Molecular and biochemical mechanisms of fludarabine and cladribine resistance in a human promyelocytic cell line

2F-Adenine arabinoside (fludarabine, Fara-A) and 2-chloro-2'-deoxyadenosine (cladribine, CdA) are nucleoside analogues with antineoplastic activity ia vitro and in vivo. Lack of clinical resistance between CdA and Fara-A has been demonstrated in patients with chronic lymphocytic leukemia (G. Juliuss on et al., N, Engl. J, Med., 327: 1056-1061, 1992). To clarify the differen ces in mechanism of resistance to CdA and Fara-A in vitro, we developed two stable, resistant cell lines, HL60/CdA and HL60/ Fara-A, by exposure to in creasing concentrations of analogues over a period of 8 months. Resistant c ells tolerated >8000 and 5-fold higher concentrations of CdA and Fara-A, re spectively. The specific activity of the nucleoside phosphorylating enzyme (using deoxycytidine as substrate) in cell extracts from HL60/CdA and HL60/ Fara-A mutants was about 10 and 60%, respectively, compared with the parent al cell line. Western blot analysis using a polyclonal antibody showed no d etectable deoxycytidine kinase (dCK) protein in CdA-resistant cells, wherea s in Fara-A-resistant cells, it was at the same level as in the parental ce lls. The mitochondrial enzyme deoxyguanosine kinase was not altered in resi stant cell lines. The HL60/CdA cells showed cross-resistance to 2-chloro-2' -arabino-fluoro-2'-deoxyadenosine, Fara-A, arabinofuranosyl cytosine, diflu orodeoxyguanosine, and difluorodeoxycytidine toxicity, most likely because of the decreased phosphorylation of these analogues by dCK. Using real-time quantitative PCR, the mRNA levels of dCK and cytosolic 5'-n ucleotidase (5'-NT), a major nucleoside dephosphorylating enzyme, were meas ured. It was shown that the dCK mRNA levels in both CdA- and Fara-A resista nt cells were decreased in parallel with the activity. The expression of 5' -NT mRNA was not significantly elevated in CdA- and Fara-A resistant cells, as compared with the parental cells, Ribonucleotide reductase maintains a balanced supply of deoxynucleotide triphosphate pools in the cell and may a lso be a major cellular target for CdA and Fara-A nucleotides. Except for t he deoxycytidine triphosphate level, the intracellular deoxynucleotide trip hosphate pools were significantly higher in Fara-A-resistant cells compared with the parental cell line, This might be a consequence of mutation or al tered regulation of ribonucleotide reductase activity and may explain the 2 -5-fold cross-resistance to several nucleoside analogues observed with HL60 /Fara-A cells. It is likely that the resistance for CdA was mainly attribut able to a dCK deficiency, and Para-A-resistant cells might have another con tributing factor to the resistance beyond the dCK deficiency.