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)

A neu/erb B2 ligand growth factor (NEL-GF) was purified to homogeneity from bovine kidney by a procedure involving ammonium sulfate fractionation (35-70% saturation) followed by sequential column chromatography on DEAE-cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). NEL-GF was found to be a 25-kDa polypeptide according to the analysis by gel filtration on Superdex 75 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NEL-GF stimulated the tyrosine-specific autophosphorylation of the neu/erb B2 gene product purified by immunoabsorbent and tyrosine-specific phosphorylation of the neu/erb B2 gene product in intact dihydrofolate reductase (DHFR/G-8 cells (NIH 3T3 cells transfected with rat c-neu). NEL-GF also down-regulated the cell surface neu/erb B2 gene product in DHFR/G-8 cells. NEL-GF was mitogenic toward NIH 3T3 cells, DHFR/G-8 cells, A431 cells (human epidermoid carcinoma cells), and SK-BR-3 cells (human breast carcinoma cells) but inactive toward bovine aorta endothelial cells. NEL-GF was sensitive to 0.1% trifluoroacetic acid but resistant to 5% beta-mercaptoethanol and appeared to be distinct from a neu protein-specific activating factor (Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991) Biochem. Biophys. Res. Commun. 179, 1536-1542) and a 30-kDa glycoprotein which competed with a monoclonal antibody for binding to the neu/erb B2 gene product (Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Slamon, D., and Lippman, M. E. (1990) Science 249, 1552-1555).
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PMID:Purification and characterization of the neu/erb B2 ligand-growth factor from bovine kidney. 135 Jul 85

In nontransformed DHFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosaccharide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endoglycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of approximately 3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa normal neu gene product was shortened to less than 30 min. In B104-1-1 cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogenically activated neu gene product was also synthesized as a 170-kDa precursor with endoglycosidase H-sensitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of less than 30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed increased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells. The mutationally activated neu gene product in B104-1-1 cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c-sis-transformed cells. These properties include: 1) accelerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in vivo tyrosine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the mutationally activated neu gene product may transform the cells by mimicking ligand-induced activation.
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PMID:Differential processing and turnover of the oncogenically activated neu/erb B2 gene product and its normal cellular counterpart. 196 62

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

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.
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PMID:Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation. 256 34

The neu gene is distantly related to the erbB gene and encodes a cell surface protein that appears to function as a growth factor receptor. To study the mechanisms that caused the conversion of the normal neu gene to an oncogenic allele, we have isolated molecular clones of the neu oncogene as well as a clone of the corresponding protooncogene. The transforming neu oncogene and the proto-neu gene clones exhibit identical restriction enzyme patterns. Amplification of the proto-neu gene in NIH 3T3 cells by means of cotransfection with a dihydrofolate reductase gene resulted in methotrexate-resistant colonies that produce high levels of normal neu-encoded p185 protein. In contrast to cells carrying low levels of the oncogene-encoded protein, these cells appeared normal. The results suggest that the lesion that led to activation of the neu gene is a minor change in DNA sequence and is apparently located in the protein-encoding region of the gene.
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PMID:Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles. 300 30

Heregulin (HRG) is a pluripotent growth factor that can stimulate the growth of some human mammary tumor cells and the differentiation of others. Two members of the epidermal growth factor receptor family of receptor/tyrosine kinases, p180erbB3 and p180erbB4, serve as receptors for the HRG ligand. While HRG appears to be capable of stimulating the autophosphorylation activity of p180erbB4, the co-expression of p185erbB2/neu with p180erbB3 is necessary for the HRG-stimulated tyrosine phosphorylation of both of these receptors. On the basis of the sequences surrounding their putative tyrosine phosphorylation sites, we predict that the different HRG-responsive receptors couple to different intracellular SH2 domain-containing proteins. Hence, the different receptors may mediate different cellular responses to the HRG ligand. In the present study we show that HRG beta 1 is mitogenic for erbB3-transfected DHFR/G8 cells, an NIH3T3 mouse fibroblast derivative that over-expresses p185erbB2/neu. HRG stimulated the incorporation of [3H]thymidine into the DNA of these cells with an EC50 of 70 +/- 7 pM. HRG was not mitogenic for parental DHFR/G8 cells that do not express the ErbB3 protein. Phosphatidylinositol (PI) 3-kinase, an enzyme believed to be important in cellular growth regulation by growth factors and oncogenes, is predicted to couple to tyrosine-phosphorylated ErbB3. We observed that HRG stimulated the association of PI 3-kinase with both p185erbB2/neu and ErbB3 in transfected DHFR/G8 cells, but not in the parental cell line. We conclude that the ErbB3 protein is capable of mediating a proliferative response of fibroblasts to HRG, and that the activation of PI 3-kinase is an integral part of the growth signaling mechanism.
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PMID:Heregulin stimulates mitogenesis and phosphatidylinositol 3-kinase in mouse fibroblasts transfected with erbB2/neu and erbB3. 753 67

