Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fibroblast growth factor (FGF) has been known to regulate the proliferation and differentiation of a variety of cell types via interaction with a specific FGF receptor on the cell surface. In the present study, Fgf8 cDNA of Mexican axolotl, Ambystoma mexicanum, was expressed in Escherichia coli as an MBP-FGF8 fusion protein. The cell proliferation activity of the recombinant FGF8 (rFGF8) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay. The addition of rFGF8 to the culture medium enhanced proliferation of BALB/c 3T3 and BHK21 cells about 1.4-1.5 fold. To analyze the binding activity of rFGF8 to the cell surface, cell surface enzyme linked immunosorbent assay was developed. Comparison of the structure of basic FGF with the computer-simulated structure of FGF8 suggested that Tyr-58, Glu-132, Tyr-139, and Leu-179 might be the potential receptor binding sites. Amino acid substitution muteins of FGF8 were constructed by PCR-derived directed mutagenesis and the muteins were overexpressed in E. coli. The rFGF8 muteins were purified and their binding activities were analyzed. Substitution of Tyr-58 or Glu-132 or Leu-179 of the FGF8 with alanine reduced the binding affinity, while substitution of Tyr-139 with alanine did not alter the binding affinity. These results imply that Tyr-58, Glu-132, and Leu-179 of FGF8 might be involved in its binding to the cell surface.
Mol Cells 2000 Dec 31
PMID:Expression and characterization of fibroblast growth factor 8 from Mexican axolotl, Ambystoma mexicanum. 1121 74

Topoisomerase II alpha is a critical gene involved in DNA replication and maintenance of genomic stability. Several chemotherapeutic agents target topoisomerase II and levels of expression are an important factor in chemosensitivity. Transcriptional regulation has been demonstrated to regulate topoisomerase II alpha levels under several circumstances, including cellular confluence, heat shock, and expression of oncogenes including ras and myb. Expression of topoisomerase II alpha is regulated by cellular proliferation; transcriptional down-regulation in confluent cells is modulated through sequences within the promoter. In this study, we examined DNA-protein interactions within the topoisomerase II alpha promoter in exponential and confluent phase NIH3T3 cells. Using electrophoretic mobility shift assay and in vitro DNase I footprint experiments, the involvement of NF-Y in transcriptional regulation was established. Incubation of the DNA minor groove-binding agents Hoechst 33342 and Hoechst 33258 with nuclear extracts revealed drug binding to regions surrounding the inverted CCAAT boxes within the topoisomerase II alpha promoter and displacement of proteins binding to these elements. Addition of both Hoechst 33342 and Hoechst 33258 to NIH3T3 cells at confluence resulted in increased expression of topoisomerase II alpha. In addition, MTT cytotoxicity assays in confluent cells showed an additive effect of incubation with Hoechst 33342 and the topoisomerase II alpha poison etoposide. Therefore, DNA binding drugs which block transcription factor activation of the promoter may deregulate topoisomerase II alpha and this strategy may be of value in modifying gene expression and modulating chemosensitivity.
Mol Pharmacol 2001 Apr
PMID:Transcriptional regulation of topoisomerase II alpha at confluence and pharmacological modulation of expression by bis-benzimidazole drugs. 1125 13

The LRM55 rat astrocytoma cell line was used to study the time and concentration effects of trimethyltin (TMT) exposure on intracellular adenosine triphosphate (ATP) levels, formazan production from (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) release. TMT concentrations of > or =50 microM produced a delayed increase in extracellular LDH from approximately 20% at 24 h to almost 70%, at 72 h. Twenty-four hours before cell lysis was detectable ATP levels decreased to less than 30% and formazan production declined to 70% (50 microM), 31% (100 microM), and 21% (200 microM) of control values. Concentrations of TMT (5 and 10 microM) that produced little or no LDH release also decreased ATP levels (62 and 49% of control, respectively) and formazan production (63 and 52% of control, respectively) by 48 h. These data support the hypothesis that TMT exposure interferes with energy production and that this event likely contributes to the delayed cell death seen in this cell line. Moreover, the declines in ATP and formazan production can occur at subtoxic concentrations in LRM55 cells.
In Vitr Mol Toxicol 2000
PMID:Trimethyltin reduces ATP levels and MTT reduction in the LRM55 rat astrocytoma cell line. 1131 77

