D-type cyclins are important cell cycle regulators that promote cellular proliferation in response to growth factors by inactivation of the retinoblastoma protein (Rb). Cyclin D1 has been shown to be overexpressed in several cancer types and to act as an oncogene in breast cancers. As D-type cyclins are rate limiting for progression into S phase, the level at which they accumulate must be carefully regulated. Several mechanisms leading to overexpression of cyclin D1 have been reported including ampli®cation, translocation and stabilization of the mRNA. Here, we present data showing elevated cyclin D1 protein in breast cancer samples in the absence of elevated mRNA level. Further, we found that in these cases, cyclin D3 protein also accumulates and that the coordinate increase in cyclin D1 and D3 occurs in 15% (7/47) of breast cancers. In addition we show that blocking the activity of the 26S proteosome results in the accumulation of cyclin D1 and D3, that both D-type cyclins are ubiquitinated and associate with Cul-1, a component of the SCF ubiquitin ligase complex. Finally, we show that the coordinated elevation of cyclin D1 and D3 is also observed in the breast cell line MCF-7 and demonstrate that the degradation of cyclin D1 and D3 is de®cient in this cell line. These results indicate that cyclin D1 and cyclin D3 share a common mechanism of degradation and we propose that the coordinate increase of D-type cyclins observed in primary breast cancers re¯ects a defect in their proteolysis.

Cyclin D1 binds and regulates the activity of cyclindependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1⅐CDK4/6 complexes during the G 1 phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3␤ (GSK-3␤) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499 -3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3␤, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.Mammalian cell cycle progression is regulated by a family of cyclin-dependent kinases (CDKs).1 Among the cyclin subunits, D-type cyclins associate with CDK4 and -6 to phosphorylate the retinoblastoma (Rb) protein during the G 1 phase of the cell cycle. Hyperphosphorylation of Rb promotes the release of the E2F family of transcription factors that then promote entry into S phase through activation of key target genes (1). Although this function of cyclin D1 is well established, several recent observations suggest that additional roles may exist for free cyclin D1 in the regulation of transcription. First, cyclin D1 has been shown to associate with the estrogen receptor and to promote its transcriptional activity (2-4). Second, cyclin D1 has been shown to associate with the transcription factor DMP1 and to antagonize its ability to induce cell cycle arrest (5). Third, cyclin D1 was shown to affect the transcriptional activity of Sp1 (6), the androgen receptor (7), and v-Myb (8). Although the impact of cyclin D1 interactions with these transcription factors remains to be demonstrated, the fact that the interactions are independent of binding to CDK4 suggests that cyclin D1 may affect cell cycle progression independently from its role in the regulation of CDKs and the phosphorylation of Rb.D-type cyclins have been suggested to be rate-limiting for the progression from G 1 into S phase of the cell cycle, based on the observation that their constitutive overexpression leads to a shorter G 1 phase (9, 10). Further, constitutive overexpression of cyclin D1 in mouse mammary glands leads to carcinoma, indicating that cyclin D1 acts as an oncogene (11). In human breast cancer, abnormal accumulation of cyc...

The relationship between the development of acute hepatitis and the production of TNF-alpha IFN-gamma and IL-6 by liver-associated T lymphocytes following intravenous injection of concanavalin A (Con A) was studied in rats. Following a single injection of Con A, there was a dose and time-dependent correlation in the serum levels of serum alanine aminotransferase (ALT), IL-6, IFN-gamma and TNF-alpha. These increases correlated with an increase in the numbers of CD4+, CD8+ and CD25+ T cells in blood and CD4+ and CD25+ T cells in the liver perfusate, but not with CD8+ T cells in liver perfusate. Increased levels of IL-6, IFN-gamma and TNF-alpha were constitutively produced by liver-associated CD4+ T cells when cultured. In Con A-stimulated cultures, liver-associated CD4+ T cells secreted increasing levels of TNF-alpha in a time-dependent manner following Con A injection, but TNF-alpha production by peripheral blood lymphocytes was transient with peak levels detected at 1 h which then declined over 24 h. Histological examination of the liver revealed fatty change, hepatocyte degeneration and necrosis, with an associated cell infiltrate of neutrophils and CD4+ T cells both in the portal areas and around the central veins. These results support the hypothesis that Con A-induced liver damage is mediated by CD4+ T cells acting within the liver, at least in part through the secretion of TNF-alpha, IFN-gamma and IL-6.

Coordinated accumulation of cyclin D1 and D3 is observed in 15% of primary breast cancers and in the breast cancer cell line MCF-7 this simultaneous overexpression is due to a defect in their ubiquitin-mediated proteolysis. The F-box protein Skp2 is a component of an SCF ubiquitin ligase complex and can associate with cyclin D1 and the cdk inhibitor p21 (Zhong-Kang et al., 1998). We extend this observation and show that cyclin D3 can also associate with Skp2 suggesting that cyclins D1, D3 and p21 may share the same SCF complex. In agreement with this hypothesis we report here that in primary breast cancers and in MCF-7 cells where cyclins D1 and D3 are elevated the level of p21 is also elevated. Further, we demonstrate that the turnover of p21 protein is reduced in MCF-7 cells. We show that p21 is active as a cdk inhibitor in this cell line but that the presence of elevated levels of cyclin D3 titrates p21 away from cyclin D1-cdk4/6 complexes and cdk2 complexes resulting in increased kinase activities. Our results suggest that a defect in the SCF complex may occur in 15 ± 20% of breast cancers and that the resulting coordinated elevation of cyclins D1 and D3 overcomes the inhibition of cell cycle progression by p21. We propose that in the context of cyclins D1 and D3 overexpression, p21 may promote cell cycle progression.

The relationship between the changes in liver pathology and the production of interleukin (IL)-1alpha, IL-6, and tumor necrosis factor-alpha (TNF-alpha) by intrahepatic mononuclear cells was studied in rats fed alcohol and subsequently exposed to lipopolysaccharide (LPS). Rats were fed 40% ethanol in drinking water, whereas control rats were provided with a chow diet with isocaloric or 2% sucrose drinking solutions for up to 20 weeks. Decreased IL-1alpha and TNF-alpha production in 24-hr culture supernatants of mononuclear cells isolated from liver perfusate was detected while IL-6 remained unchanged over 20 weeks. When animals were injected with LPS (1.0 microg/kg body weight), there was a 5-fold rise in ALT levels in the ethanol-fed group, but not in control groups. Increased IL-6 and TNF-alpha levels in the serum and supernatant of cultured intrahepatic mononuclear cells stimulated with or without LPS or concanavalin A was observed. There was a correlation between levels of ALT and TNF-alpha, but not IL-6. T cells and Kupffer cells were the major source of TNF-alpha in culture supernatants of hepatic perfusate mononuclear cells from ethanol-consuming rats injected LPS. In addition, pathological liver injury was evident, which suggests a pathogenic role for TNF-alpha in alcohol-induced liver disease.