Design and characteristics of a cavity-enhanced Fourier-transform spectrometer based on a supercontinuum source.

We report the in-house fabrication of a high-resolution Fourier-transform spectrometer (FTS) for the spectroscopy of molecules in the gas phase at resolutions down to 0.002 cm-1 working in the spectral range from 5880 cm-1 (1.7 µm) to 15 380 cm-1 (650 nm). The FTS employs a supercontinuum as a broadband light source and a He:Ne laser with a homemade frequency-stabilization scheme as the spatial reference for the sampling of the interferogram on a constant optical path difference (OPD) grid. The sampling of the two lasers is performed at constant time intervals, and the resampling process is performed at the software level. The resampling of the interferogram on a constant OPD grid relies on cubic approximations of the He:Ne interference pattern to determine its zero-crossings. The use of an invariant in the sampling process allows us to perform on-the-fly data treatment. Both the hardware aspect and the data processing are described with, in each case, an original approach. We also report the successful coupling of the FTS with a high finesse optical cavity with effective mirror reflectivities of 99.76%, allowing us to reach sensitivities down to 6.5 × 10-8 cm-1 with a root-mean-square accuracy of 0.0017 cm-1 on the position of the Doppler-broadened transitions with a mean transition width of 0.046 cm-1 for spectra recorded at a spectral resolution of 0.015 cm-1. The sensitivity of the instrument per spectral element, once normalized, represents the best sensitivity reported in the literature for Fourier-transform incoherent broadband cavity-enhanced absorption spectroscopy with a supercontinuum light source.

Cysteine deprivation promotes eumelanogenesis in human melanoma cells.

Melanocytic cells can produce two types of pigment, pheomelanin or eumelanin. We used two types of human melanoma cell lines to explore the regulation of pigmentation by biochemical and enzymatic studies. These two cell lines were previously designated as either pheomelanotic or of mixed type when cultured in a medium rich in cysteine. We analyzed the effects of L-cysteine depletion on melanin synthesis and the involvement of the tyrosinase-related proteins in the production of both eumelanin and pheomelanin. Cultures were exposed to L-cysteine concentrations ranging from 206 to 2.06 microM, and the following parameters were measured: tyrosine hydroxylase activity, intracellular L-cysteine and glutathione concentrations, eumelanin and pheomelanin formation, and tyrosinase-related protein-1 and -2 mRNA levels. Extracellular L-cysteine depletion significantly increased tyrosine hydroxylase activity and promoted both eumelanogenesis and visible pigmentation in both lines. In contrast, pheomelanogenesis was increased only in the pheomelanotic cell line. Whereas eumelanogenesis was apparent upon L-cysteine depletion, tyrosinase-related protein-1 expression was not induced in the pheomelanotic cells, and tyrosinase-related protein-2 expression remained unchanged. Thus, tyrosinase-related protein-1 mRNA expression seems to be concomitant with eumelanogenesis when the L-cysteine concentration is high, but does not appear essential for eumelanogenesis at low L-cysteine concentrations. The mechanisms governing pheomelanin to eumelanin balance are dependent on L-cysteine, glutathione, and tyrosinase-related protein-1 expression, but none of these factors alone appears to be dominant in directing the synthesis of a particular type of melanin.

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.

Glutathione depletion increases tyrosinase activity in human melanoma cells.

The aim of the present work was to estimate the effect of intracellular glutathione depletion on melanogenesis in human melanoma cells. We determined tyrosine hydroxylation activity, the rate-limiting step of the pathway, and 14C-melanin formation, an assay reflecting the global eumelanogenic pathway. Intracellular glutathione was depleted by treatment with buthionine-S-sulfoximine, a well-known inhibitor of gamma-glutamylcysteine synthetase. The intracellular depletion of glutathione was substantial after 20 h of incubation with 50 microM buthionine-S-sulfoximine, although a significant effect could be observed after 6 h. Tyrosine hydroxylase activity increased in parallel with glutathione depletion, to reach 160% with respect to the control values during 24 h of buthionine-S-sulfoximine treatment. We have found the response to buthionine-S-sulfoximine to be dose dependent and the two different human cell lines HBL and LND1 to have similar, if not identical, responses. 14C-melanin formation assay revealed even greater activation, up to 400% of the control values. This indicates that glutathione depletion may have two distinct effects: first, a direct one on tyrosinase activity and, second, an effect on the promotion of eumelanogenesis. The stimulation of tyrosine hydroxylase can be explained by a possible inactivation of the enzyme by endogenous thiol compounds rather than by a direct effect of buthionine-S-sulfoximine itself on tyrosinase. The data suggest that thiol compounds may play a role for stimulation of melanogenesis by ultraviolet radiation.

Chromosomal findings in cultured melanocytes from a giant congenital nevus.

A giant congenital pigmented nevus was observed in a newborn male. A melanocyte culture was established from nevus fragments removed at age 6 days. Analysis of 136 metaphases harvested from the primary culture showed 74% normal mitoses, 22% polyploids, and 4% mitoses with chromosome rearrangements involving in particular 1p, 12q, and 19p. In addition, the culture showed a high level of HLA-DR expression. Although histology showed no sign of malignancy, these findings may illustrate one of the first events which might eventually progress toward malignancy.

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.

Human melanoma targeting with alpha-MSH-melphalan conjugate.

A conjugate made of alpha-MSH as a drug carrier and melphalan has been designed in order to target human melanoma cells. Iodination of the alpha-MSH moiety led to a relatively stable tracer which could be easily separated and analysed by reverse phase high pressure liquid chromatography. The conjugate was found to be unstable at neutral pH and a serious denaturation can take place at concentrations exceeding 100 micrograms/ml, especially in plasma. Receptor-mediated cytotoxicity has been shown by the use of cultured alpha-MSH receptor positive/negative cells as well as in vivo B16 murine melanoma model. Body distribution and uptake of the labelled compound were unaltered as compared to those of labelled free hormone. alpha-MSH receptor recognition properties also remained unchanged with a better apparent affinity of the conjugate probably due to the alkylating activity of melphalan itself. Using human melanoma dendritic cells expressing more than 10,000 alpha-MSH binding sites per cell as an in vitro model, we were able to demonstrate higher cytotoxicities as compared to melphalan-treated cells. In contrast, melanoma cells with low receptivity did not show higher cytotoxicity. P388D1 mouse plasmocytoma cells lacking receptors were much more sensitive to melphalan than the conjugate. This phenomenon appeared to be related with the number of binding sites expressed at the time of the experiment as well as cell differentiation and the doubling time. Our findings strongly support the concept of a receptor-mediated cytotoxicity and may enable the in vivo melphalan delivery to target tissues to be increased, achieving an improvement of drug penetration inside melanoma cells.

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.

Compared cytotoxicity effects of five anticancer drugs on human (HBL) and mouse (B16) melanoma cells in vitro.

In order to assess the potential interest of replacing the murine cell lines by human cell lines for in vitro cytotoxic assays, the sensitivity and the selectivity of the murine B16 and the human HBL melanomas to five chemotherapeutic drugs were investigated in vitro. The cytotoxicities of Melphalan, Daunorubicin (DNR), Hexamethylmelamine (HMM), Hydroxymethylpentamethylmelamine (HMPMM), and Dihydroxymethyltetramethylmelamine (DHTMM), 2 HMM derivatives, were measured in the two cell lines using two different techniques: reduction of a tetrazolium derivative (MTT) and tritiated thymidine uptake into DNA. The cytotoxicity at inhibitory concentration 50 (IC50) was determined after one hour as well as after 2 days exposure of cell after one hour as well as after 2 days exposure of cells to each drug. The results indicate that the HBL human melanoma was generally more sensitive to Melphalan and DHTMM than the B16 murine melanoma cells as far as the IC50 was concerned. In contrast, no difference of sensitivity was found to DNR and DHTMM. HMM was found to be inactive in both cell lines. The analysis of variance on IC50 values showed that the sensitivity of murine and human melanoma cell lines to drugs was statistically different. Despite the identical selectivity of the two cell lines, two promising observations can be made as far as the comparison of the two cell lines is concerned: 1) the higher sensitivity of HBL human cell line to Melphalan in the in vitro assays and 2) the slightly lower sensitivity of HBL to DNR, a drug without clinical activity against human melanoma.

In vitro cytotoxicity of hexamethylmelamine (HMM) and its derivatives.

The cytotoxicity of hexamethylmelamine (HMM) and its metabolites was investigated in three murine cell lines: one in vitro naturally sensitive to HMM (RC) and two in vivo naturally resistant (P388 and P388D1). The percentage of viable cells was determined both by the in situ reduction of a tetrazolium salt (MTT assay) and by the uptake of labelled thymidine into DNA (3HTdR assay). Short (1h) and long (48h) exposures of cells to drugs were considered. In all experimental conditions used, HMM was found to be inactive, whereas its hydroxylated metabolite hydroxymethylpentamethylmelamine (HMPMM) and one analog N, N 'dihydroxymethyltetramethylmelamine (DHTMM) were found to be cytotoxic. The results further indicated that HMM must be metabolized before it can exert its cytotoxic effect. The activity of HMPMM and DHTMM was found unlikely to be related to extracellular or intracellular release of formaldehyde.

Comparison of two cytotoxicity assays--tetrazolium derivative reduction (MTT) and tritiated thymidine uptake--on three malignant mouse cell lines using chemotherapeutic agents and investigational drugs.

