Reduced drug incorporation into DNA and antiapoptosis as the crucial mechanisms of resistance in a novel nelarabine-resistant cell line
© Yamauchi et al.; licensee BioMed Central Ltd. 2014
Received: 11 February 2014
Accepted: 23 July 2014
Published: 29 July 2014
Nine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis.
In the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting.
CEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells.
An ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.
KeywordsAra-G Ara-GTP Nelarabine Resistance T-ALL
Nucleoside analogs belong to one of the most clinically useful and frequently used classes of agents for the treatment of hematological malignancies [1–6]. Nelarabine, 2-amino-9-β-D-arabinofuranosyl-6-methoxy-9H-purine, is a relatively new anticancer agent that targets T-cell malignancies, including T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma [4–6]. The Cancer and Leukemia Group B conducted a phase 2 study of nelarabine for adult patients with relapsed or refractory T-cell leukemia/lymphoma . Treatment with nelarabine resulted in a 41% response rate and a 31% complete remission rate. Although this clinical outcome is promising, nelarabine therapy should be further optimized by an improved understanding of its mechanism of action and by overcoming drug resistance.
Upon intravenous administration, nelarabine is demethylated to the active compound 9-β-D-arabinofuranosylguanine (ara-G) by adenosine deaminase in the plasma [4, 8–11]. Ara-G is transported into leukemic cells mainly via nitrobenzylthioinosine-sensitive nucleoside membrane transporter human Equilibrative Nucleoside Transporter 1 (hENT1) . Ara-G is then phosphorylated to ara-G monophosphate by cytoplasmic deoxycytidine kinase (dCK) and mitochondrial deoxyguanosine kinase (dGK) . This phosphorylation is the rate-limiting step of the intracellular activation of nelarabine. Ara-G nucleotide is partly dephosphorylated by cytosolic 5′-nucleotidase II (cN-II). Ara-G monophosphate is then phosphorylated to ara-G diphosphate and eventually to ara-G triphosphate (ara-GTP). Ara-GTP is an intracellular active metabolite, which is subsequently incorporated into both nuclear and mitochondrial DNA, thereby terminating DNA elongation. Thus, incorporation of the drug into DNA is critical for its cytotoxicity [8–10].
Nelarabine resistance is a major obstacle to improving response rates, and overcoming this drug resistance would provide new strategies for optimal nelarabine administration. In the present study, we established a novel ara-G-resistant subclone of the human T-cell lymphoblastic leukemia cell line, CCRF-CEM. Factors involved in the intracellular activation of ara-G that might be closely related to ara-G resistance [8–12], including hENT1, dCK, dGK, cN-II, and drug incorporation into DNA, were extensively investigated. Because ara-G is phosphorylated by cytoplasmic dCK and mitochondrial dGK, the contribution of both nuclear and mitochondrial DNA damage was evaluated. Moreover, because the induction of apoptosis is the final output of mechanism of ara-G cytotoxicity, the levels of apoptosis-related proteins were determined.
Ara-G was purchased from R.I. Chemicals (Orange, CA, USA) and dissolved in 100% dimethyl sulfoxide. Standard ara-GTP was provided by GlaxoSmithKline, Japan (Tokyo, Japan). [5-3H] ara-G (30 Ci/mmol) was purchased from Moravek Biochemicals, Inc (Brea, CA, USA). Nine-β-D-arabinofucanosyl-2-fluoroadenine (F-ara-A) and cytarabine (ara-C) were purchased from Sigma-Aldrich (St Louis, MO, USA).
Cell culture and development of an ara-G-resistant subclone
Human T-cell lymphoblastic leukemia CCRF-CEM cells were cultured in RPMI1640 media supplemented with 10% fetal calf serum. An ara-G-resistant variant, CEM/ara-G, was established by serial incubation of the cells with ara-G, followed by limiting dilution for cloning. In brief, the parental CEM cells were maintained with escalating concentrations of ara-G. The initial concentration (0.2 μM) was one tenth the concentration required to inhibit 50% growth of CEM cells (IC50). The cultures were observed daily and allowed to grow. In subsequent passages, the concentration of ara-G was gradually increased. Passaging was repeated for 10 months. When the ara-G concentration in the culture media reached 20 μM, one cell line resistant to ara-G (CEM/ara-G) was cloned by the limiting dilution method .
