REGULATION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR EXPRESSION IN HUMAN COLON-CARCINOMA CELLS BY ACTIVITY OF SRC KINASE

Background. The c-src protooncogene encodes a protein tyrosine Kinase, pp60(c-src), that is a mediator in many signal transduction pathways. One pathway in which pp60(c-src) protein tyrosine kinase activity is implicated involves regulation of vascular endothelial growth factor ( VEGF) an angiogenic factor important to neovascularization of growing tumors. Recently we demonstrated that decreased activity of pp60(c-src ) in colon tumor cells contributes to decreased expression of VEGF. Th is study examined the relationship between pp60(C-src) activation cell density, and VEGF production in a colon tumor cell line. Methods. Par ental HT-29 colon adenocarcinoma cells and stable subclones created by transfection with c-src antisense and sense (control) expression vect ors were plated under sparse (2 x 10(4) cells/cm(2)) and confluent (20 x 10(4) cells/cm(2)) conditions and grown for 36 hours. Protein and R NA were extracted from cells to determine pp60(c-src) levels c-Src tyr osine kinase activity, and VEGF mRNA expression. Results. The pp60(c-s rc) kinase activity of HT-29 cells and control sense-transfected clone s grown under confluent conditions was increased threefold to fivefold compared with cells grown under sparse conditions. In contrast, the a bility of confluent culture conditions to increase pp60(c-src) activit y was blunted in antisense transfectants. By regression analysis, VEGF expression was found to vary directly with pp60-(c) (src) levels (r(2 ) = 0. 886). Conclusions. Cell density contributes to the regulation o f c-src kinase activity and VEGF expression in HT-29 cells. When the s teady-state level of pp60(c-src) is reduced in antisense transfectants , not only is the steady-state level of VEGF reduced, but the ability of confluence to stimulate pp60(c-src) activity and VEGF production is too. These data suggest that c-src may be an intermediary of both con stitutive and I inducible pathways for VEGF production in colon tumor cells.