CHARACTERIZATION OF SELECTED STRONGLY AND WEAKLY INVASIVE SUBLINES OFA PRIMARY HUMAN-MELANOMA CELL-LINE AND ISOLATION OF SUBTRACTIVE CDNA CLONES

Invasion of basement membranes is a key step in systemic spread of tum our cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenot ypes from a primary human melanoma cell line by repeated passage throu gh a reconstituted basement membrane in vitro. The sublines differed a pproximately 5-fold in their invasive potential. Invasiveness correlat ed with better attachment and overexpression of the integrin alpha(v)/ beta(3) (vitronectin/laminin-receptor). Treatment with retinoic acid i nhibited proliferation in both sublines and invasion in the weakly inv asive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regard ing collagenase type-IV and retinoic-acid receptors but enhanced expre ssion of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wil d-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in in vasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melano mas which differ in their ability to invade basement membranes and int eract with components of the extracellular matrix. (C) 1995 Wiley-Liss , Inc.