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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint
Rumani Singh1, Rajkumar S Kalra1, Kamrul Hasan1
1Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
Abstract:
CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.
Insights
Cellular apoptosis regulator factor (CARF) protein is linked to the cell cycle and DNA damage response. CARF levels increase during senescence and growth arrest, suggesting its role in these critical cellular processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cellular apoptosis regulator factor (CARF) is an ARF-binding protein known to influence the p53-p21-HDM2 pathway.
- CARF overexpression induces growth arrest in cancer cells and premature senescence in normal cells via p53 activation.
Purpose of the Study:
- Investigate the association between CARF and the cell cycle.
- Determine CARF's involvement in the DNA damage response (DDR).
Main Methods:
- Assessed CARF protein half-life.
- Analyzed CARF expression levels in cycling and senescent cells.
- Examined CARF expression in response to various growth arrest and senescence-inducing agents.
- Correlated CARF levels with DDR and checkpoint protein expression (ATM, ATR, CHK1, CHK2, γH2AX, p53, p21).
Main Results:
- CARF protein has a half-life of less than 60 minutes.
- CARF levels peak in G2 and early prophase of the cell cycle.
- CARF expression is upregulated during replicative senescence in fibroblasts.
- CARF levels increase transcriptionally and translationally during drug-induced G1 arrest and senescence.
- CARF upregulation correlates with increased DDR and checkpoint signaling proteins.
- Oncogenic RAS and TRF2 knockdown also lead to CARF upregulation.
Conclusions:
- CARF protein is closely associated with cell cycle progression.
- CARF is implicated in the DNA damage response and checkpoint signaling pathways.
- CARF plays a significant role in cellular senescence and growth arrest mechanisms.
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