A family of phenotypically and biologically different transplantable hamster melanomas was derived from a single tumor more than 40 yr ago. In this work, we were seeking the differences between the abilities of the cells from two biologically heterogeneous (melanotic and amelanotic) members of this family to undergo spontaneous or camptothecin-induced apoptosis. We studied these differences by looking at three important features of the apoptotic process, i.e. binding of annexin V, DNA fragmentation and caspase-3 activity. Of these, annexin binding and DNA fragmentation were more pronounced in the parental, melanotic line while the activity of caspase-3 was stronger in the amelanotic tumor cells. We concluded that a spontaneous alteration of the original, melanotic melanoma line into an amelanotic one, associated with more aggressive tumor progression, was accompanied by significant decrease in ability to undergo spontaneous and camptothecin-induced apoptosis, and that apoptosis of these two cell types may not depend on the activity of caspase-3.

Objectives: Changes in the sensitivity of two lines of transplantable melanoma cells to the antiproliferative activity of interleukin 6 (IL-6), oncostatin M (OSM), tumor necrosis factor-α (TNF-α) during melanoma progression were the subject of this study. We were looking for a correlation between these changes and the ability of melanoma cells to undergo spontaneous apoptosis. Methods: The influence of exogenous cytokines on the proliferation of melanoma cells was measured by colorimetric methods with MTT and apoptosis of these cells was estimated by staining with annexin V/propidium iodide, measurement of DNA degradation and cell cycle analysis. Results: It was observed that during melanoma progression the sensitivity of those two melanoma line cells to the antiproliferative effect of IL-6 and OSM did not change, while a spontaneous alteration of the melanotic line into an amelanotic one seemed to be accompanied by resistance of the amelanotic melanoma cells to the antiproliferative activity of TNF-α. Simultaneously, we observed a decreased ability of amelanotic melanoma cells (in comparison with the native line) to undergo spontaneous apoptosis. Conclusions: The observed resistance to the TNF-α antiproliferative effect which appears during melanoma progression seems to be correlated with a lower ability of the amelanotic melanoma cells to undergo spontaneous apoptosis.

In the study described here we investigated the possibility of an association between the aggressiveness of melanoma and multidrug resistance phenotype by analyzing the expression and activity of P-glycoprotein (Pgp) in two genetically related transplantable hamster melanomas--a melanotic (Ma) and an amelanotic (Ab) form --which differed in aggressiveness and metastatic potential. Flow cytometric analysis of Pgp activity (using a verapamil-sensitive rhodamine R123 exclusion test) as well as Western blotting of cellular lysates showed its preferential (although not very marked) expression in the Ab melanoma cells. The Ab melanoma cells also exhibited a higher proportion of tumor-infiltrating lymphocytes (TIL), mostly of T cell phenotype, that may have reflected a higher immunogenicity of the tumor. In conclusion, Pgp activity appeared to be associated with less-differentiated more aggressively metastasizing melanoma (the Ab variant) although its role in maintaining this phenotype remains to be established.