Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are de®cient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCF Skp2 ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCF Skp2 ubiquitin ligase complex aects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCF Skp2 complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that dierential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway. Oncogene (2001) 20, 3641 ± 3650.

The mechanism by which all-trans retinoic acid (ATRA) leads to a G 1 arrest of the cell cycle remains unclear. We show here that the decrease in D-type cyclin levels observed following ATRA treatment correlates with an increase in the rate of cyclin D1 ubiquitylation in both T-47D and MCF-7 breast cancer cell lines. However, MCF-7 cells are more resistant to ATRA than T-47D cells indicating that cyclin D1 degradation is not sufficient for ATRA-mediated arrest. We found a striking difference between these cells in that while ATRA induces an elevation in the cdk inhibitor p27 in T-47D cells, this is not observed in the ATRA-resistant MCF-7 cells. Furthermore, we demonstrate that ATRA promotes the ubiquitylation of Skp2, an F-box protein that targets p27 for degradation. Moreover, overexpression of Skp2 in T-47D cells prevents accumulation of p27 and promotes resistance to ATRA. In addition, overexpression of cyclin D1 in T-47D cells also promotes ATRA resistance. We found that the mechanism of ATRA-induced ubiquitylation of cyclin D1 and Skp2 is independent of CUL-1 expression and that ATRA can rescue cyclin D1 degradation in the uterine cell line SK-UT-1, where D-type cyclins are stabilized due to a specific defect in proteolysis. These data suggest that ATRA induces a novel pathway of ubiquitylation and that the degradation of the F-box protein Skp2 is the mechanism underlying p27 accumulation and cyclin E-cdk2 inactivation following ATRA treatment.It has been known for more than 50 years that retinoids are potent agents for the control of cellular differentiation and proliferation. Several studies have shown that retinoids can suppress the process of tumorigenesis both in vitro and in vivo (1-3) and that cells exposed to all-trans retinoic acid (ATRA) 1 arrest in the G 1 phase of the cell cycle. However, the molecular mechanism underlying this arrest remains unclear.Cyclin-dependent kinases (cdk) are key cell cycle regulators. Their activities are regulated at several levels including phosphorylation, binding to their regulatory subunits, the cyclins, and binding to small inhibitory proteins called the cyclin-dependent kinase inhibitors (CKI). Among the cyclin subunits, D-type cyclins associate with CDK 4 and 6 to phosphorylate the retinoblastoma (Rb) protein. 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. Cyclin E-CDK2 complexes act downstream of cyclin D-CDK4/6 to maintain Rb phosphorylation and events leading to the premature activation of cyclin E-CDK2 complex trigger inappropriate entry into S phase. However, the activity of cyclin E-CDK2 complexes are restricted by binding to the CDK inhibitors p27 and p21. The importance of these proteins is best illustrated by the fact that mutations leading to either accumulation of cyclin D1, or elevated cyclin E-associated kinase activity due to loss of p27 expression, are both frequent events contributing to tumorigenesis (4 -7). Therefore, agents...

The three human D-type cyclins, cyclin D1, D2 and D3 share the ability to bind to and activate cdk4 and 6. MMTV-cyclin D1 transgenic mice develop mainly adenocarcinoma, while MMTV-cyclin D2 mice show a lack of alveologenesis during pregnancy and only develop carcinoma at low frequency. The effect of cyclin D3 overexpression in mammary glands remains hitherto unknown. We generated MMTV-cyclin D3 transgenic mice and report here that they develop exclusively squamous cell carcinoma. We show that although cyclin D3 transgene expression was detected early in puberty, postnatal development and mammary gland proliferation were normal in virgin animals. In contrast, multiparous mice develop multiple foci of abnormal growth that correspond to various stages of squamous metaplasia. Therefore, our results support a role for cyclin D3 in squamous differentiation. In addition, we found that p16 expression during involution is abolished, while p27 expression increased in MMTV-cyclin D3 mice, two modifications that have been reported in the other MMTV-D-type cyclin transgenic models. Our observations indicate that despite biochemical redundancy in vitro and in vivo, D-type cyclins promote distinct oncogenic pathways.

No abstract