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Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints
1The Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
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.
Insights
The ATM gene, not p53, is crucial for regulating DNA synthesis inhibition and cell cycle checkpoints after radiation. Loss of ATM abrogates G1/S checkpoint control and S-phase DNA replication inhibition.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Damage Response
Background:
- Protein p53 and ATM gene product are key regulators in cellular response to DNA damage.
- Radiation-induced cell cycle arrest is critical for DNA repair and genomic stability.
- Cyclin-dependent kinases (Cdks) play vital roles in cell cycle progression and are targets of damage-induced regulation.
Purpose of the Study:
- To investigate the distinct roles of p53 and ATM in radiation-induced DNA synthesis inhibition.
- To elucidate the regulation of cyclin E/Cdk2 and cyclin A/Cdks by p53 and ATM.
- To understand the involvement of these proteins in G1/S and S-phase checkpoints.
Main Methods:
- Utilized wild-type and p53-deficient mouse lung fibroblasts (MLFs) and human fibroblasts (normal and AT).
- Employed gamma radiation (8 Gy) to induce DNA damage.
- Assessed DNA synthesis inhibition and recovery using BrdU labeling and flow cytometry.
- Analyzed levels and interactions of cyclins (E and A) and Cdks, including p21WAF1/Cip1.
Main Results:
- p53 deficiency did not significantly affect DNA synthesis inhibition/recovery within S phase or cyclin A/Cdk inhibition.
- ATM deficiency abrogated transient cyclin E/Cdk2 inhibition at G1/S, most DNA synthesis inhibition, and cyclin A/Cdk inhibition.
- p53 was not required for cyclin E/Cdk2 inhibition at G1/S or for S-phase DNA synthesis inhibition.
Conclusions:
- Neither p53 nor p21 is essential for G1/S checkpoint regulation or S-phase DNA synthesis inhibition.
- The ATM gene product is indispensable for G1/S checkpoint control and S-phase DNA replication inhibition.
- Differential roles of p53 and ATM in DNA damage response pathways are highlighted, particularly concerning cell cycle checkpoints.