ICANCERRESEARCH57.I10-I 16,JanuaryI. I997J Overexpression of DNA Polymerase @JSensitizesMammalian Cells to 2',3'-Deoxycytidine and 3'-Azido-3' .deoxythymidine' Khalil Bouayadi, Jean-Sébastien Hoffmann, Pascal Fons, Michele Tiraby, Jean-Paul Reynes, and Christophe Cazaux2 Laboratoire de Microbiologic et de Génétique,UniversitéPaul Sabatier, 118 route de Narbonne, 31062 Toulouse cédex(K. B., P. F., C. C.]; Institut de Pharmacologic et Biologic Structurale. Centre National de Ia Recherche ScienujIque, UPR 9062, 205 route de Narbonne, 31077 Toulouse cédexfl. S. H.!; and Laboratoire CAYLA, ZI. Montaudran, 5 rue J. Rodier, 3/400 Toulouse (M. T., J. P. R.J ABSTRACT nator AZT-MP was also observed (10, 11). In vivo, AZT-MP is incorporated into cellular DNA (12), and it has been suggested that Mammalian DNA polymerase @3is a DNA repair enzyme expressed polymerase 13may play a role in this process (1 1). @ constitutively at a low level. In vitro, purified DNA polymerase (Pol) incorporates the nucleotide analogues 2'-3' deoxycytidine (ddC)-triphos AZT and ddC are antiviral agents currently used in the treatment phate and 3'-azido-3'-deoxythymidine (AZT)-triphosphate Into DNA, of AIDS. These chemotherapeutic drugs target HIV reverse tran causing chain termination. We have tested the possibility ofenhancing the scriptase which incorporates them into HIV DNA, terminating cytotoxicity of these chain terminators against mammalian cells by in DNA polymerization (13). In this study, we hypothesized that creasing the level of Pol (I. Chinese hamster ovary AA8 and murine overexpression of Pol f3, a cellular target of ddC and AZT, might melanoma B16 cell lines were stably transfected with rat pol fi cDNA render these analogues effective also against tumor cells. To ex underthe controlof a viral enhancer/promoter.Wefoundthat overex plore this possibility, we stably transfected CHO AA8 and murine pression of Pol @3sensitized the cells to ddC and AZT. To confirm the role of this polymerasein this process,we preparedcell extractsfrom the melanoma B 16 cells with rat polf3 cDNA. We found an increased control and Pol @3overexpressingChinese hamster ovary cell lines and sensitivity to ddC and AZT of the transfected cells, and we tested in vitro their capacity to incorporate ddC-triphosphate and AZT confirmed the involvement of Pol (3 in this process using an in vitro triphosphate into DNA. We found that inhibition of DNA replication by replication assay with cell extracts. To our knowledge, this is the both chain terminators was more pronounced when extracts from pol first report of a kill or “suicide―associationinvolving a polymerase @3-transfectedcellswere used, providing a direct evidence of the involve activity. ment of Pol @3inthe sensitizationprocess.In addition,we showedthat The conversion of nucleoside analogues to nucleotide triphosphates cotransfection with bacterial or viral thymidine/thymidylate kinase genes enhanced the Pol fl-mediated cytotoxicity of AZT, suggesting that phos. is necessary before their incorporation into DNA by DNA poly phorylationandpolymerizationactivitiesmightbecombinedtopotentiate merases. Analogues readily enter cells but are often poor substrates their respective effects. These observations may be useful for improving for the sequential conversion to the phosphorylated forms by cellular therapeutic efficiency of DNA chain terminators. TK and TMK which specifically convert thymidine (14). For instance, the concentration of AZT-MP is more than 50-fold higher than that of AZT-TP in human cell lines, suggesting that it is a very poor substrate INTRODUCTION for human thymidylate kinase (13). Here, we present data showing Pol j3@,a single polypeptide of 39 kDa, is one of the five known that cells coexpressing both Pol f3 and bacterial or herpes thymidine/ mammalian DNA polymerases and is highly conserved among higher thymidylate kinases are more sensitive to AZT than cells expressing eukaryotes (I). It is believed to function primarily in the repair of only kinases or polymerase. These data may have particular impor damaged DNA (2) but may also have a role in DNA replication (3, 4) tance in the perspective of new therapeutic combinations for cancer and in DNA damage processing (5). Pol /3 is expressed at a constant gene therapy. level throughout the cell cycle (6) and exposure of cells to certain DNA-damaging agents induces Pol @3expression (7, 8). Features that distinguish Pol @3fromother cellular polymerases are its small size, MATERIALS AND METHODS the lack of associated exonuclease and endonuclease activities, and its high infidelity in replicating DNA (9). Materials. All oligonucleotides were synthesized on a Cyclone Plus In vitro, it