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Molecular Oncology, Markers, Clinical Correlates |
Department of Medicine and Care, Clinical Pharmacology [K. L., C. P., B. P.], Faculty of Health Sciences, SE-581 85 Linköping, Sweden; Departments of Clinical Pharmacology [E. M., F. A.] and Clinical Oncology [J. L.], Karolinska Hospital, SE-171 76 Stockholm, Sweden; Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University of Medicine, H-1444 Budapest, Hungary [T. S.]; and Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Center, SE-751 85 Uppsala, Sweden [S. E.]
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ABSTRACT |
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INTRODUCTION |
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CAFdA is a 2'-arabino-fluoro derivative of CdA. The introduction of a fluorine at the 2'-arabino position of CdA has increased its acid stability. The solubility problems associated with the administration of Fara-A and the low oral bioavailability of CdA may be overcome by using CAFdA. We have reported previously (13) that in some patients the plasma chloroadenine, a hydrolysis product of CdA, exceeded that of the CdA concentration when patients were treated with CdA p.o. CAFdA was more stable against deglycosylation by hepatic enzymes as compared with CdA (14) .
The mechanism of action of CAFdA has been studied in detail elsewhere (15, 16, 17) . CAFdA has properties that combine the features of two clinically used drugs, CdA and Fara-A. CAFdA, like CdA and Fara-A, is toxic to nondividing human lymphocytes as well as to rapidly proliferating cells. CAFdA is phosphorylated to its monophosphate mainly by dCK and also by mitochondrial enzyme dGK (18) and, after further cellular phosphorylation, to the final active metabolite CAFdATP. CAFdATP inhibited ribonucleotide reductase to an extent similar to CdATP, and DNA polymerase to an extent similar to Fara-ATP. However, data on metabolism and the mechanism of drug resistance to CAFdA are limited in humans, and, thus far, there is only one report on CAFdA pharmacokinetics in animals (19) . A therapeutic effect was shown after a 1-week oral treatment with CAFdA, which eliminated 90% of the CLL cells transplanted into severe combined immunodeficiency mice (20) . CAFdA administered p.o. has also been shown to be an effective agent against human colon tumor xenografts (21) .
In the present study, we have characterized the intracellular metabolism of CAFdA to its mono-, di-, and triphosphate derivatives in vitro, both in human cell lines and in peripheral blood mononuclear cells isolated from CLL and AML patients. We have also determined the kinetic parameters for dCK using CAFdA as substrate in crude extracts from various human cell lines, as well as with purified recombinant dCK. The results were compared with those obtained with CdA to evaluate the therapeutic effectiveness of CAFdA as a basis for future clinical trials in humans. Moreover, human T-lymphoblastic (CCRF-CEM) and human promyelocytic leukemia (HL60) cell lines were exposed continuously to CAFdA, as a model for acquired drug resistance. The resulting cells were characterized with respect to growth inhibition, drug metabolism, cytotoxicity, and cross-resistance to other antimetabolites and clinically used anticancer drugs. These results may be important for preclinical evaluation of drug toxicity and sensitivity during CAFdA therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells were suspended in RPMI 1640 containing 25 mM HEPES [RPMI 1640-HEPES (Life Technologies, Inc., Paisley, United Kingdom) to get 1 x 106 cells/ml; and CdA (from Dr. Zygmunt Kazimierczuk, Foundation for the Development of Diagnostics and Therapy, Warsaw, Poland) or CAFdA (kindly provided by Dr. Howard Cottam, University of California, San Diego, CA) was added to the medium at a concentration 0.05–25 µM. After incubation for 2 h at 37°C, cells were centrifuged, washed twice with ice-cold PBS, and extracted for nucleotide determinations, performed as described in "HPLC Determination of CdA and CAFdA Nucleotides" section. Cells from a CLL patient were incubated with 10 µM CdA or CAFdA for 2 h. After incubation, the cells were centrifuged and resuspended in drug-free RPMI 1640-HEPES. To study the efflux kinetics of CdA and CAFdA, cells were withdrawn at desired intervals, and determinations of CdA and CAFdA nucleotides were performed with HPLC as described below.
Development of Resistant Cell Lines.
