The angiogenic switch in hamster buccal pouch keratinocytes is dependent on TGFbeta-1 and is unaffected by ras activation

This study was undertaken to investigate the mechanisms by which Syrian hamster buccal pouch keratinocytes treated in vivo with 7,12- dimethylbenza]anthracene (DMBA), switch from an angio-inhibitory to an angiogenic phenotype. Cells were cultured from pouches at various times after exposure to carcinogen and their angiogenic activity assessed. The angio-inhibitory activity present in conditioned media from normal cells was lost as early as 3 weeks after carcinogen treatment, resulting in weak expression of angiogenic activity. By 5 weeks, cells had become strongly angiogenic due to the secretion of high levels of TGFbeta-1, a potent angiogenic factor. Because the switch to high levels of secreted TGFbeta-1 occurred at the same time as the activation of the H-ras oncogene, non-angiogenic cell lines lacking an activated H-ras oncogene were stably transfected with mutant H-ras and their transformed and angiogenic phenotypes were evaluated. Although ras transfection drove two of the three cultured cell lines to anchorage independence and modestly increased their ability to clone in low serum, it had no effect on the angiogenic phenotype or on the level of secreted active TGFbeta-1. These results demonstrate that the angiogenic phenotype in the hamster buccal pouch model of oral carcinogenesis develops in a step-wise fashion with an early decrease in the production of an inhibitor of angiogenesis and a subsequent marked increase in the secretion of the inducer TGFbeta-1. Although the activation of the H-ras oncogene contributed to anchorage independence, it did not affect the expression of the angiogenic phenotype in this model system.