dTTP Pool Research Articles

Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.

Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BackgroundThe greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy.Methodology/Principal FindingsTo generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation.Conclusions/SignificanceThe resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance.

Effect of the Thymidylate Synthase Inhibitors on dUTP and TTP Pool Levels and the Activities of DNA Repair Glycosylases on Uracil and 5-Fluorouracil in DNA

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide 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 adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases 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 fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal 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.

Academic Life: The Whole Package

When I received the unexpected but welcome invitation to write a Reflections article, I wondered which aspects of my life in science are sufficiently distinctive to make the article worth reading. One aspect may be my survival as a department chair for nearly 25 years while struggling to contribute as well in teaching and research, in other words, embracing the “whole package” of academic life: teaching, research, and service. Another part may be my experience as coauthor of a biochemistry textbook. To deal with both, I will begin with my introduction to biochemistry.

Novel resveratrol analogs induce apoptosis and cause cell cycle arrest in HT29 human colon cancer cells: Inhibition of ribonucleotide reductase activity

Resveratrol (3,4',5-trihydroxy-trans-stilbene; RV), an ingredient of wine, exhibits a broad spectrum of antiproliferative effects against human cancer cells. In order to enhance these effects, we modified the molecule by introducing additional methoxyl and hydroxyl groups. The resulting novel RV analogs, M5 (3,4',5-trimethoxy-trans-stilbene), M5A (3,3',4,5'-tetramethoxy-trans-stilbene) and M8 (3,3',4,4',5,5'-hexahydroxy-trans-stilbene) were investigated in HT29 human colon cancer cells. Cytotoxicity was evaluated by clonogenic assays and the induction of apoptosis was determined using a specific Hoechst/propidium iodide double staining method. Cell cycle distribution was evaluated by FACS. The influence of M8 on the concentration of deoxyribonucleoside triphosphates (dNTPs), the products of ribonucleotide reductase (RR), was determined by high-performance liquid chromatography. M5 and M5A caused a dose-dependent induction of apoptosis and led to remarkable changes of the cell cycle distribution. After treatment with M5, growth arrest occurred mainly in the G2-M phase, whereas incubation with M5A resulted in arrest in the G0-G1 phase of the cell cycle. Incubation of HT29 cells with M8 produced a significant imbalance of intracellular dNTP pools, being synonymous with the inhibition of RR activity. The dATP pools were abolished, whereas the dCTP and dTTP pools increased. Due to these promising results, the investigated RV analogs deserve further preclinical and in vivo testing.

Synthetic Lethality by Lentiviral Short Hairpin RNA Silencing of Thymidylate Kinase and Doxorubicin in Colon Cancer Cells Regardless of the p53 Status

Intracellular supply of dTTP is a highly regulated process and has been a key target for chemotherapeutic drug development. Thymidylate kinase (TMPK) is the key enzyme for dTTP formation in both de novo and salvage pathways. In this study, we used lentiviral-based small hairpin RNA to silence TMPK expression in p53(+/+) and p53(-/-) HCT-116 colon cancer cells. This approach was sufficient to decrease the dTTP pool gradually without affecting p53 expression and generating cytotoxicity. TMPK knockdown significantly increased doxorubicin sensitivity dramatically in p53-proficient, p53-null HCT-116, and LoVo colon cancer cells. The decrease in the dTTP pool using this approach augmented the DNA damage response and enhanced apoptotic induction after exposure to low-dose doxorubicin, leading to cell death. In contrast, silencing of thymidylate synthase which blocks the de novo pathway was incapable of sensitizing p53-null HCT-116 cells to doxorubicin-induced apoptosis because of the compensation by the salvage pathway. Our results suggest the lentiviral delivery of small hairpin RNA targeting TMPK in combination with a low dose of doxorubicin as a new approach to kill colon cancer cells regardless of p53 status.

Mitochondrial Thymidine Kinase and the Enzymatic Network Regulating Thymidine Triphosphate Pools in Cultured Human Cells

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.

N-hydroxy-N'-(3,4,5-trimethoxyphenyl)-3,4,5-trimethoxy-benzamidine, a novel resveratrol analog, inhibits ribonucleotide reductase in HL-60 human promyelocytic leukemia cells: Synergistic antitumor activity with arabinofuranosylcytosine

Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, exhibits a broad spectrum of antiproliferative effects against human cancer cells. In order to develop a derivative with comparable effects, we modified the molecule by introducing additional methoxyl groups. The resulting novel RV analog, N-hydroxy-N'-(3,4,5-trimethoxyphenyl)-3,4,5-trimethoxybenzamidine (KITC), was investigated in HL-60 human promyelocytic leukemia cells. The induction of apoptosis was determined employing a specific Hoechst/propidium iodide double staining method and cell cycle distribution was evaluated by FACS. KITC's influence on the concentration of deoxyribonucleoside triphosphates, the products of ribonucleotide reductase (RR), was determined using the HPLC method. In addition, we analyzed the effects of KITC treatment on the incorporation of 14C-cytidine into the DNA of tumor cells in order to quantify the loss of RR in situ activity. To reveal a potential value of KITC for supporting conventional chemotherapy, we also examined whether a combination of KITC with arabinofuranosylcytosine (Ara-C) could yield synergistic growth inhibitory effects. KITC caused a dose-dependent induction of apoptosis, whereas no remarkable changes of the cell cycle distribution were observed. Incubation with KITC resulted in a significant depletion of intracellular dTTP and dATP pools and was also found to remarkably reduce the in situ activity of RR, the key enzyme of de novo DNA synthesis. In addition, KITC exhibited synergistic combination effects when applied sequentially with Ara-C. Due to these promising results, KITC deserves further preclinical and in vivo testing.

Mitochondrial Expression of theDrosophila melanogasterMultisubstrate Deoxyribonucleoside Kinase

The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) is studied as a candidate suicide gene for applications in combined gene/chemotherapy of cancer. We have created an engineered Dm-dNK nucleoside kinase that is targeted to the mitochondrial matrix. The enzyme was expressed in a thymidine kinase 1-deficient osteosarcoma cell line, and the sensitivity of the cells to cytotoxic nucleoside analogs was determined when the enzyme was targeted to either the nucleus or the mitochondrial matrix. Although the total deoxythymidine (dThd) phosphorylation activity was similar in cells expressing Dm-dNK in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria showed higher sensitivity to the antiproliferative activity of several pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine. Labeling studies using [3H]dThd showed that the cells expressing the mitochondrial enzyme had an increased incorporation of [3H]dThd into DNA, shown to be due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which Dm-dNK was targeted to the mitochondria. The difference in the specific activity of the dTTP pool is a result of different contributions of the de novo and the salvage pathways for the dTTP synthesis in transduced cells. In summary, these findings suggest that mitochondrial targeting of Dm-dNK facilitates nucleoside and nucleoside analog phosphorylation and could be used as a strategy to enhance the efficacy of nucleoside analog phosphorylation and concomitantly their cytostatic potential.

Evidence for distinct DNA‐ and RNA‐based mechanisms of 5‐fluorouracil cytotoxicity in Saccharomyces cerevisiae

5-Fluorouracil (5FU) is an effective chemotherapeutic drug developed as an inhibitor of thymidylate synthetase (TS). Inhibition of TS leads to 'thymine-less death', a condition resulting from depletion of dTTP pools and misincorporation of dUTP into newly synthesized or repaired DNA. 5FU is also incorporated into RNA and a growing body of evidence suggests that RNA-based effects play a significant role in its cytotoxicity. Indeed, recent experiments in yeast showed that defects in the nuclear RNA exosome subunit Rrp6p cause hypersensitivity to 5FU. The present study asked whether the 5FU hypersensitivity of an rrp6-Delta yeast strain reflects the DNA- or RNA-based effects of 5FU. Genetic analyses suggest that while a DNA repair mutation, apn1-Delta, causes sensitivity to 5FU-induced DNA damage, an rrp6-Delta mutation causes hypersensitivity, due to the RNA-based effects of 5FU. Analysis of a strain with normal DNA and RNA metabolism grown in the presence of 5FU shows that UMP suppresses the 5FU-induced defect more than dTMP, suggesting that the RNA-based toxicity of 5FU predominates in these cells. These findings underscore the importance of understanding the RNA-based mechanism of 5FU cytotoxicity and highlight the use of yeast as a model system for elucidating its details.

Mitochondria as determinant of nucleotide pools and chromosomal stability

Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochondrial DNA as models and analyzed the outcome of mitochondrial dysfunction on major cellular repair activities. We show that the deoxyribonucleoside triphosphate (dNTP) pools are affected, most prominently we detect a 3-fold reduction of the dTTP pool when normalized to the number of cells in S-phase. It is known that imbalanced dNTP pools are mutagenic and in accordance, we show that mitochondrial dysfunction results in chromosomal instability, which can explain its role in tumor development. We did not find any straightforward correlation between ATP levels and dNTP pools in cells with defective mitochondrial activity. Our results suggest that mitochondria are central players in maintaining genomic stability and in controlling essential nuclear processes such as upholding a balanced supply of nucleotides.

Mitotic control of dTTP pool: a necessity or coincidence?

The fidelity of DNA replication in eukaryotic cells requires a balanced dNTP supply in the S phase. During the cell cycle progression, the production of dTTP is highly regulated to coordinate with DNA replication. Intracellular thymidine is salvaged to dTTP by cytosolic thymidine kinase (TK1) and thymidylate kinase (TMPK), both of which expression increase in the G1/S transition and diminish in the mitotic phase via proteolytic destruction. Anaphase promoting complex/cyclosome (APC/C)-mediated ubiquitination targets TK1 and TMPK to undergo proteasomal degradation in mitosis, by which dTTP pool is minimized in the early G1 phase of the next cell cycle. In this review, we will focus on regulation of TK1 in the post-S phase and the importance of mitotic proteolysis in controlling dNTP balance, replication stress and genomic stability. Finally, we discuss how thymidine pool and oligomeric forms of TK1 can affect mitotic control of dTTP.

Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate

Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.

Cytotoxic and biochemical effects of 3,3′,4,4′,5,5′-hexahydroxystilbene, a novel resveratrol analog in HL-60 human promyelocytic leukemia cells

Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, is an inhibitor of the proliferation-linked enzyme ribonucleotide reductase (RR) and shows a broad spectrum of cytotoxic effects against human cancer cells. In order to enhance these effects, we introduced additional hydroxyl moieties into the molecule. In the present study, the activity of a novel RV analog, 3,3',4,4',5,5'-hexahydroxystilbene (M8), was investigated in HL-60 human promyelocytic leukemia cells. Cytotoxicity of M8 alone or in combination with Ara-C was assessed employing growth inhibition assays. Effects of M8 on nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) were examined by HPLC. The apoptotic potential of M8 and RV was compared using a specific double-staining method and inhibition of TNF-alpha-induced activation of NF-kappaB was studied. Cell-cycle distribution was analyzed by FACS. Addition of ascorbic acid decreased the IC(50) value of M8 from 6.25 microM to 2 microM. M8 depleted dATP and dTTP pools to 41% and 21% of control values, whereas dCTP pools increased to 199% of untreated controls. In addition, TTP, ATP, CTP, and GTP concentrations were decreased while UTP concentrations increased. M8 induced apoptosis at concentrations significantly lower than RV and could remarkably inhibit the activation of NF-kappaB. M8 arrested cells in the S phase of the cell cycle while depleting cells in the G2-M phase and exhibited synergistic combination effects when applied simultaneously with Ara-C. Due to these promising results, this novel polyhydroxylated stilbene derivative might become an additional option for the treatment of leukemia and therefore deserves further preclinical and in vivo testing.

Mitochondrial DNA Depletion and Thymidine Phosphate Pool Dynamics in a Cellular Model of Mitochondrial Neurogastrointestinal Encephalomyopathy

Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to approximately 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine.

Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Deltatrx1 Deltatrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Deltatrx1 Deltatrx2 yeast. In contrast to wild-type cells, Deltatrx1 Deltatrx2 cells were unable to accumulate or maintain high levels of dNTPs when alpha-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Deltatrx1 Deltatrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.

Induction of thymidine phosphorylase expression by AZT contributes to enhancement of 5′-DFUR cytotoxicity

Thymidine phosphorylase (TP) regulates intracellular thymidine metabolism and can enhance the anti-tumor effectiveness of 5′-deoxy-5-fluorouridine (5′-DFUR) by conversion of the pro-drug 5′-DFUR to 5-fluorouracil (5-FU) in tumor tissues. 5′-DFUR is an effective anti-tumor drug in cells expressing high levels of TP. 3′-Azido 3′-deoxythymidine (AZT) is a thymidine analog that has been proven useful in the treatment of acquired immunodefiency syndrome (AIDS). In this study, we found that AZT induces TP expression and enhances the sensitivity of human myeloid leukemia U937 cells to 5′-DFUR. Both the protein level and the activity of TP in U937 cells were elevated for 48 h after exposure to AZT (20, 100 or 300 μM). AZT enhanced TP promoter activity in a dose-dependent manner. AZT also increased TP mRNA levels in U937 cells as assayed by Real-time reverse-transcription PCR. AZT enhanced the cytotoxic effect of 5′-DFUR on U937 cells. A TP inhibitor, TPI, abrogated the cytotoxic activity of 5′-DFUR, and attenuated the combined cytotoxicity of AZT and 5′-DFUR. These results suggest that AZT enhances the cytotoxic effect of 5′-DFUR on U937 cells by upregulating TP activity in addition to its inhibition of thymidine kinase (TK) activity and reduction of intracellular dTTP pools.

Effect of Cell Cycle Arrest on the Activity of Nucleoside Analogues against Human Immunodeficiency Virus Type 1

Human immunodeficiency virus (HIV) reverse transcription can be notably affected by cellular activation, differentiation, and division. We hypothesized that changes in the cell cycle could also affect HIV susceptibility to nucleoside analogues, which compete with natural nucleotides for incorporation into viral DNA and inhibit viral replication through premature termination of reverse transcription. Proliferating HeLa-derived indicator cells were arrested in the S/G2 phase with etoposide, a topoisomerase II inhibitor, or in the G1/S phase with aphidicolin, a polymerase alpha inhibitor. Cell cycle arrest by both agents induced a remarkable decrease in HIV susceptibility to zidovudine (AZT). This decrease was seen both with a single-cycle infectivity assay and with a viral DNA quantitation assay, indicating that the effect of cell cycle arrest was exerted at the reverse transcription stage. The increase in the 50% inhibitory concentration (IC50) seen with arrested cells was strongest for AZT (23-fold) and stavudine (21-fold) but more modest for other drugs (lamivudine, 11-fold; dideoxyinosine, 7-fold; and nevirapine, 3-fold). In drug-resistant reverse transcriptase mutants, the increase in AZT IC50 (relative to that in dividing cells) was most prominent with a Q151M mutant and was comparable to the wild type in other drug-resistant mutants. Quantitation of intracellular pools of dTTP and AZT 5'-triphosphate (AZTTP) showed that etoposide treatment induced a significant increase in intracellular dTTP and consequently a decrease in AZTTP/dTTP ratios, suggesting that the decrease in viral susceptibility to AZT was caused by reduced incorporation of the analogue into nascent viral DNA. These results emphasize the importance of cellular proliferation and deoxynucleoside triphosphate metabolism in HIV susceptibility to nucleoside analogues and underscore the need to study the activities of drugs of this class with natural target cells under physiological conditions of activation and proliferation.

Control of dTTP pool size by anaphase promoting complex/cyclosome is essential for the maintenance of genetic stability

Anaphase promoting complex/cyclosome (APC/C)-mediated proteolysis is essential for chromosome segregation, mitotic exit, and G1 entry. Here, we show the importance of APC/C in the control of dTTP pool size in mammalian cells. Two enzymes, thymidine kinase 1 (TK1) and thymidylate kinase (TMPK), involved in dTTP formation are the targets of the APC/C pathway. We demonstrate that TMPK is recognized and degraded by APC/C-Cdc20/Cdh1-mediated pathways from mitosis to the early G1 phase, whereas TK1 is targeted for degradation by APC/C-Cdh1 after mitotic exit. Overexpression of wild-type TK1 and TMPK induces a four- to fivefold increase in the cellular dTTP pool without promoting spontaneous mutations in the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene. In contrast, coexpression of nondegradable TK1 and TMPK expands the dTTP pool size 10-fold accompanied by a drastic dNTP pool imbalance. Most interestingly, disruption of APC/C proteolysis of TK1 and TMPK leads to growth retardation and a striking increase in gene mutation rate. We conclude that down-regulation of dTTP pool size by the APC/C pathway during mitosis and the G1 phase is an essential means to maintain a balanced dNTP pool and to avoid genetic instability.