Thymidylate Synthetase Activity Research Articles

Inhibition of Thymidylate Synthetase Activity Induced in Varicella-Zoster Virus Infected Cells by (E)-5-(2-bromovinyl)-2′-deoxyuridine

The authors investigated the effect' of thymidylate synthetase (TS) on the antivariclllazoster virus (VZV) activity of ( E)5-(2-bromovinyl)-2′-deoxyuridine (BVDU). TS catalyses the conversion of deoxyuridylate (dUMP) to thymidylate (dTMP) and is a key enzyme in pyrimidine biosynthesis, providing the only source of dTMP synthesized de novo in mammalian cells. VZV encodes a specialized viral form of TS. TS activity in cells infected with VZV (TIO-VZV and TK−-VZV) increased proportionally with focus formation. From kinetic analysis using the Michaelis-Menten equation, the authors determined a Km value of 6.6μm for dUMP of TS induced in VZV-infected cells and a corresponding value from mock-infected cells of only 2.8μm. BVDU inhibited the induction of TS activity in TK+-VZV-infected cells at concentrations under ×10−3μm, but did not inhibit TS activity of TK−-VZV- or mock-infected cells at concentrations as high as 10μm. Inhibitory activity of BVDU against TS induced in TK+-VZV-infected cells appears to occur when BVDU is phosphorylated to BVDU monophosphate by viral pyrimidine kinase. These results suggest that the selective inhibitory action of BVDU on VZV replication depends on a specific interaction with both viral TK and TS. 5-Bromodeoxyuridine and 5-iododeoxyuridine inhibited TS activities induced in both VZV (TK+, TK−) and mock-infected cells. Other antiherpes compounds [i.e. 1-β-D-arabinofuranosyl- E-5-(2-bromovinyl)uracil, 9- 2-hydroxyethoxymethyl)-guanine, arabinosyladenine, and others] did not inhibit TS activity in VZV-infected cells at concentrations Of 10μm.

Biologically Based Dose-Response Modeling in Developmental Toxicology: Biochemical and Cellular Sequelae of 5-Fluorouracil Exposure in the Developing Rat

Mechanistically based dose-response models for developmental toxicity require elucidation of critical biological events that intervene between maternal exposure and adverse developmental outcome. We have examined some of the major events in the rat embryo/fetus following a subcutaneous injection of 5-fluorouracil (5-FU; 0-40 mg/kg) to the dam on Day 14 of gestation. This treatment resulted in reduced fetal weight that was significant at doses of 20 mg/kg and higher, generalized reduced ossification at doses above 25 mg/kg, and wavy ribs at doses of 30 mg/kg and higher. Numerous malformations including cleft palate and hindlimb defects were substantially increased at doses of 35 and 40 mg/kg. 5-FU inhibits thymidylate synthetase (TS), resulting in inhibited growth of rapidly proliferating tissues. To identify early events in the pathogenesis of hindlimb defects, we examined the effects of 5-FU on TS activity, cell cycle, growth, and morphology in the developing hindlimb as a function of dose and time. The rate of decline of TS activity following 5-FU exposure was dose related, although maximal inhibition and recovery were similar at doses within (20 and 40 mg/kg) and below (10 mg/kg) the range of detectable developmental toxicity. Flow cytometric analysis of nuclei from embryonic hindlimbs revealed a transient increase in the percentage of cells in S phase and decrease in G 0/G 1 phase 8 hr after maternal injection of 20-40 mg 5-FU/kg, but not at lower doses. Reduction in growth and morphometric changes of hindlimbs were observed only after maternal exposure to 40 mg/kg. The tissue specificity of these effects was examined by comparing the hindlimb with other embryonic tissues. There was also a dose-related decline of TS activity in the embryonic liver. However, the pattern of recovery of TS activity and cell cycle alterations were different in the liver than in the hindlimb, probably reflecting the higher cell proliferative rate in the liver at this stage. We have derived a quantitative, empirical model for induction of hindlimb defects based on TS inhibition and subsequent cellular events following 5-FU exposure. The model predicted a dose response similar to that of the observed data although the predicted curve was shifted toward lower doses. These results suggest that while this model may not capture all of the critical events involved in the induction of hindlimb defects following maternal exposure to 5-FU, it does reflect a central mechanism of its developmental toxicity. Biologically based dose-response modeling provides a framework for testing mechanistic hypotheses, and developing such models should ultimately improve our ability to perform risk assessments.

The cytotoxic activity of cyclic imido alkyl ethers, thioethers, sulfoxides, sulfones and related derivatives

Cyclic imides such as N-substituted alkyl ethers, thioethers, sulfoxides, sulfones and related derivatives were potent agents against human single cell tumors and selected solid tumor growths, eg adenocarcinoma of the colon and glioma. These agents in the L1210 lymphoid leukemia tumor model preferentially inhibited DNA synthesis. The regulatory enzyme sites in the purine pathway were targets of the agents. Other sites of inhibition were DNA polymerase alpha and thymidylate synthetase activities. d(NTP) pool levels were also reduced by the agents over 60 min.

Changes with age of mammary glands in male and female soft-furred rat, Millardia meltada, in relation to prolactin and testosterone.

The soft-furred rat, millardia (Millardia meltada), is characterized by the development of androgen-dependent mammary tumours only in males. The age-related changes of the activities of thymidylate synthetase (TS) and thymidine kinase (TK), which contribute to DNA synthesis through de novo and salvage pathways, respectively, and structure in the mammary glands were studied in both males and females of this species between 5-28 months of age. While TK activity had no relation to age, TS activity decreased with age in males. In the females, TK activity increased with age, but not TS activity. These enzyme activities were generally higher in females than in males. The mammary glands of both sexes consisted of fine ducts with small end-buds and the glands of males contained mostly black pigments at any age examined. In either males or females, serum levels of prolactin and testosterone related little with age, DNA synthesizing enzyme activities or structure of the mammary glands. Furthermore, elevation by pituitary grafting of circulating prolactin affected neither DNA synthesizing enzyme activities nor structure of mammary glands in both sexes. The histological structures of adrenal, testis, ovary, ventral prostate and uterus of millardia were essentially similar to those of mice or rats.

SUPPRESSION OF TUMORIGENESIS AND TUMOR-GROWTH OF MOUSE MAMMARY-GLANDS BY A TRADITIONAL CHINESE HERBAL MEDICINE, SHO-SAIKO-TO

Hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in mammary glands of mice. We investigated the effect of Sho-saiko-to (SST; a Japanese modified traditional Chinese herbal medicine containing seven medicinal plants) on the formation and growth of HAN and mammary tumour growth in SHN virgin mice which showed a high incidence of spontaneous mammary tumours. Chronic oral administration of SST reduced the number and the area of HAN and mammary thymidylate synthetase (TS) activity with slight decrease of serum prolactin level. In the established mammary tumours of mice, 20-day administration of SST reduced the activity of TS and thymidine kinase (TK) with slight reduction of serum prolactin level, though the significant change of mammary-tumour growth was not observed. These results indicate that SST may prevent the mammary tumourigenesis by suppression of the de novo pathway for pyrimidine nucleotide synthesis and the mammary tumour growth by suppression of the salvage pathway as well as the de novo pathway in mice.

Effects of recombinant human erythropoietin on DNA synthesis in rat hematopoietic organs.

In an attempt to define the effects of recombinant human erythropoietin (rEPO) on DNA synthesis in hematopoietic organs, we investigated DNA-synthesizing enzyme activities, i.e., thymidylate synthetase and thymidine kinase activities, and bromodeoxyuridine-immunohistochemistry during the recovery phase of hematopoietic cells in bone marrow and spleen after the hypoplastic period induced by cyclophosphamide (Cy) treatment in rats. Treatment with rEPO increased enzyme activities and cell number of erythroid series in bone marrow cells; it also increased organ weight and S-phase cells in the spleen, followed by an augmentation of the number of erythrocytes and a rise in the hemoglobin and hematocrit levels in peripheral blood with or without Cy treatment.

Suppression by kampo medicines in preneoplastic mammary hyperplastic alveolar nodules of SHN virgin mice.

Sho-saiko-to (SST), Keishi-bukuryo-gan (KBG), and Shakuyaku-kanzo-to (SKT) are Japanese modified traditional Chinese herbal medicines (Kampo medicines) consisting of 7, 5, and 2 medical plants, respectively. It is known that the hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in the mammary glands of mice. We examined the effects of SST, KBG, and SKT on the formation and growth of HAN in a high-mammary-tumor strain of SHN virgin mice. Oral administration of SST for 60 days beginning at 90 days of age reduced the number and area of HAN and mammary thymidylate synthetase activity with a reduction of serum prolactin level. There was little difference between the other experimental groups and the control in the formation and growth of HAN and the enzyme activities. These results indicate that SST may have a preventive effect on malignant mammary transformations.

Suppression of Spontaneous Development of Uterine Adenomyosis by a Chinese Herbal Medicine, Keishi-Bukuryo-Gan, in Mice

Keishi-bukuryo-gan (KBG) is a traditional Chinese herbal remedy and has been used for the treatment of gynecological disorders, such as hypermenorrhea, dysmenorrhea, and infertility. The effects of KBG on the development of uterine adenomyosis, which is characterized by an abnormal growth of glands and stroma into and beyond the smooth muscle layers of the uterus, were examined in an experimental animal model using the SHN strain of mice. Mice fed handmade chow containing relatively high doses of KBG (0.5% and 1%) showed a significantly lower incidence of adenomyosis and lower activity of thymidylate synthetase (TS) in the uteri than mice fed control handmade chow containing no KBG. The long-term exposure to KBG between 25 and 120 days of age hardly affected the estrous cycle, food intake and body weight. However, mice provided with chow containing a low dose of KBG (0.1%) showed no difference in the incidence of adenomyosis as compared with the controls. The inhibitory effects of the high doses of KBG were nullified by pituitary isografting, which has been proved to enhance the development of adenomyosis. The present mouse data support the view in humans that the oral administration of KBG is a useful tool for the treatment of uterine adenomyosis.

Effects of 5‐Fluorouracil on embryonic rat palate in vitro: Fusion in the absence of proliferation

5-Fluorouracil (5-FU) inhibits the enzyme thymidylate synthetase (TS) which results in inhibition of DNA synthesis. 5-FU is teratogenic in many species, inducing cleft palate, limb, and tail defects. In the present study, gestation day (GD) 14 embryonic rat craniofacial explants were exposed to 5-FU in organ culture with increasing concentrations and durations of exposure. Palates exposed to 5-FU were morphologically abnormal and craniofacial shape, size, and palatal fusion pattern were affected with the severity of effects dependent on concentration and duration of exposure. Cleft palate was induced in vitro as opposing palates overlapped in a narrowed oral cavity. Palates exposed to higher levels of 5-FU were growth inhibited, but fused even though proliferation ceased and few cells were available to participate in elevation and fusion. This was demonstrated as a biphasic concentration-response profile for palatal fusion in which 0.05 to 0.15 micrograms 5-FU/ml produced decreasing rates of palatal fusion, while exposure to 0.15 to 3.0 micrograms/ml resulted in progressively increasing rates of fusion. The effects of 5-FU were detected biochemically as a reduction in TS activity which was concentration and time dependent during the first 12 hours, with a return to control levels by 24 hours. During the first day, 5-FU did not alter protein levels, but DNA levels significantly decreased at the high concentration, 2.0 micrograms/ml. After 5 days in culture, both DNA and protein decreased with increasing 5-FU concentration and duration of exposure. Also by the end of the culture period, 3H-TdR incorporation had decreased in a concentration dependent manner. It is concluded that progressive inhibition of proliferation and growth in organ culture results in two different morphological outcomes: cleft palate resulting from a narrowed oral cavity and increased incidence of anterior palatal fusion under conditions of strong growth reduction. This study demonstrates that elevation and fusion can occur in the absence of growth and proliferation. Based on these observations, severe inhibition of growth or proliferation would not necessarily be sufficient to induce cleft palate.

135 Inhibition of thymidylate synthetase activity induced in varicella-zoster virus infected cells by ( E)-5-(2-bromovinyl)-2′-deoxyuridine

135 Inhibition of thymidylate synthetase activity induced in varicella-zoster virus infected cells by ( E)-5-(2-bromovinyl)-2′-deoxyuridine

Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematod́a: Metastrongyloidea)

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.

Activities of thymidylate synthetase and thymidine kinase in gastric cancer

In a previous clinical study, sequential methotrexate and 5-fluorouracil has shown improved efficiacy for treating advanced gastric cancer of the poorly differentiated type. In this study, we investigated whether difference in the levels of thymidylate synthetase (TS) and thymidine kinase (TK) activities in gastric cancer tissue account for selectivity of the treatment. Activity of TS was higher in 19 cases of the poorly differentiated type than in 16 cases of the well differentiated type ( P < 0.02), whereas TK activity was lower in the poorly differentiated type than in the well differentiated type ( P < 0.01). Thus, the TS TK ratio was significantly higher in poorly differentiated gastric cancers than in well differentiated cancers ( P < 0.001). These results suggest that a greater dependence upon the de novo pathway of pyrimidine synthesis in poorly differentiated gastric carcinomas may enhance the efficacy of sequential methotrexate and 5-fluouracil treatment.

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2′-deoxyuridine in lymphocytes

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2′-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2′-deoxyuridine, and cell concentration. 5-fluoro-2′-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2′-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.

Interferon alpha-2a shows antitumor activity in combination with 5-fluorouracil against human colon carcinoma xenografts: a study in reference to thymidylate synthetase activity inhibition.

To clarify the mode of antitumor activity shown by a combination of recombinant human interferon alpha-2a (IFN) and 5-fluorouracil (5-FU), experimental therapy was performed on human colon carcinoma (Co-4) xenografts serially transplanted into nude mice, using IFN and 5-FU, either alone or in combination. IFN alone showed dose-dependent antitumor activity and 5-FU also revealed a moderate antitumor effect. Although IFN, given as 600,000 units/mouse daily sc x 14, and 5-FU, given as 60 mg/kg q4d x 3 ip, showed additive antitumor activity against Co-4, the thymidylate synthetase (TS) inhibition rate was unchanged in the tumors treated with the IFN/5-FU combination in comparison with those treated with 5-FU alone. This suggests that the antitumor activity of IFN and 5-FU in combination does not involve augmentation of the TS inhibition by 5-FU.

Antitumor activity of fluoropyrimidines and thymidylate synthetase inhibition.

Experimental chemotherapy with 5‐fluorouracil (5‐FU; 60 mg/kg), l‐hexylcarbamoyl‐5‐fluorouracil (HCFU; 70 mg/kg), 3‐(3‐(6‐benzoyloxy‐3‐cyano‐2‐pyridyloxycarbonyI)benzoyl)‐l‐ethoxymethyl‐5‐fluorouracil (BOF‐A2; 30 mg/kg) and UFT (20 mg/kg as tegafur with uracil at a molar ratio of 1:4) was performed using human gastric (H‐111) and colon (Co‐4) carcinoma strains in nude mice. 5‐FU was administered ip with a q4d × 3 schedule and the other agents were given po daily for three weeks. Concentrations of 5‐FU in the serum and the tumor were assessed by gas chromatography‐ntass fragmentography, two hours or 12 days (5‐FU) after the last treatment, and thymidy late synthetase (TS) was assayed according to the method of Spears et al. using the same schedule. The antitumor activity of the agents was assessed in terms of the actual tumor weight at the end of the experiment. HCFU was effective against both strains and 5‐FU was effective against Co‐4, although the other agents were ineffective against either strain. Statistically significant correlations were found between the serum and tumor concentrations of 5‐FU and antitumor activity. Statistically significant correlations were also observed between the antitumor activity and TS inhibition rate (TSIR) and the activity of free thymidylate synthetase (TSfree), with higher TSIR and lower TSfree resulting in higher antitumor activity. Therefore, TSIR and TSfree were thought to be promising indicators for predicting the antitumor activity of fluoropyrimidines.

Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli

We have developed a recombinant system that directs the functional expression from Escherichia coli of both dihydrofolate reductase-thymidylate synthetase (DHFR-TS) and the isolated DHFR domain from Plasmodium falciparum. Both products are inhibitory to a number of E. coli cell lines to the extent that cell growth ceases immediately upon induction. This dramatic inhibition is not seen in strain AB 1899, in which amounts of plasmodial protein of up to 100 times the basal E. coli TS level can be accumulated. However, as well as the full-length DHFR-TS molecule, smaller proteins carrying an intact TS substrate-binding site are produced. These represent ca. 60–75% of the total plasmodial protein expressed and are observed in every E. coli strain examined. We show that they are not derived by degradation of the parent DHFR-TS molecule, but can be correlated with the sizes of proteins expected to be produced if erroneous initiation of translation were occurring at 3 internal methionine residues.

Activity of thymidylate synthetase, thymidine kinase and galactokinase in primary and xenografted human colorectal cancers in relation to their chromosomal patterns

The relationship between chromosome anomalies and metabolic modifications in human colorectal cancers grafted into nude mice was studied. Two distinct chromosomal patterns have been demonstrated i.e., monosomic type (MT) characterized by multiple chromosome losses or deletions always involving chromosome 18, and trisomic type (TT) characterized by progressive gains of chromosomes. Grafted tumors conserve original karyotypes observed on corresponding primary tumors. Most changes involve the loss of chromosomes carrying genes encoding for enzymes of the de novo pathways and the gain of chromosomes carrying genes encoding for enzymes of the salvage pathways of nucleotide synthesis. In MT tumors the long arm (q) of chromosome 17 is frequently duplicated in association with a deletion of the short arm, forming an isochromosome 17q. The activities of 3 enzymes, thymidylate synthetase (TS) mapped on chromosome 18, thymidine kinase (TK) and galactokinase (GalK), both mapped on chromosome 17q, were studied. TS is a de novo enzyme and TK and GalK are salvage enzymes. A clear correlation could be demonstrated between tumor types and enzyme activities: MT tumors had lower TS and higher TK and GalK activities than TT tumors. These differences were too large to result from a gene dosage effect only. These data suggest that serial studies on grafted colorectal cancers give a better representation of metabolic disturbances than studies on fresh tumor samples, usually contaminated by non-cancerous cells.

Increased Activities of Thymidylate Synthetase and Thymidine Kinase in Human Thyroid Tumors

Thymidylate synthetase (TS) is known to catalyse the methylation of deoxyuridine monophosphate (dUMP) for the de novo synthesis of deoxythymidine monophosphate (dTMP). Thymidine kinase (TK) catalyzes the formation of dTMP by the phosphorylation of thymidine via the pyrimidine salvage pathway. High TS and TK activities have been observed in rapidly proliferating tissues. Both TS and TK activities in human thyroid adenocarcinoma increased to approximately 2-fold that in normal thyroid tissue. The thyroid TK isozymes can be separated into two types by DEAE-cellulose column chromatography. One of them is associated with the cytosol cell fraction of rapidly proliferating cells, such as fetal or neoplastic cells. Its activity is not affected by low concentration of deoxycytidine triphosphate (dCTP), and its molecular weight and Km value for thymidine are 120 kD and 0.5 x 10(-6) M, respectively. The average activity of this cytosol-associated isozyme in thyroid adenocarcinoma was increased slightly to 2.3-fold that of normal thyroid tissue. These results obtained from the activities of TK and its isozyme may be indicative of the slow-growing property of thyroid adenocarcinoma compared to mammary or colonic adenocarcinoma, which has high activities of TK and its isozyme.

Establishment and Characterization of a Thymidine Kinase Deficient Avian Fibroblast Cell Line Derived from a Japanese Quail Cell Line, QT35.

An avian thymidine kinase deficient (TK-) fibroblast cell line (QTTK-) was established from a Japanese quail cell line, QT35, and characterized the biological properties. QTTK- could grow in the presence of 5-bromo-2'-deoxyuridine (BUdR, 100 micrograms/ml) and not in the growth medium with hypoxanthine-aminopterin-thymidine. Compared to QT35 cells, the 3H-thymidine incorporation of the QTTK- cells and the TK activity of the cell extract significantly decreased to 0.3% and 0.5%, respectively. In the thymidylate synthetase activity, the karyotype, and the sensitivities to either fowlpox virus (FPV) or herpesvirus of turkeys (HVT), OTTK- cells were similar to the parental QT35 cells. Since QTTK- cells were permissive to FPV and HVT infections and these viruses could not grow in the presence of 50 to 75 micrograms/ml of BUdR, QTTK- cells may be useful for the construction of recombinant FPV and HVT that have foreign genes within the TK gene of the virus genome.

Anticancer Effects of a Chinese Herbal Medicine, Juzen-taiho-to, in Combination with or without 5-Fluorouracil Derivative on DNA-Synthesizing Enzymes in 1,2-Dimethylhydrazine Induced Colonic Cancer in Rats

Juzen-taiho-to (JTT; [Shi-quan-da-bu-tang], a Japanese modified Chinese herbal prescription) in combination with an anticancer drug UFT (5-fluorouracil derivative) prevented the body weight loss and the induction of the colonic cancer in rats treated with a chemical carcinogen 1,2-dimethylhydrazine (DMH), and suppressed markedly the activity of thymidylate synthetase (TS) involved in the de novo pathway of pyrimidine synthesis in colonic cancer induced by DMH.