Date of Award

1-1-2010

Document Type

Campus Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Michael D Wyatt

Second Advisor

Douglas L Pittman

Abstract

Methylating agents are widely used in chemotherapy and kill cancer cells by causing DNA damage. The O6-methylating anticancer drugs procarbazine, dacarbazine, and temozolomide generate O6-MeG adducts, while a thiopurine antimetabolite 6-thioguanine (6-thioG) generates S6-MeG DNA adducts. Instead of pairing with cytosine, these modified guanines mispair with thymine during DNA replication. The Mismatch repair (MMR) pathway rectifies base mismatches. However, MMR is unable to correct these S6-MeG:T or O6-MeG:T mispairs. In fact, upon recognition of the lesions, MMR initiates a futile repair cycle resulting in apoptosis. Thus, MMR is required for the efficacy of these drugs. MMR processing of the mispaired guanines is known to invoke another DNA repair pathway known as homologous recombination (HR), but the coordination among the two pathways in resolving these lesions or eliminating damaged cells is not completely understood.

During the course of this work, I generated a mammalian system to investigate the interplay between MMR and HR in response to methylating agents and 6-thioG. Cells deficient for HR alone were 5-fold more sensitive to MNNG (N- methyl-NOe-nitro-N-nitrosoguanidine), a prototypical methylating agent, and 6-thioG. Interestingly, loss of MMR alleviated the sensitivity of HR-deficient cells, which suggests that MMR dependent processing generates damaged DNA intermediates that are recombinogenic. Thus, HR contributes to the repair of the damage caused by methylating drugs and thiopurines. Unsuccessful attempts of MMR to remove the mismatch create gaps in the daughter strand generating substrates for HR. In HR-deficient cells, lethal double-stranded breaks accumulate as a result of the inability to process the damage.

The requirement of HR to process damage induced by methylating agents and thiopurines implies that inhibition of HR would increase the efficacy of these agents. However, HR deficiency resulted in deleterious events following treatment with these drugs, specifically the formation of radial chromosomes, aneuploidy and multinucleation. This study suggests that HR-insufficiency would likely present a risk factor for the development of secondary cancers resulting from methylating chemotherapeutic and thiopurine treatment. Therefore, the novel findings presented here provide insight into the clastogenic mechanisms of methylating chemotherapy drugs and thiopurines.

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