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.
Highlights
Apoptosis is an important physiological process that is often utilized during the development and maintenance of normal tissue homeostasis in organisms
Our study reveals that Adriamycin or CARP-1 functional mimetic (CFM) promote increase of CARP-1 and γ-H2AX proteins and their co-localization
Cleavage in a time-dependent manner, with a robust cleaved PARP noted over a 12-h treatment period, Adriamycin stimulated PARP cleavage as early as 3 h of treatment (Figure 1C). These findings collectively demonstrate that both the compounds inhibit cell growth in part by stimulating apoptosis, and a robust apoptosis activation occurs at 12 h of treatments with respective compounds, co-incident with a robust increase in CARP-1 levels and activities of jun N-terminal Kinases (JNKs) and H2AX
Summary
Apoptosis is an important physiological process that is often utilized during the development and maintenance of normal tissue homeostasis in organisms. Genetic and biochemical studies identified two major pathways of apoptosis and a host of biochemical components that function to regulate these pathways in a variety of cell and organism models [2,3]. The regulators of apoptosis signaling, including the ones that are yet to be discovered, represent attractive avenues for development of intervention strategies for such pathologies where apoptosis is defective [3,4]. In this regard, elucidation of novel mechanism(s) of apoptosis signaling would be useful in further defining cellular apoptotic pathways
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