Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands

Correspondence to: R MY Barge, Leiden University Medical Center, Department of Hematology, C2-R, PO Box 9600, 2300 RC Leiden, The Netherlands; Fax: 31 71 5266755

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 vitrois 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. Leukemia (2000) 14, 1678-1684.

acute myeloid leukemia (AML); drug resistance; cytarabine (AraC); deoxycytidine kinase (dCK); RT-PCR; dCK activity

Introduction

Patients with acute myeloid leukemia (AML) are treated with a combination of drugs usually consisting of 1--arabinofuranosylcytosine (cytarabine, AraC) and an anthracyclin, occasionally supplemented by a third drug.^1,2 The clinical outcome of patients with AML is still unsatisfactory,³ due to resistance of the leukemic blasts to AraC and the anthracyclins, resulting in a low long-term leukemia-free survival of the patients (30-40%). AraC is a deoxycytidine (dC) analogue and has to be phosphorylated to Ara-CTP to compete with dCTP for incorporation into DNA, finally inhibiting DNA synthesis. The key enzyme in the phosphorylation of AraC to Ara-CTP is deoxycytidine kinase (dCK, E.C.2.7.1.74), a salvage pathway enzyme that converts AraC to Ara-CMP by transferring a -phosphate of ATP or UTP.^4,5,6 In vitro studies have demonstrated that resistance to AraC can be induced in leukemic cells by exposing the cells to increasing concentrations of AraC. Many mechanisms for AraC resistance in vitro have been postulated, but a predominant role has been ascribed to loss of dCK activity, leading to a block in the conversion of AraC to its toxic form Ara-CTP.^{7,8,9,10,11,12} In AraC-resistant rat leukemic cells, inactivation of dCK was shown to be caused by either point mutations in the coding region of dCK or genomic rearrangements of the dCK locus.^13,14 dCK activity could be restored in these cell lines by transfecting the cells with wild-type dCK constructs, reflected by sensitivity to AraC.¹⁵

Since dCK inactivation may also play a role in resistance to AraC in vivo, reliable and convenient dCK measurements in vitro and in vivo are required. Previously, dCK activity was measured in crude cellular extracts for which high numbers of cells are required (10-60 ´ 10⁶ cells).^16,17,18 This analysis of dCK activity in cellular extracts is not optimal for the measurements of dCK activities in material of patients with leukemia, since usually only small numbers of cells are available and these specimens contain a heterogeneous cell population with varying percentages of leukemic blasts.¹⁷ Studier et al¹⁹ have set up a system in which they cloned the dCK complete coding region into a vector and translated dCK in a bacterial system. dCK activity was subsequently analyzed in vitro. Since this procedure is very time-consuming for the analysis of patients with resistant leukemia, we have set up a RT-PCR-based procedure that allows direct assessment of the dCK activity and dCK protein truncations in a small number of purified cells without the necessity of cloning the dCK locus into a vector. In a two-step RT-PCR on dCK cDNA, a T7 RNA polymerase promoter sequence is introduced upstream of the coding region. This T7 RNA polymerase promoter is used in an in vitro transcription-translation system to obtain pure dCK proteins. These in vitro translated dCK proteins are analyzed on sodium dodecyl sulfate-polyacrylamide (SDS) gel for their molecular weights and dCK activities are determined using tritium-labeled substrates. The protein activity truncation assay (PAT assay) was optimized in AraC or 5-aza-2'-deoxycytidine (decitabine, DAC) sensitive and resistant rat leukemic cell lines. In addition, we applied this novel assay for dCK activity measurements in bone marrow (BM) and PHA-stimulated T cells from healthy donors, as well as in purified leukemic blasts from patients with AML.

Materials and methods

Chemicals

2-Chloro-2'-deoxyadenosine (2-CdA), bovine serum albumin, adenosine 5'-triphosphate magnesium salt, uridine 5'-triphosphate sodium salt were purchased from Sigma (Sigma Chemicals, St Louis, MO, USA). Il-2 was purchased from Roussel (Roussel, Uclaf, Paris, France). Creatine kinase and creatine phosphate were obtained from Boehringer (Mannheim, Germany) and NaF from Merck (Darmstadt, Germany). ³H-2-chloro-2'-deoxyadenosine (22 Ci/mmol) was bought from Campro Scientific (Veenendaal, The Netherlands). L-[4,5-³H] Leucine (1 mCi/ml) TRK 6830 from Amersham (Pharmacia Biotech, Buckinghamshire, UK). TNT T7 Coupled Wheat Germ Extract System and RNasin Ribonuclease Inhibitor (40 U/l) were purchased from Promega (Madison, WI, USA). TRIzol reagent for total RNA isolation was from GIBCO BRL, Life Technologies, (Gaithersburg, MD, USA). QIAGEN provided us with the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Puregene DNA isolation kit was purchased from Gentra Systems (Minneapolis, MN, USA).