To delineate the mechanism of resistance to CAFdA, the human T-lymphoblastic (CCRF-CEM) and human promyelocytic leukemia (HL60) cell lines were exposed continuously to increasing concentrations of CAFdA over a period of 8–12 months. At intervals of 2 weeks, the concentrations of the drugs were increased by 5 nM. Then, the resistant cell lines were kept at concentrations of 100 nM CAFdA in CCRF-CEM cells (CEM/CAFdA) and 150 nM CAFdA in HL60 (HL60/CAFdA) cells for 2 months. The cell lines maintained stable resistance for at least 20 passages in the absence of drugs. All of the cell lines were kept in suspension culture in exponential growth in RPMI 1640 containing heat-inactivated FCS (10%), penicillin (100 units/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM; all from Life Technologies, Inc., Paisley, United Kingdom) at 37°C in a humidified atmosphere containing 5% CO2 and were routinely tested for Mycoplasma contamination.
The number of cells in G1, S, and G2+M phase of the cell cycle were determined using a PAS II cytometer (Partec, Münster, Germany), which was equipped with a mercury arc lamp. 4,6-diamidino-2-phenylindole was excited at 365 nm and the fluorescence was measured at >435 nm. The multicycle program (Phoenix Flow Systems, San Diego, CA) was used for histogram analysis.
Before each experiment, the cell lines were subcultured in drug-free medium for at least 2 passages and then they were cultured with logarithmic growth (about 0.8–1.3 x 106) over a period of 3 days, washed twice with prewarmed RPMI 1640-HEPES, and counted. The cells were resuspended in the same medium to a final count of 1–2 x 106 cells/ml.
Cytotoxicity Assay.
The cytotoxicity assay was performed as described by Mosmann et al.(22)
. Briefly, cell lines were suspended at 2 x 105 cells/ml, and 100-µl aliquots were dispensed into 96-well round-bottomed microtiter plates that already contained 5 µl of drug dilutions in triplicate. Wells containing no drugs were used as controls, and wells containing no cells were used as blanks. The plates were then incubated at 37°C for 72 h in an atmosphere of 5% CO2. We also investigated whether a continuous or a short incubation of CAFdA might have different efficacies in CCRF-CEM and HL60 cells. The cells were incubated either with 50 nM drug continuously for 120 h or with 300 nM drug for 2 h every 24 h over five days. Afterward, 10 µl of a stock solution of 5 mg/ml MTT (Sigma, St. Louis, MO) was added to each well, and the plates were incubated for an additional 4 h at 37°C. Formazan crystals were dissolved with 100 µl of 10% SDS/10 mM HCl solution overnight at 37°C. Absorbance (A) was measured using an ELISA plate reader (Labsystems Multiscan RC) at the wavelength of 540 nm with reference at 650 nm. Cell survival (CS) was expressed as percentage of control wells:
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HPLC Determination of CdA and CAFdA Nucleotides.
Intracellular CdA and CAFdA nucleotides were determined as described previously (23)
. Briefly, 200 µl of ice-cold 0.4 M perchloric acid containing 0.08 M triethylammonium phosphate was added to the cell pellet, and the pH was adjusted to 6.2 by the addition of approximately 100 µl of ice-cold 1.2 M potassium hydroxide-0.4 M ammonium dihydrogen-phosphate. The extract was centrifuged at 10,000 x g for 5 min. at 4°C, and the final volume of the supernatant was measured. Aliquots of the supernatants were analyzed immediately by HPLC or stored at -20°C. The column was an Ultrasphere ODS (250 x 4.6 mm, 5 µm, Beckman Instruments Fullerton, CA) equipped with the Guard-Pak precolumn (µBondapak C18; Millipore, Milford, MA). The mobile phase consisted of triethylammonium phosphate buffer [TEAP, 0.08 M (pH 6.1)] and methanol (89:11, v/v), and the flow-rate was 1.8–2 ml/min. The temperature of the autosampler was kept at 8°C. The HPLC system consisted of a Milton Roy CM 4000 pump, a variable-wavelength detector (LDC Division, Riviera Beach, FL) and a CMA-240 autosampler (Carnegie Medicine, Stockholm, Sweden). CdATP was synthesized by Sierra Bioresearch (Tucson, AZ) and provided by Dr. William Plunkett (Department of Clinical Investigation, The University of Texas, Houston, TX). The retention times for CdAMP, CdATP, CAFdAMP, and CAFdATP were 12, 15.5, 18.5, and 25 min., respectively. The absorbances were detected at a wavelength of 265 nm; the concentrations, expressed in µM, were calculated by dividing the total amount of each nucleotide in the sample by the number of cells and the mean cell volume of the sample.
