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J. Biol. Chem., Vol. 275, Issue 35, 26727-26731, September 1, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/35/26727    most recent M002304200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhu, C. Articles by Karlsson, A. Search for Related Content PubMed PubMed Citation Articles by Zhu, C. Articles by Karlsson, A. Social Bookmarking                      What's this?

Incorporation of Nucleoside Analogs into Nuclear or Mitochondrial DNA Is Determined by the Intracellular Phosphorylation Site^*

Chaoyong Zhu, Magnus Johansson, and Anna Karlsson

From the Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, S-141 86 Stockholm, Sweden

Received for publication, March 20, 2000, and in revised form, April 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nucleoside analogs used in cancer chemotherapy and in treatment of virus infections are phosphorylated in cells by nucleoside and nucleotide kinases to their pharmacologically active form. The phosphorylated nucleoside analogs are incorporated into DNA and cause cell death or inhibit viral replication. Cellular DNA is replicated both in the nucleus and in the mitochondria, and nucleoside analogs may interfere with DNA replication in both these subcellular locations. In the present study we created a cell model system where nucleoside analogs were phosphorylated, and thereby pharmacologically activated, in either the nucleus, cytosol, or mitochondria of cancer cells. The system was based on the reconstitution of deoxycytidine kinase (dCK)-deficient Chinese hamster ovary cells with genetically engineered dCK targeted to the different subcellular compartments. The nucleoside analogs phosphorylated by dCK in the mitochondria were predominantly incorporated into mitochondrial DNA, whereas the nucleoside analogs phosphorylated in the nucleus or cytosol were incorporated into nuclear DNA. We further show that the nucleoside analogs phosphorylated in the mitochondria induced cell death by an apoptotic program. These data showed that the subcellular site of nucleoside analog phosphorylation is an important determinant for incorporation of nucleoside analogs into nuclear or mitochondrial DNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytotoxic nucleoside analogs such as 1--D-arabinofuranosylcytosine (AraC,¹ Cytarabine), 2-chloro-2'-deoxyadenosine (CdA, Cladribine), and 2',2'-difluorodeoxycytidine (dFdC, Gemcitabine) are commonly used in chemotherapy of cancer (1, 2). Other nucleoside analogs, such as 3'-azido-2',3'-dideoxythymidine and 2',3'-dideoxycytidine, are used in treatment of human immunodeficiency virus infection (3). The nucleoside analogs are transported across the cell membrane by nucleoside transporters and are phosphorylated in cells by nucleoside and nucleotide kinases to their pharmacologically active triphosphate forms. The nucleoside analog triphosphates compete with the deoxyribonucleotides as substrates of cellular or viral DNA polymerases. Incorporation of nucleoside analogs into the elongating DNA strands induces cell death or inhibits viral replication (3-5).

The first and rate-limiting phosphorylation step of most nucleoside analogs is catalyzed by the deoxyribonucleoside kinases as follows: deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and thymidine kinases 1 and 2 (TK1 and TK2) (3, 6). TK1 is a cytosolic enzyme, and dCK is located in the cytosol or nucleus, whereas both dGK and TK2 are mitochondrial enzymes (7-11). The rate-limiting step of nucleoside analog phosphorylation occurs accordingly in different subcellular compartments depending on which nucleoside kinase catalyzes the reaction. The effects of nucleoside analogs on mitochondria have been intensively studied since several pyrimidine nucleoside analogs used in antiviral therapy damage mitochondrial DNA and thereby interfere with mitochondrial function (12-14). The mitochondrial deoxyribonucleotide pool has been suggested to be separated from the cytosolic/nuclear pool (15-17). Accordingly, nucleoside analogs phosphorylated by mitochondrial deoxyribonucleoside kinases may become trapped within the mitochondria and affect mitochondrial, but not nuclear, DNA. We have recently shown that overexpression of human mitochondrial dGK in cancer cell lines enhances the sensitivity to cytotoxic nucleoside analogs (18). These results suggest that the mitochondrial nucleoside kinases may be important for the pharmacological activation of nucleoside analogs.

