Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

III Purine antagonists Biosynthesis of guanine nucleotides has been reported to be up regulated in tumor cells. In guanine nucleotide synthesis, there are 2 rate-limiting enzymes, i.e. inosine monophosphate dehydrogenase for de novo synthesis and hypoxanthine guanine phosphoribosyltransferase for the salvage pathway. Therefore, agents acting on these 2 enzymes to inhibit guanine nucleotide synthesis could be expected to have a superior effect on tumor proliferation. The main antitumor agents belonging to this class are thiopurines [including 6-mercaptopurine (6 MP), 6-thioguanine (6 TG) and 6-thioinosine (TIR)], thiazofurin (TZF), and arabinofuranosylfluoroadenine (F-ara-A). In the activation of 6MP to its ribotide. PRPP is the rate limiting factor. After the ribotide is produced, it is metabolized to another active form by enzymes catalyzing purine nucleotide metabolism. The antitumor effect of TZF is enhanced by the combination of TZF with allopurinol, which increases the plasma hypoxanthine level and subsequently inhibits recovery of the reduced guanine nucleotide pool by TZF. F-ara-A induces DNA strand damage as well as inhibiting DNA synthesis and is expected to have a significant antitumor effect on slowly growing tumors. These agents are mainly effective for treating hematological malignancies. IV Antifolic agents Among the antifolates, methotrexate (MTX) is the most useful drug for both hematologic malignancies and solid tumors. MTX primarily inhibits one-carbon transfer through the inhibition of dihydrofolate reductase and thus blocks the biosynthesis of both purine and pyrimidine nucleotides. Formyl polyglutamate synthetase catalyzes folate to its polyglutamate, both the active and retention forms. It is also important as an activating enzyme as well as being a target of MTX. MTX directly inhibits thymidylate synthetase, which could be the main target during high-dose therapy. High-dose MTX therapy with leucovorin (LV) rescue is effective even for tumors which are resistant to conventional treatment. During clinical use, not only MTX levels but also those of its inactive metabolites [7-hydroxy-MTX and 2, 4-diamino-N10-methylpteroic acid(DAMPA)] should be monitored. High-dose MTX therapy with LV rescue requires precise monitoring and LV rescue should be continued until the MTX level falls below 5 x 10(-8) M. MTX is also known as the safest drug which can be directly administered to into the central nervous system. Many other antifolates are under development, among which trimetrexate might be the most promising. Studies on antimetabolites have developed side by side with research on nucleotide tumor cell metabolism, which has produced a number of the antitumor agents now available for cancer chemotherapy.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Clinical pharmacology of anticancer agents [Part 5] Antimetabolites (2)]. 154 70

Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.
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PMID:Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines. 168 77

1-beta-D-Arabinofuranosylcytosine (ara-C) inhibits nuclear DNA replication in Chinese hamster ovary cells by an efficient chain termination mechanism without affecting the rate at which cells traverse G1 and enter S [Heintz, N. H., & Hamlin, J. L. (1983) Biochemistry 22, 3557-3562]. Here we have employed ara-C to enrich for replication intermediates formed during initiation of DNA synthesis in synchronized CHOC 400 cells, a methotrexate-resistant derivative of Chinese hamster ovary cells that contains approximately 1000 copies of an early replicating 150-kb chromosomal domain. This highly amplified domain includes the gene for dihydrofolate reductase (DHFR). CHOC 400 cells were collected at the G1/S boundary of the cell cycle with aphidicolin prior to release into S in the presence of both [methyl-3H] thymidine and various concentrations of ara-C. Chromatographic fractionation of restriction endonuclease digests over benzoylated naphthoylated DEAE-cellulose (BND-cellulose) showed that high concentrations of ara-C inhibited the maturation of chromosomal replication intermediates containing ssDNA (replication forks) into dsDNA for up to 60 min. The effect of ara-C on the sequence complexity of replication intermediates formed during early S phase was determined by hybridizing purified intermediates labeled with 32P in vitro to Southern blots of genomic DNA derived from both methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. In the absence of ara-C, 32P-labeled ssDNA BND-cellulose fractions from cultures released into S for 30-60 min hybridized to a spectrum of restriction fragments encompassing 40-50 kb of the amplified DHFR domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Replication intermediates formed during initiation of DNA synthesis in methotrexate-resistant CHOC 400 cells are enriched for sequences derived from a specific, amplified restriction fragment. 300 61

The mechanisms of action of MTX and 5-FU have been further elucidated. Such studies will be important for the design of drug combinations and for the development of novel antifolate and fluoropyrimidine analogs. A greater understanding of MTX and ara-C transport and drug levels required to optimize transport may also aid in these endeavors. Pharmacokinetic parameters have been found to be predictors of relapse in children with acute leukemia, particularly with respect to MTX, 6-MP and ara-C. The intracellular terminal half-life of ara-C was correlated with remission duration in AML. Assay systems aimed at uncovering response predictors through biochemical analysis of patient tumor samples are being developed, including an interesting use of NMR spectroscopy to study the pharmacokinetics of fluorine-19-labeled 5-FU in vivo. Such an approach may yield valuable information on 5-FU anabolism in tumors in situ. A high frequency of resistance to MTX apparently may be generated within a single cell cycle by transient exposures to DNA synthesis inhibitors. The resistance may be based on either target enzyme amplification or altered membrane transport. These important studies provided bases for the rapid emergence of clinical resistance. Further, the multidrug-resistant phenotype appears to be a much broader based phenomenon as MTX resistance was found to be a frequent event in cells selected for multidrug resistance. A variety of novel approaches have been proposed to overcome antimetabolite resistance and to improve the selectivity of these agents, including the use of guanosine nucleotides, leucovorin and allopurines as biochemical modulators of 5-FU. Efficient techniques for the transfection of resistant DHFR into tissues using retroviruses have been reported. These studies serve as starting point for the ultimate development of more effective strategies for the treatment of human malignancies.
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PMID:Antimetabolites. 307 79

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on DNA replication in methotrexate-resistant Chinese hamster ovary cells was examined under circumstances in which nuclear DNA synthesis could be distinguished from mitochondrial DNA synthesis. G1-arrested cells were induced to traverse G1 and enter the S phase in the presence of radiolabeled thymidine and various concentrations of the drug. ara-C did not affect the kinetics of G1 traverse and subsequent entry into S after release from isoleucine deprivation, as measured by autoradiography. However, the inhibitor reduced the net rate of thymidine incorporation into nuclear DNA in a dose-dependent fashion. Autoradiography of nuclear matrix-DNA halo structures suggests that the drug inhibits nuclear thymidine incorporation by slowing chain elongation and movement of newly replicated DNA through a matrix-bound replication apparatus. Southern blot analysis of restriction digests of DNA radiolabeled in early S in the presence of ara-C indicates that the synthesis of the early-replicating amplified dihydrofolate reductase domain in these cells begins at sequences identical with those observed in cells synchronized with aphidicolin or hydroxyurea. Progressively lower concentrations of ara-C permit proportionately greater extents of the amplified unit to be replicated. These results suggest that ara-C slows the rate of chain elongation without altering the site at which DNA replication is initiated within individual replicons.
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PMID:In vivo effects of cytosine arabinoside on deoxyribonucleic acid replication in Chinese hamster ovary cells. 2. Cytosine arabinoside affects the rate of synthesis but not the pattern of labeling of an amplified chromosomal sequence at the onset of the S period. 661 84

Antimetabolites are rational agents with specific S-phase and enzyme targets. Low levels of target enzymes in tumors are associated with innate drug sensitivity, and the general requirement for transport and metabolic activation of antimetabolites creates several loci of acquired drug resistance. Pharmacodynamic studies of TS inhibition after fluoropyrimidines clearly can predict for tumor sensitivity and response to fluoropyrimidine-based therapy or identify factors related to resistance, and ara-dCTP levels in leukemic cells can be useful for refined dosing of araC. Powerful new DHFR and TS directed agents are in advanced levels of clinical evaluation, and purine analogues directed against adenosine deaminase are newly available for treatment of indolent lymphomas. Progress in analysis of tumors, such as PCR techniques to study gene expression or immunostaining of target enzymes, offer increasing promise for individualization of patient selection. Increased experience with biochemical modulators, including biologic response modifiers, has opened the possibility for selective attack on specific mechanisms of drug resistance. Sophisticated pharmacokinetic modeling and pharmacogenetic testing of metabolic phenotypes can now be done to achieve optimal dosing with less risk of toxicity. Considerations of ultimate genetic mechanisms of antimetabolite effects, especially by programmed cell death, and relationships to mechanisms of cell cycle regulation offer exciting rationales for future drug development.
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PMID:Clinical resistance to antimetabolites. 764 70

A series of beta-alkylaminopropiophenone derivatives were demonstrated to be potent antineoplastic agents. Several compounds showed activity against Ehrlich ascites carcinoma growth in CF1 mice by demonstrating over 70% inhibition. Most of these agents proved to be potent cytotoxic agents in inhibiting the growth of a number of murine and human cancer cell lines grown in tissue culture. Their ED50 values were comparable to those of the selected standard anticancer drugs, such as 6-MP, ara-C, hydroxyurea, 5-FU, 6-aza-UMP, etoposide, antimycin A, actinomycin D and cycloheximide. In the mode of action studies in Tmolt3 cells, beta-(3",5"-dimethyl)piperidinopropiophenone was observed to reduce DNA and RNA synthesis significantly at 25 microM within 60 min incubation. The site of action of this agent appears to involve the reduction of the activities of Tmolt3 DNA polymerase alpha 1 dihydrofolate reductase, PRPP-amido transferase and ribonucleoside reductase.
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PMID:Antineoplastic activities of 2,3,4-chloro-substituted beta-alkylaminopropiophenone derivatives in CF1 mice and in murine and human tumor cells. 886 31

Transformed cells are characterized by imbalances in metabolic routes. In particular, different key enzymes of nucleotide metabolism and DNA biosynthesis, such as CTP synthetase, thymidylate synthase, dihydrofolate reductase, IMP dehydrogenase, ribonucleotide reductase, DNA polymerase, and DNA methyltransferase, are markedly up-regulated in certain tumor cells. Together with the concomitant down-modulation of the purine and pyrimidine degradation enzymes, the increased anabolic propensity supports the excessive proliferation of transformed cells. However, many types of cancer cells have maintained the ability to differentiate terminally into mature, non-proliferating cells not only in response to physiological receptor ligands, such as retinoic acid, vitamin D metabolites, and cytokines, but also following exposure to a wide variety of non-physiological agents such as antimetabolites. Interestingly, induction of tumor cell differentiation is often associated with reversal of the transformation-related enzyme deregulations. An important class of differentiating compounds comprises the antimetabolites of purine and pyrimidine nucleotide metabolism and nucleic acid synthesis, the majority being structural analogs of natural nucleosides. The CTP synthetase inhibitors cyclopentenylcytosine and 3-deazauridine, the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine, the dihydrofolate reductase inhibitor methotrexate, the IMP dehydrogenase inhibitors tiazofurin, ribavirin, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and mycophenolic acid, the ribonucleotide reductase inhibitors hydroxyurea and deferoxamine, and the DNA polymerase inhibitors ara-C, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and aphidicolin, as well as several nucleoside analogs perturbing the DNA methylation pattern, have been found to induce tumor cell differentiation through impairment of DNA synthesis and/or function. Thus, by selectively targeting those anabolic enzymes that contribute to the neoplastic behavior of cancer cells, the normal cellular differentiation program may be reactivated and the malignant phenotype suppressed.
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PMID:Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. 1041 91

A novel fusion gene consisting of the open reading frame of a double-mutant (Phe22-Ser31) dihydrofolate reductase (dmDHFR) cDNA fused to the open reading frame of cytidine deaminase (CD) was constructed and characterized for the purpose of conferring simultaneous resistance to methotrexate (MTX) and cytosine arabinoside (ara-C). The kinetic properties of purified recombinant dmDHFR-CD fusion protein were compared with those of purified CD and dmDHFR. The fusion protein was found to retain enzymatic properties of both dmDHFR and CD, in that the Km and Kcat values of purified dmDHFR-CD protein were found to be virtually identical to those of CD and dmDHFR alone. Retrovirus-mediated expression of dmDHFR-CD in NIH 3T3 cells conferred significant resistance (10- to 12-fold) against MTX and ara-C, compared with mock- and single gene-infected cells and the level of resistance obtained was similar to that of cells expressing both CD and dmDHFR from a retroviral bicistronic vector. Infection of mouse bone marrow cells with the dmDHFR-CD construct also showed high levels of resistance to MTX and ara-C in a CFU-GM assay. This fusion protein confers resistance to two antineoplastic agents that differ in their mechanism of action, and may be useful in the design of gene transfer strategies for protection of target cells against multiple drugs. Since high-dose ara-C and MTX are used in the treatment of lymphomas, this vector may be of value in protecting human hematopoietic progenitor cells from the toxicity of these antimetabolites.
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PMID:Expression of a novel double-mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside. 1054 14

An SFG-based retroviral bicistronic vector containing a double-mutant dihydrofolate reductase-cytidine deaminase fusion cDNA (F/S DHFR-CD) with IRES-eGFP confers resistance to both methotrexate (MTX) and cytarabine (ara-C). Two weeks after transplantation with marrow transduced with either a fusion or a control gene (eGFP-IRES-NeoR), human lymphoma (SKI-DLCL-1) cells were injected sc into the flanks of nonobese diabetic/severe combined immune deficiency mice. In mock-transplanted mice, maximal tolerated dose (MTD) of posttransplant MTX/ara-C (15/10 mg/kg/day, x3) was unable to control tumor growth. Transfer of the fusion gene allowed doses of MTX/ara-C (25/15 mg/kg/day, x4) twofold higher than the MTD to be tolerated. The tumor burden defined the efficiency of posttransplant chemotherapy; early treatment, 48 h after tumor inoculation, provided tumor-free survival, while starting treatment after having palpable tumor growth (7 days) delayed tumor growth a median time of 28 days. In addition, the early treated group had higher gene expression in peripheral blood and marrow cells than the late treated group (P < 0.05), suggesting that early treatment allowed for enrichment of transduced marrow progenitors. These results encourage clinical studies using this retroviral fusion gene construct.
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PMID:Methotrexate and cytarabine inhibit progression of human lymphoma in NOD/SCID mice carrying a mutant dihydrofolate reductase and cytidine deaminase fusion gene. 1533 57


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