sodium-dodecyl-sulfate and Melanoma

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.

Topics: Cathepsin B; Cathepsin L; Cathepsins; Culture Media, Serum-Free; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Electrophoresis; Humans; Hydrogen-Ion Concentration; Leucine; Lysosomes; Melanoma; Neoplasm Invasiveness; Sodium Dodecyl Sulfate; Substrate Specificity; Tumor Cells, Cultured

HMB-45 is an anti-melanoma monoclonal antibody widely used in diagnostic pathology owing to its great specificity in identifying poorly differentiated melanomas. In this study, by a series of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots with the enhanced chemiluminescent (ECL) detection method on the HU-214 melanoma cell line, we identified the antigen of HMB-45 in a protein or proteins of 30-35 kDa. Although this result is in discrepancy with the previous literature which identified the antigen as a protein of 7 or 10 kDa, a family of proteins of 25-70 kDa of as a protein of 100 kDa (gp100), the present data indicate that the antigen signal we found might be specific. Furthermore, immunoblots on neuraminidase-treated cell lysates show, in agreement with already published data, that the antigen might be a sialated glycoprotein with the sialic acid involved in the epitope. Immunoblots on partially purified melanosomes confirmed the presence of the antigen in these organelles.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Immunohistochemistry; Luminescent Measurements; Melanocytes; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Sialic Acids; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

The stability of complexes between serine proteinases and their inhibitors after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis has been claimed to indicate covalent bond formation. In this work we have investigated the effects of SDS on the stability of complexes between single-chain or two-chain tissue plasminogen activator (t-PA) and its inhibitor (PAI-1). Complexes formed by incubation of t-PA with PAI-1 for 15 min at 22 degrees C were further incubated with various amounts of SDS before being subjected to SDS-polyacrylamide gel electrophoresis. The molecular species in the gels were identified both by zymography or by autoradiography after immunoblotting with antibodies directed against either t-PA or PAI-1. It was demonstrated that the interaction of SDS with t-PA.PAI-1 complexes before electrophoresis resulted in a transition from the complexed state to the free forms of t-PA and PAI-1 in a time- and dose-dependent manner. The first-order dissociation rate constant in the presence of 35 mM SDS at 22 degrees C had a koff value of 1.4 x 10(-2) min-1, which corresponds to a half-life of 49.5 min. The t-PA released from the complexes was fibrinolytically active, whereas the released PAI-1 inhibited activator-dependent fibrinolysis. In a similar fashion, the well characterized nonacylated pair alpha 1-proteinase inhibitor-elastase was dissociated by SDS treatment, confirming the validity of our experimental approach to demonstrate the reversibility of t-PA.PAI-1 complexes. These results demonstrate that SDS-polyacrylamide gel electrophoresis traps the molecular species in the state in which the proteins existed prior to the analysis, and they suggest that under the conditions used, the interaction of t-PA with PAI-1 results in the formation of nonacylated reversible complexes. This phenomenon may be relevant to the pathophysiology of fibrinolysis and to the general mechanism of serine proteinase-inhibitor complex formation.

Topics: alpha 1-Antitrypsin; Humans; Immunoblotting; Kinetics; Leukocyte Elastase; Melanoma; Neutrophils; Pancreatic Elastase; Plasminogen Activator Inhibitor 1; Protein Binding; Sodium Dodecyl Sulfate; Tissue Plasminogen Activator; Tumor Cells, Cultured

A sensitive, specific, competitive enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of tyrosinase. Binding sites of anti-tyrosinase antibodies were competed for by purified tyrosinase adsorbed onto microtiter plates and a known (standard) or unknown (sample) amount of tyrosinase in solution. Adsorbed antibodies were detected by goat anti-rabbit IgG F(ab')2 labeled with peroxidase. A sensitivity range of 2.1 to 14 ng (30-200 fmol)/well was obtained. SDS was found to be the most suitable detergent for solubilizing the enzyme. Tyrosinase was extracted from B16 mouse melanoma and assayed by the ELISA. The tyrosinase content per mg melanoma protein was 505 +/- 106 (S.D.) ng. This assay is not only useful for measuring the content of normal tyrosinase in crude extracts but also is possibly applicable to detecting the unprocessed tyrosinases.

Topics: Animals; Catechol Oxidase; Enzyme-Linked Immunosorbent Assay; Melanoma; Mice; Monophenol Monooxygenase; Sodium Dodecyl Sulfate

The mRNA for human (tissue type) plasminogen activator from a human melanoma cell line (Bowes) was investigated in different translation systems. After translation of poly(A)-rich RNA in Xenopus oocytes a biologically active plasminogen activator was obtained. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the secreted translation products revealed a protein band precipitable with affinospecific antibody and migrating at the same position (apparent molecular mass of approximately 70000 Da) as the native melanoma cell product. Translation in rabbit reticulocyte lysate yielded an immunoprecipitable band migrating at position corresponding to a molecular mass of 52000 Da. Addition of 12-O-tetradecanoylphorbol 13-acetate to the cell cultures resulted in increased production of plasminogen activator. Concomittantly more poly(A)-rich RNA could be extracted from the cells and this RNA was more effectively translated by oocytes into biologically active plasminogen activator. Translation of poly(A)-rich RNA from phorbol-ester-treated cells in the reticulocyte lysate system yielded the 52000-Da protein also seen with RNA from untreated cells. However, in addition a prominent protein band of apparent molecular mass of 48000 Da was detectable. Its intensity increased with increasing doses of tetradecanoylphorbol acetate. This phorbol-ester-induced protein was not precipitable with the affinospecific antibody against plasminogen activator.

Topics: Animals; Cell Line; Electrophoresis, Polyacrylamide Gel; Female; Humans; Melanoma; Oocytes; Phorbols; Plasminogen Activators; Protein Biosynthesis; Rabbits; Reticulocytes; RNA, Messenger; RNA, Neoplasm; Sodium Dodecyl Sulfate; Tetradecanoylphorbol Acetate; Xenopus laevis

Topics: Angiomatosis; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Histiocytoma, Benign Fibrous; Humans; Intracellular Membranes; Melanoma; Membrane Proteins; Osteitis Fibrosa Cystica; Skin Neoplasms; Sodium Dodecyl Sulfate

The purpose of this study is to classify the membrane proteins from some cutaneous tumours (basal cell epitheliomas, squamous epitheliomas, melanomas) and possibly detect the ones which are characteristic of each type of tumour. The microsomal, mitochondrial and nuclear fractions were purified following the usual techniques. The membrane proteins were solubilized with sodium dodecyl sulfate, treated with iodoacetamide and chromatographed in SDS-acrylamide slab-gel.

Topics: Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Humans; Intracellular Membranes; Melanoma; Membrane Proteins; Skin Neoplasms; Sodium Dodecyl Sulfate

The microsomal, mitochondrial, and nuclear fractions of cutaneous tumors have been investigated. Cutaneous tumors were homogenized and microsomal, mitochondrial, and nuclear fractions were purified. The membrane proteins were solubilized with sodium duodecyl sulfate (SDS). The membrane lipids were removed and membrane proteins were solubilized again in a small volume of SDS solution and chromatographed in SDS-acrylamide slab-gel. The plates were stained with Coomassie Brilliant Blue to show protein bands. The preliminary results show that the electrophoretic profiles of microsomal proteins are characteristic of some tumors.

Topics: Carcinoma; Carcinoma, Squamous Cell; Chromatography, Gel; Fibroma; Humans; Intracellular Membranes; Melanoma; Membrane Proteins; Microsomes; Mitochondria; Neoplasm Proteins; Nuclear Envelope; Skin Neoplasms; Sodium Dodecyl Sulfate

Topics: Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Humans; Male; Melanoma; Plasminogen Activators; Skin; Sodium Dodecyl Sulfate

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Chromatography, Affinity; Chromatography, Gel; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immune Sera; Immunosorbent Techniques; Leukocytes; Male; Melanoma; Sodium Dodecyl Sulfate

Topics: Animals; Cell Fractionation; Chick Embryo; Chromatophores; Electrophoresis, Polyacrylamide Gel; Hair; Melanoma; Mice; Molecular Weight; Organoids; Proteins; Retinal Pigments; Skin Neoplasms; Sodium Dodecyl Sulfate