Studies on factors influencing human plasma alpha-MSH.

The various physiological effects of alpha-MSH, mainly on the CNS and on pigmentation in animal models, are well documented in the literature. Only a few investigators have confirmed similar properties in the human. However, the possible physiopathological role played by this hormone in human melanoma is still poorly defined. In order to approach this subject in a manner as complete as possible, we have performed, during the past four years, three different series of experiments: 1) alpha-MSH measurements in plasma samples from: a. melanoma and other cancer patients, b. whole body UVA irradiated healthy adults, c. circadian rhythm determinations in melanoma patients and in healthy male adults; 2) alpha-MSH measurements in human melanoma tumours; 3) alpha-MSH receptor expression on human melanoma cells in culture involving: a. alpha-MSH radio-binding assays and b. tyrosinase assay. Our results so far show 1) increased alpha-MSH levels in melanoma patients' plasma, alpha-MSH responsiveness to UVA stimulated skin, large immunoreactive alpha-MSH content in melanoma metastases and an alpha-MSH circadian rhythm in some individuals different from cortisol; 2) alpha-MSH receptor expression in melanoma cells could be increased by various effectors able to stimulate melanogenesis.

Production of interleukin 1 by activated blood monocytes and alveolar macrophages from melanoma patients.

The production of Interleukin 1 (Il1) by circulating blood monocytes and alveolar macrophages was studied in melanoma patients. There were 144 patients in the monocytes study and 5 patients in the alveolar macrophages study. The Il1 activity was tested by a bioassay and reported in units based on the integration of the area under the curve. This was shown to be preferable to the standard method, i.e. probit analysis. Results showed that there was no depression of Il1 activity in melanoma patients as compared to control (98 + 32 units, versus 100 units). There was no difference when the values were compared according to sex, age and stage of the disease. However, a significant difference was found between phototype I and phototype IV. Alveolar macrophages, in all experiments (n = 5), had a significantly lower Il1 activity than the autologous monocytes. It is concluded that we can question the relevance of Il1 production by peripheral blood monocytes to the state of the immunity of melanoma patients.

Evidence for alpha-melanocyte-stimulating hormone (alpha-MSH) receptors on human malignant melanoma cells.

The presence of alpha-MSH receptors on human melanoma has so far been suggested in the literature but not proved. We describe a reproducible and specific binding assay of alpha-MSH on human melanoma cells, using a high-specific-activity 125I-labelled hormone (1.5 to 2 mCi/micrograms) with consistent receptor binding (usually exceeding 2 pg/10(6) cells) and stable for 3 weeks. Asynchronized cells in suspension were incubated for 15 min at 37 degrees C with the tracer and various concentrations of unlabelled hormones. Synthetic alpha-MSH was compared to beta-MSH, ACTH1-24, ACTH4-10, beta-LPH, CLIP, CRF, MIF I, A8VP and beta-endorphin. Out of a panel of 8 human melanoma cell lines, 3 showed specific and reproducible alpha-MSH binding curves. No significant binding to human fibroblast and human carcinoma cells was seen. alpha-MSH, beta-MSH and, to a lesser extent ACTH4-10 (a part of the alpha-MSH sequence) were the only peptides able to displace labelled alpha-MSH from its binding sites, indicating the high specificity of the MSH receptor. Affinity constants (Ka) ranged from 10(8) to 10(9) l/mole and the estimated receptor number was 1,000 to 2,000 per cell. We conclude that some human melanoma cell lines expressed specific MSH receptors with stable affinity but which are low in number.

Production of the effector molecule thymidine by human lung alveolar macrophages.

Cultured human alveolar macrophages (HAM phi) were found to produce soluble factors which inhibit tritiated thymidine [( 3H]-TdR) incorporation into tumour cells in vitro. We present evidence that thymidine (TdR) can be detected in HAM phi culture supernatants by thin-layer chromatography. Moreover, TdR secretion by HAM phi is an active process. Using [6-14C]-orotic acid as an early precursor to TdR and [3H]-TMP as the phosphorylation product, we were able to show that cultured HAM phi transformed them into labelled TdR. The very efficient thin-layer chromatography of the labelled metabolites was backed up by high-pressure liquid chromatography of nucleotides. HAM phi produce TdR by de novo synthesis and dephosphorylation. This phenomenon is due to the lack of thymidine kinase in normal mature macrophages. Since TdR, in high concentrations, can inhibit DNA synthesis through the 'TdR blockade' phenomenon, it is suggested that TdR secretion by HAM phi is a mechanism of non-specific modulation of tumour cell growth but highly restricted to the immediate macrophage microenvironment in vivo. The effectiveness of the thymidine gradient may thus be quite narrow, but is worthy of interest.

A new test for phagocytosis of human alveolar macrophages in suspension, using 99mTc labelled human serum albumin particles.

Current studies on human alveolar macrophages (HAM phi) indicate that two subpopulations may exist: adherent and nonadherent HAM phi. Most works in the literature were done exclusively on adherent HAM phi. In order to obviate to the loss of nonadherent HAM phi, we elaborated a new test for measuring endocytosis in HAM phi suspensions. According to this method, HAM phi in suspension were allowed to phagocytize millimicrospheres of human serum albumin labelled with 99m Tc (0.2 less than 0 less than 0.5 m; TcK9R - Cis Sorin Biomedica). Bound activity was separated from free activity by centrifugation on PercollR. The resulting internatant ring was found to contain activity bound to HAM phi which had phagocytized human serum albumin millimicrospheres. HAM phi were characterized by morphology and cytochemistry. The method here presented allows the functional study of both adherent and non-adherent HAM phi populations.

