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In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo. [ABSTRACT FROM AUTHOR]

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML. (Blood. 2000;96:1517-1524)

We have investigated whether cytarabine (AraC) or decitabine (DAC) induce deficiency of deoxycytidine kinase (DCK) through different mutations of the dck gene, related to their distinct interference with DNA replication. Also, it is not known whether mutations of the dck gene are the result of selection of mutants or de novo induction. To address these issues, three subclones of a rat leukemic cell line (RCL/O), sensitive to cytotoxicity mediated by AraC and DAC, were exposed to gradually increasing concentrations (from 0.1 to 10 microM) of either AraC or DAC over a 140 days vs a 180 days period. During the course of resistance induction DCK activity was monitored. We found that all clones acquired irreversible cross-resistance, at marginally cytotoxic AraC or DAC concentrations of 0.1 to 0.4 times the IC50 for the parental clones. Furthermore, all resistant cell lines were DCK deficient and harbored different mutations in the dck gene. AraC induced both rearrangements and point mutations in the dck gene when administered over 140 days and 180 days, respectively. 140 days DAC induction yielded point mutations only. All point mutations detected were nonrandomly distributed within the dck coding region. SSCP analysis showed that in the majority of resistant clones more than one bandshift was present. The data suggest the presence of multiple resistant clones, originating from one sensitive clone and thus arguing against selection of mutants as a mechanism for the development of AraC and DAC resistance.

Deoxycytidine kinase activity (dCk) was monitored in cell lines from a rat acute myeloid leukemia model of acquired resistance to cytosine arabinoside (AraC) and decitabine (DAC). In both AraC-resistant cell lines (RCL/A and its subclone RA/7), as well as in a DAC-resistant cell line (RCL/D) which we generated from the drug-sensitive RCL/0 cell line, a total deficiency of dCk activity and a cross-resistance for AraC and DAC was demonstrated. Furthermore, the metabolization of deoxycytidine (dC) was severely impaired in all these cell lines. Km values for dC (9.4 microM in RCL/0 cells) had increased 70- to 100-fold in RCL/D (Km = 673.2 microM), in RCL/A (Km = 947.2 microM) and in RA/7 (Km = 817.5 microM). Vmax values were unaltered in RCL/D and RA/7, and twofold increased in RCL/A. Addition of hydroxyurea (HU) to cell cultures stimulated dCk salvage pathway activity in RCL/0 cells for dC, AraC, and DAC by increasing Vmax values approximately 160% leaving Km constants unchanged. In all resistant cell lines, HU pre-incubation did not influence the level of dCk activity, leaving Km and Vmax values unaltered. These data indicate that deficiency of dCk activity is crucial in the mechanism of drug resistance in this model.

In this study we describe the establishment of a leukemic cell line (BNML-CL/ara-C), originating from the 1-beta-D-arabinofuranosylcytosine (ara-C)-resistant brown Norway rat myelocytic leukemia model (BNML/ara-C), that retains the in vivo generated ara-C resistance. Its biological and biochemical characteristics have been compared with a cell line, derived from the ara-C-sensitive BNML model (BNML-CL/O). Resistance to ara-C was attributed to a decrease in phosphorylation of ara-C. Deoxycytidine (dCyd) kinase activity in crude cell extracts with dCyd as substrate showed similar enzyme activities in both cell lines, whereas with ara-C as substrate no dCyd kinase activity was detectable in the ara-C-resistant cell line. Two isoenzymes of dCyd kinase with different substrate specificities have been described (Cheng, Y.C., Domin, B., and Lee, L.S. Biochim. Biophys. Acta, 481: 481-492, 1977), cytoplasmic (dCyd kinase I, substrates: dCyd and ara-C) and mitochondrial (dCyd kinase II, substrates: dCyd and thymidine). In the ara-C-sensitive BNML model, thymidine induced a reduction of dCyd kinase activity when dCyd was used as substrate. However, thymidine did not affect kinase activity with ara-C was used as substrate. In the BNML-CL/ara-C, thymidine even induces a dCyd kinase inhibition of 85% with dCyd as substrate. It is likely that the ara-C-specific dCyd kinase deficiency in BNML-CL/ara-C cells was due to a selective loss of dCyd kinase I, whereas dCyd kinase II activity remained intact.

In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.

In this study, it has been shown that in 21 patients with AML the dCyd kinase and dCyd deaminase activities correspond closely to the clinical response to ara-C remission induction therapy. Patients with primary disease were treated with a conventional-dose ara-C regimen whereas nonresponders and relapsed patients followed an ID ara-C regimen (1 g/m2 X 12). Of these 21 patients (11 with primary disease and ten relapsed), seven had ara-C resistant disease (three primary and four relapsed patients). Five of the seven patients had a very low dCyd kinase and normal dCyd deaminase activity, whereas the other two had a normal dCyd kinase and an increased dCyd deaminase activity.