Oncogene (2007) 26, 7414–7422 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE DNA damage response mediators MDC1 and 53BP1: constitutive activation and aberrant loss in breast and lung cancer, but not in testicular germ cell tumours

J Bartkova1,8, Z Horˇ ejs˘ ı´ 1,2,8, M Sehested3, JM Nesland4, E Rajpert-De Meyts5, NE Skakkebæk5, M Stucki6, S Jackson7, J Lukas1 and J Bartek1,2

1Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Copenhagen, Denmark; 2Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 3Department of Pathology, Copenhagen University Hospital, Copenhagen, Denmark; 4Department of Pathology, The Norwegian Radium Hospital, University of Oslo, Oslo, Norway; 5Department of Growth and Reproduction, Copenhagen University Hospital, Copenhagen, Denmark; 6Institute of Veterinary Biochemistry and Molecular Biology, Universiy of Zu¨rich, Zu¨rich, Switzerland and 7The Gurdon Institute, Cambridge, UK

MDC1 and 53BP1 are critical components of the DNA Introduction damage response (DDR) machinery that protects genome integrity and guards against cancer, yet the tissue expression Maintenance of genomic integrity and protection patterns and involvement of these two DDR adaptors/ against harmful mutagenic effects of DNA damage rely mediators in human tumours remain largely unknown. Here on DNA damage response machinery, a complex we optimized immunohistochemical analyses of human network of signalling and effector pathways that 53BP1 and MDC1 proteins in situ and identified their coordinate cell cycle checkpoints with DNA repair and virtually ubiquitous expression, both in proliferating and cell death mechanisms (Shiloh, 2003; Kastan and quiescent, differentiated tissues. Focus formation by 53BP1 Bartek, 2004; Lukas et al., 2004b; Bartek and Lukas, and/or MDC1 in human spermatogenesis and subsets of 2007). In response to DNA lesions, cells activate the breast and lung carcinomas indicated physiological and apical signalling kinases ATM, ATRor DNA-PK ‘pathological’ activation of the DDR, respectively. Further- (Shiloh, 2003; Bakkenist and Kastan, 2004), which then more, aberrant reduction or lack of either protein in phosphorylate a plethora of substrates within the DNA significant proportions of carcinomas supported the candi- damage response (DDR) network, including the effector dacy of 53BP1 and MDC1 for tumour suppressors. kinases Chk1 and Chk2 (Bartek and Lukas, 2003) that Contrary to carcinomas, almost no activation or loss of become activated upon their phosphorylations by ATR MDC1 or 53BP1 were found among testicular germ-cell and ATM, respectively, and further amplify and spread tumours (TGCTs), a tumour type with unique biology and the alert signal to downstream effectors. An emerging exceptionally low incidence of p53 mutations. Such con- class of important regulators operating near the top of comitant presence (in carcinomas) or absence (in TGCTs) of the DDRcascade consists of the so-called checkpoint DDR activation and DDR aberrations supports the roles of mediators (also known as adaptors), a group of large MDC1 and 53BP1 within the ATM/ATR-regulated check- multidomain proteins currently including BRCA1, point network which, when activated, provides an early anti- 53BP1, MDC1/NFBD1 (MDC1), TopBP1 and claspin cancer barrier the pressure of which selects for DDR defects (Petrini and Stracker, 2003; Kastan and Bartek, 2004; such as p53 mutations or loss of 53BP1/MDC1 during Mochan et al., 2004; Bartek and Mailand, 2006; Stucki cancer progression. and Jackson, 2006). Whereas claspin and TopBP1 are Oncogene (2007) 26, 7414–7422; doi:10.1038/sj.onc.1210553; involved in ATR/Chk1-mediated signalling (Kumagai published online 4 June 2007 and Dunphy, 2000; Kumagai et al., 2006; Liu et al., 2006), the remaining mediator proteins contribute to Keywords: DNA damage response; breast cancer; lung efficient responses to DNA double strand breaks (DSBs) cancer; testicular cancer; MDC1 and 53BP1 defects; mediated by ATM (DiTullio et al., 2002; Goldberg tumour suppressors et al., 2003; Mochan et al., 2003; Stewart et al., 2003; Ward et al., 2003; Xu and Stern, 2003; Huyen et al., 2004; Lukas et al., 2004a, b; Bekker-Jensen et al., 2005; Stucki et al., 2005; Lou et al., 2006). Although the Correspondence: Professor J Bartek, Institute of Cancer Biology and precise role(s) and mechanisms of action of the mediator Centre for Genotoxic Stress Research, Danish Cancer Society, proteins remain to be elucidated, they appear to Strandboulevarden 49, Copenhagen DK-2100, Denmark. facilitate interactions between ATM/ATRand their E-mail: [email protected] 8These authors contributed equally to this work. substrates and contribute to spatiotemporal modulation Received 5 February 2007; revised 14 March 2007; accepted 26 April of chromatin around, processing of, and signalling from, 2007; published online 4 June 2007 the sites of DNA damage (Bartek et al., 2004; Kastan DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7415 and Bartek, 2004; Mochan et al., 2004; Stucki and normal tissues remain largely unknown. Our present Jackson, 2006). study was designed to help fill such major gaps in the A large body of experimental and clinical evidence current knowledge about MDC1 and 53BP1, two implicates defects of various components of the DDR important DDRcomponents that protect genomic machinery, including DNA damage sensors, signalling stability and whose malfunction may contribute to kinases and diverse effectors involved in DNA repair, tumourigenesis. cell