Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:024500 (thymidylate synthase)
 2,970 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biochemical and biological studies have been carried out with 2-desamino-2-methylaminopterin (dmAMT), which inhibits tumor cell growth in culture but is only a weak inhibitor of dihydrofolate reductase (DHFR). Since it was possible that the species responsible for growth inhibition are polyglutamylated metabolites, the di-, tri-, and tetraglutamates of dmAMT were synthesized and tested as inhibitors of purified recombinant human DHFR, murine L1210 leukemia thymidylate synthase (TS), chicken liver glycinamide ribonucleotide formyltransferase (GARFT), and murine L1210 leukemia aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT). The compounds with three and four gamma-glutamyl residues were found to bind two orders of magnitude better than dmAMT itself to DHFR, TS, and AICARFT, with 50% inhibitory concentration values in the 200 to 300 nM range against all three enzymes. In contrast, at a concentration of 10 microM, dmAMT polyglutamates had no appreciable effect on GARFT activity. These findings support the hypothesis that dmAMT requires intracellular polyglutamylation for activity and indicate that replacement of the 2-amino group by 2-methyl is as acceptable a structural modification in antifolates targeted against DHFR as it is in antifolates targeted against TS. In growth assays against methotrexate (MTX)-sensitive H35 rat hepatoma cells and MTX-resistant H35 sublines with a transport defect, dmAMT was highly cross-resistant with MTX, but not with the TS inhibitors N10-propargyl-5,8-dideazafolic acid and N-(5-[N-(3,4-dihydro-2-methyl-4-ox-oquinazolin-6-yl)-N- methylamino]thenoyl)-L-glutamic acid, implicating DHFR rather than TS as the principal target for dmAMT polyglutamates in intact cells. On the other hand, an H35 subline resistant to 2'-deoxy-5-fluorouridine by virtue of increased TS activity was highly cross-resistant to N10-propargyl-5,8-dideazafolic acid and not cross-resistant to MTX, but showed partial cross-resistance to dmAMT. Both thymidine and hypoxanthine were required to protect H35 cells treated with concentrations of dmAMT and MTX that inhibited growth by greater than 90% relative to unprotected controls. In contrast, N10-propargyl-5,8-dideazafolic acid and N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)-N-methylamino] thenoyl)- L-glutamic acid required only thymidine for protection. Like MTX, therefore, dmAMT appears to inhibit purine as well as pyrimidine de novo synthesis, and its effect on cell growth probably reflects the ability of dmAMT polyglutamates to not only block dihydrofolate reduction but also interfere with other steps of folate metabolism, either directly or indirectly via alteration of reduced folate pools.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical and biological studies on 2-desamino-2-methylaminopterin, an antifolate the polyglutamates of which are more potent than the monoglutamate against three key enzymes of folate metabolism. 131 37

Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies, we identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. We now report on the use of DNA transfer techniques to examine directly the effects of each TS form on drug response. TS cDNA constructs, corresponding to the normal or variant TS mRNA, were expressed in Chinese hamster lung cells or in Escherichia coli, and response to 5-fluoro-2'-deoxyuridine was determined. We observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in the mammalian cells, as well as in bacteria. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.
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PMID:A naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxyuridine in mammalian and bacterial cells. 135 60

A human fibrosarcoma cell line, HT-1080, and four new cell lines (HS-16, HS-28, HS-30, and HS-42) were established from untreated patients with mesenchymal chondrosarcoma, peripheral nerve sheath sarcoma, malignant hemangiopericytoma, and mixed mesodermal tumor, respectively, and were used for analysis of mechanisms of intrinsic resistance to methotrexate. All four new cell lines were resistant to methotrexate as determined by inhibition of thymidylate synthase in whole cells and by growth inhibition, as compared with HT-1080, a methotrexate sensitive cell line. Methotrexate uptake, level of dihydrofolate reductase, and inhibition of this enzyme by methotrexate in the four cell lines were comparable to HT-1080 cells. However, levels of long chain polyglutamates (glu3-5) of methotrexate achieved after a 24-h incubation with this drug were much lower in the four new cell lines as compared to the HT-1080 cell line (5- to 20-fold lower). The low levels of methotrexate polyglutamates formed is likely the major cause of intrinsic methotrexate resistance in these new sarcoma cell lines.
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PMID:Intrinsic resistance to methotrexate in human soft tissue sarcoma cell lines. 137 1

This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide plus 5-fluorouracil, in CD8F1 mice bearing spontaneous, autochthonous, breast tumors or first-passage advanced transplants of these spontaneous tumors. The combination and sequence of administration of these drugs were selected on the basis of known potentiating biochemical interactions. High performance liquid chromatography and nuclear magnetic resonance spectroscopy measurements of biochemical changes resulting from treatment with N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide indicated a severe depletion of cellular energy levels in the treated tumors. 6-Aminonicotinamide produced a severe block of the pentose shunt, and 5-fluorouracil severely inhibited both thymidylate synthase and thymidine kinase in the treated tumors. This quadruple drug combination, administered on a 10-11-day schedule, produced an impressive partial tumor regression rate of 67% of large, spontaneous, autochthonous, murine breast tumors and a tumor regression rate of 74% of first-passage transplants of the spontaneous breast tumors.
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PMID:Biochemical modulation of tumor cell energy: regression of advanced spontaneous murine breast tumors with a 5-fluorouracil-containing drug combination. 137 19

Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical anti-tumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU +/- leucovorin (LV) +/- brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC50) for brequinar, FU, and FU+LV (100 microM) were 0.4, 20, and 10 microM, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC10 (0.1 microM) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 microM brequinar and FU +/- LV did not increase the cytotoxicity of FU +/- LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 microM brequinar for 24 h alone or followed by a 2-h exposure to FU (25 microM) +/- LV (100 microM). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU+brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 microM hypoxanthine and 25 microM FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).
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PMID:Biochemical modulation of 5-fluorouracil with or without leucovorin by a low dose of brequinar in MGH-U1 cells. 138 Apr 7

Antitumor effect and active metabolites of fluoropyrimidines were examined in mice with transplantable colon adenocarcinoma 38 (Co 38). 5-Fluoro-2'-deoxyuridine (FUdR) treatment resulted in a much higher level of free 5-fluoro-2'-deoxyuridine-5'-monophosphate in the tumor than 5-fluorouracil (5-FU) did, and thymidylate synthase was almost completely inhibited after FUdR treatment, but FUdR showed weaker antitumor activity than 5-FU did. Moreover, 5-fluorouridine (FUR) also hardly inhibited tumor growth. A more marked tumor inhibition was obtained when FUdR and FUR were administered together. The antitumor activity of 5-FU was similar to that of the combination of FUdR and FUR. In combination with 2,2'-anhydro-5-ethyluridine, a uridine phosphorylase inhibitor, FUdR lost its antitumor activity, but that of FUR was somewhat potentiated. On the other hand, in combination with leucovorin (LV), 5-FU showed markedly potentiated antitumor activity, while the antitumor activity of FUdR or FUR was not potentiated. Addition of LV to the combination of FUdR and FUR enhanced the inhibitory effect of the drugs. From these results, the combination of FUdR and FUR together with LV, and the combination of 5-FU and LV seem to be highly efficacious against Co 38.
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PMID:In vivo antitumor effects of fluoropyrimidines on colon adenocarcinoma 38 and enhancement by leucovorin. 138 27

We have previously shown that treatment of the HT29 human colorectal tumor (HCT) cell line with 100 nM 5-fluorodeoxyuridine (FdUrd) induces DNA fragments ranging from 50 kilobases to 5 megabases. The studies reported here were conducted to characterize the kinetics, concentration dependence, and pharmacologic specificity of this process and to determine if such fragmentation varies among HCT cell lines. HT29 and SW620 cells yielded similar fragment size distributions upon treatment with either FdUrd or CB3717 [a folate analog inhibitor of thymidylate synthase (TS)]. With either of these agents the SW620 line required higher drug concentrations or longer incubation times than HT29 cells to achieve a given level of fragmentation or cytotoxicity, even though the two cell lines are equally sensitive to FdUrd-induced TS inhibition. These data indicate that SW620 resistance is not due to a lesion in the events leading up to TS inhibition but it may be due to a difference in the steps following TS inhibition. Aphidicolin, a DNA polymerase inhibitor, did not cause substantial fragmentation or cytotoxicity in these two cell lines, demonstrating that the fragmentation response to the other two drugs is not a general consequence of DNA synthesis inhibition. A third HCT line, HuTu80, gave rise only to a smaller and more discrete population of DNA fragments, ranging from approximately 50 to 200 kilobases, following exposure to FdUrd. Similar patterns were seen in this line upon treatment with CB3717 or aphidicolin, indicating that this fragmentation pattern is not specific to TS inhibition and may be characteristic of a more general response than that seen in the other two cell lines. DNA fragments induced by FdUrd in HuTu80 cells did not degrade into smaller pieces, demonstrating that the process by which they are formed is distinct from apoptosis. We conclude that the responses of HCT cells to FdUrd-induced TS inhibition vary significantly, that these differences may reflect heterogeneity in the mechanism of DNA damage formation, and that, in some cases, FdUrd resistance may be due to alterations in the fragmentation process.
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PMID:Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity. 143 36

Research efforts over the past year further elucidate the determinants of sensitivity and mechanisms of resistance to the antimetabolites fluorouracil, methotrexate, and cytarabine. Progress has been made in clarifying the complex regulation of target enzyme expression for these antimetabolites. Advances in analytical methodology should facilitate quantitation of thymidylate synthase content in tumor tissue prior to and following fluorouracil-based therapy. Information concerning the basis for certain drug interactions may guide rational dose rates and schedules for clinical trials. A better understanding of the clinical pharmacology of these agents has suggested strategies to minimize their toxicity while maintaining therapeutic activity.
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PMID:Antimetabolites. 145 23

We assayed thymidylate synthase (TS) activity in normal and malignant gynecologic tissues. TS activity in the normal cervix, secretory endometrium, and ovaries from a total of 61 patients was 1.00 +/- 0.52 pmol/g tissue (mean +/- SD). Thus the upper limit (mean + 2SD) of TS activity in normal gynecologic tissue was 2.00 pmol/g tissue. TS activity was generally higher in malignant tissue than in normal tissue, and particularly high activity was detected in cervical cancer. TS activity in endometrial cancer was relatively low, but activity in poorly and moderately differentiated tumors was significantly elevated compared to well differentiated tumors. These findings suggest that cervical cancer is a highly proliferative tumor and that in endometrial cancer, cell proliferation is more rapid in poorly differentiated tumors than in well differentiated ones.
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PMID:Thymidylate synthase activity in normal and malignant gynecologic tissues. 149 11

The inhibition of thymidylate synthase (TS) by the 5-fluorouracil (5FU) metabolite 5-fluorodeoxyuridine monophosphate can be biochemically modulated by leucovorin (LV). LV administration increases the level of reduced folates in tissues, which promotes the inhibition of TS. We have studied the antitumor effect, free 5-fluorodeoxyuridine monophosphate levels, and inhibition of TS in two murine colon tumors at several time points after weekly 5FU or LV and 5FU administration. The antitumor effect of 5FU alone could be potentiated by LV in both tumors. 5-Fluorodeoxyuridine monophosphate levels (212 and 46 pmol/g wet wt. after 2 h for Colon 26 and Colon 38, respectively) were sufficient to mediate TS inhibition, but the levels were not related to antitumor activity. Untreated controls of the 5FU-sensitive tumor Colon 38 had 3 times lower TS levels than did those of the more resistant tumor Colon 26. One course of treatment resulted in a comparable extent and retention of TS inhibition for 5FU and LV/5FU therapy in both tumors. After 1 week there was complete recovery of TS inhibition, but the TS levels in tumors from 5FU-treated mice tended to be higher than the controls, which was more pronounced after three courses of therapy. A 4-fold increase of TS levels was seen in Colon 26 after 5FU therapy. The elevation of TS in this tumor affected the extent of TS inhibition. Tumors treated with 5FU and LV also showed an increase of TS, but to a lesser extent, while the absolute effect on TS inhibition remained the same. This might be related to the potentiating effect of LV on 5FU antitumor activity in vivo in these tumors.
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PMID:Elevation of thymidylate synthase following 5-fluorouracil treatment is prevented by the addition of leucovorin in murine colon tumors. 151 48


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