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Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells' ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration. These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.

Reverse transcriptase polymerase chain reaction for the detection of tyrosinase-mRNA-positive cells in peripheral blood of melanoma patients, as a possible marker of hematogenous dissemination, has demonstrated varying detection rates. This study examined the sensitivity and reproducibility of the technique using a protocol of multiple polymerase chain reaction to determine circulating melanocytic cells. For each of the 123 melanoma patients included in this study, four nested polymerase chain reactions were performed from two blood specimens requiring both polymerase chain reactions from at least one blood sample to be positive to consider a patient as positive. Thus, a definitive result was obtained in 98% of the cases, whereas only 1.6% lacked conclusive findings. Thus, we found a correlation between the tyrosinase detection rate and the clinical stage. Circulating tyrosinase-mRNA-positive cells were detected in 13% of patients with primary tumor, 17% with regional skin/lymph node metastasis, and 44% with distant metastasis. Positivity also correlated with known melanoma progression markers such as gender, tumor thickness, and histologic type. Positive results were obtained more frequently in (i) men compared with women, (ii) patients with thick primary melanomas (> 4 mm: 38%) compared with those with thinner tumors (1.1-4 mm, 22%; < or = 1 mm, 5%), and (iii) patients with nonclassifiable (38%), nodular (34%), and occult primary melanomas (30%) compared with those with acrolentiginous (17%), superficial spreading (9%), or lentigo maligna melanoma (0%). These findings suggest that detection of tyrosinase-mRNA-positive cells in peripheral blood is not an adequate marker for identifying melanoma patients with distant metastasis. Reverse transcriptase polymerase chain positivity in early melanoma stages, however, as corresponding to other prognostic parameters, may indicate increased risk for the development of hematogenous metastasis and may be of value as a progression marker.

For serial cultivation of normal human melanocytes media supplemented with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) are largely employed. By using a culture medium that permits cultivation of melanocytes without TPA, the effects of TPA on melanocyte proliferation, phenotype, and susceptibility to lymphokine-activated killer cells were studied. Addition of 50 ng/ml TPA to the medium induced rapid dendrite formation and increased the cell proliferation rate by 16-63% in mitogen-rich media (four of seven cultures, p < 0.01), and by 237% in mitogen-reduced media (p < 0.001). Furthermore, several phenotypic changes indicating early stages of melanocyte transformation were induced by 50 ng/ml TPA. These included increased expression of melanoma progression-associated antigens such as A.1.43 and A.10.33, upregulation of nerve-growth factor receptor as well as of the melanocyte-activation marker HMB-45 and of histocompatibility class I antigens. In contrast, the expression of the differentiation marker K.1.2 and of intercellular adhesion molecule-1 was decreased in TPA-treated cultures. Most of these changes persisted even after removal of TPA from the culture medium (> or = 2 weeks). Staurosporine, a protein kinase C inhibitor, modulated melanocyte-antigen expression similar to TPA, suggesting that protein kinase C downmodulation rather than activation by TPA is involved. In addition to the antigenic alterations, the susceptibility of TPA-treated melanocytes to lymphokine-activated killer cell cytotoxicity decreased by 40% (p < 0.01), possibly due to their altered surface antigen expression. The presented data reveal that the tumor promoter TPA hitherto used as a supplement of melanocyte culture media induces profound phenotypic and functional changes of the cultured cells, indicating incipient transformation of normal human melanocytes in vitro.

