The pyrimidine nucleoside phosphorylases (PyNP), uridine (UP; EC 2.4.2.3) and thymidine (TP; EC 2.4.2.4) phosphorylase, are involved in the salvage pathway of pyrimidine nucleosides, but also in the activation of fluoropyrimidines. They are responsible for the conversion of the pro-drug 5′-deoxy-5-fluorouridine (5′-DFUR, doxifluridine) to its active form 5-fluorouracil (5-FU) (Armstrong and Diasio, 1980). Because of their reversible phosphorolytic activity, UP and TP also assume the conversion of 5-FU to its anabolites, respectively 5-fluorouridine (5-FUrd) and 5-fluoro-2′-deoxyuridine (5-FdUrd) (Ishitsuka et al, 1980;Peters et al, 1986).PyNP gene transfer into cancer cells in order to increase the metabolic activation of fluoropyrimidines and thus the sensitivity of transfected cells to these anti-metabolites could be a new strategy in cancer gene therapy. Encouraging data have been obtained with TP gene transfer (Patterson et al, 1995;Schwartz et al, 1995;Kato et al, 1997;Evrard et al, 1999aEvrard et al, , 1999b. Unlike TP, the data concerning UP are not numerous. NIH-3T3-derived cells expressing high levels of UP are more sensitive to fluoropyrimidines than parental cells . The increase of 5′DFUR antitumor activity by an extract from calf thymus (thymosin fraction 5) was attributed to an increase of UP activity (Ikemoto et al, 1999). In this paper, we studied the effects of UP overexpression by gene transfer in human breast cancer cells on the metabolic activation and on the cytotoxicity of fluoropyrimidines.
MATERIALS AND METHODS
Chemicals
Cell linesMCF-7 human breast adenocarcinoma cells (ATCC number HTB-22) were cultured at 37˚C in a fully humidified 5% CO 2 atmosphere in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine. COS-7 cells (ATCC number CRL-1651) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 5% FCS and 2 mM glutamine.
UP overexpression in COS-7 and MCF-7 cellsThe pUP-SG5 plasmid, containing the full length human UP cDNA (Watanabe and Uchida, 1995), was kindly provided by Dr SH Watanabe (Nippon Roche Research Center, Japan). The labelling of UP cDNA by a short myc sequence was performed by PCR using pUP-SG5, pFU polymerase (Stratagene, Amsterdam, The Netherlands) and specific primers. The myc/UP cDNA was ligated into the mammalian expression vector pcDNA3 (Invitrogen, NV Leek, The Netherlands) to produce pcmyc/UP. Cells were transfected with pcmyc/UP, pUP-SG5 or pcDNA3 (control) using Lipofectamine™ (Life Technologies, CergyPontoise, France). COS-7 cells were lysed after 48 h incubation. Stable MCF-7 transfectants were selected for 250 µg ml -1 geneticin (Life Technologies, Cergy-Pontoise, France) resistance.
ImmunoblottingProteins from cell lysates were electrophoresed on SDS-polyaclamide gel, electroblotted onto nitrocellulose membrane, labelled with an anti-myc antibody (Invitrogen, France) and visualized using X-ray films after incubation with ECL reagents as previously described (Evrard et al, 1999b). Summary The relationship between uri...