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p53-dependent cell cycle arrests are preserved in DNA-activated protein kinase-deficient mouse fibroblasts
L C Huang1, K C Clarkin, G M Wahl
1Gene Expression Laboratory, The Salk Institute, La Jolla, California 92037, USA.
Abstract:
p53 is involved in at least three cell cycle checkpoints in normal cells: two in G1, activated by either DNA damage or by ribonucleotide pool depletion in the absence of damage, and one in metaphase/anaphase activated by an incomplete mitotic spindle. We tested whether any of these checkpoints require the DNA-activated protein kinase (DNAPK), since data indicate that it is activated by damaged DNA to modify p53 in cultured cells and in cell-free systems. Fibroblasts isolated from mice with severe combined immune deficiency (SCID) were used because the sole genetic defect underlying this syndrome lies within the DNAPK gene. This report shows that age-matched SCID and isogenic wild-type embryonic fibroblasts arrested in response to DNA damage, ribonucleoside triphosphate depletion, and spindle poisons, whereas p53-/- fibroblasts failed to do so. Therefore, DNAPK-deficient scid cells preserve normal p53-dependent cell cycle checkpoints. The data provide one explanation of why scid mice are not tumor prone though they are deficient in double-strand break repair.
Insights
DNA-activated protein kinase (DNAPK) is not required for p53-dependent cell cycle checkpoints. SCID mouse cells lacking DNAPK function maintain normal cell cycle arrest in response to DNA damage or spindle issues.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The p53 protein is crucial for regulating cell cycle checkpoints in response to various cellular stresses.
- DNA-activated protein kinase (DNAPK) is implicated in DNA damage response pathways and p53 modification.
- Mice with severe combined immune deficiency (SCID) harbor a genetic defect in the DNAPK gene.
Purpose of the Study:
- To investigate the role of DNAPK in p53-dependent cell cycle checkpoints.
- To determine if DNAPK is essential for cell cycle arrest induced by DNA damage, nucleotide depletion, or spindle abnormalities.
Main Methods:
- Utilized fibroblasts from SCID mice (DNAPK-deficient) and age-matched wild-type controls.
- Employed p53-/- fibroblasts as a control for p53-dependent checkpoint function.
- Assessed cell cycle arrest in response to DNA damage, ribonucleotide triphosphate depletion, and spindle poisons.
Main Results:
- SCID and wild-type fibroblasts exhibited normal cell cycle arrest under all tested conditions.
- p53-/- fibroblasts failed to arrest, confirming p53's essential role in these checkpoints.
- DNAPK-deficient SCID cells retained functional p53-dependent cell cycle checkpoints.
Conclusions:
- DNAPK is not required for the activation of p53-dependent cell cycle checkpoints.
- The data suggest that DNAPK-independent pathways mediate cell cycle arrest.
- This finding offers a potential explanation for the lack of tumor predisposition in SCID mice despite their DNA repair deficiency.