This review summarizes recent information on the effects of immunomodulatory cytokines on human melanoma cells. The action of interferon (IFN)-alpha, -beta, and -gamma has been extensively examined in melanoma and melanocyte cultures in vitro, and increasing information on the action of other cytokines is now available. All IFNs revealed a dose-dependent antiproliferative effect on melanoma cells with the highest growth inhibition caused by IFN-beta. Proliferation was also inhibited by interleukin (IL) 1-alpha and -beta, and tumor necrosis factor (TNF)-alpha. For IL-4, both growth-stimulatory and -inhibitory properties have been reported. Cellular differentiation in terms of melanin synthesis, formation of dendritelike structures, and antigenic changes was not affected by IFN-alpha or -beta. IFN-gamma, however, induced a more dedifferentiated and biologically more aggressive phenotype of melanoma cells. Histocompatibility antigen (HLA) class I molecules were found upregulated by all IFNs and by TNF-alpha, associated with a marked increase of melanoma cell lysis by tumor infiltrating lymphocytes in vitro. HLA class II molecules were de novo expressed or enhanced by IFN-gamma and TNF-alpha. The adhesion molecules ICAM-1, LFA-3, and VLA-2 were upregulated by IFN-gamma, TNF-alpha, and IL-1-beta, whereas melanoma-associated antigens were hardly affected by cytokines. It seems that both antiproliferative and immunomodulatory effects may contribute to the antitumoral activity of cytokines in vivo. In vivo application of cytokines as well as combinations with cytotoxic drugs, therefore, may be promising for future treatment strategies.

The effects of human recombinant interferon-alpha-2a (rIFN-alpha), natural interferon-beta (nIFN-beta) and recombinant interferon-gamma (rIFN-gamma) on the proliferation, morphology and antigen expression of cultured human melanocytes were studied in vitro. The investigations were performed in 12-O-tetradecanoylphorbol-13-acetate (TPA)- and serum-containing melanocyte growth medium (MGM), in TPA- and serum-free complete melanocyte medium (CMM) and its mitogen reduced variant (RMM). In MGM, none of these interferons inhibited the growth of normal melanocytes at concentrations 1-10,000 international units (IU)/ml over a period of 5 d. Only nIFN-beta, dose dependently, inhibited melanocyte proliferation in CMM and RMM in a 6- and 12-d assay (growth inhibition at 10,000 IU/ml; 77-80% of the controls, p less than 0.001). In contrast, rIFN-alpha and rIFN-gamma exerted no (RMM), or minor effects (CMM) on melanocyte proliferation (only in 12-d assays at 10,000 IU/ml: 24% and 21% of the controls respectively, p less than 0.01). In parallel experiments performed on melanoma cells, all three interferons were potent inhibitors of proliferation in a 5-d serum-free assay (growth inhibition at 10,000 IU/ml; rIFN-alpha 59%, nIFN-beta 78%, rIFN-gamma 56%, all p less than 0.001). In addition, nIFN-beta and also rIFN-gamma caused striking morphologic changes of normal melanocytes in vitro. Especially under greater than or equal to 10 IU/ml rIFN-gamma cytoplasmic spreading and flattening of the cultured melanocytes and their nuclei were seen, thus resembling melanoma cells in vitro. Untreated human melanocytes grown in MGM showed high expression of the melanoma-associated antigens HMB-45 (95-100%) and K.1.2 (40-100%), whereas the progression marker A.1.43 was present only on less than 5% of the cells. Cultured melanocytes were 95-100% positive for histocompatibility antigen class I (HLA-I), 30-75% were positive for ICAM-1, whereas they were negative for HLA-DR. After treatment with rIFN-alpha, increased expression of HLA-I antigens was found; nIFN-beta and rIFN-gamma decreased the labeling with HMB-45 (75-100%) and with K.1.2 (25-80%), whereby the expression of A.1.43 was found slightly increased (5-15%). The HLA class I antigens were upregulated by both nIFN-beta and rIFN-gamma, nIFN-beta being the most potent agent. Also, both nIFN-beta and rIFN-gamma increased the expression of ICAM-1 (nIFN-beta, 75-90%; rIFN-gamma, 90-95%) and induced de novo expression of HLA-DR antigen (nIFN-beta, 15-20%; rIFN-gamma, 65-95%).(ABSTRACT TRUNCATED AT 400 WORDS)

