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)

The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-alpha, is up-regulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF)-kappaB consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducible mechanisms for transcription of this gene are fairly well delineated, the mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood. Recently, we demonstrated an increased rate of IkappaB-alpha degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kappaB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkappaB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkappaB-alpha phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kappaB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkappaB-alpha in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkappaB that is phosphorylated at Ser-32 reacted with IkappaB-alpha in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-alpha or IKK-beta are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappaB, the IkappaB-alpha wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-alpha transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kappaB.
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PMID:Elevated constitutive IkappaB kinase activity and IkappaB-alpha phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-alpha transcription. 1009 73

The lineage-specific human tyrosinase promoter has been used to successfully target gene expression at the transcriptional level to melanoma cells. The tyrosinase promoter, alone and in combination with a single, or a dual, tandem melanocyte-specific enhancer, was used to regulate expression of the firefly luciferase reporter gene. Transient transfections of these tissue-specific luciferase constructs in human and murine melanoma (Pmel, B16mel) and colon carcinoma (WiDr, MC38) cell lines resulted in melanoma-specific luciferase expression that was amplified 5- and 500-fold with the addition of a single or double enhancer, respectively, to the tyrosinase promoter. When the double enhancer-promoter construct expressed the highly toxic Escherichia coli purine nucleoside phosphorylase (PNP) gene, transfection of the same cell lines followed by administration of the prodrug 6-methyl purine deoxyriboside (6-MPDR) at a concentration of 50 microM caused melanoma-specific in vitro cell killing. Within 5 days after prodrug administration methylthiazol-tetrazolium (MTT) cytotoxicity assays showed that only 15 and 9% of Pmel and B16mel cells, respectively, remained viable compared with controls. This effect was highly specific, as 90 and 96% of WiDr and MC38 colon carcinoma cells remained viable 5 days after identical treatment. This effect was a direct result of increased tissue-specific conversion of 6-MPDR to the toxic metabolite 6-methylpurine (6-MP), as documented by HPLC analysis of culture supernatants. These results show that the dual tandem melanocyte-specific enhancer provides powerful amplification of the transcriptional targeting of gene expression afforded by use of the tyrosinase promoter. This amplification translates into increased, highly specific cytotoxicity to melanoma by the PNP/6-MPDR enzyme/prodrug system and, therefore, has potential efficacy in the use of gene therapy for the treatment of metastatic melanoma.
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PMID:Augmentation of melanoma-specific gene expression using a tandem melanocyte-specific enhancer results in increased cytotoxicity of the purine nucleoside phosphorylase gene in melanoma. 1022 23

The tetracycline regulatory (TET) system provides a useful means of controlling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100-200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.
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PMID:Use of a recombinant parvovirus to facilitate screening for human melanoma cell clones expressing tetracycline-responsive transactivators. 1033 32

We sequenced 3201 bp upstream from the ATG translation start codon of the human melanocortin-1 receptor (MC1R). A number of transcriptional initiation sites were detected over a region of approximately 600 base pairs upstream of the receptor coding region. These consist of GC-rich regions, each including SP-1 consensus binding motifs. Neither a TATA nor a CAAT box was found in this region. The 5'-flanking region also contains the consensus regulatory elements for AP-1, AP-2, and several E-boxes. Gel shift assays targeting the three GC boxes confirmed binding of SP-1. A promoter assay revealed that the minimal region exhibiting promoter activity was located between nucleotides -517 and -282 in human melanoma SK-Mel-2 cells. Further deletion from -517 to -447, which removed an SP-1 site, completely abolished luciferase activity. In conclusion, the MC1R promoter shares the characteristics of many other GPCR promoters. These characteristics include GC-rich sequence, lack of a TATA box, and binding of SP-1.
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PMID:Characterization of the promoter region of the human melanocortin-1 receptor (MC1R) gene. 1046 96

The enhancement of gene transfection by ultrasound (US) was evaluated in vitro and in vivo using the B16 mouse melanoma model. Cultured cells were either exposed in suspensions in vitro or implanted subcutaneously in female C57BL/6 mice for 10-14 days and, subsequently exposed, in vivo. For comparison to results with a luciferase plasmid, a reporter plasmid for green fluorescent protein (GFP) was used to evaluate transfection efficiency. US was supplied by a system, similar to a Dornier HM-3 lithotripter, that produced shock waves (SW) of 24.4 MPa peak positive and 5.2 MPa peak negative pressure amplitudes at the focus. The plasmids were mixed with the suspensions to achieve 20 ,microL mL(-1), or were injected intratumorally to provide 0.2 mg DNA per mL of tumor. Acoustic cavitation was promoted by retaining 0.2 mL of air in the 1.2-mL exposure chambers in vitro and by injecting air at 10% of tumor volume in vivo. In vitro, cell counts declined to 5.3% of shams after 800 SW exposure, with 1.4% of the cells expressing GFP after 2 days of culture. In vivo, 2 days after 400 SW exposure, viable-cell recovery from excised tumors was reduced to 4.2% of shams and cell transfection was enhanced by a factor of about 8, reaching 2.5% of cell counts (p < 0.005 in t-test). These results show that strong tumor ablation induced by US shock wave treatment can be coupled with simultaneous enhancement of gene transfection.
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PMID:Ultrasonic enhancement of gene transfection in murine melanoma tumors. 1062 30

Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.
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PMID:Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. 1067 58

A new series of cationic lipids has been synthesized for gene delivery using 3,5-dihydroxybenzyl alcohol as the backbone and starting material. Using CMV driven expression system and luciferase gene as a reporter, we demonstrated that the transfection activity of these new lipids when formulated with Tween 80 as co-lipid is comparable to that of DOTAP, one of the most commonly used cationic lipids for transfection. Among the four different cell lines tested including murine melanoma BL-6 cells, human embryonic kidney 293 cells, HepG2 and HeLa cells, the highest transgene expression was seen in 293 cells. Results from in vivo experiments using mice as an animal model show that these cationic lipids preferentially transfect the cells in the lung upon tail vein administration. The cationic lipid, N,N,N-trimethyl-N-[3,5-bis(tetradecyloxy)benzyl] ammonium bromide 4c(di-C14:0) with two 14-hydrocarbon chains exhibits the best transfection activity. These results suggest that these new aromatic ring-based cationic lipids are useful transfection reagents for both in vitro and in vivo gene transfer studies.
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PMID:Synthesis and characterization of aromatic ring-based cationic lipids for gene delivery in vitro and in vivo. 1068 7

We describe the comparison of non-viral polycation transfection reagents, adenovirus-enhanced transferrinfection (AVET), polyethylenimine (PEI800) and transferrin-conjugated PEI800 (Tf-PEI800) in their ability to transfect murine and primary human melanoma cell lines. Expression of a reporter gene, cell surface marker and secreted protein (interleukin-2) was assessed for each vector system. Testing for luciferase reporter gene expression in murine and primary human cell lines, AVET and Tf-PEI800, both showed high levels of expression and comparable activity. Furthermore, when the melanoma cell line B16F10 was transfected with a cell surface marker up to approximately 97% of the cells expressed the protein on the cell surface. Assessing the levels of secreted IL-2 in murine cell lines, AVET/IL-2, Tf-PEI800/IL-2 and PEI800/IL-2 all expressed high levels of the cytokine (up to 20 microg IL-2/10(6) cells/24 h). In primary human melanoma cell lines, AVET/IL-2 transfected cells secreted more IL-2 than cells transfected with either Tf-PEI800/IL-2 or PEI800/IL-2. In murine melanoma cell culture experiments, positively charged PEI800/DNA and Tf-PEI800/DNA complexes gave similar transfection efficiencies. However, when subcutaneous tumors in mice were injected with the luciferase reporter gene complexed with either Tf-PEI800 or AVET, higher transfection activity was measured in the tumors as compared to ligand free PEI800/DNA complexes.
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PMID:Development of transferrin-polycation/DNA based vectors for gene delivery to melanoma cells. 1068 8

