Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints.
We investigated the requirements for protein p53 and the ATM gene product in radiation-induced inhibition of DNA synthesis and regulation of the cyclin E/ and cyclin A/cyclin dependent kinases (Cdks). Wild type (WT) mouse lung fibroblasts (MLFs), p53(-/-) knock-out MLFs, normal human skin fibroblasts (HSF-55), and human AT skin fibroblasts (GM02052) were used in the investigations. The absence of p53 had no significant effect on the inhibition or recovery of DNA synthesis throughout the S phase, as determined from BrdU labeling and flow cytometry, or the rapid inhibition of cyclin A/Cdks. Gamma radiation (8 Gy) inhibited DNA synthesis and progression into G2 during the first 3 h after irradiation, and the recovery of these processes occurred at similar rates in both WT and p53(-/-) MLFs. The cyclin A/Cdks were inhibited 55-70% at 1 h after irradiation in both cell types, but p21WAF1/Cip1 levels or p21 interaction with Cdk2 did not increase in the irradiated p53(-/-) MLFs. Although p53(-/-) MLFs do not exhibit prolonged arrest at a G1 checkpoint, radiation did induce a rapid 20% reduction and small super-recovery of cyclin E/Cdk2 within 1-2 h after irradiation. Similar inhibition and recovery of cyclin E/Cdk2 previously had been associated with regulation of transient G1 delay and the inhibition of initiation at an apparent G1/S checkpoint in Chinese hamster cells. In contrast, loss of the ATM gene product abrogated transient cyclin E/Cdk2 inhibition, most inhibition of DNA synthesis and all, but a 10-15% inhibition, of the cyclin A/Cdks. The results indicate that neither p53 nor p21 is required for transient inhibition of cyclin E/Cdk2 associated with the G1/S checkpoint or for inhibition of DNA synthesis at 'checkpoints' within the S phase. Conversely, the ATM gene product appears to be essential for regulation of the G1/S checkpoint and for inhibition of DNA replication associated with the inhibition of cyclin A/Cdk2. Differential aspects of DNA synthesis inhibition among cell types are presented and discussed in the context of S phase checkpoints.
Xie
G
G
The Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Habbersett
R C
RC
Jia
Y
Y
Peterson
S R
SR
Lehnert
B E
BE
Bradbury
E M
EM
D'Anna
J A
JA
D000818:Animals; D001260:Ataxia Telangiectasia / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D016203:CDC2 Protein Kinase / Q000378:metabolism / Q000528:radiation effects; D042846:CDC2-CDC28 Kinases; D016466:CHO Cells; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D006224:Cricetinae; D019927:Cyclin E; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism / Q000528:radiation effects; D016213:Cyclins / Q000378:metabolism / Q000528:radiation effects; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D016193:G1 Phase; D017353:Gene Deletion; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011494:Protein Kinases / Q000378:metabolism / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000528:radiation effects; D011506:Proteins / Q000378:metabolism*; D016196:S Phase; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004247:DNA; D011494:Protein Kinases; C102333:histone H1 kinase; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases