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)

The neu proto-oncogene product has been found to exist in two interconvertible forms in G8/DHFR mouse fibroblasts. The 185-kilodalton form (p185) present in growing cells is replaced by a 175-kilodalton form (p175) under conditions of serum starvation. This low molecular weight form accounts almost exclusively for the phosphotyrosine content of the receptor and is associated with increased tyrosine kinase activity. Addition of serum, platelet-derived growth factor or tumor promoter induces conversion of p175 to p185 within minutes, and this increase in molecular weight is associated with phosphorylation of serine and threonine; removal of serum growth factors is followed by replacement of p185 with p175 over several hours. Unlike G8/DHFR cells, the human breast cancer cell line SK-Br-3 expresses a high molecular weight neu/HER2 receptor with unchanged phosphotyrosine content in both serum-starved and serum-stimulated cultures. These findings indicate that activation of the neu proto-oncogene product in G8/DHFR cells may be regulated in part by protein kinase C-mediated receptor transmodulation rather than by ligand availability alone.
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PMID:Modulation of a Mr 175,000 c-neu receptor isoform in G8/DHFR cells by serum starvation. 197 80

In an attempt to find potent antifolates with selectivity against tumor cells with intrinsic or acquired resistance to methotrexate, eleven nonclassical 2,4-diaminoquinazoline antifolates were synthesized and tested as inhibitors of dihydrofolate reductase from L5178Y cells. Several compounds appeared to be good enzyme inhibitors, with I50 values around 1 nM. Two of the compounds were also good inhibitors of cell growth in vitro. One of these (PKC-32, 9-(2,4-diamino-5-methylquinazoline-6-methylene)aminophenanthren e) appeared to be 100-fold more potent than methotrexate as an inhibitor of growth of a methotrexate-resistant cell line with impaired transport for methotrexate. PKC-32 and PKC-155 were also tested against mouse tumors in vivo. PKC-32 was modestly active in vivo as compared with methotrexate. This drug may be a useful agent in the treatment of methotrexate-resistant tumors.
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PMID:Synthesis and evaluation of 2,4-diaminoquinazoline antifolates with activity against methotrexate-resistant human tumor cells. 648 72

The c-erbB-2 receptor tyrosine kinase is often overexpressed in human tumors, but the functional implications of this phenotype remain unclear. We previously used phosphorylation-specific antibodies to define major differences in c-erbB-2 tyrosine kinase activity between overexpressing human tumor cell lines (Epstein, R. J., Druker, B. J., Roberts, T. M., and Stiles, C. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10435-10439). Here we extend this approach to define the relationship between c-erbB-2 tyrosine phosphorylation and protein kinase C (PKC)-dependent transmodulation. Phosphorylation-specific antibodies to the juxtamembrane PKC site Thr686 recognize tyrosine-dephosphorylated wild-type c-erbB-2 following G8/DHFR 3T3 cell treatment with PKC agonists. B104-1-1 cells transformed by activated c-erbB-2 express a subset of tyrosine-phosphorylated receptors that are homologously phosphorylated on Thr686, indicating that Thr686 phosphorylation alone is insufficient to abrogate receptor tyrosine phosphorylation. Similarly, the c-erbB-2-overexpressing human cancer cell lines SK-Ov-3 and BT-474 express constitutively Thr686-phosphorylated receptors. SK-Ov-3 cells express predominantly kinase-inactive c-erbB-2 that is heavily Thr686-phosphorylated, indicating that Thr686 phosphorylation in this line is heterologous in origin. In contrast, BT-474 cells express constitutively autophosphorylated c-erbB-2 despite Thr686 phosphorylation. These results indicate that Thr686 phosphorylation does not directly abolish c-erbB-2 activity and suggest that such phosphorylation reflects constitutive PKC activity induced by either receptor-activating mutations or heterologous growth factors. The latter possibility suggests in turn that c-erbB-2 interacts in an as yet undefined way with heterologous growth factor receptors in human tumor cells.
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PMID:Human cancer cells exhibit protein kinase C-dependent c-erbB-2 transmodulation that correlates with phosphatase sensitivity and kinase activity. 870 75

Amine-carboxyboranes with varying alkyl chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 microM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.
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PMID:Effects of alkyl amine carboxyboranes on L1210 DNA fragmentation and nucleic acid metabolism. 969 Dec 46

