Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Drug resistance is often a limiting factor in successful chemotherapy. Our laboratory has been interested in studying mechanisms of resistance to drugs that are targeted to the thymidylate biosynthesis pathway especially those that target thymidylate synthase (TS) and dihydrofolate reductase (DHFR). We have used leukemia as a model system to study resistance to methotrexate (MTX) and colorectal cancer as the model system to study 5-fluorouracil (5-FU) resistance. In leukemias, we and others have shown that transport, efflux, polyglutamylation and hydrolase activities are major determinants of MTX resistance. We have further reported that some leukemic cells have an increase in DHFR gene copy number possibly contributing to the resistant phenotype. Recently, we have begun to study in detail the molecular mechanisms that govern translational regulation of DHFR in response to MTX as an additional resistance mechanism. Studies thus far involving colorectal tumors obtained from patients have focused predominantly on the predictive value of levels of TS expression and p53 mutations in determining response to 5-FU. Although the predictive value of these two measures appears to be significant, given the variety of resistance to 5-FU observed in cell lines, it is not likely that these are the only measures predictive of response or responsible for acquired resistance to this drug. The enzyme uridine-cytidine monophosphate kinase (UMPK) is an essential and rate-limiting enzyme in 5-FU activation while dihydropyrimidine dehydrogenase (DPD) is a catabolic enzyme that inactivates 5-FU. Alterations in UMPK and DPD may therefore explain failure of 5-FU response in the absence of alterations in TS or p53. Transcription factors that regulate TS may also influence drug sensitivity. We have found that mRNA levels of the E2F family of transcription factors correlates with TS message levels and are higher in lung metastases than in liver metastases of colorectal cancers. Moreover, gene copy number of the E2F-1 gene appears to be increased in a significant number of samples obtained from metastases of colorectal cancer. We have also generated mutants of both DHFR and TS that confer resistance to MTX as well as 5-FU by random as well as site-directed mutagenesis. These mutants used alone or as fusion cDNAs of the mutants have proven to be useful in transplant studies where transfer of these mutant cDNAs to bone marrow cells have been shown to confer drug resistance to recipients. The fusion cDNAs of DHFR such as the DHFR-herpes simplex virus type 1 thymidine kinase (HSVTK) are also useful for regulation of gene expression in vivo using MTX as the small molecule regulator that can be monitored by positron emission tomography (PET) scanning or by optical imaging using a fusion construct such as DHFR-EGFP.
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PMID:Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase. 1208 58

Pemetrexed (Alimta; Eli Lilly and Co, Indianapolis, IN) is a novel antifolate/antimetabolite with activity in breast cancer and has well-defined molecular targets, including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. In a phase II trial in patients with T3-4, N0-2 breast cancer, expression of thymidylate synthase, dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, p53, and c-erb-B2 (at the mRNA or protein level) is being examined before and 24 hours after the first dose of neoadjuvant pemetrexed and after three cycles of single-agent treatment to establish correlations of biomarker levels and changes with clinical outcome and toxicity. Full biomarker and clinical data are not yet available from this study; however, clinical responses to pemetrexed treatment have been observed in patients. Results of this trial should provide both an idea of the activity of neoadjuvant pemetrexed in breast cancer and information on biomarker association with clinical performance that can be used in the design of additional clinical studies to assess the predictive value of these markers.
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PMID:Pemetrexed: translational research in breast cancer. 1209 37

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an approximately 16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.
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PMID:Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition. 1238 95

Patients with ultraviolet-sensitive syndrome (UV(S)S) are sensitive to sunlight, but present neither developmental nor neurological deficiencies. Complementation studies with hereditary DNA repair syndromes show that UV(S)S is distinct from all known xeroderma pigmentosum (XP) and Cockayne syndrome (CS) groups. UV(S)S cells exhibit some characteristics typical of CS, including normal global genomic (GGR) repair of UV-photoproducts, poor clonal survival and defective recovery of RNA synthesis after UV exposure. Those observations have led us to suggest that UV(S)S cells, like those from CS, are defective in transcription-coupled repair (TCR) of cyclobutane pyrimidine dimers (CPD). We have now examined the repair of CPD in the transcribed and non-transcribed strands of the active dihydrofolate reductase (DHFR) and p53 genes, and of the silent alpha-fetoprotein (AFP) and mid-size neurofilament (NF-M) genes in normal human cells and in cells belonging to UV(S)S and CS complementation group B. Our results provide compelling evidence that the UV(S)S gene is essential for TCR of CPD and probably other bulky DNA lesions. As a possible distinction between UV(S)S and CS patients, we postulate that the UV(S)S gene may not be required for TCR of oxidative lesions. We have also found that repair of CPD in either DNA strand of the genomic fragments examined, occurs at a slower rate in TCR-deficient cells than in the non-transcribed strands in normal cells; we suggest that in the absence of TCR, global repair complexes have hindered access to lesions in genomic regions that extend beyond individual transcription units.
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PMID:Ultraviolet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers. 1250 86

Gene amplification is a common mechanism that contributes to the drug resistance. To explore the molecular genetic background related to the MTX resistance in the mouse MTX-resistant cells, differential PCR was used to determine the amplification and overexpression of DHFR gene. In addition, the correlations between c-myc, p53 status and dhfr amplification were studied. Amplification and overexpression of dhfr suggested its role in MTX-resistant cells. However, no amplification and overexpression of c-myc were detected. On the other hand, no alteration of p53 copy number was found. The increased mRNA level of p53 suggested the normal function of p53. These results implicated the status of c-myc and p53 had no correlation with dhfr amplification, therefore some other molecular genetic alterations may exist to permit the dhfr amplification in MTX-resistant cells.
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PMID:[Study on the genetic alterations in MTX-resistant cells by differential polymerase chain reaction]. 1281 55

The p14(ARF) protein, the product of an alternate reading frame of the INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2 to target p53 for proteosomal degradation and causes an increase in steady-state p53 levels, leading to a G(1) and G(2) arrest of cells in the cell cycle. Although much is known about the function of p14(ARF) in the p53 pathway, not as much is known about its function in human tumor growth and chemosensitivity independently of up-regulation of p53 protein levels. To learn more about its effect on cellular proliferation and chemoresistance independent of p53 up-regulation, human HT-1080 fibrosarcoma cells null for p14(ARF) and harboring a defective p53 pathway were stably transfected with p14(ARF) cDNA under the tight control of a doxycycline-inducible promoter. Induction of p14(ARF) caused a decrease in cell proliferation rate and colony formation and a marked decrease in the level of dihydrofolate reductase (DHFR) protein. The effect of p14(ARF) on DHFR protein levels was specific, because thymidylate kinase and thymidylate synthase protein levels were not decreased nor were p53 or p21WAF1 protein levels increased. The decrease in DHFR protein was abolished when the cells were treated with the proteasome inhibitor MG132, demonstrating that p14(ARF) augments proteasomal degradation of the protein. Surprisingly, induction of p14(ARF) increased resistance to the folate antagonists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine in the medium reversed this resistance, indicating that p14(ARF) induction increases the reliance of these cells on thymidine salvage.
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PMID:p14ARF expression increases dihydrofolate reductase degradation and paradoxically results in resistance to folate antagonists in cells with nonfunctional p53. 1520 49

The comet assay is a sensitive method for measuring DNA strand breaks in eukaryotic cells. After embedding in agarose, cells are lysed and electrophoresed at high pH. DNA loops containing breaks (in which supercoiling is relaxed) escape from the nucleoid comet head to form a tail. Oligonucleotide probes were designed for 5' and 3' regions of the genes for dihydrofolate reductase (DHFR) and O6-methylguanine DNA methyltransferase (MGMT), both from the Chinese hamster, and the human tumour suppressor p53 gene. Alternate ends were labelled with either biotin or fluorescein. These probes were hybridized to the DNA of comets from Chinese hamster ovary (CHO) cells or human lymphocytes treated with H2O2 or photosensitizer plus light to induce oxidative damage. Amplification with Texas red- and fluorescein-tagged antibodies led, in the case of p53 in human cells, to red and green signals located in the comet tail (as well as in the head), indicating the presence of breaks in the vicinity of the gene. However, only one end of the MGMT gene appeared in the tail and almost no signals from the DHFR gene, either red or green, were in the tail of comets from CHO cells. Restriction on movement from the head to tail may result from the presence of a 'matrix-associated region' in the gene. The kinetics of repair of oxidative damage were followed; strand breaks in the p53 gene were repaired more rapidly than total DNA. Thus, fluorescent in situ hybridization in combination with the comet assay provides a powerful method for studying repair of specific genes in relation to chromatin structure.
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PMID:DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. 1521 25

Methionine deprivation imposes a metabolic stress, termed methionine stress, that inhibits mitosis and induces cell cycle arrest and apoptosis. The methionine-dependent central nervous system tumor cell lines DAOY (medulloblastoma), SWB61 (anaplastic oligodendroglioma), SWB40 (anaplastic astrocytoma), and SWB39 (glioblastoma multiforme) were compared with methionine-stress resistant SWB77 (glioblastoma multiforme). The cDNA-oligoarray analysis and reverse transcription-PCR verification indicated common changes in gene expression in methionine-dependent cell lines to include up-regulation/induction of cyclin D1, mitotic arrest deficient (MAD)1, p21, growth arrest and DNA-damage-inducible (GADD)45 alpha, GADD45 gamma, GADD34, breast cancer (BRCA)1, 14-3-3sigma, B-cell CLL/lymphoma (BCL)1, transforming growth factor (TGF)-beta, TGF-beta-induced early response (TIEG), SMAD5, SMAD7, SMAD2, insulin-like growth factor binding protein (IGFBP7), IGF-R2, vascular endothelial growth factor (VEGF), TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha converting enzyme (TACE), TRAIL receptor (TRAIL-R)2, TNFR-related death receptor (DR)6, TRAF interacting protein (I-TRAF), IL-6, MDA7, IL-1B convertase (ICE)-gamma, delta and epsilon, IRF1, IRF5, IRF7, interferon (IFN)-gamma and receptor components, ISG15, p65-NF-kappaB, JUN-B, positive cofactor (PC)4, C/ERB-beta, inositol triphosphate receptor I, and methionine adenosyltransferase II. On the other hand, cyclins A1, A2, B1 and B2, cell division cycle (CDC)2 and its kinase, CDC25 A and B, budding uninhibited by benzimidazoles (BUB)1 and 3, MAD2, CDC28 protein kinase (CKS)1 and 2, neuroepithelial cell transforming gene (NET)1, activator of S-phase kinase (ASK), CDC14B phosphatase, BCL2, TGF-beta activated kinase (TAK)1, TAB1, c-FOS, DNA topoisomerase II, DNA polymerase alpha, dihydrofolate reductase, thymidine kinase, stathmin, and MAP4 were down-regulated. In the methionine stress-resistant SWB77, only 20% of the above genes were affected, and then only to a lesser extent. In addition, some of the changes observed in SWB77 were opposite to those seen in methionine-dependent tumors, including expression of p21, TRAIL-R2, and TIEG. Despite similarities, differences between methionine-dependent tumors were substantial, especially in regard to regulation of cytokine expression. Western blot analysis confirmed that methionine stress caused the following: (a) a marked increase of GADD45alpha and gamma in the wt-p53 cell lines SWB61 and 40; (b) an increase in GADD34 and p21 protein in all of the methionine-dependent lines; and (c) the induction of MDA7 and phospho-p38 in DAOY and SWB39, consistent with marked transcriptional activation of the former under methionine stress. It was additionally shown that methionine stress down-regulated the highly active phosphatidylinositol 3'-kinase pathway by reducing AKT phosphorylation, especially in DAOY and SWB77, and also reduced the levels of retinoblastoma (Rb) and pRb (P-ser780, P-ser795, and P-ser807/811), resulting in a shift in favor of unphosphorylated species in all of the methionine-dependent lines. Immunohistochemical analysis showed marked inhibition of nuclear translocation of nuclear factor kappaB under methionine stress in methionine-dependent lines. In this study we show for the first time that methionine stress mobilizes several defined cell cycle checkpoints and proapoptotic pathways while coordinately inhibiting prosurvival mechanisms in central nervous system tumors. It is clear that methionine stress-induced cytotoxicity is not restricted by the p53 mutational status.
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PMID:Modulation of gene expression in human central nervous system tumors under methionine deprivation-induced stress. 1549 78

The role of various enzymes in folate dependent one-carbon metabolism, which are involved in mobilizing the folate pool for DNA synthesis and the DNA methylation reaction, was investigated. Male Swiss mice (6 weeks old) were subjected to 2, 5 and 7 Gy total body gamma-irradiation. The animals were killed at intervals of 24, 48, 72, 96, 120 and 192 h and the livers were removed. Using a 12000 x g supernatant of 10% tissue homogenate, the activities of dihydrofolate reductase, thymidylate synthase and methylenetetrahydrofolate reductase were determined. The profiles of these folate enzymes were correlated to DNA damage by monitoring p53 protein profile and by comet tail moment analysis. A significant increase in activity of dihydrofolate reductase and thymidylate synthase was observed up to 96 h post-irradiation and the activity subsided thereafter, reaching control value after 192 h. A sharp decline in methylenetetrahydrofolate reductase activity was observed until 192 h after irradiation. Total folates declined by 54% after 96 h following irradiation, and p53 protein concentration in nuclei increased after irradiation, proportionate to radiation dose, and subsided slowly. Thus results indicate a significant drop in total folate levels and rise in p53 protein concentration in the liver after total body gamma-irradiation. It may appear that, under radiation stress conditions, levels of enzymes involved in one-carbon metabolism for DNA repair, are modulated up to a certain time interval, in a dose specific manner. It may also appear that the requirements of folate for nucleotide base synthesis seem to be met at the expense of other one-carbon transfer reactions.
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PMID:Modulation of enzymes involved in folate dependent one-carbon metabolism by gamma-radiation stress in mice. 1563 62

Pemetrexed (Alimta) is a novel folate antimetabolite that primarily inhibits the enzymes thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyl transferase (GARFT), all of which are involved in pyrimidine and purine synthesis. In a phase II trial of patients with T3/4, N0-2 breast cancer, expression of thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), p53, and c-erb-B2 (at the mRNA or protein level) was examined in tumor biopsy specimens before and 24 hours after the first dose of pemetrexed and after three cycles of single-agent treatment to establish correlations of biomarker levels and changes with clinical outcome and toxicity. Although final data are not available, initial indications are that clinical response may correlate with decreased or low TS expression. The results obtained from clinical data are supported by laboratory results in three cell lines (MDA-231, MCF-7, and ZR-75). These results suggest that in vitro transcript profiling to identify which genes are important predictors of successful cytotoxic chemotherapy, followed by a focused clinical trial to confirm the in vitro results, may be the best approach for translational research.
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PMID:Translational research with pemetrexed in breast cancer. 1565 41


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