2002 AACR.
1811
A NOVEL APPROACH TO ISOLATE CELL CYCLE REGULATORS: FUNCTIONAL CHARACTERIZATION OF LETM-1 WHICH CAUSES G0/G1 CELL CYCLE ARREST
Intracellular Signalling II (Poster Session), Exhibit Hall A-C, Sec 4, Mon. 08 April,2002 8:00AM – 12:00PM
Yasumichi Hitoshi, Denise Pearsall, Jeff Quast, Susan Demo, Rigel Inc, South San Francisco, CA; Michel Janicot, Jorge Vialard, Walter Luyten, Walter Woulters, Janssen Research Insitute, Beerse Belgium; Donald Payan, James Lorens, Susan Molineaux, Mark Bennett, Rigel Inc, South San Francisco, CA
In recent years, there have been major developments in understanding of the cell cycle. Normal cell proliferation is tightly regulated by activation and inactivation of a series of proteins that constitute the cell cycle machinery. Expression and activity of components of the cell cycle can be altered during development of a variety of human diseases such as cancer, cardiovascular disease, and psoriasis where aberrant proliferation contributes to pathology of the illness. Cell cycle regulation is highly conserved among all eukaryotic cells and genetic screens in various organisms such as yeast, worms, and flies, have yielded critical regulators of this cellular process. However there is a need to establish screens in human cells to better understand how disruption of cell cycle regulation causes disease. Using a retroviral cDNA/peptide library-based functional screen with a Tet-regulatable gene expression system, we have identified several molecules causing cell cycle arrest in human cells. Here we report the identification and molecular characterization of a surface molecule, leucine zipper-EF-hand containing transmembrane protein 1 (LETM-1), through a retroviral cDNA library-based functional screen. LETM-1 was originally identified as the gene flanking the critical region of the Wolf-Hirschhorn syndrome (WHS), a chromosomal disorder characterized by a 4p monosomy that results in certain facial dysmorphisms and neurological manifestations. Our results demonstrate that LETM-1 overexpression causes potent G0/G1 arrest in a p53-independent and Rb-dependent manner, suggesting cell cycle regulation through LETM-1. They also suggest the existence of a LETM-1 signaling pathway that may play an important role in neuromuscular development.
376
ISOLATION AND CHARACTERIZATION OF COMPOUNDS THAT INHIBIT SCF AND APC COMPLEX E3 UBIQUITIN LIGASE ACTIVITIES
Novel Agents: Novel Targets and Modulation of Activity (Poster Session), Exhibit Hall A-C, Sec 13, Sun. 07 April,2002 8:00AM – 12:00PM
Mark Bennett, Jaining Huang, Julie Sheung, Robert Daly, Cindy Coquilla, Ruby Daniel, Todd Pray, Susan Demo, Takaria Gururaja, Liming Dong, Dave Anderson, Guy Laidig, Dane Goff, Raj Singh, Susan S Molineaux, Don Payan, rigel, inc, south san francisco, CA
Orderly progression through the sequential stages of the eukaryotic cell cycle requires the accumulation and turnover of both positive and negative cell cycle regulatory proteins. The stability of these cell cycle regulatory proteins, which often act at cell cycle checkpoints, is commonly determined by ubiquitination and proteasome targeting. In an effort to identify novel therapeutic compounds that modulate cell proliferation with potential application in oncology, we have focused on two classes of E3 ubiquitin ligases: the SCF (skp/cullin/F-box) and APC (anaphase promoting complex) complexes. A plate-based in vitro ubiquitination assay utilizing purified E1, E2 (Ubc5), E3 (Roc1/Cul1 or APC11/APC2), and ubiquitin was established and used to screen for small molecule inhibitors of the E3 activities. The inhibitors were characterized for activity on E2 charging and for E3 complex selectivity. Several structurally distinct classes of compounds were identified with a range of in vitro biochemical activities that included inhibition of E2 charging, inhibition of both the APC and SCF E3 activities, and selective inhibition of the APC-catalyzed ubiquitination reaction. The ability of the E3 ubiquitin ligase inhibitory compounds to influence a panel of cell-based assays in tumor cell lines was also investigated. Among the cell-based assays examined were proliferation, cell cycle progression, apoptosis, chemosensitization, and accumulation or turnover of key cell cycle regulatory proteins. One class of compound was found to dramatically inhibit the ability of both HeLa and A549 cells to progress through the cell cycle following release from either nococazole- or thymidine-induced arrest. These results demonstrate that compounds can be identified that both display in vitro biochemical selectivity for E3 ubiquitin ligase inhibition and exhibit cell-based activities relevant to cell cycle progression and therefore confirm ubiquitin ligases as potential therapeutic targets for oncology.
808
MI-2 HISTONE DEACETYLASE COMPLEX PROTEIN INVOLVED IN P21(WAF-1/CIP-1)-INDUCED GROWTH ARREST
Transcription Regulation (Poster Session Discussion), Exhibit Hall E, Room 131, Sun. 07 April,2002 1:15PM – 5:15PM
Susan Demo, Yasumichi Hitoshi, Jerry Garnica, Jeff Quast, Denise Pearsall, Rigel, Inc, South San Francisco, CA; Jorge Vialard, Jimmy Van Heusden, Walter Luyten, Michel Janicot, Walter Woulters, Janssen Research Foundation, Beerse Belgium; James Lorens, Mark Bennett, Susan Molineaux, Donald G Payan, Rigel, Inc, South San Francisco, CA
p21 is an important regulator of the cell cycle that acts to inhibit cyclin-dependent kinase activity. It is thought to mediate p53-induced G1 cell cycle arrest. In order to elucidate other effectors involved in p21-dependent growth arrest, we undertook a genetic screen utilizing retroviral introduction of human cDNA libraries into A549 cells engineered to express p21 in a tetracycline-regulatable manner. We isolated several cDNAs that were capable of overcoming growth arrest induced by p21. One such cDNA encoded a fragment of a novel gene that has 50% homology to the Xenopus p66 Mi-2 histone deacetylase complex protein (Nat Genet 1999 Sep;23(1):62-6). Overexpression of this fragment termed, B5-8, allowed cells to continue to cycle after induction of p21 as measured by BrdU incorporation. Ribonuclease protection (RPA) assays showed that clone B5-8 did not effect the level of p21 mRNA. A GST-B5-8 fusion protein was expressed in e. coli and used for analysis of proteins that associate with B5-8. GST-B5-8 associated with Mi-2, HDAC1, and HDAC2, consistent with observations on p66 in Xenopus extracts. These results suggest that the Mi-2 chromatin remodeling complex is involved in p21-mediated growth arrest. Several possible mechanistic models are considered. |
