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

Daniel R. McNeill and David M. Wilson III

Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland

Abstract analogue B-D-arabinofuranosylcytosine (also known as Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the cytarabine), the topoisomerase inhibitors camptothecin primary enzyme in mammals for the repair of abasic and etoposide, or the cross-linking agents mitomycin C sites in DNA, as well as a variety of 3¶ damages that arise and cisplatin. Transient expression of ED in the human upon oxidation or as products of enzymatic processing. cancer cell line NCI-H1299 enhanced cellular sensitivity If left unrepaired, APE1 substrates can promote to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and mutagenic and cytotoxic outcomes. We describe dideoxycytidine, demonstrating the potential usefulness herein a dominant-negative form of APE1 that lacks of this strategy in the treatment of human tumors. detectable nuclease activity and binds substrate DNA (Mol Cancer Res 2007;5(1):61–70) with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two 96 amino acid substitutions at active site residues Glu Introduction 210 (changed to Gln) and Asp (changed to Asn). Genetic damage can arise via a variety of means, most In vitro biochemical assays reveal that ED impedes notably via spontaneous decay, reactions with intracellular wild-type APE1 AP site incision function, presumably chemicals, aberrant enzymatic processing events, and modifi- by binding AP-DNA and blocking normal lesion cation by environmental or clinical DNA-damaging agents processing. Moreover, tetracycline-regulated (tet-on) (1-4). The most common intermediates or forms of DNA expression of ED in Chinese hamster ovary cells damage include base modifications, abasic sites, and single- enhances the cytotoxic effects of the laboratory strand breaks. These lesions are typically recognized and DNA-damaging agents, methyl methanesulfonate removed by the concerted effort of proteins that function in the (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). base excision repair (BER)pathway (5).More complex lesions, This MMS-induced, ED-dependent cell killing coincides such as DNA double-strand breaks, are corrected by recombi- with a hyperaccumulation of AP sites, implying that national repair responses (6). BER involves base excision by excessive DNA damage is the cause of cell death. a DNA glycosylase, phosphodiester bond cleavage at the Because an objective of the study was to identify a resulting abasic site by an apurinic/apyrimidinic (AP)endonu- protein reagent that could be used in targeted gene clease, gap filling and termini cleanup by a DNA polymerase/ therapy protocols, the effects of ED on cellular lyase, and nick ligation by a DNA ligase. Defects in participants sensitivity to a number of chemotherapeutic of BER have been associated with increased sensitivity to compounds was tested. We show herein that ED DNA-damaging agents, genomic instability, cancer susceptibil- expression sensitizes Chinese hamster ovary ity, neurodegeneration, premature aging phenotypes, and other cells to the killing effects of the alkylating agent human diseases (7). 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as The major repair protein for abasic sites in mammals is AP carmustine) and the chain terminating nucleoside endonuclease 1 (APE1; refs. 8, 9). This enzyme recognizes AP analogue dideoxycytidine (also known as zalcitabine), sites in DNA and cleaves immediately 5¶ to the lesion, but not to the radiomimetic bleomycin, the nucleoside generating a DNA strand break with a priming 3¶-hydroxyl group and a 5¶-abasic residue. Prior biochemical and structural biology studies have indicated a model (known as ‘‘passing the baton’’)whereby human APE1 cooperates with the next protein Received 10/4/06; revised 11/21/06; accepted 11/28/06. h Grant support: Intramural Research Program of the NIH, National Institute on in BER (i.e., DNA polymerase )to coordinately execute the Aging. next steps of the corrective response (10). In addition to AP The costs of publication of this article were defrayed in part by the payment of sites, APE1 exhibits repair activity on a variety of modified page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (nonconventional)3 ¶-DNA termini, including, but not limited Note: Supplementary data for this article are available at Molecular Cancer to, phosphates and phosphogycolyates (ref. 11 and references Research Online (http://mcr.aacrjournals.org/). therein). Because abasic lesions are formed spontaneously at a Requests for reprints: David M. Wilson III, Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, frequency of roughly 10,000 times per mammalian genome per Baltimore, MD 21224. Phone: 410-558-8153; Fax: 410-558-8157. E-mail: day, perhaps not surprisingly, knockouts in APE1 in mice lead [email protected] Copyright D 2007 American Association for Cancer Research. to embryonic lethality (12-14), and sufficient depletion of doi:10.1158/1541-7786.MCR-06-0329 APE1 in cells leads to cell inviability (15, 16).

