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c‐Ha‐ ras EJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype
Author(s) -
Sadhu D. N.,
Lundberg M. S.,
Burghardt R. C.,
Ramos K. S.
Publication year - 1994
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041610312
Subject(s) - transfection , epidermal growth factor , biology , vascular smooth muscle , growth factor , cell culture , microbiology and biotechnology , cell growth , fetal bovine serum , endocrinology , medicine , smooth muscle , genetics , receptor
Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P 4 ) were transfected by lipofection with c‐Ha‐ ras EJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6‐week period. Oncogene‐transfected cells ( ras ‐LF‐1) exhibited differences in morphology and growth pattern relative to vector controls (neo‐LF‐1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF‐1 cells. Transfection of c‐Ha‐ ras EJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchor‐age independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum‐deprived SMCs with 5% fetal bovine serum (FBS) increased steady‐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. α‐smooth muscle (SM) actin gene expression was markedly reduced in ras‐transfected cells relative to vector controls. These results show that transfection of c‐Ha‐ ras EJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor‐related signal transduction and tumorigenic potential. © 1994 Wiley‐Liss, Inc.