c-ras-Ha gene expression is regulated by insulin or insulinlike growth factor and by epidermal growth factor in murine fibroblasts.

Abstract

Although much is known about the structure of ras-encoded proteins, little is known about how expression is regulated. In serum-stimulated murine fibroblasts, c-ras-Ha mRNA levels fluctuated with the growth state but not with the position in the cell cycle. Two types of growth factors regulated c-ras-Ha expression: insulin (IN) Qr insulinlike growth factor I, each apparently acting through its cognate receptor, and epidermal growth factor (EGF). In quiescent cells, IN or insulinlike growth factor I induced c-ras-Ha mRNA three-to fivefold within 4 h, but thereafter the mRNA declined. By contrast, EGF had little effect in 4 h but induced the mRNA after 4 to 6 h. When quiescent cells were given serum c-ras protooncogenes are normal cellular genes that are expressed in most cell types, both proliferating and terminally differentiated, but have the potential to play a causative role in the neoplastic transformation of certain cells (2). ras genes encode a family of membrane-associated proteins (p21 proteins) that bind and hydrolyze GTP. Structural and biochemical analyses suggest that p21 proteins function analogously to GTP-binding proteins (G proteins) that couple occupied receptors to intracellular signalling systems (20). The receptors coupled to ras are unknown, but they are presumed to deliver positive mitogenic signals.The primary mechanism by which ras protooncogenes are converted to transforming genes is a point mutation at one of three major sites in the coding sequence (2). In most cases, these point mutations diminish or abolish GTPase activity, which by analogy with other G proteins, should enhance receptor-generated signals. Indeed, when ras proteins bearing one of these point mutations are introduced into mammalian fibroblasts, the cells initiate DNA synthesis and undergo phenotypic changes characteristic of growth factorstimulated or transformed cells (15,22,36).Depending upon the cell type, the oncogenic potential of ras genes can also be manifest when the protooncogene is overexpressed (7,31,34). For example, a point mutation in an intron of the c-ras-Ha gene is apparently responsible for elevated expression of this gene in the human bladder carcinoma cell line T24/EJ; although the c-ras-Ha gene in T24/EJ cells also carries a mutation that diminishes GTPase activity, the mutation that confers overexpression is at least partially responsible for the transforming activity of the c-ras-Ha gene in these cells (10 sis when microinjected into fibroblasts that have been stimulated to proliferate by serum growth factors (26). Together, these findings suggest that the level of c-ras-Ha expression must be carefully controlled for regulated proliferation of normal cells.Despite the importance of c-ras expression for normal cell proliferation, the physiological signals that regulate these genes are largely unknown. This is in striking contrast to c-fos and c-mnc protooncogenes, which encode nuclear proteins (3) and for which the mechanisms of regulation by growth factors and intracellular mediators of growth ...

Ras proteins are essential and selective for the action of insulin-like growth factor 1 late in the G1 phase of the cell cycle in BALB/c murine fibroblasts.

“…1%) and 25% polyethylene glycol were added, and the precipitates were collected, washed, and counted as described (22). A displacement curve was generated by LIGAND as described (19 (12), and showed the greatest growth inhibitory effect in initial studies, it was used in subsequent experiments and is referred to as the anti-mas IgG.…”

“…When the cells were in middle to late G, (17)(18)(19), they were shifted to serum-free medium, injected with an antibody, and incubated further with IGF-I and/or [3 EGF-induced c-fos expression. By contrast, the anti-G a-subunit IgG inhibited c-fos induction by PDGF or EGF.…”

“…A31 and BPA31 cells were obtained, grown, and made quiescent as described (18,19 (21). For immunofluorescence, cells were fixed in methanol, incubated with anti-ras or anti-G a-subunit IgG and fluorescein-rabbit anti-mouse IgG, and viewed by epifluorescence microscopy.…”

Serum and v-src increase the level of a CCAAT-binding factor required for transcription from a retroviral long terminal repeat.

“…The magnitude of the induction compares favorably with the threefold induction by TPA of SV2CAT and even of a promoter with multimers of the TRE-responsive AP-1 element (Lee et al 1987). This level of induction is also comparable to the response of the Hsp70 promoter to serum (10-fold after 10-18 hr) and of the c-Ha-ras promoter to insulin-like growth factor 1 and epidermal growth factor (EGF) (5-to 7-fold after 16 hr) (Wu et al 1986;Lu et al 1989). However, the m o s t significant finding about the serum i n d u c t i o n of the LTR, however, is that it is mediated by a ubiquitous transcriptional e l e m e n t formerly believed to play a role only in basal level of transcription.…”

“…A similar prediction is made for the c-Ha-ras promoter because the CCAAT element from this promoter bound the serum-induced CCAAT factor and because this promoter is also responsive to serum with kinetics, comparable to the RSV LTR (Lu et al 1989).…”