Past studies using either antisense, small interfering RNA, or substitution), resulted in a dominant-negative form of the human small molecule inhibitor strategies have revealed that APE1- and yeast protein that bound substrate DNAwith high affinity and deficient cells exhibit hypersensitivity to a number of DNA- blocked subsequent nucleic acid processing in vivo (25, 26). Prior damaging agents, including the laboratory agents methyl studies have established the importance of the acidic residues methanesulfonate (MMS), hydrogen peroxide, menadione, and E96 and D210 to the enzymatic repair activity of APE1 (Fig. 1A; paraquat, and the anticancer agents ionizing radiation, thiotepa, ref. 27 and references therein). We postulated that simultaneous 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; also known as mutation of these two amino acids would create a catalytically carmustine), temozolomide, gemcitabine, and the nucleoside inactive APE1 protein with improved DNA-binding capacity. analogue h-L-dioxolane-cytidine (also known as troxacitabine; After the introduction of E96Q and D210N substitutions into refs. 17-24). These findings reveal a prominent role for APE1 in the APE1 coding sequence, we expressed and purified the the repair of specific oxidative, alkylation, and enzymatic DNA resulting double mutant protein, which we have termed ED, and intermediates, and identify this protein as a potential target for biochemically characterized the product (Fig. 1B). As shown in certain therapeutic paradigms. As a novel means of inhibiting Fig. 1C, the ED protein exhibited no AP site incision activity at APE1 function in mammalian cells, we have used a dominant- a concentration 10-fold higher (i.e., at 10 ng)than required to negative strategy. Specifically, we created a mutant form of incise all DNA substrate by the wild-type enzyme (i.e., at 1 ng). APE1, termed ED, which is catalytically inactive but possesses a Based on prior knowledge of the effect of independent higher DNA substrate binding affinity than the wild-type substitutions at E96 or D210 (28), we estimate that the ED protein. The studies herein further identify the primary protein is >56,000,000-fold reduced in nuclease capacity. biological substrates of APE1 and reveal that ED sensitizes Furthermore, using a previously established electrophoretic cells to the cytotoxic effects of the therapeutic DNA-damaging mobility shift assay (see Materials and Methods), we found that agents BCNU and dideoxycytidine, but not to bleomycin, h-D- ED displayed an f13-fold higher affinity for AP-DNA arabinofuranosylcytosine (ara-C; also known as cytarabine), compared with wild-type APE1 protein (Fig. 1D); this camptothecin, etoposide, mitomycin C, or cisplatin. enhanced affinity is presumably due to the loss of the repulsion between the negatively charged acidic residues and the Results phosphate backbone of DNA. In studies not shown, ED did AP Endonuclease and DNA Binding Properties of the ED not bind undamaged DNA with increased stability in electro- Protein phoretic mobility shift assays, indicating that the improved Previous work had found that neutralization of a conserved affinity was specific for substrate DNA. (negatively charged)acidic residue within the active site of certain nucleases can produce an enzymatically inactive protein ED Interferes with Wild-type APE1 Repair Activity In vitro molecule with enhanced DNA-binding activity. For example, As an initial means of assessing the potential of ED to act as such a mutation in the flap endonuclease, FEN1 (i.e., a D to A a dominant-negative protein, we examined the effect of ED on

Figure 1. ED protein. A. Schematic ofthe APE1-AP-DNA interface.Aqua ribbon, APE1 protein. White, bound abasic substrate. Gold, abasic residue. Several key active site residues are denoted, as are the primary recognition loops a5, a8, and a11. Yellow, acidic residues mutated in ED [i.e., glutamic acid 96 (E96) and aspartic acid 210 (D210)]. B. Comparison ofwild-type APE1 and the ED protein. Proteins were separated on a 12% SDS-polyacrylamide gel and stained with Coomassie blue. Arrow, position ofthe two proteins. Molecular weight ( MW) standards from top to bottom are f103, 81, 48, 36, 27, and 19 kDa. C. Endonuclease activity ofAPE1 and ED. Proteins were assayed forAP site incision activity at 0, 0.01, 0.1, 1, and 10 ng as described in Materials and Methods. Representative incision gel. S, substrate; P, product. D. Binding activity ofAPE1 and ED. Electrophoretic mobility shiftassays were done with radiolabeled GFA-C (100 fmol) and 0.1, 0.3, 1, or 3 ng of protein as described in Materials and Methods. Top, a representative binding gel. Bottom, points, average ofsix independent binding experiments; bars, SD.

