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A H2AX-CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage
John D. Dingell Veterans Administration Medical Center, Detroit, USA; Department of Oncology, Karmanos Cancer Institute, Detroit, USA.
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2019 (English)In: Cancers, ISSN 2072-6694, Vol. 11, no 2, article id 221Article in journal (Refereed) Published
Abstract [en]

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 (gamma 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 gamma H2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and gamma H2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished gamma 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 (Delta 600-652) mutant. Moreover, cells expressing CARP-1 (Delta 600-652) mutant were resistant to apoptosis, and had diminished levels of gamma H2AX, 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 (K-d) 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.

Place, publisher, year, edition, pages
MDPI, 2019. Vol. 11, no 2, article id 221
Keywords [en]
CCAR1/CARP-1, gamma H2AX, apoptosis, chemotherapeutics, cancer cells
National Category
Cancer and Oncology
Identifiers
URN: urn:nbn:se:umu:diva-162511DOI: 10.3390/cancers11020221ISI: 000460747200095PubMedID: 30769864OAI: oai:DiVA.org:umu-162511DiVA, id: diva2:1344692
Available from: 2019-08-21 Created: 2019-08-21 Last updated: 2019-08-21Bibliographically approved

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Muthu, Magesh

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