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Overexpression of DNA Polymerase ß Sensitizes Mammalian Cells to 2',3'-Deoxycytidine and 3'-Azido-3'-deoxythymidine¹

Laboratoire de Microbiologie et de Génétique, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cédex [K. B., P. F., C. C.]; Institut de Pharmacologie et Biologie Structurale, Centre National de la Recherche Scientifique, UPR 9062, 205 route de Narbonne, 31077 Toulouse cédex [J. S. H.]; and Laboratoire CAYLA, Z.I. Montaudran, 5 rue J. Rodier, 31400 Toulouse [M. T., J. P. R.]

Mammalian DNA polymerase ß is a DNA repair enzyme expressed constitutively at a low level. In vitro, purified DNA polymerase (Pol) ß incorporates the nucleotide analogues 2'-3' deoxycytidine (ddC)-triphosphate and 3'-azido-3'-deoxythymidine (AZT)-triphosphate into DNA, causing chain termination. We have tested the possibility of enhancing the cytotoxicity of these chain terminators against mammalian cells by increasing the level of Pol ß. Chinese hamster ovary AA8 and murine melanoma B16 cell lines were stably transfected with rat pol ß cDNA under the control of a viral enhancer/promoter. We found that overexpression of Pol ß sensitized the cells to ddC and AZT. To confirm the role of this polymerase in this process, we prepared cell extracts from the control and Pol ß overexpressing Chinese hamster ovary cell lines and tested in vitro their capacity to incorporate ddC-triphosphate and AZT-triphosphate into DNA. We found that inhibition of DNA replication by both chain terminators was more pronounced when extracts from pol ß-transfected cells were used, providing a direct evidence of the involvement of Pol ß in the sensitization process. In addition, we showed that cotransfection with bacterial or viral thymidine/thymidylate kinase genes enhanced the Pol ß-mediated cytotoxicity of AZT, suggesting that phosphorylation and polymerization activities might be combined to potentiate their respective effects. These observations may be useful for improving therapeutic efficiency of DNA chain terminators.

¹ This research was supported by the Région Midi-Pyrénées and the Ligue Nationale Contre le Cancer (axe thérapie génique).

² To whom requests for reprints should be addressed, at Institut de Pharmacologie et de Biologic Structurale, Centre National de la Recherche Scientifique UPR A9062, 205 route de Narbonne, 31077 Toulouse cédex, France. Fax: 33-5-61-17-59-94; E-mail: cazaux@ipbs.fr.

Received 7/15/96. Accepted 11/ 1/96.

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