STUDY OF DNA FRAGMENTATION AND APOPTOSIS-RELATED GENE PRODUCTS IN HUMAN BRAIN TUMORS AND ESTABLISHMENT OF APOPTOSIS-INDUCING THERAPY.

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1. We detected the internucleosomal DNA fragmentation by modified TUNEL staining and analyzed the expression of apoptosis-related gene products in cultured glioma cells and biopsied brain tumor specimens. The fragmented and condensed nuclei were clearly stained for TUNEL and the percentages of stained nuclei (apoptotic index) were ranging from 0% to 8.9%. Fas, Bcl-2 families (Bcl-2, Bcl-x and Bax) and ICE families (ICE,Ich-1) were found to be involved in tumorigenesis of certain brain tumors.2. We investigated the role of the Fas ligand-Fas system in human glioblastoma cell lines (T98G,U251 and A172).Immunohistochemical and flow cytometrical analyzes revealed that all three cell lines expressed Fas highly, however, apoptotic changes such as nuclear fragmentation, the DNA ladder pattern on gel electrophoresis, and reduction of viable cells were observed only in T98G cells by treatment of Fas mAb (50ng/ml).3. We have been applying an adoptive immunotherapy protocol to patients with malignant brain tumors using OK-432-activated peripheral blood mononuclear cells (OK-MCs). To elucidate the mechanism of OK-MCs' cytotoxicity, we examined the cytocidal activity of OK-MCs against Fas expressing T98G glioma cells.Apoptosis of T98G cells induced by treatment with OK-MCs was unequivocally inhibited by the treatment of T98G cells with ZB4 monoclonal antibody, which binds to Fas and blocks the binding of Fas ligand to Fas. These results indicate that the cytotoxic activity of OK-MCs via apoptosis seems to be partly mediated by the Fas ligand/Fas system.