Epidemiological studies have shown that there exists some correlation between cadmium exposure and human cancers. The evidence that cadmium and cadmium compounds are probable human carcinogens is also supported by experimental studies reporting induction of malignant tumors formation in multiple species of laboratory animals exposed to these compounds. In vitro studies with mammalian cells have also shown that cadmium is clastogenic, but its mutagenic potential is rather weak. In this research, we performed the MTT assay for cell viability to assess the cytotoxicity of cadmium chloride (CdCl2), and the CAT-Tox (L) assay to measure the induction of stress genes in thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2), by creating stable transfectants of different mammalian promoter-chloramphenicol acetyltransferase (CAT) gene fusions. Cytotoxicity experiments with the parental cell line yielded a LC50 of 6.1 +/- 0.8 microg/mL, upon 48 h of exposure. Four (metallothionein--HMTIIA, 70-kDa heat shock protein--HSP70, xenobitic response element--XRE, and cyclic adenosine monophosphate response element--CRE) out of the 13 constructs evaluated showed statistically significant inductions (p < 0.05). The induction of these genes was concentration-dependent. Marginal inductions were also recorded for the c-fos, and 153-kDa growth arrest DNA damage (GADD153) promoters, indicating a potential for CdCl2 to damage DNA. However, no significant inductions (p > 0.05) of gene expression were recorded for cytochrome P4501A1--CYP1A1, glutathion-S-transferase Ya subunit--GST Ya, nuclear factor kappa (B site) response element--NFkappaBRE, tumor suppressor protein response element--p53RE, 45-kDa growth arrest DNA damage--GADD45, 78-kDa glucose regulated protein--GRP78, and retinoic acid response element--RARE. As expected, these results indicate that metallothioneins and heat shock proteins appear to be excellent candidates for biomarkers for detecting cadmium-induced proteotoxic effects at the molecular and cellular levels. Induction of XRE indicates the potential involvement of CdCl2 in the biotransformation process in the liver, while activation of CRE indicates stimulation of cellular signaling through the protein kinases pathway.
Mol Cell Biochem 2001 Jun
PMID:Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride. 1167 4

Recent studies in our laboratory indicated that arsenic trioxide has the ability to cause significant cytotoxicity, and induction of a significant number of stress genes in human liver carcinoma cells, HepG2. However, similar investigations with atrazine did not show any significant effects of this chemical on HepG2 cells, even at its maximum solubility of 100 microg/mL in 1% dimethyl sulfoxide (DMSO). Further cytogenetic studies were therefore carried out to investigate the combined effects of arsenic trioxide and atrazine on cell viability and gene expression in immortalized human hepatocytes. Cytotoxicity was evaluated using the MTT-assay for cell viability, while the CAT-Tox (L) assay was performed to measure the induction of stress genes in thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2), by creating stable transfectants of different mammalian promoter-chloramphenicol acetyltransferase (CAT) gene fusions. Cytotoxicity experiments yielded LC50 values of 11.9 +/- 2.6 microg/mL for arsenic trioxide in de-ionized water, and 3.6 +/- 0.4 microg/mL for arsenic trioxide in 100 microg/mL atrazine; indicating a 3 fold increase in arsenic toxicity associated with the atrazine exposure. Co-exposure of HepG2 cells to atrazine also resulted in a significant increase in the potency of arsenic trioxide to upregulate a number of stress genes including those of the glutathione-S-transferase Ya subunit--GST Ya, metallothioneinIIa--HMTIIA, 70-kDa heat shock protein--HSP70, c-fos, 153-kDa growth arrest and DNA damage (GADD153), 45-kDa growth arrest and DNA damage (GADD45), and 78-kDa glucose regulated protein--GRP78 promoters, as well as the xenobiotic response element--XRE, tumor suppressor protein response element--p53RE, cyclic adenosine monophosphate response element--CRE, and retinoic acid response element--RARE. No significant changes were observed with respect to the influence of atrazine on the modulation of cytochrome P450 1A1-CYP 1A1, and nuclear factor kappa (B site) response element--NFkappaBRE by arsenic trioxide. These results indicate that co-exposure to atrazine strongly potentiates arsenic trioxide-induced cytotoxicity and transcriptional activation of stress genes in transformed human hepatocytes.
Mol Cell Biochem 2001 Jun
PMID:Atrazine potentiation of arsenic trioxide-induced cytotoxicity and gene expression in human liver carcinoma cells (HepG2). 1167 11

Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol.
Cell Mol Life Sci 2001 Nov
PMID:Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons. 1176 90

1. Cell death mode switch of cortical neurons from E17 rats was studied. Cells rapidly died under the serum-free condition. The time-course of cell death was markedly delayed by increasing cell density for primary culture in the trypan blue exclusion, LDH release, and MTT assays. 2. By analyzing cell death by the use of double staining using PI/TUNEL and PI/Annexin V combinations, the mode in the low density culture was found to be necrosis, while that in the high density culture was apoptosis. 3. The intracellular ATP level after the start of serum-free culture rapidly decline to 25% of 0-time level in the low density culture, but it was 60% in the high density culture. Both oligomycin and zVAD-fmk markedly decreased ATP levels and the population of TUNEL-positive neurons, while 3-aminobenzamide slightly increased these indices. 4. Thus. it is strongly suggested that the cell death mode switch from necrosis to apoptosis is closely related to intracellular ATP levels, and some conditioned medium factors observed in the high density culture may affect both ATP level and cell death mode switch.
Cell Mol Neurobiol 2001 Aug
PMID:Cell density-dependent death mode switch of cultured cortical neurons under serum-free starvation stress. 1177 63

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.
J Mol Neurosci 2001 Dec
PMID:A vasoactive intestinal peptide antagonist inhibits the growth of glioblastoma cells. 1185 29

Amyloid beta protein (Abeta) increases free radical production and lipid peroxidation in PC12 nerve cells, leading to apoptosis and cell death. The effect of ursolic acid from Origanum majorana L. on Abeta-induced neurotoxicity was investigated using PC12 cells. Pretreatment with isolated ursolic acid and vitamin E prevented the PC12 cell from reactive oxygen species (ROS) toxicity that is mediated by Abeta. The ursolic acid resulted in decreased Abeta toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and trypan blue assay. Thus, treatment with these antioxidants inhibited the Abeta-induced neurotoxic effect. Therefore, these results indicate that micromolar Abeta-induced oxidative cell death is reduced by ursolic acid from Origanum majorana L.
Mol Cells 2002 Feb 28
PMID:Ursolic acid of Origanum majorana L. reduces Abeta-induced oxidative injury. 1191 74

To evaluate whether leptin plays a putative role in breast tumorigenesis, we studied the expression of its long and short receptor isoforms in various tumoral breast tissues. We applied semiquantitative RT-PCR method to RNA extracted from 20 breast cancer biopsies and two human breast cancer cell lines (T47-D and MCF-7). Our results showed the expression of both leptin receptor transcripts in all tumoral tissues examined. By in situ hybridization experiments, we localized leptin receptors in proliferating epithelial cells. Study of leptin effects on human breast cancer cells growth was performed by [3H]-thymidine incorporation method and colorimetric MTT assay. We demonstrated that leptin (50-100 ng/ml) significantly stimulates proliferation of the human breast cancer cell line T47-D (P<0.05). Western blot analysis indicated that leptin induces a time-dependent activation of mitogen-activated protein kinases (MAPKinase) 1 and 2 in T47-D cell line. Moreover, the specific MAPK-inhibitor PD 98059 blocked cell proliferation induced by leptin. In conclusion, we demonstrate that leptin receptors are expressed in breast cancer and that leptin induces proliferation in the T47-D cell line via activation of the MAPKinases pathway. These data suggest that leptin and its receptors may be implicated in mammary cell proliferation and in breast cancer pathogenesis.
Mol Cell Endocrinol 2002 Feb 25
PMID:Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line. 1191 59


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