title: Antagonists of anaphase-promoting complex (APC)-2-cell cycle and apoptosis regulatory protein (CARP)-1 interaction are novel regulators of cell growth and apoptosis.
abstract: CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Although CARP-1 is a regulator of chemotherapy-dependent apoptosis, it is also a part of the NF-κB proteome and a co-activator of steroid/thyroid nuclear receptors as well as β-catenin signaling. Our yeast two-hybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. CARP-1 also binds with anaphase-promoting complex/cyclosome co-activators Cdc20 and Cdh1. Following mapping of the minimal epitopes involved in CARP-1 binding with APC-2, a fluorescence polarization assay was established that indicated a dissociation constant (K(d)) of 480 nm for CARP-1/APC-2 binding. Fluorescence polarization assay-based high throughput screening of a chemical library yielded several small molecule antagonists of CARP-1/APC-2 binding, termed CARP-1 functional mimetics. CFM-4 (1(2-chlorobenzyl)-5'-phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one), a lead compound, binds with and stimulates CARP-1 expression. CFM-4 prevents CARP-1 binding with APC-2, causes G(2)M cell cycle arrest, and induces apoptosis with an IC(50) range of 10-15 μm. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc20 proteins. Depletion of CARP-1, however, interferes with CFM-4-dependent cell growth inhibition, activation of caspases, and apoptosis. Because CFM-4 also suppresses growth of drug-resistant human breast cancer cells without affecting the growth of human breast epithelial MCF-10A cells, elevating CARP-1 by CFM-4 and consequent apoptosis could in principle be exploited to further elucidate, and perhaps effectively target, often deregulated cell cycle pathways in pathological conditions, including cancer.
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Vineshkumar Thidil
authors: VT
authors: Puliyappadamba
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Wenjuan
authors: W
authors: Wu
authors: Michigan High-throughput Screening Center, Kalamazoo Valley Community College, Kalamazoo, Michigan 49003.
authors: Debra
authors: D
authors: Bevis
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201; John D. Dingell Veterans Affairs Medical Center, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Liyue
authors: L
authors: Zhang
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Lisa
authors: L
authors: Polin
authors: Michigan High-throughput Screening Center, Kalamazoo Valley Community College, Kalamazoo, Michigan 49003.
authors: Robert
authors: R
authors: Kilkuskie
authors: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Russell L
authors: RL
authors: Finley
authors: College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109.
authors: Scott D
authors: SD
authors: Larsen
authors: John D. Dingell Veterans Affairs Medical Center, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Edi
authors: E
authors: Levi
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201; Breast Cancer Program, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Fred R
authors: FR
authors: Miller
authors: John D. Dingell Veterans Affairs Medical Center, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors: Anil
authors: A
authors: Wali
authors: Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201; John D. Dingell Veterans Affairs Medical Center, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan 48201; Breast Cancer Program, Wayne State University School of Medicine, Detroit, Michigan 48201. Electronic address: Rishia@Karmanos.org.
authors: Arun K
authors: AK
authors: Rishi
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019556:COS Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D056744:Cyclin B1 / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D025941:Protein Interaction Mapping / Q000379:methods; D015398:Signal Transduction; D013141:Spiro Compounds / Q000737:chemistry*; D013830:Thiadiazoles / Q000737:chemistry*; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: C569107:1-(2-chlorobenzyl)-5'-phenyl-3'H-spiro(indoline-3,2'-(1,3,4)thiadiazol)-2-one; D051017:Apoptosis Regulatory Proteins; C477529:CCAR1 protein, human; C540324:CCAR1 protein, mouse; D018797:Cell Cycle Proteins; D056744:Cyclin B1; D013141:Spiro Compounds; D013830:Thiadiazoles; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases