Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death.

Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.

Control of protein function through oxidation and reduction of persulfidated states.

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Mammalian deoxyribonucleoside kinases.

The mammalian deoxyribonucleoside kinases are deoxycytidine kinase, thymidine kinase 1 and 2 and deoxyguanosine kinase. These enzymes phosphorylate deoxyribonucleosides and thereby provide an alternative to de novo synthesis of DNA precursors. Their activities are essential for the activation of several chemotherapeutically important nucleoside analogues. In recent years, these enzymes have been thoroughly characterised with regard to structure, substrate specificity and patterns of expression. In this review, these results are reviewed and furthermore, the physiologic metabolic role of the anabolic enzymes is discussed in relation to catabolic pathways. The significance of this information for the development of therapeutic protocols and choice of animal model systems is discussed. Finally, alternative pathways for nucleoside analogue phosphorylation are surveyed, such as the phosphotransfer capacity of 5'-nucleotidase.

High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.

Mammalian thioredoxin reductase (TrxR) catalyzes reduction of thioredoxin and many other substrates, and is a central enzyme for cell proliferation and thiol redox control. The enzyme is a selenoprotein and can therefore, like all other mammalian selenoproteins, not be directly expressed in Escherichia coli, since selenocysteine-containing proteins are synthesized by a highly species-specific translation machinery. This machinery involves a secondary structure, SECIS element, in the selenoprotein-encoding mRNA, directing selenocysteine insertion at the position of an opal (UGA) codon, normally conferring termination of translation. It is species-specific structural features and positions in the selenoprotein mRNA of the SECIS elements that hitherto have hampered heterologous production of recombinant selenoproteins. We have discovered, however, that rat TrxR can be expressed in E. coli by fusing its open reading frame with the SECIS element of the bacterial selenoprotein formate dehydrogenase H. A variant of the SECIS element designed to encode the conserved carboxyterminal end of the enzyme (-Sec-Gly-COOH) and positioning parts of the SECIS element in the 3'-untranslated region was also functional. This finding revealed that the SECIS element in bacteria does not need to be translated for full function and it enabled expression of enzymatically active mammalian TrxR. The recombinant selenocysteine-containing TrxR was produced at dramatically higher levels than formate dehydrogenase O, the only endogenous selenoprotein expressed in E. coli under the conditions utilized, demonstrating a surprisingly high reserve capacity of the bacterial selenoprotein synthesis machinery under aerobic conditions. Co-expression with the selA, selB and selC genes (encoding selenocysteine synthase, SELB and tRNA(Sec), respectively) further increased the efficiency of the selenoprotein production and thereby also increased the specific activity of the recombinant TrxR to about 25 % of the native enzyme, with as much as 20 mg produced per liter of culture. These results show that with the strategy utilized here, the capacity of selenoprotein synthesis in E. coli is more than sufficient for making possible the use of the bacteria for production of recombinant selenoproteins.

The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.

Reactive oxygen species (ROS) are known mediators of intracellular signaling cascades. Excessive production of ROS may, however, lead to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis. A balance between oxidant and antioxidant intracellular systems is hence vital for cell function, regulation, and adaptation to diverse growth conditions. Thioredoxin reductase (TrxR) in conjunction with thioredoxin (Trx) is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles. In mammals, extracellular forms of Trx also have cytokine-like effects. Mammalian TrxR has a highly reactive active site selenocysteine residue resulting in a profound reductive capacity, reducing several substrates in addition to Trx. Due to the reactivity of TrxR, the enzyme is inhibited by many clinically used electrophilic compounds including nitrosoureas, aurothioglucose, platinum compounds, and retinoic acid derivatives. The properties of TrxR in combination with the functions of Trx position this system at the core of cellular thiol redox control and antioxidant defense. In this review, we focus on the reactions of the Trx system with ROS molecules and different cellular antioxidant enzymes. We summarize the TrxR-catalyzed regeneration of several antioxidant compounds, including ascorbic acid (vitamin C), selenium-containing substances, lipoic acid, and ubiquinone (Q10). We also discuss the general cellular effects of TrxR inhibition. Dinitrohalobenzenes constitute a unique class of immunostimulatory TrxR inhibitors and we consider the immunomodulatory effects of dinitrohalobenzene compounds in view of their reactions with the Trx system.

Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex.

Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.

Thioredoxin reductase 1 is upregulated in atherosclerotic plaques: specific induction of the promoter in human macrophages by oxidized low-density lipoproteins.

Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p <.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.

Expression of deoxycytidine kinase and phosphorylation of 2-chlorodeoxyadenosine in human normal and tumour cells and tissues.

Deoxycytidine kinase (dCK) activates several clinically important drugs, including the recently developed antileukaemic compound 2-chlorodeoxyadenosine (CdA). The distribution of dCK in cells and tissues has previously been determined by activity measurements, which may be unreliable because of the presence of other enzymes with overlapping substrate specificities. Therefore we have measured dCK polypeptide levels in extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas, using a specific immunoblotting technique, as well as the phosphorylation of CdA in the same extracts. High levels of dCK were found in all major subpopulations of normal mononuclear leucocytes (120 +/- 19 ng dCK/mg protein) and in B-cell chronic lymphocytic leukaemia (81 +/- 30 ng/mg, n = 23). Hairy-cell leukaemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did three samples of T-cell chronic lymphocytic leukaemia (18 +/- 14 ng/mg). Phytohaemagglutinin stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or protein expression (less than 2.5-fold). The human CEM wt T-lymphoblastoid cell line contained 56 +/- 1 ng/dCK/mg protein, while in the CEM ddC50 and AraC8D mutants that lack dCK activity, no dCK polypeptide could be detected. In colon adenocarcinomas, the dCK content was significantly higher (20 +/- 9 ng/mg, n = 20) than in normal colon mucosa (8 +/- 3.5 ng/mg, n = 19, P < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal stomach mucosa (6 +/- 5 ng/mg, n = 5, P < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed dCK levels comparable with those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg, respectively), while other sarcoma samples contained lower levels, comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). Thus, dCK is expressed constitutively and predominantly in lymphoid cells, but it is also found in solid non-lymphoid tissues, with increased levels in malignant cells. The phosphorylation of CdA in crude extracts showed a close correlation to the dCK polypeptide level.

On the phosphorylation of 2-chlorodeoxyadenosine (CdA) and its correlation with clinical response in leukemia treatment.

The nucleoside analog 2-chlorodeoxyadenosine (CdA, Cladribine) is a chemotherapeutic agent for treatment of leukemias and lymphomas, most successfully used in hairy cell leukemia and B-cell chronic lymphocytic leukemia. CdA is phosphorylated intracellularly to its monophosphate derivative by the enzymes deoxycytidine kinase and deoxyguanosine kinase. Cell lines deficient in deoxycytidine kinase were shown to be resistant to CdA and a high deoxycytidine kinase level in combination with low 5'-nucleotidase has been proposed to partly explain the selectivity in CdA toxicity for lymphoid cells. In this report biochemical properties in CdA phosphorylation mediated by deoxycytidine kinase and deoxyguanosine kinase are reviewed and discussed in relation to the further metabolism of CdA 5'-monophosphate, the different possible mechanisms of action and the correlation with clinical response. It is concluded that much is known about the metabolism and mechanisms of action of CdA, but that the remarkable therapeutic effect in hairy cell leukemia has yet to be explicitly explained.

Superoxide production by dinitrophenyl-derivatized thioredoxin reductase--a model for the mechanism and correlation to immunostimulation by dinitrohalobenzenes.

Mammalian thioredoxin reductase catalyzes NADPH dependent reduction of a wide variety of substrates and plays a central role in redox regulation and antioxidant defence. Recently the enzyme was discovered to be a selenoprotein with a catalytically active penultimate selenocysteine residue. Dinitrohalobenzenes irreversibly inhibit the enzyme with a concomitant induction of an NADPH oxidase activity, producing superoxide. A model explaining the reactivity of dinitrohalobenzenes with thioredoxin reductase is presented, involving dinitrophenyl-derivatization of both the selenocysteine residue and its neighboring cysteine residue, reduction by NADPH of the enzyme-bound flavin in dinitrophenyl-alkylated enzyme (dnp-TrxR), followed by two consecutive one-electron transfers from the flavin to nitro groups of the dnp-moieties in dnp-TrxR, forming nitro anion radicals. The nitro radicals react with oxygen to form superoxide, again generating dnp-TrxR with an oxidized flavin, which may then follow another cycle of NADPH-dependent superoxide production. Dinitrohalobenzene compounds are well known for their immunostimulatory properties. Here it is proposed that the inflammatory components of this immunostimulation can be mediated by interaction with the thioredoxin system, via effects on cell function by superoxide production, oxidative stress and increased extracellular levels of thioredoxin.

Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H2O2.

It is commonly recognized that diabetic complications involve increased oxidative stress directly triggered by hyperglycemia. The most important cellular protective systems against such oxidative stress have yet remained unclear. Here we show that the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the Txnrd1 gene, is an essential enzyme for such protection. Individually grown Txnrd1 knockout (Txnrd1(-/-)) mouse embryonic fibroblasts (MEFs) underwent massive cell death directly linked to glucose-induced H2O2 production. This death and excessive H2O2 levels could be reverted by reconstituted expression of selenocysteine (Sec)-containing TrxR1, but not by expression of Sec-devoid variants of the enzyme. Our results show that Sec-containing TrxR1 is absolutely required for self-sufficient growth of MEFs under high-glucose conditions, owing to an essential importance of this enzyme for elimination of glucose-derived H2O2. To our knowledge, this is the first time a strict Sec-dependent function of TrxR1 has been identified as being essential for mammalian cells.

ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis.

Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53.

APR-246/PRIMA-1MET inhibits thioredoxin reductase 1 and converts the enzyme to a dedicated NADPH oxidase.

The low-molecular-weight compound APR-246 (PRIMA-1(MET)) restores wild-type conformation and function to mutant p53, and triggers apoptosis in tumor cells. We show here that APR-246 also targets the selenoprotein thioredoxin reductase 1 (TrxR1), a key regulator of cellular redox balance. APR-246 inhibited both recombinant TrxR1 in vitro and TrxR1 in cells. A Sec-to-Cys mutant of TrxR1 was not inhibited by APR-246, suggesting targeting of the selenocysteine residue in wild-type TrxR1. Preheated APR-246 and its conversion product methylene quinuclidinone (MQ) were much more efficient TrxR1 inhibitors than APR-246 itself, indicating that MQ is the active compound responsible for TrxR1 enzyme inhibition. TrxR1 inhibited by MQ was still functional as a pro-oxidant NADPH oxidase. Knockdown of TrxR1 caused a partial and reproducible attenuation of APR-246-induced tumor cell death independently of p53 status. Cellular TrxR1 activity was also inhibited by APR-246 irrespective of p53 status. We show that APR-246 can directly affect cellular redox status via targeting of TrxR1. Our findings provide an explanation for the previously observed effects of APR-246 on tumor cells lacking mutant p53.

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney.

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.

Physiological functions of thioredoxin and thioredoxin reductase.

Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases. Glutaredoxins catalyze glutathione-disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase. Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system. Plants have chloroplast thioredoxins, which via ferredoxin-thioredoxin reductase regulates photosynthetic enzymes by light. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF-kappaB or the Ref-1-dependent AP1 require thioredoxin reduction for DNA binding. The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co-cytokine and chemokine activities. Thioredoxin reductase is a specific dimeric 70-kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol. In contrast, thioredoxin reductases of higher eukaryotes are larger (112-130 kDa), selenium-dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite. All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C-terminal elongation with a cysteine-selenocysteine sequence forming a redox-active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.

Catabolism of deoxycytidine in human peripheral blood mononuclear cells and its interference with the determination of in situ thymidylate synthase activity.

Resting and stimulated human peripheral blood lymphocytes and monocyte-derived macrophages were incubated with tritiated deoxycytidine labeled at the 5-position. Release of tritiated water into the medium was thereupon detected utilizing its lack of binding to active charcoal, which is an established technique to measure in situ thymidylate synthase activity. It was found that tritiated dihydrouracil, a deoxycytidine catabolite, was formed during incubation with tritiated deoxycytidine. Like water, dihydrouracil does not bind to active charcoal, and its presence in the cell medium can result in an overestimation of the in situ thymidylate synthase activity. The catabolism of dCyd was highest in macrophages where 25% of the added dCyd (0.5 microM, 0.5 nmol/million cells) had been converted to dihydrouracil after 30 min, and 90% after 12 h. The in situ thymidylate synthase activity was found to be the highest in stimulated lymphocytes. If the interference of dihydrouracil had not been considered, the activity in macrophages would have been greatly overestimated and would have appeared to be higher than that of stimulated lymphocytes.

Deoxycytidine and 2',3'-dideoxycytidine metabolism in human monocyte-derived macrophages. A study of both anabolic and catabolic pathways.

In the present study we have explored the catabolism of dCyd and 2',3'-dideoxycytidine (ddC) in mature monocyte-derived macrophages (M/M) and focused on its relation to the accumulation of intracellular anabolites. We found that dCyd catabolism in M/M was significant in 1-week old cultures (15% of 0.5 microM dCyd, 0.5 nmole/million cells, catabolized within one hour of incubation) and further increased in more mature 3-week and 5-week old cultures (> 85% catabolized within one hour). Dihydrouracil (DHU) was irreversibly formed as end product of the dCyd catabolism, with dUrd and Ura as intermediate metabolites. Intracellularly, dCyd nucleotides were detected up to three hours of incubation, with dCTP maintained at steady levels between one and three hours. However, after twelve hours of incubation DHU was the only detectable intracellular as well as extracellular metabolite. In contrast to what was observed with dCyd, we found that ddC was resistant to catabolism, with no detectable catabolites formed within twenty-four hours of incubation. Therefore ddC anabolism was unopposed by catabolic pathways and consequently intracellular anabolites of ddC accumulated throughout twenty-four hours of incubation. Based on these results, we propose that lack (or inhibition) of catabolism is at least as important as efficient anabolic phosphorylation for obtaining therapeutic effects of pyrimidine nucleoside analogs.