Stromelysins (SL) are growth factor- and cytokine-responsive- matrix metalloproteinases (NM) that cleave a wide range of extracellular matrix proteins. The SL subgroup of MMP genes encodes two highly homologous (80%) enzymes, SL-1 and SL-2, and a more distantly related member, SL-3, which exhibits about the same levels of homology with SL-1/2 and interstitial collagenase (40%). A body of evidence suggests that SLs are intimately involved in remodeling and destruction of the extracellular matrix both in health and disease. SL transcripts have been identified with relatively high frequency in malignant tumors and to a more limited extent in non-tumor tissues. This proposal has two objectives: (i) to determine the frequency with which SL genes are expressed in oral squamous carcinomas, and (ii) to determine whether the two most recently identified SL transcripts (SL-2 and SL-3) give rise to synthesis of fully functional proteins either by tumor cells or stromal cells. Studies in our laboratory have recently led to the discovery that SL-1 and SL-2 genes are expressed in a cell type specific manner in that fibroblasts express exclusively SL-1 whereas keratinocytes express exclusively SL-2. We have also obtained evidence that the SL-2 gene is constitutively expressed in at least some intraoral squamous cell carcinomas. It still remains unknown whether these tumors also express SL-3 and whether SL-3 transcripts are translated and a functional protein synthesized and secreted. I propose to address these questions primarily at the protein (rather than at the mRNA) level since any biologic effects of these enzymes are mediated by the functional enzyme protein. The strategy will be to raise murine monoclonal antibodies that are capable of discriminating between the three SL proteins. The frequency of SL gene expression in human intraoral squamous cell carcinomas will be determined by Northern analysis of tumor mRNA using SL-1, SL-2 and SL-3 specific cDNA probes. Synthesis and secretion of SL-1, SL-2 or SL-3 proteins by tumors that contain the corresponding transcripts, and by matching samples of healthy oral mucosa, will be examined by Western analysis and by immunoprecipitation of [35S]-methionine-labeled culture media derived from short term cultivation experiments. In order to determine whether SL genes are expressed by stromal cell, by tumor cells or by both, fresh frozen tumor tissue samples as well as explant cultures which contain both the stromal and the epithelial components will be analyzed by immunolocalization methods along with serially propagating isolated (epithelial/stromal) cell lines.
