Overexpression of
N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize
tumor cells to the cell-killing effects of
temozolomide, an imidazotetrazine-class chemotherapeutic
alkylating agent. In the present study, we show that both elevated MPG expression and
short hairpin RNA-mediated loss of
DNA polymerase beta (Pol beta) expression in human
breast cancer cells increases cellular sensitivity to
temozolomide. Resistance to
temozolomide is restored by complementation of either wild-type human Pol beta or human Pol beta with an inactivating mutation specific to the polymerase active site yet functional for 5'-deoxyribose-phosphate (5'
dRP) lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in base excision repair (BER), leading to an accumulation of cytotoxic 5'dRP lesions, and that the 5'
dRP lyase activity of Pol beta is required to restore resistance to
temozolomide. These results imply that Pol beta-dependent 5'
dRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as
N7-methylguanine and
N3-methyladenine. Furthermore, we find that 5'dRP-mediated cell death is independent of
caspase-3 activation and does not induce the formation of autophagosomes, as measured by
green fluorescent protein-light chain 3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the
temozolomide-mediated resistance seen in some
tumors and that balanced BER
protein expression and overall BER capacity may help predict sensitivity to
temozolomide.