In order to study the compartmentalization of nucleoside analogs phosphorylated in the mitochondria, we reconstituted a dCK-deficient cell line with genetically engineered dCK mutants targeted to either the cytosol, nucleus, or mitochondria. This cell model system allowed us to study subcellular compartmentalization of nucleoside analogs phosphorylated in the three different compartments. In summary, we showed that nucleoside analogs phosphorylated in the mitochondria became trapped in this compartment and that the subcellular location of nucleoside analog phosphorylation is an important determinant for the incorporation of nucleoside analogs into nuclear or mitochondrial DNA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Plasmid Vectors-- The pEGFP-N1 vector (CLONTECH) was used for all plasmids constructed. The wild-type nucDCK-GFP and the mutant cytDCK-GFP were constructed as described (7). The cDNA sequence encoding the 31-amino acid N-terminal mitochondrial import signal of cytochrome c oxidase subunit VIII was cloned upstream of GFP or cytDCK-GFP cDNA to create COX-GFP and mitDCK-GFP (19). The plasmids were purified with the maxi-prep kit (Qiagen), and the DNA sequences were verified by automated laser fluorescence sequencing (Amersham Pharmacia Biotech).

Culture and Transfection of Cell Lines-- The dCK-deficient CHO cell line (a gift from Dr. W. Plunkett, Houston, TX) (20) was cultured at 37 °C in McCoy 5A modified medium supplemented with 10% (v/v) fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. The cells were transfected using LipofectAMINE (Life Technologies, Inc.), and GFP fluorescence was observed in the living cells 48 h after transfection (7). For generation of stably transfected cells, the cells were subcultured 1:5 3 days after transfection, and 0.5 mg/ml Geneticin (Life Technologies, Inc.) was added. Geneticin-resistant cell colonies with green fluorescence were isolated and continuously cultured (7).

Western Blot Analysis and Enzyme Assays-- Protein extracts of the mitochondrial and cytosolic subcellular fractions were prepared as described (10). The protein extracts were separated by 12% SDS-polyacrylamide gel electrophoresis and electrotransferred to the nitrocellulose membrane. The membrane blots were probed for 1 h with a polyclonal anti-GFP antibody (CLONTECH). A secondary alkaline phosphatase conjugate anti-IgG antibody (Sigma) was applied for 1 h, and the alkaline phosphatase was visualized by incubation with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). The dCK assays were performed as described (10, 21). 2 µM [8-³H]CdA (20 Ci/mmol, Moravek Biochemicals Inc.) and 8 µM unlabeled CdA (Sigma) were used in each reaction.

Autoradiography-- The cells were cultured on poly-L-lysine-coated chamber slides (Nunc Inc.) for 24 h. Cells were labeled with 0.8 µM [³H]2-chloro-2'-deoxyadenosine (Moravek Biochem), 0.7 µM [³H]1--D-arabinofuranosylcytosine (Moravek Biochem), or 0.2 µM [³H]thymidine (DuPont) for 6-20 h. The slides were rinsed with phosphate-buffered saline, fixed 10 min in methanol:acetic acid (3:1), and washed three times with ice-cold 10% trichloroacetic acid, once with water, and once with methanol. The slides were coated with Hypercoat photoemulsion (Amersham Pharmacia Biotech) and exposed 1-3 weeks at 4 °C. The autoradiographs were developed using D-11 developer (Eastman Kodak Co.).

Cell Proliferation Assays-- 2 × 10³ cells were plated in 96-well microtiter plates, and AraC (Sigma), CdA (Sigma) or dFdC (Lilly) was added after 24 h. Cell survival was determined by the metrotetrazolium assay (Cell Proliferation Kit I, Roche Molecular Biochemicals) after 4 days of drug exposure. Each experiment was performed in triplicate. Statistical analysis was performed using the Student's paired t test.