Figure 2. In vitro competition assays with APE1 and ED. A. Competition incision assay. ED (at 0, 0.67, 2, 6.7, or 20 ng) was preincubated with radiolabeled GFA-C substrate (200 fmol) on ice. APE1 (0.3 ng or 9 fmol) was then added, and incision was allowed to proceed at 37jC for 2 min. Following incubation, conversion ofsubstrate to product was determined on a denaturing polyacrylamide gel (shown above) as described in Materials and Method s. Columns, relative incision efficiency at the indicated amount of ED. B. Competition ratio assay. APE1 and ED (at the specified ratio; at 100 fmol total) were incubated with the GFA-C substrate (50 fmol) for 10 min on ice. MgCl2 was then added, and incision was allowed to proceed at 37jC for 2 min. Relative incision efficiency was determined as in A. Columns (A and B), average ofsix independent incision experiments; bars, SD. NE, no enzyme. wild-type APE1 repair capacity using in vitro reconstitution in our hands, none of the cell lines exhibited a reliable tet assays. We define dominant-negative here as a protein that concentration–dependent response (data not shown); that is, binds cognate DNA substrates but prevents subsequent repair ED expression was either on (at tet concentrations of 0.1-10 Ag/ processing (in this case, AP site incision). In the first set of mL)or off (in the absence of tet),with no clear titratable in vitro experiments, an increasing amount of ED protein was expression being observed. Moreover, there was no obvious preincubated with 200 fmol of AP-DNA. After 10 min on ice, departure in growth characteristics (such as doubling time or wild-type APE1 protein was added and cleavage efficiency was survival), adhesion properties, or cellular morphologies of the immediately measured via a 2-min incubation at 37jC. As ED-expressing clones upon tet induction (data not shown). shown in Fig. 2A, prebinding of ED to AP-DNA inhibited wild- As a first means of determining whether ED expression type APE1 incision capacity, with nearly 100% inhibition seen might affect in vivo AP site repair, we generated whole-cell at a 1:1 ratio of ED (6.7 ng or 200 fmol)to substrate DNA. In extracts from noninduced and tet-induced ED5, ED6, and ED8 the second set of experiments, ED and APE1 were preincubated cells and measured total AP site incision capacity. We reasoned at varying ratios with abasic DNA, and then MgCl2 was added that if ED was expressed at sufficient levels and was indeed and the cleavage efficiency was measured as above. As reported inhibitory of wild-type APE1 function, extracts containing ED in Fig. 2B, a 4:1 ED to APE1 ratio resulted in roughly 80% protein would exhibit reduced incision efficiency. Consistent inhibition of wild-type incision activity. The results, in total, with this hypothesis, we found that both ED5 and ED8 (the indicate that ED can bind AP-DNA with high affinity and high-expressing clones; Fig. 3A)displayed a tet/ED-dependent interfere with normal APE1 endonuclease activity in vitro. reduction (f75%)in total AP site incision capacity (compare tet minus with tet plus), whereas ED6 (the low-expressing Establishment of ED-Expressing Mammalian Cell Lines clone)and the T-REx control extracts did not (Fig. 3B).Given Given that ED was able to inhibit wild-type APE1 nuclease these results, we postulated that sufficient ED expression would activity in the biochemical assays above, we next determined if enhance cellular sensitivity to DNA-damaging agents that ED expression would affect APE1 repair capacity in vivo. generate APE1 (or BER)DNA substrates by inhibiting normal Toward this end, we created T-REx Chinese hamster ovary repair processing. (CHO)-K1 cell lines that expressed ED protein in a tetracycline- inducible (tet-on)manner (see Materials and Methods). ED Expression Enhances Sensitivity to Laboratory DNA- Specifically, after selection and isolation of clonal integrates Damaging Agents that stably harbor the recombinant pcDNA4/TO-ED plasmid, To evaluate whether ED potentiates the cytotoxic effects of we identified four cell lines that differentially expressed ED in a DNA-damaging agents, we first examined the sensitivity of the tightly regulated, tet-on manner: ED8 > ED11 > ED5 >> ED6 stably integrated CHO cell lines to the monofunctional (from highest to lowest; Fig. 3A). Because ED11 displayed alkylating agent MMS, a classic BER substrate–generating intermediate expression similar in nature to ED8 and ED5, we compound, which produces mainly alkylated base damage and omitted this line from most subsequent analysis. We note that, AP sites (29). Initial colony formation assays were done with

Figure 3. Establishment ofCHO cell lines stably expressing ED. A. CHO clonal isolates expressing ED in a tet-dependent manner. A subset ofclones (ED8, ED11, ED5, and ED6) expressing the ED protein were identified by Western blot analysis. The experiment was carried out without (À) or with (+) tet addition. APE1, Western blot control protein. MW, lane with protein standards. Note that no cross-reactivity of the antibody was observed to the endogenous CHO APE1 protein. B. Total AP incision activity ofCHO ED-expressing cells. Incision assays were done on whole- cell extracts produced from the indicated ED-expressing lines, which had either not been exposed to tet (tet minus) or were exposed to tet (tet plus). T-REx is the nontransfected control cell line. Columns, averages of at least five data points for each test variable; bars, SD.

