Mechanisms of Uptake and Resistance to Troxacitabine, a Novel Deoxycytidine Nucleoside Analogue, in Human Leukemic and Solid Tumor Cell Lines

[CANCER RESEARCH 61, 7217–7224, October 1, 2001] Mechanisms of Uptake and Resistance to Troxacitabine, a Novel Deoxycytidine Nucleoside Analogue, in Human Leukemic and Solid Tumor Cell Lines Henriette Gourdeau,1,2 Marilyn L. Clarke,1 France Ouellet, Delores Mowles, Milada Selner, Annie Richard, Nola Lee, John R. Mackey, James D. Young,3 Jacques Jolivet, Ronald G. Lafrenie`re, and Carol E. Cass4 Shire BioChem Inc., Laval, Que´bec, H7V 4A7 Canada [H. G., F. O., A. R., N. L., J. J., R. G. L.]; Departments of Oncology [M. L. C., J. R. M., C. E. C.] and Physiology [J. D. Y.], University of Alberta, Alberta T6G 1Z2 Canada; and Cross Cancer Institute, Edmonton, Alberta T6G 1Z2 Canada [M. L. C., D. M., M. S., J. R. M., C. E. C.] ABSTRACT oxycytidine analogues such as gemcitabine (2Ј,2Ј-difluorodeoxycyti- dine; dFdC) and cytarabine (1-␤-D-arabinofuranosylcytosine; araC), -Troxacitabine (Troxatyl; BCH-4556; (-)-2؅-deoxy-3؅-oxacytidine), a de which are in the ␤-D configuration, troxacitabine has a nonnatural ␤-L oxycytidine analogue with an unusual dioxolane structure and nonnatural configuration. Troxacitabine, which shares the same intracellular ac- L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was under- tivation pathway as gemcitabine and cytarabine, undergoes a series of taken to identify potential biochemical mechanisms of resistance to trox- phosphorylation reactions through a first rate-limiting step catalyzed 5 acitabine and to determine whether there are differences in resistance by dCK (EC 2.7.1.74) to form the active triphosphate nucleotide. mechanisms between troxacitabine, gemcitabine, and cytarabine in hu- Troxacitabine triphosphate is incorporated into DNA (2), which is man leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell believed to be the main mechanism of cytotoxicity of deoxycytidine line was highly sensitive to the antiproliferative effects of troxacitabine, analogues, although there are differences in their DNA incorporation gemcitabine, and cytarabine with inhibition of proliferation by 50% ob- patterns. The incorporation of troxacitabine causes immediate chain served at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase termination (6), whereas gemcitabine incorporation allows the addi- dCK)-deficient variant (CEM/dCK؊) was resistant to all three drugs. In) contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhib- tion of a single deoxynucleotide (7, 8), and cytarabine incorporation ited high levels of resistance to cytarabine (1150-fold) and gemcitabine allows the addition of multiple deoxynucleotides before chain termi- (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis nation (9, 10). Troxacitabine also differs from gemcitabine and cyt- of troxacitabine transportability by the five molecularly characterized arabine in that it is resistant to CDA (EC 3.5.4.5; Ref. 2). Although human nucleoside transporters [human equilibrative nucleoside trans- cellular uptake of troxacitabine has been reported to occur via the porters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, equilibrative nucleoside transport processes (11), it is not known hCNT2, and hCNT3] revealed that short- and long-term uptake of 10–30 whether functional plasma membrane nucleoside transporters are re- ␮ 3 M [ H]troxacitabine was low and unaffected by the presence of either quired for troxacitabine cytotoxicity, as has been reported for gem- nucleoside transport inhibitors or high concentrations of nonradioactive citabine (12, 13) and cytarabine (14, 15). troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that Understanding of nucleoside transport processes of human cells has deficiencies in nucleoside transport were unlikely to impart resistance to advanced considerably during recent years (reviewed in Refs. 16–18). troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145R; Seven distinct nucleoside transport processes have been demonstrated 6300-fold) that exhibited reduced uptake of troxacitabine was cross- in human cells on the basis of permeant selectivities, inhibition by resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK diagnostic agents, and mechanisms of transport. The proteins respon- activity toward deoxycytidine in DU145R cell lysates was <20% of that in sible for the five major nucleoside transport processes have been DU145 cell lysates, and no activity was detected toward troxacitabine. identified by molecular cloning. The human transporter proteins com- Sequence analysis of cDNAs