Breast cancer cell proliferation is inhibited by BAD: regulation of cyclin D1.
Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.
Fernando
Romaine
R
Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920.
Foster
James S
JS
Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920.
Bible
Amber
A
Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920.
Ström
Anders
A
Department of Biosciences, Karolinska Institute, Novum, S-14157, Sweden.
Pestell
Richard G
RG
Departments of Cancer Biology and Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Rao
Mahadev
M
Department of Oncology, NCI, National Institutes of Health, Washington, D. C. 20057.
Saxton
Arnold
A
Department of Animal Science, University of Tennessee, Knoxville, Tennessee 37996.
Baek
Seung Joon
SJ
Department of Pathobiology, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996.
Yamaguchi
Kiyoshi
K
Department of Pathobiology, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996.
Donnell
Robert
R
Department of Pathobiology, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996.
Cekanova
Maria
M
Department of Pathobiology, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996.
Wimalasena
Jay
J
Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920. Electronic address: jwimalas@mc.utmck.edu.
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D056741:Cyclin D; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D016193:G1 Phase / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D020218:Response Elements; D016196:S Phase / Q000187:drug effects; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism*
C494819:BAD protein, human; D056741:Cyclin D; D016213:Cyclins; D004967:Estrogens; D016755:Proto-Oncogene Proteins c-jun; D051025:bcl-Associated Death Protein