Oncogene (2009) 28, 3880–3891 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Anticancer DNA intercalators cause p53-dependent mitochondrial DNA nucleoid re-modelling N Ashley and J Poulton Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, UK Many anticancer drugs, such as doxorubicin (DXR), altering its molecular topology. Consequently, DNA intercalate into nuclear DNA of cancer cells, thereby polymerases and other DNA-related proteins are inhib- inhibiting their growth. However, it is not well understood ited and DNA replication is reduced, causing the death of how such drugs interact with mitochondrial DNA rapidly dividing cancer cells. Unfortunately, anthracy- (mtDNA). Using cell and molecular studies of cultured clines can cause severe cardiotoxicity (Singal and Iliskovic, cells, we show that DXR and other DNA intercalators, 1998), often manifesting months or even years after such as ethidium bromide, can rapidly intercalate into treatment (Steinherz and Steinherz, 1991). The reason for mtDNA within living cells, causing aggregation of this delayed toxicity remains unknown, but one possibility mtDNA nucleoids and altering the distribution of nucleoid is that anthracyclines induce a lesion capable of damaging proteins. Remodelled nucleoids excluded DXR and the cell over a long period. A potential candidate for such maintained mtDNA synthesis, whereas non-remodelled a lesion is the mitochondrion, as cardiac-specific mito- nucleoids became heavily intercalated with DXR, which chondrial dysfunction is an early and specific feature of inhibited their replication, thus leading to mtDNA anthracycline cardiotoxicity (Jung and Reszka, 2001; depletion. Remodelling was accompanied by extensive Wallace, 2003; Conklin, 2005; Tokarska-Schlattner mitochondrial elongation or interconnection, and was et al., 2006), which can persist even after drug cessation suppressed in cells lacking mitofusin 1 and optic atrophy (Zhou et al., 2001). The mitochondria contain a 16.5-kb 1 (OPA1), the key proteins for mitochondrial fusion. In multicopy genome known as mitochondrial DNA contrast, remodelling was significantly increased by p53 or (mtDNA), and extensive mtDNA damage in the form ataxia telangiectasia mutated inhibition (ATM), indicat- of depletion and deletions has been found in the cardiac ing a link between nucleoid dynamics and the genomic tissue of DXR-treated patients (Lebrecht et al., 2005), as DNA damage response. Collectively, our results show that well as in rodent and cell culture models (Cullinane et al., DNA intercalators can trigger a common mitochondrial 2000; Lebrecht et al., 2004; Suliman et al., 2007). Using response, which likely contributes to the marked clinical the fluorescent DNA dye PicoGreen, which labels toxicity associated with these drugs. specifically nuclear and mtDNA within living cells, as Oncogene (2009) 28, 3880–3891; doi:10.1038/onc.2009.242; we have shown earlier (Ashley et al., 2005, 2008), we published online 17 August 2009 recently showed that anthracyclines are capable of rapidly penetrating into the mitochondria to directly intercalate Keywords: anthracyclines; mtDNA; mitochondria; into mtDNA (Ashley and Poulton, 2009). nucleoids; doxorubicin; mitofusin In vivo, mtDNA is arranged in punctate nucleoid structures (Iborra et al., 2004; Garrido et al.,2003;Legros et al., 2004), which consist of several copies of mtDNA bound to ‘nucleoid’ proteins, such as mitochondrial Introduction single-stranded DNA-binding protein (mtSSB), mito- chondrial transcription factor A (TFAM) and polymerase DNA-intercalating drugs form the cornerstone of many gamma (POLG). Although the functional significance of anticancer regimes. Prominent among them are the nucleoids remains obscure, yeast nucleoids can undergo anthracyclines, a group of compounds including doxo- structural remodelling in response to metabolic stress to rubicin (DXR) and daunorubicin (Hande, 1998). The protect mtDNA from damage (Chen et al., 2005b; Kucej DNA intercalators function by inserting themselves et al., 2008). The loss of the TFAM homologue and between the base pairs of the DNA double helix, thereby nucleoid protein Abf2p sensitizes yeast mtDNA to damage by the DNA intercalator ethidium bromide Correspondence: Dr N Ashley or J Poulton, Nuffield Department of (Chen et al., 2005b). Within human cells, the tumour Obstetrics and Gynaecology, University of Oxford, Level 3, Women’s suppressor p53 can translocate to the mitochondria and Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. interact with POLG to protect mtDNA from damage by E-mails: [email protected] or ethidium bromide (Achanta et al., 2005). [email protected] Received 20 January 2009; revised 7 June 2009; accepted 8 July 2009; The mitochondria are dynamic organelles that can published online 17 August 2009 undergo rapid transient fission and fusion, mediated by Anthracyclines and mitochondrial DNA N Ashley and J Poulton 3881 pro-fusion proteins such as optic atrophy 1 (OPA1) methyl ester (TMRM) revealed that the mitochondria and mitofusins 1/2 (MFN1 and MFN2), and pro-fission within DXR-treated cells had undergone marked inter- proteins such as dynamin-related protein 1 (DRP1) linking, with highly interconnected mitochondria (Chen et al., 2005a). Recently, the anticancer DNA (Figure 1aiii and Supplementary Figure 1b). The intercalator actinomycin D has been shown to trigger remodelled nucleoids frequently colocalized with small OPA1 and MFN1-mediated stress-induced mitochon- mitochondrial swellings that were not commonly drial hyperfusion, which has a protective antiapoptotic observed in controls. Similar results were obtained for effect within cells (Tondera et al., 2009). Collectively, DXR-treated A549 cancer cells and H9C2 cardio- this evidence suggests that mitochondria and mtDNA myocytes (Supplementary Figures 1a and c), although may undergo a coordinated stress response in the H9C2s did not show mitochondrial interlinking (Sup- presence of DNA-intercalating drugs. plementary Figure 1d). In this study, we sought to analyse the effects of Anti-DNA and Mitotracker Red labelling (which anticancer DNA-intercalating drugs on mtDNA and detects mitochondria in fixed cells) showed that mitochondria. We show that anthracyclines can directly ethidium bromide also caused marked nucleoid remo- interact with mtDNA at clinically relevant concentra- delling accompanied by mitochondrial interlinking tions, producing major alterations of nucleoids and (Figure 1aiv). mitochondrial morphology and ultimately resulting in Quantification of an experiment similar to that shown the depletion of mtDNA. in Figure 1a showed that DXR could induce mitochon- drial interlinking and remodelling at doses below 1 mM. H9C2 rat cardiomyocytes were more sensitive at low Results concentrations of DXR than human fibroblasts, but the level of remodelling was unaffected by incubating cells Doxorubicin and ethidium bromide alter nucleoid and with the well-known reactive oxygen species scavenger mitochondrial morphology N-acetylcysteine (Figure 1b). DXR significantly reduced To determine the long-term effect of DXR on mtDNA, nucleoid numbers within individual fibroblasts we used PicoGreen fluorescence quenching to monitor (Figure 1c) and significantly increased the average DXR–DNA interactions within normal primary human nucleoid fluorescence (Figure 1d). fibroblasts, as described earlier (Ashley and Poulton, 2009). In untreated cells, nuclear and mtDNA were brightly labelled by PicoGreen (Figure 1ai), and Remodelled nucleoids are enriched with TFAM, mtSSB both signals were substantially quenched by addition and POLG of DXR, indicating the intercalation of DXR into Within nucleoids, mtDNA is complexed with ‘core’ nuclear and mtDNA. After 24 h of DXR exposure, nucleoid proteins, including mtSSB, POLG and PicoGreen fluorescence partially recovered in both the TFAM, and associated, perhaps indirectly, with tumer- nucleus and mitochondria, the latter containing a small ous imaginal disc 1 (Tid1) and ATAD3 (He et al., 2007; number of brightly labelled and grossly enlarged Bogenhagen et al., 2008). To determine whether DXR nucleoids. The average nucleoid diameter of these affected these proteins, we immunolabelled fibroblasts enlarged nucleoids was 1–3 mm, compared with treated with DXR or vehicle. In untreated cells, TFAM B0.8 mm of normal-sized nucleoids. Although normal- was concentrated into small dots (nucleoids) distributed sized nucleoids remained within the DXR-treated cells, evenly along mitochondria, but was heavily concen- they were often heavily quenched by DXR (t ¼ 24 h trated into large mitochondrial blobs after DXR shown by the arrow in the inset). Hereafter, we term exposure (Figure 2a). Large swathes of the mitochon- these DXR-induced nucleoid alterations as ‘nucleoid dria thus became depleted of detectable TFAM. The remodelling’. After DXR exposure, the surviving focal accumulations of TFAM colocalized with mito- cells, which were allowed to recover in drug-free chondrial swellings and with remodelled nucleoids medium for 72 h, showed a marked reduction in (detected using anti-DNA IgM1—not shown). Similar nucleoid PicoGreen labelling, despite recovery of the results were obtained using ethidium (not shown). Note nuclear signal, suggestive of mtDNA depletion, which that the strong red nuclear signal in DXR-treated was confirmed using quantitative