Thymidylate Synthetase Activity Research Articles

The effect of cyclic nucleotides on DNA polymerase, thymidylate synthetase, thymidine kinase and deoxynucleoside levels of Walker carcinoma

Monobutyryl adenosine 3′,5′ monophosphate (mbcAMP) caused an inhibition of the thymidylate synthetase activity of Walker rat mammary carcinoma cells in tissue culture, which could be reversed by concomitant treatment with N 2,O 2′ dibutyryl guanosine 3′,5′ monophosphate (dbcGMP). There was no effect on thymidine kinase activity. The DNA polymerase activity of whole cells, but not broken-cell preparations was markedly inhibited by a dose of mbcAMP (100 μg/ml) having little effect on growth rate. This inhibition could be reversed to some extent by simultaneous treatment of the cells with caffeine. Treatment with mbcAMP produced a decrease in the level of dTTP and a concomitant rise in the levels of dATP, dGTP and dCTP. This situation was reversed in combination with dbcGMP, with levels of the deoxyribonucleoside triphosphates tending to revert back to control values. The effect of mbcAMP on thymidylate synthetase and DNA polymerase may account for its growth inhibitory effect.

Regulation of thymidylate synthetase in mouse leukemia cells (L1210).

L1210 mouse leukemia cells were synchronized by isoleucine deprivation followed by hydroxyurea treatment. Changes of thymidylate synthetase activity both in cell extracts and in the intact cells (in situ) were followed throughout the cell cycle. The enzyme activity assayed in situ, by measuring tritium released from [5-3H]deoxyuridine, changed markedly in relation to the DNA synthesis pattern, whereas that assayed in cell extracts remained unchanged. In the S phase of the cell cycle, activity of the enzyme was much higher as measured in situ than as measured in cell extracts. Hydroxyurea (1 mM) inhibited DNA synthesis in exponentially growing L1210 cells and was found to inhibit thymidylate synthetase activity in situ. However, the enzyme activity in cell extracts was not inhibited by the drug. In cells with DNA synthesis inhibited by hydroxyurea, the pattern of change of in situ activity with time suggested product inhibition. Results of isotope-dilution analysis of the release of tritium from [5-3H]deoxyuridine in the intact cells indicated that thymidine added to the medium caused strong inhibition of thymidylate synthetase, the most likely limiting step in this pathway. These data allow the suggestion that, during cell growth, thymidylate synthetase activity is regulated by thymine nucleotide(s), and is dependent on the rate of DNA synthesis as well as the availability of the salvage pathway substrate, thymidine.

Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase.

A tritium-suicide method for isolating thymidine auxotrophic mutants is described. Mutagenized mouse FM3A cells were cultured in medium containing [3H] deoxyuridine. Most of the surviving clones examined showed a phenotype of absolute thymidine auxotrophy. This phenotype is very stable and was found to be genetically recessive in cell-cell hybridization experiments. The growth of these variant clones was not supported by various pyrimidine nucleosides other than thymidine. The activity of thymidylate synthetase in crude extracts of these clones was less than 1% of that of FM3A cells. These results strongly indicate that the thymidine auxotrophic phenotype resulted from a genetic defect in thymidylate synthetase.

Role of thymidylate synthetase activity in development of methotrexate cytotoxicity.

Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.

Thymidylate synthetase activity in normal and Brugia pahangi-infected Aedes aegypti

Thymidylate synthetase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase; EC 2.1.2.45; TMPS) activity was detected in crude extracts of 4 to 5 day-old adult male and uninfected female Aedes aegypti. Mosquito TMPS was strongly inhibited by Mg 2+ and Ca 2+ ( id 50 values of 0.25 and 0.12 μM, respectively) but not by other divalent cations. The K m values for dUMP and 5, 10-methylene-tetrahydrofolate were 55 and 100 μM, respectively. It appeared that mosquito TMPS would not be subject to regulation by physiological concentrations of thymidine mono-, di- and triphosphates or di- and triphosphates of cytidine, uridine, adenosine or guanosine. When subjected to Sephadex G-200 gel filtration, TMPS activity occurred in three peaks, corresponding to molecular weights of 210,000, 310,000 and 450,000. Among a variety of compounds tested as inhibitors of the enzyme, FdUMP was the most potent, with an id 50 value of 0.5 nM; by contrast, FUdR was inactive at 1.0 mM. The polycyclic sulfonated anionic drug, suramin, was also a strong inhibitor of mosquito TMPS, with an id 50 value of 0.3 μM. Various 2,4-diaminoheterocyclic antifols, including methotrexate, either were inactive or were relatively weak inhibitors of this enzyme. The total activity of TMPS did not change in extracts of female mosquitoes prepared 7 and 12 days after they fed upon either normal or Brugia pahangi-infected jirds.