Two different techniques [reduction of a tetrazolium derivative (MTT) and 3HTdR uptake assays] were compared in order to evaluate the cytotoxic effects of different chemotherapeutic agents in vitro. The cytotoxicities of Melphalan, hexamethylmelamine and seven derivatives, and daunorubicin were measured on P388D1 mouse macrophage-like cell line, RC mouse renal carcinoma cell line, and B16 mouse melanoma cell lines. Growth inhibition was determined after one hour as well as after continuous (48 hours) exposure to drugs. The IC50 was calculated using an appropriate algorithm (FADHA) which allowed within and between run variabilities to be taken into account. Optimal conditions had to be elucidated for culture conditions before assay: number of cells/well and assay duration for each line. The MTT and 3HTdR uptake assays were found to be similar in terms of sensitivity and reproducibility, both for adherent and floating cell lines. However, the MTT assay has the advantages of low cost and time saving. It also avoids problems related to radioactivity manipulation and counting. Both techniques rank the same chemicals as active or inactive. The algorithm Fadha was found to be a very powerful mathematical tool for comparing the IC50 values obtained by both assays.

Human medullary thyroid carcinoma: cell cultures and xenotransplants in nude mice. Immunocytochemistry and calcitonin secretion.

Occult primary and recurrent medullary thyroid carcinomas (MTC) detected only by elevated calcitonin levels in the peripheral blood, generally after pentagastrin-test stimulation, are difficult to localize. Some new imaging procedures with radionuclide tracers or radiolabelled monoclonal antibodies against carcinoembryonic antigen seem to bring some potentially therapeutic benefits. We report our results with cell cultures and xenotransplants of human MTC with the intention of establishing reproducible models in vitro and in vivo. Cell cultures secrete calcitonin at up to 1200 pg/ml for periods ranging from 3 to 13 weeks. Immunocytochemistry detects cytoplasmic granules positive for calcitonin in polygonal epithelioid cells with dendritic processes. Xenotransplants in nude mice fare better in the subcutaneous axilla than in the subrenal capsule assay. In the former location the tumor-take is good and calcitonin is detected in the blood of the tumor-bearing animals, at levels ranging from 286 to more than 20,000 pg/ml. These models would be potentially usable as targets for radionuclide tracers and/or radiolabelled monoclonal antibodies.

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.

Use of an alpha-melanocyte-stimulating hormone analogue to improve alpha-melanocyte-stimulating hormone receptor binding assay in human melanoma.

In order to optimize the detection and measurement of alpha-melanocyte-stimulating hormone (alpha-MSH) receptivity in human melanoma cells, and the authors replaced the natural hormone by [Nle4,D-Phe7]-alpha-MSH, a more stable and potent analogue in the receptor binding assay commonly performed with alpha-MSH. The following parameters were investigated: temperature, incubation time, number of cells, and ratio of labelled to unlabelled hormone. Optimal conditions for each assay were determined. The results demonstrate that the analogue has identical binding sites to alpha-MSH, as similar reciprocal displacements of each labelled (125I) hormone by serial dilutions of unlabelled alpha-MSH or [Nle4,D-Phe7]-alpha-MSH (10(-12) M to 10(-6) M) were obtained. To further compare the two hormones, we performed a screening of various human cell lines: ten melanomas and five nonmelanomas. The assay with [Nle4,D-Phe7]-alpha-MSH yielded more receptor expression on six of ten melanoma lines against only four of ten with the natural hormone. In conclusion, the use of radiolabelled [Nle4,D-Phe7]-alpha-MSH analogue instead of labelled alpha-MSH improved both sensitivity and reproducibility in this receptor binding assay on human melanoma lines.

Alpha-melanocyte-stimulating hormone immunoreactivity in human melanoma metastases extracts.

Seven human melanoma metastases were extracted in order to check the possible presence of any alpha-melanocyte stimulating hormone (MSH) immunoreactivity. The aim of that study was to provide some explanation for, mainly, two observations that we have already made and reported: 1) increased plasma alpha-MSH levels in melanoma of tumour-bearing patients as compared with tumour-free patients; 2) the presence of specific alpha-MSH receptors on human melanoma cells in culture. We could measure large amounts of immunoreactive alpha-MSH in all tumours ranging from 0.31 to 4.27 pmoles per g of wet tissue. Further identification of the extracted material by high-performance liquid chromatography revealed compounds of higher molecular weight and more hydrophobic than synthetic alpha-MSH. In addition, purified extracts could also displace 125I-labelled alpha-MSH from its cellular binding sites in an alpha-MSH specific radio-receptor binding assay. Our findings would suggest a possible presence of some hormone precursor(s) inside the melanoma tumours.

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.