Cooperating oncogenes converge to regulate cyclin/cdk complexes.

Abstract

The cooperation of oncogenes in the transformation of primary rat Schwann cells is a strikingly synergistic process. We have explored the molecular mechanisms involved. Activation of an inducible Raf kinase results in morphologically transformed cells that are arrested in G1 via the induction of p21 ¢ipl and subsequent inhibition of cyclin/cdk activity. In contrast, coexpression of SV40 large T (LT) or a dominant-negative mutant of p53 abolishes p21Cipl induction and alleviates the growth arrest. Moreover in this scenario, Raf activation results in an increase in the specific activity of cyclin/cdk complexes with Raf and LT cooperating to superinduce cyclin A/cdk2 activity and stimulate proliferation in the absence of mitogens. Thus, signaling by Raf and its cooperating partners converges at the regulation of cyclin/cdk complexes, with the cellular responses to Raf modulated by p53.

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…; n l 3, in agreement with data in [11,12] (Figure 2), and did not induce p21 Cip-"/WAF" expression ( Figure 3). To mimic in hepatocytes the effect of chronic MAP kinase cascade activation as induced by NGF in PC12 cells, hepatocytes were infected with a poly--lysine conjugated adenovirus to express a modified oestrogen receptor-Raf-1 (' ∆Raf-1 : ER ') fusion protein, which has been shown in fibroblasts to be regulatable by oestradiol treatment [11,13]. The oestrogen receptor portion of our constructs contains a modified form of the hormone binding domain of the oestrogen receptor that has become non-responsive to oestradiol, and is now activated by the anti-oestrogen, 4-hydroxy-tamoxifen.…”

“…In general agreement with these data and those in Table 1, treatment of PC12 cells with NGF inhibited cdk2 activity by 39p5 %, whereas NGF treatment of hepatocytes caused a stimulation in cdk2 activity by 31p3 % (pS.E.M. ; n l 3, in agreement with data in [11,12] (Figure 2), and did not induce p21 Cip-"/WAF" expression ( Figure 3). To mimic in hepatocytes the effect of chronic MAP kinase cascade activation as induced by NGF in PC12 cells, hepatocytes were infected with a poly--lysine conjugated adenovirus to express a modified oestrogen receptor-Raf-1 (' ∆Raf-1 : ER ') fusion protein, which has been shown in fibroblasts to be regulatable by oestradiol treatment [11,13].…”

“…Several studies have suggested that chronic activation of protein kinase C and the MAP kinase cascade can lead to increased expression of cyclin dependent kinase (cdk) inhibitor proteins, in particular p21 Cip-" and p27 Kip-" [11,12]. To examine further the mechanism(s) by which NGF inhibits DNA synthesis in PC12 cells and promotes DNA synthesis in hepatocytes, we immunoblotted NGF-treated cells to examine the expression of the cdk inhibitor proteins p15 INK%b , p16 INK%a , p21 Cip-"/WAF" , and p27 Kip-" (Figure 3).…”

“…Indeed, the role of signalling by the MAP kinase cascade in terms of either cellular proliferation or cellular differentiation is currently under intensive study and recent reports using other cell types have suggested a role for chronic MAP kinase activation in causing exit from the cell cycle followed by cellular differentiation [11][12][13][14][15][16].…”

Extinction of Rac1 and Cdc42Hs signalling defines a novel p53-dependent apoptotic pathway

“…It has been reported that ERK activation by the mos oncogene results in p53 activation, cell cycle arrest and apoptosis of mouse embryo ®broblasts (Fukasawa and Vande Woude, 1997). Similarly, constitutively active Ras or Raf will induce both ERK and p53 in primary cells (Lloyd et al, 1997;Palmero et al, 1998;Serrano et al, 1997). However, in our experimental system MAPK signalling is apparently not required for p53 activation, since both Rac1N17 and Cdc42HsN17 expression give rise to p53 activation even in the presence of the PD-098059 MEK1 inhibitor (data not shown).…”