DIFFERENTIAL EXPRESSION OF MATRIX METALLOPROTEINASES IN ACTIVATED C-RAS(HA)-TRANSFECTED IMMORTALIZED HUMAN KERATINOCYTES

Elevated expression of matrix metalloproteinases (MMPs), a family of s ecreted proteinases that degrade matrix components of basement membran es and connective tissues, is strongly correlated with malignant expre ssion in various human epithelial cancers and epithelial cancer cell l ines. We have tested whether elevated levels of MMP expression are als o associated with malignant progression in human cutaneous squamous ce ll carcinoma. Constitutive levels of expression of steady-state mRNA a nd of secreted protein encoded by three MMP genes (matrilysin, gelatin ases A and B) were compared in a unique in vitro model of human skin c arcinogenesis. This model is composed of the parental immortalized non -tumorigenic human keratinocyte line (HaCaT), and three activated c-Ha rvey-ras-oncogene transfected variants (A-4, I-7 and II-4). Although c lone A-4 is non-tumorigenic, clones I-7 and II-4 exhibit benign and ma lignant tumorigenic phenotypes, respectively, after subcutaneous injec tion into athymic nude mice. Northern blot, Western blot, and zymogram analyses revealed three MMP-specific patterns of expression. Constitu tive matrilysin mRNA expression was markedly increased in the I-7 cell s compared with HaCaT, A-4 or II-4 cells. Secreted promatrilysin was d istinctly increased in the tumorigenic I-7 and II-4 cells compared wit h the nontumorigenic HaCaT and A-4 cells. Gelatinase A mRNA and secret ed gelatinase A protein levels were increased in each transfectant com pared with HaCaT. Both active and inactive forms of gelatinase A were detected, Gelatinase B transcripts were not detected, but an EDTA-inhi bitable gelatinase activity comigrating with gelatinase B was moderate ly enhanced in both tumorigenic variants compared with the non-tumorig enic cells. Because promatrilysin and 92-kDa gelatinase secretion were increased in both benign and malignant tumorigenic cells, and not rel ated to invasiveness in this model, it is concluded that enhanced cons titutive expression of these two MMPs is associated with acquisition o f the tumorigenic phenotype, before acquisition of the malignant pheno type.