cycle checkpoints, chromatin remodeling as well as cell death pathways, in tumourigenesis (Shiloh, 2003; Kastan and Bartek, 2004; Nevanlinna and Bartek, 2006; Bartek and Lukas, 2007). This notion is further Results supported by tumour-prone phenotypes of mouse To assess tissue expression patterns by immunohisto- models with deletions or inactivating mutations in DDR- chemistry on paraffin sections, we first sought to identify related genes (Kastan and Bartek, 2004). In human reliable antibodies for such application. Whereas a tumour pathogenesis, germ-line mutations of genes that mouse monoclonal antibody suitable for immunoper- encode components of the DDRnetwork, such as oxidase detection of 53BP1 in archival tissue specimens BRCA1, BRCA2, p53, Chk2 or ATM, predispose to is available and was previously used to analyse this various types of familial cancer, while somatic mutations protein in human lung and skin tissues (Gorgoulis et al., in one or more DDRgenes contribute to the multistep 2005), there was no appropriate reagent to examine development of the majority, if not all, sporadic tumours MDC1. To generate such antibody, rabbits and mice (Bartek and Lukas, 2003; Kastan and Bartek, 2004; were immunized with purified, recombinant polypep- Nevanlinna and Bartek, 2006). tides corresponding to distinct domains of human The intimate link between DDRand tumourigenesis MDC1 and the resulting polyclonal (rabbit) and has recently been further strengthened by discoveries of monoclonal (mouse) antibodies characterized in various constitutive activation of the DNA damage checkpoints assays. The mouse antibody DCS-380.1 raised against in early precursor lesions in several types of human the C-terminal portion of MDC1 was chosen for malignancies (Bartkova et al., 2005b; Gorgoulis et al., subsequent immunohistochemical analyses, based on 2005). Together with functional analyses of xenograft its ability to detect MDC1 by immunofluorescence and and cell culture models of oncogene activation, these immunoblotting (Supplementary Figure S1) and its results suggest that the DDRmachinery may serve as an applicability in immunoperoxidase staining of forma- inducible barrier against tumorigenic transformation lin-fixed, paraffin tissue sections (see below). Impor- and/or progression of human cancer. This concept also tantly, staining of cultured irradiated cells, but not implies that activated DNA damage checkpoints create mock-treated control cultures, with DCS-380.1 showed an environment in which inactivating mutations within the characteristic formation of radiation-induced foci the DDRnetwork are selected for in lesions that previously reported for MDC1 (Goldberg et al., 2003; eventually overcome the DDR-induced cell death or Stewart et al., 2003). Also, cells in which MDC1 was senescence pathways and progress towards malignancy depleted by siRNA-mediated knockdown lacked the (Bartkova et al., 2005b, 2006; Gorgoulis et al., 2005; Di immunostaining signal irrespective of fixation method Micco et al., 2006). used (Supplementary Figure S1A and data not shown), Given such fundamental involvement of the DDRin thus further supporting the specificity of this reagent for tumourigenesis, and the fact that major therapeutic immunostaining applications. modalities currently used to treat cancer, including To establish the physiological expression patterns of ionizing radiation and many chemotherapeutics, operate 53BP1 and MDC1 in vivo, we next performed an through causing DNA damage, it is imperative to immunohistochemical analysis of a wide spectrum of understand better the mechanisms and biological role(s) normal human tissues, using the established antibody to of the DDRmachinery. Here, we attempt to gain more 53BP1 (Schultz et al., 2000), two antibodies against insights into tissue biology and relation to human cancer MDC1 (Goldberg et al., 2003) and the Ki67 prolifera- of two DNA damage checkpoint mediators, MDC1 and tion marker. Both the 53BP1 and MDC1 proteins were 53BP1. Previous work has documented critical roles of expressed in all tissues examined, including various both MDC1 and 53BP1 in cellular responses to DSBs, epithelial, muscle, stromal and neuronal cell types and including their requirement for efficient detection and/or regardless of the proliferation and differentiation state. timely signalling of DNA lesions, and effects on multiple Some typical examples of the staining patterns for cell-cycle checkpoints. Preliminary evidence also indi- 53BP1 and MDC1 are shown in Figures 1 and 2, cates that the lack of 53BP1 in mice can predispose to respectively. Whereas 53BP1 was always nuclear tumours (Ward et al., 2005; Morales et al., 2006), and (Figure 1), including neurons (Figure 1g), MDC1 was that a subset of human melanomas and lung carcino- localized to nuclei in the majority of the tissues mas, the only cancer types examined so far, show defects (Figure 2), yet showed pan-cellular or even preferentially of 53BP1 at the protein level (Gorgoulis et al., 2005). On cytoplasmic localization in neuronal cells (Figure 2e). the other hand, there are as yet no reports of analyses of An exceptional granular (focal) nuclear staining MDC1 in either human or animal tumours. In addition, pattern for both 53BP1 and MDC1 was found in the tissue distribution and cell type-related expression scattered lymphoid cells in normal bone marrow and in patterns of either of these two checkpoint mediators in adult testis, particularly spermatocytes (Figures 3a–d).

Oncogene DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7416

Figure 2 Immunohistochemical detection of MDC1 in normal tissues. (a and b) Adjacent sections of human rectum stained with antibodies to MDC1 and Ki67, respectively; Sections of human thyroid (c), human kidney (d), human brain (e) and rat myocardium (f) stained for MDC1. Original magnification  40.

Figure 1 Immunoperoxidase detection of 53BP1 in normal human tissues. (a and b) Adjacent sections of colon stained with antibodies MDC1 (Figure 5) in a series of normal human breast to 53BP1 and Ki67, respectively; (c and d) adjacent sections of tissues (n ¼ 56), carcinoma in situ (n ¼ 55) and invasive proximal oesophagus stained for 53BP1 and Ki67, respectively. breast carcinomas (n ¼ 81). Both 53BP1 (Figure 4a) and Prostate (e), thyroid gland (f), brain (g) and skeletal muscle (h) MDC1 (Figure 5a) were expressed in nuclei of both stained for 53BP1. Original magnification  40. normal luminal and basal epithelial cells, as well as in stromal cells and there were no apparent foci formed by either protein. In contrast to almost homogeneous staining in all normal breast tissues and in the majority This contrasted with other tissues and cell types, which of the breast carcinomas, we found subsets of tumours showed homogeneous nuclear signal without apparent in which MDC1 (in 30% of cases) or 53BP1 (in 26%) foci. The focal pattern likely reflects the physiological were aberrantly reduced or lost, as compared to response to DNA double-strand breaks that occur surrounding normal cells (Figures 4e, f and 5c, d). In during meiotic recombination of spermatocytes and addition, among tumours with detectable staining, the immunoglobulin gene rearrangements in lymphocytes pattern was often focal when viewed under higher (Bassing and Alt, 2004; Bartkova et al., 2005a). To magnification, reminiscent of cells with activated DNA confirm that foci which form at the site of DSBs, for damage response due to presence of unrepaired DSBs example in response to ionizing radiation and which are (Figure 4d). commonly evaluated by immunofluorescence techniques, To compare the breast tumours with other types of are detectable also by means of enzyme-based immuno- cancer, we also examined panels of lung cancer and histochemistry, we examined several cultured human cell testicular germ cell tumours, using the same techniques types by immunoperoxidase staining. Examples shown in and antibodies and compared with corresponding Figures 3e–h demonstrate that, indeed, both the radia- normal tissues. The results obtained with invasive lung tion-induced foci and the constitutive foci reported cancer (n ¼ 55) were analogous to those seen in breast previously in diverse cancer cell lines (DiTullio et al., tumours, in that both 53BP1 (Figures 6a–c) and MDC1 2002) are detectable by the immunoperoxidase method (Figures 7a and b) proteins were detectable and formed that we employ here to analyse human tissues and a homogeneous, diffuse (non-focal) pattern in nuclei of tumour specimens. normal bronchial and alveolar epithelial cells in all With the suitable antibodies and the immunohisto- normal lung tissues examined (n ¼ 30). Furthermore, chemical approach validated, we next examined in more subsets of the lung carcinomas showed abnormally detail the staining patterns for 53BP1 (Figure 4) and reduced or lost staining for 53BP1 (in 24% of cases;

Oncogene DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7417