Rat leukemic cell lines

The AraC and 5-aza-2'-deoxycytidine (decitabine, DAC)-sensitive rat leukemic cell line RCL/O was originally purchased from TNO (Rijswijk, The Netherlands).^20,21 The rat leukemic cell lines were cultured as described previously.^13,14 The AraC resistant cell line RO/1-A was generated as described by Stegmann et al.¹⁴ The DAC resistant cell line RD/1 has been shown to be cross resistant to AraC¹³ (Table 1). A third AraC resistant cell line K7 was generated by ex vivo exposure of leukemic Brown Norway rats with AraC. A deletion of the dCK locus was observed by RT-PCR, resulting in AraC resistance in vitro.

Isolation of leukemic blasts from BM or PB of patients with AML

All specimens were collected as part of protocols that had been approved by the Medical Ethical Committee of the Leiden University Medical Center. Mononuclear cells were isolated from total bone marrow (BM) or peripheral blood (PB) samples by sedimentation on Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) (density 1.077 g/cm³) density gradient centrifugation and cryopreserved in liquid nitrogen until use. Post-Ficoll samples from PB (PB-PF) or BM (BM-PF) were thawed and leukemic blasts were purified by FACS sorting using at least two different patient specific leukemic cell markers (Table 2). Cell pellets were resuspended into 0.5 ml RPMI-1640 medium, and incubated with PE- or FITC-labeled moAb for 60 min at 4°C. Labeled leukemic blasts were washed three times, resuspended in RPMI-1640 + 2% FCS and sorted on a FACSvantage (Becton Dickinson, Mountain View, CA, USA). The purity of the leukemic blasts was always over 95% as confirmed by FACS analysis. PHA-stimulated T cells were generated as described previously.²²

Protein Activity Test (PAT assay): RNA isolation, cDNA synthesis, PCR amplifications

Total cellular RNA was isolated from a minimum of 2.5 ´ 10⁵ cells with TRIzol (GIBCO BRL, Life Technologies) according to the manufacturer's protocol. After precipitation with isopropanol, the RNA pellet was dissolved in 50 l RNase-free milliQ water. Single-stranded cDNA was prepared from 0.5-2 g RNA using M-MLV reverse transcriptase (Gibco BRL, Eggenstein, Germany) for 60 min at 37°C, as described.²² To determine the yield after cDNA synthesis, a control PCR was performed on the HPRT housekeeping gene for human and GAPDH housekeeping gene for rat, using gene specific primers generating a fragment of 351 and 450 bp respectively (forward primer HPRT: 5'-TGA CCA GTC AAC AGG GGA CA-3', reverse primer HPRT: 5'-CTT GAA CTC TGA TCT TAG GC-3', forward primer GAPDH: 5'-ACC ACA GTC CAT GCC ATC AC-3', reverse primer GAPDH: 5'-TCC ACC ACC CTG TTG CTG TA-3'). The yield was determined on a 1% agarose gel. The amount of cDNA for dCK-PCR amplifications was standardized to the HPRT-PCR yield from one healthy donor (dCO). The complete coding region of dCK was amplified using two dCK specific primers (rat A7; 5'-CCT GAG GTC CCG CGT CCT TA-3', human A7; 5'-TCT TTG CCG GAC GAG CTC TG-3', rat B5; 5'-TTG CCT GTT GTC TCC TGT GC-3', human B5; 5'-TGG AAC CAT TTG GCT GCC TG-3'), generating a 857 bp PCR fragment for human and 827 bp for rat. The PCR reaction was performed in a reaction mixture containing 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl pH 8.4, 0.2 mg BSA, 0.25 mM of each dNTP, 50 pmol of each primer and 1 U Taq Polymerase (Perkin Elmer Cetus, Foster City, CA, USA). The PCR was started after denaturation for 5 min at 95°C, followed by 30-33 cycles consisting of 48 s at 95°C, 48 s at 65°C and 48 s at 72°C, and a final elongation at 72°C for 5-10 min. A nested PCR was subsequently performed to introduce a T7 RNA polymerase promoter upstream of the start codon of the dCK locus generating an 823-bp construct for both human and rat. Six separate reactions were set up using 50 pmol of the T7 and B6 dCK primers (rat T7; 5'-GGA TCC TAA TAC GAC TCA CTA TAG GAA CAG ACC ACC ATG GCC ACC CCA CCT AAG AGG T-3', human T7; 5'-GGA TCC TAA TAC GAC TCA CTA TAG GAA CAG ACC ACC ATG GCC ACC CCG CCC AAG AGA A-3', rat B6; 5'-TGC AAT CAC AAA GTA CTC AA-3', human B6; 5'-CAA GAT CAC AAA GTA CTC AA-3'), and annealing temperatures of 55°C. PCR fragments were analyzed on a 1-1.5% agarose gel. Pooled reactions were purified by using the QIAgen PCR purification kit and concentration determined.

