Death Receptor-Induced Activation of the Chk2- and Histone H2AX-Associated DNA Damage Response Pathways

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2008; 29(1):68-82. DOI: 10.1128/MCB.00581-08
Source: PubMed


TRAIL is an endogenous death receptor ligand also used therapeutically because of its selective proapoptotic activity in cancer
cells. In the present study, we examined chromatin alterations induced by TRAIL and show that TRAIL induces a rapid activation
of DNA damage response (DDR) pathways with histone H2AX, Chk2, ATM, and DNA-PK phosphorylations. Within 1 h of TRAIL exposure,
immunofluorescence confocal microscopy revealed γ-H2AX peripheral nuclear staining (γ-H2AX ring) colocalizing with phosphorylated/activated
Chk2, ATM, and DNA-PK inside heterochromatin regions. The marginal distribution of DDR proteins in early apoptotic cells is
remarkably different from the focal staining seen after DNA damage. TRAIL-induced DDR was suppressed upon caspase inhibition
or Bax inactivation, demonstrating that the DDR activated by TRAIL is downstream from the mitochondrial death pathway. H2AX
phosphorylation was dependent on DNA-PK, while Chk2 phosphorylation was dependent on both ATM and DNA-PK. Downregulation of
Chk2 decreased TRAIL-induced cell detachment; delayed the activation of caspases 2, 3, 8, and 9; and reduced TRAIL-induced
cell killing. Together, our findings suggest that nuclear activation of Chk2 by TRAIL acts as a positive feedback loop involving
the mitochondrion-dependent activation of caspases, independently of p53.

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    • "In fact, BLEO can induce the expression of TNF receptor family genes, triggering caspase- 8 dependent apoptosis (Mungunsukh et al. 2010). Moreover, TRAIL, a ligand which also activates TNF receptors, elicits an apoptotic process accompanied by γH2AX ring/pan-nuclear signaling and subsequent canonical apoptotic morphology (Solier et al. 2009), as detected in our case. "
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    ABSTRACT: Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2'-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases. In order to map DNA damage with respect to replicating domains, the distribution of γH2AX foci induced by the radiomimetic agent bleomycin was analyzed in CHO9 interphase nuclei by delineating euchromatic (H3K4me3+) and replicating (EdU+) regions. Quantification of overlapping pixels and 3D inter-object overlap in binary masks revealed colocalization between γH2AX foci and EdU + domains both in ES and MS/LS nuclei, indicating that primary damage distribution is modulated by DNA synthesis. Further, we verified that EdU incorporation by itself did not influence BLEO-induced γH2AX nuclear patterns. Our results also revealed a repeated localization of γH2AX foci in replicating/nonreplicating interfaces which could reflect short-range chromatin migration following DNA insult.
    Full-text · Article · Jul 2014 · Chromosome Research
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    • "Cisplatin and doxorubicin are known to activate the nuclear ABL tyrosine kinase, which stimulates the p53-family of transcription factors to activate mitochondria-dependent intrinsic apoptosis [15-22]. Recent studies have shown that TRAIL can activate the DNA damage response (DDR) as a result of caspase-dependent DNA fragmentation, and this DDR response contributes to TRAIL-induced apoptosis [23,24]. This caspase-induced DDR may explain the recently reported activation of the ABL-JNK-apoptosis pathway by TRAIL [25]. "
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    ABSTRACT: TNF-Related Apoptosis Inducing Ligand (TRAIL) binds to and activates death receptors to stimulate caspase-8 and apoptosis with higher efficiency in cancer than normal cells but the development of apoptosis resistance has limited its clinical efficacy. We found that stable, but not transient knockdown of the ABL tyrosine kinase enhanced the apoptotic response to TRAIL. Re-expression of Abl, but not its nuclear import- or kinase-defective mutant, in the ABL-knockdown cells re-established apoptosis suppression. TRAIL is known to stimulate caspase-8 ubiquitination (Ub-C8), which can facilitate caspase-8 activation or degradation by the lysosomes. In the ABL-knockdown cells, we found a higher basal level of Ub-C8 that was not further increased by lysosomal inhibition. Re-expression of Abl in the ABL-knockdown cells reduced the basal Ub-C8, correlating with apoptosis suppression. We found that lysosomal inhibition by chloroquine (CQ) could also enhance TRAIL-induced apoptosis. However, this pro-apoptotic effect of CQ was lost in the ABL-knockdown cells but restored by Abl re-expression. Interestingly, kinase inhibition at the time of TRAIL stimulation was not sufficient to enhance apoptosis. Instead, persistent treatment for several days with imatinib, an ABL kinase inhibitor, was required to cause the enhanced and the CQ-insensitive apoptotic response to TRAIL. Together, these results show that persistent loss of nuclear ABL tyrosine kinase function can sensitize cells to TRAIL and suggest that long-term exposure to the FDA-approved ABL kinase inhibitors may potentiate apoptotic response to TRAIL-based cancer therapy.
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    • "Increased phosphorylation of the DNA damage-related proteins (e.g. pH2AX, p53BP1, pBRCA1, pChk1) proteins indicates greater DNA breakage and damage [27]. Salinomycin increases DNA strand breaks and induces phosphorylation of the DNA damage-related protein, H2AX [28]. "
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    ABSTRACT: A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5-5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.
    Full-text · Article · Jun 2013 · PLoS ONE
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