Figure 3 Immunohistochemical detection of 53BP1 and MDC1 in Figure 4 Immunoperoxidase detection of 53BP1 in normal human normal human testes and in cultured human cells. (a and b) Normal breast and breast carcinomas. (a) Normal mammary tissue with seminiferous tubules show positive nuclear staining for MDC1 in nuclear staining for 53BP1; the inset shows higher magnification of all stages of spermatogenesis; note the characteristic focal staining normal breast ductule, documenting a characteristic difuse (non- pattern and prominent staining of XY bodies in primary focal) staining pattern for 53BP1 in nuclei of luminal epithelial spermatocytes (b, higher magnification). (c and d) 53BP1 in nuclei cells, myoepithelial cells, as well as stromal cells. (b) Invasive ductal of male germ cells in normal seminiferous tubules; note the focal carcinoma with homogeneous nuclear staining for 53BP1. (c and d) pattern in primary spermatocytes (d, higher magnification). (e and Mucinous carcinoma of the breast with pronounced focal staining f) Normal human fibroblasts (BJ strain) without (e) and 1 h after pattern of 53BP1, shown at lower (c) and higher (d) magnification. irradiation (f; 10 Gy), stained for 53BP1; note the focal staining (e and f) Examples of aberrant gross reduction of 53BP1 staining in pattern in (f). (g) Focal staining for 53BP1 in non-irradiated human two ductal carcinomas. Note the selective loss of 53BP1 staining in breast cancer cell line MDA-MB-468. (h) Human osteosarcoma cell tumour cells, compared with preserved staining of stromal cells. line Saos2 also shows the ‘constitutive’ foci of 53BP1 without Original magnification  40 (a, b, c, e and f);  100 (inset in a exposure to radiation. Original magnification  40 (a and c);  100 and d). (b and d–h).

Figure 6h) and MDC1 (in 28%; Figure 7d), and similar to breast tumours, the staining pattern was sometimes focal, indicating constitutive activation of the DNA damage response (Figure 6f). Importantly, among the 12 tumours of the breast or lung that showed the most pronounced defect of either 53BP1 or MDC1, the aberrant lack of staining was mutually exclusive for these two DNA damage mediators in all but one case. In sharp contrast to either breast or lung tumours, we found no evidence of aberrant lack of either 53BP1 or MDC1 in any of the human testicular germ cell tumours (n ¼ 43), except for only moderate patchy reduction, especially for 53BP1, in three embryonal carcinomas. Also different from the breast and lung carcinomas was the apparent lack of any testicular tumours with focal staining patterns, except for rare foci among the intratubular malignant cells of the carcinoma in situ of Figure 5 Immunoperoxidase detection of MDC1 in normal the testis and a small subset of embryonal carcinomas human breast and breast carcinomas. (a and b) Almost homo- (see Figure 8 for examples). When compared among the geneous nuclear staining of MDC1 in normal mammary tissue (a) three types of human tumours examined in this study, and comedo carcinoma in situ (b). (c and d) Examples of aberrantly reduced staining for MDC1 in an invasive lobular (c) and ductal/ both carcinomas of the breast and lung showed subsets lobular carcinoma (d). Note the selective loss of MDC1 staining in of cases with aberrations of the 53BP1 and MDC1 tumour cells, compared with preserved staining of stromal fibro- proteins, while such defects were virtually absent in blasts and infiltrating lymphoid cells. Original magnification  40.

Oncogene DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7418

Figure 7 Immunoperoxidase detection of MDC1 in normal human lung and lung tumours. (a and b) Normal bronchus (a) and normal lung parenchyme (b) show nuclear staining for MDC1. (c) Squamous invasive carcinoma with homogeneous nuclear staining for MDC1. (d) Aberrantly reduced staining for MDC1 in tumour cells of a bronchioalveolar carcinoma. Original magnification  40.

Figure 6 Immunoperoxidase detection of 53BP1 in normal human lung and lung tumours. (a and b) Normal bronchus (a) and normal lung parenchyme (b) show nuclear staining for 53BP1. (c) Higher magnification of normal bronchus, documenting a characteristic difuse (non-focal) staining pattern for 53BP1. (d) Squamous invasive carcinoma of the lung with near homogeneous (non-focal) staining pattern of 53BP1. (e and f) Small cell lung carcinoma shown at lower (e) and higher (f) magnification, documenting a pronounced focal staining pattern of 53BP1. (g) Foci of 53BP1 in nuclei of a squamous invasive carcinoma of the lung. (h) Example of aberrant lack of 53BP1 staining in another case of squamous carcinoma. Original magnification  40 (a, b, d, e and h);  100 (c, f and g). Figure 8 Immunoperoxidase staining for 53BP1 and MDC1 in human testicular germ-cell tumours. (a and b) Preinvasive stage of carcinoma in situ of the testis shows positivity for both 53BP1 (a) and MDC1 (b) in the nuclei of intratubular malignant cells, with rare MDC1 foci. (c) Embryonal carcinoma with homogeneous, testicular tumours (Figures 9a and b). The potential non-focal pattern of 53BP1 protein expression. (d) Seminoma with reasons for such pronounced differences, their relation- moderately variable expression of MDC1. Original magnification ship with the cells of origin and the role of DNA  40 (a, b and d);  100 (c). damage response in the pathogenesis of these types of malignancies are discussed below.

insights into these aspects of 53BP1 and MDC1 biology and pathology. First, our data show that both proteins Discussion are coexpressed in virtually all normal human tissues, and in both proliferating and terminally differen- Despite the fact that 53BP1 and MDC1 are emerging as tiated, quiescent cells. This global tissue expression important spatiotemporal regulators of the genome pattern is reminiscent of the ATM kinase (Bartkova maintenance machinery close to the top of the DNA et al., 2005a), and similar to, but marginally broader damage signalling and lesion processing cascades (Bartek than, the expression pattern of the Chk2 kinase (Lukas et al., 2004; Stucki and Jackson, 2006), very little is et al., 2001; Latella et al., 2004). Such similarities are known about their tissue and cell-type expression consistent with the shared roles of ATM, 53BP1, MDC1 patterns, and their potential involvement in tumouri- and Chk2 in response to DSBs, and with the function of genesis. We believe our present study provides significant this pathway also in G1 and even G0 phases of the cell

