Overexpression of the human HAP1 protein sensitizes cells to the lethal effect of bioreductive drugs
Maria José Prieto-Alamo and
Francioise Laval1
Unité 347 INSERM, 80 Rue du Général Leclerc, 94276 Le Kremlin Bicêtre, France
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Abstract
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Abasic sites (AP sites) are generated in DNA either directly by DNA-damaging agents or by DNA glycosylases acting during base excision repair. These sites are repaired in human cells by the HAP1 protein, which, besides its AP-endonuclease activity, also possesses a redox function. To investigate the ability of HAP1 protein to modulate cell resistance to DNA-damaging agents, CHO cells were transfected with HAP1 cDNA, resulting in stable expression of the protein in the cell nuclei. The sensitivity of the transfected cells to the toxic effect of various agents, e.g. methylmethane sulfonate, bleomycin and H2O2, was not modified. However, the transfected cells became more sensitive to killing by mitomycin C, porfiromycin, daunorubicin and aziridinyl benzoquinone, drugs that are activated by reduction. To test whether the redox function of HAP1 protein was involved in this increased cytotoxicity, we have constructed a mutated HAP1 protein endowed with normal AP-endonuclease activity but deleted for redox function. When this mutated protein was expressed in the cells, elevated AP-endonuclease activity was measured, but sensitization to the lethal effects of compounds requiring bioreduction was no longer observed. These results suggest that HAP1 protein, besides its involvement in DNA repair, is able to activate bioreduction of alkylating drugs used in cancer chemotherapy.
Abbreviations: AP sites, apurinic/apyrimidinic sites; DTD, DT-diaphorase; DZQ, 3,6-diaziridinyl-1,4-benzoquinone; GSH, reduced glutathione; MMC, mitomycin C; MMS, methylmethane sulfonate; NAC, N-acetylcysteine.
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Introduction
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Apurinic/apyrimidinic sites (AP sites) arise in DNA either spontaneously, during base excision repair following exposure to a variety of DNA-damaging agents, by the action of endogenous factors such as oxygen species or by the action of ionizing radiation (for a review see ref. 1). These sites are stable at physiological pH (2) and, as they preferentially code for an adenine during replication, are promutagenic and cytotoxic lesions (3). Therefore, the repair of AP sites is essential to cell viability.
AP sites are repaired in human cells by HAP1 protein, which is a structural and functional homolog of the Escherichia coli exonuclease III protein (xthA gene product) (46). HAP1 participates in the repair of AP sites via endonucleolytic cleavage of the DNA backbone 5' of the AP site. Besides its AP-endonuclease activity, HAP1 protein also possesses RNase H and 3'-phosphodiesterase activity removing lesions blocking the 3'-side of DNA strand breaks generated by ionizing radiation or bleomycin (5). HAP1 also functions as a redox factor, altering the reduction/oxidation state of the c-Fos and c-Jun proteins, and is involved in activation of transcription factors, including AP-1 (6), p53 (7) and NF-
B (6). The redox and AP-endonuclease activities of HAP1 are encoded by distinct regions of the protein. AP site recognition has been determined by site-directed mutagenesis (8) and it has been shown that Cys65 is essential for the reduction/oxidation process, since mutation to alanine eliminates the redox activity (9). The crystal structure of HAP1 has recently been reported (10).
Heterologous expression studies have shown that HAP1 protein protects E.coli xthA nfo mutants against the toxicity of methylmethane sulfonate (MMS) and
-rays and to a lesser extent of H2O2 (11). As expression of DNA repair proteins in cells allowed to determine the biological consequences of DNA damage (12), we have overexpressed HAP1 protein in CHO cells and measured its influence on cell resistance to various DNA-damaging agents. The results show that overexpression of this protein increases the toxic effect of drugs used in cancer chemotherapy [e.g. mitomycin C (MMC) and porfiromycin] that need bioreductive activation. Overexpression of a mutated protein, with normal AP-endonuclease activity but with deleted redox function, indicates that this effect is due to the redox function of HAP1 protein.
