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Access Type

WSU Access

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Ashok S. Bhagwat

Abstract

Abasic (AP) sites are the most common type of lesions in DNA. Numerous endogenous and exogenous agents and cellular processes can induce the formation of AP sites in DNA. If left unrepaired, the deleterious AP sites cause mutagenesis and cytotoxicity. Methoxyamine is known to react with AP sites and block base excision repair. Another alkoxyamine, aldehyde-reactive probe (ARP) tags AP sites with a biotin and has been widely used to quantify these sites. In this study, I have combined both these abilities into one alkoxyamine, AA3, which reacts toward AP sites with better reactivity than ARP at physiological pH. Additionally, AA3 containing an alkyne functionality is able to tag AP sites with a biotin and a fluorescent molecule through click chemistry. AA3 is used for AP site quantification with greater sensitivity and lower background than ARP. AA3 also inhibits the first enzyme in the repair of AP sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of alkylating agents to kill cancer cells and is more efficient in such combination chemotherapy than methoxyamine.

The majority of B-cell malignancies originate from germinal centers, where the processes of somatic hypermutation (SHM) and class switch recombination (CSR) for antibody maturation are initiated by activation-induced deaminase (AID). AID deaminates cytosines in DNA creating uracils. It has been previously shown that B-cell lymphoma cell lines and patient tumors express AID at high levels and contain high levels of uracils in their genome. These cells also contain uracil-DNA glycosylases that remove uracils forming AP sites. I have demonstrated that B-cell lymphoma cell lines contain much higher levels of AP site accumulation than normal B cells and non-B cell lines. AP sites are able to react with alkoxyamines forming a stable oxime. This principle was used to block the repair of an excess of AP sites in B-cell cancers by alkoxyamines. AA3 effectively kills B-cell lymphoma cell lines that show high levels of endogenous AP sites. In contrast, AA3 is not toxic to normal human B cells, as well as non-B cell lines. However, neither MX nor ARP displays cytotoxicity in these B-cell cancers, although both of them can target to AP sites. Additionally, AA3 links covalently to B-cell cancer genome, and AA3-DNA adducts cause cell death by blocking DNA replication and elevating DNA strand breaks. To determine which functionality of AA3 is responsible for its cytotoxicity, I designed and synthesized a series of AA3 analogs. Using this approach, I have demonstrated the alkyne functional group is required for AA3 toxicity in B-cell cancers. Overall, this new family of chemicals could be further developed as novel anti-cancer drugs.

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