and without tet induction, and included the various ED- using an established aldehyde-reactive probe–based method expressing lines (ED5, ED6, ED8, and ED11)and the T-REx (see Materials and Methods). In these experiments, we found parental cells. As shown in Supplementary Fig. S1 (and that upon tet induction (i.e., ED expression), all of the cell lines Fig. 4A), the presence and level of ED expression corresponded exhibited identical levels of endogenous AP sites (Fig. 4C); this qualitatively with the degree of MMS sensitivity. Specifically, finding is consistent with the fact that the various cell lines the high-expressing ED5, ED8, and ED11 clones displayed display comparable cell growth and viability characteristics decreased cell survival after MMS challenges, whereas the after ED production. Conversely, after MMS treatment, the high low-expressing ED6 cell line and the T-REx control exhibited ED–expressing clones (ED5 and ED8)accumulated roughly ‘‘normal’’ sensitivity. Moreover, the impaired cell survival seen twice as many AP lesions (over background)than the low- with ED5, ED8, and ED11 was not detected in the absence of tet expressing ED6 clone or the T-REx control. Consistent with (denoted by ‘‘À’’), indicating that ED expression was required this observation, we also found (using an alkaline Comet assay) for MMS-induced cell death; the ED6 and T-REx cells exhibited that the ED5 and ED8 cells exhibited a 38% to 47% increase in wild-type resistance independent of tet exposure (Supplemen- alkaline-labile sites and single-strand breaks after a 0.4 mmol/L tary Fig. S1). A more quantitative analysis of the MMS MMS treatment, compared with the 20% to 30% increase response documented an f5- to 6-fold increased sensitivity over background seen with the ED6 strain and T-REx control (as determined by the relative LD37)of the high-expressing ED (data not shown). These data indicate that accumulating DNA clones (ED5, LD37 0.20 mmol/L; ED8, LD37 0.26 mmol/L), damage is likely responsible for the enhanced cell death seen in compared with the low-expressing ED6 line (LD37 1.15 the MMS-challenged, high ED–expressing cells. mmol/L)and the T-REx control (1.25 mmol/L; Fig. 4A). In addition to MMS, we also examined the cellular ED Expression Sensitizes Cells to Clinical DNA-Damaging sensitivity of the ED5, ED6, and ED8 lines to the laboratory Agents chemical H2O2. This agent leads to the formation of Because an emphasis of our effort was to generate a resource predominantly oxidized bases, AP sites, and single-strand that would enhance cellular sensitivity to anticancer agents in breaks with refractory 3¶-termini, primarily 3¶-phosphate ends future targeted gene therapy paradigms, we determined the (30). Although not as pronounced as seen with MMS (Fig. 4A), comparative cell survival of the ED-expressing clones ED5, a similar trend of increased sensitivity was observed in the ED6, and ED8, as well as the T-REx control, to a variety of high-expressing ED clones (ED5 and ED8), whereas no chemotherapeutic agents. In particular, we tested the following differential survival was seen between the low-expressing compounds: the alkylating agent BCNU, which is commonly ED6 clone and the T-REx control after H2O2 treatment used for treatment of brain tumors; the radiomimetic bleomycin, (Fig. 4B). We note that at the highest H2O2 concentration an antibiotic with potent antitumor activity against a range (160 Amol/L), there were no colonies formed with the ED5 and of lymphomas, head and neck cancers, and germ cell tumors; ED8 lines, unlike ED6 and T-REx. The comparative LD37 the chain terminating nucleoside analogues, dideoxycytidine, values for H2O2 were as follows: T-REx control, 100 Amol/L; which is used mainly to block the infectivity of the HIV; ED5, 73 Amol/L; ED6, 100 Amol/L; and ED8, 65 Amol/L. and ara-C, which is used in the treatment of leukemias; the To develop a more precise picture of the effect of ED on topoisomerase I inhibitor camptothecin, shown to have DNA repair, we measured the steady-state level of AP sites significant antitumor activity against lung, ovarian, breast,

pancreas, and stomach cancers; the topoisomerase II inhibitor etoposide, used to treat a wide spectrum of human cancers; and the DNA cross-linking agents mitomycin C and cisplatin, both of which are used in the systemic treatment of a range of malignancies. Because these anticancer agents elicit their cell killing effects via the generation of largely distinct cytotoxic DNA intermediates, our cell survival analysis also provided a means of identifying primary DNA substrates of APE1 in vivo. As shown in Supplementary Fig. S2, no obvious increased sensitivity to bleomycin, ara-C, camptothecin, etoposide, mitomycin C, or cisplatin was observed for any of the

Figure 5. Colony formation analysis with specific chemotherapeutics. A. BCNU cytotoxicity. T-REx, ED5, ED6, and ED8 were exposed to BCNU at various concentrations (as in Fig. 4A, except for exposure times; concentrations 0, 30, 100, 150, 200, 250, and 300 Amol/L). B. Dideoxycytidine (ddC) cytotoxicity. T-REx, ED5, ED6, and ED8 were exposed to dideoxycytidine at various concentrations (as in Fig. 4A, except for exposure times; concentrations 0, 1.5, 3, 6, 9, 10, and 12 mmol/L). Points, mean of at least four data points taken from two independent experimental runs; bars, SD.

