MECHANISTIC STUDY OF APOPTOTIC CELL DEATH AND INVASION POTENTIAL USING SIGNAL TRANSDUCTION INHIBITORS IN THE ONCOGENE-TRANSFECTED HUMAN CELLS

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Although src or ras oncogene induces transformation, their precise roles in the apoptotic machinery and invasion remain to be clarified. We examined the role of these oncogenes on the detachment-induced apoptosis termed anoikis and invasion potential, using HAG-1 human epithelial cell lines transfected with v-src or activated ras. Non-tumorigenic parental, mock-transfected, and ras-transfected HAG-1 cells underwent anoikis. By contrast, tumorigenic, v-src-transformed cells did not exhibit such apoptotic features. pp125^<FAK> focal adhesion kinase (FAK), was constitutively activated only in the v-src-transformed cells. Moreover, herbimycin A, a Src tyrosine kinase inhibitor, reduced tyrosyl phosphorylation of pp125^<FAK>, and reversed resistance to anoikis. These data suggest that pp60^<v-src> may substitute for integrin in the activation of pp125^<FAK>, thereby evading anoikis, and that the association of pp60^<src> with FAK might be required for acquisition of anchorage-independence. … More The functional role of ras in the acquisition of fully neoplastic potentials by v-src-transformed human gallbladder HAG/src3-1 cells, were investigated by introducing adenoviral vector containing dominant negative Ras (DN/Ras) into these cells. The anchorage-independent growth of the HAG/src3-1 was inhibted by DN/Ras by 93%. The HAG/src3-1 cells transfected with DN/ras failed to form tumors. Thus, v-src might requires a Ras-MAP kinase signaling cascade in the acquisition of tumorigenic potential. To examine the potential role of Rac, a small GTPase binding protein which acts downstream of Ras, experiments using adenovirus vectors containing DN/Ras or dominant negative Rac (DN/Rac), were performed. The data indicated that both DN/Ras and DN/Rac reduced the invasive potential of src-transfected cells by 60% and 99%, respectively, as compared to AdCALacZ containing β-gal gene as a control. Moreover, DN/Rac suppressed completely the tumorigenic potentials of src-transfected cells in nude mice. These results suggest that Src, Ras, Rac, all appeared to participate in the invasion processes, and that Rac may act downstream of these signaling pathways of invasion and tumorigenicity. Less