Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines.

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.

Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib).

The human concentrative (Na(+)-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na(+)-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na(+)-dependent cib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na(+) concentration indicated a Na(+):uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element.

Differential transport of cytosine-containing nucleosides by recombinant human concentrative nucleoside transporter protein hCNT1.

The transportability of cytosine-containing nucleosides by recombinant hCNT1 was investigated in transfected mammalian cells. Apparent K(m) values for hCNT1-mediated transport of uridine, cytidine and deoxycytidine were, respectively, 59, 140 and 150 microM. Uridine transport was inhibited 89, 32 and 11%, respectively, by 500 microM gemcitabine, cytarabine and lamivudine, demonstrating that, unlike gemcitabine (a high-affinity hCNT1 permeant), cytarabine and lamivudine are poor hCNT1 permeants.

Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines.

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and (b) treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es approximately = cit > ei > cib >>> cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.

Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells.

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the 'equilibrative sensitive' or 'es' transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximately Km values of 21 microM, 12.5 microM and 15 microM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Stability of Liposyn II fat emulsion in total nutrient admixtures.

Compatibility and safety of a safflower oil-soybean oil lipid emulsion (Liposyn II, Abbott) with amino acids and dextrose in total nutrient admixtures (TNAs) were studied. Sixty-two admixtures representing 31 different combinations of fat emulsion, amino acid injection, and dextrose injection were tested. Both 10% and 20% concentrations of the fat emulsion and three concentrations each of amino acid injection and dextrose injection were used; the core admixture components were placed in empty flexible plastic bags in three different sequences: fat, amino acids, dextrose; fat, then dextrose and amino acids simultaneously; and amino acids and dextrose simultaneously, then fat. One of two mixtures of electrolytes and trace metals was added to each sample at the end of mixing. Six samples were tested after one day at 25 degrees C, 35 after two days at 5 degrees C plus one day at 30 degrees C, and 21 after nine days at 5 degrees C plus one day at 25 degrees C. Multivitamin injections were added to each TNA just before the 24-hour room-temperature storage. pH, emulsion particle size, and zeta potential (electrostatic surface charge of lipid particles) were measured after visual inspection of each sample. Amino acids were separated by high-performance liquid chromatography and measured. Dextrose was measured by size-exclusion chromatography. In a controlled study of 24 dogs, six-hour infusions of TNAs containing Liposyn II 20% were administered for 14 days, after which all major organs and tissues were studied microscopically. At all storage times in the compatibility study, all TNAs retained a uniform, milk-like appearance.(ABSTRACT TRUNCATED AT 250 WORDS)

The DNA content of human plasma cells.

The DNA content of human plasma cells from myeloma patients relative to that of leukocytes was determined by flow and microscopic cytofluorometry after propidium iodide and fluorescent Feulgen staining, respectively. Mononucleated myeloma plasma cells from all of the 17 patients studied contained more DNA (17 to 58%) than the leukocytes from the patient. The binucleated and trinucleated plasma cells, which were more prevalent in advanced cases, contained up to two and three times, respectively, the amount of DNA determined in the mononucleated plasma cells. These observations suggest that the ploidy abnormalities of myeloma plasma cells are even more extensive than the numerous karyotypic studies have indicated.