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 in vitro and in vivo. Lack of clinical resistance between CdA and Fara-A has been demonstrated in patients with chronic lymphocytic leukemia (G. Juliusson et al., N. Engl. J. Med., 327: 1056-1061, 1992). To clarify the differences 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 increasing concentrations of analogues over a period of 8 months. Resistant cells tolerated >8,000 and 5-fold higher concentrations of CdA and Fara-A, respectively. 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 parental cell line. Western blot analysis using a polyclonal antibody showed no detectable deoxycytidine kinase (dCK) protein in CdA-resistant cells, whereas in Fara-A-resistant cells, it was at the same level as in the parental cells. The mitochondrial enzyme deoxyguanosine kinase was not altered in resistant cell lines. The HL60/CdA cells showed cross-resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, Fara-A, arabinofuranosyl cytosine, difluorodeoxyguanosine, 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'-nucleotidase (5'-NT), a major nucleoside dephosphorylating enzyme, were measured. It was shown that the dCK mRNA levels in both CdA- and Fara-A resistant 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 also be a major cellular target for CdA and Fara-A nucleotides. Except for the deoxycytidine triphosphate level, the intracellular deoxynucleotide triphosphate pools were significantly higher in Fara-A-resistant cells compared with the parental cell line. This might be a consequence of mutation or altered 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 attributable to a dCK deficiency, and Fara-A-resistant cells might have another contributing factor to the resistance beyond the dCK deficiency.
Månsson
E
E
Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
Spasokoukotskaja
T
T
Sällström
J
J
Eriksson
S
S
Albertioni
F
F
D000970:Antineoplastic Agents / Q000633:toxicity*; D001692:Biological Transport; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects*; D017338:Cladribine / Q000493:pharmacokinetics* / Q000633:toxicity*; D004273:DNA, Neoplasm / Q000096:biosynthesis; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D018922:HL-60 Cells; D006801:Humans; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000633:toxicity
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D017338:Cladribine; D014740:Vidarabine; C024352:fludarabine