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: UMLS:C0025202 (melanoma)
69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control ('TET ON' system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.
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PMID:Positive tetracycline control of expression of p15INK4B from an Epstein-Barr autonomous plasmid in a human melanoma cell line. 1072 18

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, mediates many of its effects through the intranuclear vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of nuclear receptors. Vitamin D receptor can directly regulate gene expression by binding to vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1alpha,25(OH)(2)D(3) have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the biologic activity of many naturally occurring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1alpha,24R, 25-trihydroxyvitamin D(3) (1alpha,24R,25(OH)(3)D(3)), 1alpha, 25-dihydroxy-3-epi-vitamin D(3) (1alpha,25(OH)(2)-3-epi-D(3)), 1alpha,23S,25-trihydroxyvitamin D(3) (1alpha,23S,25(OH)(3)D(3)), and 1alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D(3) (1alpha(OH)-24,25,26,27-tetranor-23-COOH-D(3); calcitroic acid) using the human G-361 melanoma cell line. Cells were cotransfected with a VDR expression plasmid and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1alpha,25(OH)(2)D(3) 24-hydroxylase (CYP24) promoter. Treatment with 1alpha,25(OH)(2)D(3) or the metabolites 1alpha,24R,25(OH)(3)D(3), 1alpha,25(OH)(2)-3-epi-D(3), and 1alpha,23S,25(OH)(3)D(3) resulted in transactivation of both constructs in a time- and dose-dependent manner, and a postitive regulatory effect was observed even for calcitroic acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24 mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent hormone. A ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [(3)H]-labeled 1alpha,25(OH)(2)D(3) from VDR than the parent hormone. Thus, we report that several natural metabolites of 1alpha,25(OH)(2)D(3) retain significant biologic activity mediated through VDR despite their apparent low affinity for VDR.
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PMID:Natural metabolites of 1alpha,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor. 1079 70

One critical factor in melanoma progression is the change from radial growth phase to vertical growth phase. We previously showed a high incidence of ras mutations in progressing but not early human melanomas. We also found that stable expression of activated Ras in a primary human melanoma cell line (WM35) led to enhanced proliferation, anchorage-independent survival, migration and invasion in vitro and enhanced subcutaneous tumor formation in vivo, transforming the melanoma phenotype from the radial growth phase to the vertical growth phase. Inhibitory cytokines, especially transforming growth factor-beta, are important in homeostasis of normal human melanocytes. Proliferation of early melanoma cells can be inhibited by transforming growth factor-beta, whereas more aggressive stages lose this response. Using a transforming growth factor-beta activated luciferase reporter transiently transfected into WM35, WM35N-ras, and WM35H-ras (WM35 transfected with mutant N-ras or H-ras genes), we demonstrated significant decreases (p < 0. 04) in transforming growth factor-beta induced reporter expression in both ras transfected cell lines. Transforming growth factor-beta also induced significant decreases (p < 0.002) in the proportion of WM35 cells in S-phase of the cell cycle; this effect was not observed in WM35N-ras cells. Furthermore, we demonstrated that an important controlling factor in transforming growth factor-beta inhibition of cell cycle progression, the phosphorylation of the Rb protein, was altered in WM35N-ras; transforming growth factor-beta caused a marked relative increase in hypophosphorylated pRb in WM35 cells, but not in WM35N-ras. These data suggest that activated Ras plays an important part in melanoma progression from the radial growth phase to the vertical growth phase by counteracting inhibition by cytokines such as transforming growth factor-beta, thus providing a growth advantage.
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PMID:Expression of activated N-ras in a primary melanoma cell line counteracts growth inhibition by transforming growth factor-beta. 1084 67

We have previously isolated the protein MIA, which is secreted from melanoma cells, and identified highly restricted expression patterns in melanocytic tumors. Preliminary studies of the human MIA gene provided evidence that the promoter is specifically activated in melanoma cells but is silent in nonmelanocytic cells and benign melanocytes. In this study we aimed to identify cis-regulatory promoter elements that mediate promoter activation during malignant transformation of melanocytes. We therefore subcloned 1.4 kb of the murine MIA promoter and a series of 5' deletion constructs into a luciferase reporter plasmid and identified the most active cis-regulatory element between nucleic acids -230 and -130. Cloning oligomeric fragments of this promoter region in front of a minimal TK promoter revealed a 30 bp enhancer element mediating expression of the reporter gene in melanoma cells but not in melanocytes or nonmelanocytic cells. Gel mobility shift assays and southwestern blots led to the identification of specific DNA-protein complexes in melanoma cells. Fine mutational analysis of the cis-regulatory promoter element showed that two critical nucleic acid residues are essential for both transcriptional activity and formation of the band shift complexes. By an initial small-scale affinity purification we were able to isolate a protein approximately 32 kDa in size from melanoma cells, which we refer to as MATF ("melanoma-associated transcription factor"). Our study identified for the first time MATF, a transcription factor that is upregulated or activated during malignant transformation of melanocytes.
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PMID:Characterization of a transcription factor binding site, specifically activating MIA transcription in melanoma. 1088 6