Tumour cell growth may be accelerated by protein kinase C (PKC) agonists such as phorbol esters and receptor tyrosine kinases, but receptor tyrosine kinases are in turn desensitized to growth factors by PKC agonists. To clarify this apparent PKC bifunctionality, we have used phosphoantibodies to determine the relationship between PKC-dependent phosphorylation events affecting the ErbB2 oncoprotein in G8/DHFR 3T3 cells. Neither the kinetics nor the extent of phorbol-induced juxtamembrane domain (Thr686) phosphorylation vary directly with C-terminal (Tyr1222) dephosphorylation, with Tyr1222 continuing to be dephosphorylated long after Thr686 phosphorylation has also declined. Platelet-derived growth factor (PDGF) mimics the short-term effects of phorbol on Thr686 and Tyr1222 phosphorylation, and confocal microscopy reveals that both of these PKC agonists induce rapid internalization of PKC-modified ErbB2. Phorbol causes sustained cytoplasmic accumulation of PKC-phosphorylated receptors, however, whereas PDGF triggers the appearance of this ErbB2 subset only briefly. Metabolic labelling and co-precipitation studies fail to implicate heterologous molecules in either the tyrosine dephosphorylation or internalization of PKC-modified ErbB2. Taken in the context of earlier juxtamembrane domain mutagenesis studies, these findings indicate that phorbol-activated PKC may desensitize growth factor receptors to extracellular ligands solely by triggering sustained receptor internalization. We submit that PKC-dependent juxtamembrane domain phosphorylation represents a physiological mechanism for shortening the duration and enhancing the specificity of growth factor signalling by promoting internalization of liganded and unliganded receptors, respectively.
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PMID:The duration of phorbol-inducible ErbB2 tyrosine dephosphorylation parallels that of receptor endocytosis rather than threonine-686 phosphorylation: implications for the physiological role of protein kinase C in growth factor receptor signalling. 985 18

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.
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PMID:Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells. 1138 17

We examined the effect of suboptimal concentrations of cyclin-dependent kinase inhibitors, which do not interfere with cell proliferation, on retinoblastoma expression in hamster (Chinese hamster ovary K1) and human (K562 and HeLa) cells. To achieve this, we used the chemical inhibitors roscovitine and olomoucine (which inhibit CDK2 preferentially), UCN-01 (which also inhibits CDK4/6) and p21 (as an intrinsic inhibitor). All chemical inhibitors and overexpression of p21 strongly induced retinoblastoma protein expression. UCN-01-mediated retinoblastoma expression was caused by an increase in both the levels of retinoblastoma mRNA and the stability of the protein. The expression of the transcription factor Sp1, a retinoblastoma-interacting protein, was also enhanced by all the cyclin-dependent kinase inhibitors tested. However, Sp1 expression was caused by an increase in the levels of Sp1 mRNA without modification in the stability of the protein. By using luciferase experiments, the transcriptional activation of both retinoblastoma and Sp1 promoters by UCN-01 was confirmed. Bisindolylmaleimide I, at concentrations causing a similar or higher inhibition of protein kinase C than UCN-01, provoked a lower activation of retinoblastoma and Sp1 expression. Finally, the effects of cyclin-dependent kinase inhibitors on dihydrofolate reductase gene expression were evaluated. Treatment with UCN-01 increased cellular dihydrofolate reductase mRNA levels, and dihydrofolate reductase enzymatic activity was enhanced by UCN-01, roscovitine, olomoucine and p21, in transient transfection experiments. These results support a mechanism for the self-regulation of retinoblastoma expression, and point to the need to establish the appropriate dose of cyclin-dependent kinase inhibitors as antiproliferative agents in anticancer treatments.
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PMID:The expression of retinoblastoma and Sp1 is increased by low concentrations of cyclin-dependent kinase inhibitors. 1465 8

Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.
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PMID:Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation. 1598 34

Drinking green tea is associated with decreased frequency of cancer development. This review outlines the wide range of mechanisms by which epigallocatechin gallate (ECGC) and other green and black tea polyphenols inhibit cancer cell survival. EGCG suppressed androgen receptor expression and signalling via several growth factor receptors. Cell cycle arrest or apoptosis involved caspase activation and altered Bcl-2 family member expression. EGCG inhibited telomerase activity and led to telomere fragmentation. While at high concentrations polyphenols had pro-oxidative activities, at much lower levels, anti-oxidative effects occurred. Nitric oxide production was reduced by EGCG and black tea theaflavins by suppressing inducible nitric oxide synthase via blocking nuclear translocation of the transcription factor nuclear factor-kappaB as a result of decreased IkappaB kinase activity. Polyphenols up- or down-regulated activity of a number of key enzymes, including mitogen-activated protein kinases and protein kinase C, and increased or decreased protein/mRNA levels, including that of cyclins, oncogenes, and tumor suppressor genes. Metastasis was inhibited via effects on urokinase and matrix metalloproteinases. Polyphenols reduced angiogenesis, in part by decreasing vascular endothelial growth factor production and receptor phosphorylation. Recent work demonstrated that EGCG reduced dihydrofolate reductase activity, which would affect nucleic acid and protein synthesis. It also acted as an aryl hydrocarbon receptor an-tagonist by directly binding the receptor's molecular chaperone, heat shock protein 90. In conclusion, green and black tea polyphenols act at numerous points regulating cancer cell growth, survival, and metastasis, including effects at the DNA, RNA, and protein levels.
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PMID:Mechanisms of cancer prevention by green and black tea polyphenols. 1701 50

We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3-4 mg (15)N-labeled protein suitable for NMR experiments. The (1)H-(15)N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.
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PMID:Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells. 1893 31


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