Coupled in vitro transcription-translation for dCK protein synthesis

The coupled in vitro transcription-translation reaction was performed according to the manufacturer's protocol (TNT coupled wheat germ extract system from Promega) on 1 g of the nested dCK-PCR construct, making use of the T7 RNA polymerase promoter introduced upstream of the dCK start codon. In vitro transcription-translation was performed at 30°C for 90 min and stored at -20°C.

³H-Leucine (3 Ci) was added to 20% of the transcription-translation reaction mixture to determine the yield of dCK protein as described in the TNT protocol with minor modifications. Briefly, ³H-Leu labeled protein was incubated in 1 M NaOH and 25% TCA respectively, spotted on glass fiber filters (Whatman GF/C glass fiber filters, Maidstone, UK), and washed thoroughly with 5% TCA and acetone. Filters were than incubated with filtercount (Packard Bioscience, Groningen, The Netherlands). ³H-incorporation was estimated using a scintillation counter. The protein concentration (mg/ml) was calculated as follows: (c.p.m./c.p.m. standard) ´ (mol ³H-Leu) ´ (1/number Leu amino acids in dCK) ´ (molecular weight dCK) ´ 10⁶.

Deoxycytidine kinase activity assay

The dCK activity was analyzed in duplicate experiments using a dCK activity protocol as originally described by Cheng and co-workers²³ with minor modifications. Briefly, dCK activity was determined using ³H-labeled 2-CdA as substrate, with substrate concentrations ranging from 0.2 to 0.002 mM. Assays were performed in reaction mixture containing 20 mM Tris/HCl pH 7.4, 5 mM MgUTP, 27 U/ml creatine phosphokinase, 7.5 mM creatine phosphate, 19 Ci/ml ³H-labeled 2CdA (specific activity 4 Ci/mmol), 10 mM unlabeled 2CdA, 7 mM NaF, 0.2% bovine serum albumin, and 0.2 mM tetrahydrouridine to block dC-deaminase activity. The reactions were initiated by the addition of 2.5 ng dCK protein per reaction and incubated at 37°C for 0-(10)-20-40-(60) min, whereafter, at each time incubation, 50 l aliquots were spotted on DEAE-coated paper discs (Whatman DE-81, Whatman, Maidstone, UK). Filters were dried and washed in 1 mM ammonium formate four times. Phosphorylated substrates bound to the filters were eluted from the filters by 0.6 M HCl/1.5 M NaCl, and ³H-labeled reaction products were determined by scintillation counting in Atomlight using a LKB Rackbeta liquid scintillation counter. Enzyme kinetic properties were calculated by linear regression analysis using Michaelis-Menten plots. K_m was expressed in mM and V_max in nmol reaction product formed per min per mg dCK protein.

SDS-PAGE

Approximately 150 ng ³H-Leu labeled dCK protein was separated on a 12.5% sodium dodecyl sulfate-polyacrylamide denaturing gel to check for dCK protein truncations and purity. The gel was fixed for 30 min in 10% methanol/10% acetic acid and after fixation incubated for 30 min in Amplifier (Amersham). The gel was dried on a vacuum slab gel dryer and autoradiography was determined after exposure to Kodak film overnight at -80°C.

