Melanoma Cell Extracts Research Articles

Enzymatic Synthesis of Resveratrol α-Glucoside by Amylosucrase of Deinococcus geothermalis.

Glycosylation of resveratrol was carried out by using the amylosucrase of Deinococcus geothermalis, and the glycosylated products were tested for their solubility, chemical stability, and biological activities. We synthesized and identified these two major glycosylated products as resveratrol-4'-O-α-glucoside and resveratrol-3-O-α-glucoside by nuclear magnetic resonance analysis with a ratio of 5:1. The water solubilities of the two resveratrol-α-glucoside isomers (α-piceid isomers) were approximately 3.6 and 13.5 times higher than that of β-piceid and resveratrol, respectively, and they were also highly stable in buffered solutions. The antioxidant activity of the α-piceid isomers, examined by radical scavenging capability, showed it to be initially lower than that of resveratrol, but as time passed, the α-piceid isomers' activity reached a level similar to that of resveratrol. The α-piceid isomers also showed better inhibitory activity against tyrosinase and melanin synthesis in B16F10 melanoma cells than β-piceid. The cellular uptake of the α-piceid isomers, which was assessed by ultra-performance liquid chromatography (UPLC) analysis of the cell-free extracts of B16F10 melanoma cells, demonstrated that the glycosylated form of resveratrol was gradually converted to resveratrol inside the cells. These results indicate that the enzymatic glycosylation of resveratrol could be a useful method for enhancing the bioavailability of resveratrol.

Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2.

Reliance on aerobic glycolysis is one of the hallmarks of cancer. Although pyruvate kinase M2 (PKM2) is a key mediator of glycolysis in cancer cells, lack of selective agents that target PKM2 remains a challenge in exploiting metabolic pathways for cancer therapy. We report that unlike its structural analog shikonin, a known inhibitor of PKM2, lapachol failed to induce non-apoptotic cell death ferroxitosis in hypoxia. However, melanoma cells treated with lapachol showed a dose-dependent inhibition of glycolysis and a corresponding increase in oxygen consumption. Accordingly, in silico studies revealed a high affinity-binding pocket for lapachol on PKM2 structure. Lapachol inhibited PKM2 activity of purified enzyme as well as in melanoma cell extracts. Blockade of glycolysis by lapachol in melanoma cells led to decreased ATP levels and inhibition of cell proliferation. Furthermore, perturbation of glycolysis in melanoma cells with lapachol sensitized cells to mitochondrial protonophore and promoted apoptosis. These results present lapachol as an inhibitor of PKM2 to interrogate metabolic plasticity in tumor cells.

Lanthanide-based peptide biosensor to monitor CDK4/cyclin D kinase activity.

We describe a lanthanide biosensor that responds to CDK4 kinase activity in melanoma cell extracts through a significant and dose dependent increase in luminescence, thanks to sensitization of a DOTA[Tb3+] complex incorporated into a CDK4 substrate peptide by a unique tryptophan residue in an adjacent phosphoaminoacid binding moiety.

Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies

Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16INK4a-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16INK4a leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity.Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

Epigenetic impacts of ascorbate on human metastatic melanoma cells.

In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of ascorbate for melanoma therapy.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is expressed in melanoma cells

Sperm-specific glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is normally expressed only in sperms, but not in somatic tissues. Analysis of the expression of GAPDS mRNA in different cancer cell lines shows that the content of GAPDS mRNA is enhanced in some lines of melanoma cells. The purpose of the study was to assay melanoma cells for the expression of protein GAPDS. Three different lines of melanoma cells were investigated. By data of Western blotting, all investigated cells contain a 37-kDa fragment of GAPDS polypeptide chain, which corresponds to the enzyme GAPDS lacking N-terminal amino acid sequence that attaches the enzyme to the cytoskeleton of the sperm flagellum. The results suggest that GAPDS is expressed in melanoma cells without N-terminal domain. The immunoprecipitation of proteins from melanoma cell extracts using rabbit polyclonal antibodies against native GAPDS allowed isolation of complexes containing 37-kDa subunit of GAPDS and full-length subunit of somatic glyceraldehyde-3-phosphate dehydrogenase (GAPD). The results indicate that melanoma cells express both isoenzymes, which results in the formation of heterotetrameric complexes. Immunocytochemical staining of melanoma cells revealed native GAPDS in the cytoplasm. It is assumed that the expression of GAPDS in melanoma cells may facilitate glycolysis and prevent the induction of apoptosis.

Effect of human cell malignancy on activity of DNA polymerase ι

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.

A Simulated MS/MS Library for Spectrum-to-spectrum Searching in Large Scale Identification of Proteins

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.

HShroom1 links a membrane bound protein to the actin cytoskeleton

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with beta-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton.

Association between high activity of DNA polymerase iota and the development of human uveal melanoma

Enzymatic activity of DNA polymerase iota (Pol t) was analyzed in human uveal melanoma cell extracts, using an earlier elaborated approach. The Pol t activity was observed in seven out of eight malignant tumors, while it was absent in the normal uveal tract cells of the same patients. These findings serve as an additional confirmation of the Pol t oncogenic potential.

Inhibition of telomerase activity by a distamycin derivative: effects on cell proliferation and induction of apoptosis in human cancer cells

In this study, we evaluated the potential of the distamycin derivative MEN 10716 as a telomerase inhibitor. Exposure of human melanoma cell extracts to MEN 10716 induced a dose-dependent inhibition of telomerase activity, with an IC50 of 24±3 μM. When intact JR8 melanoma cells were chronically exposed to the drug (200 μM every other day for 50 days), a marked inhibition (>80%) of the enzyme's catalytic activity was consistently observed starting from day 1. At later points in time, MEN 10716 inhibited melanoma cell proliferation and induced apoptosis. Cells surviving MEN 10716 exposure were characterised by a higher melanin content and a greater expression of p16INK4A protein than control cells. The effects of MEN 10716 were subsequently evaluated in different tumour cell systems. In particular, even in the H460 non-small cell lung cancer cell line, chronic exposure of the cells to the drug (100 μM every other day for 50 days) induced a consistent inhibition (>85%) of telomerase activity, a reduction of cell proliferation potential, and apoptosis. Conversely, MEN 10716 treatment did not appreciably inhibit cell proliferation in the U2-OS telomerase-negative human osteogenic sarcoma cell line. Interestingly, no variation in the mean telomere length was observed in MEN 10716-treated JR8 melanoma cells, whereas an appreciable increase in the mean telomere length was found in H460 lung cancer cells after drug exposure. Overall, the results of the study indicate that MEN 10716 is a possible telomerase inhibitor and suggest that abrogation of telomerase activity can affect cell proliferation even through pathways that are not dependent on telomere erosion.

Immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of IL-10 production

Cultured melanoma cells release soluble factors that influence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunoreactive plaque, which encoded heavy-chain ferritin (H-ferritin). Previous studies have drawn attention to the immunosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma. These studies demonstrated, firstly, that H-ferritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by Western blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and consequently of the ratio of H- to L-ferritin expression. Suppression of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin. Similar results were obtained with H- and L-ferritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-10, which suggested that the immunosuppressive effect of H-ferritin was mediated by IL-10. Assays of cytokine production from anti-CD3-stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN-gamma and had only slight effects on IL-2 and IL-4 production. Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma.

Transglutaminase-Mediated Oligomerization of Galectin-3 Modulates Human Melanoma Cell Interactions with Laminin

Tumor cell adhesion and migration to laminin are important events during invasion and metastatic spread. Galectin-3, a multifunctional member of the galectin family, binds specifically the poly-N-acetyllactosamine residues of laminin and has been implicated in tumor invasion and metastasis. Galectin-3 is multimerized by transglutaminase, an enzyme that catalyzes cross-linking between glutamine and other aminoacid residues. In this study, we examined the consequences of transglutaminase-mediated galectin-3 oligomerization on the interactions between cancer cells and laminin. We first demonstrated that human galectin-3 is cross-linked by guinea pig liver transglutaminase, forms oligomers, and incorporates the marker 5-(biotinamido) pentylamine. Expression of transglutaminase activity in the A375 and A2058 human melanoma cell extracts was revealed by its ability to induce galectin-3 oligomerization and 5-(biotinamido) pentylamine incorporation. Transglutaminase-treated galectin-3 did not affect adhesion or migration of the melanoma cells to laminin but consistently induced a significant increase of the percentage of cell spreading compared to the control (23.5 +/- 2.3%, vs. 10.6 +/- 1.9% at 180 min, p < 0.05), or to untreated galectin-3 or transglutaminase alone. Our study is the first demonstration that human galectin-3 is oligomerized by transglutaminase with, as a consequence, a specific effect of melanoma cell spreading on laminin. This phenomenon could be of significance in the modulation of cancer cell interactions with laminin during tumor invasion and metastasis.

N-Oct 5 is generated by in vitro proteolysis of the neural POU-domain protein N-Oct 3.

The neural POU-domain proteins N-Oct 3 and N-Oct 5 were first identified in electrophoretic mobility retardation assays through their ability to bind to the octamer sequence ATGCAAAT. These two N-Oct factors are detected in extracts from tumor-derived and normal neural cells. They are present differentially, however, in extracts from melanocytes and melanoma cells: N-Oct 3 is present in extracts from both melanocytes and melanoma cells, whereas N-Oct 5 is more evident in extracts from metastatic melanoma cells. We show here that a cDNA encoding N-Oct 3 directs synthesis of both the N-Oct 3 and N-Oct 5 proteins and that the N-Oct 5 protein in neural and melanoma-cell extracts is also related to N-Oct 3. N-Oct 5, however, is apparently not expressed in vivo: It is not detected if cells are rapidly lysed in SDS or if extracts are prepared with a cocktail of protease inhibitors that includes the serine-protease inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). These data suggest that N-Oct 5 is a specific in vitro proteolytic cleavage product of N-Oct 3 and is not directly related to melanocyte malignancy.

High-Molecular-Weight Forms of Tyrosinase and the Tyrosinase-Related Proteins: Evidence for a Melanogenic Complex

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.

Plasmodium falciparum: Pfalhesin and CD36 Form an Adhesin/Receptor Pair That Is Responsible for the pH-Dependent Portion of Cytoadherence/Sequestration

The cytoadherent behavior of two Plasmodium falciparum (human malaria) cell lines, FCR-3 and IT04 (a cell line with elevated ICAM-1 adherence), was studied using CHO cells transfected with CD36 or ICAM-1 receptors as target cells. ICAM-1-mediated adherence was found to be relatively pH insensitive, whereas CD36-mediated adherence was pH sensitive and inhibited by monoclonal antibodies and peptides based on a region found in human band 3 protein and named pfalhesin. Immobilized pfalhesin was used as an affinity matrix to purify CD36 from extracts of C32 amelanotic melanoma cells, which have ICAM-1 as well as CD36 receptors, and bind both parasite cell lines. We conclude that pfalhesin and CD36-constitute an adhesin/receptor pair.

Cellular uptake and tubulin binding properties of four vinca alkaloids

The in vitro effects of four Vinca alkaloids, vinblastine (VLB), vincristine (VCR), vindesine (VDS) and vinepidine (VPD), on B16 melanoma proliferation, binding to bovine brain tubulin and B16 melanoma cell extracts, and uptake by the B16 cells were compared. The relative binding affinities to bovine brain tubulin were VPD > VCR ⋍ VDS > VLB with the K a for VPD being about 4-fold higher than that for VLB. On the other hand, the relative effects on B16 cell proliferation were exactly the opposite. Differences were found in the degree of concentration of the four alkaloids by the cells: 100-fold for VLB, 50-fold for VCR and VDS, and 20-fold for VPD. At the extracellular concentrations of drugs which inhibit proliferation by 50%, the intracellular concentration would still be far less than the tubulin concentration. Thus, it is likely that all of the Vinca alkaloids would be bound to tubulin and difference in uptake rather than K a values is the major factor in determining the relative effectiveness of the drugs. L cells showed 50% the sensitivity of B16 melanoma cells toward VLB and 30% the sensitivity toward VPD. The L cells also concentrated these drugs to a lesser extent than did the B16 cells.

Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine

Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase). 145C chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding 14C chloroethylthioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated β-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with NADPH as electron donor ad DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intracellularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.

Determination of catechol O-methyltransferase activity in relation to melanin metabolism using high-performance liquid chromatography with fluorimetric detection

A new sensitive method for the determination of catechol O-methyltransferase activity has been developed. The method is based on the O-methylation of the indolic intermediates of melanin metabolism. The substrate, 5,6-dihydroxyindole-2-carboxylic acid, is converted by the enzyme to two O-methylated products, which can be separated by high-performance liquid chromatography and measured with fluorimetric detection. The physiological presence of both substrate and products could be detected in crude melanoma cell extracts. The limit of sensitivity for detection of the O-methylated products is less than 0.5 pmol per injection. The method was compared with an earlier described HPLC method which makes use of uv detection of O-methylated products of 3,4-dihydroxybenzoic acid. The described method will be used to study the importance of catechol O-methyltransferase as a protective enzyme in (malignant) melanocytes.

Western blot analysis of serological responses following immunization with vaccinia viral lysates of melanoma cells

Western blot analysis was used to characterize the antibody response of melanoma patients during immunization with vaccinia-virus-induced melanoma cell lysates (VMCL). Strong antibody responses were detectable within 4 weeks from commencement of immunization against different fractions (from 3 to 10) in blots of VMCL. The approximate MW of the main fractions identified were 100, 91, 85, 77, 64-66, 49, 46, 43 and 34-36 kDa. Comparison of the reactivity of the sera with extracts of vaccinia virus suggested that the reactivity against VMCL was not against viral antigens in the lysates. Studies on autologous extracts from 7 patients immunized with VMCL indicated that the majority of the fractions identified in extracts of VMCL were also identified in autologous extracts of melanoma cells. This indicated vaccine-induced responses against the patients' tumor cells and not merely against alloantigens in the vaccine. Sera from the immunized patients appeared to identify a number of fractions of similar MW in extracts of colon and glioma cells, suggesting that the responses were not specific for melanoma. Possible exceptions were reactivity against melanoma fractions of 85, 64-66 and 36 kDa. Our studies provide evidence for the effectiveness of VMCL in inducing antibody responses against autologous melanoma cells and form a basis for subsequent biochemical and genetic analysis of the antigens identified by antibody responses in immunized patients.