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Materials and methods
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Drugs
MMS (Aldrich, Milwaukee, WI) was dissolved in Dulbecco's medium. H2O2 (Merck, Darmstadt, Germany), MMC (Sanofi, Paris, France), daunorubicin (Rhône-Poulenc Rorer, Paris, France), aziridine (Fluka, Buchs, Switzerland), bleomycin (Roger Bellon, Paris, France), melphalan (Wellcome, London, UK) and porfiromycin (a gift from Dr G.Krishna, Vion Pharmaceuticals, New Haven, CT) were dissolved in water. 3,6-Diaziridinyl-1,4-benzoquinone (DZQ) was dissolved in dimethyl sulfoxide then diluted in culture medium.
Cell culture
CHO-9 cells were grown in Dulbecco's medium supplemented with 5% horse serum and 5% fetal calf serum in a 5% CO2 humidified atmosphere. They were transfected by electroporation, using a Bio-Rad (Hercules, CA) gene pulser apparatus, as described (13). The transfected cells were grown in G418-containing medium (750 µg/ml) until appearance of clones. Cell survival was measured by incubating exponentially growing cells for 60 min at 37°C in culture medium containing increasing concentrations of the drugs. They were then rinsed, trypsinized and aliquots of the suspension were cultured until appearance of clones. Subcellular fractionation was performed by disrupting the cells in a hypotonic buffer and centrifugation, as described (14).
Plasmid construction
The pcDM8-HAP1 plasmid, carrying the human HAP1 cDNA (4) was provided by Dr I.D.Hickson. The coding sequence was excized with XbaI, purified by gel electrophoresis and ligated into the HindIII site of the psV2-neo vector. Plasmids with the insert in the correct orientation were called psV2-HAPl. In order to suppress the redox activity of HAP1 protein, mutagenesis was performed by a technique using uracil-containing DNA and a phagemid vector (15). The HAP1 cDNA was cloned in the XhoI/SpeI site of the pBlueScript II SK() plasmid (Stratagene, La Jolla, CA), which was then introduced into E.coli RZ1032 (dut ung). The uracil-containing ssDNA was isolated and used as template for in vitro synthesis of a complementary strand, primed by a mutagenic oligonucleotide (5'-ACACTCAAGATCGCCTCTTGGGAATGTG-3'). The resulting double-stranded DNA was tranformed in E.coli JM 109 which possesses a proficient uracil-DNA glycosylase able to inactivate the uracil-containing strand. This mutant form of HAP1 cDNA, where cytosine 195 was replaced by an adenine (9), was ligated into the HindIII site of the psV2-neo vector, yielding the psV2-HAPl:C65A plasmid. The site-specific mutant cDNA was checked by DNA sequencing using the Applied Biosystem (ABI Prism 310 genetic Analyzer) Taq DyeDeoxy Terminator Cycle sequencing procedure (Perkin Elmer, Norwalk, CT) according to the manufacturer's specifications.
RTPCR analysis of HAP1 mRNA expression
Total cellular RNA was isolated by guanidinium thiocyanate cell lysis. It was reverse transcribed using the First Strand Synthesis kit (Boehringer Mannheim, Mannheim, Germany). HAPl-specific PCR was performed using 20 µl of the reverse transcription mixture using specific primers and the Expand Synthesis kit (Boehringer) in a final volume of 100 µl. PCR conditions were established to obtain exponential amplification (16). For analysis, 10 µl of the PCR products were separated through a 1.8% agarose gel and visualized by ethidium bromide staining.
AP-endonuclease activity determination
Cells were harvested by trypsinization and suspended (108 cells/ml) in a buffer containing 70 mM HEPES, pH 7.6, 400 mM NaCl, 1 mM DTT and 10% glycerol. They were disrupted by sonication at 0°C in the presence of protease inhibitors (13). Cell debris were removed by centrifugation (10 000 g, 5 min, 4°C). Cell extracts were incubated (final volume 100 µl) in a buffer containing 20 mM glycineKOH and 3 mM MgC12 with E.coli [3H]DNA containing apurinic sites (12 ng, sp. act. 340 c.p.m./ng) prepared as described (17). This depurinated substrate contained ~6 AP sites/kb DNA. After increasing incubation times at 37°C, the radioactivity present in the acid-soluble fraction was quantitated by scintillation spectroscopy.