Results

Assessment of the PAT assay in PHA-stimulated T cells and bone marrow from healthy donors

A schematic representation of the PAT-assay of one healthy donor (dCO) is given in Figure 1. RNA was isolated from a range of 2.5 ´ 10⁵ to 3 ´ 10⁶ PHA-stimulated T cells and used for cDNA synthesis. In a RT-PCR with A7-B5 primers on cDNA, the complete coding region of dCK was amplified generating an 857-bp PCR fragment. A nested PCR with T7-B6 primers was performed on this A7B5 PCR fragment to introduce a T7 RNA polymerase promoter sequence, upstream of the start codon of dCK, generating an 823-bp fragment (Figure 1a). Subsequently an in vitro transcription-translation reaction was performed on the T7B6-PCR constructs to generate dCK proteins. To estimate the amount of PCR product for optimal protein synthesis, a titration of 0.003-0.03 mg/ml T7B6-PCR construct was performed. At 0.01 mg/ml T7B6-PCR product, a maximal amount of dCK protein was synthesized as analyzed by ³H-leucine incorporation (data not shown). The in vitro translated dCK proteins were analyzed for their molecular weights (30.5 kDa for wild-type dCK) by gel electrophoresis on a 12.5% SDS-PAGE gel (Figure 1b). In parallel, dCK activity tests (Figure 1c) were performed on the in vitro translated dCK proteins with 0.01 mM ³H-labeled 2-CdA as a substrate. K_m and V_max values were determined using Michaelis-Menten plots. K_m and V_max values at protein concentrations ranging from 50-150 ng/ml varied from 1.52 ´ 10^-2-3.21 ´ 10^-2 mM and 601.7-1009.4 nmol/min ´ mg, respectively (Figure 2). For further dCK activity studies we standardized the dCK concentration at 125 ng/ml.

With this standardized dCK protocol, we determined dCK expression and activities of unfractionated BM samples and PHA-stimulated T cells from five healthy donors. From all samples, T7B6-PCR constructs of normal length (823 bp) were obtained coding for 30.5 kDa dCK proteins after in vitro transcription-translation (data not shown). The mean yield of dCK protein synthesis was 2.09 ´ 10^-2 g per reaction (range 1.85 ´ 10^-2-2.34 ´ 10^-2 g). dCK activities in BM ranged from 101-230 nmol/min ´ mg and in PHA-T cells from 81-218 nmol/min ´ mg dCK protein (Table 3).

PAT assay in primary human leukemic blasts

Bone marrow and peripheral blood samples of patients with acute myeloid leukemia do contain a heterogeneous population of cells. To measure dCK activity specifically in leukemic blasts, we purified the leukemic blast samples by FACS sorting using at least two patient specific cell markers (Table 2). Figure 3 shows the results of the PAT assay in (purified) AML blasts of 10 patients with AML. All PCR amplifications revealed T7B6-PCR constructs of 823 bp (Figure 3a) which could be translated in vitro to 30.5 kDa dCK proteins (Figure 3b). All 10 samples were shown to be able to phosphorylate 2-CdA with activities ranging from 80.0-508.0 nmol/min ´ mg (Figure 3c).

PAT assay in AraC resistant rat leukemic cell lines

We used the PAT assay in rat leukemic cell lines to test whether this protocol can also be used to analyze dCK involvement in AraC resistant cells. In our laboratory, we previously generated three different AraC resistant rat leukemic cell lines, RD/1, RO/1-A, and K7 (Table 1). In all three resistant cell lines, the resistant phenotype was caused by inactivation of dCK. In the parental AraC sensitive cell line RO/1, dCK activity was measured both in cellular extracts, as well as on in vitro translated dCK proteins (Table 1). dCK activities of 8.18 ´ 10^-2 nmol/min ´ ml in cellular extracts and 1.76 ´ 10^-2 nmol/min ´ ml in wheat germ lysates were observed. In the AraC-resistant cell line RD/1, wild-type T7B6 dCK-PCR fragments were amplified that coded for dCK proteins of 30.5 kDa. Although dCK proteins of normal molecular weight were detected, no dCK activity on in vitro translated could be measured using 2-CdA as a substrate. These findings were in agreement with the dCK activity measurements on total cellular extracts of this cell line (Table 1). In the second AraC-resistant rat leukemic cell line (RO/1-A) we detected additional PCR fragments in co-expression with wild-type dCK in the nested PCR reaction (Figure 4a). Analyzing these aberrant PCR fragments of approximately 500 and 200 bp in the in vitro transcription-translation system did reveal dCK proteins of approximately 7.5 kDa on SDS-PAGE for both PCR constructs (Figure 4b). These truncated dCK proteins were shown to be inactive in our dCK activity tests using ³H-labeled 2-CdA as substrate (Figure 4c). In a third AraC resistant cell line K7, it was shown that no dCK-PCR fragment could be amplified, corresponding with loss of dCK activity in cellular extracts.