Apoptosis Detection-- Apoptosis was determined by caspase-3 activity and DNA laddering. 50 µM dFdC, CdA, or cisplatin was used for all experiments. For caspase-3 assays, 2 × 10⁶ cells were harvested after 6 h of drug incubation. Caspase-3 activity assay was performed with the ApoAlert Colorimetric assay kit (CLONTECH). For the DNA laddering analysis, 2 × 10⁵ cells were harvested after 48 h of drug incubation, and the DNA was extracted (22).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Targeting of dCK to Three Different Subcellular Compartments-- We constructed three plasmids to express the cDNA of human dCK targeted to the nucleus, cytosol, or mitochondria (Fig. 1A). dCK was expressed as a fusion protein to the green fluorescent protein (GFP) in order to visualize easily the subcellular location of the recombinant enzyme in vivo. Wild-type dCK-GFP (nucDCK-GFP) is predominantly located in the cell nucleus, and a mutant lacking the N-terminal nuclear localization signal (cytDCK-GFP) is retained in the cytosol (7). The N-terminal mitochondrial import signal of cytochrome c oxidase subunit VIII can be fused to heterologous proteins, such as GFP (COX-GFP) (19), to target these proteins to the mitochondria. We fused the cytochrome c oxidase mitochondrial import signal to the N terminus of the cytosolic dCK mutant to target dCK to the mitochondria (mitDCK-GFP). dCK-deficient Chinese hamster ovary (CHO) cells were transfected with the plasmids, and stably transfected cell clones were isolated. Visualization of GFP by fluorescence microscopy showed predominantly nuclear fluorescence for the nucDCK-GFP construct and cytosolic fluorescence for the cytDCK-GFP construct (Fig. 1B). The cells transfected with the mitDCK-GFP plasmid showed an irregular dotted fluorescence pattern around a non-fluorescent nucleus (Fig. 1B). This pattern is similar to the fluorescence pattern of the mitochondrial COX-GFP protein (Fig. 1B) as well as to other mitochondrial proteins fused to GFP (18, 19). To verify further the mitochondrial location of mitDCK-GFP, we separated cytosolic and mitochondrial protein fractions and used an anti-GFP antibody to detect the fusion proteins by Western blot (Fig. 2A). A major band of 54 kDa was detected in the cytosolic, but not mitochondrial, fraction of the cells expressing nucDCK-GFP or cytDCK-GFP. In contrast, a band of similar size was detected in the mitochondrial, but not cytosolic, fraction of cells expressing mitDCK-GFP. We conclude that the three plasmids target dCK-GFP to the three different subcellular compartments.

View larger version (79K): [in this window] [in a new window]   Fig. 1.   Targeted overexpression of dCK in three different subcellular compartments. A, plasmid constructed to target the fusion protein of human dCK and GFP to the nucleus (nucDCK-GFP), cytosol (cytDCK-GFP), and mitochondria (mitDCK-GFP). Mutations are shown in lowercase letters. COX8, cytochrome oxidase c subunit VIII; MTS, mitochondrial targeting signal. CMV, cytomegalovirus promoter. B, fluorescence microscopy images of CHO cells transfected with the GFP plasmids. wt, wild type.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Expression and activity of dCK-GFP in the transfected cells. A, Western blot analysis with anti-GFP antibodies of cytosolic (C) and mitochondrial (M) subcellular protein fractions of CHO cells expressing nucDCK-GFP, cytDCK-GFP, and mitDCK-GFP. B, dCK activity, determined as CdA phosphorylation, in crude extracts of the untransfected and transfected CHO cells. wt, wild type.

Phosphorylation of Nucleoside Analogs by dCK in Different Subcellular Compartments-- We determined the dCK activity in the transfected cells to verify its enzymatic activity. The dCK activity, determined as phosphorylation of the nucleoside analog CdA, was 4-fold higher in the dCK-expressing cells than in the dCK-deficient parent cell line (Fig. 2B). There was no significant difference in dCK activity between the cells overexpressing dCK-GFP in the cytosol, nucleus, or mitochondria. Phosphorylation of CdA detected in the dCK-deficient parent cells is probably due to mitochondrial dGK, because CdA phosphorylation is catalyzed by both dCK and dGK (23, 24).

