A dominant-negative form of the major human abasic endonuclease enhances cellular sensitivity to laboratory and clinical DNA-damaging agents.
Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3' damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu(96) (changed to Gln) and Asp(210) (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue beta-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.
McNeill
Daniel R
DR
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA.
Wilson
David M
DM
D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D016466:CHO Cells; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000187:drug effects*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D016877:Oxidants; D004247:DNA; D008741:Methyl Methanesulfonate; D006861:Hydrogen Peroxide; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase