SV40 Large T-Antigen Disturbs the Formation of Nuclear DNA-Repair Foci Containing MRE11

Oncogene (2002) 21, 4873 – 4878 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc REVIEW SV40 large T-antigen disturbs the formation of nuclear DNA-repair foci containing MRE11

Martin Digweed*,1, Ilja Demuth1, Susanne Rothe1, Regina Scholz2, Andreas Jordan2, Carsten Gro¨ tzinger3, Detlev Schindler4, Markus Grompe5 and Karl Sperling1

1Institut fu¨r Humangenetik, Charite´ – Campus Virchow-Klinikum, Humboldt Universita¨t zu Berlin, Germany; 2Klinik fu¨r Strahlenheilkunde, Charite´ – Campus Virchow-Klinikum, Humboldt Universita¨t zu Berlin, Germany; 3Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charite´ – Campus Virchow-Klinikum, Humboldt Universita¨t zu Berlin, Germany; 4Institut fu¨r Humangenetik, Theodor-Boveri-Institut fu¨r Biowissenschaften (Biozentrum), Bayerische Julius-Maximilians- Universita¨tWu¨rzburg, Germany; 5Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon, USA

The accumulation of DNA repair proteins at the sites of Keywords: ionizing irradiation; Fanconi anaemia; im- DNA damage can be visualized in mutagenized cells at mortalization the single cell level as discrete nuclear foci by immunofluorescent staining. Formation of nuclear foci in irradiated human fibroblasts, as detected by antibodies directed against the DNA repair protein MRE11, is Introduction significantly disturbed by the presence of the viral oncogene, SV40 large T-antigen. The attenuation of foci The two major mechanisms for DNA double strand formation was found in both T-antigen immortalized break (DSB) repair in mammalian cells are nonhomo- cells and in cells transiently expressing T-antigen, logous end joining (NHEJ) and homologous indicating that it is not attributable to secondary recombination (HR). Many genes involved in these mutations but to T-antigen expression itself. ATM- two processes have been identified: DNA-PKCS, KU70, mediated nibrin phosphorylation was not altered, thus the KU80, RAD50, MRE11, NBS1, DNA-Ligase IV and disturbance of MRE11 foci formation by T-antigen is XRCC4 in NHEJ; RAD51, RAD52, RAD54, XRCC2 independent of this event. The decrease in MRE11 foci and XRCC3 in HR (reviewed in Kanaar et al., 1998). was particularly pronounced in T-antigen immortalized Some of these proteins, such as the trimeric MRE11/ cells from the Fanconi anaemia complementation group RAD50/nibrin complex (involved in both HR and FA-D2. FA-D2 cells produce essentially no MRE11 NHEJ) and RAD51 (specific for HR), accumulate at DNA repair foci after ionizing irradiation and have a the sites of DSBs and become visible as discrete nuclear significantly increased cellular radiosensitivity at low foci by immunofluorescence staining after ionizing radiation doses. The gene mutated in FA-D2 cells, irradiation (Maser et al., 1997; Nelms et al., 1998; FANCD2, codes for a protein which also locates to Tashiro et al., 2000). Nibrin is the protein mutated in nuclear foci and may, therefore, be involved in MRE11 cells from patients with the DNA-repair disorder, foci formation, at least in T-antigen immortalized cells. Nijmegen Breakage Syndrome (NBS; Varon et al., This finding possibly links Fanconi anaemia proteins to 1998) characterized at the cellular level by chromoso- the frequently reported increased sensitivity of Fanconi mal instability and radiosensitivity (reviewed in anaemia cells to transformation by SV40. From a Digweed et al., 1999). FANCD2, the protein mutated practical stand point these findings are particularly in patients of Fanconi anaemia complementation group relevant to the many studies on DNA repair which FA-D2, also forms nuclear foci in response to ionizing exploit the advantages of SV40 immortalized cell lines. irradiation (IR) but only after modification to the The interference of T-antigen with DNA repair monoubiquitinated FANCD2-L isoform by a complex processes, as demonstrated here, should be borne in of several other proteins which themselves are mutated mind when interpreting such studies. in the other Fanconi anaemia complementation groups Oncogene (2002) 21, 4873 – 4878. doi:10.1038/sj.onc. (Garcia-Higuera et al., 2001). At the cellular level, 1205616 Fanconi anaemia (FA) is characterized by chromoso- mal instability and extreme sensitivity to bifunctional alkylating agents which can form DNA interstrand crosslinks (reviewed in Joenje and Patel, 2001). Interestingly, HR, NHEJ and the FANCD2 path- *Correspondence: M Digweed, Institut fu¨ r Humangenetik, Charite´ – ways are linked to each other by BRCA1 which binds Campus Virchow, Augustenburger Platz 1, 13353 Berlin, Germany; E-mail: [email protected] directly to DNA (Paull et al., 2001) and is part of a Received 21 February 2002; revised 17 April 2002; accepted 26 supercomplex, BASC, implicated in genome surveil- April 2002 lance (Wang et al., 2000). BRCA1 can modulate the SV40 large T-antigen and DNA-repair foci M Digweed et al 4874 nuclease activity of MRE11/RAD50/nibrin (Paull et gene which codes for nibrin (Varon et al., 1998). al., 2001), interacts with RAD51 in common nuclear Primary fibroblasts from FA patients show apparently foci after IR or crosslinking treatments (Bhattacharyya normal production of MRE11 foci after ionizing et al., 2000; Scully et al., 1997) and associates with irradiation. FANCD2-L in response to DNA damage (Garcia- In order to be able to quantify the formation of Higuera et al., 2001). MRE11 foci after ionizing irradiation we examined the dose response of foci formation. Primary fibroblasts growing asynchronously were irradiated with increas- MRE11 foci are formed in fibroblasts as a response to ing doses and examined for MRE11 foci by counting ionizing irradiation the number of foci per nucleus. Figure 2a shows the distribution of foci/nucleus measured in this experi- Figure 1 shows examples of the nuclear foci which are ment. The majority of unirradiated cells show no foci, found in fibroblasts after ionizing irradiation and only 5% of the cells show one or two foci presumably immunofluorescence staining for MRE11. The brightly reflecting the repair of endogenous DNA damage. fluorescing 0.5 – 1.5 mm foci are clearly distinguishable With increasing irradiation, the foci-negative fraction from the diffuse speckled staining of the remaining reduces and the distribution of foci per nucleus in the nucleus. This is particularly clear if the irradiated and foci-positive fraction shifts to the right. After 12 Gy unirradiated control cells are compared or the irradiation, essentially all cells have moved from the irradiated control cells with irradiated cells from a foci-negative to the foci-positive fraction, those cells Nijmegen Breakage Syndrome (NBS) patient. NBS not yet showing foci probably move into the foci- cells lack not only foci but even the correct nuclear positive fraction at a slightly later timepoint since localization of MRE11 due to mutation of the NBS1 MRE11 foci are observed for up to 24 h after