Oncogene DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7419

Figure 9 Summary of 53BP1 and MDC1 immunohistochemical data from three types of human tumours: carcinomas of the breast and lung, and testicular germ-cell tumours. (a and b) Graphical summaries of the frequencies of reduced/lacking MDC1/53BP1 proteins and activation (foci formation), respectively. The 90 normal tissues include 56 breast, 30 lung tissues and four normal testes samples (the testicular germ-cell tumours (TGCTs) originate from early gonocytes, while in adult testes, the DDRmachinery is activated during meiotic recombination, as shown in Figure 4). Among TGCTs, there was a moderate patchy reduction of MDC1/ 53BP1 staining and focus formation in three of the embryonal carcinomas tested, but of lower degree than the aberrations scored in lung or breast tumours). (c) Schematic illustration of the major differences between breast and lung cancer on one hand, and TGCTs on the other, in terms of the DDRactivation and 53BP1/MDC1 loss, considered within the framework of the DDRmachinery as an anti-cancer barrier and selective pressure for DDRdefects that facilitate tumour progression.

cycle (Bartek et al., 2004; Latella et al., 2004). These Second, we provide evidence for aberrant reduction or features contrast with those of numerous other DDR loss of 53BP1 and MDC1 in subsets of human proteins whose function is restricted to proliferating carcinomas of the breast and lung, thereby supporting cells, and often limited to the S and G2 phases, such as the notion of 53BP1 and MDC1 as emerging candidate the ATR-Chk1 signalling module or the homologous tumour suppressors. For 53BP1, such a notion is so far recombination machinery, for example (Lukas et al., supported by the tumour-prone phenotype of 53BP1- 2001; Jazayeri et al., 2006). Since no cell-cycle check- deficient mice (Ward et al., 2005; Morales et al., 2006), point role is needed in response to DNA damage in non- as well as aberrant absence of the protein in subsets of proliferating cells, the ATM/MDC1/53BP1 pathway human lung tumours and melanomas (Gorgoulis et al., operating in quiescent tissues probably promotes repair 2005). On the other hand, the recently reported MDC1 of DSBs to maintain the transcribed genome functional, gene knockout mice have not been analysed for tumour and/or eliminates cells with irreparable damage through incidence so far (Lou et al., 2006) and, owing to the lack apoptosis. of suitable reagents, there have been no reports on Apart from response to external genotoxic insults, this MDC1 protein expression in tissues or tumours. Clearly, pathway also contributes to processing of the DSBs that additional work is required to examine the candidacy of occur as parts of the physiological programmes of 53BP1 and MDC1 as tumour suppressors. On the other meiotic recombination in spermatogenesis and immu- hand, such a possibility is also consistent with the fact noglobulin class switch or antigen receptor gene that aberrations in other components of the DSB recombination during lymphocyte differentiation (Bas- response pathway, including ATM, Chk2, H2AX, the sing and Alt, 2004). It is reassuring that activation Mre11/Rad50/Nbs1 complex, BRCA1 or p53 are all events of the DSB response machinery, such as known to predispose to, or contribute to progression of, autophosphorylation of ATM and phosphorylation of various forms of cancer (Kastan and Bartek, 2004; histone H2AX (Bartkova et al., 2005a, b), as well as foci Nevanlinna and Bartek, 2006). formation of 53BP1 and MDC1 (this study and Third, our present results further strengthen and Gorgoulis et al., 2005) can be conveniently monitored extend the recently proposed concept of DDRactivation in vivo by immunohistochemistry. From this point of as an inducible biological barrier against progression of view, antibodies specific for such activatory phospho- human tumours beyond their early, preinvasive stages epitopes and/or those detecting the foci formed by (Bartkova et al., 2005b; Gorgoulis et al., 2005; Bartek the DDRproteins at the sites of DNA lesions, might and Lukas, 2007). This concept reflects the documented be regarded as a kind of ‘bio-dosimeter’ applicable for frequent constitutive activation of the DDRnetwork in in situ analyses of biopsy specimens. various types of major human tumours, and the ability

