<span style="font-variant: small-caps">A H2AX–CARP-1 </span>Interaction Regulates Apoptosis Signaling Following DNA Damage

• Main Authors: Sreeja C. Sekhar, Jaganathan Venkatesh, Vino T. Cheriyan, Magesh Muthu, Edi Levi, Hadeel Assad, Paul Meister, Vishnu V. Undyala, James W. Gauld, Arun K. Rishi
• Format: Article
• Language: English
• Published: MDPI AG 2019-02-01
• Series: Cancers
• Subjects: CCAR1/CARP-1; γH2AX; apoptosis; chemotherapeutics; cancer cells
• Online Access: https://www.mdpi.com/2072-6694/11/2/221

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 (&#947;H2AX) 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 &#947;H2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and &#947;H2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished &#947;H2AX, 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 (&#916;600&#8315;652) mutant. Moreover, cells expressing CARP-1 (&#916;600&#8315;652) mutant were resistant to apoptosis, and had diminished levels of &#947;H2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1&#8315;35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636&#8315;650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636&#8315;650) peptide bound with H2AX (1&#8315;35) peptide with a dissociation constant (K_d) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1&#8315;35) peptide or EGFP-tagged CARP-1 (636&#8315;650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636&#8315;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.