Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G 1 -specific cell cycle arrest through induction of p21 Cip1 . This in turn leads to inhibition of cyclin D-and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracyclinedependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.The serine-threonine protein kinase Raf is an essential component of the signal transduction pathway leading to mitogenactivated protein (MAP) kinase activation by growth factors which work through Ras (for reviews see references 32 and 53). Raf is a direct downstream effector of Ras (58, 90, 91) and phosphorylates MAP kinase-extracellular signal-regulated kinase (ERK) (MEK) (10, 38), which in turn activates the p42 and p44 ERK kinases (1,25,37,60), resulting in their translocation into the nucleus (8,42). This cascade of events leads to the activation of transcription factors and eventually to cell proliferation (for reviews see references 52 and 88). Blocking endogenous raf mRNA expression with antisense constructs or dominant negative Raf mutants interferes with mitogen-induced proliferation of NIH 3T3 cells (30,36,77), while microinjection of bacterially expressed c-Raf-1 protein is sufficient to induce DNA synthesis in quiescent cells (81).Raf was first discovered as the transforming principle of avian and murine retroviruses (2, 34). Oncogenic activation of Raf can be achieved by truncation of its N-terminal regulatory domain (2, 33) or by constitutive translocation of Raf to the plasma membrane by adding a farnesylation signal, such as the CAAX motif (41). Activated Raf proteins readily transform established fibroblast cell lines and can confer growth factor independence to avian and murine macrophages (6, 26). Moreover, similar to Ras (39, 73), Raf cooperates with other cellular and viral oncogenes, such as the Myc and simian virus 40 large T antigen genes, and has been shown to be transforming in the absence of p53 function in various cell types (3,26,46,54).In addition to the notion that Raf signalling plays a k...

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G 1 cell cycle inhibitors p27 Kip1 and p21 Cip1 (Ckis) via a Myc-induced protein. We provide biochemical and genetic evidence to show that this sequestration is mediated via induction of cyclin D1 and/or cyclin D2 protein synthesis rates. Consistent with this conclusion, primary cells from cyclin D1 -/-and cyclin D2 -/-mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1 -/-cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant forming kinasedefective, Cki-binding cyclin-cdk complexes. The sequestration function of D cyclins thus appears essential for Myc-induced cell cycle progression but dispensable for apoptosis.

Induction of the Myc-oestrogen receptor fusion protein (MycER) by 4-OH-tamoxifen (OHT) leads to the activation of Cyclin E/Cyclin-dependent kinase 2 (CycE/Cdk2) complexes followed by the induction of DNA synthesis. As CycE/Cdk2 activity is essential for G1/S transition, we have investigated the mechanism by which Myc can activiate CycE/Cdk2. Our results suggest that this activation may involve at least two Myc-dependent steps: the induction of cyclin E gene transcription followed by accumulation of cyclin E mRNA in a protein synthesis-independent manner and the inhibition of p27 Kip1 association with CycE/Cdk2 complexes containing newly synthesised CycE. As a consequence phosphorylation of CycE-bound Cdk2 by cyclin activating kinase (CAK) is accelerated. We propose a model in which the active newly synthesised CycE/Cdk2 complexes trigger a positive feed-back mechanism to activate preexisting complexes through phosphorylation-dependent p27 Kip1 release.

The aim of the present study was to test the hypothesis that immobilization of bone morphogenic protein (BMP2) on the surface of titanium implants can enhance peri-implant bone formation. Ten adult female foxhounds received experimental titanium screw implants in the mandible 3 months after removal of all premolar teeth. Three types of implant surfaces were evaluated in each animal: (i) implants with machined titanium surface, (ii) implants coated with collagen I, (iii) implants coated with collagen I, chondroitin sulphate (CS) and BMP2. Peri-implant bone regeneration was assessed using histomorphometry after 1 and 3 months in five dogs each by measuring bone-implant contact (BIC) and the volume density of the newly formed peri-implant bone (BVD). After 1 month, there was no significant enhancement in BIC values but volume density of the newly formed peri-implant bone was significantly higher in the two groups of coated implants. No significant difference was found between collagen and BMP2 coating. After 3 months, BIC was significantly higher in both collagen and BMP2-coated implants compared with implants with machined surfaces. Peri-implant BVD was also significantly increased in coated implants in comparison with machined surfaces. It was concluded that collagen coating of dental screw implants can enhance BIC and peri-implant bone formation. Addition of BMP2 does not increase peri-implant bone formation in the present application.

The aim of the present study was to test the hypothesis that organic coating of titanium screw implants that provides binding sites for integrin receptors can enhance periimplant bone formation. Ten adult female foxhounds received experimental titanium screw implants in the mandible 3 months after removal of all premolar teeth. Four types of implants were evaluated in each animal: (1) implants with machined titanium surface, (2) implants coated with collagen I, (3) implants with collagen I and cyclic RGD peptide coating (Arg-Gly-Asp) with low RGD concentrations (100 micromol/mL), and (4) implants with collagen I and RGD coating with high RGD concentrations (1000 micromol/mL). Periimplant bone regeneration was assessed histomorphometrically after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the volume density of the newly formed periimplant bone (BVD). After 1 month, BIC was significantly enhanced only in the group of implants coated with the higher concentration of RGD peptides (p = 0.026). Volume density of the newly formed periimplant bone was significantly higher in all implants with organic coating. No significant difference was found between collagen coating and RGD coatings. After 3 months, BIC was significantly higher in all implants with organic coating than in implants with machined surfaces. Periimplant BVD was significantly increased in all coated implants in comparison to machined surfaces also. It was concluded that organic coating of machined screw implant surfaces providing binding sites for integrin receptors can enhance bone implant contact and periimplant bone formation.