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.