The role of base excision repair in the sensitivity and resistance to temozolomide-mediated cell death.
DNA-alkylating agents have a central role in the curative therapy of many human tumors; yet, resistance to these agents limits their effectiveness. The efficacy of the alkylating agent temozolomide has been attributed to the induction of O6-MeG, a DNA lesion repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT). Resistance to temozolomide has been ascribed to elevated levels of MGMT and/or reduced mismatch repair. However, >80% of the DNA lesions induced by temozolomide are N-methylated bases that are recognized by DNA glycosylases and not by MGMT, and so resistance to temozolomide may also be due, in part, to robust base excision repair (BER). We used isogenic cells deficient in the BER enzymes DNA polymerase-beta (pol-beta) and alkyladenine DNA glycosylase (Aag) to determine the role of BER in the cytotoxic effect of temozolomide. Pol-beta-deficient cells were significantly more susceptible to killing by temozolomide than wild-type or Aag-deficient cells, a hypersensitivity likely caused by accumulation of BER intermediates. RNA interference-mediated pol-beta suppression was sufficient to increase temozolomide efficacy, whereas a deficiency in pol-iota or pol-lambda did not increase temozolomide-mediated cytotoxicity. Overexpression of Aag (the initiating BER enzyme) triggered a further increase in temozolomide-induced cytotoxicity. Enhanced Aag expression, coupled with pol-beta knockdown, increased temozolomide efficacy up to 4-fold. Furthermore, loss of pol-beta coupled with temozolomide treatment triggered the phosphorylation of H2AX, indicating the activation of the DNA damage response pathway as a result of unrepaired lesions. Thus, the BER pathway is a major contributor to cellular resistance to temozolomide and its efficacy depends on specific BER gene expression and activity.
Trivedi
Ram N
RN
Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213-1863, USA.
Almeida
Karen H
KH
Fornsaglio
Jamie L
JL
Schamus
Sandra
S
Sobol
Robert W
RW
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D045647:DNA Glycosylases / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism; D019951:DNA Polymerase beta / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006657:Histones / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D000077204:Temozolomide; D014162:Transfection
D018906:Antineoplastic Agents, Alkylating; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D003606:Dacarbazine; D019951:DNA Polymerase beta; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D000077204:Temozolomide