Epidermal growth factor increases the level of the cyclin-dependent kinase (CDK) inhibitor p21/CIP1 (CDK-interacting protein 1) in A431 cells by increasing the half-lives of the p21/CIP1 transcript and the p21/CIP1 protein.
DNA synthesis was inhibited in A431 cells by epidermal growth factor (EGF) in a p21/CIP1-dependent manner [where CIP1 is cyclin-dependent kinase (CDK)-interacting protein 1]. When 1 or 10 nM EGF was added, the level of p21/CIP1 was increased to the same extent, and the protein level peaked after approx. 5 h of incubation. The increase in p21/CIP1 mRNA upon addition of EGF was rapid, and was enhanced in the presence of cycloheximide. The half-life of p21/CIP1 mRNA in EGF-treated A431 cells was increased approx. 2-fold; this is in contrast with the case in MCF-7 cells with normal p53, in which the half-life of p21/CIP1 mRNA was not increased upon addition of EGF. This increased stability accounts for most of the increase in mRNA levels observed in A431 cells during short incubation periods. Additionally, upon prolonged incubation of A431 cells with EGF, the half-life of the protein was also increased compared with that in untreated cells and in cells treated with EGF for short time periods. Nuclear run-on assays demonstrated only marginal stimulation of transcription by 10 or 1 nM EGF, or by 10 ng/ml tumour necrosis factor alpha. Our results indicate that the most important mechanisms by which EGF increases p21/CIP1 protein levels in A431 cells are post-transcriptional and post-translational stabilization.
Johannessen
L E
LE
Institute of Pathology, The National Hospital, The University of Oslo, 0027 Oslo, Norway.
Knardal
S L
SL
Madshus
I H
IH
D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D004815:Epidermal Growth Factor; D004247:DNA; D003513:Cycloheximide; D018844:Cyclin-Dependent Kinases