Author ORCID Identifier
https://orcid.org/0000-0003-3311-8120 : Magesh Muthu
https://orcid.org/0000-0002-1836-0093 : Paul Meister
https://orcid.org/0000-0002-2956-9781 : James W. Gauld
CCAR1/CARP-1, γH2AX, apoptosis, chemotherapeutics, cancer cells
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.
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Sekhar, Sreeja C.; Venkatesh, Jaganathan; Cheriyan, Vino T.; Muthu, Magesh; Levi, Edi; Assad, Hadeel; Meister, Paul; Undyala, Vishnu V.; Gauld, James W.; and Rishi, Arun K.. (2019). A H2AX–CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage. Cancers, 11 (2), 221.