Measurement of Activities of Deoxynucleoside Kinases in Crude Extracts and Determination of the Kinetic Parameters of Purified Recombinant Enzyme.
dCK activities were measured in crude extracts according to a procedure described previously by Spasokoukotskaja et al., (24)
. The pure recombinant dCK was prepared as described elsewhere (25)
. Briefly, cells were suspended to 106 cells/ml in an extraction buffer containing 50 mM Tris-HCl (pH 7.6), 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol, and 0.5% NP40. The suspended cells were then frozen and thawed three times and centrifuged at 11,800 x g in an Eppendorf centrifuge for 5 min. at 4°C to remove cell debris. The supernatant was collected and used as a source of protein for the enzyme assays. The substrates were [5-3H]-dCyd (specific activity: 16.7 Ci/mmol, Moravek Biochemicals, Brea, CA), [8-3H]-CdA (specific activity: 4 Ci/mmol, Moravek Biochemicals), and [8-3H]-CAFdA specific activity: 2.77 Ci/mmol, a gift from Dr. William Plunkett, labeled at Moravek Biochemicals). The activity of dGK was determined by using [8-3H]-CdA in the presence of 500 µM dCyd to block the dCK enzyme. The activity of thymidine kinase was measured using methyl-[3H]dThd (specific activity: 24 Ci/mmol, Amersham, Little Chalfont, England) as substrate. The radiolabeled substances, which were dissolved in 50% methanol, were dried under nitrogen and redissolved in RPMI 1640-HEPES before use. For each substrate, the concentration was consistently chosen to be 10 times higher than the Michaelis-Menten constant value (Km) for the principal kinase phosphorylating that substrate. The assays were initiated by the addition of 2–3 µg protein to a reaction mixture containing 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 5 mM ATP, 4 mM DTT, 10 mM sodium fluoride, and substrates in a total volume of 25 µl. After incubation at 37°C for 15 and 30 min., 10-µl aliquots were withdrawn and spotted on WHATMAN DE-81 filter discs, which were then washed as described by Spasokoukotskaja et al.(24)
. Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined over various CdA (1–8 µM) and CAFdA (0.36–3 µM) concentrations. The specific activities of the enzymes were expressed as pmol of nucleoside phosphorylated by extract material from 106 cells over 1 min.
Measurement of dCK activity with purified enzyme with dCyd, CdA, and CAFdA as substrates was performed using the phosphoryl transfer assay containing 100 µM [32P]ATP (10 µCi/µl), 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 125 mM KCl, 10 mM DTT, 0.5 mg/ml BSA, dCK, and various concentrations of nucleoside in a total volume of 50 µl. The phosphorylated products were separated by TLC and quantitated as described previously (26) . Vmax and Km were calculated using a commercially available program, GraphPad PRISM Version 2.0 (Intuitive Software for Science, San Diego, CA).
Western Blotting.
The amount of dCK protein was determined in HL60 and CCRF-CEM cell extracts by Western blot. A peptide anti-dCK antibody was used as previously described (27)
. Approximately 40 µg of crude cell extract and 2 µg of purified recombinant dCK were analyzed, and the protein bands were detected by the enhanced chemiluminescence method as described by the suppliers (Amersham).
Protein Determination.
The protein content in cell extracts was determined according to the method of Lowry et al. (28
; DC protein assay, Bio-Rad Laboratories, CA).
Kinetic and Statistical Calculations.
The areas under the cellular concentration versus time curve (AUC) from time 0 up to the time that the last measured CdA and CAFdA nucleotides concentration were calculated according to the linear trapezoidal rule. Statistics were calculated using StatView software (Abacus Concepts, Berkeley, CA). The differences between CdA and CAFdA nucleotides were analyzed by paired t test analysis and between CLL and AML patients by unpaired t test analysis. A P of less than 0.05 was considered to be statistically significant.
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RESULTS |
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. In CLL cells at 1-µM exogenous concentration of analogues, the mean ratio of CAFdAMP:CdAMP concentrations was 1.6 ± 0.5 (P = 0.05), and an even higher CAFdATP:CdATP ratio was achieved (4.3 ± 3.5; P = 0.001). In cells from AML patients, the kinetic of CAFdA and CdA monophosphates accumulation showed practically no differences, whereas CAFdATP concentrations considerably exceeded those of CdATP at all of the concentrations of analogues (P = 0.04; Fig. 1B
). Besides this, in AML patients, the triphosphate:monophosphate ratios, as well as the absolute triphosphate concentrations of both analogues were 3- to 4-fold higher compared with CLL patients (Fig. 1, A and B)
. The retention of CAFdA and CdA nucleotides accumulated in CLL cells after a 2-h exposure to 10-µM concentrations of analogues was followed up to 48 h after the exposure. The decay of CAFdA mono- and triphosphates in cells after resuspension in drug-free medium were biphasic with a prolonged terminal phase for CAFdATP. The intracellular half-life of CAFdATP was longer than that for CdATP (t1/2, 7.3 and 4.3 h, respectively). The CAFdAMP exposure, expressed as the area under the nucleotide concentration versus time curve (AUC0–48), to the CLL cells was higher for CAFdA (302 µM x h) when compared with corresponding values of CdA (117 µM x h). The differences in the AUC for triphosphates were even more pronounced (250 and 66 µM x h for CAFdATP and CdATP, respectively). More than 90% of the CdATP and CAFdATP was eliminated from the cell within 48 h of incubation in drug-free medium (data not shown).
