, agonists of nuclear receptor PPARg and inhibitors of retinoblastoma protein (pRb) play important roles in the development and activation of beige cells ( Auffret et al . 2012 , Ohno et al . 2012 , Harms & Seale 2013 ). The inactivation of pRb may
Diana Vargas, Noriaki Shimokawa, Ryosuke Kaneko, Wendy Rosales, Adriana Parra, Ángela Castellanos, Noriyuki Koibuchi and Fernando Lizcano
K Hofman, JV Swinnen, F Claessens, G Verhoeven and W Heyns
In search of potential androgen receptor coregulators we performed a yeast two-hybrid screening using the androgen receptor ligand-binding domain as bait and a human prostate cDNA library as prey and found that the carboxy-terminal domain of retinoblastoma-associated Kruppel protein (RbaK), a member of the Kruppel zinc finger protein family, interacts in a ligand-dependent way with the ligand-binding domain of the androgen receptor. RBaK was recently identified as a transcriptional regulator that interacts with the retinoblastoma protein and thereby influences E2F regulated transcription. The interaction of RBaK with the androgen receptor was further documented using mammalian two-hybrid experiments, in vitro binding studies and coimmunoprecipitation. Finally, we demonstrated that both RBaK and the retinoblastoma protein coactivate androgen receptor-mediated transcription in cotransfection experiments. In conclusion, our data show that RBaK interacts with the androgen receptor and increases its transcriptional activity. Moreover, the double interaction of RBaK with the retinoblastoma protein and with the androgen receptor provides a novel link between the androgen receptor and the regulation of the cell cycle.
Victor Quereda and Marcos Malumbres
to CDK4–cyclin D or CDK6–cyclin D complexes is unclear ( Malumbres & Barbacid 2005 ). The primary substrates of the CDKs in G1 progression are the members of the retinoblastoma protein family (pRB). pRB negatively regulates entry into the cell cycle
Monica Fedele and Alfredo Fusco
retinoblastoma protein (pRB) negatively regulates G 1 entering, progression, and transition to S by blocking the activity of factors responsible for cell cycle progression, which include the E2Fs. CDK4, CDK6, and CDK2, complexed to D- and E-type cyclins, mediate
Aniello Cerrato, Valentina De Falco and Massimo Santoro
-suppressor genes RB1 (retinoblastoma: pRB protein) and TP53 (p53 protein) are frequently mutated in human cancer, and several lines of evidence indicate that both pathways must be inactivated in cancer to overcome senescence or apoptosis ( Hahn & Weinberg 2002
Chun-Che Yen, Ya-Hui Huang, Chu-Yu Liao, Cheng-Jung Liao, Wan-Li Cheng, Wei-Jan Chen and Kwang-Huei Lin
kit (Amersham Inc., Piscataway, NJ, USA) as described previously ( Shih et al. 2004 ). The antibodies used were rabbit polyclonal antibodies to cyclin E, and retinoblastoma (Rb) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal
JS Lewis, TJ Thomas, CM Klinge, MA Gallo and T Thomas
It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.
C Green, R Chatterjee, HH McGarrigle, F Ahmed and NS Thomas
Cells are maintained in a quiescent state by members of the retinoblastoma protein family, pRb and p130. Both are phosphoproteins and hypophosphorylated forms of pRb and p130 bind and repress the activity of E2F transcription factors, thereby preventing entry into the cell cycle. Mitogenic stimulation causes activation of cyclin dependent kinases (cdk) that phosphorylate both pRb and p130, thereby releasing E2F factors which stimulate the transcription of a number of genes that are required for DNA synthesis and for regulating the cell cycle. In non-dividing cells, cdks are maintained in an inactive state by cdk inhibitor proteins such as p27(Kip1). The aim of our study was to determine how E2F complexes are regulated during the differentiation of human primary granulosa lutein cells (GLC) of the corpus luteum (CL). The CL is formed in the ovary after ovulation at the terminal stage of folliculogenesis after completion of maturation and differentiation of Graafian follicles. As shown by flow cytometry GLC are not dividing, being predominantly in the G(0)/G(1) phase of the cell cycle and, consistent with this, they contain the cdk inhibitor protein, p27(Kip1), but not E2F-1 which is normally expressed only in proliferating cells. The GLC do express E2F-4, hypophosphorylated pRb, p130 forms 1 and 2 and, surprisingly, hypophosphorylated p107. p107 is normally present only in dividing cells where it regulates E2F activity during the cell cycle. These forms of pRb, p130 as well as p107, together with E2F-4 are all active in that they can bind an E2F DNA-binding site in a pull-down assay. Immunocytochemistry shows that these proteins are expressed in almost all GLC but have different sub-cellular distribution: p107 is concentrated in nucleoli, while p130 and E2F-4 show relatively even nuclear and cytoplasmic distributions. Both pRb and p130 have been implicated previously in repressing E2F activity in many different cell types during cell cycle arrest in G(0)/G(1). We conclude that p107 is active in human primary GLC but its nucleolar localisation would suggest that it represses ribosomal RNA synthesis rather than E2F activity.
Jack-Michel Renoir, Céline Bouclier, Amélie Seguin, Véronique Marsaud and Brigitte Sola
-dependent kinase (Cdk) and the phosphorylation of retinoblastoma protein (pRb; Fig. 2 ; Altucci et al . 1996 , Foster & Wimalasena 1996 , Hurd et al . 1997 , Prall et al . 1997 , Doisneau-Sixou et al . 2003 ). Evidence for a key role of c-myc in E 2
Masaki Shiota, Akira Yokomizo and Seiji Naito
and hypophosphorylated form of retinoblastoma protein (pRb; Johnson et al . 1995 , Darbinian et al . 1999 ), both of which have been implicated in PCa progression ( Yeh et al . 1998 , Davis et al . 2006 ). NFκB NFκB (p65) is a protein complex