Discussion

Mutational inactivation of dCK has been thought to play an important role in AraC resistance in vitro,^{7,8,9,10,13,14} however, limited in vivo data are available.²⁴ Previously, dCK activities were measured in cellular extracts or in bacterial lysates using the pET system. A disadvantage of the analysis of dCK activity in cellular extracts is the requirement of high amounts of cells. Moreover, it is known that dCK expression is strongly variable among different cell sources and moments of sampling,^25,26 resulting in changes in dCK activity measurements that are difficult to compare between various experiments. The cloning of dCK into the pET vector avoids these variables, but is very time-consuming since many cloned dCK-cDNA fragments will necessarily be tested to give a good representation of the whole sample when a heterogeneous cell population is present such as in leukemias. In this paper, we describe a novel method that allows direct assessment of dCK activity in a small number of (leukemic) cells. In this procedure, dCK complete coding region is amplified by RT-PCR and dCK proteins are synthesized using a T7 RNA polymerase promoter sequence introduced upstream of the coding region in a nested PCR reaction. The dCK protein can be analyzed for truncations on SDS-PAGE and activity measurements are performed on 125 ng/ml in vitro translated dCK proteins. Our RT-PCR-based dCK assay can be rapidly performed since cloning is not necessary. In addition, for this test only small numbers of cells are required (2.5 ´ 10⁵ cells). Even more, the dCK input in the activity assays is equal and therefore dCK activity measurements are not influenced by fluctuations in the dCK expression among cell sources. Another advantage is the ability to determine the molecular weight of the in vitro translated dCK proteins on SDS-PAGE gel.

We have demonstrated that this protein activity truncation assay (PAT assay) can easily be used to determine dCK activity and expression in primary human cells, purified leukemic blasts and rat leukemic cell lines. K_m (mean 25.4 M) and V_max (mean 794.2 nmol/min ´ mg) studies in PHA-stimulated T cells from a healthy donor showed that the dCK proteins obtained in the in vitro transcription-translation reaction exhibited similar enzyme kinetics as described by others (K_m values ranging from 0.99-163 M and V_max between 1-760 nmol/min ´ mg).^16,26,27 That the V_max values we observed are relatively high as compared to the literature, may be explained by the fact that there are no inhibitory factors in the wheat germ extract mixture, which may be present in cellular extract.^16,17,23,27

Enrichment of the leukemic blasts is necessary in order to analyze dCK in leukemic blasts specifically, since bone marrow or peripheral blood samples of patients with AML contain a heterogeneous cell population consisting of leukemic blasts and non-leukemic cells. Leukemic blasts were enriched by FACS sorting using two patient specific cell surface markers, increasing the blast percentage in the samples to over 95%. It was shown that FACS sorting did not influence the dCK activity since comparable dCK activities were observed in both purified (AML 2, 3, 4, 6, 7, 8, 9, and 10; mean activity 281.6 nmol/min ´ mg) and unpurified material (AML 1 and 5; mean activity 405.5 nmol/min ´ mg).

In three AraC-resistant rat leukemic cell lines exhibiting three different mechanisms of dCK inactivation, we demonstrated that the PAT assay can also simply be used to detect total loss of dCK activity either caused by point mutations (RD/1), internal deletions (RO/1-A) and gross genomic alterations (K7).

In conclusion, this PAT assay has shown to be a relatively rapid method for the analysis of dCK in small numbers of primary human cells, without the need of cloning the coding region of dCK into the pET vector. Total loss of dCK by mutational inactivations can easily be detected including truncations of the dCK protein on SDS-PAGE. Due to the simplicity of this assay, this assay is a convenient method to analyze more fundamental features of the dCK protein in vitro. This PAT assay might also be used to monitor the involvement of dCK inactivation in patients with resistant vs sensitive AML that are treated with AraC. In this respect, we have to stress that a FACS-sorted leukemic blast sample might still exist of a heterogeneous blast population, in which both wild-type as well as mutated dCK proteins are expressed. In addition, this method may also be used for analyzing the involvement of dCK inactivation in resistance to other drugs which need to be phosphorylated by dCK, such as gemcitibine resistance in solid tumors.¹¹

Acknowledgements

This project was supported by the Dutch Cancer Society (grant RUL 96-1347).