Both CEM and CEM/ara-G cells (2 × 106 cells/ml, 10 ml) were incubated at 37°C with various concentrations of radiolabeled or non-labeled ara-G for the time periods indicated. Cells were then washed twice with PBS and centrifuged (500 × g, 5 min, 4°C) to collect the cell pellet.
Growth inhibition effects were determined by the sodium 3′-(1-[(phenylamino)-carbonyl-3,4-tetrazolium])-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay according to the manufacturer’s instructions (Roche, Indianapolis, IN, USA) with slight modifications .
Alternatively, the number of viable cells were quantitated as of the ATP present, which signals the presence of metabolically active cells, by using The CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega Corp., Madison, WI, USA). Briefly, the cell suspension having been treated were added to the reagent (1:1, v/v). The sample was mixed for 2 min for cell lysis, and allowed to stand for 10 min to stabilize the luminescent signal. The luminescence intensity of the sample was measured thereafter. This method was applied to assess the viability of mitochondrial DNA-depleted ρ0CEM cells.
Measurement of analog triphosphate concentrations in leukemic cells
Intracellular concentrations of ara-GTP, F-ara-A triphosphate (F-ara-ATP), and ara-C triphosphate (ara-CTP) were determined by using the HPLC assay method that we previously established [13, 14]. Briefly, cells (1 × 106 cells/ml, 10 ml) were incubated for 6 h with 10 μM ara-G, F-ara-A, or ara-C. The acid-soluble fraction, the nucleotide pool, was extracted from the cells by the addition of perchloric acid followed by neutralization. An aliquot of the sample was subjected to HPLC analysis. Chromatography was performed with the TSK gel DEAE-2 SW column (250 mm length × 4.6 mm inside diameter; Tosoh, Tokyo, Japan) and 0.06 M Na2HPO4 (pH 6.9) - 20% acetonitrile buffer at a constant flow rate of 0.7 ml/min. Each analog triphosphate concentration was quantitated by its peak area and expressed as pmol/107 cells.
Western blot analysis
Protein levels of dCK, dGK, caspase-3, caspase-9, Bcl2, Bcl-xL, Bax, Bad, Bid, Bim, AKT, and p-AKT were determined by using standard western blotting techniques . Mouse monoclonal anti-dCK was developed in the Department of Pediatrics of Mie University School of Medicine . Rabbit polyclonal anti-dGK antibody (Abgent, San Diego, CA, USA), rabbit polyclonal anti-caspase-3 (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-caspase-9 (Cell Signaling Technology), rabbit polyclonal anti-Bcl-2 (Cell Signaling Technology), rabbit polyclonal anti-Bcl-xL (Cell Signaling Technology), rabbit polyclonal anti-Bax (Cell Signaling Technology), rabbit polyclonal anti-Bad (Cell Signaling Technology), rabbit polyclonal anti-Bid (Cell Signaling Technology), rabbit polyclonal anti-Bim (Cell Signaling Technology), rabbit polyclonal anti-AKT (Cell Signaling Technology), rabbit polyclonal anti-P-AKT (Santa Cruz Biotechnology, Inc. Dallas, TX, USA), and anti-actin antibodies (Sigma-Aldrich) were used as primary antibodies .
Determination of hENT1 and cN-II transcripts
To evaluate mRNA levels of hENT1 (accession: NM_001078177) and cN-II (accession: NM_012229), real-time RT-PCR was performed by using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA) as previously described [13, 15]. Primers for hENT1 and cN-II were purchased from Applied Biosystems. The relative quantification method was used. The expression level of hENT1 or cN-II was normalized using β-Actin as a house-keeping gene in each cell line. The final value was expressed as the ratio of the expression level of hENT1 or cN-II of CEM/ara-G cells to that of CEM cells (the expression level of hENT1 or cN-II of CEM cells was set as 1).
