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Experimental Therapeutics |
Departments of Experimental Therapeutics [C. O. R., M. A., V. G.] and Leukemia [V. G.], The University of Texas M. D. Anderson Cancer Center, and The Graduate School of Biomedical Sciences, Houston, Texas 77030-4095; Departments of Pharmacology and Medicine, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599 [B. S. M.]; and Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, S-751 23 Uppsala, Sweden [S. E.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Metabolically, the nontoxic prodrug nelarabine is demethoxylated by adenosine deaminase to ara-G (2) , which permeates the cell via nitrobenzylthioinosine-sensitive and -insensitive equilibrative nucleoside transporters (5) . The rate-limiting step in the formation of the active form, ara-GTP, is the phosphorylation of ara-G to its monophosphate form (ara-GMP) by nucleoside kinase (6) . The ara-GMP is converted to di- and triphosphate forms. In vitro investigations using T-lymphoid, B-lymphoid, or myeloid human leukemia cell lines demonstrated that the differential accumulation of ara-GTP in these cells might be responsible for T-cell-selective cytotoxicity (7, 8, 9, 10) . Lineage-specific accumulation of ara-GTP was also observed in primary leukemia cells obtained from patients (6 , 11) . Cellular pharmacokinetic studies during a Phase I trial demonstrated that accumulation of ara-GTP was higher in circulating T lymphoblasts than in B lymphoblasts or myeloblasts (3) . Correlation of the cellular pharmacokinetics and clinical response revealed that patients who achieved a complete or partial response accumulated significantly higher peak ara-GTP levels (3) than did nonresponding patients. These findings clearly demonstrate the importance of the intracellular accumulation of ara-GTP in achieving clinical responses to nelarabine therapy.
Once phosphorylated, the fraudulent nucleoside triphosphate competes with the native deoxynucleotide as a substrate for incorporation into DNA by DNA polymerases (12 , 13) . These polymerases halt at the site of analogue incorporation, which results in inhibition of DNA synthesis and the initiation of programmed cell death (14) . The incorporation of the analogue into DNA and subsequent apoptotic response is essential for the antineoplastic activity of clinically tested and effective nucleoside analogues such as cladribine (15) , cytarabine (16) , fludarabine (17 , 18) , and gemcitabine (18 , 19) , as well as nelarabine (14) .
As mentioned above, the accumulation of intracellular ara-GTP is important for clinical responses. Clinically relevant analogues such as cytarabine (20) , cladribine (20) , fludarabine (20) , gemcitabine (21) , and decitabine (22) are activated by dCK. However, in the case of ara-G, controversy exists as to which enzyme is responsible for this critical first step. Nucleoside kinase assays in cell-free systems or in cell lines with dCK or without dCK have demonstrated that ara-G serves as a substrate for cytosolic dCK (2 , 10 , 20) . Purified mitochondrial dGK has also been shown to use ara-G with an affinity similar to dGuo (23, 24, 25) . Until now, no investigations have been done with whole-cell system to address the roles of each of these kinases in ara-G phosphorylation. To determine the role of each of these kinases, we used a mutant form of T-cell lymphoblastic cell line CCRF-CEM that was made resistant to ara-C by chronic exposure to ara-C (26) . This cell line lacks dCK activity and has very low level of endogenous dGK activity. These cells were infected with retrovirus containing cDNA of dCK and dGK, and were used to determine the role of each enzyme in ara-G phosphorylation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Line.
The CCRF-CEM T-lymphoblast cell line and its Ara-C8D derivative (CCRF-CEM, dCK-) cell line have been described previously (26)
. The latter cell line contains mutated dck gene with no detectable protein or functional dCK activity; the dgk gene is unaffected, and functional dGK activity are present (26)
. These cell lines were cultured in DMEM-low glucose supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc., Grand Island, NY) and maintained in mid-log phase growth at 37°C in 5% CO2 in a fully humidified incubator. Under these conditions, the cell doubling time was 24 h. Cells were routinely tested for Mycoplasma by using a kit (Gen-Probe Inc., San Diego, CA).
