METODOLOGIA DEL PROCESO EXPERIMENTAL

In addition to rapid events in the membrane and cytosol, serum and growth factors rapidly and transiently induce the expression of the protooncogenes c-fos and c-myc (Reviewed in Rozengurt and Sinnett-Smith, 1988). Protooncogenes represent the cellular counterparts of transforming DNA sequences initially isolated from acutely transforming retroviruses. Thus, c-fos and c-myc are the normal cellular homologues of the transforming genes of the FBJ osteosarcoma (fos) and avian myelocytomatosis (myc) viruses, respectively. Since these cellular genes are highly conserved during evolution and because many protooncogenes are expressed in developing embryonic tissues, it is widely thought that they may play a role in the regulation of normal growth and differentiation. Indeed, expression of a transfected myc gene (Armelin etal., 1984, Mougneau etal., 1984, Sorrentino e tal., 1986) or microinjection of the c-myc protein (Kaczmarek etal., 1985) into quiescent 3T3 cells stimulates entry into DNA synthesis in the presence of other growth factors (reviewed in Cole, 1986). Similarly,

expression of fos antisense RNA (Nishikura and Murray, 1987) or microinjection of fos- specific antibodies (Riabowol etal., 1988) blocks DNA synthesis in fibroblasts stimulated by serum.

Both c-fos and c-myc are thought to play a role in the regulation of gene expression. The demonstration that the product of the protooncogene c-jun, identified as the trans-acting factor A P -1, forms a tight complex (dimer) with fos protein is consistent with a role for c-fos in the regulation of gene transcription (reviewed in Curran, 1988 ). Dimerization is mediated by the leucine repeat (or leucine zipper) with two a-helices forming a coiled coil structure. In the heterodimer form both fos and jun contribute to transcriptional activation. While jun-jun homodimers are also able bind DNA, fos-fos dimers are not stable and have no DNA-binding activity (reviewed in Karin and Smeal, 1992). These dimers with DNA-binding abilities are thought to contribute to AP-1 activity and participate to varying extents in its regulation by extracellular stimuli ( Karin and Smeal, 1992). The transcriptional regulation of the c-fos gene itself is complex. The c-fos promoter contains several upstream enhancer elements which

bind sequence-specific protein factors and thereby control transcription of the gene

(Treisman, 1986, Cun-an, 1988, Prywes etal., 1988, Sassone-Corsi et a!., 1988a). Using in vitro assays, a number of cis-acting DNA sequences have been Identified which stimulate c- fos expression in a signal-specific manner. In different cell types, the serum response element (SHE) is required for the induction of the c-fos gene by serum, EGF, PDGF or insulin (Treisman, 1986, Prywes et al., 1988). Similarly, the TRE, also known as the A P-1

consensus sequence, is required for c-fos induction by phorbol esters, presumably acting through a PKC-dependent pathway, whereas the cyclic-AMP-responsive element (CRE) controls the transcription of c-fos induced by cAMP (Sassone-Corsi et al., 1988a). Transcription expression of c-fos appears to be negatively autoregulated and the

phosphorylation of c-fos protein (c-Fos) in Its cartxjxyl-region is implicated in converting c-Fos to a repressor of its own expression (Sassone-Corsi, et a!., 1988b, Offir, et a!., 1990). The target genes regulated by fos still remain to be identified.

Recent evidence also strongly suggests that the c-myc protein (c-Myc) is a transcription factor (Blackwood and Eisenman, 1991). It possesses a number of functional domains found in other proteins modulating transcription, specifically the leucine zipper characteristic of the fos- jun-CREB transcription families (Landschulz eta!., 1988) and the basic helix loop helix motif found in enhancer binding proteins (Murre et a/., 1989). Recently both a heterodimeric partner called Max (Blackwood and Eisenman, 1991) and a consensus DNA-binding

sequence for Myc (Blackwell etal., 1990, Prendergast and Ziff, 1991) have been identified. It has also been demonstrated that the oncogenic activity of the c-myc protein requires

dimerization with Max (Amati etal., 1993). Transcriptional expression of c-myc has been shown to be autoregulated by c-myc protein (c-Myc) but also requires additional trans-acting factors (Penn, et al., 1990). These observations also support a role for c-myc in the

regulation of cellular gene transcription. However, it is still unknown precisely which genes are regulated by myc.

As stated previously, serum and growth factors transiently induce the expression of c-fos and c-myc. c-fos mRNA is detectable as early as 10 minutes after stimulation of quiescent fibroblasts with serum or polypeptide growth factors, such as PDGF or EGF, and is maximally increased after 30 minutes ( Cochran etal., 1984, Greenberg and Ziff, 1984, Kruljer etal.,

1984, Muller etal., 1984, Curran, 1988). In contrast c-myc is detectable within 30 minutes and reaches a maximum after 3 hours stimulation (Kelly etal., 1983,Muller e ta l., 1984). Bombesin also induces c-fos and c-myc expression with similar kinetics (Letterio et al., 1986, Palumbo etal., 1986, Bravo etal., 1987a, Rozengurt and Sinnett-Smith, 1987). Since both genes encode nuclear proteins it was reasoned that their transient expression in early G-| may play a role in the transduction of the mitogenic signal in the nucleus (Abrams etal.,

1982, Alitalo etal., 1983, Perssonand Leder, 1984, Curran, 1988).

Crucially, the increase in c-fos and c-myc mRNA levels is not prevented by inhibitors of protein synthesis (Kelly etal., 1983, Greenberg e ta l., 1986). In fact these genes are

overexpressed when growth factors are added together with drugs such as cycbheximide or anisomycin (Kelly etal., 1983, Greenberg etal., 1986). These results indicate that the increase in c-fos and c-myc mRNA levels is not secondary to the growth response and suggests that in quiescent cells regulatory factors are present and poised to respond to environmental stimuli.