title: Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines.
abstract: Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine and to determine whether there are differences in resistance mechanisms between troxacitabine, gemcitabine, and cytarabine in human leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell line was highly sensitive to the antiproliferative effects of troxacitabine, gemcitabine, and cytarabine with inhibition of proliferation by 50% observed at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase (dCK)-deficient variant (CEM/dCK(-)) was resistant to all three drugs. In contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhibited high levels of resistance to cytarabine (1150-fold) and gemcitabine (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis of troxacitabine transportability by the five molecularly characterized human nucleoside transporters [human equilibrative nucleoside transporters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, hCNT2, and hCNT3] revealed that short- and long-term uptake of 10-30 microM [(3)H]troxacitabine was low and unaffected by the presence of either nucleoside transport inhibitors or high concentrations of nonradioactive troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that deficiencies in nucleoside transport were unlikely to impart resistance to troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145(R); 6300-fold) that exhibited reduced uptake of troxacitabine was cross-resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK activity toward deoxycytidine in DU145(R) cell lysates was <20% of that in DU145 cell lysates, and no activity was detected toward troxacitabine. Sequence analysis of cDNAs encoding dCK revealed a mutation of a highly conserved amino acid (Trp(92)-->Leu) in DU145(R) dCK, providing a possible explanation for the reduced phosphorylation of troxacitabine in DU145(R) lysates. Reduced deamination of deoxycytidine was also observed in DU145(R) relative to DU145 cells, and this may have contributed to the overall resistance phenotype. These results, which demonstrated a different resistance profile for troxacitabine, gemcitabine, and cytarabine, suggest that troxacitabine may have an advantage over gemcitabine and cytarabine in human malignancies that lack or have low nucleoside transport activities.
authors.affiliation: Shire BioChem Inc., Laval, Québec, H7V 4A7 Canada. hgourdeau@ca.shire.com
authors.forename: H
authors.initials: H
authors.lastname: Gourdeau
authors.forename: M L
authors.initials: ML
authors.lastname: Clarke
authors.forename: F
authors.initials: F
authors.lastname: Ouellet
authors.forename: D
authors.initials: D
authors.lastname: Mowles
authors.forename: M
authors.initials: M
authors.lastname: Selner
authors.forename: A
authors.initials: A
authors.lastname: Richard
authors.forename: N
authors.initials: N
authors.lastname: Lee
authors.forename: J R
authors.initials: JR
authors.lastname: Mackey
authors.forename: J D
authors.initials: JD
authors.lastname: Young
authors.forename: J
authors.initials: J
authors.lastname: Jolivet
authors.forename: R G
authors.initials: RG
authors.lastname: Lafrenière
authors.forename: C E
authors.initials: CE
authors.lastname: Cass
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D003561:Cytarabine / Q000493:pharmacokinetics; D003564:Cytidine Deaminase / Q000378:metabolism; D003596:Cytosine / Q000031:analogs & derivatives / Q000493:pharmacokinetics* / Q000494:pharmacology; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics; D003842:Deoxycytidine Kinase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004148:Dioxolanes / Q000493:pharmacokinetics* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D033703:Nucleoside Transport Proteins; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D013237:Stereoisomerism; D014316:Tritium; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000493:pharmacokinetics; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D002352:Carrier Proteins; D004148:Dioxolanes; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D003561:Cytarabine; D003841:Deoxycytidine; D014316:Tritium; C074908:troxacitabine; D003596:Cytosine; D012964:Sodium; D003842:Deoxycytidine Kinase; D003564:Cytidine Deaminase; D014529:Uridine; D000093542:Gemcitabine