Transient Transfections, Preparation of Cell Extracts, and siRNA Experiments Altogether, these results indicate that cyclin D1 is part of a transcriptional complex that controls the activation of the p21waf1 gene, suggesting that this could play an important role during cell transformation. Importantly, the cyclin D1-mediated inhibition of p21waf1 was also observed in breast cancer cells that contain elevated levels of cyclin D1. In the presence of cyclin D1, STAT3 and its transcriptional cofactor NcoA/SRC1a are normaly recruited to DNA, but the histone acetylase CBP and the RNA polymerase II do not associate with the promoter. In the present study, using chromatin immunoprecipitation (ChIP) experiments, we show that cyclin D1 is recruited to the p21waf1 promoter by STAT3 proteins to down-regulate its activity. After tyrosine phosphorylation, STAT3 proteins dimerize, translocate into the nucleus, and activate the expression of cell cycle genes such as p21waf1, fos, cyclin D, myc, and pim-1 ( Bromberg et al., 1999 Catlett-Falcone et al., 1999 Kiuchi et al., 1999 Shirogane et al., 1999 Ivanov et al., 2001). STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine stimulation. We have previously shown that cyclin D1 interacts with STAT3 proteins to inhibit their transcriptional activity ( Bienvenu et al., 2001). However it remains to be determined if cyclin D1 is directly present on the DNA and if its effects are observed under physiological conditions or during carcinogenesis. These studies suggest that cyclin D1 is contained within transcriptional regulatory complexes. Additionally, it can also interact with the histone deacetylase HDAC3 ( Lin et al., 2002), such that its inhibitory effects on the androgen and thyroid hormone receptors are lifted when cells are exposed to trichostatin A (TSA Lin et al., 2002 Petre et al., 2002). Cyclin D1 also regulates the activity of SP1 and Rb through its association with TAF II250, a component of TFIID ( Adnane et al., 1999 Siegert et al., 2000). A direct association with NcoA/SRC1a or GRIP-1 likely explains the effect of cyclin D1 on ER activation (Zwijsen et al., 1997, 1998) and on the activity of the MEF muscle factor ( Lazaro et al., 2002).

Moreover, cyclin D1 interacts with several transcriptional cofactors such as the histone acetylase P/CAF ( McMahon et al., 1999) and members of the p160 family of coactivators including NcoA/SRC1a or GRIP-1 ( Zwijsen et al., 1998 Lazaro et al., 2002). Cyclin D1 regulates the activity of various transcription factors such as MyoD, STAT3, Beta2/NeuroD, the estrogen, thyroid and androgen receptors or the v-myb, and DMP1 proteins ( Bernards, 1999 Coqueret, 2002). Therefore, cyclin D1 is involved in a large complexity of network interactions that regulate cell proliferation as well as cell growth.Р”В®xtending its cell cycle functions, cyclin D1 has been reported to have new functions that are independent of cyclin-dependent kinases. In Arabidopsis, D-type cyclin expression is induced in the presence of sucrose, the major source of carbon that plays a central role in regulating cellular metabolism and physiology in plants. Overexpression of Arabidopsis cyclin D2 in transgenic tobacco leads to an increased rate of biomass accumulation, enhanced root growth, and a rapid attainment of flowering size ( Cockcroft et al., 2000). In addition, inactivation of the cyclin D1 gene produced a small mouse phenotype ( Fantl et al., 1995) and led to a disturbance of growth in Drosophila ( Datar et al., 2000 Foley and Sprenger, 2000 Meyer et al., 2000) and Caenorhabditis elegans ( Park and Krause, 1999). However, cyclin D1-deficient mice are viable but show developmental abnormalities to restricted tissues such as the retina, the nervous system, and the breast epithelium ( Sicinski et al., 1995). In light of these observation, it was predicted that the inactivation of the cyclin D1 gene would lead to important defects due to dysregulated cell proliferation.

Cyclin D then binds cdk4 or cdk6 to activate the kinase activity of these proteins, initiate Rb phosphorylation and induce cell cycle progression toward S phase. After mitogen stimulation of quiescent cells, genes encoding D-type cyclins get activated at the beginning of the G1 phase.

D1 form