Histone H2AX phosphorylation on a serine four residues from the carboxyl terminus (producing γH2AX) is a sensitive marker for DNA double-strand breaks (DSBs). DSBs may lead to cancer but, paradoxically, are also used to kill cancer cells. Using γH2AX detection to determine the extent of DSB induction may help to detect precancerous cells, to stage cancers, to monitor the effectiveness of cancer therapies and to develop novel anticancer drugs.
Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, c-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and c-H2AX signals were reduced by RNase H1 transfection, which removes transcriptionmediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.
The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.
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.
Apoptosis is a fundamental process for metazoan development. It is also relevant to the pathophysiology of immune diseases and cancers, and the outcome of cancer chemotherapies as well as being a target for cancer therapies. Apoptosis involves intrinsic pathways typically initiated by DNA damaging agents and engaging mitochondria, and extrinsic pathways typically initiated by “death receptors” and their ligands TRAIL and TNF at the cell surface. Recently, we discovered the apoptotic ring, which microscopically looks like a nuclear annular staining early in apoptosis. This ring is in three-dimensional space a thick intranuclear shell consisting of epigenetic modifications including histone H2AX and DNA damage response (DDR) proteins. It excludes the DNA repair factors usually associated with γ-H2AX in the DDR nuclear foci. Here, we summarize our knowledge of the apoptotic ring, and discuss its biological and pathophysiological relevance, as well as its value as potential pharmacodynamic biomarker for anticancer therapies.
The “apoptotic ring” is characterized by the phosphorylation of histone H2AX at serine 139 (γ-H2AX) by DNA-dependent protein kinase (DNA-PK). The γ-H2AX apoptotic ring differs from the nuclear foci patterns observed in response to DNA-damaging agents. It contains phosphorylated DNA damage response proteins including activated Chk2, activated ATM, and activated DNA-PK itself but lacks MDC1 and 53BP1, which are required to initiate DNA repair. Because DNA-PK can phosphorylate heat shock protein 90α (HSP90α) in biochemical assays, we investigated whether HSP90α is involved in the apoptotic ring. Here we show that HSP90α is phosphorylated by DNA-PK on threonines 5 and 7 early during apoptosis and that both phosphorylated HSP90α and DNA-PK colocalize in the apoptotic ring. We also show that DNA-PK is a client of HSP90α and that HSP90α is required for full DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. In contrast, HSP90 inhibition by geldanamycin markedly enhances TRAIL-induced DNA-PK and H2AX activation. Together, our results reveal that HSP90α is a substrate and chaperone of DNA-PK in the apoptotic response. The response of phosphorylated HSP90α to TRAIL and its localization to the γ-H2AX ring represent epigenetic features of apoptosis that offer insights for studying and monitoring nuclear apoptosis.
RNA splicing is required to remove introns from pre-mRNA, and alternative splicing generates protein diversity. Topoisomerase I (Top1) has been shown to be coupled with splicing by regulating serine/ arginine-rich splicing proteins. Prior studies on isolated genes also showed that Top1 poisoning by camptothecin (CPT), which traps Top1 cleavage complexes (Top1cc), can alter RNA splicing. Here, we tested the effect of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. The RNA of HCT116 cells treated with CPT for various times was analyzed with ExonHit Human Splice Array. Unlike other exon array platforms, the ExonHit arrays include junction probes that allow the detection of splice variants with high sensitivity and specificity. We report that CPT treatment preferentially affects the splicing of splicing-related factors, such as RBM8A, and generates transcripts coding for inactive proteins lacking key functional domains. The splicing alterations induced by CPT are not observed with cisplatin or vinblastine and are not simply due to reduced Top1 activity, as Top1 downregulation by short interfering RNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) blocked the splicing alteration induced by CPT, which suggests that the rapid Pol II hyperphosphorylation induced by CPT interferes with normal splicing. The preferential effect of CPT on genes encoding splicing factors may explain the abnormal splicing of a large number of genes in response to Top1cc. Cancer Res; 70(20); 8055-65. ©2010 AACR.
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