On the release and half-life of S100B protein in the peripheral blood of melanoma patients.

The aim of the present work was to investigate the origin and half-life of endogenous S100B protein reported by many investigators as a useful melanoma serum marker. Within cells, S100B protein exists in homo- or heterodimer form containing mainly Ca(++), having a substantial fraction bound to membranes. As such, S100B is believed to be involved in the regulation of cytoskeleton. Also, a role in the cell cycle progression has been suggested. Although S100B appears having important intracellular functions, proofs of its secretion, at least at concentrations such as the ones measured in melanoma patients, are still lacking. Consistent with this view is the fact that immunohistology for S100 protein reported by numerous authors clearly indicate an exclusive intracellular staining. For these reasons, it was of a major interest to investigate how and when S100B is shed to the blood. Knowing that significant S100B levels are seen only in stage IV patients, we hypothesized that cell death may be the major source of circulating S100B protein in these patients. This hypothesis was studied in an in vitro model simulating cell death and in vivo in melanoma and other cancer patients undergoing highly cytotoxic regional immunochemotherapy using isolated limb perfusion with tumor necrosis factor and melphalan, as well as in tumor exudates and pleural fluids. Our results strongly suggest melanoma and endothelial cell death and subsequent continuous drainage to the blood as the major mechanism behind S100B release to the blood circulation. We estimated the endogenous S100B protein half-life to be about 30 min.

Expression of the MC1 receptor gene in normal and malignant human melanocytes. A semiquantitative RT-PCR study.

Alpha melanocyte-stimulating hormone (MSH) and related proopiomelanocortin-derived (POMC) peptides bind to the melanocortin 1 receptor (MC1-R) of mammalian melanocytes and stimulate proliferation and melanogenesis. POMC transcripts and alpha-MSH-like immunoreactivity have been found in melanoma cells and a possible autocrine loop involving MC1-R and POMC-derived products has been proposed. Therefore, the alpha-MSH/MC1-R system plays a major role in the biology of melanocytes, and provides targets for melanoma diagnosis and therapy. However, the relative levels of MC1-R expression in normal melanocytes (NM) and melanoma cells are unknown, and it is still debated whether or not all human melanomas express the MC1-R. We describe a semiquantitative RT-PCR assay for MC1-R expression, using a competition vector generated by deleting 164 bp of the native gene. The competitor was employed to analyse a panel of human melanoma cells, tumour samples, giant congenital nevus cells (CNM) and normal melanocytes (NM). All samples were positive for MC1-R expression, but expression of the receptor gene did not correlate with that of tyrosinase. Expression levels were about 10 and 20 times higher for surgical specimens and cultured melanoma cells, respectively, than for NM, but comparable for CNM and NM. Thus, high MC1-R expression is a frequent event in malignant melanocytes, and might lead to a higher activity of the alpha-MSH/MC1-R system in melanoma cells as compared to normal melanocytes, for equal local concentrations of the hormone or related melanocortins.

alpha-Melanotropin immunoreactivity in human melanoma exudate is related to necrosis.

We have previously reported high immunoreactive alpha-MSH (IR-alpha-MSH) concentrations in melanoma patients' plasma, as well as significant amounts in melanoma metastases and cells grown in culture. Necrosis within the melanoma tumour leads to a massive proteolysis of intracellular proteins and release of cell content: this might significantly contribute to the elevated IR-alpha-MSH plasma levels measured in melanoma patients. To test this hypothesis, we studied the necrosis-related release of MSH from human melanoma cells, using a specific radioimmunoassay. The studies of fine-needle biopsies indicated that most of the human melanoma tumour exudates tested contained very high MSH concentrations (> 500 pg/ml; 14/15), while plasma levels were generally normal (< or = 25 pg/ml; 10/15). The level in an exudate from a non-melanoma tumour type was < 40 pg/ml. In vitro studies showed that release of the IR-alpha-MSH was time- and temperature-dependent, and related to cell death.

Immunoreactive alpha-melanotropin as an autocrine effector in human melanoma cells.

