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Updated: Jan 29, 2026
Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response
Published on: August 2, 2024
A H2AX⁻CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage
Sreeja C Sekhar1,2, Jaganathan Venkatesh3,4, Vino T Cheriyan5,6
1John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201, USA. sreejacsekhar@gmail.com.
Abstract:
Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and γH2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished γH2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Δ600⁻652) mutant. Moreover, cells expressing CARP-1 (Δ600⁻652) mutant were resistant to apoptosis, and had diminished levels of γH2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1⁻35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636⁻650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636⁻650) peptide bound with H2AX (1⁻35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1⁻35) peptide or EGFP-tagged CARP-1 (636⁻650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636⁻650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds.
Insights
Cell Cycle and Apoptosis Regulatory Protein (CARP-1) directly binds with Histone H2AX, a crucial interaction for Adriamycin and CFM compound-induced apoptosis in cancer cells. This binding is essential for CARP-1
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein regulating apoptosis.
- Adriamycin (doxorubicin) and CARP-1 functional mimetic (CFM) compounds induce apoptosis in cancer cells.
- Histone 2AX phosphorylation (γH2AX) is implicated in DNA damage repair and apoptosis signaling.
Purpose of the Study:
- To investigate the role of CARP-1 interaction with H2AX in apoptosis signaling induced by Adriamycin and CFM compounds.
- To identify the specific binding sites between CARP-1 and H2AX.
- To assess the impact of this interaction on cancer cell apoptosis and drug sensitivity.
Main Methods:
- Comet assays to detect DNA damage.
- Western blotting to detect CARP-1 and γH2AX protein levels.
- Immunofluorescence microscopy for co-localization studies.
- CARP-1 knockdown experiments.
- Site-directed mutagenesis to identify binding epitopes.
- Surface Plasmon Resonance (SPR) for binding kinetics.
- Treatment with various peptides and EGFP-tagged constructs.
Main Results:
- Adriamycin and CFM-4.16 induced DNA damage, inhibited cell growth, and elevated CARP-1 and γH2AX levels in human breast and cervical cancer cells.
- CARP-1 and γH2AX co-localized in apoptotic cells, and CARP-1 knockdown diminished γH2AX and apoptosis.
- CARP-1 directly binds to H2AX, with specific binding epitopes identified on both proteins (H2AX residues 1-35 and CARP-1 amino acids 636-650).
- A CARP-1 mutant (Δ600⁻652) resistant to apoptosis showed diminished γH2AX levels.
- Peptides and constructs representing the binding interface conferred resistance to Adriamycin and CFM-4.16, and a TAT-tagged CARP-1 peptide partially abrogated Adriamycin-induced growth inhibition.
Conclusions:
- CARP-1 interaction with H2AX is essential for apoptosis signaling induced by Adriamycin and CFM compounds.
- The identified binding interface between CARP-1 and H2AX is critical for mediating apoptosis.
- Targeting this CARP-1/H2AX interaction may represent a novel therapeutic strategy for sensitizing cancer cells to chemotherapy.
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