CLONING AND CHARACTERIZATION OF A cDNA FOR THE 72 KD TYPE Iv COLLAGENASE AND DEMONSTRATION OF ITS INCREASED EXPRESSION IN HUMAN COLONIC ADENOCARCINOMAS

Abstract

Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. A 3.1 Kb cDNA clone for the 72 Kd type IV collagenase was identified by screening a human placental cDNA library in lambda gt11. Primer extension reactions with messenger RNA from A2058 melanoma cells revealed a single transcriptional start site for 72 Kd type IV collagenase that is located 50 bp upstream of the 5' end of the clone. Expression of the enzyme in human colon carcinoma was examined by blot hybridizations of total RNA from 19 primary colon tumors diagnosed as adenocarcinoma, and corresponding adjacent normal mucosa. The filters were probed with the cDNA clone. The results were expressed as the ratio of the mRNA levels in the tumor tissue to that in the adjacent normal mucosa (R value). In thirteen of eighteen cases (72%), the level of 72 Kd type IV collagenase was higher in the primary tumor than in the normal tissue. The cases were divided into high expression (R values 4.50 to 29.34) and intermediate expression (R values 2.54 to 3.31) subgroups. Both groups showed statistically significant (p<0.05) elevations when compared with the five cases showing the lowest levels of 72 Kd type IV collagenase mRNA expression (low expression subgroup, R values 0.96 to 1.48). The remaining case, a tubulovillous adenoma (benign polyp) had the lowest R value, 0.49. Immunoperoxidase staining of invasive colorectal tumor tissue and adjacent normal tissue confirmed the elevated levels of 72 Kd type IV collagenase associated with the tumor samples based on mRNA analysis.