To investigate epithelial cell proliferation and oncoprotein expression of the serrated adenoma, a term that has been used synonymously with mixed hyperplastic and adenomatous polyp, immunohistochemical staining using polyclonal antibodies against Ki-67 and p53, and a Bcl-2 monoclonal antibody, was performed and the results compared with those in hyperplastic polyps and tubular adenomas. A total of 20 serrated adenomas all characterized by a serrated glandular pattern, contained immature goblet cells, upper crypt zone mitotic figures, and a few nucleoli within the epithelial cells. Twenty hyperplastic polyps and 20 tubular adenomas (all with low-grade dysplasia) were examined, and lesions that contained separate areas of hyperplastic and adenomatous glands were excluded. The Ki-67-positive rate in the middle zone of the crypts in serrated adenomas was significantly higher than in hyperplastic polyps but lower than in tubular adenomas; a similar tendency was also noted for the upper zone. Both serrated adenomas and hyperplastic polyps demonstrated Bcl-2-positive reactivity that was essentially limited to the lower crypt zone, while in contrast, involvement in tubular adenomas often extended to the middle zone. No p53 overexpression was found in any category. These results suggest that serrated adenomas may be committed to independent growth.
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.
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