ED-expressing clones. Conversely, colony formation assays did reveal that ED expression enhanced the cell killing effects of BCNU and dideoxycytidine by up to 1.4- to 2.2-fold and 2.1- to 2.8-fold, respectively (Supplementary Fig. S2; Fig. 5).

ED Expression Enhances the Killing of MMS, BCNU, and Dideoxycytidine in NCI-H1299 Cells To assess the potential value of ED in the treatment of human cancer, we determined the effect of ED expression on MMS, BCNU, and dideoxycytidine sensitivity in the non– small cell lung cancer line NCI-H1299 (31). This cell line was selected largely due to its ability to be transfected efficiently (z96%), a feature that allowed us to evaluate the effect of ED without a significant number of background (nontransfected) cells. Thus, after transient transfection of either the pcDNA4/ TO control vector or the pcDNA4/TO-ED expression construct, Figure 4. Cell survival analysis and AP site damage. A. MMS cytotoxicity. After tet induction, T-REx, ED5, ED6, and ED8 were exposed we plated, challenged with either MMS, BCNU, or dideox- to MMS at various concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mmol/L) for ycytidine, and permitted colony formation of the NCI-H1299 1 h, then grown and stained for colony formation (see Materials and cells (see Materials and Methods). As with the CHO line, Methods). B. H2O2 cytotoxicity. Procedure was done as in A, except for using 0, 20, 40, 80, 120, and 160 Amol/L H2O2 as the challenge. Points ED expression enhanced cellular sensitivity to MMS, BCNU, (A and B), mean ofat least fourindependent data points; bars, SD. C. and dideoxycytidine relative to the controls, with the increase Steady-state AP site levels. After 24 h tet exposure, T-REx, ED5, ED6, in sensitivity for the human cancer line being f2.6-, 3.2-, and and ED8 cells were exposed to 0.4 mmol/L MMS for 1 h (or not treated), then washed and collected. After genomic DNA was isolated, the number 1.6-fold, respectively (Fig. 6). A similar effect of ED was ofAP sites (expressed as AP sites per 1 Â 106 bp) was quantified using observed on cellular sensitivity to MMS and BCNU (dideox- an aldehyde reactive probe – based colorimetric assay (see Materials and Methods). Columns, average ofat least fiveindependent data points; ycytidine was not tested)in the human osteosarcoma cell line bars, SD. U-2 OS (Supplementary Fig. S3).

Figure 6. Effect of ED expression on sensitivity ofNCI-H1299 cells to MMS, BCNU, and dideoxycytidine. A. MMS cytotoxicity. NCI-H1299 cell line was transfected with pcDNA4/TO (Vector)or pcDNA4/TO-ED (ED), and subsequently exposed to MMS at various concentrations (0, 0.5, 1, 2, and 3 mmol/L) for 1 h, then grown and stained for colony formation (see Materials and Methods). B. BCNU cytotoxicity analysis. Procedure was done as in A at 0, 25, 50, 100, and 150 Amol/L. C. Dideoxycytidine cytotoxicity analysis. Procedure was done as in A, except exposure was for 24 h, at 0, 1.5, 3, 6, and 9 mmol/L. Points (A-C), mean percentage survival values ofat least four data points.

Discussion FEN1 mutant causes genetic instability and increased sensitivity Prior laboratory studies have documented the value of to MMS, likely by preventing processing of DNA replication active site nuclease mutants specifically and dominant-negative intermediates and promoting the formation of recombinogenic DNA binding proteins in general. For instance, Resnick and DNA double-strand breaks. A DNA binding fragment (lacking colleagues (25, 26)have used a FEN1 mutant that harbors a the catalytic domain)of the critical strand break response D to A substitution at a residue found to be critical for DNA protein, poly(ADP-ribose)polymerase 1, has also been shown to processing to determine the in vivo substrates of the flap act as dominant-negative by blocking subsequent repair steps endonuclease. Using either genetically engineered yeast strains (32, 33). Upon tissue-specific expression, the poly(ADP-ribose) or exogenously expressing yeast systems, they show that the polymerase 1 fragment (its DNA-binding domain)was found to

