Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA Sophie Curbo a,⁎, Magnus Johansson a , Jan Balzarini b , Lionel D. Lewis c , Anna Karlsson a a

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, S-141 86 Stockholm, Sweden Rega Institute for Medical Research, B-3000 Leuven, Belgium c Section of Clinical Pharmacology, Department of Medicine, Dartmouth Hitchcock Medical Center and Dartmouth Medical School, Lebanon, NH, USA b

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

The nucleoside analogs 9-β-D-arabinofuranosylguanine (araG) and 1-β-D-arabinofuranosylthymine

Received 29 January 2009

(araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be

Revised version received 6 April 2009

predominantly incorporated into mtDNA of cells, but the pharmacological importance of their

Accepted 25 May 2009

accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to

Available online 28 May 2009

araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ0 cells (lacking mtDNA). The mRNA levels and activities of

Keywords:

deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and

Deoxyribonucleoside kinase

thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-

ρ0 MOLT-4 cells

4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p < 0.01) in the ρ0 cells,

9-β-D-arabinofuranosylguanine

whereas the TK1 mRNA level was lower (p < 0.05). The enzyme activity of the S-phase restricted

1-β-D-arabinofuranosylthymine

TK1 was also lower (p < 0.05) in the MOLT-4 ρ0 cells, whereas the activities of dGK, dCK and TK2

Mitochondrial DNA

were similar in MOLT-4 wt and ρ0 cell lines. The sensitivities to different cytotoxic nucleoside

Nucleoside analogs

analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells. © 2009 Elsevier Inc. All rights reserved.

Introduction Since the first mammalian ρ0 cell line was generated by King and Attardi [1] a number of mtDNA-depleted cell lines have been produced by long-term exposure to reagents, such as ethidium bromide (EtBr), that inhibit replication of mitochondrial genes by DNA polymerase γ. All the enzymes involved in mtDNA synthesis and regulation are encoded in the nuclear DNA, synthesized in the cytosol and then imported into the mitochondria. The mtDNA

encodes genes for 2 mitochondrial rRNAs, 22 tRNAs and 13 protein subunits required for oxidative phosphorylation and a functional electron transport chain. Decreased mtDNA content results in decreased synthesis of mtDNA encoded subunits of the respiratory chain and ultimately in defective electron transport and oxidative phosphorylation [2,3]. Nucleoside analogs are a group of compounds commonly used in anti-cancer and anti-viral treatment that require intracellular phosphorylation for pharmacological activity. The deoxyribonu-

⁎ Corresponding author. Fax: +46 8 7795383. E-mail address: [email protected] (S. Curbo). Abbreviations: araT, 1-β-D-arabinofuranosylthymine; araG, 9-β-D-arabinofuranosylguanine; EtBr, Ethidium bromide; BvDU, (E)-5-(2bromovinyl)-2′-deoxyuridine; CdA, 2-chloro-2′-deoxyadenosine, Thd; Thymidine, dGK; deoxyguanosine kinase; dCK, deoxycytidine kinase; TK1, thymidine kinase 1; TK2, thymidine kinase 2 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.05.021

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cleoside kinases involved in the first phosphorylation step are the rate-limiting enzymes for the conversion of most nucleoside analogs into active nucleotide analogs. Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are the initial mono phosphorylating enzymes located in the nucleus and cytoplasm whereas the enzymes deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2) are located in the mitochondria. Several nucleoside analogs such as 2′,3′-dideoxycytidine (ddC) and 2′,3′-dideoxyinosine (ddI) interfere with mtDNA replication and cause delayed mitochondrial toxicity in chronically treated HIV infected patients, although they are phosphorylated outside the mitochondrial compartment and transported into mitochondria via mitochondrial transporters [4–7]. We have previously demonstrated that the nucleoside analogs 9-β-D-arabinofuranosylguanine (araG) and 1-β-D-arabinofuranosylthymine (araT) which are substrates of the mitochondrial dGK and TK2, respectively, are predominantly incorporated into mtDNA of cells [8–10]. However, although araG was predominantly incorporated into mitochondrial DNA the total amount of mtDNA remained unchanged [9]. It is currently not known if the mtDNA of cells contribute to the acute cytotoxicity of nucleoside analogs such as araG and araT. Furthermore, it is not known whether the loss of mtDNA content in ρ0 cells leads to changes in deoxyribonucleoside kinase activities or mRNA expression. To address these questions we investigated the cytotoxicity of several nucleoside analogs in MOLT-4 wild-type and ρ0 petite mutant cells [11] and measured mRNA levels and enzyme activities of TK1, TK2, dCK and dGK in the cells. In summary we found that the mRNA levels of dGK and dCK were similar whereas the mRNA level of TK2 was slightly higher in the ρ0 cells although the corresponding enzyme activities were unaffected by the lack of mtDNA. Both the TK1 mRNA level and enzyme activity were significantly lower in the ρ0 cells compared to the MOLT-4 wt cells. We conclude that the presence of mtDNA is not a prerequisite for the acute cytotoxicity of araG and araT in T-lymphoblastoid MOLT-4 cells.

Enzyme assays The cells were seeded at the same density each time they were subcultured and given fresh medium. The day before harvest they were seeded at the same density (4 × 105 cells/ml). The cells were suspended at 60 × 106 cells/ml in an extraction buffer containing 50 mM Tris–HCl, pH 7.6, 2 mM dithiothreitol, 5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol and 0.5% Nonidet P40. The cells were further disrupted by three freeze–thaw cycles in liquid nitrogen and subsequently centrifuged at 4 °C at 13,000 rpm for 20 min, whereupon the supernatants were collected and stored at −80 °C until analyzed. The protein concentrations were determined using the Bio-Rad protein assay reagent with bovine serum albumin as a protein concentration standard. The enzymatic assays were carried out as described previously [12,13]. Briefly they were performed in 50 mM Tris–HCl, pH 7.6, 5 mM MgCl2, 5 mM ATP, 2 mM dithiothreitol, 15 mM NaF, 0.5 mg/ ml bovine serum albumin, 40–150 μg protein and finally labeled (hot) and unlabeled (cold) substrates were added to a total volume of 50 μl. In the different assays the mixture of hot and cold substrates were as follows: 3 μM [8-3H]araG (Moravek Biochemicals Inc.) and 1 μM unlabeled araG in the presence or absence of 1 mM dCyd; 3 μM [methyl-3H]thymidine (DuPont) and 7 μM unlabeled thymidine; 2.5 μM [8-3H]CdA (Moravek Biochemicals Inc.) and 2.5 μM unlabeled CdA either with or without addition of 1 mM dCyd (Moravek Biochemicals Inc.); 2.5 μM [methyl-3H]BvDU (Moravek Biochemicals Inc.). Ten microliters was spotted on Whatman DE-81 filter disks after 0-, 10-, 20- and 30-min incubation at 37 °C. The filters were then washed three times in 5 mM ammonium formate. The filter-bound monophosphorylated nucleotide product of the reaction was then eluted from the filter with 0.1 M KCl and 0.1 M HCl, and the radioactivity quantified by scintillation counting. The data presented represent the mean± S.D. from three experiments.

RNA extraction and cDNA synthesis

Materials and methods Cell culture The MOLT-4 T-lymphoblast cell line and the MOLT-4 ρ0 cell line, generated from the MOLT-4 wild-type cells by long-term exposure to ethidium bromide [11], were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 4.5 mg/ml glucose, 110 μg/ml sodium pyruvate and 50 μg/ml uridine. The cells were cultured at 37 °C in a humidified 5% CO2 atmosphere.

Inhibition of tumor cell growth by drugs 2.0–2.5 × 105 cells/ml were seeded in 200 μl-wells of 96-well microtiter plates in the presence of serial dilutions of the test compounds. The cells were allowed to proliferate for 72 h in the presence of the test compounds. Cell proliferation was assayed by counting the cell number in a Coulter Counter. The data are presented as the inhibitory concentration (IC)50, which was defined as the concentration of a drug that is required to inhibit cell proliferation by 50%. Each experiment was performed at least two to three times.