Construction of CEM Cell Lines Overexpressing dCK or dGK.
The dCK cDNA cloning and expression have been described before (27
, 28)
. The dGK cDNA was obtained by PCR from polyadenylated RNA isolated from Raji B lymphoblasts. The full-length cDNA (dGK+) was obtained by primers dGK-1FW (5'-TAGGGATCCGAATCGTGGGAATGG) and dGK-3RV (5'-CGGGATCCTTACAGATTCTTT). All of the primers were checked for sequence analysis as described previously (27)
.
The Moloney murine leukemia/sarcoma-based retroviral vector LNPO-dCK has been described (27 , 28) . LNPO-dGK contained mitochondrial leader sequence for mitochondrial localization. LNPO-dCK and LNPO-dGK were made by inserting the dCK or dGK cDNA into the BamH1 site of the pLNPO vector. The dCK- cell line was infected with either LNPO-dCK or LNPO-dGK (27) .
Immunoblot Assays.
Cells (1 x 107) were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM DTT. One protease inhibitor mixture tablet was added to each 10 ml lysis buffer (Boehringer Mannheim, Manheim, Germany). Cell lysates (50 µg protein) were resolved by 10% SDS-PAGE and then electrophoretically transferred onto Immobilon P membranes (Millipore, Bedford, MA). After being blocked with 4% nonfat dry milk in PBS-T (PBS with 0.05% Tween 20) for 1 h, the membranes were probed with either rabbit polyclonal anti-dCK peptide antibodies (29)
or affinity purified rabbit polyclonal anti-dGK antibodies (30)
. The membranes were washed in PBS-T and incubated with horseradish peroxidase-conjugated goat antirabbit antibodies for 1 h before visualization with an enhanced chemiluminescence detection system (NEN Life Science Products, Inc., Boston, MA).
The relative expression of dCK and dGK was quantitated using a densitometer and normalized to the value obtained for actin within the same samples on the same blot. The amount of dCK found in dCK- CEM cells served as the control, and that amount was given a value of 1. The background density from an exposed and developed portion of the film was determined from pixels within an area identical to that containing the bands of interest. This value was subtracted from the density value of each sample.
Enzyme Extraction Protocol.
Exponentially growing cells (2 x 107) were lysed by three freeze-thaw cycles (31)
. For dCK, the extraction buffer contained 50 mM Tris-HCl (pH 7.6), 20% glycerol, 0.5% NP40, and 2 mM DTT. One protease inhibitor mixture tablet was added for every 10 ml of extraction buffer (Boehringer Mannheim). For dGK, 0.05% Triton X-100 was substituted for the NP40. The lysates were cleared by centrifugation, and the protein concentration was determined by the Bradford method as per manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA).
Enzyme Activity Assays.
The functional activity of dCK or dGK was determined as described previously (31)
. For dCK, 1 µM [3H]ara-C was used as the substrate. Deaminase inhibitor, tetrahydrouridine, was added to the reaction mixture. For dGK, 20 µM [3H]dGuo was used as the substrate and to prevent its phosphorolysis, 100 µM 8-aminoguanosine was included in the reaction mixture. For the relative ara-G kinase activity, reaction mixtures containing either 10 µM or 100 µM ara-G were used. To compare and inhibit phosphorylation of dGuo or ara-G by dCK, kinase assays were performed in the absence or presence of high concentrations (150 µM) of deoxycytidine (the preferred substrate of dCK) in the assay mixture.
Aliquots (3 µl) of enzyme extract were mixed and the reactions allowed to progress. Aliquots (20 µl) of the reaction were spotted on DE-81 filter papers (Whatman, Maidstone, United Kingdom) and allowed to dry. The filter papers were washed in 5-mM ammonium formate, then in ethanol, were air-dried, and the radioactivity was quantitated. Under these conditions, the lower limit of detection was 
0.05 nmol of ara-C/mg of protein or 0.05 pmol of dGuo or ara-G/mg of protein. The results were expressed as nmol (for dCK) or pmol (for dGK) of phosphorylated product per mg protein.