enhance the sensitivity of prostate carcinoma cells to ionizing combining transient ED expression with MMS, BCNU, or radiation and etoposide, highlighting its potential usefulness in dideoxycytidine treatment. certain gene therapy–based anticancer treatment protocols (34). BCNU is an alkylating agent that reacts mainly with the N-7 We report herein the design of a dominant-negative APE1 or O-6 position of guanine, and the N-3 position of cytosine protein, which we have termed ED. (40). Although the O-6 alkylation product is a major cytotoxic ED harbors two active site mutations at acidic residues (E96 intermediate (primarily upon the formation of DNA interstrand and D210)previously shown to be essential for efficient cross-links), many of the other base modifications likely give catalytic activity (ref. 27 and references therein). Notably, this rise to increased abasic sites via either the enhanced mutant protein exhibits a >56,000,000-fold reduced nuclease destabilization of the N-glycosylic linkage or removal by capacity, while displaying a better than wild-type AP-DNA DNA glycosylases (41, 42). This amplified level of AP sites binding affinity (Fig. 1). In vitro reconstituted assays also and accompanying inhibition of repair by ED is likely revealed that ED interferes with (or inhibits)wild-type APE1 responsible for the enhanced cell killing observed with BCNU incision activity (Fig. 2). Given these biochemical results, we (Fig. 5). It is thus tempting to speculate that simultaneous did a series of experiments to determine the effects of ED inhibition of the major repair protein for O-6 alkylation damage expression in mammalian cells, postulating that ED (by binding (i.e., the O-6 methylguanine DNA methyltransferase; ref. 43), DNA with high affinity and blocking subsequent wild-type and a central BER component such as APE1, would elicit a APE1 repair activity)would ( a)facilitate the identification of more pronounced cytotoxic response to BCNU than single the primary biological substrates of APE1 and (b)serve as a protein–targeted paradigms. Notably, the results of others resource in gene-therapy paradigms to selectively sensitive support the notion that the APE1 repair nuclease capacity is a cancer cells to certain chemotherapeutics. determinant in BCNU resistance, particularly in brain tumors We have shown within that ED production leads to a (21, 44). markedly increased sensitivity to MMS and a concomitant The enhanced cell killing exhibited by the ED-expressing accumulation of AP site damage (Fig. 4). Because abasic sites cells after treatment with dideoxycytidine, but not ara-C, is are prominent DNA intermediates of MMS exposure (29), our consistent with the known 3¶ to 5¶ exonuclease preferences results underscore the critical importance of APE1 in the repair of APE1 for these chain-terminating D-configured nucleoside of AP lesions (and potentially, certain DNA single-strand analogues. In particular, APE1 exhibits a f3-fold more breaks). The less dramatic hypersensitivity of ED-expressing robust excision activity for dideoxycytidine than for ara-C cells to H2O2 implies a lesser role for APE1 in the repair of (45). Because APE1 harbors an even more proficient removal oxidative 3¶ blocking lesions, such as 3¶-phosphate groups, yet activity for L-configured nucleoside analogues, such as h-L- still indicates a significant contribution of APE1 to the efficient dioxolane-cytidine (excised roughly 8-fold faster than processing of oxidative DNA damage. This sensitivity profile dideoxycytidine; ref. 45), future studies will need to of the ED-expressing cells is consistent with APE1 exhibiting a determine the effect of ED on the cytotoxic potential of robust AP endonuclease activity in vitro (even for oxidized AP these (not readily available)agents. Prior investigations sites ref. 35), yet a comparably weak 3¶-phosphodiesterase have indeed revealed that APE1 repair capacity correlates function (36), and with the polynucleotide kinase/phosphatase with sensitivity to certain L-configured nucleoside analogues protein operating as the main human DNA 3¶-phosphatase (37). (22, 46). The data also suggest that AP sites are effective cytotoxic The lack of enhanced sensitivity to bleomycin was lesions, a conclusion that is consistent with the observation that surprising given that APE1 was found to be the primary sufficient APE1 depletion in mammalian cells leads to an activity for excising 3¶-phosphoglycolates (a major product of accumulation of AP sites and an accompanying cell inviability this antibiotic)from single-strand breaks in DNA (47).This (15). We propose that the T-REx CHO system described within observation may suggest that the main toxic intermediates could be a useful asset in characterizing the functional formed by bleomycin are those related to clustered DNA activities of other human APE1 mutants and/or population damage, including juxtaposed AP sites and/or 3¶ damage– variants (38). containing single-strand breaks, or complex DNA double- Emerging work has documented the potential of regulating strand breaks (48). Although APE1 has been shown to excise a specific DNA damage responses as a means of promoting synthetic mimic of a trapped 3¶-topoisomerase I protein (i.e., a selective cell survival or cell death in the clinic (3, 4, 39). 3¶-DNA tyrosyl residue; ref. 11), the fact that ED expression This concept has particular usefulness in anticancer (and in does not enhance sensitivity to camptothecin exposure suggests some instances antiviral)treatment paradigms, where geno- that APE1 is not a major player in the removal of this form of toxic agents are often used to eradicate cancerous (or rapidly DNA damage. Indeed, evidence indicates that tyrosyl DNA dividing)tissue. As noted throughout, a goal of our effort phosphodiesterase serves as the primary repair enzyme for such was to create a protein source with potential therapeutic 3¶-blocking lesions in mammalian cells (49, 50). The value. We therefore examined whether ED expression observation that ED induction does not alter cellular sensitivity enhanced the cell killing of a variety of chemotherapeutic to etoposide, mitomycin C, and cisplatin is consistent with agents, initially in the various CHO cell lines. We found APE1 not playing a known role in the repair of 5¶-trapped that the most pronounced effect on cell survival upon ED topoisomerase-DNA intermediates or interstrand/intrastrand induction was seen with BCNU and dideoxycytidine DNA cross-links. In closing, the data herein not only shed (Fig. 5). This increased cell killing was subsequently important insights into the in vivo nucleic acid substrates of observed in the human cancer cell line NCI-H1299 when APE1 but also highlight the fact that APE1 is a rational

