Báo cáo khoa học: Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts docx

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts Miriam Frangini 1 , Chiara Rampazzo 1 , Elisa Franzolin 1 , Mari-Carmen Lara 2 , Maya R. Vila ` 2 , Ramon Martı ´ 2 and Vera Bianchi 1 1 Department of Biology, University of Padova, Italy 2 Institut de Recerca Hospital Universitari Vall d’Hebron, Barcelona, Spain Mitochondrial DNA depletion syndromes (MDSs) are a group of heterogeneous mitochondrial diseases characterized by reduced copy numbers of mtDNA, insufficient synthesis of the mitochondrially encoded components of the respiratory chain complexes, and impairment of energy metabolism. MDSs are inherited as autosomal recessive traits and present striking tissue- specific phenotypes. The underlying genetic defects have been identified only for a small fraction of the reported cases [1]. Interestingly, out of nine genes involved in MDS, four code for enzymes of deoxy- nucleotide metabolism, namely the two mitochondrial deoxynucleoside kinases [thymidine kinase (TK2)] [2] and deoxyguanosine kinase [3], cytosolic thymidine phosphorylase (TP) [4] and p53R2 [5], the stable isoform of ribonucleotide reductase small subunit. Whereas TP is a catabolic enzyme that degrades thymidine and deoxyuridine and participates in the Keywords dTTP pool turnover; mitochondrial DNA depletion syndrome; mitochondrial DNA precursors; p53R2; thymidine phosphorylase Correspondence V. Bianchi, Department of Biology, Via Ugo Bassi 58B, 35131 Padova, Italy Fax: +39 0498276280 Tel: +39 0498276282 E-mail: vbianchi@bio.unipd.it (Received 16 October 2008, revised 1 December 2008, accepted 11 December 2008) doi:10.1111/j.1742-4658.2008.06853.x Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP syn- thesis in noncycling cells, where cytosolic dTTP synthesis via R1 ⁄ R2 ribo- nucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adap- tation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activi- ties of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleo- tidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [ 3 H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibro- blasts with 5% residual TK2 activity synthesized dTTP at an almost nor- mal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity. Abbreviations BVDU, 5-bromovinyl-2-deoxyuridine; KIN109, 1-(6-[1,1-(diphenyl)-1-(4-pyridyl)methoxy]hexyl)thymine; MDS, mitochondrial DNA depletion syndrome; TK1, thymidine kinase 1; TK2, mitochondrial thymidine kinase; TP, thymidine phosphorylase. 1104 FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS regulation of thymidine phosphate pools, the other three are synthetic enzymes needed for the maintenance of dNTP pools in nonproliferating cells. During cell growth, the main source of dNTPs for nuclear and mtDNA replication is S-phase-specific de novo synthesis catalyzed by the canonical R1 ⁄ R2 form of ribonucleotide reductase. Regulated proteoly- sis of R2 in late mitosis [6] turns off R1 ⁄ R2-dependent ribonucleotide reduction, leading to a drop in dNTP pool sizes in G 1 and postmitotic cells. The dNTPs required for DNA repair and mtDNA synthesis during the whole lifespan of cells are instead produced by salvage of deoxynucleosides by cytosolic and mito- chondrial kinases and by R1 ⁄ p53R2-dependent ribonu- cleotide reduction. As compared to the rate of de novo synthesis during cell proliferation, the de novo synthesis of dNTPs catalyzed by R1 ⁄ p53R2 amounts to only a few per cent [7], but it is essential to supply precursors for mtDNA. In humans, genetic inactivation of p53R2 causes a very severe form of MDS that affects multiple organs, leading to death within a few weeks after birth [5]. The broad spectrum of affected tissues in p53R2- mutated individuals [5] suggests a general, fundamental function of p53R2 for dNTP synthesis in nonprolifer- ating cells. Conversely, mutations in either of the two mitochondrial deoxynucleoside kinases have more restricted effects. Deficiency of deoxyguanosine kinase causes a hepatocerebral MDS [3], and TK2 deficiency was originally discovered to cause acute fatal mito- chondrial myopathy [2]. The phenotypic spectrum of TK2 deficiency has turned out to be wider than the myopathy observed originally [8], and also the recently described mouse models of TK2 deficiency show mtDNA depletion in multiple organs [9,10]. A further variable is the speed of the MDS progression. In most cases of TK2 defi- ciency, the disease has early onset and rapid develop- ment, with death occurring during early childhood. In two cases of TK2 deficiency, a severe reduction of TK2 enzymatic activity was associated with late onset and slow progression of the myopathy and long sur- vival [11–14]. The two patients were both compound heterozygotes, harbored two different pairs of TK2 mutations, i.e. T77M ⁄ R161K [13] and R152G ⁄ K171del [12] (according to the latest GenPept entry, NP_004605.3, the mutations are T150M ⁄ R234K and R225G ⁄ K244del), and presented contrasting and dis- tinct features of myopathic damage. Although the overall condition of the patients deteriorated with time, in both cases an apparent negative selection of abnormal muscle fibers suggested that compensatory molecular mechanisms restored respiratory chain func- tions or mtDNA levels in the surviving fibers. In one case, compensatory adaptations of nucleotide meta- bolism were hypothesized [12]. In nonproliferating fibroblasts, the dTTP pool is maintained by a network of interlocked synthetic and catabolic enzyme activities. TK2 operates in parallel with R1 ⁄ p53R2 in the synthesis of thymidine nucleo- tides, and mitochondrial and cytosolic deoxynucleotid- ases and cytosolic TP are active in their degradation [15]. The balance between the competing enzyme activ- ities sets the level of dTTP in cytosolic and mitochon- drial pools. The availability of thymidine in the extracellular milieu is an additional factor, enhancing the influence of salvage relative to de novo synthesis [15]. A rapid exchange of nucleotides across the mito- chondrial inner membrane maintains the cytosolic and mitochondrial compartments in equilibrium. We were interested in investigating whether in the fibroblasts of the two TK2 patients the operation of the network was altered to compensate for the reduced TK2 activity. In extracts from quiescent cultures of patient and control fibroblasts, we measured the amount of TK2 and other enzymes of the network from their catalytic activity, by western blotting or, indirectly, by mRNA quantification. We then deter- mined in situ activities from the phosphorylation of [ 3 H]thymidine in intact cells. Surprisingly, although the activity of TK2 in extracts from quiescent patient cells was only 5–40% of that of control cells, the in situ activity of the enzyme was hardly affected, with no changes being seen in the composition of the dNTP pools. We detected no major change in the expression of other enzymes involved in dTTP regulation. These observations suggest that TK2 activity in wild-type fibroblasts largely exceeds the basic requirements for the maintenance of mitochondrial dTTP. Results Low TK2 activity in extracts of patient fibroblasts Cycling cells are unaffected by loss of TK2 activity, because the bulk of mitochondrial dTTP is produced de novo in the cytosol by ribonucleotide reduction and by thymidine kinase 1 (TK1)-catalyzed salvage of thy- midine [15]. Therefore, we perfomed all experiments with quiescent fibroblasts where, in the absence of S-phase-specific de novo synthesis, TK2 contributes to the maintenance of the dTTP pool together with the R1 ⁄ p53R2 variant of ribonucleotide reductase. We first compared the activity of TK2 in protein extracts of fibroblasts from patients Pa [12] and Pb [13] and from two control lines (Table 1). We M. Frangini et al. Thymidine metabolism in TK2-mutated fibroblasts FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS 1105 employed both a specific assay based on the phosphor- ylation of 5-bromovinyl-2-deoxyuridine (BVDU), a deoxyuridine analog that is a good substrate for TK2 but not for TK1 [16], and the phosphorylation of thymidine, used in earlier determinations of TK2 in the same patient cells [12,13]. A specific inhibitor of TK2 [1-(6-[1,1-(diphenyl)-1-(4-pyridyl)methoxy]hexyl) thymine (KIN109)] [16] strongly inhibited thymidine phosphorylation, demonstrating that the reaction depended on TK2 in all cell lines (Table 1). The assays with the two substrates gave concordant results, show- ing lower TK2 activity in Pa extracts (about 5% of the control) than in Pb extracts (40% of the control). Con- sidering that the mutated enzymes might be unstable during preparation or storage, we tested, by the BVDU phosphorylation assay, whether the TK2 activity of Pa and control extracts was enhanced by glycerol, 15 mm MgCl 2 or increasing concentrations (0.2–5 mm) of ATP, and whether freezing decreased the activity of the cell lysate. None of these modifica- tions changed the specific activity of TK2, which in Pa extracts remained below 10% of the control value (not shown). Unchanged dNTP pools in patient fibroblasts We next compared the size of the dTTP pools in quies- cent cultures of control and TK2-mutated fibroblasts. No significant difference was observed. The total cellu- lar pool contained 1.72 ± 0.2 pmol dTTP per 10 6 cells in the controls, 1.67 ± 0.3 pmol dTTP per 10 6 cells in Pa cells, and 1.33 ± 0.2 pmol dTTP per 10 6 cells in Pb cells. Also, the mitochondrial dTTP pools were similar in control and patient fibroblasts: 0.11 ± 0.03 pmol per 10 6 cells and 0.08 ± 0.03 pmol dTTP per 10 6 cells respectively. These data differ from the only published report on dTTP pools in human TK2- deficient fibroblasts, where the sizes of the mitochon- drial dTTP pools of two mutant lines were 50% and 30% smaller than that of the controls, and also the dCTP pool was reduced, albeit not significantly, caus- ing an imbalance of the dNTP pools as compared to the controls [17]. We determined the amounts of all four dNTPs in the cytosolic and mitochondrial pools of the two patient lines and of two additional controls. The relative sizes of the pools were those observed pre- viously in quiescent skin and lung fibroblasts [18], and we found no dNTP pool imbalance in the patient cells (not shown). Thus, the strong reduction of TK2 activ- ity in Pa and Pb fibroblasts did not lead to detectable modifications of mitochondrial and cytosolic dTTP during 10 days of quiescence in culture. There is no increase of dNTP de novo synthesis in patient fibroblasts We reasoned that in the patient fibroblasts, the R1 ⁄ p53R2-dependent de novo pathway might be over- expressed to compensate for the TK2 defect. We there- fore determined the mRNA levels of the three ribonucleotide reductase subunits and of p53, the tran- scription factor that controls the expression of p53R2, during proliferation and after 10 days of quiescence (Table 2). As compared with proliferating cells, quies- cent cells contained between 20% and 50% of R1 mRNA, whereas R2 mRNA decreased to 10%. Both p53 and p53R2 mRNAs increased during quiescence. Table 1. TK2 activity in cell extracts from quiescent cultures of control and TK2-mutated fibroblasts. Two enzyme assays specific for TK2 were used, as detailed in Experimental procedures. In one assay, TK2 activity was measured using [ 3 H]BVDU, a TK2-specific substrate [16]. In the second assay, the phosphorylation of [ 3 H]thy- midine was measured in the absence or presence of 100 l M KIN109, a TK2-specific inhibitor [16]. The more than 80% inhibition of thymidine phosphorylation caused by KIN109 indicates that TK2 is the kinase involved in the reaction. Pa and Pb, TK2-mutated cells. TK2 enzyme activity is expressed in picomol of substrate phosphor- ylated ⁄ min of incubation ⁄ mg of protein. Values are means of dupli- cate determinations from three to six separate experiments ± standard deviation of the mean. TK2 activity (pmolÆmin )1 Æmg )1 protein) Cell line [ 3 H]BVDU (0.2 l M) [ 3 H]Thymidine (1 lM) )KIN109 +KIN109 Controls 5.2 ± 1.2 12.1 ± 5.8 0.4 ± 0.2 Pa 0.2 ± 0.1 0.9 ± 0.3 0.2 ± 0.1 Pb 2.1 ± 0.4 4.8 ± 1.8 0.3 ± 0.1 Table 2. Comparison of expression of the three ribonucleotide reductase subunits and p53 in quiescent and cycling cultures of control and TK2-mutated fibroblasts. The levels of the mRNAs for ribonucleotide reductase subunits R1, R2 and p53R2 and for p53 were evaluated by real-time RT-PCR on cDNAs prepared from total RNA extracted from proliferating and quiescent fibroblasts. Cyclo- philin A was the internal control used for normalizations. The level of each mRNA is expressed as fold increase relative to that mea- sured in cycling cells. Data are means of measurements from three different experiments run in triplicate ± standard deviation of the mean. Pa and Pb, TK2-mutated lines. mRNA fold increase (quiescent ⁄ cycling cells) Cell line R1 R2 p53R2 p53 Control 0.35 ± 0.06 0.06 ± 0.01 2.82 ± 0.77 3.40 ± 0.59 Pa 0.18 ± 0.03 0.10 ± 0.03 5.10 ± 0.53 8.53 ± 1.57 Pb 0.47 ± 0.04 0.12 ± 0.03 3.78 ± 0.23 5.47 ± 0.90 Thymidine metabolism in TK2-mutated fibroblasts M. Frangini et al. 1106 FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS Whereas the changes of ribonucleotide reductase subunits were similar in all lines, the induction of p53 mRNA was more variable, with the largest increase being seen in Pa fibroblasts. At the protein level, western blotting experiments (Fig. 1) confirmed the large increase of p53 in Pa cells. For p53R2, we found no clear variations between patient and control cells. Thus, the R1 ⁄ p53R2 form of ribonucleotide reduc- tase was present at comparable levels in patient and control fibroblasts during quiescence. To assess whether, in the former case, ribonucleotide reduction contributed to the synthesis of dTTP more than in the controls, we used hydroxyurea, a specific inhibitor of ribonucleotide reductase. In all cell lines, 2 h of treat- ment with 3 mm hydroxyurea induced, at most, a 20% decrease of dTTP, with no preferential effect on the TK2-mutated fibroblasts (not shown). De novo synthesis of dTMP occurs mostly by deami- nation of dCMP followed by methylation of dUMP [19]. dCMP is derived from dephosphorylation of dCDP produced by ribonucleotide reductase and from phosphorylation of deoxycytidine by deoxycytidine kinase. We found no difference in the activity of deoxycytidine kinase in extracts from patient and control cells, ruling out the possibility that enhanced deoxycytidine salvage contributed to the synthesis of dTTP in the TK2-mutated cells (not shown). There is no downregulation of deoxynucleotide catabolism in patient fibroblasts The size of dNTP pools results from the interplay between synthesis and degradation of their components [20]. Pa and Pb fibroblasts maintained apparently nor- mal dTTP pools without upregulating the de novo pathway. A possible mechanism might be the down- regulation of catabolic enzymes such as TP or the two deoxynucleotidases, cytosolic deoxynucleotidase and mitochondrial deoxynucleotidase [21]. In protein extracts from quiescent cultures of the two control and two TK2-mutated lines, we measured the activity of TP and the combined activity of the two deoxynucleo- tidases (Table 3). Whereas deoxynucleotidase activity was identical in all lines, TP levels were more variable, but the range of variation was the same in patient and control cells. Thus, reduced breakdown of deoxynucle- otides or thymidine did not account for the mainte- nance of dTTP in patient fibroblasts. Thymidine metabolism in TK2-mutated fibroblasts To measure the in situ activity of the metabolic path- way that leads, in intact cells, to the synthesis of dTTP, we incubated quiescent TK2-mutated and con- trol fibroblasts with 25 nm [ 3 H]thymidine for 5 and 20 min, and measured changes in the size of the dTTP Fig. 1. Western blot analysis of ribonucleotide reductase subunits and p53 in extracts from quiescent and cycling cultures of control and TK2-mutated fibroblasts. We examined by immunoblotting the abundance of p53R2, R1, R2 and p53 in extracts of control (Ca, Cb) and TK2-mutated (Pa, Pb) fibroblasts in proliferating cultures (P) and after 10 days of quiescence (Q). The relative amount of each pro- tein was normalized using b-actin as loading control. Q ⁄ P, ratio between levels of each protein in quiescent and proliferating cul- tures of each cell line. The blot for p53 in Cb extracts was obtained from a separate electrophoresis run. Table 3. TP and deoxynucleotidase activity in cell extracts from quiescent cultures of control and TK2-mutated fibroblasts. TP acti- vity was assayed with 1 m M thymidine as substrate, and total (mitochondrial + cytosolic) deoxynucleotidase activity with 5 m M [ 3 H]dUMP. Enzyme activities are expressed as nanomol of product formed ⁄ hours of incubation ⁄ mg of protein. Data are mean values of three different experiments ± standard deviation. Subject TP activity (nmol thymineÆh )1 Æmg )1 ) Deoxynucleotidase activity (nmol deoxyuridineÆh )1 Æmg )1 ) Ca 273 ± 52 2340 ± 240 Cb 587 ± 125 2160 ± 540 Pa 244 ± 87 2160 ± 300 Pb 731 ± 67 2340 ± 240 M. Frangini et al. Thymidine metabolism in TK2-mutated fibroblasts FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS 1107 pool and its specific radioactivity (Fig. 2A,B). As found earlier in quiescent skin fibroblasts [15], the addition of thymidine even at such a low concentration caused an immediate, albeit modest, expansion of the dTTP pool, with only minor differences being seen between control and patient cells (Fig. 2A). During the incubation with 25 nm [ 3 H]thymidine, the presence of 1 lm BVDU prevented the expansion of the pool with the same efficiency in Ca and Pa cells (Fig. 2A), con- firming the involvement of TK2 in the phosphorylation of thymidine in both cell lines inferred above (Table 1) from the inhibition of thymidine phosphorylation in vitro caused by KIN109. The specific radioactivity of the dTTP pool reflects the efficiency with which the salvage of extracellular thymidine competes with the endogenous de novo synthesis of dTTP [15,22]. The [ 3 H]thymidine supplied in the medium had a specific radioactivity of 20 000 c.p.m.Æpmol )1 . After 20 min of incubation, the dTTP pool had reached a specific radioactivity of about 7000 c.p.m.Æpmol )1 in Ca and Pb cells, and about 5000 in Cb and Pa cells (Fig. 2B), indicating that one-third of the dTTP of Ca and Pb cells and between one-quarter and one-fifth of the dTTP of Cb and Pa cells was derived from salvage of extracellular thymidine. BVDU decreased the specific radioactivity of dTTP after 20 min of incubation to 75% in Ca cells and to 25% in Pa cells (Fig. 2B). As BVDU inhibited the expansion of the dTTP pool in the two lines to the same extent (Fig. 2A), the difference in specific radio- activity suggests that TK2 competed less efficiently with the de novo synthesis by p53R2 in the TK2- mutated cells. To determine the turnover of the dTTP pool, we performed a pulse–chase experiment with Ca and Pa fibroblasts. We incubated the cultures with 0.1 lm [ 3 H]thymidine for a total of 90 min (pulse). In the chase, after 60 min we shifted one-half of the cultures to medium containing 0.1 lm nonradioactive thymi- dine and the second half to medium without thymi- dine. At 5, 15 and 30 min after medium change, we analyzed, in both series of cultures, the size of the dTTP pool and its specific radioactivity (Fig. 3). The pool size doubled during the first 60 min of pulse (Fig. 3A) and returned to the prepulse value during the 30 min chase in both control and TK2-mutated cells in the absence of thymidine. When thymidine was present during the chase, the decline of the dTTP pool was only transitory. Under both conditions of chase, the specific radioactivity of dTTP declined progres- sively, indicating a turnover of the pool (Fig. 3B). In the presence of extracellular nonradioactive thymidine, the half-life was about 14 min in Ca fibroblasts and 18 min in Pa fibroblasts, similar to values observed earlier in normal lung fibroblasts [7]. In thymidine-free medium, the half-life of dTTP-specific radioactivity exceeded 30 min. The slow decay reflected the de novo synthesis of unlabeled dTTP. From the changes of the specific radioactivity and the concentration of dTTP during the chase, we could calculate the rate of resynthesis of dTTP from thymi- dine by the procedure described earlier in similar pulse–chase experiments with TK2-proficient lung fibroblasts [7]. During the first 15 min of chase and in A B Fig. 2. In situ phosphorylation of [ 3 H]thymidine by control and TK2- mutated fibroblasts. Quiescent cultures of control (Ca and Cb) and TK2-mutated (Pa and Pb) fibroblasts were incubated for 0 min (open bars), 5 min (gray bars) and 20 min (black bars) with 25 n M [ 3 H]thymidine at a specific radioactivity of 20 000 c.p.m.Æpmol )1 .To confirm the involvement of TK2 in the in situ phosphorylation of thymidine, Ca and Pa lines were also incubated in the presence of 1 l M BVDU added 15 min before [ 3 H]thymidine, and the incubation was continued for 5 min (dotted bars) or 20 min (striped bars). (A) Size of the total cellular dTTP pool (pmol dTTP per 10 6 cells). (B) Specific radioactivity of dTTP in the cellular pool (c.p.m.Æ pmol )1 ). Data are means of determinations from five separate experiments. Bars indicate standard deviations. Thymidine metabolism in TK2-mutated fibroblasts M. Frangini et al. 1108 FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS the absence of added thymidine, dTTP was synthesized at a rate of 0.045 pmolÆ10 )6 cellsÆmin )1 in Ca cells and 0.054 pmolÆ10 )6 cellsÆmin )1 in Pa cells. When thymi- dine was present in the medium, the corresponding rates were 0.066 and 0.090 pmolÆmin )1 , respectively. The higher rates observed in the presence of thymidine reflect the salvage activity of TK2. These data confirm once more that the synthesis of dTTP in the wild-type and TK2-mutated fibroblasts occurs with virtually the same efficiency, although the in vitro determination of TK2 activity in cell extracts shows a 15-fold difference (Table 1). The assay in Table 1 was performed with 1 lm [ 3 H]thymidine. To better compare the in vivo rate of TK2 activity shown in Fig. 3 with that measured in vitro , we repeated the TK2 assay using [ 3 H]thymidine at 0.1 lm, i.e. the con- centration employed in the pulse–chase experiment of Fig. 3, and expressed the measured enzyme activity in pmolÆ10 )6 cells. We obtained values of 0.5 pmolÆ min )1 with Ca extracts and 0.02 pmolÆmin )1 with Pa extracts, the same difference between patient and control fibro- blasts that was observed with higher concentrations of substrate in Table 1. However, the in vitro value of TK2 activity in the control extract was 10-fold higher than the rate of dTTP synthesis measured during the chase in vivo, whereas this did not occur with the patient fibroblasts. This remarkable difference suggests that TK2-proficient fibroblasts contain an excess of TK2 enzyme whose potential is not fully employed under normal conditions in intact cells. Discussion Thymidine kinases catalyze the rate-limiting step of the salvage pathway that converts thymidine to dTTP. The cell contains two such enzymes, the S-phase-specific cytoplasmic TK1 and mitochondrial TK2. Both kinases are encoded by nuclear genes present in all cells of the body. However, the mtDNA depletion caused by mutations of the TK2 gene appears only in selected types of cells, especially in skeletal muscle [2]. The consequent mtDNA depletion syndrome is a severe disease, generally characterized by early onset and death in infancy. All the known cases of the syndrome are caused by mutations that impair but do not com- pletely abolish TK2 enzyme activity, suggesting that Fig. 3. Analysis of dTTP pool turnover in control and TK2-mutated fibroblasts by a pulse–chase experiment with [ 3 H]thymidine. Quiescent cultures of control (Ca, squares) and TK2-mutated (Pa, triangles) fibroblasts were incubated for 90 min with 100 n M [ 3 H]thymidine. After a 60 min pulse (black lines), the chase (dotted lines) was started by shifting part of the cultures to fresh med- ium without thymidine (open symbols) or with 100 n M nonradioactive thymidine (closed symbols) for 5, 15 and 30 min. (A) Size of the total cellular dTTP pool (pmol dTTP ⁄ 10 6 cells). (B) Specific radio- activity of the dTTP pool (c.p.m. Æ pmol )1 ). M. Frangini et al. Thymidine metabolism in TK2-mutated fibroblasts FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS 1109 the latter condition is incompatible with life in humans. In the mouse, complete TK2 knockout leads to death within 2–4 weeks after birth [9]. The same reduction of lifespan was observed in mice expressing a mutated but partially functional TK2 [10]. The fibroblasts analyzed in the present investigation were derived from patients affected by an unusual form of TK2 deficiency, characterized by late onset of the syndrome, relatively slow progression, and long survival [11–14]. Phenotypic changes during the devel- opment of the disease suggested the operation of some undefined molecular mechanism partially compensating for the metabolic defect. Skin fibroblasts are, as a rule, unaffected by gene mutations causing mtDNA depletions in other organs. They seemed to be a promising cellular system in which to investigate how cells can cope with a strongly reduced activity of TK2. The enzyme becomes relevant for the maintenance of the mitochondrial dTTP pool when cells have left the cell cycle and the synthesis of dNTPs in the cytosol is strongly decreased [7,15] due to proteasome-dependent degradation during mitosis of TK1 and R2, the S-phase-specific small subunit of ribonucleotide reductase. Fibroblasts can be main- tained in a quiescent state in culture for extended times [7,23], and have been used as in vitro models of TP or TK2 deficiency [7,15,23]. Only one report exists on the mitochondrial dNTP pools in TK2-mutated human fibroblasts [17]. The cells were from two patients homozygous for TK2 mutations different from those present in the fibroblasts examined here. In that case, both patient lines contained smaller mitochondrial dTTP and dCTP pools relative to the controls, and presented a moderate imbalance of the overall mitochondrial dNTP pool. No analysis of thymi- dine metabolism was performed in those cells. The two mutated fibroblast lines examined here carry two distinct heterozygote genotypes [12,13] that produce a TK2 with strongly reduced activity as deter- mined in whole cell extracts (Table 1). TK2 activity in extracts of the same patients’ fibroblasts has been reported previously [12,13]. However, enzyme activity was determined by an assay based on the competition between thymidine and deoxycytidine, in one case in whole cell extracts [13], and in the other [12] in mitochondrial extracts. Although it is not possible to compare directly these data with ours, the < 10% residual TK2 activity that we detected with thymidine as substrate in Pa extracts (Table 1) agrees with the 1% residual activity measured in mitochondrial extracts from the same cell line in [12], although the absolute values differed. In [13], the range of TK2 activity in the controls was identical to ours, but the activity in patient extracts (corresponding to our Pb extracts) was 0.5 pmolÆmg )1 proteinÆmin )1 , 10-fold lower than here (Table 1). The recombinant TK2 proteins encoded by the two mutated alleles of patient Pb were produced in bacteria and characterized by Wang et al. [13]. Both mutated forms of the enzyme showed increased K m and reduced V max values, resulting in a 98% reduction of activity as compared with the recombinant wild-type enzyme [13], which appeared to correlate directly with the development of disease in the patient. Our analysis of the in situ activity of the compound heterozygous mutant enzyme in the patient fibroblasts provides a different picture, with no difference between Pb and control fibroblasts in the phosphorylation of radioac- tive thymidine (Fig. 2). Despite their decreased TK2 enzymatic activity, in both TK2-mutated lines we did not detect any signifi- cant change in dNTP pool composition either in the cytosol or in the mitochondria. This normal pool pheno- type did not result from a metabolic adaptation of the enzymatic network that regulates the dTTP pools in qui- escent fibroblasts [15]. We did not find an increased dependence of the mutated cells on R1 ⁄ p53R2-depen- dent de novo synthesis, and nor did we observe downre- gulation of the catabolic enzymes directly involved in dTTP regulation, i.e. the cytosolic and mitochondrial deoxynucleotidases and TP (Table 3). When we com- pared the dynamics of the dTTP pool in mutated and control fibroblasts by incubating the cells with radio- active thymidine, in striking contrast to the low TK2 activity measured in cell extracts, the mutated cells phos- phorylated thymidine as efficiently as the wild-type cells. This was particularly unexpected for the Pa line, which apparently contained less than 10% of the control TK2 activity. We concentrated on this cell line, and com- pared, in a pulse–chase experiment, the turnover of the dTTP pool with that of the Ca control line, which expresses a similar level of TP (Table 3). This experi- ment revealed that the control cells in vivo synthesized dTTP with the same efficiency as the mutant cells, i.e. at a rate 10-fold lower than that observed in an in vitro assay for the phosphorylation of thymidine by TK2, which is the rate-limiting reaction in the conversion of thymidine to dTTP. Thus, in the control quiescent cells, most of the TK2 present is not active, possibly due to feedback inhibition by dTTP and dCTP [24]. The expression of TK2 increases when cultured fibro- blasts become quiescent [15], but the enzyme does not work at its full potential. The present data show that a small fraction of such potential TK2 activity is sufficient to maintain the dTTP pools of wild-type fibroblasts. In the mutants, however, the in vivo and Thymidine metabolism in TK2-mutated fibroblasts M. Frangini et al. 1110 FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS in vitro rates of TK2 activity coincide, and by fully exploiting their TK2 complement, the cells preserve their dTTP pools, which might explain the lack of mtDNA depletion in these cells. How the mutated enzymes escape from the inhibitory mechanism operat- ing on the wild-type enzyme remains to be established. We were unable to detect feedback inhibition of TK2 by dTTP and dCTP in assays with crude cell extracts. The TK2-mutated fibroblasts described by Saada et al. [17] had normal mtDNA content and cytochrome oxidase activity, and yet were reported to contain lower pyrimidine dNTP pools in mitochondria. This pheno- type contrasts with our present finding, and might be linked to the kind of TK2 mutations involved. We earlier silenced TK2 by small interfering RNA transfection of normal skin fibroblasts (the Cb line used here), obtaining a small (10%) reduction of the dTTP pool. When thymidine was added to the med- ium, however, the silenced cells were not able to sal- vage the nucleoside as efficiently as control cells [15]. The reduction of TK2 activity in the silenced cells in those experiments was comparable to that found here in the Pa cells, but it affected the dTTP pool size, in contrast to the present findings. We believe that this may depend on the multifactorial control of dNTP metabolism. A complex network of enzymes modulates the pools. Interindividual variations in specific compo- nents of the network between control and affected lines may mask the effects of single enzyme mutations. The differences in TP activities detected among the four fibroblast lines shown in Table 3 exemplify this point. How do our present data relate to the tissue-specific phenotype of TK2-associated mtDNA depletion? Skel- etal muscle is known to contain low levels of TK2 activity [25,26]. Indeed, when myoblasts differentiate into myotubes, there is no induction of TK2 expres- sion (C. Rampazzo, unpublished observations). Thus, it is possible that the physiological level of TK2 in muscle cells is very close to the minimum required for pool maintenance, and that when the enzyme is par- tially inactivated by gene mutations its activity may fall below the required threshold. Experimental procedures Cell lines and cell growth The TK2-mutated fibroblast lines were derived from two patients with different compound heterozygous geno- types, i.e. R152G ⁄ K171del in the case of Pa [12] and T77M ⁄ R161K in the case of Pb [13]. The cell lines were obtained by M. R. Vila ` from skin biopsy specimens taken from patients at ages 14 years (Pa) and 10 years (Pb). The age-matched control fibroblast lines were available in the Padova laboratory. Cells were grown in DMEM with 10% heat-inactivated fetal bovine serum and antibiotics. Conflu- ent, contact-inhibited cultures were shifted to fresh medium with 0.1% serum, and maintained in a quiescent state for 10 days before the experiments. Cells were periodically checked for mycoplasma contamination by a PCR-based method (Minerva Biolabs GmbH, Berlin, Germany). We determined cell numbers with a Coulter counter, and cell cycle distribution by flow cytometry. Enzymatic assays We prepared whole cell extracts as described previously [16], by adding protease inhibitor mixture to the lysis buffer. The supernatants were aliquoted and stored at )80 °C until use. We measured protein concentration by the colorimetric pro- cedure of Bradford [27], with BSA as standard. All enzyme assays were done with two different aliquots of extracts to check for proportionality. We assayed TK2 activity with 0.2 lm [ 3 H]BVDU (Moravek Biochemicals and Radiochemi- cals, Brea, CA, USA) as the substrate [16] in the presence of 50 lm 5-bromouracil (Sigma Aldrich, St Louis, MO, USA) to inhibit TP, or with 1 lm [ 3 H]thymidine (Perkin-Elmer Life Sciences, Waltham, MA, USA) in the absence or presence of 100 lm KIN109, a specific TK2 inhibitor [16]. We deter- mined TP activity according to Martı ´ et al. [28], with the modifications detailed in [15], and total deoxynucleotidase (cytoplasmic deoxynucleotidase + mitochondrial deoxynu- cleotidase) activity with 5 mm [ 3 H]dUMP as substrate, as described in [29]. We expressed TK2 activity as pmol prod- uctÆmin )1 Æmg )1 protein, and TP and deoxynucleotidase activities as nmolÆh )1 Æmg )1 protein. mRNA expression analysis by real-time RT-PCR To quantify mRNA expression of ribonucleotide reductase subunits and p53, we performed real-time RT-PCR using the Applied Biosystems 7500 Real Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). Total RNA was obtained from cycling and quiescent cultures of the four cell lines. Cells were treated with RNase-free DNase, and the RNA was quantified by spectrophotometry. Two micrograms of RNA were reversed transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems Inc.), following the manufacturer’s instructions. A 5 lL aliquot of cDNA diluted 1 : 50 was mixed with the TaqMan Universal PCR Master Mix and the following Gene Expres- sion Assays Taqman probes: p53 (TP53, Hs00153349), ribo- nucleotide reductase R1 subunit (RRM1, Hs00168784_m1), ribonucleotide reductase R2 subunit (RRM2, Hs Hs00357247_g1), and the p53-dependent subunit 2 of ribo- nucleotide reductase (RRM2B, Hs00153085_m1). The rela- tive quantity of mRNA was normalized using cyclophilin A (PPIA, Hs99999904_m1) as endogenous control. The PCR M. Frangini et al. Thymidine metabolism in TK2-mutated fibroblasts FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS 1111 cycle consisted of an initial step of 50 °C for 2 min, fol- lowed by 10 min at 95 °C, and 40 repetitions of two-step cycles of 50 °C for 15 s and 60 °C for 1 min. All assays were performed at least in triplicate. PCR data were pro- cessed by genamp 7500 SDS software. Immunoblotting The cell protein lysates were prepared and quantified as described in [23]. The conditions for gel electrophoresis and immunoblotting and the antibodies employed were as detailed in [15]. Isotope experiments and dNTP pool analyses The procedures for the isotope experiments and the separa- tion of cytosolic and mitochondrial dNTP pools were detailed previously [22]. Before starting the incubation with [ 3 H]thymidine (20 000 c.p.m.Æpmol )1 ), we substituted the medium with fresh medium with 0.1% dialyzed serum and left the cultures to equilibrate for 1–2 h. All manipulations were performed in a 37 °C room to avoid thermal shocks. Incubations with 25 nm [ 3 H]thymidine were performed for 5 and 20 min. In one protocol, 1 lm BVDU (Sigma) was added 15 min before the radioactive nucleoside. For the pulse–chase experiment with 100 nm [ 3 H]thymidine, we applied the protocol described in [7]. In all experiments, [ 3 H]thymidine incubations were ended by moving the cells on ice to a cold room, and total dNTP pools were extracted with ice-cold 60% methanol for 1 h from the cells still attached to the plates [18]. Cytosolic and mitochondrial dNTPs were separated by differential centrifugation of cell homogenates [22] and extracted with 60% methanol. The sizes of dNTP pools and the specific radioactivity of dTTP were determined with a DNA polymerase-based assay [22,30] with the modifications reported in [22], incubating two different aliquots of pool extracts for 1 h at 37 °Cina reaction mix containing 0.25 lm [ 32 P]dATP and 0.2 units of Klenow enzyme. Acknowledgements This work was supported by grants from Italian Telethon (Grant GGP05001), AIRC, the Italian Association for Cancer Research, and the Cariparo Foundation to V. Bianchi, and from the Spanish Insti- tuto de Salud Carlos III (PI 06 ⁄ 0735 and CP 04 ⁄ 0240 to R. Martı ´ and PI 04 ⁄ 0415 to M. R. Vila ` ). References 1 Copeland WC (2008) Inherited mitochondrial diseases of DNA replication. Annu Rev Med 59, 131–146. 2 Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S & Elpeleg O (2001) Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 29, 342–344. 3 Mandel H, Szargel R, Labay V, Elpeleg O, Saada A, Shalata A, Anbinder Y, Berkowitz D, Hartman C, Barak M et al. (2001) The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 29, 337–341. 4 Nishino I, Spinazzola A & Hirano M (1999) Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 283, 689–692. 5 Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, Aubert S, Chretien D, de Lonlay P, Paquis-Flucklinger V, Arakawa H et al. (2007) Mutation of RRM2B, encod- ing p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 39, 776–780. 6 Chabes AL, Pfleger CM, Kirschner MW & Thelander L (2003) Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis. Proc Natl Acad Sci USA 100, 3925–3929. 7 Pontarin G, Ferraro P, Valentino ML, Hirano M, Rei- chard P & Bianchi V (2006) Mitochondrial DNA deple- tion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy. J Biol Chem 281, 22720–22728. 8 Oskoui M, Davidzon G, Pascual J, Erazo R, Gurgel- Giannetti J, Krishna S, Bonilla E, De Vivo DC, Shan- ske S & DiMauro S (2006) Clinical spectrum of mito- chondrial DNA depletion due to mutations in the thymidine kinase 2 gene. Arch Neurol 63, 1122–1126. 9 Zhou X, Solaroli N, Bjerke M, Stewart JB, Rozell B, Johansson M & Karlsson A (2008) Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice. Hum Mol Genet 17, 2329–2335. 10 Akman HO, Dorado B, Lo ´ pez LC, Garcı ´ a-Cazorla A, Vila ` MR, Tanabe LM, Dauer WT, Bonilla E, Tanji K & Hirano M (2008) Thymidine kinase 2 (H126N) knoc- kin mice show the essential role of balanced deoxy- nucleotide pools for mitochondrial DNA maintenance. Hum Mol Genet 17, 2433–2440. 11 Barthe ´ le ´ my C, Ogier de Baulny H, Diaz J, Cheval MA, Frachon P, Romero N, Goutieres F, Fardeau M & Lombe ` s A (2001) Late-onset mitochondrial DNA deple- tion: DNA copy number, multiple deletions, and com- pensation. Ann Neurol 49 , 607–617. 12 Vila ` MR, Segovia-Silvestre T, Gamez J, Marina A, Naini AB, Meseguer A, Lombe ` s A, Bonilla E, Di Mauro S, Hir- ano M et al. (2003) Reversion of mtDNA depletion in a patient with TK2 deficiency. Neurology 60, 1203–1205. 13 Wang L, Limongelli A, Vila MR, Carrara F, Zeviani M & Eriksson S (2005) Molecular insights into mitochon- drial DNA depletion syndrome in two patients with Thymidine metabolism in TK2-mutated fibroblasts M. Frangini et al. 1112 FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS novel mutations in the doexyguanosine kinase and thy- midine kinase 2 genes. Mol Genet Metab 84, 75–82. 14 Vila ` MR, Villaroya J, Garcia-Arumi E, Castellote A, Meseguer A, Hirano M & Roig M (2008) Selective mus- cle fiber loss and molecular compensation in mitochon- drial myopathy due to TK2 deficiency. J Neurol Sci 267, 137–141. 15 Rampazzo C, Fabris S, Franzolin E, Crovatto K, Fran- gini M & Bianchi V (2007) Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells. J Biol Chem 282, 34758–34769. 16 Franzolin E, Rampazzo C, Perez-Perez MJ, Hernandez AI, Balzarini J & Bianchi V (2006) Bromovinyl-deoxy- uridine: a selective substrate for mitochondrial thymi- dine kinase in cell extracts. Biochem Biophys Res Commun 344, 30–36. 17 Saada A, Ben-Shalom E, Zyslin R, Miller C, Mandel H & Elpeleg O (2003) Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency. Biochem Biophys Res Commun 310, 963–966. 18 Ferraro P, Pontarin G, Crocco L, Fabris S, Reichard P & Bianchi V (2005) Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). J Biol Chem 280, 24472–24480. 19 Bianchi V, Pontis E & Reichard P (1987) Regulation of pyrimidine deoxyribonucleotide metabolism by sub- strate cycles in dCMP-deaminase deficient V79 hamster cells. Mol Cell Biol 7, 4218–4224. 20 Reichard P (1988) Interactions between deoxyribonucleo- tide and DNA synthesis. Annu Rev Biochem 57, 349–374. 21 Bianchi V & Spychala J (2003) Mammalian 5¢-nucleo- tidases. J Biol Chem 278, 46195–46198. 22 Pontarin G, Gallinaro L, Ferraro P, Reichard P & Bianchi V (2003) Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools. Proc Natl Acad Sci USA 100, 12159–12164. 23 Pontarin G, Ferraro P, Ha ˚ kansson P, Thelander L, Reichard P & Bianchi V (2007) p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage. J Biol Chem 282, 16820– 16828. 24 Wang L, Saada A & Eriksson S (2003) Kinetic proper- ties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopa- thy. J Biol Chem 278, 6963–6968. 25 Saada A, Shaag A & Elpeleg O (2003) mtDNA deple- tion myopathy: elucidation of the tissue specificity in the mitochondrial thymidine kinase (TK2) deficiency. Mol Genet Metab 79, 1–5. 26 Rylova SN, Mirzaee S, Albertioni F & Eriksson S (2007) Expression of deoxynucleoside kinases and 5¢-nucleotidases in mouse tissues: implications for mito- chondrial toxicity. Biochem Pharmacol 74, 169–175. 27 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254. 28 Martı ´ R, Spinazzola A, Tadesse S, Nishino I, Nishigaki Y & Hirano M (2004) Definitive diagnosis of mitochon- drial neurogastrointestinal encephalomyopathy by bio- chemical assays. Clin Chem 50, 120–124. 29 Rampazzo C, Mazzon C, Reichard P & Bianchi V (2002) 5 ¢-Nucleotidases: specific assays for five different enzymes in cell extracts. Biochem Biophys Res Commun 293, 258–263. 30 Sherman PA & Fyfe JA (1989) Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers. Anal Biochem 180, 222–226. M. Frangini et al. Thymidine metabolism in TK2-mutated fibroblasts FEBS Journal 276 (2009) 1104–1113 ª 2009 The Authors Journal compilation ª 2009 FEBS 1113 . Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts Miriam Frangini 1 , Chiara Rampazzo 1 , Elisa Franzolin 1 , Mari-Carmen. caus- ing an imbalance of the dNTP pools as compared to the controls [17]. We determined the amounts of all four dNTPs in the cytosolic and mitochondrial pools of the two patient lines and of two. quiescent and cycling cultures of control and TK2-mutated fibroblasts. We examined by immunoblotting the abundance of p53R2, R1, R2 and p53 in extracts of control (Ca, Cb) and TK2-mutated (Pa, Pb) fibroblasts