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Results
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AP-endonuclease activity in transfected cells
The G418-resistant clones, obtained after transfection of CHO cells with the psV2-HAP1 plasmid, expressed an increased AP-endonuclease activity, spanning a broad range of activity level. One clone (CHO-HAP1 cells) showed an activity ~7-fold higher than the endogenous level for CHO cells (Figure 1
). This clone was chosen for further analysis, because cells expressing a similar level of AP-endonuclease activity were selected after transfection with the psV2HAP1:C65A plasmid, containing the mutated HAP1 cDNA (CHO-HAP1:C65A cells) (Figure 1
).
In order to determine the cellular localization of the expressed protein, nuclei and mitochondria were purified and the AP-endonuclease activity measured in the subcellular fractions. The foreign protein was expressed in the cell nuclei. Although the results show a constitutive activity in the mitochondria, no increased activity was detected in the mitochondrial fraction of the transfected cells (Table I
).
To check that this increased activity was actually due to expression of the foreign cDNA, HAP1 mRNA was detected in CHO-HAP1 cells by RTPCR. First-strand cDNA was generated with total RNA from control and transfected cells then amplified with specific primers. As shown in Figure 2
, HAP1 mRNA was detected in the transfected cells, while the low amount of amplified mRNA observed in control cells was probably due to a sequence homology between the human and hamster cDNA.

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Fig. 2. Expression of the HAP1 mRNA in CHO-HAP1 cells. Total RNA isolation from parental CHO and CHO-HAP1 cells, first-strand cDNA synthesis and PCR amplification were performed as described in Materials and methods. The products were analyzed by agarose gel electrophoresis. Lane 1, CHO cells; lane 2, CHO-HAP1 cells; lane 3, markers.
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Effect of HAP1 gene expression on drug toxicity
The survival of CHO and CHO-HAP1 cells was measured after exposure to increasing concentrations of different DNA-damaging agents, either alkylating (MMS, aziridine, MMC, etc.) or oxidative (bleomycin or H2O2) compounds. Expression of the HAP1 protein did not significantly modify cell survival after treatment with MMS, aziridine, melphalan, H2O2 or bleomycin, as shown by the D10 values calculated from the survival curves (Table II
). However, when the cells were treated with MMC, porfiromycin, daunorubicin or DZQ, the transfected cells were more sensitive than the parental CHO cells (Table II
).
It was checked that the doubling time for the two cell lines was identical (~16 h), indicating that growth rate was not influencing cell sensitivity and no modification of survival was observed in treated cells expressing the psV2neo vector.
Role of the redox function of the HAP1 protein
Survival was modified when CHO-HAP1 cells were treated with drugs that need enzymatic reduction to form reactive metabolites. This suggests that the redox activity of HAP1 protein, expressed in the transfected cells, could increase the formation of such toxic intermediates. Two sets of experiments were performed to test this hypothesis.
Drug toxicity was measured in cells grown for 1 h in the presence of N-acetylcysteine (NAC), as this compound is rapidly converted to reduced glutathione (GSH) and modifies the redox capacity of the cells (18). An increased toxicity of MMC and porfiromycin was observed in control cells grown in the presence of NAC (Figure 3
), showing activation of these drugs by a reducing compound. Expression of HAP1 protein increased cell sensitivity to a lesser extent than NAC treatment. However, CHO-HAP1 cells grown in the presence of NAC and treated with MMC or porfiromycin showed a greater sensitivity than CHO cells under the same conditions (Figure 3
and Table III
). This suggests that expression of HAP1 protein in the cell nuclei and NAC treatment had additional effects to enhance the toxicity of these drugs.
In a second set of experiments, the HAP1 cDNA was mutated in order to delete the redox activity of the protein and was ligated in the psV2neo vector, yielding the psV2-HAP1:C65A plasmid. One clone of transfected cells was chosen (CHO-HAP1:C65A cells), because the APendonuclease activity was identical in CHO-HAP1 and CHO-HAP1:C65A cells (Figure 1
). The MMC and porfiromycin cytotoxicities were measured in the different cell lines and were identical in CHO and CHO-HAP1:C65A cells (Figure 4
), in the presence or absence of NAC (Table III
). Identical survivals were also measured for CHO and CHO-HAP1:C65A cells treated with either daunorubicin or DZQ (data not shown). These results show that the mutated protein has lost its capacity to increase the lethal effect of the drugs. They strongly suggest that the increased sensitivity observed in the case of CHO-HAP1 cells is due to the redox function of the expressed HAP1 protein.