Calculation of ara-G incorporation into both nuclear and mitochondrial DNA
Both nuclear and mitochondrial DNA fractions were isolated from cells after incubation with tritiated ara-G for the indicated time periods at 37°C. For nuclear DNA isolation, the acid-insoluble fraction (obtained as described above) was used. To solubilize RNA, the acid-insoluble fraction was resuspended in 100 μl of 0.4 N KOH and kept at room temperature for 4 h. The sample was then mixed with 100 μl of 5% perchloric acid and 20 μl of 4 N HCl, followed by centrifugation (15,000 × g, 30 sec, 4°C). After removal of the supernatant (RNA), the precipitate was mixed with 100 μl of 5% perchloric acid and heated at 92°C for 20 min to solubilize DNA. After centrifugation (15,000 × g, 30 sec, 4°C), the supernatant was isolated as DNA, and the precipitate (protein) was discarded . The mitochondrial fraction was extracted by using the Qproteome Mitochondria Isolation Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Radioactivity was determined in both fractions by using a liquid scintillation counter.
Evaluation of nuclear and mitochondrial DNA synthesis
The inhibition of DNA synthesis by ara-G was evaluated by assessing the incorporation of tritiated thymidine into DNA . Cells (2 × 106 cells) were pre-incubated with or without 10 μM ara-G for 3 h, followed by washing in fresh media and subsequent incubation with tritiated thymidine for 4 h. The nuclear and mitochondrial DNA fractions were extracted as described above and evaluated for radioactivity by using a liquid scintillation counter.
Quantitation of apoptotic cell death
To evaluate cytotoxicity, apoptotic cell death was determined by staining for phosphatidylserine externalization by using annexin V (Roche Applied Science, Indianapolis, IN, USA) or for the sub-G1 cell cycle population by using propidium iodide (Beckman Coulter, Fullerton, CA, USA) and performing flow cytometry 72 h after treatment . To confirm the induction of mitochondrial apoptosis, the cleavage of caspase-3 and caspase-9 was detected by western blotting as described above.
Derivation of mitochondrial DNA-depleted cells (ρ0CEM cells)
CEM cells were cultured in the presence of 100 ng/ml ethidium bromide to inhibit mitochondrial DNA replication for more than 20 generations (almost 1 month) . ρ0 cells were derived and maintained in the presence of 50 mg/ml uridine. The total cellular enzyme activity of cytochrome c oxidase, subunits of which are encoded by mitochondrial DNA, was tested by using the Mitochondrial Activity Assay Kit (BioChain, Institute, Inc., Hayward, CA, USA) according to the manufacturer’s instructions.
All statistical analyses were performed with Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA, USA). All graphs were generated using GraphPad Prism (version 5.0; GraphPad Software, San Diego, CA, USA).
Establishment of ara-G-resistant CEM (CEM/ara-G) cells
Drug sensitivities of CEM and CEM/ara-G cells
Cross-resistance in CEM/ara-G cells
Evaluation of factors (hENT1, dCK, dGK, and cN-II) essential for intracellular ara-GTP production
Inhibition of DNA synthesis by the incorporation of ara-G into DNA
Derivation of mitochondrial DNA-depleted cells (ρ0CEM cells)
Drug sensitivity of CEM cells after the loss of mitochondrial DNA
In the present study, we developed a new cell line variant of the T lymphoblastic leukemia CCRF-CEM cell line, which was resistant to ara-G, an active compound of nelarabine (Figures 1 and 2, Table 1), and investigated its mechanism of drug resistance. Reduced transporter hENT1 transcript level and decreased dCK and dGK protein levels (Figure 4) resulted in decreased ara-GTP production (Figure 1) in CEM/ara-G cells. The subsequent incorporation of ara-G into nuclear and mitochondrial DNA was reduced (Figure 5), and unable to inhibit DNA synthesis in both fractions of CEM/ara-G cells (Figure 5). Importantly, the cytotoxic effect of ara-G was almost unchanged on CEM cells that were depleted of mitochondrial DNA (Figure 6, Table 2), suggesting that mitochondrial DNA damage was unlikely to contribute greatly to ara-G cytotoxicity. Thus, the reduced triphosphate production (Figure 1) and the subsequent reduction of drug incorporation into nuclear DNA (Figure 5) were closely associated with the development of ara-G resistance in CEM/ara-G cells. The anti-apoptotic nature was also related to the drug resistance in this cell line (Figure 7).
