Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-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 designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated 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 silent 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 activity 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 enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.
Goan
Y G
YG
Department of Surgery, Veterans General Hospital-Kaohsiung, Taiwan, Republic of China.
Zhou
B
B
Hu
E
E
Mi
S
S
Yen
Y
Y
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003845:Deoxycytosine Nucleotides / Q000032:analysis; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007624:KB Cells; D012264:Ribonucleotide Reductases / Q000502:physiology*; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003841:Deoxycytidine; C024107:2'-deoxycytidine 5'-triphosphate; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate; D000093542:Gemcitabine