Enhancement of transgene expression is an important issue in human gene therapy. Here we describe a novel system for enhancing transgene expression by cointroduction of plasmid DNA with FR901228, a water-soluble histone deacetylase inhibitor. When a luciferase expression vector was cointroduced into cells with FR901228, luciferase gene expression was enhanced 50-fold in the mouse melanoma cell line B16-F1 and 5200-fold in NIH3T3 cells in comparison to cells without the drug. Luciferase gene expression enhancement was dependent on both drug dose and treatment time. Acetylated histones increased in accordance with drug dose, and the activation of gene expression occurred at the transcriptional level. The stimulation of luciferase gene expression by FR901228 was also observed in a B16-F1 clone stably expressing luciferase. Cointroduction of the luciferase plasmid with FR901228 into a B16-F1 tumor mass activated luciferase gene expression 3- to 4-fold. Thus, activation of transgene expression by FR901228 may serve as a new tool for gene therapy.
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PMID:Amplification of transgene expression in vitro and in vivo using a novel inhibitor of histone deacetylase. 1093 82

Treatment of human melanoma cells with a combination of recombinant fibroblast interferon (IFN-beta) and the protein kinase C (PKC) activator mezerein (MEZ) causes a rapid and irreversible suppression in growth and terminal cell differentiation. Temporal subtraction hybridization combined with random clone selection, reverse Northern hybridization, high throughput microchip cDNA array screening, and serial cDNA library arrays permit the identification and cloning of genes that are differentially expressed during proliferative arrest and terminal differentiation in human melanoma cells. A specific melanoma differentiation associated (mda) gene, mda-7, exhibits reduced expression as a function of melanoma progression from melanocyte to metastatic melanoma. In contrast, treatment of metastatic melanoma cells with IFN-beta + MEZ results in expression of mda-7 mRNA and protein. To evaluate the mechanism underlying the differential expression of mda-7 as a function of melanoma progression and induction of growth arrest and differentiation in human melanoma cells the promoter region of this gene has been isolated from a human placental genomic library and characterized. Sequence analysis by GCG identifies multiple recognition sites for the AP-1 and C/EBP transcription factors. Employing a heterologous mda-7 luciferase gene reporter system, we demonstrate that ectopic expression of either AP-1/cJun or C/EBP can significantly enhance expression of the mda-7 promoter in melanoma cells. In contrast, a dominant negative mutant of cJun, TAM67, is devoid of promoter-enhancing ability. Western blot analyses reveals that cJun and the C/EBP family member C/EBP-beta are physiologically relevant transcription factors whose expression corresponds with mda-7 mRNA expression. Electrophoretic mobility shift assays (EMSA) performed using nuclear protein extracts from terminally differentiated human melanoma cells document binding to regions of the mda-7 promoter that correspond to consensus binding sites for AP-1 and C/EBP. These results provide further mechanistic insights into the regulation of the mda-7 gene during induction of terminal cell differentiation in human melanoma cells.
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PMID:AP-1 and C/EBP transcription factors contribute to mda-7 gene promoter activity during human melanoma differentiation. 1094 17

Gene therapy as a form of molecular medicine is expected to have a major impact on medical treatments in the future. However, the clinical use of gene therapy today is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. In this study, we show that focused ultrasound provides an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the beta-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55- (Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro between 2% (Mewo) and 12% (AT1). The in vivo stimulation was assessed in the Dunning prostate tumor R3327-AT1 implanted subcutaneously in Copenhagen rats using the beta-galactosidase reporter. After intratumoral DNA injection, focused ultrasound induced a 10-fold increase of beta-galactosidase positive cells in histology and a 15-fold increase of beta-galactosidase protein expression in the ELISA assay. In contrast, ultrasound was not found to enhance reporter gene expression after intravenous plasmid application. Because ultrasound waves can be focused on different anatomical locations in the human body without significant adverse effects, the control of DNA transfer by focused ultrasound is a promising in vivo method for spatial regulation of gene-based medical treatments.
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PMID:In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound. 1100 72

The MGSA/GRO protein is endogenously expressed in almost 70% of the melanoma cell lines and tumors, but not in normal melanocytes. We have previously demonstrated that over-expression of human MGSA/GROalpha, beta or gamma in immortalized murine melanocytes (melan-a cells) enables these cells to form tumors in SCID and nude mice. To examine the possibility that the MGSA/GRO effect on melanocyte transformation requires expression of other genes, differential display was performed. One of the mRNA's identified in the screen as overexpressed in MGSA/GRO transformed melan-a clones was the newly described M-Ras or R-Ras3 gene, a member of the Ras gene superfamily. Over-expression of MGSA/GRO upregulates M-Ras expression at both the mRNA and protein levels, and this induction requires an intact glutamine-leucine-arginine (ELR)-motif in the MGSA/GRO protein. Western blot examination of Ras expression revealed that K- and N-Ras proteins are also elevated in MGSA/GRO-expressing melan-a clones, leading to an overall increase in the amount of activated Ras. MGSA/GRO-expressing melan-a clones exhibited enhanced AP-1 activity. The effects of MGSA/GRO on AP-1 activation could be mimicked by over-expression of wild-type M-Ras or a constitutively activated M-Ras mutant in control melan-a cells as monitored by an AP-1-luciferase reporter, while expression of a dominant negative M-Ras blocked AP-1-luciferase activity in MGSA/GRO-transformed melan-a clones. In the in vitro transformation assay, over-expression of M-Ras mimicked the effects of MGSA/GRO by inducing cellular transformation in control melan-a cells, while over-expression of dominant negative M-Ras in MGSA/GROalpha-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes.
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PMID:MGSA/GRO-mediated melanocyte transformation involves induction of Ras expression. 1103 Jan 54

Calcyclin (S100A6) is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity. The calcyclin gene promoter fragment -361/-167 activates transcription several fold when compared to the basal -167/+134 promoter fragment indicating the presence of enhancer element within -361/-167 bp region. By means of the electrophoretic mobility shift assay (EMSA) we found that this region contains a protein-binding site and mapped it to an E-box sequence at position -283/-278. Using antibodies against USF1 we identified the upstream stimulatory factor as the transcription factor bound to the E-box sequence in EMSA. This factor was also enriched in protein fractions obtained from Ehrlich ascites tumor cells nuclear extract by affinity chromatography using the E-box sequence as a ligand. Cotransfection of the USF1 expression vector with a plasmid carrying the luciferase gene under control of the -361/+134 calcyclin gene promoter fragment resulted in several fold activation of luciferase activity. On the other hand, mutations within the E-box led to a marked decrease in the efficiency of calcyclin gene promoter fragment. The results indicate that USF1 binds to an E-box sequence of the calcyclin gene promoter and enhances its transcription activity. This mechanism might be responsible for the upregulation of calcyclin gene expression in response to various stimuli and in tumors.
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PMID:Upstream stimulatory factor is involved in the regulation of the human calcyclin (S100A6) gene. 1111 18

Dendritic cell (DC)-mediated cancer immunotherapy is a very promising alternative approach to cancer treatment. In a previous study, we successfully transfected bone marrow-derived dendritic progenitors (BMDDPs) with a T7 vector--a nonviral, cytoplasmic-based autogene expression system--encoding a model tumor antigen, firefly luciferase, and subsequently stimulated the transfected cells to differentiate into DCs. When injected into experimental mice, those DCs generated a strong immune response against tumor cells bearing luciferase, which not only prevented occurrence of metastasis but also eradicated existing tumors. In the present study, we constructed a T7 vector encoding mouse tyrosinase, a well--known melanoma associated tumor antigen, and used it to transfect BMDDPs. Reverse transcriptase polymerase chain reaction and Western analysis confirmed the expression of tyrosinase by DCs differentiated from transfected BMDDPs. Two immunizations of these DCs at a dose of 2 x 10(6) of each successfully prevented tumor growth. More importantly, one injection of 2 x 10(6) of these DCs into mice followed by five doses of recombinant human interleukin-2 administration effectively eradicated existing tumors as indicated by pulmonary metastasis assay.
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PMID:Murine tyrosinase expressed by a T7 vector in bone marrow-derived dendritic progenitors effectively prevents and eradicates melanoma tumors in mice. 1112 87


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