Phosphorylation of CAFdA and CdA by Pure Recombinant dCK and by the Kinases in Crude Cell Extracts.
An explanation for the higher intracellular levels of CAFdATP can be found in the more efficient phosphorylation of CAFdA by dCK as compared with CdA. Table 1
shows the Km and relative Vmax values for CdA, CAFdA, and natural substrate, dCyd, with recombinant dCK using the phosphoryl transfer assay. CAFdA was three times more efficient than CdA and showed nearly 8-fold higher efficiency than the natural substrate dCyd. In crude extracts of CCRF-CEM cells, the Km values—measured with radiolabeled dCyd, CdA, and CAFdA—were 2.5, 3.4, and 2.3 µM, respectively. In extracts of HL60 cells, the Km values were 2.0, 2.9, and 1.4 µM, respectively. Thus both the recombinant dCK and the enzyme from leukemic cells had higher affinity to CAFdA, than to CdA.
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. We found a similar toxicity toward CCRF-CEM and HL60 when cells were exposed to CAFdA as continuous treatment (50 nM for 120 h) or short treatment (300 nM for 2 h every 24 h for 5 days; data not shown). The choice of CAFdA concentration was based upon the concentration achievable clinically in plasma in patients treated with CdA as continuous infusion (steady state level) or as short infusions (maximum concentration), respectively (11)
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; Table 2
). Resistant cells also showed a considerable degree of cross-resistance to analogues such as CdA, Fara-A, 1-ß-D-arabinofuranosylcytosine, difluorodeoxyguanosine, and difluorodeoxycytidine (data not shown). We found no differences in the sensitivity of resistant cells to tubercidin, vincristine, daunorubicin, idarubicin, mitoxantron, 5-fluorouracil, or paclitaxel (data not shown). The percentage of cells in S phase and the thymidine kinase activities did not differ substantially between the CAFdA-resistant cells and wild-type cells (Table 2)
. Resistant cell lines accumulated lower levels of CAFdA nucleotides (Table 2)
as well as CdA metabolites (data not shown). To investigate the mechanisms for this reduced anabolism, we measured the dCK activities using dCyd as substrate in the four cell lines. CEM/CAFdA and HL60/CAFdA had 98 and 76% reduced dCK activity, respectively, as compared with the wild-type cells (Table 2)
. The dCK activity measured with CdA as substrate was slightly higher than for the natural substrate, dCyd, probably because the latter is also a good substrate for the mitochondrial enzyme, dGK but showed a similar decrease. The dCK protein levels were also determined using immunoblotting. There was no detectable dCK protein in the CAFdA-resistant cell line extracts, whereas it was easily measured in CEM and HL60 wild-type cell extracts (Fig. 3)
. Thus, the protein levels appeared to be proportional to the dCK activity measurements in these cells. The relative activity of dGK (an enzyme that also phosphorylates CdA and CAFdA) cells was not changed in HL60/CAFdA cells, but it was slightly elevated in CEM/CAFdA (Table 2)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro incubation of mononuclear cells from CLL and AML patients with identical exogenous drug concentrations leads to a higher intracellular accumulation of CAFdATP compared with CdATP. In these cells, the major metabolite was CAFdAMP, and the accumulation of CAFdADP represented less than 10% of the total nucleotides, indicating that dCK is not the rate-limiting step in the generation of CAFdATP, as has been shown for Fara-A, arabinofuranosylcytosine, and difluorodeoxycytidine. However, we found several differences in the metabolism of CdA and CAFdA in human cell lines. The retention of CAFdA metabolites were higher compared with CdA metabolites. The 48-h concentration of CAFdATP was more than 2-fold higher than the concentration measured for CdATP. These results indicate that intermittent treatment with CAFdA may result in a higher level of CAFdATP as compared with CdA treatment and CdATP levels. The more efficient phosphorylation of CAFdA in leukemic cells from patients at low nucleoside concentrations is most likely due to the fact that CAFdA is a better substrate for dCK than CdA. This was observed both with pure recombinant dCK and with enzyme measurement of endogenous dCK in crude cell extracts.
