A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

Abstract

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 B-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.

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) in human ovarian cancer and indentification of the therapeutic potential of APE1/Ref-1 inhibitor

“…IC 50 concentrations of cisplatin were 9.09-(A2780) or 4.63-fold (CP70) lower than those for cisplatin (MTT assay). Our results are supported by those of others in that the suppression of APE1 results in hypersensitivity to chemotherapeutic agents (10,26,27). Additionally, targeted reduction of Ape1/Ref-1 protein by specific anti-sense oligonucleotides renders mammalian cells hypersensitive to methylmethane sulfonate (MMS), H 2 O 2 , bleomycin, temozolomide (TMZ) and gemcitabine (22,28,29).…”

Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia

“…DNA repair activity, in particular activation of APE1, represents an interesting point of control for cell death. Long understood to lead to genetic mutations associated with spontaneous tumor formation, AP sites have also been found to induce cell death in yeast (5), and deficiency in APE1 expression and activity exacerbates oxidative injury in multiple models, including neurons (12,20,21). Unrepaired AP sites accumulate following cerebral ischemia (4,6), and sustained decrease in APE1 protein expression has been observed to precede cell death in injured cell populations (10,22,23) (Fig.…”

Human Apurinic/Apyrimidinic Endonuclease 1

“…A similar damaging agent rescue pattern was observed when the human protein was expressed in yeast-deficient in the major AP endonuclease, APN1 (72). Chinese hamster ovary cells designed to inducibly express a dominant-negative form of APE1 (termed ED, due to E96Q and D210N mutations), which displays better than wild-type binding affinity for substrate DNA, but is devoid of nuclease activity, exhibited pronounced hyper-sensitivity to MMS, yet a more mild increase in sensitivity to hydrogen peroxide (138). The combined data are consistent with APE1 maintaining a powerful AP endonuclease function that participates in the repair of AP sites formed by the direct and indirect action of MMS, and a lesser, albeit significant, 3¢-repair activity for removal of 3¢-blocking groups (e.g., 3¢-phosphates) generated by the oxidizing agent hydrogen peroxide.…”

“…Such a role is consistent with the biochemical activities of APE1, as alkylating agents, particularly mono-functional versions, induce a high number of abasic sites through both direct and indirect mechanisms, and certain anti-metabolites generate exonuclease 3¢-end substrates. In addition, expression of the dominant-negative ED protein (introduced in ''APE1 Biological Roles'' section) in Chinese hamster ovary cells increases the toxicity of the anticancer agents, 5-fluorouracil and 5-fluorodeoxyuridine, with the latter causing greater cell death, presumably because its effects are targeted explicitly to DNA, and not to RNA as well (138). This observation is generally consistent with the recent findings that PARP1 and other BER enzymes, including APE1, play a role in resistance to 5-fluorodeoxyuridine more so than 5-fluorouracil, likely due to the higher levels of BER substrate base lesions, that is, 5-fluorouracil and uracil, introduced into genomic DNA by the former compound (60).…”