Total RNA was extracted concomitantly from the same cell culture from which the crude cell extracts were made. The Trizol reagent (Invitrogen) was used for extraction and thereafter the total RNA was treated with DNase I. Only RNA samples for which the ratio of 28S to 18S RNA was between 1.8 and 2.2 were used. To amplify the cDNA, a 20 μl reaction volume was used containing 2 μg of total RNA, 4 μl 5×RT buffer (Invitrogen), 200 ng random hexamer primer (Invitrogen), 1 μl of dNTP mix (10 mM each), 1 μl DTT (0.1 M), 40 U RNaseOUT (Invitrogen) and 1 μl SuperScript RT (Invitrogen). A mix of the RNA, dNTPs, hexamer primer and water was heated at 65 °C for 5 min to denature the RNA and then chilled on ice during the addition of the other compounds. The transcription of the cDNA was then performed at 37 °C for 50 min followed by 15 min at 70 °C to heat-inactivate the RT.

Real-time quantitative PCR (relative quantification of dGK, dCK, TK1 and TK2 mRNAs) For each RNA extract, the mRNA of the nuclear genes for β-actin, dGK, dCK, TK1, and TK2 was quantified separately by a two-step RT-PCR. The primers for the real-time PCR were designed using Primer Express (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) and are documented in Table 1.

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Table 1 – Primers and probes used in the real-time PCR. Gene β-actin

dGK

dCK

TK1

TK2

Sequence Forward primer Reverse primer Probe Forward primer Reverse primer Probe Forward primer Reverse primer Probe Forward primer Reverse primer Probe Forward primer Reverse primer Probe

TET was chosen as reporter dye for β-actin and FAM for dGK, dCK, TK1 and TK2. TaqMan Universal PCR master kit was used (PE Applied Biosystems) to prepare the reactions. The reaction volume was 25 μl containing 12.5 μl 2× TaqMan buffer, 2.5 μl of the cDNA, and for βactin: 0.2 μM Fwd primer, 0.4 μM Rev primer, 0.1 μM probe; dGK and TK2: 0.4 μM Fwd primer, 0.4 μM Rev primer, 0.2 μM probe; dCK and TK1: 0.4 μM Fwd primer, 0.4 μM Rev primer, 0.1 μM probe. The PCR cycling parameters were 50 °C for 2 min to activate the AmpErase UNG enzyme, 95 °C for 10 min to denature the UNG enzyme and to activate the polymerase, then 50 cycles of 95 °C for 15 s and 60 °C for 1 min. Real-time detection of fluorescence emissions was performed with an ABI PRISM 7700 (Applied Biosystems). Data acquisition and analysis were performed based on the method described by Pfaffl [14]. The data presented represent the mean ±S.D. from three (dCK, TK1 and TK2) or two (dGK) experiments.

5′-TCCTCCTGAGCGCAAGTACTC-3′ 5′-GCATTTGCGGTGGACGAT-3′ 5′-TGTGGATCAGCAAGCAGGAGTATGACGAGT-3′ 5′-ACCAGAATGGCACGTAGCAT-3′ 5′-CCTGATAGATATGCCACTCGATG-3′ 5′-CAGCTGGAGCCCTTCCCTGAGAAACT-3′ 5′-GGACCCGCATCAAGAAAATC-3′ 5′-AGTACTTTGAACATTGCACCATCTG-3′ 5′-AACATCGCTGCAGGGAAGTCAACATTTGT-3′ 5′-GTGGCTGTCATAGGCATCGA-3′ 5′-CCAAATGGCTTCCTCTGGAA-3′ 5′-CCAACGCCGGGAAGACCGTAATTG-3′ 5′-GGAATGGTTTGACTGGATCTTGAG-3′ 5′-GCGGAATGACCTTCTCCTCTT-3′ 5′-CCTTCGGCACAATCCTGAGACTTGTTACCA-3′

two cell lines. The data are expressed as percent mRNA expression in MOLT-4 ρ0 cells compared to the mRNA expression in MOLT-4 wt cells with β-actin used as an internal control (Table 2). The mRNA expression of TK1 was lower (62%, p < 0.05) in the ρ0 cells than in the wt cells. The mRNA expression of TK2 was markedly elevated in the ρ0 cells (189%, p < 0.01) compared to the wt cells whereas the mRNA levels of dGK and dCK were only moderately altered in the ρ0 cells (141 and 119%, respectively, although p > 0.05).