Monolayer cultures of the human melanoma cell lines StML-12, StML-11, StML-14 (third, respectively, twenty-fifth subculture), and SKMel-28 derived from specimens representing different stages of tumor progression were treated with 10-10,000 U/ml rTNF-alpha applied for 72 h. The effects of rTNF-alpha on cell proliferation, DNA synthesis, cell viability, cloning efficiency, cell division, cell morphology, and the immunophenotype were studied in triplicate experiments. The cell line StML-14(3) revealed a significantly dose-dependent reduction of growth due to both cytostatic and cytotoxic activities of rTNF-alpha as well as a decrease of CE. Increased numbers of cells in prophase were observed 24 h after addition of r-TNF-alpha. In addition, dislocation of chromosomes in the metaphase, formation of micronuclei, and dose-dependent increases of cells exhibiting micronuclei and the DNA amount per cell were detected at the end of treatment. On the other hand, only a slight sensitivity to the anti-proliferative effect of rTNF-alpha was observed with StML-14(25) and SKMel-28, whereas StML-12 and StML-11 were significantly resistant. The last four cell lines were serially subcultivated and presented common phenotypic patterns with more malignant characteristics than the cell line StML-14(3) before treatment. Overall, rTNF-alpha enhanced the malignant immunophenotype of the cell lines tested. It increased the expression of the "late" melanoma progression markers A.10.33 and A.1.43, and Ki67, and it decreased the expression of the "early" progression marker K.1.2. The expression of HLA-I, HLA-DR, and ICAM-1 was also enhanced after rTNF-alpha treatment, whereas in contrast to other cytokines, rTNF-alpha did not induce the de novo expression of HLA-DR in HLA-DR-negative melanoma cell lines. These findings indicate that rTNF-alpha induces cytostasis and decreases cell viability of certain rTNF-alpha-sensitive melanoma cells. These effects may result in selection of rTNF-alpha-non-sensitive human melanoma cell populations with higher proliferation rates and a more aggressive immunophenotype in vitro.

The antitumor activities of human interferon (IFN) alpha, beta, and gamma alone or in combination were studied on four human melanoma cell lines (StML-11, StML-12, StML-14, and SKMel-28) in various concentrations (1-50,000 IU/ml IFN alpha, 0.1-1000 IU/ml IFN beta, 1-10,000 IU/ml IFN gamma) in vitro. In all experiments IFN beta exhibited the most potent antiproliferative effect of all IFN tested. After 3 d of incubation a 50% growth inhibition was achieved with 20-40 IU/ml for natural IFN beta and with 600-1200 U/ml for recombinant IFN gamma. Substantially higher doses (7,000 to more than 50,000 IU/ml) of recombinant IFN alpha 2a were required to achieve a 50% growth inhibition. A strong synergistic antiproliferative activity resulted from the combination of IFN alpha with IFN gamma and IFN beta with IFN gamma. None of the IFN tested induced terminal differentiation of melanoma cells in vitro. The formation of dendrites was inhibited, and the portion of differentiated cells in vitro was reduced after treatment with IFN in comparison to the untreated controls (untreated controls: 100%; portion of differentiated cells after treatment with IFN alpha: 58%-74%, IFN beta: 48%-96%, IFN gamma: 10%-33%). The melanin synthesis was slightly elevated after treatment with IFN alpha (untreated controls: 100%; after treatment with IFN alpha: 103%-157%, ns.) and decreased significantly after treatment with IFN beta (49%-71%, p less than 0.05) as well as with IFN gamma (80%-88%, ns.). Cell surface markers were modulated varyingly by the IFN: HLA-I antigens were enhanced by all IFN, with IFN beta emerging as the most potent inducer. Only IFN gamma, however, induced a de novo expression of HLA-DR and -DQ antigens and increased the expression of the ICAM-1 molecule and of the melanoma progression marker A.1.43. Possibly, these findings indicate a biologically more aggressive phenotype of melanoma cells.