1 might present a good target for inhibition during 5-FU therapy in clinic.

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