Deoxyribonucleoside kinase activity of dCK, dGK, TK1 and TK2 in crude cell extracts of MOLT-4 wt and ρ0 cells

We used real-time detection of fluorescence emissions from an ABI PRISM 7700 to determine the mRNA of TK1, TK2, dCK and dGK in the

The DE81 filter disc assay [12] was used to determine the level of nucleoside kinase activity in crude protein cell extracts from the two cell lines (Table 3). The total araG phosphorylating activity was similar in the two cell lines. Since araG is a substrate of both dGK and dCK the assay was also performed in the presence of 1 mM deoxycytidine (dCyd), which has been shown to inhibit the dCK catalyzed activity. The araG phosphorylating activity in the presence of excess dCyd was similar to the activity acquired without dCyd present (data not shown) and there was no difference in araG phosphorylating activity between the two cell lines. 2-chloro-2′deoxyadenosine (CdA) is phosphorylated mainly by dCK but also by dGK [15]. The CdA phosphorylating activity was compared between the MOLT-4 wt and the MOLT-4 ρ0 cells. In the presence of excess of dCyd the CdA phosphorylating activity, corresponding to the dGK catalyzed enzyme activity, was also similar in the two cell lines and of

Table 2 – The mRNA levels for dGK, dCK, TK1 and TK2 were quantified with two-step real-time PCR (RQ-PCR) in MOLT-4 wt and ρ0 cells, using β-actin as an internal control.

Table 3 – Kinase activities in crude cell extracts of MOLT-4 wt and MOLT-4 ρ0cells were measured with the DE81 filter disc assay [12].

Statistical analysis Statistical significance was calculated using two-tailed Student's t-test.

Results mRNA content of dGK, dCK,TK1 and TK2 in MOLT-4 wt and ρ0 cells

Gene

dGK dCK TK1 TK2

MOLT-4 ρ0 Percent mRNA level compared to wt (100%) 141 ± 34 119 ± 21 62 ± 22⁎ 189 ± 13⁎⁎

The data are presented as percent mRNA in MOLT-4 ρ0 cells compared to the control in the MOLT-4 wt cells (100%) and represent the mean±S.D. from three (TK1, TK2 and dCK) or two experiments (dGK), (⁎p< 0.05, ⁎⁎p<0.01).

Enzyme dGK dGK dCK TK1⁎ TK2

Substrate

MOLT-4 wt (pmol/mg/min)

MOLT-4 ρ0 (pmol/mg/min)

araG + dCyd CdA + dCyd CdA dThd BvDU

1.4 ± 0.5 1.2 ± 0.3 39 ± 19 34 ± 10 0.91 ± 0.11

1.5 ± 0.6 1.2 ± 0.4 38 ± 15 15 ± 3 0.87 ± 0.09

The phosphorylating activities of different substrates are shown in pmol/mg/min and represent the mean ± S.D. from at least three experiments (⁎p < 0.05).

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Table 4 – The sensitivity of MOLT-4 wt and MOLT-4 ρ0 cells to several anti-viral and anti-cancer nucleoside analogs and cytotoxics was determined through a cell proliferation assay. Compound

FdUrd⁎ BvDU BvAraU AraT⁎⁎ AZT⁎ dFdC ddC HPMPC AraA⁎⁎ CldA⁎⁎ PMEA⁎⁎ dGuo AraG⁎ GCV⁎⁎ dFdG Abacavir⁎ PMEG⁎ ddI Etoposide

IC50 (μM) MOLT-4 wt

MOLT-4 ρ0

0.019 ± 0.002 357 ± 159 ≥ 500 252 ± 36 299 ± 107 0.014 ± 0.007 3.9 ± 0.6 ≥ 500 24 ± 7 0.17 ± 0.01 57 ± 12 36 ± 0 1.6 ± 0.5 228 ± 23 0.012 ± 0.001 112 ± 29 0.93 ± 0.25 ≥ 500 0.24 ± 0.17

0.0082 ± 0.0002 463 ± 64 > 500 8.0 ± 2.6 42 ± 25 0.020 ± 0.008 4.2 ± 1.1 > 500 3.6 ± 0.4 0.061 ± 0.014 14 ± 6 27 ± 9 0.24 ± 0.07 ≥ 500 0.043 ± 0.012 32 ± 4 0.15 ± 0.00 > 500 0.26 ± 0.19

