Metastatic NIH 3T3 x LTA cell hybrids express 72 kDa type IV collagenase

We previously reported that the murine fibroblast cell line LTA is tumorigenic but non-metastatic, and is non-responsive to a transfected H-ras oncogene. In contrast, NIH 3T3 cells are non-tumorigenic but are ras-responsive and become metastatic when transfected with ras. Somatic cell hybrids between LTA and NIH 3T3 cells are tumorigenic, metastatic and ras-responsive. Here we examined expression of type IV collagenases in parental LTA and NIH 3T3 cells (with and without ras) and four metastatic LTA x NIH 3T3 hybrids (also with and without ras). Parental NIH 3T3-derived cells had both 72 kDa and 92 kDa gelatinase activities, and LTA-derived cells had either aberrantly sized approximately 90 kDa activity alone or neither enzyme activity. All four metastatic hybrids expressed 60-72 kDa gelatinase activity, while three of them also had 92 kDa activity and one had only minimal 92 kDa activity. Thus the metastatic phenotype of the hybrids was associated with expression of 72 kDa gelatinase. Levels of RNA for tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) were relatively constant, suggesting independent regulation of type IV collagenases and their inhibitors. Southern blotting and probing with PCR-synthesized cDNA fragments of the mouse 72 kDa type IV collagenase gene showed that this gene was present in all cells although the structure of this gene in one of the three LTA cell lines differed from that of the other cells. Our results suggest that a key change in the metastatic hybrids, relative to non-metastatic parental LTA cells, is induction of expression of 72 kDa type IV collagenase.