Melanotropin is a peptide having several functions, including the stimulation of melanogenesis and the modulation of proliferation of melanocytes and melanoma cells. It acts through binding to high-affinity receptors of the melanocortin-1 subtype, exclusively expressed in cells of the melanocytic lineage. Elevated levels of immunoreactive alpha-melanotropin were previously reported in melanoma cell lines, tumours and plasma from patients with melanoma. Here, we show that this high ectopic production of melanotropin is restricted to melanoma and non-pituitary tumours with the same neuroectodermic origin. The occurrence of a melanotropin-specific autocrine loop was further investigated in human melanoma cells. Immunoreactive alpha-melanotropin was spontaneously released from a melanoma cell line (HBL) expressing melanotropin receptors on the cell surface. This release was significantly increased in the presence of melanotropin-related peptides such as corticotropin-(4-10)-peptide and beta-melanotropin, competing for binding to the melanotropin receptor and was directly correlated to the displacement potential of these peptides. Both spontaneous and induced releases of immunoreactive alpha-melanotropin could be blocked at low temperatures, suggesting the involvement of intracellular protein movement in the release mechanism. The release of immunoreactive alpha-melanotropin was not significant in melanoma cells expressing very low levels of melanotropin receptors (IGR3) or in non-melanoma cells (SCC1). However, upon expression of the melanocortin-1 receptor cDNA into IGR3 cells, spontaneous and competition-induced releases of immunoreactive alpha-melanotropin were both increased and also blocked at low temperatures. This observation further underlines a role for the melanotropin receptor in the release of immunoreactive alpha-melanotropin. These experiments indicate that an autocrine loop between the melanocortin-1 receptor and immunoreactive alpha-melanotropin may be functional in human melanoma cells.

Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human melanoma cells due to the presence of alpha-MSH-specific receptors. This mechanism appeared to be both peptide- and cell-type-specific.

Partial characterization of IR-alpha-MSH peptides found in melanoma tumors.

Our previous work indicated that IR-alpha-MSH (immunoreactive alpha-melanocyte-stimulating hormone) plasma levels are three times as high in melanoma patients with progressing disease than in disease-free patients, and that the melanoma tumor itself may be the source of IR-alpha-MSH. Further identification of the material in tumor extracts has been carried out in this study, and the results presented here show that the immunoreactivity is associated with a major fraction of about 16 kDa and another of 5-9 kDa. Significant amounts of the immunoreactive material were also found in human melanoma cells but not in culture supernatants. The presence of this material may be related to the melanogenic status of the tumor cells. We have estimated the intracellular IR-alpha-MSH to be within a 0.4 to 2.3 nM range in melanoma tumor cells. We have investigated the melanogenic effect of the IR-alpha-MSH material and its relationship to alpha-MSH. Purified extracts both from metastases and cultured cells were found to promote frog skin darkening as well as tyrosinase activity in Cloudman S91 melanoma cells. The IR material could also displace labeled alpha-MSH from its binding sites in human melanoma cells. Our data clearly indicate that melanoma cells engage in an autocrine production of alpha-MSH-like bioactive peptides by melanoma cells, of larger mol.wt., which are able to bind to MSH receptors. These peptides may be involved in the regulation of melanogenesis and possibly in the growth and proliferation of melanoma cells by an autocrine/paracrine mechanism.

Degradation of alpha-melanocyte stimulating hormone (alpha-MSH) by CALLA/endopeptidase 24.11 expressed by human melanoma cells in culture.

The common acute lymphoblastic leukemia antigen (CALLA) is identical to human endopeptidase 24.11 (E-24.11) and is expressed on certain human melanoma lines. This work was conducted in order to investigate whether alpha-melanocyte-stimulating hormone (alpha-MSH) could be a substrate for E-24.11, its degradation leading to the negative alpha-MSH radiobinding assay results observed with some CALLA-positive cell lines. We used 3 human melanoma cell lines (GLL-19, Mel Juso and G361) which lack receptors to alpha-MSH and express CALLA, and, as a control, one CALLA-negative melanoma cell line (HBL) with specific receptors for alpha-MSH. Radioimmunoassays give evidence that alpha-MSH was degraded in the presence of the 4 melanoma cell lines and that disappearance of the peptide was significantly reduced by phosphoramidon in 2 lines (GLL-19 and G361). Upon incubation of alpha-MSH with GLL-19 and G361 cell membranes, 3 degradation products were completely abolished in the presence of phosphoramidon. Amino acid content analysis of alpha-MSH fragments produced by purified E-24.11 permitted identification of 6 peptide bonds in the sequence of alpha-MSH susceptible to cleavage by the enzyme. It is concluded that alpha-MSH is a substrate in vitro for purified E-24.11 and for the enzyme present on the human melanoma cell lines GLL-19 and G361, expressing a high level of endopeptidase activity. However, hydrolysis of alpha-MSH by this enzyme does not seem to represent the main factor responsible for the apparent absence of receptors for the hormone on some cell lines.