Suppression of malignant growth potentials of v-Src-transformed human gallbladder epithelial cells by adenovirus-mediated dominant negative H-Ras

Although Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function, the signaling mechanism whereby Src induces malignant transformation of human epithelial cells still remains unclear. In the present study, we analyzed the functional role of Ras, which acts downstream of Src in intracellular signaling, in the acquisition of fully neoplastic potentials by v-Src-transformed human gallbladder epithelial cells (HAG/src3-1) by infecting these cells with replication-defective adenovirus vector expressing dominant negative H-Ras (AdCARasY57). High efficiency of gene transduction was demonstrated with the adenovirus vector containing beta-gal gene insert (AdCALacZ). On infection with AdCARasY57, the activity of mitogen-activated protein (MAP) kinase, a major downstream event triggered by Ras, was markedly inhibited over 7 days, indicating that the inhibition of Ras function by AdCARasY57 remains active during this period. AdCARasY57 did not inhibit the monolayer growth of HAG-1 cells transfected with activated H-ras, but inhibited the HAG/src3-1 cells by 30%, as compared with cells infected with AdCALacZ as a control. This growth inhibition by AdCARasY57 was strengthened nearly twofold on surfaces coated with an antiadhesive polymer (poly 2-hydroxyethylmethacrylate) that can quantitate anchorage-independent growth, and was much more pronounced up to 95% when assayed in soft agar. The HAG/src3-1 cells transfected with beta-gal gene produced tumors in nude mice within 4 weeks after implantation, whereas cells infected with AdCARasY57 failed to form tumors during this period. These findings show that Ras function is essential for v-Src-induced anchorage-independent growth in vitro as well as tumorigenesis in vivo, and that mitogenic activity driven by v-Src is not solely dependent on MAP kinase pathway. Because anchorage-independent growth correlates with tumor growth in vivo as well as metastatic potential, targeting Ras would be potentially useful for the treatment of human tumors with elevated Src tyrosine kinase activity.