encoding dCK revealed a mutation of a prise two functionally and molecularly distinct groups: (a) the hENTs; highly conserved amino acid (Trp923Leu) in DU145R dCK, providing a and (b) the sodium-dependent hCNTs. The proteins and their func- possible explanation for the reduced phosphorylation of troxacitabine in DU145R lysates. Reduced deamination of deoxycytidine was also observed tional activities are: (a) hENT1, broadly selective and mediates es in DU145R relative to DU145 cells, and this may have contributed to the (equilibrative NBMPR-sensitive) activity; (b) hENT2, broadly selec- overall resistance phenotype. These results, which demonstrated a differ- tive and mediates ei (equilibrative NBMPR-insensitive) activity; (c) ent resistance profile for troxacitabine, gemcitabine, and cytarabine, sug- hCNT1, pyrimidine nucleoside selective and mediates cit (concentra- gest that troxacitabine may have an advantage over gemcitabine and tive, insensitive to NBMPR and thymidine-selective activity); (d) cytarabine in human malignancies that lack or have low nucleoside trans- hCNT2, purine nucleoside and uridine selective and mediates cif port activities. (concentrative, insensitive to NBMPR and formycin B-selective) activity; and (e) hCNT3, broadly selective and mediates cib (concentra- INTRODUCTION tive, insensitive to NBMPR and broadly selective) activity. The proteins responsible for csg (concentrative, sensitive to NBMPR and Ј Ј Troxacitabine (Troxatyl; BCH-4556; (-)-2 -deoxy-3 -oxacytidine) guanosine-selective)- and cs (concentrative, sensitive to NBMPR)- has potent antitumor activity against human leukemic (1) and solid mediated transport activities, which have only been reported in a few tumor (2–5) xenograft animal models and is in clinical trials against cell types, have not been identified. human malignancies. Unlike naturally occurring nucleosides and de- The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine. The antiproliferative ef- Received 5/4/01; accepted 7/26/01. fects of troxacitabine against human cell lines that either possessed or The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with lacked the capacity for nucleoside transport or phosphorylation of 18 U.S.C. Section 1734 solely to indicate this fact. deoxycytidine were compared to determine whether the loss of either 1 H. G. and M. L. C. contributed equally to this work. 2 To whom requests for reprints should be addressed, at Shire BioChem Inc., 275 Boulevard Armand-Frappier, Laval, Que´bec H7V 4A7, Canada. E-mail: hgourdeau@ 5 The abbreviations used are: dCK, deoxycytidine kinase; CDA, cytidine deaminase; ca.shire.com. hCNT, human concentrative nucleoside transporter; hENT, human equilibrative nucleo- 3 J. D. Y. is a Heritage Scientist of the Alberta Heritage Foundation for Medical side transporter; NBMPR, nitrobenzylmercaptopurine ribonucleoside; THU, 3,4,5,6- Research. tetrahydrouridine; DMDC, 2Ј-deoxy-2Ј-methylidenecytidine; HPLC, high-performance 4 C. E. C. is Canada Research Chair in Oncology. liquid chromatography. 7217 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. UPTAKE AND RESISTANCE TO TROXACITABINE process gave rise to resistance to troxacitabine. The transportability of Resistance to troxacitabine was induced by exposing DU145 cells stepwise troxacitabine was then examined in human cell lines that either lacked to 2-fold increments of troxacitabine (0.01–10 ␮M) that were increased only nucleoside transport activity altogether or exhibited a single nucleo- when the proliferation rates of drug-treated cultures were similar to those of side transporter activity by measuring the rates of uptake of [3H]trox- untreated cultures. After 8 months of continuous exposures, the resistant R ␮ acitabine in the presence and absence of transport inhibitors or high variant (designated DU145 ) was maintained in 2 M troxacitabine. concentrations of nonradioactive troxacitabine. These studies indi- Chemosensitivity Testing. The relative cytotoxicities of troxacitabine, gemcitabine, and cytarabine against CCRF-CEM, CEM/ARAC8C, and CEM/ cated that troxacitabine, a poor permeant of nucleoside transporters, dCK cells were assessed using the CellTiter 96 proliferation assay. This assay enters cells primarily by passive diffusion, suggesting that a lack of is based on the reduction of a tetrazolium compound to a soluble formazan nucleoside transport capability was unlikely to be involved in the derivative by the dehydrogenase enzymes of metabolically active cells. The development of resistance. This was confirmed in a troxacitabine- absorbance (490 nm) is directly proportional

Load more