Thymidylate Synthetase Activity Research Articles

Relative activities of thymidylate synthetase and thymidine kinase in 1,2-dimethylhydrazine-induced colon carcinomas in rats.

Thymidylate synthetase (TS) and thymidine kinase (TK) are known to catalyse the methylation of dUMP for the de novo synthesis of dTMP and the phosphorylation of thymidine for the salvage synthesis of dTMP in the pyrimidine pathway, respectively. High TS and TK activities and the existence of TK isozymes have been observed in rapidly proliferating tissues. TS and TK activities in 1,2-dimethylhydrazine (DMH)-induced colon carcinomas in rats increased significantly to 331 and 207% of the activities in normal colon, respectively, and were well correlated inversely (y = -0.93x + 5.24), with a correlation coefficient of -0.787. The colonic TK isozymes were separated into two types by DEAE-cellulose column chromatography. The TK isozyme eluted from the column by the elution buffer alone without NaCl was markedly higher (23.6-fold) in activity in DMH-induced colon carcinoma than in normal control colon and was not affected by deoxycytidine triphosphate. This isozyme, whose mol. wt is 100,000 by h.p.l.c., is thought to be closely involved in rapid DNA replication. These results indicate that early biochemical changes in DMH-induced colon carcinoma in rats may serve as a useful model and provide valuable insight into the mechanisms involved in colonic carcinogenesis.

Effects of estrogen and prolactin on thymidylate synthetase and thymidine kinase activities in N-nitrosomethylurea-induced rat mammary tumors

Methylglyoxal bis(butylamidinohydrazone) (MGBB) inhibited S-adenosylmethionine decarboxylase (SAMDC) activity competitively with S-adenosylmethionine (SAM) showing the Ki value of 1.8 × 10−5 M. MGBB showed less SAMDC-stabilizing effect in rat liver in vivo than did methylglyoxal bis(guanylhydrazone) (MGBG). MGBB inhibited the growth of human erythroid leukemia K 562 cells. Putrescine, spermidine and spermine concentrations in MGBB-treated cells were depressed to 56%, 58% and 88% of the values of control cells, respectively. [35S]Methionine incorporation into trichloroacetic acid-insoluble fraction was decreased in the inhibitor-treated cells.

Effect of 5-fluorouracil on the cell growth and cell cycle kinetics of a mouse ascites tumor growing in vivo.

The effect of 12, 24 and 36 mg/kg body weight doses of fluorouracil (5-FU) on the Bp 8 ascites sarcoma growing in vivo was studied. From sequential studies of the total number of cells together with the composition of cells in the cell cycle, the cell cycle flow was calculated and correlated to the pharmacokinetics, which was determined by using 3H-5-FU. The dose of 12 mg/kg 5-FU affected cell growth between 24 and 72 hours, while the effect of higher doses was immediate. An early block in outflow of cells from G1 was followed by an increased outflow, indicating an early inhibition followed by an enhancement of the initiation of the DNA synthesis. This increased outflow from G1 together with the decrease in outflow from the early S-phase, i.e. decreased DNA synthesis, resulted in an accumulation of cells in the early part of the S-phase. The prolonged effects on the cell growth and the cell cycle flow despite the very fast decline in the drug concentration both in the ascites fluid and within the cells, together with a constant level of the drug in the macromolecular fraction, suggest an interaction between 5-FU and RNA/DNA at later times rather than an inhibition of the thymidylate synthetase activity.

Regulation of Pancreatic Islet Cell Replication: An Inquiry into the Controversy Regarding the Effects of Steroid Hormones*

The controversial issue of the effects of prednisolone and 17 beta-estradiol on replication of fetal rat pancreatic islets in culture was studied using 32P and [3H]thymidine as probes for studying DNA synthesis. DNA synthesis was not affected by the steroid hormones, as was evident from the rate of incorporation of 32P into total DNA. Decreased incorporation of [3H]thymidine into DNA found in islets treated with either of these steroids seemed to reflect an inhibitory effect of these hormones on thymidine kinase, leading to decreased phosphorylation of labeled thymidine. In addition, the hormones stimulated the activity of thymidylate synthetase, thus enhancing the endogenous synthesis of thymidine and thereby diluting the specific activity of the [3H]thymidine added to the cultured islets. Further support for a lack of inhibition of growth of islet cells treated with steroid hormones was provided by the observation that prednisolone increased uridine kinase activity and RNA biosynthesis, both of which may participate in the growth of cells preceding mitosis and (the latter) in protein hormone biosynthesis.

α-Adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given α-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a β-adrencoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.

Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts.

2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus-infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1-infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells.