The table shows the inhibitory concentration (IC)50 in μM and represents data mean ± SD from two to three independent experiments (⁎p < 0.05, ⁎⁎p < 0.01).

a similar level to the araG phosphorylating activity. At 2.5 μM (E)-5(2-bromovinyl)-2′-deoxyuridine (BvDU) is primarily a substrate of TK2 and its phosphorylating activity was similar in the two cell lines. Although the phosphorylation of Thymidine (Thd) is catalyzed by both TK1 and TK2 the Thd phosphorylating activity in cycling cells mainly corresponds to the TK1 activity since TK1 is found in high amounts in S-phase cells compared to TK2 which is constitutively expressed but in low amounts. The Thd phosphorylating activity was substantially higher in the MOLT-4 wt cells compared to the ρ0 cells (p < 0.05). The expression of TK1 mRNA in the ρ0 cells was also found to be lower compared to the wt cells consistent with a relationship between TK1 mRNA expression and enzyme activity was observed in these cells. However, no such relationship was found between the mRNA expression of TK2 and its corresponding enzyme activity.

Sensitivity to anti-cancer and anti-viral compounds in MOLT-4 wt and ρ0 cells The cells were tested for sensitivity to several anti-cancer and antiviral compounds (Table 4). The MOLT-4 ρ0 cells were equally sensitive or more sensitive to most of the compounds tested except for ganciclovir (GCV) which was 2-fold less toxic (p < 0.01) in these cells. AraG and araT that have been shown to be predominately incorporated into mtDNA of MOLT-4 wild-type cells were found to exhibit increased toxicity, 6- (p < 0.05) and 32-fold (p < 0.01), respectively, to the ρ0 cells compared to the MOLT-4 wt cells demonstrating that the presence of mtDNA is not necessary for araG and AraT to exert toxicity. AZT, which is known to be incorporated into mtDNA of cells, also exhibited 7-fold (p < 0.05) increased toxicity in the ρ0 cells. Overall, the MOLT-4 ρ0 cell line exhibited increased or similar sensitivity to most of the nucleoside analogs tested in this study.

Discussion This is the first study of the deoxyribonucleoside kinase mRNA levels and enzymatic phosphorylation activities of dGK, dCK, TK1 and TK2 in ρ0 T-lymphoblast cells. The enzyme activities of dGK, dCK and TK2 did not differ between the cell lines, whereas the ρ0 MOLT-4 cells showed significantly lower TK1 activity. The mRNA expression of TK1 was also lower, indicating that there was likely to be a concordant relationship between the expression of mRNA and enzyme activity of TK1 in these cells. The reduction of expression of TK1 mRNA and enzyme activity in the MOLT-4 ρ0 cells could possibly be explained by the difference in doubling time between the two cell lines, which is 24 h for the MOLT-4 wt cells and 42 h for the MOLT-4 ρ0 cells [11], and the fact that TK1 expression is restricted to S-phase. The expression of TK2 mRNA was elevated in MOLT-4 ρ0 cells, in contrast the TK2 enzyme activity was similar in the MOLT-4 wt- and ρ0 cells. Although the activities of dGK and dCK were similar in the cell lines the mRNA levels were slightly elevated, although not statistically significant, in the ρ0 cells. These results are in agreement with previous studies showing no direct correlation between the mRNA expression of dGK, dCK and TK2 and their respective enzyme activities. The lack of correlation between mRNA levels and enzyme activity has been suggested to be due to post-translational mechanisms [16–19]. The nucleoside analog araG has previously been shown to be predominantly incorporated into mitochondrial DNA of both lymphocytic and non-lymphocytic cell lines [8,9]. The biochemical and pharmacological importance of the accumulation of araG in mtDNA is presently not known. However, it has been suggested that the acute cytotoxicity of araG in CEM T-lymphoblastic cells is not mediated by incorporation of araG into mtDNA, but rather by the incorporation of araG into nuclear DNA [9]. Other studies also suggest an S-phase-specific cytotoxicity of araG [20]. To address the role of mtDNA in the toxicity of araG and other anti-viral and anticancer agents in T-lymphoblast cells the toxicity of these agents was compared in MOLT-4 wt and ρ0 cells. In comparison with the wt cells, the ρ0 cells showed increased sensitivity to araG and araT, both of which have been shown to be predominantly incorporated into mtDNA of MOLT-4 wt cells, demonstrating that mtDNA is not a prerequisite for the acute toxicity of these agents. Although pyrimidine analogs are mainly active in dividing cells and the acute toxicity of these agents is likely caused by effects on nuclear DNA some pyrimidine nucleoside analogs, such as AZT and ddC, are known to cause delayed mitochondrial toxicity after prolonged treatment [6,21–23]. The mode of delayed toxicity of these nucleoside analogs is different. For ddC it has been shown that delayed toxicity is caused by depletion of mtDNA, whereas accumulated data argue that delayed AZT toxicity is not due to affects on mtDNA replication, but rather due to competitive inhibition of TK2 [22–27]. In our experimental setting the cytotoxicity of AZT was increased in the ρ0 cells whereas the sensitivity to ddC was similar in both cell-types, demonstrating that the mtDNA was not necessary for their ability to exert acute toxicity. We do not know the mechanism by which the sensitivity was increased to certain nucleoside analogs, in particular in the arabinosylnucleoside derivatives, in the ρ0 MOLT-4 cells. The activities of the deoxyribonucleoside kinases were similar except for TK1 which was decreased in the ρ0 cells and could not explain the increase in sensitivity in these cells. Besides depleting the ρ0 cells of mtDNA the EtBr-treatment potentially altered the

