Requirements for p53 and the ATM Gene Product in the Regulation of G1/S and S Phase Checkpoints

Oncogene. 1998 Feb 12;16(6):721-36. doi: 10.1038/sj.onc.1201793.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Ataxia Telangiectasia / pathology
  • Ataxia Telangiectasia Mutated Proteins
  • CDC2 Protein Kinase / metabolism
  • CDC2 Protein Kinase / radiation effects
  • CDC2-CDC28 Kinases*
  • CHO Cells
  • Cell Cycle Proteins
  • Cells, Cultured
  • Cricetinae
  • Cyclin E
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclin-Dependent Kinases / metabolism
  • Cyclin-Dependent Kinases / radiation effects
  • Cyclins / metabolism
  • Cyclins / radiation effects
  • DNA / biosynthesis*
  • DNA Replication / radiation effects
  • DNA-Binding Proteins
  • Fibroblasts / cytology
  • Fibroblasts / metabolism*
  • Fibroblasts / radiation effects
  • G1 Phase
  • Gene Deletion
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Protein Kinases / metabolism
  • Protein Kinases / radiation effects
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Serine-Threonine Kinases / radiation effects
  • Proteins / metabolism*
  • S Phase
  • Signal Transduction*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Tumor Suppressor Proteins

Substances

  • CDKN1A protein, human
  • Cdkn1a protein, mouse
  • Cell Cycle Proteins
  • Cyclin E
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • DNA-Binding Proteins
  • Proteins
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • DNA
  • Protein Kinases
  • histone H1 kinase
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Atm protein, mouse
  • Protein-Serine-Threonine Kinases
  • CDC2 Protein Kinase
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cdk2 protein, mouse
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases