Overexpression of ribonucleotide reductase as a mechanism of resistance to2,2-difluorodeoxycytidine in the human KB cancer cell line

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designate d the KB-Gem clone. The KB parental cell line IC50 was 0.3 mu M dFdCyd, as compared with the KB-Gem clone IC50 of 32 mu M dFdCyd. The KB-Gem clone dem onstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA ( 9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK), Resulting sequences revealed two silen t mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme acti vity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzy me activities of the KB-Gem clone and parental cells. Thus, contrary to pre vious hypotheses, DCK deficiency does not play the primary role in the resi stance mechanism of dFdCyd, accepting a secondary role to the overexpressio n of the target gene, RR, and pool expansion.