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 cytokine melanoma growth-stimulating activity (MGSA) is a growth factor for melanoma cells and a chemotaxin for neutrophils. Known purification procedures of MGSA from human sources or expression systems give a low yield and require multiple chromatography steps. Here, a fast and high-yield method for the purification of recombinant MGSA is described. Approximately 500 micrograms MGSA were recovered from the bacterial lysate of a 10 liter culture within a day. For this purpose, total mRNA of Hs294T melanoma cells was isolated and cDNA of MGSA was obtained by reverse transcription and polymerase chain reaction. The cDNA of MGSA was subcloned into the expression vector pGEX-2T, generating a fusion with the Schistosoma japonicum glutathione S-transferase gene. The fusion protein was expressed in E. coli DH5a and purified from the bacterial lysate using glutathione-sepharose beads. MGSA was cleaved from the complex of fusion protein and glutathione-sepharose beads with thrombin and purified to homogeneity by anion-exchange high-performance liquid chromatography with a Mono-S-column. The bioactivity of the recombinant MGSA was assessed by chemotactic migration and triggered [Ca2+]i-transients in human neutrophils. In addition, [125I]MGSA bound specifically to undifferentiated human leukemia cells HL-60 transfected with the cDNA of the interleukin-8 (IL-8) receptor beta with similar properties as [125I]IL-8. Thus, this described method might be a powerful tool to generate large amounts of cytokines in a short time.
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PMID:Single-step purification of recombinant melanoma growth-stimulating activity by anion-exchange high-performance liquid chromatography. 792 55

The nm23-H1 gene is regarded as a human homologue of the mouse nm23 gene, which was expressed in a non-metastatic subline of mouse melanoma K-1735. The expression levels of nm23-H1 mRNA and the levels of protein during induced differentiation of human leukemia cell lines were analysed. mRNA levels of the megakaryoblastic leukemia line MEG-01, which were induced to differentiate into megakaryocyte by TPA, decreased rapidly from 2 days after the start of treatment and became almost undetectable at day 4. Similar down-regulation of nm23-H1 mRNA was also observed in the induced differentiation of the promyelocytic leukemia line HL-60 by TPA, or DMSO into monocyte-macrophage lineage or granulocytes, respectively. The amount of Nm23-H1 protein was analysed by Western immuno-blot analysis using mouse antiserum raised against a recombinant fusion protein with glutathione S-transferase. The amount of Nm23-H1 protein also decreased during the induced differentiation of these leukemia cell lines. On the other hand, in the differentiation of the erythroleukemia line K562 by hemin, levels of both mRNA and protein of Nm23-H1 elevated transiently, then reduced to the original level. When MEG-01 and K562 were stably transfected with nm23-H1 cDNA, MEG-01 transfectants showed reduced sensitivity to the induction of differentiation, whereas K562 transfectants were better induced to synthesize hemoglobin than controls. These findings suggest the possibility that Nm23-H1 protein plays an important role to maintain the proliferation of immature leukemic cells in MEG-01 and HL-60, but it may also play a role in the early stage of K562 differentiation, possibly in the different manner.
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PMID:Alteration of nm23 gene expression during the induced differentiation of human leukemia cell lines. 805 9

Cellular drug resistance is believed to involve P-glycoprotein-related drug efflux as well as xenobiotic detoxification. In the present study, we analyzed five human melanoma cell lines with 1- to 6-fold doxorubicin resistance for doxorubicin retention and MDR-1 and GST pi gene expression. All the cell lines had high doxorubicin retention, and efflux blockers such as trifluoperazine and verapamil did not have a major effect on drug retention or cytotoxicity. Even though all the cell lines carried the MDR-1 and GST pi genes, gene amplification was not associated with drug resistance. Both laser flow cytometry and immunoperoxidase staining showed high expression of C-219 reactive P-glycoprotein in some of the resistant cells which was not accompanied by either high drug efflux or sensitivity to doxorubicin efflux blockers.
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PMID:Doxorubicin resistance in human melanoma cells: MDR-1 and glutathione S-transferase pi gene expression. 809 41

Glutathione transferases (GSTs) are enzymes involved in the resistance of tumor cells to bifunctional alkylating cytostatic drugs. We investigated the melphalan sensitivity together with activity and cellular concentration of GST isoenzymes of human melanoma cell line RPMI 8322 in different phases of the cell cycle. By centrifugal elutriation three cell fractions containing different proportions of cells in the G1 phase were isolated. Melphalan sensitivity was estimated by the colony formation assay. The cell fraction with the largest proportion of G1 cells was more sensitive to the drug than the fractions enriched in S and G2 cells. The GST activity of the cell fractions was measured with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate and the concentrations of GST P1-1, GST M1-1 and GST A1-1 were quantitated by use of isoenzyme-specific ELISA. The results show that there were less GST activity and lower GST P1-1 and A1-1 concentrations in the G1 cell enriched fraction, demonstrating a cell cycle dependence of GST expression. Thus, the cell fraction most sensitive to melphalan had the highest proportion of G1 cells and displayed the lowest GST activity, suggesting that the cell cycle dependent sensitivity to melphalan may at least partially depend on the expression of GSTs.
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PMID:Cell cycle dependent sensitivity of human melanoma cells to melphalan is correlated with the activity and cellular concentration of glutathione transferases. 829 55

Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.
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PMID:Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line. 861

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.
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PMID:Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin. 891 Apr 76

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.
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PMID:Inhibition of glutathione S-transferase activity in human melanoma cells by alpha,beta-unsaturated carbonyl derivatives. Effects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid, and trans-2-hexenal. 895 Feb 26

The incidence rate of testicular cancer has been steadily increasing during the last 50 years, and only cryptorchidism, i.e. undescended testes, has been identified as an important risk factor. An interplay between changing environmental factors and genetic susceptibility e.g. in foreign compound metabolizing enzymes, may have important influences on the risk. The aim of this study was to investigate if glutathione S-transferase mu (GST mu) deficiency, which in previous studies has been associated with malignant melanoma and cancers of the lung and bladder, is a risk factor of testicular cancer. Three hundred and seventy-eight men participated (80 seminomas, 104 non-seminomas and 194 controls) in a population-based case-control study. The phenotype of GST mu was determined in 366 men by ELISA, the genotype was determined in 324 men by polymerase chain reaction. The concordance between geno- and phenotype was 94.4%. The odds ratio of having the GST mu negative phenotype and testicular cancer was 1.08, (0.72-1.64; 95% confidence interval (CI)), and the odds ratio of having the GSTM1 null genotype and testicular cancer was 1.10; CI95% (0.71-1.70). This study provides no evidence of an association between phenotypically determined GST mu deficiency or GSTM1 null genotype and testicular cancer. The narrow confidence intervals rule out GST mu as a major single risk factor for testicular cancer.
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PMID:Genotype and phenotype of glutathione S-transferase mu in testicular cancer patients. 911 Mar 58

L-buthionine-S,R-sulfoximine (BSO) selectivley inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC50 values (microM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC90 for melanoma was 25.5 microM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 microM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-mu protein and mRNA levels were significantly reduced in both cell lines. GST-pi expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.
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PMID:Selective and synergistic activity of L-S,R-buthionine sulfoximine on malignant melanoma is accompanied by decreased expression of glutathione-S-transferase. 926 31

LIMPII (lysosomal integral membrane protein II) is one of a family of proteins structurally related to the cell surface glycoprotein CD36. We recently defined a single structural domain on CD36 that mediates binding to adhesive glycoprotein thrombospondin-1 (TSP1). The CD36-TSP1 interaction is known to play a role in platelet-tumor and platelet-monocyte adhesion, angiogenesis, and in monocyte uptake of apoptotic cells. To test whether LIMPII also binds TSP1, a LIMPII peptide corresponding to the TSP1 binding domain of CD36 was expressed as a recombinant glutathione S-transferase (GST) fusion protein. In solid phase binding assays, purified 125I-TSP1 bound to immobilized GST/LIMPII in a time-dependent and saturable manner. Inhibition by excess unlabeled TSP1 or EDTA demonstrated specificity. LIMPII.TSP1 complex formation was specifically blocked by soluble LIMPII fusion protein, by monospecific rabbit IgG directed against the LIMPII peptide and by CD36 fusion proteins containing the TSP1 binding domain. Transfection of Bowes melanoma cells with a chimeric LIMPII cDNA that targets expression to the plasma membrane conferred the ability to bind 125I-TSP1 and to adhere to TSP1-coated surfaces. This study defines a TSP1 binding site conserved between LIMPII and CD36 and suggests that cell surface LIMPII may function in some circumstances as an adhesion receptor for TSP1. Computer-assisted homology searches suggest that the TSP1 recognition motif identified from study of CD36 family members may be widely expressed in nature.
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PMID:Lysosomal integral membrane protein II binds thrombospondin-1. Structure-function homology with the cell adhesion molecule CD36 defines a conserved recognition motif. 947 26


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