1 Willemze R, Suciu S, Archimbaud E, Muus P, Stryckmans P, Louwagie EA, Berneman Z, Tjean M, Wijermans P, Dohner H, Jehn U, Labar B, Jaksic B, Dardenne M, Zittoun R. A randomized phase II study on the effects of 5-aza-2'-deoxycytidine combined with either amsacrine or idarubicin in patients with relapsed acute leukemia: an EORTC Leukemia Cooperative Group phase II study (06893). Leukemia 1997; 11: (Suppl. 1) S24-S27), MEDLINE

2 Rowe JM. What is the best induction regimen for acute myelogenous leukemia? Leukemia 1998; 12: (Suppl. 1) S16-S19,

3 Schiller GJ. Treatment of resistant disease. Leukemia 1998; 12: (Suppl. 1) S20-S24, MEDLINE

4 Momparler RL, Fischer GA. Mammalian deoxynucleoside kinase. I. Deoxycytidine kinase: purification, properties, and kinetic studies with cytosine arabinoside. J Biol Chem 1968; 243: 4298-4304,

5 Harrington C, Perrino FW. The effects of cytosine arabinoside oil RNA-primed DNA synthesis by DNA polymerase alpha-primase. J Biol Chem 1995; 270: 26664-26669,

6 Major PP, Egan EM, Beardsley GP, Minden MD, Kufe DW. Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA. Proc Natl Acad Sci USA 1981; 78: 3235-3239,

7 Owens JK, Shewach DS, Ullman B, Mitchell BS. Resistance to1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene. Cancer Res 1992; 52: 2389-2393,

8 Bhalla K, Nayak R, Grant S. Isolation and characterization of a deoxycytidine kinase-deficient human promyelocytic leukemic cell line highly resistant to 1-beta-D-arabinofuranosylcytosine. Cancer Res 1984; 44: 5029-5037, MEDLINE

9 Kees UR, Ford J, Dawson VM, Piall E, Aherne GW. Development of resistance to 1-beta-D-arabinofuranosylcytosine after high-dose treatment in childhood lymphoblastic leukemia: analysis of resistance mechanism in established cell lines. Cancer Res 1989; 49: 3015-3019,

10 Bergman AM, Pinedo HM, Jongsma AP, Brouwer M, Ruiz v HV, Veerman G, Leyva A, Eriksson S, Peters GJ. Decreased resistance to gemcitabine (2',2'-difluorodeoxycitidine) of cytosine arabinoside-resistant myeloblastic murine and rat leukemia cell lines: role of altered activity and substrate specificity of deoxycytidine kinase. Biochem Pharmacol 1999; 57: 397-406,

11 Ruiz van Haperen VW, Veerman G, Eriksson S, Boven E, Stegmann AP, Hermsen M, Vermorken JB, Pinedo HM, Peters GJ. Development and molecular characterization of a 2',2'-difluorodeoxycytidine-resistant variant of the human ovarian carcinoma cell line A2780. Cancer Res 1994; 54: 4138-4143,

12 Dumontet C, Fabinowska-Majewska K, Mantinicic D, Callet BE, Tigaud I, Gandhi V, Lepoivre M, Peters GJ, Rolland MO, Wyczechowska D, Fang X, Gazzo S, Voorn DA, Vanier-Viornery A, MacKey J. Common resistance mechanisms to deoxynucleoside analogues in variants of the human erythroleukaemic line K562. Br J Haematol 1999; 106: 78-85, MEDLINE

13 Stegmann AP, Honders MW, Nagemeijer A, Hoebee B, Willemze R, Landegent JE. In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene. Ann Hematol 1995; 71: 41-47,

14 Stegmann AP, Honders MW, Willemze R, Landegent JE. De novo induced mutations in the deoxycytidine kinase (dck) gene in rat leukemic clonal cell lines confer resistance to cytarabine (AraC) and 5-axa-2'-deoxycytidine (DAC). Leukemia 1995; 9: 1032-1038,

15 Stegmann AP, Honders WH, Willemze R, Ruiz v HV, Landegent JE. Transfection of wild-type deoxycytidine kinase (dck) cDNA into an AraC- and DAC-resistant rat leukemic cell line of clonal origin fully restores drug sensitivity. Blood 1995; 85: 1188-1194,