Antineoplastic Activity of a Series of Boron Analogues of α-Amino Acids

A series of amine cyanoboranes, amine carboxyboranes, and boron analogues of α-amino acids have been investigated for antineoplastic activity against the growth of Ehrlich ascites cells. Additional studies demonstrated that the boron analogues inhibited DNA and RNA synthesis at 300 μM. The suppression of DNA synthesis of Ehrlich ascites cells correlated with the reduction of DNA polymerase, 5-phosphoribosyl-1-pyrophosphate amidotransferase, and dihydrofolate reductase activities afforded by the boron compounds. These derivatives did not suppress protein synthesis, thymidylate synthetase, or thymidine monophosphate kinase activities as previously reported for some boron antineoplastic agents.

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.

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts.

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

Mechanism of pyrimethamine resistance in recent isolates of Plasmodium falciparum.

Clones of Plasmodium falciparum prepared from recent isolates of infected blood were studied to determine the molecular mechanism of naturally occurring pyrimethamine resistance. Total DNA, as well as thymidylate synthetase and dihydrofolate reductase activities, were characterized from these lines. Restriction analysis of DNA from pyrimethamine-susceptible and -resistant lines of the parasite showed no obvious amplification of any DNA fragment. Further, analysis of DNA from resistant and susceptible lines by centrifugation in cesium chloride-ethidium bromide revealed no extrachromosomal amplification in the resistant line. Comparison of the dihydrofolate reductase enzyme activity in the two lines revealed similar KmS for substrate but a large difference in the inhibition constant for pyrimethamine. Additionally, the enzyme from the resistant line was considerably more stable in vitro than the corresponding enzyme from the susceptible line. The thymidylate synthetase activity in the two lines was similar and unaffected by pyrimethamine. The mechanism of drug resistance in this isolate involves altered properties of the dihydrofolate reductase conferring both a different affinity for the drug and increased stability.

A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates.

Methotrexate (MTX)-resistant human breast cancer cells (MTXR ZR-75) were obtained following serial passage of the wild-type ZR-75-1 cells (wild-type ZR-75) in MTX. The resistant cell line contains neither quantitative nor qualitative changes in dihydrofolate reductase compared to the parental line. Resistance is associated with a 3-fold decrease in MTX transport into MTXR ZR-75 cells as well as a 3-fold decrease in the activity of thymidylate synthetase in the resistant subline. Moreover, marked differences were observed between the wild-type and MTXR ZR-75 cells in their ability to convert MTX to its polyglutamate derivatives. Wild-type ZR-75 cells accumulate significant intracellular levels of antifolates during prolonged (24 h) exposure to 2 microM MTX, due to the formation of MTX polyglutamates. In contrast, essentially no polyglutamates are formed in the MTXR cells even during conditions which result in a vast excess of free intracellular drug in these cells. This defect is not associated with any apparent change in the activity of the enzyme folylpolyglutamyl synthetase, nor is there any alteration in the apparent Km of this enzyme for MTX in the resistant cells. Further studies demonstrate that the MTXR ZR-75 cells are cross-resistant to antifolate analogues which can be converted to polyglutamate derivatives (aminopterin and dichloromethotrexate), yet they are relatively sensitive to antifolate analogues such as 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, triazinate, and trimetrexate, which cannot be converted to polyglutamate forms. These studies identify a new mechanism (diminished accumulation of MTX polyglutamates) associated with resistance to MTX and lend additional support to the hypothesis that the formation of these derivatives is an important determinant of MTX cytotoxicity.

Mutator phenotype in a mutant of S49 mouse T-lymphoma cells with abnormal sensitivity to thymidine.

We have selected and characterized a thymidine-sensitive S49 mutant line, MC-3-3. MC-3-3 cells are 35-fold more sensitive to the cytotoxic effects of thymidine and 15-fold more sensitive to the cytotoxic effects of 5-bromodeoxyuridine than wild type S49 cells. In contrast, the MC-3-3 mutant line does not exhibit increased sensitivity to the cytotoxic action of 5-fluorodeoxyuridine. The MC-3-3 mutant line possesses levels of thymidylate synthetase and thymidine kinase activity which are equivalent to the levels in wild type S49 cells, but the ribonucleotide reductase activity in MC-3-3 cells, using CDP as a substrate, is only 10-30% of that in wild type cells. Using ADP as a substrate, the ribonucleotide reductase activity in permeabilized MC-3-3 cells is slightly higher than that in wild type S49 cells. The deoxyribonucleotide pools in exponentially growing MC-3-3 cells are approximately 40-50% of those in wild type S49 cells. By hybrid analysis, we determined that the thymidine sensitivity of the MC-3-3 cells is recessive. The MC-3-3 mutant line displays a rate of spontaneous mutation which is 15-30-fold higher than that of wild type S49 cells. The MC-3-3 mutant cells are also 5-10-fold more sensitive than wild type cells to the cytotoxic effects of tunicamycin and compactin. These results suggest that the MC-3-3 mutant line possesses a mutation in the dTTP binding site in ribonucleotide reductase; abnormal regulation of this enzyme results in an increase in the rate of spontaneous mutation.

Mechanism of cytotoxic activity of 5'-deoxy-5-fluorouridine.

The purpose of these studies was to characterize the effect of the new fluoropyrimidine nucleoside 5'-deoxy-5-fluorouridine (5'dFUrd) on macromolecular processes in correlation with its cytotoxicity in Ehrlich ascites tumor cells. Following a 2-h exposure, 5'-dFUrd exhibited an LD50 (as determined by clonogenicity) of 48 microM. In cells supplemented with 10 microM dThd, the LD50 for 5'-dFUrd increased to 660 microM. DNA synthesis was markedly and rapidly suppressed by all cytotoxic concentrations of 5'-dFUrd. There was no apparent direct measurable effect of 5'-dFUrd on either RNA or protein synthesis, although both were suppressed 24 h after the drug exposure. Thymidylate synthetase activity was completely inhibited by all cytotoxic concentrations of 5'-dFUrd. FUra incorporation into RNA was also measured and appeared to correlate with the dThd-nonreversible toxicity of 5'-dFUrd. These studies indicate that the mechanism of 5'-dFUrd cytotoxicity is directly analogous to that reported for 5-fluorouracil. The inhibition of thymidylate synthetase leading to an inhibition of DNA synthesis was the most potent cytotoxic mechanism (i.e., dThd-reversible) for 5'-dFUrd, and was found to be highly time-dependent. Higher concentrations of 5'-dFUrd resulted in dThd-nonreversible toxicity, which appeared to be related to the incorporation of FUra into RNA.

Cobalamin increased activity of thymidylate synthetase in the stimulated human lymphocyte

Using PHA stimulated normal human lymphocytes, it is shown that when cobalamin is added on day 2 to the cultures of these cells it causes a highly significant increase in the activity of the enzyme thymidylate synthetase if the cells are harvested on day 3. The results support and extend a previous study where the enzyme was shown to be absent or low in patients with cobalamin deficiency. It is suggested that the action of cobalamin in preventing the megaloblastic development relates to its capability to induce thymidylate synthetase.

2'-deoxyribosyl analogues of UDP-N-acetylglucosamine in cells treated with methotrexate or 5-fluorodeoxyuridine.

dUDP-GlcNAc, the 2'-deoxyribosyl analogue of UDP-GlcNAc, has been identified in human lymphoid cells treated with the dihydrofolate reductase inhibitor, methotrexate. It was shown previously that elevation of dUTP accompanies the gross expansion in intracellular deoxyuridylate pools that results from the methotrexate-induced block in thymidylate synthetase activity (1). dUDP-GlcNAc presumably is formed from dUTP acting in place of UTP in the normal pathway for formation of UDP-GlcNAc. Neither dUTP nor dUDP-GlcNAc has been detected in untreated cells. Inhibition of thymidylate synthetase by treatment of cells with 5-fluorodeoxyuridine (5-FdUrd) also causes the appearance of dUDP-GlcNAc, and, in addition, 5-FdUDP-GlcNAc, synthesized from 5-FdUTP. The metabolic effects, if any, of these analogues are not known. Synthesis of the analogues may help to limit accumulation of dUTP and 5-FdUTP under circumstances in which the deoxyuridine triphosphatase mechanism is insufficient.

Inverse relationship between thymidylate synthetase and cytidine triphosphate synthetase activities during pyrimidine limitation inSalmonella typhimurium

Inverse relationship between thymidylate synthetase and cytidine triphosphate synthetase activities during pyrimidine limitation in<i>Salmonella typhimurium</i>

Inverse relationship between thymidylate synthetase and cytidine triphosphate synthetase activities during pyrimidine limitation in Salmonella typhimurium

Inverse relationship between thymidylate synthetase and cytidine triphosphate synthetase activities during pyrimidine limitation in Salmonella typhimurium

Roles of thymidylate synthetase activity in herpes simplex virus-infected HeLa cells

Thymidylate synthetase was not essential for herpes simplex virus replication. However, it appeared to contribute to the formation of a portion of thymidine nucleotides for DNA synthesis in virus-infected cells. Therefore, it is possible that the enzyme plays an important role in determining the potency of several selective antiviral thymidine analogs. Since synergistic effects of 5-fluoro-2′-deoxyuridine with other analogs on virus reproduction were observed, it is suggested that the effects of these analogs depend to a certain degree on the abilities of the monophosphate derivatives to inhibit thymidylate synthetase.