therapeutic target (depending on the agent used and damage 10 min. Subsequently, 0.3 ng (or 9 fmol)of wild-type APE1 generated)and that ED is a potential resource for future gene was added, and the reaction was placed at 37jC for 2 min. The therapy paradigms. reaction was stopped and the percentage of substrate converted to product was determined as above. The competition ratio assay was conducted using the AP Materials and Methods endonuclease reaction conditions above with a few exceptions. Reagents Specifically, a defined ratio of APE1 to ED was first incubated DNA oligonucleotides were obtained from Midland Certi- 32 [at amounts of 0%, 20%, 50%, 80%, or 100% wild-type APE1 fied Reagent Company (Midland, TX). [g- P]ATP (3,000 Ci/ (totaling 100 fmol of combined protein)] with 50 fmol of mmol)was purchased from Roche Pharmaceuticals (Indian- radiolabeled GFA-C DNA in the presence of 0.5 mmol/L EDTA apolis, IN), and T4 polynucleotide kinase was from New at 0jC for 10 min. Subsequently, MgCl2 was added to a final England BioLabs (Beverly, MA). All chemicals were purchased concentration of 10 mmol/L, and the reaction immediately from Sigma-Aldrich (St. Louis, MO)unless otherwise speci- transferred to 37jC for 2 min. As above, the reaction was fied. Denaturing polyacrylamide gel reagents were acquired stopped and the percentage of substrate converted to product from National Diagnostics (Atlanta, GA). Acrylamide and the was determined. low-range prestained protein standards were obtained from Bio- For the DNA binding electrophoretic mobility shift assay, Rad (Hercules, CA). GetpureDNA kit and the DNA Damage protein (either APE1 or ED at 0.1, 0.3, 1, or 3 ng)was Quantification kit were purchased from Dojindo Molecular incubated with 5¶-32P-end labeled duplex GFA-C DNA (100 Technologies, Inc. (Gaithersburg, MD). T-REx CHO-K1, fmol)for 10 min at 0 jCin1Â electrophoretic mobility shift pcDNA4/TO, and DMEM were obtained from Invitrogen assay buffer [25 mmol/L MOPS (pH 7.2), 100 mmol/L KCl, Corporation (Carlsbad, CA). The human cancer cell lines 1mmol/LDTT,50Ag/mL BSA, 20% glycerol, and NCI-H1299 and U-2 OS were purchased from American Type 4 mmol/L EDTA]. The binding reactions were subsequently Culture Collection (Manassas, VA). analyzed on an 8% nondenaturing gel [which contained 8% acrylamide (37.5:1), 2.5% glycerol, 20 mmol/L Tris-HCl APE1 Wild-type and Mutant Protein (pH 7.2), 10 mmol/L sodium hydroxide, and 0.5 mmol/L The bacterial expression system for producing wild-type EDTA] at 4jC (54). The percentage of labeled DNA bound by recombinant human APE1 protein (i.e., pETApe)was gener- protein was determined by phosphorimager analysis as above. ated as described (51). Overlapping PCR mutagenesis was used (see refs. 28, 52)to sequentially introduce two codon changes (i.e., the E96Q and D210N amino acid substitutions) ED-Expressing CHO Cell Lines into the pETApe construct to create the double mutant APE1 The ED cDNA was amplified from the pET-ED expression Bam protein, ED. ED was expressed and purified from the construct described above and subcloned into the HI and Eco recombinant pET-ED plasmid as detailed for the wild-type RI restriction sites of pcDNA4/TO. Accuracy of the APE1 protein (51). nucleotide sequence and the presence of the appropriate nucleotide substitutions for ED were verified. To create putative ED-expressing CHO cell lines, the pcDNA4/TO-ED construct AP Endonuclease and DNA Binding Assays was transfected into the T-REx CHO cell line using the Amaxa AP endonuclease activity was measured using a duplex Nucleofactor System (Amaxa Biosystems, Gaithersburg, MD). substrate termed GFA-C essentially as described (53). In brief, The T-REx CHO line harbors an endogenous copy of pcDNA6/ an F (tetrahydrofuran)–containing 26-mer oligonucleotide TR, which permits tet-regulatable (tet-on)gene expression from [GFA; 5¶- AATTCACCGGTACG(F)ACTAGAATTCG-3¶] was pcDNA4/TO. Stable integrates were selected by growth in 5¶-32P-end labeled and annealed to an unlabeled complementary DMEM [supplemented with 1% penicillin, streptomycin and strand (GFA-C comp; 5¶-CGAATTCTAGTCCGTACCGGT- glutamate, and 10% fetal bovine serum (tet minus)] containing GAATT-3¶). Subsequent incision reactions consisted of zeocin (pcDNA6/TR)and blasticidin (pcDNA4/TO).ED 25 mmol/L MOPS (pH 7.2), 100 mmol/L KCl, 1 mmol/L expression was subsequently evaluated by standard Western MgCl , 1 mmol/L DTT, and 50 Ag/mL bovine serum albumin; 2 blot analysis using whole-cell extracts (100 Ag)and polyclonal either APE1 or ED protein at 0, 0.01, 0.1, 1, or 10 ng; and antibodies to the human APE1 protein (Trevigen, Gaithersburg, 1 pmol of GFA-C in a final volume of 10 AL. Reactions were MD), after exposure of several putative T-REx-ED isolates to carried out for 10 min at 37jC, stopped by the addition of 1 Ag/mL tet (see more below). Detection was carried out using 10 AL formamide buffer, and analyzed by electrophoresis in an goat anti-rabbit horseradish peroxidase–conjugated secondary 18% denaturing polyacrylamide gel. AP endonuclease activity antibody (Pierce Biotechnology, Rockford, IL). is the amount of radiolabeled F-DNA converted to the shorter incised DNA product per minute. Percentage conversion was determined on a Molecular Dynamics Typhoon phosphorim- Whole-Cell Extract Assays ager using ImageQuant software (version 5.2, Amersham CHO cell lines were cultured in DMEM (as above). Once Biosciences Corp., Piscataway, NJ). cells reached 80% confluence, they were treated with tet The ED competition incision assay was conducted using the (final concentration 1 Ag/mL)for 24 h. For extract preparation, above AP endonuclease reaction conditions with a few cells were trypsinized, washed with PBS, and harvested by modifications. ED (in the amount of 0, 0.67, 2, 6.7, or 20 ng) centrifugation. Cell pellets were frozen at À80jC for 1 h before was first preincubated with 200 fmol GFA-C DNA at 0jC for extract preparation. Cells were resuspended in 1 mL lysis buffer

