C-HA-RAS(EJ) TRANSFECTION OF RAT AORTIC SMOOTH-MUSCLE CELLS INDUCES EPIDERMAL GROWTH-FACTOR RESPONSIVENESS AND CHARACTERISTICS OF A MALIGNANT PHENOTYPE

Although the role of several protooncogenes, including sis, myc, and m yb in the regulation of growth and differentiation of vascular cells h as been examined in some detail, limited information is available on t he contribution of ras genes to these processes. In the present studie s the influence of oncogenic ras transfection on the phenotypic expres sion of rat aortic smooth muscle cells (SMCs) was examined. Cultured r at aortic SMCs during early passage (P-4) were transfected by lipofect ion with c-Ha-ras(EJ) in a pSV2 neo vector or with pSV2 neo vector alo ne. Stable transfectants were selected in G418 over a 6-week period. O ncogene-transfected cells (ras-LF-1) exhibited differences in morpholo gy and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appear ance of focal cellular arrangements giving rise to latticelike structu res. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transiection of c-Ha-ras(EJ) was associated with a twofold increase in p21 levels relative to pSV2 vector controls demons trating that exogenous ras was expressed in these cells. Overexpressio n of ras p21 afforded SMCs a lower serum requirement for growth compar ed to vector controls, anchorage independent growth on soft agar, and acquisition of epidermal growth factor (ECF) responsiveness. Stimulati on of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased s teady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. alpha-smoo th muscle (SM) actin gene expression was markedly reduced in ras-trans fected cells relative to vector controls. These results show that tran sfection of c-Ha-ras(EJ) into aortic SMCs induces an altered phenotypi c state characterized by alterations in growth factor-related signal t ransduction and tumorigenic potential. (C) 1994 Wiley-Liss, Inc.