Monoclonal antibodies to human melanoma associated antigens: study of their specificity and visualization at the cellular level of the antigenic distribution.

Six monoclonal antibodies(Mabs) including 4 anti-melanoma, one anti-glioma, and one anti-HLA-DR have been tested in a 125I-protein A antibody binding assay using a panel of 34 different cell lines. This panel included 19 melanomas from different clinical and geographical origins, 10 fibroblast lines out of which 9 were established from melanoma patients, 2 glial cell lines, 1 osteosarcoma, 1 teratocarcinoma, and 1 murine melanoma. The reactivity pattern of the 4 anti-melanoma Mabs showed that they were not directed against antigens strictly restricted to melanoma, but rather against antigenic structures preferentially expressed on melanoma cells. These Mabs were found to crossreact with gliomas, thus they seem to recognize neuroectoderm associated differentiation antigens. The high crossreactivity of the anti-glioma Mab for melanoma was confirmed in this study. As expected from the literature, HLA-DR antigens were found to be expressed on more than 50% of the melanoma lines tested. The cellular distribution of the antigens recognized by two anti-melanoma Mabs on melanoma cells could be visualized by an autoradiographic procedure. From the labeling pattern it was concluded that only a proportion of the cells, varying from 13 to 38%, expressed the relevant antigen.

Effects of 5-fluorouracil on various melanoma cell metabolisms. Its use for assessing the cold thymidine pool.

5-Fluorouracil (5FU) increases the rate of incorporation of tritiated thymidine (3HTdR) and iododeoxyuridine (125 IUdR) into the cell and its DNA. 5FU has differential effect on metabolism of DNA through its specific action on thymidylate synthetase. Effects of 5FU on the incorporation of other metabolic precursors were studied, as well as the sensitivity of different cells to 5FU. This multiparametric approach enables us to predict the effect of 5FU on a cell type depending on its cold thymidine (TdR) pool size and the permeation of the cell to the drug. Furthermore, 5FU was used as a tool to recognize the difference between the effects of TdR on the 3HTdR and 125IUdR incorporation. Three parameters are superimposed: isotope dilution, inhibition of metabolic activity of the pyrimidine synthesis and metabolic competition between TdR and UdR. The measurement of the TdR pool size of the cell can be obtained using a specific equation, which is a function of the slope of the inhibition curve of the labelled precursors (3HTdR and 125IUdR) incorporation.

alpha-Melanocyte-stimulating hormone binding and biological activity in a human melanoma cell line.

Synthetic alpha-melanocyte-stimulating hormone (alpha-MSH) was found to bind to the plasma membrane of the HM6A human melanoma cell line, using an immunocytochemical method. When treated with 10(-7) to 10(-9) M alpha-MSH, melanoma cells exhibited an increase of intracellular cyclic adenosine 3':5'-monophosphate, followed by stimulation of tyrosinase activity. Significant inhibition of DNA synthesis measured by [3H]thymidine uptake and inhibition of cell growth was found. A retrovirus expression was detected in the supernatant of HM6A cells as assayed by the KC cell syncytium-forming test. In he presence of 10(-7) M alpha-MSH, the number of syncytium-forming units was increased 15-fold. These results demonstrate that alpha-MSH modulates human melanoma differentiation and virus expression in vitro.

Suppressor cell induction and reticuloendothelial cells activation produced in the mouse by beta 1-3 glucan.

We made a sequential study of the proliferative and functional changes occurring in RE cells after beta 1-3 glucan administration in BDF1, and C57BL mouse. beta 1-3 glucan was administered by single i.v. 50 mg/kg or i.p. 15 mg/kg injection. This successively induced changes in RE cells as follows: on day 3 a rise of acid phosphatase activity in peritoneal macrophages; on day 6 an increase of 3H-TdR incorporation in spleen and peritoneal lymphocytes together with an intense suppression of PHA and LPS responses by spleen cells; on day 10 a 5-fold increase of the percentage of peroxidase rich monocytes in the peritoneum. Thereafter all the values went back to or below control. Our results indicate that beta 1-3 glucan is an in vivo mitogen and a macrophage activator.

[Formation of pseudotypes of VSV after culture in a human melanoma]. / Formation de pseudotypes de VSV après culture dans un mélanome humain.

Electron microscope studies showed a high production of melanosomes and viral particles budding into the cisternae of the endoplasmic reticulum in cells derived from a subcutaneous metastasis. The history of this subline (HM6B-A) has been summarized elsewhere. An amelanotic subline (provided by the same patient) did not show similar virus particles. When infected with a vesicular stomatitis virus (VSV) thermolabile mutant (tl), these cells produced a VSV pseudotype in which a thermosensitive antigen was modified. Modification of surface VSV antigens was also detected by neutralisation tests. Using these tests, the VSV-pseudotype particles could be used as a tool to detect one or more antigens to a "putative" human melanomavirus, which might be only partially expressed.