[50 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 1 mmol/L DTT, efficiency, NCI-H1299 cells were transfected with the pEYPF- 10% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride] C1 plasmid (Clontech, Mountain View, CA)and grown on a and then sonicated using a Misonix Microson sonicator six-well chamber slide for 2 days to f80% confluence. Cells (Farmingdale, NY). Following centrifugation, a Bradford assay were then washed twice with 1Â PBS and analyzed for YFP (Bio-Rad)was run to determine the protein concentration of the expression using an epifluorescence Zeiss (Berlin, Germany) supernatant (whole-cell extract). Total AP endonuclease activity microscope. At least 50 cells were analyzed for 4¶,6-diamidino- was measured using radiolabeled 34F double-stranded DNA 2-phenylindole and FITC staining to determine transfection (55). AP endonuclease reactions consisted of 50 mmol/L efficiency. Cell survival experiments with U-2 OS were done HEPES (pH 7.5), 100 mmol/L KCl, 1 mmol/L MgCl2, 10 pmol essentially as above, except that Amaxa Nucleofactor kit V was of the oligonucleotide duplex, and 1 Ag whole-cell extract in a used for transfection and cells were cultured in DMEM final volume of 10 AL. Reactions were done for 10 min at (supplemented as above). The transfection efficiency of U-2 37jC, stopped, and analyzed as above. OS was z94%.

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Mol Cancer Res 2007;5(1). January 2007 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

Daniel R. McNeill and David M. Wilson III

Mol Cancer Res 2007;5:61-70.

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