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Discussion
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AP sites in DNA result from spontaneous depurination or from the action of DNA glycosylases during the repair of alkylated or oxidized bases. In human cells, the first step of AP site repair is performed by HAP1 protein, which also possesses redox activity (46). Heterologous expression studies have shown that the HAP1 protein protects xth nfo E.coli mutants against alkylating agents and
-rays and to a lesser extent against H2O2 (11). This is consistent with the weak 3'-diesterase activity of this protein (19), which is necessary for the repair of H2O2-induced damage. Similar results were obtained by complementation of apn1-deficient yeast with HAP1 protein (20). This protective effect was also suggested by expression of antisense HAP1 RNA in HeLa cells, which renders the cells hypersensitive to the toxic effects of MMS, H2O2, menadione and paraquat (21). Furthermore, generation, by gene targeting, of mice lacking a functional ref-1 (or hap1) gene indicates that this gene is necessary for early embryonic development of the mouse and shows the functional importance of HAP1 protein (22).
We have constructed HAP1-expressing transgenic CHO cells to study the role of this protein in conferring drug resistance. Under our experimental conditions, HAP1 expression does not modify the cell sensitivity to different agents, although the protein is expressed in the cell nuclei. This suggests that the constitutive level of AP-endonuclease activity is high enough to repair the AP sites formed during repair of damage induced by these compounds and confirms the results obtained by expressing HAP1 cDNA in HeLa cells (23).
However, expression of HAP1 protein renders the cells more sensitive to the toxic effects of MMC, porfiromycin, daunorubicin or DZQ, drugs that need bioreductive activation. MMC preferentially kills hypoxic tumor cells and requires bioreduction to exert its cytotoxic action. MMC alkylates DNA monofunctionally (24) and generates interstrand crosslinks by bifunctional alkylation (25). It is activated by various enzymes, e.g. DT-diaphorase (DTD) (26,27) or NADPH:cytochrome b15 reductase (28). In the presence of GSH, MMC forms predominantly bis-adducts in DNA in vitro, suggesting that GSH participates in the bifunctional activation of the drug in vivo (29). Porfiromycin also forms mono- and bis-adducts in cellular DNA (30) and the toxicity of this drug is higher in hypoxic than in aerobic cells (31). DZQ is activated by DTD and its cytotoxicity is inhibited by dicumarol, a DTD inhibitor (32). Cell sensitivity to the lethal effects of MMC and porfiromycin was enhanced in the presence of NAC, which is readily transformed to GSH in the cells, and expression of HAP1 protein was able to further enhance this sensitivity. As the foreign protein is mostly expressed in the cell nuclei, this suggests that the localization of the reducing agent plays an important role in the exertion of its activity. We have deleted the redox function of HAP1 protein and expressed this mutated enzyme in the cells. Although the AP-endonuclease activity was identical in cells expressing the wild-type or the mutated protein, the sensitivity was not modified when the mutated protein was expressed. This strongly suggests that the redox function of HAP1 protein may play a role in the activation of these drugs.
Hypoxic cells of solid tumors represent a resistant population that limits the curability by X-irradiation or by chemical compounds. Therefore, the role of enzymes in the reductive activation of chemotherapeutic agents is an area of interest to better understand and increase the selective toxicity of these treatments. Our results show that a protein, although not directly involved in oxidative metabolism, is able to influence cellular sensitivity to drugs requiring bioreduction used in cancer therapy.
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Acknowledgments
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This work was supported by grants from INSERM, ARC (Villejuif) and Ligue Nationale contre le Cancer. M.J.P.A. was supported by a grant from the Ministry of Education and Culture of Spain.
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Notes
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1 To whom correpondence should be addressed Email: laval{at}kb.inserm.fr 
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Received October 5, 1998;
revised November 30, 1998;
accepted November 30, 1998.