Influence of bcl-2-Related Proteins on Matrix Metalloproteinase Expression in a Rat Glioma Cell Line Lisa Oliver, Karine Tremblais, Nathalie Guriec, Ste ´phane Martin,* Khaled Meflah, Jean Menanteau, and Franc ¸oise-Marie Vallette IFR 26, U 419 INSERM, 9 quai Moncousu, 44035 Nantes Cedex 01, France; and Clinique de Neurochrirugie, CHU, Nantes, France Received May 22, 2000 The expression of bcl-2-related proteins has been shown to be a key element in tumoral malignancy. The degradation of the extracellular matrix (ECM) by spe- cialized matrix metalloproteinases (MMPs) is another major step in tumor invasion and metastasis. We have examined, in a rat glioma cell line A15A5, the effect of the stable transfection of human bcl-2, bax and bcl-xl on MMPs expression. Using a zymographic assay, we found that all transfected cell lines expressed a gela- tinase activity which is predominantly associated with MMP-9. In bcl-2 and bcl-xl transfected cells, the transcription of MMP-9 was decreased compared to that of control or bax transfected cells. In addition, in bax transfected A15A5, we observed a down regulation of TIMP-1, the inhibitor of MMP-9. These results sug- gest that the ratio between MMP-9 and its inhibitor TIMP-1 is tightly controlled in cells overexpressing bcl-2 related proteins (i.e., high ratio in bax trans- fected A15A5 and low ratio in bcl-2 transfected A15A5). However, MMPs secreted by bcl-2 transfected cells were still capable of hydrolyzing FasL present on human lym- phocytes. Our results suggest that the expression of bcl-2 related proteins could participate in the regulation of MMP-9/TIMP-1 in gliomas. © 2000 Academic Press Key Words: apoptosis; bcl-2 related proteins; gliomas; metalloproteinases. The major reason for the therapeutic failure in gli- oma treatment is their poor response to adjuvant che- motherapy or radiotherapy. In addition, the rapid spreading and subsequent seeding of glioma cells from the original site of the tumor impairs the efficacies of surgical resection (1). The resistance of gliomas to con- ventional therapies is not only due to the difficulty of most drugs to cross the brain-blood barrier but also due to the intrinsic chemo- and radio-resistance of these tumors (2). The expression of antiapoptotic molecules has been shown to be an important obstacle in conven- tional anticancer treatments such as radio- or chemo- therapies of tumors (3). Apoptosis or programmed cell death is the process used by metazoans to eliminate unnecessary or potentially harmful cells. This program is tightly controlled by a family of proto-oncogenes the prototype of which is the product of the BCL-2 gene. Members of this family can be either anti-apoptotic (e.g., bcl-2, bcl-xl) or pro-apoptotic (e.g., bax, bid . . .) and can form homor- or heterodimers (4). Pro- and antiapoptotic bcl-2 related proteins are often coex- pressed within the same cell and the ratio between pro- and antiapoptotic proteins is a major determinant of the fate of the cell. Dysregulation of apoptotic control is believed to be a major step in tumor progression, at least, in the final steps of malignant transformation (5). Degradation of the ECM by MMPs appears to be involved in tumor invasion and metastasis. Experi- mental evidence has directly implicated MMPs in the remodeling of the stromal tissue surrounding tumors (6). These proteases have been implicated in a number of developmental and pathological processes. MMPs can generate active matrix protein fragments, influ- ence the release and the activation of growth factors which in turn modulate angiogenesis (see for example 7), tumor cell growth and apoptosis (8). MMPs are also involved in the processing or shedding of cell surface proteins such as CD44 or TNF (8). Among the proteins thought to be the target of MMPs is the protein, FasL expressed at the surface of cytotoxic lymphocytes (9, 10). In the latter case, the shedding of Fas L by MMPs could be an important factor in the prevention of the killing of tumoral cells by cytotoxic T cells. In addition to the action of these proteases, their receptors and/or inhibitors can also be directly involved in cell migra- tion, although their precise role is not clearly estab- lished (8). Abbreviations used: CNS, central nervous system; ECM, extracel- lular matrix; FasL, Fas ligand; LDH, lactate dehydrogenase; MMP, matrix metalloprotease; TIMP, inhibitor of MMP. Biochemical and Biophysical Research Communications 273, 411– 416 (2000) doi:10.1006/bbrc.2000.2952, available online at http://www.idealibrary.com on 411 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.