has been demonstrated that purified Pol (3 efficiently DNA synthesizer from MilliGenlBiosearch and purified on 20% polyacryl amide gel. For DNA synthesis assays, the 60-mer templates were hybrid incorporates the deoxynucleotide analogue ddCMP, an inhibitor of ized to the 5' 32P-labeled 17-mer primer. Pol 13cDNA harboring plasmid DNA synthesis, with efficiency comparable to that of dCMP when an pRSET was kindly furnished by Dr. Wilson (Galveston, TX). Highly oligonucleotide template is used (10). Under similar conditions, but purified calf thymus DNA polymerase a-primase, @3,8, and a were gener using a higher amount of enzyme, incorporation of the chain termi ous gifts from Dr. HUbscher (Zurich, Switzerland) and polyclonal antibod ies against Pol @3were provided by Dr. Sweasy (Yale University, New Received 7/I 5196;accepted 11/1/96. Haven, CT). DNA plasmids pUT526& pUT599 (VECT 4991), pZEOSGO, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with and pZEOSG1 were obtained from Cayla (Toulouse, France). Analogues 18 U.S.C. Section 1734 solely Io indicate this fact. were obtained from Weilcome (AZT), Moravek Biochemicals, Inc. (AZT @ This research was supported by the Region Midi-Pyrénéesandthe Ligue Nationale TP), Sigma (ddC), and Pharmacia (ddCTP). Restriction and DNA modifi Contre Ic Cancer (axe thdrapie génique). cation enzymes were obtained from New England Biolabs and Boehringer 2 To whom requests for reprints should be addressed, at Institut de Pharmacologic et de Biologic Structurale, Centre National de Ia Recherche Scientifique UPR A9062, 205 Mannheim. route de Narbonne, 31077 Toulouse cédex,France.Fax: 33-5-61-17-59-94; E-mail: Bacteria and Mammalian Cell Culture. Bacteria were grown in LB broth [email protected]. complemented when required with zeocin (Cayla) at 20 p.g/ml. CHO AA8 and 3 The abbreviations used are: Pol (3, DNA polymerase @;HSV tic, herpes simplex virus type-I thymidine kinase gene; TK, thymidine kinase; TMK, thymidylate kinase; Sh, ble Bl6 BL/6 parental cells were grown in L-glutamine containing a-MEM and Sh encoded protein conferring resistance to zeocin; ddC, 2'-3' deoxycytidine; AZT, RPMI 1640 (BioWhittaker), respectively, complemented with 10% FCS (Bio 3'-azido-3'-deoxythymidine; AZT-MP, AZT monophosphate; AZT-TP, AZT triphos Whittaker), penicillin G/streptomycin sulfate (BioWhittaker), and amphoteri phate; ddCMP, ddC monophosphate; GCV. ganciclovir [(2-amino-1,9-(2-hydroxy-l-hy droxymethyl)ethoxy)methyl)-6H-purine-6-one]; CHO, Chinese hamster ovary cell; HSV, cm B (Sigma). Media for growth of stably transfected CHO and Bl6 cells were @ herpes simplex virus: HSV-l, herpes simplex virus type I ddCTP, ddC triphosphate. supplemented by 100 gxg/mland 20 g@g/m1zeocin(Cayla), respectively. 110 Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1997 American Association for Cancer Research. CYTOTOXICACTIONOF DNA POLYMERASE@3 Construction of Expression Plasmids. Pol @3-overexpressing plasmid mm and were disrupted in a Dounce homogenizer. Nuclei were harvested by pUTpolIJ was constructed from a pUT687 plasmid4 carrying the thymidine centrifugation for 10 mm at 3300 X g at 0°C.The nuclei pellet was suspended kinase gene of Escherichia coli fused in frame with the bacterial Sh ble gene in hypotonic buffer containing 350 mtvi NaCI. After 30 mm of extraction at conferring resistance to the broad-spectral zeocin xenobiotic of the phleomycin 0°C,thenuclear extracts were centrifuged at 15,000 X g for 20 mm. Proteins family (15). The pol @3genegenerated primers TA'VFCCATGOCACTCGTG from cytosol and nuclear extracts were precipitated by addition of ammonium GAACFCGCAAAC1TF and UAAGCTAGCTCACTCCTGTCmGGGC sulfate, resuspended in dialysis buffer [50 mt@iTris-HC1 (pH 7.5), 1 mM DTT, TC. Upon restriction enzyme digestion, a NcoI-NheI fragment containing pol 100 mt@imono-K glutamic acid, and 10% glycerol], and dialyzed for 2 h at 0°C. @3wasobtainedandthenrecombinedwiththeNcoI-AvrIIfragmentofpUT687 Extracts were frozen in liquid nitrogen and stored at —70°C.Theywere used to give pUTpolf3(Fig. IA). for both Western blotting and in vitro DNA replication assays. The E. coli tmk gene was fused with E. coli tk and Sh ble by using a 4-kb Western Blot Analysis. Sixty @igof cell extracts were electrophoresed in PCR-generated fragment obtained as already described (16) by amplification a 12% SDS-polyacrylamide gel. Proteins in the gel were transferred electro of the acpP-hoIB intergenic region of the Kohara phage AE9G1(17). First, this phoretically to nitrocellulose membrane. Pol (3 was revealed by incubating the tmk-containing PCR product digested with NcoI and MluI (treated with Kle membrane with purified rabbit anti-PoI (3 polyclonal antibody