Previously, 3 independent studies investigated the mechanisms of ara-G resistance in leukemic cell lines. Shewach et al. first developed an ara-G-resistant leukemic clone from T lymphoblastic leukemia MOLT-4 cells and demonstrated decreased production of intracellular ara-GTP . However, they did not determine the mechanisms for the reduced ara-GTP production. Curbo et al. generated 2 ara-G-resistant CEM subclones that were 132-fold and 260-fold more ara-G resistant than CEM . They demonstrated a decrease in ara-G incorporation into mitochondrial DNA and loss of dCK activity. However, they showed that the drug incorporation into mitochondrial DNA was not associated with the acute cytotoxicity induced by ara-G in their later study . Their latest study further demonstrated that the depletion of mitochondria DNA does not attenuate the cytotoxicity of ara-G in MOLT-4 cells . They concluded that the loss of dCK activity is the critical factor responsible for ara-G resistance. Our study demonstrated that ara-G inhibited both nuclear and mitochondrial DNA synthesis in CEM cells (Figure 5). However, the result showing that ρ0CEM cells were similarly sensitive to ara-G (Figure 6) suggests that the critical event should be the inhibition of nuclear DNA synthesis not mitochondrial DNA damage. Lotfi et al. developed 2 ara-G-resistant MOLT-4 variants that were 108-fold and 184-fold more ara-G resistant than MOLT-4 . They showed that dGK deficiency was the most prominent change in these cells and that a dCK defect was associated with increased ara-G resistance . They further identified increases in Bcl-xL in these ara-G-resistant clones . The alteration of the kinases and anti-apoptotic Bcl-xL indicate a possible contribution of these factors to ara-G resistance, which is consistent with our present findings. Nevertheless, apart from these reports, we clearly showed all of the successive changes in the transporter hENT1, kinases (dCK and dGK), ara-GTP production, ara-G incorporation into nuclear and mitochondrial DNA, inhibition of DNA synthesis, and induction of mitochondria-mediated apoptosis. Thus, unlike previous studies, the present study was comprehensive and systematic in investigating the mechanism of resistance to ara-G in leukemic cells.
CEM/ara-G cells demonstrated cross-resistance to F-ara-A and ara-C. However, the resistance to the purine analog F-ara-A was much greater than that to the pyrimidine analog ara-C (Table 1). Because F-ara-A and ara-C share an identical pathway for their intracellular activation, the difference in resistance might be due to a structural difference between the 2 agents, but this possibility was not investigated in detail here. Nevertheless, one strategy to overcome ara-G resistance may be a high-dose ara-C therapy that can achieve 50-fold higher plasma ara-C concentrations than regular-dose ara-C, which would surpass the level of cross-resistance to ara-C [35, 36].
An ara-G-resistant CEM variant was successfully established. The mechanism of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.
Sodium 3′-(1-[(phenylamino)-carbonyl-3,4-tetrazolium])-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate
Human Equilibrative Nucleoside Transporter 1
Cytosolic 5′-nucleotidase II
- IC50 :
50% growth-inhibitory concentration.
This work was supported in part by grants from the Gout Research Foundation (2008, 2009, 2010). The role of the funding body was in design, in the collection, analysis, and interpretation of data, and in the writing and the submission of the manuscript.
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