Measurement of Intracellular Nucleoside Triphosphates by High-pressure Liquid Chromatography.
To quantify ara-GTP accumulation in whole cells, we incubated exponentially growing cells (5 x 106-1 x 107) with 100 µM ara-G. Nucleotides were extracted by perchloric acid, neutralized with KOH, and analyzed using high-pressure liquid chromatography (32)
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Cell Proliferation Assay.
Briefly, CEM cells were cultured into 96-well dishes at a concentration of 20,000 cells/well and incubated with ara-G at indicated concentrations. After 24-hours of incubation, cell proliferation was determined using the MTS Cell Titer Aqueous assay, (Promega, Madison, WI), which measured the conversion of a tetrazolium compound into formazan by a mitochondrial dehydrogenase enzyme in live cells. The amount of formazan was measured spectrophotometrically and was linear with the cell number. Each data point was the average of three independent determinations.
Calculations and Statistical Analysis.
Student’s two-tailed unpaired t test was used to determine the significance of differences in ara-GTP accumulation in whole cells between cell lines. The rates of substrate phosphorylation and ara-GTP accumulation were determined by linear regression analysis that included the 0-h time point. The rates of substrate phosphorylation obtained during in vitro assays (mean with SE) were compared between cell lines by two-tailed Student’s t tests.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The parental wild-type CEM cells had high endogenous dCK levels and relatively low endogenous dGK contents. The ara-C-resistant cell line (dCK-) showed little or no expression of the dCK protein but maintained endogenous levels of the dGK protein. Given the absence of a cell line that lacks dGK, we used this dCK- cell line to reconstitute dCK and overexpress dGK. Cells containing transfer of dck (dCK+; Fig. 1
, Lane 3) produced about half of the dCK protein and the same amounts of dGK as did the parental cell line. Cells infected with dgk (dGK+) produced little or no dCK (Fig. 1
, Lane 4) and about five times the amount of dGK as did the parental cells or the other two mutant cell lines.
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). This increase in phosphorylation was because of exogenously expressed dCK rather than endogenous dGK, because dCK- cells could not phosphorylate ara-C to detectable levels under similar conditions. As expected, overexpression of dGK+ in the absence of dCK did not result in any phosphorylated products of ara-C (Fig. 2A)
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or presence (Fig. 2C)
of the dCK substrate, deoxycytidine. The extract prepared from dCK- cells, which express endogenous levels of dGK, phosphorylated [3H]dGuo at a constant rate of 0.91 ± 0.15 pmol/mg protein/min (r2 = 0.96), a rate similar to that of the reconstituted dCK (dCK+) cell line (0.93 ± 0.19 pmol/mg protein/min; r2 = 0.89). Thus for dGuo, dGK is the major phosphorylating enzyme at this concentration of substrate. Consistent with that postulate, extracts from the cell line overexpressing dGK (dGK+) produced much higher levels of phosphorylated dGuo (Fig. 2, B and C)
. By 60 min, >150 pmol of phosphorylated products were formed. The presence of dCyd did not inhibit the dGuo-phosphorylating activity of dGK, additionally confirming that dGuo was the preferred substrate for the dGK enzyme.