Initial studies with CAFdA indicated that mutant lymphoblasts with reduced dCK activity and with elevated cytoplasmic 5'-nucleotidase, or with expanded dNTP pools, were resistant to CAFdA toxicity (20)
. However, the mechanism of acquired resistance to CAFdA has not been investigated thus far. In an attempt to achieve this goal, we developed CAFdA-resistant CCRF-CEM and HL60 cells. We found no cross-resistance or changes in sensitivity to a marker for nucleoside transport, tubercidin, or multidrug resistance-related drugs, like vincristine, daunorubicin, idarubicin and paclitaxel or to other types of drugs such as 5-fluorouracil. This indicates that the mechanism of acquired resistance to CAFdA is specific and not a general phenomenon. The CAFdA-resistant cell lines accumulated less CAFdA nucleotides and had a lower levels of the phosphorylating enzyme dCK as well as lower dCK protein than the wild-type cells (Table 2
; Fig. 3
). Recent experiments showed that the efficiency of CAFdA as substrate for the mitochondrial enzyme dGK was similar to CdA and it was 60% of that of the natural substrate, deoxyguanosine (18)
. In cells and tissues where dCK is absent, dGK is most likely to be the main CdA- and CAFdA-activating enzyme. It is notable that some increase in the relative activity of dGK was observed in the CCRF-CEM mutant cell line (Table 2)
. These cells were shown to be more sensitive to CdA than to CAFdA (Fig. 2A)
. Thus, dGK may have a significant role in the cytotoxicity of nucleoside analogues in leukemic cells lacking dCK (29)
.
Taken together, the mechanisms of acquired resistance to CAFdA are similar to those leading to CdA resistance. Compared with CdA, CAFdA is phosphorylated more efficiently and eliminated more slowly, leading to differences in the cellular pharmacokinetics and pharmacology of the two drugs. On the basis of these results, one would expect not only greater antitumoral effects but also higher hematological toxicity of CAFdA compared with CdA because dCK is constitutively and highly expressed in bone marrow and normal lymphocytic cells (24) . Moreover, the substitution of hydrogen by fluorine at the C-2'-arabino (up) position in CAFdA not only increased the chemical stability of the glycosidic bond of the compound to acidic or enzymatic hydrolysis but also resulted in a higher lipophilicity of the nucleoside (30) . This would favor its penetration through the blood-brain barrier into the brain, in which the expression of dCK is low but the activity of dGK is about 10-fold higher than in other tissues (25) . Surprisingly, in mice, the concentration of CAFdA in the brain was found to be lower than that of CdA (31) . Whether CAFdA will be an effective drug against lymphoproliferative diseases remains to be investigated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was financially supported in part by grants from the Swedish Children Cancer Foundation, the Swedish Medical Research council, and the Swedish Cancer Foundation. 
2 To whom requests for reprints should be addressed, at Department of Clinical Pharmacology, Karolinska Hospital, SE-171 76, Stockholm, Sweden. Phone: 46-8-5177-5832; Fax: 46-8-331343; E-mail: fral{at}mb.ks.se
3 The abbreviations used are: Fara-A, fludarabine; CdA, 2-chloro-2'-deoxyadenosine; CdATP, CdA 5'-triphosphate; CdAMP, CdA 5'-monophosphate; CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; CAFdA, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine; CAFdAMP, CAFdA monophosphate; CAFdATP, CAFdA triphosphate; dCK, deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; HPLC, high-pressure liquid chromatogaphy; AUC, area(s) under (the cellular concentration versus time) curve; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Received 3/ 4/99; revised 5/20/99; accepted 5/24/99.
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), CAFdAMP (
), and CAFdATP (
) in the cells of patients with CLL (A, n = 14) and AML (B, n = 4). Peripheral blood leukemic cells were incubated with the indicated concentrations of CAFdA and CdA for 2 h. They were washed, extracted, and analyzed for CdA and CAFdA nucleotide content as described in "Materials and Methods." The accumulation of CdA 5'-diphosphate and CAFdADP represented less than 5–10% of the total nucleotides and could not be quantitated adequately. The cellular accumulation rate is expressed as µM per h.