E XP E RI ME N TAL C ELL R E SE A RC H 315 ( 2 0 0 9 ) 25 3 9– 2 5 43

expression of several nuclear genes in these cells, rendering the cells more sensitive to certain nucleoside analogs. One possible mechanism underpinning the increased sensitivity could be altered expression of nucleoside/nucleotide transporter proteins. A mitochondrial nucleotide carrier has been reported and thus nucleotides may be synthesized in the cytoplasm or mitochondria and subsequently imported into or exported from the mitochondria [28]. It is likely that there are other mitochondrial carriers yet to be identified and perhaps they could be implicated in the altered sensitivity. The difference in growth rates between the MOLT-4 wt and ρ0 cells is an additional factor which might have contributed to our findings. Taken together, the cell lines exhibit similar sensitivity to most of the tested nucleoside analogs. The most important finding in this study was that mtDNA was not necessary for the cytotoxic activity of araG and araT and the other tested nucleoside analogs to exert their acute toxic effect in T-lymphoblastoid cells.

Acknowledgments This work was supported by grants from the Swedish Research Council, the Swedish Cancer Society, the Karolinska Institute, the K. U.Leuven (GOA 05/19) and the O'Haus Foundation. The authors would like to thank Marjan Amiri and Lizette van Berckelaer for excellent technical help.

REFERENCES

[1] M.P. King, G. Attardi, Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation, Science 246 (1989) 500–503. [2] D.C. Wallace, C. Stugard, D. Murdock, T. Schurr, M.D. Brown, Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 14900–14905. [3] J.M. Shoffner, A. Kaufman, D. Koontz, N. Krawiecki, E. Smith, M. Topp, D.C. Wallace, Oxidative phosphorylation diseases and cerebellar ataxia, Clin. Neurosci. 3 (1995) 43–53. [4] J.Y. Feng, A.A. Johnson, K.A. Johnson, K.S. Anderson, Insights into the molecular mechanism of mitochondrial toxicity by AIDS drugs, J. Biol. Chem. 276 (2001) 23832–23837. [5] Y. Lai, C.M. Tse, J.D. Unadkat, Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs, J. Biol. Chem. 279 (2004) 4490–4497. [6] W. Lewis, M.C. Dalakas, Mitochondrial toxicity of antiviral drugs, Nat. Med. 1 (1995) 417–422. [7] C.H. Chen, Y.C. Cheng, The role of cytoplasmic deoxycytidine kinase in the mitochondrial effects of the anti-human immunodeficiency virus compound, 2',3'-dideoxycytidine, J. Biol. Chem. 267 (1992) 2856–2859. [8] C. Zhu, M. Johansson, A. Karlsson, Differential incorporation of 1-beta-D-arabinofuranosylcytosine and 9-beta-Darabinofuranosylguanine into nuclear and mitochondrial DNA, FEBS. Lett. 474 (2000) 129–132. [9] S. Curbo, B. Zhivotovsky, M. Johansson, A. Karlsson, Effects of 9beta-D-arabinofuranosylguanine on mitochondria in CEM Tlymphoblast leukemia cells, Biochem. Biophys. Res. Commun. 307 (2003) 942–947. [10] C. Zhu, M. Johansson, A. Karlsson, Incorporation of nucleoside analogs into nuclear or mitochondrial DNA is determined by the intracellular phosphorylation site, J. Biol. Chem. 275 (2000) 26727–26731.

