DEVELOPMENT AND MOLECULAR CHARACTERIZATION OF A 2',2'-DIFLUORODEOXYCYTIDINERESISTANT VARIANT OF THE HUMAN OVARIAN-CARCINOMA CELL-LINE A2780

2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine a nalogue with promising antitumor activity. In order to be active it mu st be phosphorylated by deoxycytidine kinase (dCK). We induced resista nce to dFdCyd in the human ovarian carcinoma cell line A2780 by exposu re to increasing concentrations of dFdCyd. The IC50, defined as the co ncentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 mu M in the resistant vari ant, named AG6000. Although the resistant cell line is routinely cultu red in 6 mu M dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd. AG6000 is cross-resistant to other drugs which require activation by dCK, such as 1-beta-D-arabinofuranos ylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. There was no specific dCK activity in extracts from AG6000 cells. Western bl ot analysis using a polyclonal anti-dCK antibody did not reveal any dC K protein in AG6000 cell extracts. Reverse-transcribed and PCR-amplifi ed mRNA, using specific dCK primers, demonstrated that AG6000 expresse d a normal length amplicon of 701 base pairs, besides an aberrant ampl icon of 500 base pairs. Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000. The latter cell line wa s also cross-resistant to 2',2'-difluorodeoxyuridine, the deamination product of dFdCyd. Additionally, cross-resistance to the multidrug res istance drugs doxorubicin and vincristine was observed. This was not a ssociated with the induction of P-glycoprotein, as determined by the R Nase protection assay. Injection of AG6000 cells s.c. into nude mice d emonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors. No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors. The se results indicate that the dFdCyd resistance phenotype is stable, an d mainly due to dCK deficiency.