Relationships between G1 arrest and stability of the p53 and p21Cip1/Waf1 proteins following gamma-irradiation of human lymphoma cells.
We investigated temporal relationships between ionizing radiation-induced G1 arrest and induction of the p53-regulated genes GADD45, CIP1/WAF1, and MDM2 in a series of Burkitt's lymphoma and lymphoblastoid cell lines that differed in p53 gene status. Emphasis was placed on characterization of the EW36 cell line, which despite expressing wild-type p53 genes, is defective in G1 arrest following gamma-irradiation (P. M. O'Connor et al., Cancer Res., 53: 4776-4780, 1993). Induction of CIP1/WAF1, GADD45, and to a lesser extent MDM2 mRNA was observed in all wild-type p53 lines that arrested in G1. Cell lines that contained only mutant p53 genes or were heterozygous for p53 mutations failed to induce appreciable levels of these p53-regulated transcripts and did not arrest in G1. G1 arrest in the wild-type p53 cell line WMN was more prolonged than elevation of CIP1/WAF1, GADD45, or MDM2 transcripts, suggesting that G1 arrest duration must be dependent upon stability of these newly synthesized proteins. In agreement, we found that p21Cip1/Waf1, a potent inhibitor of G1-S phase cyclin-dependent kinases, was maintained at elevated levels throughout the period that WMN cells remained arrested in G1. EW36 cells exhibited normal induction of CIP1/WAF1, GADD45, and MDM2 mRNA following gamma-irradiation, suggesting that the defect in G1 arrest must reside downstream of p53 transactivation. Investigations into the stability of p53 and p21Cip1/Waf1 revealed that EW36 cells failed to maintain elevated levels of these proteins following irradiation. p53 levels decreased within 4 h of irradiation, and p21Cip1/Waf1 levels decreased shortly after the normal decline of CIP1/WAF1 mRNA levels. Degradation of p21Cip1/Waf1 coincided with the escape of EW36 cells from G1 arrest. Our studies suggest that p21Cip1/Waf1 stability may determine G1 arrest duration and that premature degradation of this protein could provide an alternative route to subversion of the G1 checkpoint in cancer cells.
Bae
I
I
Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Fan
S
S
Bhatia
K
K
Kohn
K W
KW
Fornace
A J
AJ
O'Connor
P M
PM
D002051:Burkitt Lymphoma / Q000235:genetics* / Q000473:pathology* / Q000532:radiotherapy; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000502:physiology* / Q000528:radiation effects; D004355:Drug Stability; D016193:G1 Phase / Q000502:physiology* / Q000528:radiation effects*; D005720:Gamma Rays / Q000627:therapeutic use*; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects*; D016158:Genes, p53; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology / Q000528:radiation effects; D009687:Nuclear Proteins; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000528:radiation effects; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000502:physiology* / Q000528:radiation effects; D000097642:GADD45 Proteins
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D000097642:GADD45 Proteins; C493956:CDKN1A protein, human; C497206:MDM2 protein, human