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[11] R. Armand, J.Y. Channon, J. Kintner, K.A. White, K.A. Miselis, R.P. Perez, L.D. Lewis, The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells), Toxicol. Appl. Pharmacol. 196 (2004) 68–79. [12] E.S. Arner, T. Spasokoukotskaja, S. Eriksson, 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. 188 (1992) 712–718. [13] L. Wang, S. Eriksson, 5-Bromovinyl 2'-deoxyuridine phosphorylation by mitochondrial and cytosolic thymidine kinase (TK2 and TK1) and its use in selective measurement of TK2 activity in crude extracts, Nucleosides Nucleotides Nucleic Acids 27 (2008) 858–862. [14] M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) e45. [15] L. Wang, A. Karlsson, E.S. Arner, S. Eriksson, Substrate specificity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2-chlorodeoxyadenosine, J. Biol. Chem. 268 (1993) 22847–22852. [16] L. Wang, S. Eriksson, Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria, Biochem. J. 351 (Pt 2) (2000) 469–476. [17] K. Lotfi, K. Karlsson, A. Fyrberg, G. Juliusson, V. Jonsson, C. Peterson, S. Eriksson, F. Albertioni, The pattern of deoxycytidineand deoxyguanosine kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia, Biochem. Pharmacol. 71 (2006) 882–890. [18] F. Albertioni, S. Lindemalm, S. Eriksson, G. Juliusson, J. Liliemark, Relationship between cladribine (CdA) plasma, intracellular CdA5'-triphosphate (CdATP) concentration, deoxycytidine kinase (dCK), and chemotherapeutic activity in chronic lymphocytic leukemia (CLL), Adv. Exp. Med. Biol. 431 (1998) 693–697. [19] C. Smal, D. Vertommen, L. Bertrand, M.H. Rider, E. van den Neste, F. Bontemps, Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells, Nucleosides Nucleotides Nucleic Acids 25 (2006) 1141–1146. [20] C.O. Rodriguez Jr., V. Gandhi, Arabinosylguanine-induced apoptosis of T-lymphoblastic cells: incorporation into DNA is a necessary step, Cancer. Res. 59 (1999) 4937–4943. [21] M.C. Dalakas, I. Illa, G.H. Pezeshkpour, J.P. Laukaitis, B. Cohen, J.L. Griffin, Mitochondrial myopathy caused by long-term zidovudine therapy, N. Engl. J. Med. 322 (1990) 1098–1105. [22] M.C. Dalakas, Peripheral neuropathy and antiretroviral drugs, J. Peripher. Nerv. Syst. 6 (2001) 14–20. [23] A.A. Johnson, A.S. Ray, J. Hanes, Z. Suo, J.M. Colacino, K.S. Anderson, K.A. Johnson, Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase, J. Biol. Chem. 276 (2001) 40847–40857. [24] E.E. McKee, A.T. Bentley, M. Hatch, J. Gingerich, D. Susan-Resiga, Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity, Cardiovasc. Toxicol. 4 (2004) 155–167. [25] M.D. Lynx, E.E. McKee, 3'-Azido-3'-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria, Biochem. Pharmacol. 72 (2006) 239–243. [26] L. Wang, A. Saada, S. Eriksson, Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy, J. Biol. Chem. 278 (2003) 6963–6968. [27] H. Lee, J. Hanes, K.A. Johnson, Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase, Biochemistry 42 (2003) 14711–14719. [28] V. Dolce, G. Fiermonte, M.J. Runswick, F. Palmieri, J.E. Walker, The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 2284–2288.