The results presented above verified that the transduced enzymes were functional with pure substrates. Next, to test the postulate that dGK phosphorylates ara-G at a Km similar to that of its natural substrate, dGuo, and dCK phosphorylates ara-G with lower affinity, we measured the in vitro ara-G-kinase activity in the transfectants that overexpressed dCK or dGK (Fig. 3)
. To mimic the kinetic concentrations, [3H]ara-G was used at a concentration of 10 µM (Fig. 3, A and B)
, which is near the Km for dGK or at 100 µM (Fig. 3, C and D)
, which is near the Km for dCK. To compare and determine the role of dCK, the ara-G phosphorylation assay was performed in the absence (Fig. A and C) or presence (Fig. 3, B and D)
of the inhibitor deoxycytidine. In experiments with dCK- cells, endogenous levels of dGK could phosphorylate ara-G at the relatively low concentration of 10 µM ara-G (Fig. 3A
; 0.08 ± 0.01 pmol/mg protein/min; n = 10); the addition of dCyd did not affect this rate (Fig. 3B
; 0.10 ± 0.01 pmol/mg/min; n = 8). Extracts from dGK+ cells phosphorylated ara-G about six times faster (Fig. 3A
; 0.48 ± 0.03 pmol/mg protein/min) than the extracts from dCK- cells (P = 0.0237), and again the addition of dCyd did not affect this rate (Fig. 3B
; 0.39 ± 0.06 pmol/mg protein/min; n = 8). Moreover, when dCK was restored, the rate of ara-G phosphorylation was significantly faster (Fig. 3A
; 0.15 ± 0.01 pmol/mg/min; n = 8) than that of cells containing only endogenous dGK (P = < 0.0001). As expected, the addition of dCyd inhibited the activity of the transferred dCK+, resulting in ara-G being phosphorylated at the same rate as in dCK- cells (0.07 ± 0.02; P = 0.34; n = 4). An identical pattern of ara-G phosphorylation was observed when 1 µM ara-G was tested (data not shown).
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). Again, the addition of dCyd (Fig. 3D)
had no effect on the rate of ara-G phosphorylation in either dCK- (4.12 ± 0.20 pmol/mg/min) or dGK+ (4.95 ± 0.37 pmol/mg/min). At 100 µM ara-G, dCK+ phosphorylated ara-G at the maximum rate in the absence of deoxycytidine (24.02 ± 0.67 pmol/mg/min). The addition of dCyd inhibited the enzyme activity and decreased the rate of ara-G phosphorylation by 3-fold (9.68 ± 1.60 pmol/mg/min).
Whole Cell Accumulation of Ara-GTP.
Next, to determine whether the expressed enzymes would phosphorylate ara-G in a whole-cell milieu, we incubated dCK-, dCK+, and dGK+ cells with 100 µM ara-G for 3–12 h (Fig. 4)
. The lowest accumulation of ara-GTP (<5 µM) was found in dCK- cultures, which have low endogenous dGK levels. Although there was a time-dependent increase in ara-GTP levels, the accumulation reached a plateau in these cells by 3–6 h. At 12 h, the concentration was 8.0 ± 0.8 µM. However, when dGK was overexpressed, the intracellular accumulation of ara-GTP increased to 48.3 µM ± 7.4 µM by 12 h (Fig. 4)
. Similarly, when dCK was overexpressed the accumulation of ara-GTP also increased and reached 57.7 µM ± 16.2 µM by 12 h. Moreover, in both of these cell lines, ara-GTP accumulation did not reach a plateau in the dCK or dGK cells suggesting that longer incubation periods would result in continuing accumulation. This observation also indicates that at these concentrations of ara-GTP, the enzymes are not feedback inhibited. Given the correlation between ara-GTP and response to treatment with nelarabine (3)
, we have explored this pharmacological strategy to increase ara-GTP in freshly isolated leukemia cells (6)
and designed a protocol to infuse higher levels of nelarabine (33)
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. In the dCK- cell line the accumulation of ara-GTP at any ara-G concentration was low (2.7–6.7 µM). Compared with dCK- cell line, expression of either dGK or dCK resulted in significant accumulation of ara-GTP at all four of the concentrations of ara-G (see Ps in Table 1
for dCK- versus dCK+ and dCK- versus dGK+). When dCK was expressed, incubation with 10–100 µM ara-G resulted in proportional increases in ara-GTP, which ranged from 8.5 ± 1.4 to 35.7 ± 4.5 µM. In dGK+, cells that had no endogenous dCK and overexpressed dGK, 3-h incubations with 10 and 30 µM ara-G resulted in accumulations of 18.2 ± 1.5 µM and 21.7 ± 1.8 µM ara-GTP that were higher than dCK+ cells (P = 0.0008 at 10 µM; and P = 0.0304 at 30 µM). At higher ara-G concentrations, dGK+ cells accumulated ara-GTP at similar levels as did the dCK+ cells (P = 0.5529 at 100 µM and 0.5659 at 300 µM; Table 1
). These findings strongly suggest that both dCK and dGK can phosphorylate ara-G in intact cells and that this phosphorylation step eventually results in the intracellular accumulation of ara-GTP.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A corollary to high expression of dGK is the fact that leukemia cells show differential cytotoxicity to nucleoside analogs that are phosphorylated by only dCK compared with that with ara-G. A recent trial of fludarabine with nelarabine demonstrated a bimodal accumulation of ara-GTP but not fludarabine triphosphate (40) in circulating leukemia cells. The 70-fold variation in the level of ara-GTP found in cells from the patients in this trial probably reflected heterogeneity in ara-G phosphorylation. The variation in accumulation of fludarabine triphosphate was much lower (6–7-fold). Because both of these analogues are substrates for dCK, these results cannot be explained by an increase in the specific activity of dCK. It is tempting to speculate, then, that the population of cells that accumulated more ara-GTP may be those that expressed a high specific activity of dGK. Prospective evaluation of the expression of each of these kinases in primary leukemia cells may reveal a biochemical basis for the pharmacologic differences in the accumulation of ara-GTP. These differences may identify patients who can benefit from nelarabine as opposed to traditional analogs that use only dCK.
As mentioned before, the location of endogenous dGK enzyme is in the mitochondria (23 , 25 , 30 , 37 , 41) . In our system, consistent with previous report (42) , overexpression of dGK results in accumulation of protein in mitochondrial fraction (data not shown). On the basis of cytotoxicity observed with analogs that are phosphorylated by overexpressed dGK, it is postulated that the cells incorporate these analogs and result in cytotoxicity. At present, it is not clear how the triphosphate of nucleoside analogs, such as ara-G, generated in mitochondria transport to nucleus for incorporation into DNA to elicit cell death. Nonetheless, the accumulation of ara-GTP in the cells lacking dCK and overexpressing dGK (dGK+), and subsequent cytotoxicity provide an evidence for such a postulate.
As is true for dGK, dCK is constitutively expressed in many tissues. However, unlike dGK, the highest specific activity of dCK is found in lymphoid tissues, particularly immature T cells (38
, 43)
. In our study, a 3-h incubation of parental CEM cells with 100 µM ara-G resulted in the intracellular accumulation of 100 µM ara-GTP (14)
, whereas in dCK- cells, the loss of this low-affinity high-specific activity enzyme resulted in the intracellular accumulation of only 5 µM ara-GTP. Although in dCK- cells, the rate-limiting phosphorylation of ara-G reflects the contribution of only dGK, the low accumulated concentration of ara-GTP underscores the importance of dCK in the dose-dependent intracellular accumulation of ara-GTP at clinically achievable levels of nelarabine. Because dCK+ cells contained less dCK than did the parental CEM cells, the intracellular accumulation of ara-GTP in the dCK+ cells was about five times less than in the parental cells with unchanged dCK, a finding that additionally strengthens the association between dCK and ara-GTP accumulation. Because the Km of dCK for ara-G (>100 µM) is much greater than for its natural substrate deoxycytidine (<1 µM; Refs. 2
, 20
), we would expect that the dose-dependent phosphorylation of ara-G would become saturated only at higher levels of ara-G than those needed to saturate dGK. Our findings are consistent with this notion. Furthermore, cells with the highest specific activity of dCK, such as immature T cells, would also be expected to be sensitive to the cytotoxic effects of nelarabine. Indeed, this particular cell type has been shown to be sensitive to nelarabine in vitro (7, 8, 9, 10)
and in a Phase I trial of this prodrug (3)
. Another cell lineage that has been identified as possessing high dCK activity is that of B-cell chronic lymphocytic leukemia. Among patients with a variety of indolent leukemias, patients with B-cell chronic lymphocytic leukemia achieved objective responses. The fact that circulating leukemia lymphocytes from these patients accumulated high levels of ara-GTP additionally strengthens our hypothesis that cells that express high dCK would be particularly susceptible to nelarabine therapy. Taken together, these data strongly suggest that in these cells (CEM and patient cells), because of the high specific activity of dCK, dGK may play a minimal role for ara-GTP accumulation.
In contrast to mitochondrial localization of dGK, endogenous native dCK is a cytosolic enzyme (29
, 44)
; however, overexpression of the protein may result in nuclear localization (29
, 42)
. Irrespective of the location of overexpressed enzyme, the phosphorylated product accumulates in the cell and incorporates into DNA to initiate cytotoxicity. This has been demonstrated both in cell lines (27
, 28)
and in vivo in tumor model system (28)
. In the present study also, expression of dCK in the dCK-deficient cell line results in accumulation of ara-GTP in the cytosol (Table 1
; Fig. 4
), and this was associated with cell death (data not shown).
In conclusion, we have demonstrated here that ara-G was phosphorylated by both mitochondrial dGK and cytosolic dCK. In the absence of dCK, the accumulation of ara-GTP was low, maybe because of the low endogenous level of dGK. However, when the specific activity of dGK was increased, the accumulation of ara-GTP was augmented. Because dGK is present in mitochondria (30) , cells that express greater numbers of mitochondria are vulnerable to high accumulation of ara-GTP and ara-G-induced cytotoxicity. Similarly, when the specific activity of dCK was amplified, a proportional increase in the accumulation of intracellular ara-GTP was observed. Despite the low affinity of dCK for ara-G, when this enzyme is present, the accumulation of intracellular ara-GTP was also augmented. Finally, these studies illustrate the role of each kinase for phosphorylation of ara-G in intact cells; at low concentrations of the substrate dGK is the preferred enzyme; however, at high concentrations of ara-G dCK phosphorylates it efficiently. The relative abundance of each kinase in primary leukemia cells may predict for phosphorylation of ara-G.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant CA57629 from the National Cancer Institute, Department of Health and Human Services. 
2 To whom requests for reprints should be addressed, at Department of Experimental Therapeutics, Box 71, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Phone: (713) 792-2989; Fax: (713) 794-4316; E-mail: vgandhi{at}mdanderson.org
3 The abbreviations used are: ara-G, 9-ß-D-arabinosylguanine; ara-C, arabinosylcytosine; ara-GMP; ara-G monophosphate; ara-GTP, ara-G triphosphate; dCK, deoxycytidine kinase; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine.
Received 4/11/01. Accepted 3/26/02.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß. J. Biol. Chem., 265: 4033-4040, 1990.This article has been cited by other articles:
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  M. H. Cohen, J. R. Johnson, R. Justice, and R. Pazdur FDA Drug Approval Summary: Nelarabine (Arranon(R)) for the Treatment of T-Cell Lymphoblastic Leukemia/Lymphoma Oncologist, June 1, 2008; 13(6): 709 - 714. [Abstract] [Full Text] [PDF]
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 V. Gandhi, C. Tam, S. O'Brien, R. C. Jewell, C. O. Rodriguez Jr, S. Lerner, W. Plunkett, and M. J. Keating Phase I Trial of Nelarabine in Indolent Leukemias J. Clin. Oncol., March 1, 2008; 26(7): 1098 - 1105. [Abstract] [Full Text] [PDF]
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), dCK+ (
), and dGK+ cells (
) were prepared and used as described in "Materials and Methods." The phosphorylation of ara-C (A) or dGuo in the absence (B) or presence (C) of deoxycytidine was quantified as a function of time and expressed as nmol/mg protein or pmol/mg protein, respectively. Data points are the means of three separate experiments conducted in quadruplicate; bars, ± SE. Where the bars are not visible, they are contained within the symbol.
