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The 92-kDa Gelatinase B Is Expressed by Advanced Stage Melanoma Cells: Suppression by Somatic Cell Hybridization with Early Stage Melanoma Cells¹

Division of Cancer Research, Sunnybrook Health Science Centre, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5 [J. R. M., M. R. B., Y. L., J. R., R. S. K.], and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M4S 1K9 [J. R. M., R. S. K.]

The production and local release of various proteolytic enzymes, either by tumor cells or tumor-associated stromal cells, is thought to facilitate the malignant behavior of solid tumors. Human cutaneous melanoma offers an excellent clinical model to study the possible contribution of such proteases to solid tumor progression because melanoma goes through a series of well defined stages in its pathogenesis; moreover, permanent cell lines have been established from these various stages. As a first step to analyzing the gelatinolytic enzymes in melanoma pathology, we examined cell lines derived from early stage primary melanomas in which patients were cured of their disease and compared the results to those obtained with cell lines established from advanced stage primary lesions or metastases (i.e., from patients who eventually succumbed to the disease). We found that 80% of cell lines examined from early stage lesions constitutively produced only the 72-kDa gelatinase A but never the 92-kDa gelatinase B. In contrast, the majority of advanced stage cell lines examined produced both the 72-kDa gelatinase A and the 92-kDa gelatinase B. Advanced stage cell lines that did not constitutively produce the 92-kDa gelatinase B could be induced to do so with transforming growth factor ß, interleukin 1ß or 12-O-tetradecanoyl-phorbol-13-acetate. In total, 0 of 5 early stage cell lines constitutively expressed the 92-kDa gelatinase B, and only 2 of 5 could be induced to produce this activity. In contrast, all advanced stage cell lines that were evaluated either constitutively or inducibly produced the 92-kDa gelatinase B. To analyze the mechanism by which 92-kDa gelatinase B production is switched on in the advanced stage melanoma cell lines, somatic cell hybrids were constructed using an advanced stage melanoma cell line as one partner and either one of two early stage cell lines as the other. Constitutive production of the 92-kDa gelatinase B in such hybrids was lost and could not be induced in such hybrids. Coculture of the early and advanced stage cell lines failed to recapitulate what was seen after somatic hybridization, and zymographic analysis of lysates from hybrid cell lines demonstrated no 92-kDa gelatinase B activity. Reverse transcription-PCR analysis demonstrated that the loss of 92-kDa gelatinase B production occurred at the level of steady-state mRNA for the enzyme. The results indicate that malignant progression of human cutaneous melanoma is accompanied by the constitutive or induced ability to produce the 92-kDa gelatinase B, and that this switch in production is most likely related to the loss of a negative regulatory activity present in early stage nonmalignant melanomas.

¹ This work has been supported by NIH Grant R01-CA-42133 and The Medical Research Council of Canada. J. R. M. is supported by a Studentship from the Medical Research Council of Canada. M. R. B. is supported by a Fellowship from the Sunnybrook Trust Foundation. R. S. K. is a Terry Fox Research Scientist of the National Cancer Institute of Canada.

² Present address: Department of Cell Biology, Vanderbilt University, Nashville, TN 37232-2175.

³ To whom requests for reprints should be addressed, at Sunnybrook Health Science Centre, Division of Cancer Biology Reserach, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5.

Received 4/24/95. Accepted 7/12/95.

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