PLC-gamma, ras-GAP and Shc have been proposed to be in vivo substrates for the neu-encoded p185neu receptor tyrosine kinases. We compared the tyrosine phosphorylation levels of PLC-gamma, ras-GAP and Shc in two NIH3T3 derived cell lines, transformed B104-1-1 and non-transformed DHFR/G8 cells in which point-mutation activated and normal rat neu genes were transfected and expressed, respectively. We found that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex were more significant in B104-1-1 cells than in DHFR/G8 cells, while no obvious difference could be detected for the tyrosine phosphorylation levels of ras-GAP and PLC-gamma between these two cell lines. Furthermore, we observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated neu. The truncated neu product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. Our results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role for cell transformation induced by the point mutation-activated neu, and that stable binding to mutant p185neu may not be necessary for Shc to mediate this signaling pathway.
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PMID:Tyrosine phosphorylation of Shc proteins and formation of Shc/Grb2 complex correlate to the transformation of NIH3T3 cells mediated by the point-mutation activated neu. 778 91

Chemotherapeutic drug resistance is a major clinical problem and cause for failure in the therapy of human cancer. One of the goals of molecular oncology is to identify the underlying mechanisms, with the hope that more effective therapies can be developed. Several mechanisms have been suggested to contribute to chemoresistance: 1) amplification or overexpression of the P-glycoprotein family of membrane transporters (eg, MDR1, MRP, LRP) which decrease the intracellular accumulation of chemotherapy; 2) changes in cellular proteins involved in detoxification (eg, glutathione S-transferase pi, metallothioneins, human MutT homologue, bleomycin hydrolase, dihydrofolate reductase) or activation of the chemotherapeutic drugs (DT-diaphorase, nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase); 3) changes in molecules involved in DNA repair (eg, O6-methylguanine-DNA methyltransferase, DNA topoisomerase II, hMLH1, p21WAF1/CIP1; 4) activation of oncogenes such as Her-2/neu, bcl-2, bcl-XL, c-myc, ras, c-jun, c-fos, MDM2, p210 BCR-abl, or mutant p53. An overview of these resistance mechanisms is presented, with a particular focus on the role of oncogenes. Some current strategies attempting to reverse their effects are discussed.
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PMID:Role of oncogenes in resistance and killing by cancer therapeutic agents. 909 Apr 98

The retinoblastoma tumor suppressor gene product (pRb) is involved in controlling cell cycle progression from G1 into S. pRb functions, in part, by regulating the activities of several transcription factors, making pRb involved in the transcriptional control of cellular genes. Transient-transfection assays have implicated pRb in the transcription of several genes, including c-fos, the interleukin-6 gene, c-myc, cdc-2, c-neu, and the transforming growth factor beta2 gene. However, these assays place the promoter in an artificial context and exclude the effects of far 5' upstream regions and chromosomal architecture on gene transcription. In these experiments, we have studied the role of pRb in the control of cell cycle-related genes within a chromosomal context and within the context of the G1 phase of the cell cycle. We have used adenovirus vectors to overexpress pRb in human osteosarcoma cells and breast cells synchronized in early G1. By RNase protection assays, we have assayed the effects of this virus-produced pRb on gene expression in these cells. These results indicate that pRb is involved in the transcriptional downregulation of the E2F-1, E2F-2, dihydrofolate reductase, thymidine kinase, c-myc, proliferating-cell nuclear antigen, p107, and p21/Cip1 genes. However, it has no effect on the transcription of the E2F-3, E2F-4, E2F-5, DP-1, DP-2, or p16/Ink4 genes. The results are consistent with the notion that pRb controls the transcription of genes involved in S-phase promotion. They also suggest that pRb negatively regulates the transcription of two of the transcription factors whose activity it also represses, E2F-1 and E2F-2, and that it plays a role in downregulating the immediate-early gene response to serum stimulation.
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PMID:Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein. 967 66

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