Oncogene DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7420 of an ever increasing spectrum of known oncogenes and Looijenga, 2005; Rajpert-De Meyts, 2006). Impor- (Bartkova et al., 2005b, 2006; Gorgoulis et al., 2005; Di tantly, whatever the reasons for the low incidence of Micco et al., 2006; Pusapati et al., 2006; Tort et al., spontaneous DDRactivation in TGCTs may be, this 2006), as well as telomere attrition (d’Adda di Fagagna correlates well with the low frequency of defects in DDR et al., 2003; Takai et al., 2003), to elicit the DDR components such as p53 (Lutzker and Levine, 1996; activation in both cell culture and mouse tumour Oosterhuis and Looijenga, 2005) or the loss of 53BP1/ models. The concept also postulates that the cell death MDC1 (this study). The difference in terms of consti- and cell cycle arrest/senescence that impose the barrier tutive DDRactivation and its impact in TGCTs, as against tumour progression as a result of the activated opposed to other solid human tumours (such as DNA damage checkpoints represent a major source of carcinomas of the breast or lung), is schematically selection pressure for tumour-promoting mutations such depicted in Figure 9c. The overall correlation between as those in p53 and other DDRcomponents, that allow DDRactivation and defects in DDRcomponents such rescue of tumour cell proliferation at the expense of as p53, as seen in carcinomas, or the concomitant lack of increasing genetic instability and cancer progression DDRactivation and defects (as seen in TGCTs) further (Bartkova et al., 2005b, 2006; Gorgoulis et al., 2005; Di supports the concept of the DDRbarrier as a selection Micco et al., 2006). What is also common to human pressure to favour DDRdefects such as p53 mutations tumours is that the extent of the constitutive DDR during tumour progression. activation and signalling is greater in the preinvasive Last but not least, the ability to monitor the status of lesions compared with the overtly malignant tumours. activation and defects within the DDRmachinery may This decreased DDRsignalling in later stages of tumour help optimize therapeutic decisions in the clinic. As the progression may be caused by several factors, such as major non-surgical treatment modalities used in con- the tumour-acquired defects within the upstream DDR temporary oncology, ionizing radiation and chemother- sensing or signalling machinery, aberrantly enhanced apy mostly rely on causing DNA damage, individual DNA repair and/or activation of telomerase. The assessment of the cellular machinery that primarily advanced malignancies with such persistent DDR responds to such insults, both in the patient and the signalling had likely suffered inactivating mutations tumour, may prove beneficial for personalized medicine downstream within the DDRmachinery, such as in p53 approaches in the future. or other apoptosis or cell-cycle arrest effectors, which allow survival and tumour growth despite the ongoing futile checkpoint signalling. Materials and methods So how do the present data on 53BP1 and MDC1 fit the above concept? The scenario observed in breast Antibodies and lung tumours fits very well, as we observed Mouse monoclonal antibodies against human MDC1 were activation (foci formation) in subsets of cases, and also generated by immunizing Balb/c mice with the recombinant, aberrant reduction or lack of these DDRcomponents, bacterially expressed and gel-purified C-terminal fragment of consistent with the idea of selection for such defects to human MDC1/NFBD1 protein. Fusion with NS2 mouse escape the imposed barrier. In this context, it is also myeloma cells yielded a number of hybridomas from which significant that among the 12 cases with the most the clone DCS380.1 was selected for further studies (see pronounced defects of either MDC1 or 53PB1, such Results). The additional immunoreagents used in this study defects (except in one case) were always mutually included rabbit (#889 and #1987) and sheep (#3835) poly- clonal antibodies against distinct domains of human MDC1 exclusive, suggesting that one defect in the common (Goldberg et al., 2003; and unpublished data), mouse pathway of MDC1-53BP1 (Bekker-Jensen et al., 2005; monoclonal antibody to 53BP1 (Schultz et al., 2000; a gift Stucki and Jackson, 2006) was sufficient to deregulate from TD Halazonetis), and rabbit polyclonal antibody to the this mechanism. Ki67 proliferation marker (Dako, Glostrup, Denmark). The testicular germ cell tumours are exceptional among the types of human malignancies tested for Cell culture, RNA interference, irradiation and constitutive DDRactivation so far, in that this immunofluorescence phenomenon occurs only rarely. This conclusion is The human U-2-OS sarcoma cell line and WI38 primary supported by the apparent lack of 53BP1/MDC1 foci in fibroblast cell strain were cultured in Dulbecco’s modified these tumours seen in the present study, as well as by the Eagle’s medium (Biowhittaker, Copenhagen, Denmark) paucity of ATM activation and gH2AX in both the supplemented with 10% fetal bovine serum (HyClone, Logan, human testicular carcinoma in situ precursor lesions as UT, USA) and antibiotics. Depletion of MDC1 in live well as among the invasive testicular germ-cell tumours cells was achieved by siRNA mediated knockdown approach, (TGCTs) (Bartkova et al., 2005a). This difference using previously published siRNA duplexes and protocols between TGCTs and the other major solid cancers (Goldberg et al., 2003; Lukas et al., 2004a). In some may reflect several factors, including the unique origin experiments, cells were exposed to ionizing radiation (IR, 15Gy). IRwas delivered by an X-ray generator (Pantak from early germ cells (as opposed to somatic cells), HF160, 150 kV; 15 mA; dose rate 2.18 Gy/min). One hour after distinct spectra of oncogenes involved and longer irradiation, mock-treated and irradiated cells grown on glass telomeres, all specific features of the former tumours coverslips were fixed in 4% buffered formaldehyde and (Albanell et al., 1999; Nowak et al., 2000; Bartkova examined by immunofluorescence as described (Bekker-Jensen et al., 2003; Rajpert-De Meyts et al., 2003; Oosterhuis et al., 2005).

Oncogene DNA damage response mediators MDC1 and 53BP1 J Bartkova et al 7421 Immunoblotting slides. For antigen unmasking, the deparaffinized sections were The cell lysates were separated on sodium dodecyl sulphate– boiled in a microwave oven for 10–15 min in citrate buffer polyacrylamide gel electrophoresis and electroblotted onto (0.01 M citric acid monohydrate, pH 6.0) before initiating the nitrocellulose membrane. Membranes were incubated with staining procedure. The sensitive immunoperoxidase staining anti-ATM 1981S-P or anti-Flag M5 (Sigma, Vallensbaek, method was performed as described (Lukas et al., 2001), using Denmark). Immunoblots of soluble cell extracts were prepared the Vectastain Elite kit (Vector Laboratories, Burlingame, CA, and probed with antibodies against MDC1 as reported earlier USA) to detect the primary antibodies, and a nickel sulphate- (Horejsi et al., 2004). enhancement step without nuclear counterstaining to visualize the chromogenic (diaminobenzidine) reaction and detect even Normal tissues and tumours moderate nuclear signals. The immunostaining patterns were Tumors and normal tissues were provided by the tissue banks evaluated by an experienced pathologist. Since at least 90 and of the participating Institutes in Denmark and Norway, with a 80–85% of cells in normal tissues were positive for 53BP1 and consent from the local ethical committees. Multiple biopsy MDC1, respectively, we considered the results in cancer specimens of the majority of normal tissue types were included specimens as still normal when more than 70% (for 53BP1) in the study, resected either for cosmetic reasons or benign or over 65% (for MDC1) cancer cells were stained. The staining conditions, or taken from a margin of the specimen as far from patterns of tumours were regarded as aberrantly reduced the tumour as possible when the biopsy was taken for a when only 40–70% of cancer cells were positive for 53BP1 tumour. To ensure optimal preservation of the analysed and 35–65% for MDC1, while we considered as aberrant loss protein epitopes, no necropsy specimens were included in our of 53BP1 and MDC1 when 0–39 and 0–34% of cancer cells study. The testicular tumour specimens were from routine were stained, respectively, in contrast to positive staining of the diagnostic or therapeutic surgery, while the fetal testes samples surrounding normal or stromal cells on the same section. were from approved abortions and the normal adult testes were from a preserved tissue margin removed during Acknowledgements orchidectomy due to a non-germinal cell testicular tumour or for prostate cancer. This work was supported by grants from the Danish Cancer Society, the Danish National Research Fund, the Danish Immunohistochemistry on paraffin sections Centre for Translational Breast Cancer Research, the Eur- Indirect immunoperoxidase staining was carried out using opean Commission (integrated projects ‘Active p53’, ‘DNA formaldehyde-fixed, paraffin embedded tissue sections repair’ and ‘Mutant p53’), MSM (grant no. 6198959216) and mounted on poly-L-lysine-coated or electrostatically treated Cancer Research UK.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene