Deoxycytidine transport and pyrimidine deoxynucleotide metabolism in phytohaemagglutinin-stimulated pig lymphocytes.

Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.     

Regulation of uridine kinase activity in BALB/C-3T3 cells by serum components

After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels or uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce and induction of thymidine kinase activity in late G1.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

Differences in the induction of thymidine kinase isozymes in estrogen-treated immature and adult rats

Thymidine kinase activity in immature and castrated adult rat uterus has been examined in respose to estrogen treatment. Following estrogen administration. it was found that immature uterine thymidine kinase activity was increased 30-fold after 24 h, but almost no effect was produced on castrated or non-castrated adult uterus. Uterine thymidine kinase activity was separated into three peaks (peak 1, 2 and 3) by means of DEAE-cellulose column chromatography. In response to estrogen, the thymidine kinase isozymes differed in adult and immature uteri. In immature uteri, marked and selective increase of the activity was found in peak I, whereas in adult only a slight increase in peak 2 activity was observed. The thymidine kinase activity in peak 1 and peak 2 were found to have different enzymatic properties and molecular weight, as determined by gel filtration of 125 000 for peak 1 and 100 000 for peak 2.From these results, it is suggested that estrogen induces specific thymidine kinase isozyme in immature uterus and that the isozyme may be involved in DNA synthesis. Such a induction mechanism seems to be lost during the development.     

Regulation of uridine kinase activity in BLAB/C-3T3 cells by serum components

Summary After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels of uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce an induction of thymidine kinase activity in late G₁. This work was supported by NCI Grants CA24913 and CA16084. W. W. was supported by NIH Postdoctoral Fellowship AM 1477. W. J. P. was supported by JFRA32 from the American Cancer Society. A preliminary report of this research was given at the Eighth International Cell Cycle Conference held at Research Triangle Park, NC, May 15–16, 1980.     

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.     

A biochemical investigation of the adenovirus-induced G1 to S phase progression: Thymidine kinase,ornithine decarboxylase,and inhibitors of polyamine biosynthesis

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was respnsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5- infected cells, insensitive to 0.01 μ/ml actinomycin D and relatively resistant to reduced Ca²⁺ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5. α-Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5-induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1-arrested cells by a mechanism different from serum, and by passes events in the normal G1 to S phase progression.     

The role of zinc ions in the transformation of lymphocytes by phytohaemagglutinin

1. Incorporation of [(3)H]thymidine into DNA was inhibited by EDTA and diethylenetriamine-NNN'N'N'-penta-acetate but not by nitrilotriacetate even when Ca(2+) was present at more than twice the concentration of the chelators. 2. The inhibition caused by EDTA was most effectively reversed by Zn(2+) but also to a lesser extent by Cd(2+). Very little if any activation of the EDTA-inhibited system was obtained with Co(2+), Cu(2+), Fe(3+), Mn(2+) or Ni(2+) added alone. 3. Fe(3+) added to the Zn(2+)-activated lymphocytes in the presence of EDTA markedly increased thymidine incorporation. Addition of Cd(2+) prevented the inhibition of incorporation which occurred at high Zn(2+) concentrations. 4. If EDTA was added more than 15h after phytohaemagglutinin, the inhibition of incorporation was less than that obtained by its addition at zero time. If Zn(2+) was added later than 12h after EDTA and phytohaemagglutinin, the inhibition of incorporation by EDTA was not fully reversed. A study of the time-course of the effects of delayed additions of EDTA and Zn(2+) suggested that, on average, the cells required Zn(2+) between 20 and 30h after phytohaemagglutinin addition to allow the full rate of thymidine incorporation at 37h. 5. The increase in the rate of protein synthesis caused by phytohaemagglutinin was not inhibited by EDTA until about 8h. The further increase after this was totally inhibited by EDTA but this inhibition was fully reversible with Zn(2+). The rate of protein synthesis in EDTA-inhibited cultures at 40h was the same as that at 10h. 6. There was a close similarity between the effects of EDTA on lymphocyte stimulation and those reported by Kay et al. (1969) with low doses of actinomycin D.     

Biochemical paramters of BHT-induced cell growth in mouse lung.

Male mice of 7 different strains were injected i.p. with 400 mg/kg of butylated hydroxytoluene (BHT). 2 and 4 days later, the incorporation of thymidine into pulmonary DNA was significantly increased in all treated animals and this was accompanied by an increase in lung weight and pulmonary DNA. Thymidine kinase activity and DNA polymerase activity were enhanced in the lungs of BHT-treated animals and maximum activity of these enzymes appeared to precede maximum thymidine incorporation by 24 h. 3 days after BHT a good correlation was found between administered dose and thymidine kinase activity. Measuring the activity of this enzyme might serve as a convenient biochemical marker to follow and to quantitate BHT-produced cell proliferation in lung. The concentrations of cyclic AMP and the activity of adenylate cyclase were not altered by BHT on days 1-9 after administration. BHT produced also some dose-dependent, time-dependent increases in the activities of pulmonary 5'-nucleotidase and glucose-6-phosphate dehydrogenase (G6PDH), but had little effect on isocitric dehydrogenase (ICDH), pyruvate kinase (PK) and lactic dehydrogenase (LDH).     

Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) at concentrations of 0.01-0.1mum. The pattern of incorporation of label from [5-(3)H]uridine and [6-(3)H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1mum) prevented the stimulation of [5-(3)H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular P(i) pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate (32)P from [(32)P]P(i). 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3':5'-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.     

Uptake of orotate and thymidine by normal and regenerating rat livers

1. At 1h after operation livers from partially hepatectomized rats showed a 60-100% increase in the capacity to concentrate (3)H radioactivity from orotate, thymidine or uridine with respect to the radioactivity in plasma. Uptake of [(3)H]cytidine into liver was unaffected, as was entry of any precursor studied into any tissue other than liver. 2. This increase in intracellular radioactivity was detectable 10min after operation with both orotate and thymidine. With orotate the augmentation had disappeared by 3 days, but with thymidine it was still evident 8 days after partial hepatectomy, when [(3)H]thymidine incorporation into DNA was no longer increased. Competition studies established that orotate was not entering the liver by the same mechanism as thymidine. 3. In the soluble fraction of the liver all the (3)H radioactivity from orotate was present as uridine nucleotides. Thymidine was not phosphorylated, and was believed to be catabolized.     

Thymidine phosphorylase, thymidine kinase and thymidylate synthetase activities in cerebral hemispheres of developing chick embryos

The changes in activities of thymidine phosphorylase (EC 2.4.2.4), thymidine kinase (EC 2.7.1.75) and thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, EC 2.1.1.-) in the cerebral hemispheres of developing chick embryos were determined and compared with the course of DNA synthesis and of natural cell death in this organ. Thymidine phosphorylase activity reaches a broad maximum at the 12th to 14th day of incubation, followed by a rapid decrease. Thymidine kinase and thymidylate synthetase activities are highest at the earliest time studied (day 10) and decrease until day 14, followed by an increase from day 14 to 16 and a further decrease from day 16 through 1 day post-hatching. The rate of DNA synthesis essentially follows these activities, but the increase at day 16 is not discernible. Our previous study revealed high DNA synthesis at day 10, with natural cell death concurring on days 12-14, followed by another peak after day 16 (glial proliferation) and a decrease after day 16. It appears that thymidine phosphorylase activity reaches a maximum (days 12-14) at the time of maximum cell death, which may be correlated with the degradative function of this enzyme. This was also the time for minimum activities of thymidine kinase and thymidylate synthetase; on the other hand, these activities reach a first (day 10) and second (day 16) maximum at the time of maximum DNA synthesis; this may be correlated with the synthetic functions of these enzymes.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor

The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the lambda phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the lambda cI857 temperature-sensitive repressor. Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the lambda N protein. Heat inactivation of the lambda repressor by growth at 42 degrees C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein. Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography. Thymidine kinase has a subunit Mr = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a dimer during Sephadex G-100 chromatography and glycerol gradient centrifugation. Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5. The enzyme is protected from heat inactivation by thymidine and has a half-life at 40 degrees C of 30 min in the presence of thymidine and 3 min in its absence. Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 microM for thymidine and ATP, respectively. Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 microM. The anti-herpes drug acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) also appears to be a competitive inhibitor of thymidine (Ki of approximately 300 microM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition. Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.     

The effect of 5-azacytidine on the growth and thymidine kinase activity of coleoptile sections ofTriticum

The growth of coleoptile sections ofTriticum and its stimulation by indole-3-acetic acid (IAA) are inhibited by 5-azacytidine added into the cultivation medium. 50 per cent depression of the elongation was observed at 2×10^?3M 5-azacytidine concentration. Thymidine kinase activity in cell-free extracts prepared from coleoptile sections treated with 5-azacytidine, and caleulated per 10 mg of their wet weight, is increased while IAA administration resulted in its depression. The observed changes in thymidine kinase activity can be explained assuming the different uptake of water due to 5-azacytidine and IAA treatment.     

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: effect of hypophysectomy

The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.     

Mechanism of the elevation in cardiolipin during HeLa cell entry into the S-phase of the human cell cycle

CL (cardiolipin) is a key phospholipid involved in ATP generation. Since progression through the cell cycle requires ATP we examined regulation of CL synthesis during S-phase in human cells and investigated whether CL or CL synthesis was required to support nucleotide synthesis in S-phase. HeLa cells were made quiescent by serum depletion for 24 h. Serum addition resulted in substantial stimulation of [methyl-(3)H]thymidine incorporation into cells compared with serum-starved cells by 8 h, confirming entry into the S-phase. CL mass was unaltered at 8 h, but increased 2-fold by 16 h post-serum addition compared with serum-starved cells. The reason for the increase in CL mass upon entry into S-phase was an increase in activity and expression of CL de novo biosynthetic and remodelling enzymes and this paralleled the increase in mitochondrial mass. CL de novo biosynthesis from D-[U-(14)C]glucose was elevated, and from [1,3-(3)H]glycerol reduced, upon serum addition to quiescent cells compared with controls and this was a result of differences in the selection of precursor pools at the level of uptake. Triascin C treatment inhibited CL synthesis from [1-(14)C]oleate but did not affect [methyl-(3)H]thymidine incorporation into HeLa cells upon serum addition to serum-starved cells. Barth Syndrome lymphoblasts, which exhibit reduced CL, showed similar [methyl-(3)H]thymidine incorporation into cells upon serum addition to serum-starved cells compared with cells from normal aged-matched controls. The results indicate that CL de novo biosynthesis is up-regulated via elevated activity and expression of CL biosynthetic genes and this accounted for the doubling of CL seen during S-phase; however, normal de novo CL biosynthesis or CL itself is not essential to support nucleotide synthesis during entry into S-phase of the human cell cycle.