We used an autoradiography method to distinguish between incorporation of nucleoside analogs into nuclear or mitochondrial DNA (Fig. 3). [³H]dThd was used as a control, and it was incorporated into nuclear DNA of all cell lines independently of dCK expression as expected. A lightly dotted [³H]dThd autoradiography pattern was detected outside the nucleus, representing dThd incorporation into mitochondrial DNA (16). AraC is predominantly phosphorylated by dCK (24), and as expected, no incorporation of [³H]AraC into DNA was detected in the dCK-deficient cells. The cells expressing nucDCK-GFP and cytDCK-GFP both efficiently incorporated [³H]AraC into nuclear DNA. In contrast, the cells expressing mitDCK-GFP showed an irregularly dotted autoradiography pattern distributed throughout the cells, indicating that the nucleoside analog was incorporated into mitochondrial DNA. Cells incubated with [³H]CdA showed a similar pattern as those incubated with AraC, except for dCK-deficient cells that incorporated CdA into mitochondrial DNA, probably due to CdA phosphorylation catalyzed by mitochondrial dGK (23, 24). In summary, our data showed that nucleoside analogs phosphorylated in either the cytosol or the nucleus were incorporated into nuclear DNA, whereas nucleoside analogs phosphorylated within the mitochondria became trapped in this compartment and were not incorporated into nuclear DNA.

View larger version (119K): [in this window] [in a new window]   Fig. 3.   Autoradiography of dCK-GFP-transfected CHO cells incubated with [3H]dThd, [3H]AraC, and [3H]CdA.

Sensitivity to Nucleoside Analogs-- The sensitivity of the transfected cells to the nucleoside analogs dFdC, AraC, and CdA was determined (Fig. 4). The three dCK overexpressing cell lines were more sensitive to the nucleoside analogs than the dCK-deficient parent cell line. The cell lines overexpressing dCK in the nucleus or in the cytosol cells exhibited 50-400-fold lower IC₅₀ for the investigated compounds compared with the untransfected cells. There was no difference in nucleoside analog sensitivity between the cells expressing dCK in the nucleus or the cytosol. However, the cells expressing mitochondrial dCK were less sensitive compared with the cells expressing cytosolic or nuclear dCK and exhibited only a 2-30-fold decrease in IC₅₀ compared with the dCK-deficient parent cell line.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   AraC, CdA, and dFdC sensitivity of dCK-deficient CHO cells expressing GFP () or dCK-GFP targeted to the nucleus (), cytosol (×), or mitochondria ().

Induction of Apoptosis-- Nucleoside analogs that interfere with nuclear DNA replication induce cell death by apoptosis similar to several other drugs that damage nuclear DNA (25-27). To address the question, whether nucleoside analogs phosphorylated in the mitochondria induce apoptosis, we determined the presence of inter-nucleosomal DNA fragmentation and caspase-3 activity after incubation with nucleoside analogs. Cells expressing dCK in either the cytosol, nucleus, or mitochondria that were incubated with either CdA (Fig. 5) or dFdC (data not shown) showed caspase-3 activation and DNA laddering indicating cell death by an apoptotic pathway.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Induction of apoptosis by the nucleoside analog CdA. A, caspase-3 activity of cells exposed to CdA or cisplatin for 6 h in the presence or absence of the caspase-3 inhibitor DEVD. B, DNA laddering of cells exposed to CdA or cisplatin for 48 h.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have in the present study demonstrated that the subcellular site of nucleoside analog phosphorylation is an important determinant for the incorporation of these compounds into nuclear or mitochondrial DNA. Nucleoside analogs phosphorylated in the mitochondria became trapped in this compartment and were incorporated into mitochondrial but not nuclear DNA. A major implication of these findings is that nucleoside analogs phosphorylated by the mitochondrial nucleoside kinases dGK and TK2 are likely to affect mitochondrial DNA rather than nuclear DNA. Several antiviral nucleoside analogs such as 3'-azido-2',3'-dideoxythymidine and fialuridine damage mitochondrial DNA and thereby cause adverse effects (12-14). 3'-Azido-2',3'-dideoxythymidine and fialuridine are substrates of both cytosolic TK1 as well as mitochondrial TK2 (28, 29). Based on the data presented in the current paper, it is likely that the intramitochondrial phosphorylation of these compounds catalyzed by TK2 results in selective mitochondrial DNA damage. However, the anti-human immunodeficiency virus nucleoside analogs 2',3'-dideoxycytidine also cause mitochondrial DNA damage, although this compound is phosphorylated by dCK and not by the mitochondrial nucleoside kinases (14, 30). Thus, there appears to exist a mechanism for import of phosphorylated nucleoside analogs into mitochondria. Recently, a mitochondrial dCTP transport protein was identified that further supports this hypothesis (31). Based on these findings, we believe that nucleoside analogs phosphorylated in the cytosolic/nuclear compartment can be imported into the mitochondria, whereas no mechanism exists for export of phosphorylated nucleoside analogs from the mitochondria to the cytosol.

The mitochondrial genome encodes enzymes involved in the respiratory chain, and defective expression of these enzymes results in disruption of cellular aerobic metabolism and cell death. The pyrimidine antiviral nucleoside analogs that damage mitochondrial DNA have been reported to cause either depletion of mitochondrial DNA or become incorporated into mitochondrial DNA and thereby interfere with mitochondrial DNA replication or transcription (12-14). In contrast to the rapid induction of cell death caused by incorporation of nucleoside analogs into nuclear DNA, the effects on mitochondria by the antiviral nucleoside analogs are delayed and occur after weeks of drug exposure. In the present study, we detected cell death by an apoptotic pathway already within days of exposure to the anti-cancer nucleoside analogs that were phosphorylated in mitochondria and incorporated into mitochondrial DNA. Nucleoside analogs phosphorylated in the cytosolic/nuclear compartment, such as AraC and CdA, have previously been shown to induce apoptosis (25-27), but little is known about induction of apoptosis by mitochondrial DNA lesions. An early event in apoptosis caused by nuclear DNA-damaging agents is the release of cytochrome c from the mitochondrial intermembrane space to the cytosol. The released cytosolic cytochrome c binds, together with dATP, to the apoptotic protease-activating factor-1, and the complex activates the caspase cascade that executes the apoptotic program (32). The mechanism and regulation of cytochrome c release from mitochondria are still to a large extent unknown, and we do not yet know whether apoptosis induced by nucleoside analog phosphorylation in mitochondria is associated with this phenomena. However, we cannot presently exclude that there is a small leakage of phosphorylated nucleoside analogs from mitochondria to the nucleus that we were unable to detect by the methods used in the present study, and it is possible that the activation of caspases results from nuclear DNA damage. We are currently initiating studies to resolve this issue.

The cytosolic/nuclear nucleoside kinases have different substrate specificities compared with the mitochondrial nucleoside kinases. Our data suggest that it may be possible to minimize mitochondrial DNA damage by designing novel nucleoside analogs that are not phosphorylated by the mitochondrial enzymes. It may also be possible to develop cytotoxic nucleoside analogs that specifically target mitochondrial DNA. There are important differences between nuclear and mitochondrial DNA physiology. Nucleoside analogs that affect nuclear DNA replication are primarily effective in the S-phase of the cell cycle when nuclear DNA replication occurs. However, their efficiency is decreased by the large increase in deoxyribonucleotide synthesis via the de novo pathway in S-phase cells that compete with the nucleoside analogs as substrates for the DNA polymerases. In contrast, mitochondrial DNA is replicated independently of nuclear DNA throughout the cell cycle (33), and the mitochondrial dNTP pools are separated from the cytosolic pool where the de novo deoxyribonucleotide synthesis occurs (15-17). Furthermore, the cell nucleus contains several efficient DNA repair systems that remove incorporated nucleoside analogs that cause chain termination or mismatch base pairing. In contrast, mitochondrial DNA repair systems are inefficient in repairing DNA strand breaks as compared with the nuclear system (34). The differences described above suggest that nucleoside analogs targeting nuclear and mitochondrial DNA may exhibit different pharmacological profiles that could be used for the development of novel anti-cancer chemotherapy regimes.

	FOOTNOTES

^* This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Foundation, and the Swedish Foundation for Strategic Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 46-8-58587932; Fax: 46-8-58587933; E-mail: anna.karlsson@mbb.ki.se.

Published, JBC Papers in Press, May 25, 2000, DOI 10.1074/jbc.M002304200

	ABBREVIATIONS

The abbreviations used are: AraC, 1--D-arabinofuranosylcytosine; dCK, deoxycytidine kinase; dGK, deoxyguanosine kinase; TK1 and TK2, thymidine kinase 1 and 2; GFP, green fluorescent protein; CdA, 2-chloro-2'-deoxyadenosine; dFdC, 2',2'-difluorodeoxycytidine; CHO, Chinese hamster ovary.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/35/26727    most recent M002304200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhu, C. Articles by Karlsson, A. Search for Related Content PubMed PubMed Citation Articles by Zhu, C. Articles by Karlsson, A. Social Bookmarking                      What's this?