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)

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

We determined the cell cycle-dependent fluctuation of mRNAs that encode different enzymes of the deoxynucleotide metabolism in permanent cell lines of human and murine origin. In normal growing cells, dihydrofolate reductase, thymidine kinase, and both subunits of ribonucleotide reductase all show exactly the same variation. The mRNAs rise near the G1-S boundary, peak in early S phase, and return in G2 to approximately the level of early G1. Deoxycytidine kinase mRNA does not follow this pattern, but remains essentially unchanged. Conversely, in DNA tumor virus-transformed cells, the levels of all these mRNAs remain relatively constant throughout all phases. These data provide evidence that DNA tumor viruses suppress a transcriptional down-regulation common to enzymes responsible for the DNA precursor pathway. The usefulness of analysis of mRNA levels of these genes for the detection of DNA tumor virus transformation is indicated.
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PMID:A common regulation of genes encoding enzymes of the deoxynucleotide metabolism is lost after neoplastic transformation. 769 88

We have investigated the drug resistance and gene amplification potential of NIH3T3 cells transfected with sequences coding for K-FGF, a known oncogene product, or bFGF, a non-oncogene member of the fibroblast growth factor family. Resistance to methotrexate, N-(phosphonacetyl)-L-aspartate and hydroxyurea was observed with K-fgf transfectants, due to amplification of dihydrofolate reductase, CAD or ribonucleotide reductase R2 genes, respectively. In keeping with the increase in gene amplification frequency, cells transfected with the K-fgf gene also exhibited a marked increase in CAD gene amplification rate, as determined by fluctuation analysis in the presence of N-(phosphonacetyl)-L-aspartate. Cells transfected with bFGF encoding cDNA also exhibited a significant elevation in N-(phosphonacetyl)-L-aspartate resistance, and CAD gene amplification. Treatment with suramin, which interferes with the interaction of fibroblast growth factors with their cell surface receptors, did not decrease the drug resistance properties of K-fgf transfected cells. These observations with suramin and the findings with bFGF, which lacks a conventional signal sequence for secretion, suggests that the growth factor-mediated effects on drug resistance and gene amplification occur through an intracellular as opposed to autocrine mode of action. The finding that aberrant growth factor expression regulates gene amplification opens up new possibilities for investigating intracellular mechanisms relevant to this process and also describes new functions for the altered expression of K-FGF and bFGF, which are relevant to mechanisms of malignant progression.
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PMID:Fibroblast growth factor mediated alterations in drug resistance, and evidence of gene amplification. 790 43

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.
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PMID:Global change of gene expression at late G1/S boundary may occur in human IMR-90 diploid fibroblasts during senescence. 807 91

We studied inhibition of growth of the malaria parasite Plasmodium falciparum in in vitro culture using antisense (AS) oligodeoxynucleotides (ODNs) against different target genes. W2 and W2mef strains of drug-resistant parasites were exposed to AS ODNs over 48 hr, and growth was determined by microscopic examination and [3H]hypoxanthine incorporation. At ODN concentrations of 1 microM, phosphorothioate (PS) ODNs inhibited growth in a target-independent manner. However, between 0.5 and 0.005 microM, ODNs against dihydrofolate reductase, dihydropteroate synthetase, ribonucleotide reductase, the schizont multigene family, and erythrocyte binding antigen EBA175 significantly inhibited growth compared with a PS AS ODN against human immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was approximately 0.05 microM, whereas that for non-sequence-specific controls was 15-fold higher. PS AS ODNs against DNA polymerase alpha showed less activity than that for other targets, whereas a single AS ODN against triose-phosphate isomerase did not differ significantly from controls. We conclude that at concentrations below 0.5 microM, PS AS ODNs targeted against several malarial genes significantly inhibit growth of drug-resistant parasites in a nucleotide sequence-dependent manner. This technology represents an alternative method for identifying malarial genes as potential drug targets.
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PMID:Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides. 855 72

We have analysed relative DHFR gene copy numbers in nine cell lines of various cell type and species origins. The cells studied expressed either low, low and inducible or constitutively elevated levels of c-Myc protein. DHFR gene amplification was observed only when c-Myc protein levels were upregulated. The amplification of the DHFR gene was transient in inducible cell lines. Cell lines exhibiting constitutively deregulated c-Myc protein levels, however, showed both DHFR gene amplification and ongoing rearrangements of the DHFR locus. In contrast, the relative gene copy numbers of ribonucleotide reductase R1 subunit, ornithine decarboxylase, syndecan 2, glyceraldehyde-3-phosphate-dehydrogenase, and cyclin C remained unaffected irrespective of c-Myc protein levels, suggesting a locus-specific genomic instability of the DHFR gene in cells with deregulated c-Myc protein levels. Overall, the results of the present study support the notion that DHFR gene amplification as a consequence of c-Myc deregulation may occur in a variety of cell lines irrespective of their cell type and species origins.
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PMID:c-Myc overexpression associated DHFR gene amplification in hamster, rat, mouse and human cell lines. 857 Feb 5

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.
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PMID:T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex. 862 61

A number of alpha-, beta-, and gamma-alkylaminopropiophenone derivatives were found to be potent antineoplastic agents in CF(I) mice by inhibiting the growth of Ehrlich ascites carcinoma at 8 mg/kg/day and in vitro cytotoxic agents against murine and human cancer cell growth. Two analogs, beta-dimethylaminopropiophenone (1) and beta-pyrrolidinopropiophenone (3), were further tested for their in vitro effects on the metabolism of Tmolt3 cells. beta-Dimethylaminopropiophenone demonstrated potent reduction of DNA synthesis, RNA synthesis, and the pool levels of the dNTPs. Enzyme activities, such as DNA polymerase a, ribonucleotide reductase, PRPP amidotransferase, and most significantly, dihydrofolate reductase, were reduced by the agents from 25 to 100 microM after 60 min.
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PMID:Antineoplastic activities of alpha-, beta-, and gamma-alkylaminopropiophenone derivatives in mice and in murine and human tissue culture cells. 868 3

Antimetabolic anticancer agents possess their own target enzymes: that of methotrexate is dihydrofolate reductase; 5-fluorouracil and ZD1604, thymidylate synthase; hydroxyurea, ribonucleotide reductase; 2'-deoxycoformycin, adenosine deaminase; N-(phosphonacetyl)-L-aspartate, aspartate transcarbamylase. Overproduction of each target enzyme has been observed with various animal and human cell lines which acquired resistance to all these agents. These facts suggest that this is a common mechanism for resistance to these agents. Most of these resistant cells showed amplification of the corresponding genes in double minute chromosome or homogeneously stained region of the chromosome. The relation between the degree of resistance and those of enzyme overproduction, the expression and amplification of the gene coding for each enzyme protein in various resistant cell lines are demonstrated and discussed.
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PMID:[Acquisition of resistance to anticancer agents by overproduction of target enzymes]. 915 48

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.
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PMID:Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability. 935 52


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