Characterization of an amber suppressor in Pneumococcus.

Partial revertant has been isolated, with resistance to aminopterin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su+) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su+ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidylate synthetase gene are suppressible. Thymidylate synthetase activity is partially restored by su+. Optochin mutants can also be suppressed. Thus su+ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycin is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor. The t-RNA extracted from the suppressor strain su+ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su+ gene is amber specific. Thus su+ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su+; this is additional evidence that both categories contain point mutations.

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions

1. 1. Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing Tetrahymena pyriformis GL-1 and ST strains, contrary to previous reports. 2. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2–3 × 10 4 cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3–5 × 10 5 cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2–6 × 10 4 cells/ml . 4. 4. In the PPL medium, the final cell numbers reached are about 6.5 × 10 5 cells/ml , while in the Neff's medium, the cell numbers increase further (to about 2 × 10 6 cells/ml ). 5. 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transfered from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.

Antineoplastic Agents III: Effects of Dibromoethyl and Vinyl Esters of N-Benzyloxycarbonyl-l-phenylalanine on Ehrlich Ascites Tumor Cell Metabolism

Evidence is presented that N-benzyloxycarbonyl-l-phenylalanine vinyl ester and 1,2-dibromoethyl ester are inhibitors of Walker 256 carcinosarcoma and Ehrlich ascites carcinoma tumor growth. The major effects of these two agents on Ehrlich ascites cell metabolism were the inhibition of deoxyribonucleic acid and protein synthesis and the alteration of cellular regulatory processes controlling cytokinetics. Deoxynucleotide (purine) kinase enzymes appeared to be the focal site for inhibition of deoxyribonucleic acid synthesis with marginal inhibition of thymidylate synthetase activity. Cyclic adenosine monophosphate levels were elevated by drug treatment whereas chromatin protein phosphorylation, cell respiration, and lysosomal activities were inhibited. N-Benzyloxycarbonyl-l-phenylalanine 1,2-dibromoethyl ester was a latent in vitro chymotrypsin inhibitor. Some preliminary evidence suggests that these activated esters may inhibit cellular enzymatic activity by alkylating imidazole and lysine residues of proteins.

Mechanism of resistance of Pediococcus cerevisiae strains to 5-fluorodeoxyuridine

The Pediococcus cerevisiae strains, the wild-type (ATCC 8081), a folate-permeable strain, PteGlu, and a folate-permeable amethopterin-resistant strain, AMr, were made resistant to 5-fluoro-2′-deoxyuridine (FdUrd) by successive transfer in increasing concentrations of the drug. The selected mutant strains, wild-type FdUrd, PteGluFdUrd and AMeFdUrd exhibited a 10 7-fold greater tolerance to FdUrd than the parents. The activities of folate-related enzymes did not change with the development of resistance. Dihydrofolate reductase (5, 6,7,8-tetrahydrofolate: NADP + oxidorductase, EC 1.5.1.3) was of almost the same activity in the FdUrd-susceptible parents as in the resistant mutants. Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) activity and its susceptibility to inhibition by 5-fluoro- 2′-deoxyuridine-5′-monophosphate (FdUMP) were the same in the parents as in the mutants. Two factors were found to be responsible for the resistance to FdUrd: (1) a decrease or lack of activity of thymidine kinase (ATP; thymidine- 5′-phosphotransferase, EC 2.7.1.21) and (2) an impaired cell permeability to FdUrd. After incubation for 1 min the susceptibility strains accumulated radioactive FdUrd up to 45-fold (wild-type) and 90-fold (PteGlu and AMr strains) higher intracellular concentration than that of the medium. About 50% of the accumulated radioactivity was FdUMP. The wild-type FdUrd mutant could not accumulate the drug against the concetration gradient, whereas the PteGlu FdUrd and AMr FdUrd strains accumulated the substrate up to an 8 times higher intracellular concentration than in the medium. The radioactivity identified in the medium after incubation with the susceptibility and resistant cells was intact FdUrd. Thus, no degradation of FdUrd to fluorouracil (FUra) occured prior to uptake, nor was FUra released from the cells into the medium. The uptake of FdUrd in the parents and in the mutants was shown to be glucose-dependent and inhibited by deoxyuridine and thymidine.

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

Abstract—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.

Antitumor Agents XXIX: Effects of Eupahyssopin on Nucleic Acid, Protein, Anaerobic, and Aerobic Metabolism of Ehrlich Ascites Tumor Cells

Evidence is presented that the antitumor agent eupahyssopin, a germacranolide, inhibits deoxyribonucleic acid, ribonucleic acid, protein, and cholesterol synthesis of Ehrlich ascites tumor cells in CF1 male mice. Eupahyssopin appears to bind to nucleotide bases of deoxyribonucleic acid. The drug also inhibits deoxyribonucleic acid and messenger ribonucleic acid polymerase activities and, marginally, thymidylate synthetase activity. Template activity, as regulated by phosphorylation of histones and nonhistones, and cyclic 3ʹ,5ʹ-adenosine monophosphate levels also were affected by drug therapy. Lysosomal enzymes, e.g., deoxyribonuclease and ribonuclease, and cathepsin activities were inhibited by eupahyssopin administration. A number of sulfhydryl-bearing enzymes of the glycolytic and Krebs cycles were inhibited by drug treatment, as were enzymes required for glycogen synthesis and breakdown and for phosphatase hydrolysis. Eupahyssopin also inhibited both basal respiration and coupled oxidative phosphorylation processes of the tumor cell. It was proposed that eupahyssopin, which contains the O=CC=CH2 as an α-methylene γ-lactone moiety within its allylic ester side chain, has the ability to undergo a rapid Michael-type addition with sulfhydryl groups of these enzymes and thus to inhibit their activities in a manner analogous to that described for other sesquiterpene antitumor agents containing the O=CC=CH2 moiety.

Thymidylate synthetase and dihydrofolic acid reductase in the stimulated human lymphocyte.

The phytohemagglutinin (PHA)-stimulated human lymphocytes demonstrated trace or no activity (dihydro) folate acid reductase using three methods including a radioassay, but demonstrated ample activity of thymidylate synthetase. This was true regardless of the day of harvest, (first through seventh) of the stimulated lymphocyte. The lymphocyte extracts revealed no inhibitor to the reductase enzyme when these extracts were added before the liver extracts to the assay system. When methotrexate (MTX) was added to the culture media of the lymphocytes, there was, as expected, an increase in the synthetase activity, but the expected rise in the reductase activity did not occur, it remained nil. On the other hand, MTX did influence the incorporation of nucleosides by the stimulated lymphocytes in a fashion similar to its action on the incorporation of the same nucleosides by other cells.

Mode of action of 5-fluorocytosine

A 5-fluorocytosine-sensitive isolate of Candida albicans was examined for the presence of thymidylate synthetase activity and 5-fluoro-2′-deoxyuridylic acid in the presence and absence of a growth-inhibiting concentration of 5-fluorocytosine. Initial studies demonstrated thymidylate synthetase activity in a crude homogenate prepared from this isolate. This activity could be inhibited by preincubating the homogenate with 5-fluoro-2′-deoxyuridylic acid. Evidence is presented suggesting that the inhibition of this enzyme is irreversible. Later studies measured thymidylate synthetase activity and 5-fluoro-2′-deoxyuridylic acid levels in homogenates prepared from harvested C. albicans after varying intervals of exposure to 100 μg/ml of 5-fluorocytosine. Thymidylate synthetase activity was observed to be 20 per cent of control after 1 hr of exposure to 5-FC, remaining decreased through the remainder of the 24 hr. 5-fluoro-2′-deoxyuridylate was measurable at all of the time points examined in the fungal cells exposed to 5-fluorocytosine in levels from 2 to 40 pmoles/μg of DNA while no evidence was found for this compound in the controls. In light of these findings, it is suggested that inhibition of thymidylate synthetase activity by endogenously synthesized 5-fluoro-2′-deoxyuridylic acid is a possible mechanism by which 5-fluorocytosine exerts its antifungal effect.

Correlation of DNA accumulation rate with thymidylate synthetase activity in developing rat cerebellum: effect of hypothyroidism.

Experimental hypothyroidism in the neonatal rat is known to result in a delay in cerebellar DNA biosynthesis. Because of the known high correlation between rate of DNA biosynthesis and thymidylate synthetase (TS) activity in neonatal hyperthyroidism in the rat, we studied this correlation under conditions of neonatal hypothyroidism. The equation for the least-squares logistic curve describing cerebellar DNA content in neonatal rats as a function of postnatal age (day of birth is day 0), was differentiated to give an expression for the instantaneous rate of cerebellar DNA accumulation in units of nanograms per hour per milligram wet cerebellum. The rate of accumulation of cerebellar DNA in hypothyroid neonatal rats from litters of propylthiouracil-treated dams was significantly (p < 0.05) decreased below control values at 3, 5, and 9 days of age, and significantly elevated at 15 and 21 days of age; the maximum decrease (78% of control) occurred on day 5, and the maximum elevation (266% of control) occurred on day 21. The developmental pattern of cerebellar TS activity in units of picomoles per hour per milligram wet cerebellum, closely paralleled the pattern for DNA accumulation rate in cerebella of both normal and hypothyroid neonatal rats, with cerebellar TS activity in hypothyroid pups significantly decreased below normal on postnatal days, 5 and 9, and significantly elevated on days 15 and 21. During this early developmental time period, a linear correlation of the rates of cerebellar DNA accumulation with the values of cerebellar TS activity gave correlation coefficients of 0.98 and 0.95 for normal and hypothyroid animals, respectively. These data thus support results from our previous study of thyroxine-induced hyperthyroidism, further suggesting that TS may be critical for maintenance of replicative DNA synthesis in the developing rat cerebellum.

Altered thymidine kinase or thymidylate synthetase activities in 5-fluoro deoxyuridine resistant variants of mouse neuroblastoma.

Abstract: Abstract‐We have previously described a 5‐fluorodeox yuridine (FUdR) resistant neuroblastoma variant, possessing normal levels of ATP: thymidine‐5‐phosphotransferase (EC 2.7.1.21) [trivial name: thymidine kinase (TK)] but an 8‐fold elevation in methy1enetetrahydrofolate:dUrd‐5′P C‐methyltransferase (EC 2.l.l.b) [trivial name: thymidylate synthetase (TS)] relative to the drug‐sensitive parental clone. This variant possesses elevated levels of the parental TS species, 30% of which is uninhibitable by in vivo pulses of FUdR, suggesting the subcellular compartmentalization of this enzyme. We contrast this variant with a second FUdR resistant clone isolated from an ethyl‐methane‐sulfonate mutagenized population of the parental clone. This variant displays a 96% reduction in TK specific activity, despite normal FUdR and thymidine uptake rates, demonstrating the independence of thymidine phosphorylation and uptake. Grown without drug, its resistance declines (half‐life of 15 cell divisions) with its TK specific activity rising to a plateau of 16% of the parental level after 56 cell divisions. Thymidine (1.0μM) protects the TK+ but not the TK‐ variants from FUdR induced growth inhibition but is without effect on TS specific activity. Unlike Tetrahymena (DICKENS et al., 1975), neuroblastoma TS activities appear not to be regulated by adenosine or guanosine cyclic nucleotide levels.

Thymidine 5′-Monophosphate-Requiring Mutants of Saccharomyces cerevisiae Are Deficient in Thymidylate Synthetase

Thymidylate synthetase activity was measured in crude extracts of the yeast Saccharomyces cerevisiae by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (tup1, -2, or -4), which were permeable to thymidine 5'-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying tup mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5'-monophosphate as high as 50 mug/ml, or by subsequent removal of thymidine 5'-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (cdc8), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5'-monophosphate auxotroph arrested with a morphology identical to the cdc21 strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5'-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that tmp1 and cdc21 are the same locus. These results identify tmp1 as the structural gene for yeast thymidylate synthetase.

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.

Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td(+), whereas particles produced after infection with td(-) had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td(ts) to td(+) simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td(ts) and frd(ts) genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd(ts) mutants to frd(+) not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.

Developmental Modifications Induced by DNA Base Analogs

Pulse treatment of Drosophita melanogasler larvae with thymidine analogs in the presence of inhibitors of thymidylate synthetase activity induces a variety of growth lesions in the adult flies. We determined the frequency of the developmental lesions induced for a variety of treatment conditions and examined the incorporation of the analogs into DNA of larvae treated in a similar manner for each of these conditions. Lesion induction is correlated with bromodeoxyundine incorporation into DNA. The distribution of incorporated bromodeoxyuridine in the pyrimidine oligonucleotides released from DNA by formic acid-diphenylamine hydrolysis was compared with the incorporation pattern of exogenous thymidine. This comparison shows that bromodeoxyuridine is not randomly accepted at all thymine sites for incorporation into DNA under the feeding conditions used in these experiments. There were also differences between the patterns of bromodeoxyuridine- incorporated DNAs from larvae receiving the analog under conditions slightly effective and highly effective for morphogenic lesions. Examination of the size and frequency of lesions as a function of larval age at the time of treatment indicates there is a clonal pattern of propagation of the analog-affected cells. Since several cell generations separate the analog pulse and the time of eventual differentiation of the imaginal discs, we conclude that following the incorporation of bromodeoxyuridine into morphogenically critical base sequences of DNA, a cell undergoes an informational change that is transmitted to its progeny cells which differentiate during metamorphosis as a cluster of phenotypically altered cells. The presence of bromodeoxyuridine per se in each cell of a clone is not necessary for expression of the altered patterns of differentiation.

Inhibition of thymidylate synthetase of walker carcinoma by chlorambucil. a possible mechanism of action

Effects of chlorambucil (CA) on growth of Walker carcinoma 256 and on enzymes involved in the synthesis of thymidine monophosphate are described. A single intraperitoneal dose (25 mg kg ) or multiple doses (8mg/kg/day for 3 days) of CA were “curative” for drug-sensitive (WS) tumors. Thymidylate (dTMP) synthetase activity of WS tumors was significantly decreased (approximately 25%) at 3 hr, reached its lowest level (approximately 50% loss) at 12 hr. and remained at this level up to 60 hr after administration of 25 mg kg of CA. Dihydrofolate (FAH 2) rcductase activity did not change significantly up to 36 hr and then slowly decreased (approximately 20% loss) by 60 hr. Thymidine (TdR) kinase activity of WS tumors was not affected. Treatment of WS tumors with multiple doses of CA also resulted in pronounced inhibition of dTMP synthetase activity (approximately 50% loss). some decrease (approximately 15% loss) in FAH 2 reductasc activity and no change in TdR kinase activity. A Chlorambucil-resistant strain (WR) of Walker carcinoma was developed. In contrast to WS, after treatment with CA the enzyme activities of WR tumors remained essentially the same as those from untreated animals. In vitro incubation of partially purified dTMP synthetase enzymes from either WS or WR tumors with CA inhibited both enzymes to the same extent (approximately 50% loss at 1.25 × 10 −6 M). TdR kinase and FAH 2 reductasc activities were not inhibited up to 1.25 × 10 −4 M CA. The results of these studies support the concept that CA exerts cytotoxic activity by inhibition of dTMP synthetase. Activities of dTMP synthetase and TdR kinase were found to be significantly altered in WR tumors as compared to WS tumors. The activity of dTMP synthetase was decreased approximately 20% and that of TdR kinase was increased approximately 35% in WR tumors. Resistance to CA may be due. in part, to increased dependence of WR tumors on the salvage pathway for synthesis of dTMP.