16 Johansson M, Karlsson A. Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase. Biochem Pharmacol 1995; 50: 163-168,

17 Arner ES, Spasokoukotskaja T, Juliusson G, Liliemark J, Eriksson S. Phosphorylation of 2-chlorodeoxyadenosine (CdA) in extracts of peripheral blood mononuclear cells of leukaemic patients. Br J Haematol 1994; 87: 715-718, MEDLINE

18 Riva CM, Rustum YM. 1-beta-D-arabinofuranosylcytosine metabolism and incorporation into DNA as determinants of in vivo murine tumor cell response. Cancer Res 1985; 45: 6244-6249,

19 Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Meth Enzymol 1990; 185: 60-89,

20 Hagenbeek A, Martens AC, Colly LP. In vivo development of cytosine arabinoside resistance in the BN acute myelocytic leukemia. Semin Oncol 1987; 14: 202-206, MEDLINE

21 Colly LP, Willemze R, Honders W, Hoorn F, Edelbroek PM. In vivo studies on high-dose 1-beta-D-arabinofuranosylcytosine (HDara-C) and 1-beta-D-arabinofuranosyluracil (ara-U) with respect to pharmacokinetics, cell kinetics, and cytotoxicity in a rat myelocytic leukemia model (BNML). Semin Oncol 1985; 12: 49-54,

22 Kaashoek JG, Mout R, Falkenburg JH, Willemze R, Fibbe WE, Landegent JE. Cytokine production by the bladder carcinoma cell line 5637: rapid analysis of mRNA expression levels using a cDNA-PCR procedure. Lymphokin Cytokin Res 1991; 10: 231-235,

23 Cheng YC, Domin B, Lee LS. Human deoxycytidine kinase. Purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients. Biochim Biophys Acta 1977; 481: 481-492,

24 Flasshove M, Strumberg D, Ayscue L, Mitchell BS, Tirier C, Heit W, Seeber S, Schutte J. Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside. Leukemia 1994; 8: 780-785,

25 Momparler RL, Onetto N, Momparler LF, Gyger M, Leclerc JM, Rivard GE. Drug sensitivity test for patients with accute leukemia on high-dose ara-C therapy. Semin Oncol 1985; 12: 31-33,

26 Arner ES, Spasokoukotskaja T, Eriksson S. Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues. Biochem Biophys Res Commun 1992; 188: 712-718, MEDLINE

27 Usova EV, Eriksson S. The effects of high salt concentrations on the regulation of the substrate specificity of human recombinant deoxycytidine kinase. Eur J Biochem 1997; 248: 762-766,

Figure 1  Schematic representation of the protein activity truncation assay (PAT assay). (a) A7B5 and T7B6 PCR fragments were analyzed on a 1.5% agarose gel. (b) In vitro translated dCK proteins analyzed on a 12.5% SDS-PAGE gel. (c) Activity test on 125 ng in vitro translated dCK proteins.

Figure 2  Analysis of the optimal amount of dCK protein for accurate K_m studies. Kinetic constants for dCK activity isolated from total BM samples from a pool of five healthy donors were calculated with 2-CdA as substrate. (a) K_m and V_max values were calculated by fitting to the Michaelis-Menten model. (b) The K_m and V_max values given in (mM) and (nmol/min ´ mg), respectively.

Figure 3  PAT assay on primary human leukemic cells. (a) T7B6 dCK-PCR constructs analyzed on a 1.5% agarose gel. T7B6 RT-PCR analysis performed on cDNA from (purified) leukemic blasts isolated from patients with AML. (b) Corresponding dCK proteins obtained by in vitro transcription-translation. (c) Corresponding dCK activities as determined on 2.5 ng dCK protein using 0.002 mM ³H-labeled 2-CdA as substrate.

Figure 4  PAT assay on truncated dCK-PCR constructs generated from rat leukemic cells. (a) T7B6 dCK-PCR fragments as detected on a 1.5% agarose gel. Lane 1, wild-type dCK from the RO/1 cell line; lane 2, truncated dCK-PCR construct of approximately 500 bp; lane 3, aberrant dCK-PCR product of about 200 bp. (b) In vitro translated dCK proteins of these truncated PCR constructs. (c) Corresponding dCK protein activities.

Table 1  Characteristics of rat leukemic cell lines

Table 2  Characteristics of patients with AML

Table 3  dCK activity values of in vitro translated proteins from primary human cells with 0.002 mM 2-CdA as substrate