Genetic education of dendritic cells (DCs) with tumor-associated antigens is an encouraging development in DC-mediated tumor immunotherapy. In this study, to increase the transgene expression by DCs using nonviral vectors, a cytoplasmic T7 vector (T7T7/T7Luc) was used to transfect bone marrow-derived DCs with the firefly luciferase gene as a reporter and as a model tumor antigen. As a result, the luciferase activity of T7T7/T7Luc-transfected DCs was more than four times greater than that of DCs transfected with pCMVLuc, a commonly used nonviral vector. Furthermore, the luciferase activity was increased three times more when dendritic progenitor cells rather than mature DCs were transfected. In vivo tumor studies showed that T7T7/T7Luc-transfected DCs, which express high levels of luciferase (model tumor antigen), stimulated a stronger immune response than did pCMVLuc-transfected DCs, which express relatively low levels of luciferase, as indicated by the cytotoxic T lymphocyte assay. T7T7/T7Luc transfected DCs, when injected into recipient mice, evoked an antigen-specific immune response that can effectively eradicate implanted metastasis and prevent new tumor development by murine melanoma cells genetically modified to express luciferase. Therefore, the T7 system is a powerful nonviral vector that can be used to genetically educate DCs with tumor-associated antigens for tumor immunotherapy.
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PMID:Enhanced transgene expression and effective in vivo antitumor immune responses initiated by dendritic progenitors transfected with a nonviral T7 vector expressing a model tumor antigen. 1068 40

Induction of irreversible growth arrest and terminal differentiation in human melanoma cells following treatment with recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ) results in elevated expression of a specific melanoma differentiation associated gene, mda-7. Experiments were conducted to define the mechanism involved in the regulation of mda-7 expression in differentiating human melanoma cells. The mda-7 gene is actively transcribed in uninduced HO-1 human melanoma cells and the rate of transcription of mda-7 is not significantly enhanced by treatment with IFN-beta, MEZ or IFN-beta+MEZ. The high basal activity of the mda-7 promoter in uninduced melanoma cells and the absence of enhancing effect upon treatment with differentiation inducers is corroborated by transfection studies using the promoter region of mda-7 linked to a luciferase reporter gene containing the SV40 polyadenylation signal sequence. RT - PCR analysis detects the presence of low levels of mda-7 transcripts in uninduced and concomitant increases in differentiation inducer treated HO-1 cells. However, steady-state mda-7 mRNA is detected only in IFN-beta+MEZ and to a lesser degree in MEZ treated cells. We show that induction of terminal differentiation of HO-1 cells with IFN-beta+MEZ dramatically increases the half-life of mda-7 mRNA while treatment with cycloheximide results in detectable mda-7 mRNA in control and inducer treated cells. These observations confirm constitutive activity of the mda-7 promoter in HO-1 cells irrespective of differentiation status suggesting posttranscriptional processes as important determinants of mda-7 expression during terminal differentiation. The 3' UTR region of mda-7 contains AU-rich elements (ARE) that contribute to rapid mda-7 mRNA turnover during proliferation and reversible differentiation, a process controlled by a labile protein factor(s). Substitution of the SV40 polyadenylation signal sequence in the luciferase reporter plasmid with the mda-7-ARE-3'-UTR renders the Luciferase message unstable when expressed in proliferating and reversibly differentiated melanoma cells. In contrast, the luciferase message is stabilized when the mda-7-ARE-3'-UTR construct is expressed in terminally differentiated HO-1 cells. These results provide compelling evidence that mda-7 expression during terminal differentiation in human melanoma cells is regulated predominantly at a posttranscriptional level.
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PMID:Regulation of mda-7 gene expression during human melanoma differentiation. 1071 78


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