CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein.
C/EBPalpha has a role in growth arrest and differentiation of mouse preadipocytes. To study the mechanism of C/EBPalpha-induced growth arrest, we developed a cell line, HT1, that contained the human C/EBPalpha gene under Lac repressor control. IPTG-induced C/EBPalpha caused inhibition of cell proliferation and DNA synthesis as measured by colony growth assays, cell counting, and BrdU uptake. A number of proteins that are known to be involved in the regulation of the cell cycle, such as cyclin-dependent kinase (CDK)2 and CDK4, proliferating cell nuclear antigen (PCNA), p53, c-fos, and the CDK inhibitor p16 and p27 were investigated by Western analysis. No change in their expression was observed. However, the p21 (WAF-1/CIP-1/SDI-1) protein was significantly elevated in growth-arrested HT1 cells. Elevation of p21/SDI-1 mRNA (threefold) and activation of the p21/SDI-1 promoter by C/EBPalpha did not account for the 12- to 20-fold increase in p21/SDI-1 protein. Protein synthesis inhibition by cycloheximide (CHX) treatment indicated that the half-life of p21/SDI-1 in dividing HT1 cells was approximately 30 min. However, in C/EBPalpha growth-arrested cells, the level of the p21/SDI-1 did not change for > 80 min after CHX addition. Our studies demonstrate that C/EBPalpha activates p21/SDI-1 by increasing p21/SDI-1 gene expression and by post-translational stabilization of p21/SDI-1 protein. Furthermore, induction of p21/SDI-1 is responsible for the ability of C/EBPalpha to inhibit proliferation because transcription of antisense p21/SDI-1 mRNA eliminated growth inhibition by C/EBPalpha.
Timchenko
N A
NA
Department of Pathology, Baylor College of Medicine, Houston, Texas 77071, USA.
Wilde
M
M
Nakanishi
M
M
Smith
J R
JR
Darlington
G J
GJ
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D002455:Cell Division / Q000502:physiology*; D003001:Cloning, Molecular; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D022762:CCAAT-Enhancer-Binding Proteins; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase