The retinoblastoma susceptibility gene product p105Rb (RB) is generally believed to be an important regulator in the control of cellular proliferation and in regulating the cell cycle. The biochemical mechanisms for RB's action remain unclear. The transforming properties of several DNA tumor virus oncoproteins are dependent, at least in part, on their ability to bind to RB and presumably to sequester RB from its cellular counterparts. Recent reports have suggested that RB directly interacts with three important transcription factors, E2F, ATF-2 and ElF- 1. With these, it is clear that RB binds a number of cellular proteins, which may be directly or indirectly involved in transcriptional regulation of a set of genes required for controlling cell growth. Indeed, Six cellular genes have been identified as targets of transcriptional regulation by RB. These results reveal a new mechanism by which RB constrains cellular proliferation. Recently, we have demonstrated that RB activates transcription of c-jun gene through the Spl binding site within the c-jun promoter. The mechanism by which RB stimulates Spl-mediated transactivation is liberation of Spl from an inhibitor, Spl-I. This exciting observation warrants further investigation. The specific objectives of this proposal are: l) To isolate and characterize the cellular factor(s) that mediate inhibition of Spl binding activity. 2.) To study biochemical mechanisms by generation of antisera against Spl inhibitor (Spl-I). 3.) To determine the molecular events leading to stimulation of Sp l -mediated transactivation by retinoblastoma gene product. 4.) To study whether Spl- I expression or its binding to RB is cell cycle dependent. These objectives can be accomplished by purification of protein by using heparin-agarose, gel filtration and affinity chromatography, CAT assay, gel retardation assays, deletion, linker scanning mutant and site- directed mutagenesis, bacterially expressed GST fusion proteins, cell- free transcription-translation system, immunoprecipitation, 32p- orthophosphate labeled cells and synchronized cells. The study of the molecular mechanisms of Spl-I as a cellular target for RB is critically important. The cloning of this Spl inhibitor will likely provide insight into its identity, function and regulation. We believe that this proposal will not only identify a biochemical function for RB, but also will have a significant impact on our understanding of a functional link between two distinct classes of oncoproteins, RB and c- Jun, that are involved in the control of cell growth, as well as defining a novel mechanism for the regulation of c-jun expression.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Pathology B Study Section (PTHB)
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University of California Los Angeles
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Calhoun, Colonya C; Lu, Ying-Chun; Song, Jun et al. (2009) Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype. Mol Cell Biochem 320:35-43
Lu, Ying-Chun; Song, Jun; Cho, Hee-Yeon et al. (2006) Cyclophilin a protects Peg3 from hypermethylation and inactive histone modification. J Biol Chem 281:39081-7
Song, Jun; Lu, Ying-Chun; Yokoyama, Kazunari et al. (2004) Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells. J Biol Chem 279:24414-9
Song, Jun; Pang, Shen; Lu, Yingchun et al. (2004) Gene silencing in androgen-responsive prostate cancer cells from the tissue-specific prostate-specific antigen promoter. Cancer Res 64:7661-3
Song, Jun; Pang, Shen; Lu, Yingchun et al. (2004) Poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNA from a pol II promoter. Biochem Biophys Res Commun 323:573-8
Chiu, Robert; Rey, Osvaldo; Zheng, Jun-Qi et al. (2003) Effects of altered expression and localization of cyclophilin A on differentiation of p19 embryonic carcinoma cells. Cell Mol Neurobiol 23:929-43
Cui, Yukun; Mirkia, Kirash; Florence Fu, Yu-Hsieh et al. (2002) Interaction of the retinoblastoma gene product, RB, with cyclophilin A negatively affects cyclosporin-inhibited NFAT signaling. J Cell Biochem 86:630-41
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Nishitani, J; Nishinaka, T; Cheng, C H et al. (1999) Recruitment of the retinoblastoma protein to c-Jun enhances transcription activity mediated through the AP-1 binding site. J Biol Chem 274:5454-61
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