Figure 1 MRE11 nuclear foci observed in irradiated cells. Primary fibroblasts and SV40 T-Ag immortalized fibroblasts were irradiated at 378C on a thermostated water bed with 12 Gy using the X-ray apparatus Muller MG 150 (UA=100 kV, I=10 mA, filter 0.3 mm Ni, dose rate: 2.1 Gy/min) and 8 h later were fixed for 10 min in 4% paraformaldehyde, permeabilized for 5 min at 48Cin 0.5% Triton X-100 and blocked by incubation overnight at 48C in 2% BSA in phosphate-buffered saline. Slides were then incubated with a primary rabbit antibody directed towards MRE11 (Novus Biologicals) followed by detection with a secondary Cy2-conjugated goat anti rabbit-Ig antibody. Cell nuclei were counterstained with 4’,6-diamidino-2-2-phenylindole (DAPI). Digital microscopy was performed with the Zeiss Axiophot microscope equipped with a CCD camera (SensiCam) using the Zeiss filter set 13 (excitation 470, emission 505 – 530) for Cy2 stains and filter set 20 (excitation 546, emission 575 – 640) for Cy3 stains. Fluorescent signals were pseudo-coloured by the AxioVision software. Typical digital images are shown. LN9: primary control fibroblasts; LN9i: SV40 T-Ag immortalized control fibroblasts; NBS558: primary fibroblasts from an NBS patient; PD20: primary FA-D2 fibroblasts: PD20i: T- Ag immortalized FA-D2 fibroblasts; PD733: primary FA-D2 fibroblasts; PD733i: T-Ag immortalized FA-D2 fibroblasts; FAG326: primary FA-G fibroblasts; FAG326i: T-Ag immortalized FA-G fibroblasts; FAE548: primary FA-E fibroblasts; FAE548i: T-Ag immortalized FA-E fibroblasts. Unirradiated control primary fibroblasts (LN9) are also shown (picture top left)

Oncogene SV40 large T-antigen and DNA-repair foci M Digweed et al 4875 irradiation (Maser et al., 1997). Since the culture response to ionizing irradiation as reported previously examined was growing logarithmically the foci positive (Maser et al, 1997). As both measurements reflect the fraction must be made up of cells in all compartments same phenomenon we chose percentage positive cells of the cell cycle at the time of irradiation. This is a for quantification of further experiments since this reflection of the involvement of MRE11 in both the allows a more rapid assessment of larger numbers of cell cycle-independent NHEJ and the initial steps of cells. cell cycle-dependent HR (Bressan et al., 1999; de Jager et al., 2001). Figure 2b compares the measurement of foci per Attenuation of MRE11 foci formation in SV40 nucleus with the measurement of the proportion of T-antigen immortalised fibroblasts foci-positive cells in the population. Clearly, both measurements exhibit a dose-dependent foci-forming An examination of the cells shown in Figure 1 would suggest that primary fibroblasts and fibroblasts immortalized by the SV40 large T-antigen (T-Ag), an oncogenic viral transcription factor, differ in their foci forming response to ionizing irradiation: after 12 Gy irradiation, far fewer of the T-Ag transformed cells display MRE11 foci in comparison to primary fibroblasts. The effect is clearly different to that seen in cells from NBS patients since the correct nuclear localization of MRE11 is preserved in T-Ag transformed cells. The quantification by foci-counting, shown in Figure 3, confirmed the observation of reduced foci formation in T-Ag immortalized cells. There are consistently more cells displaying IR- induced MRE11 foci in the primary strains, in comparison to their T-Ag immortalized derivatives, regardless of whether the cells are controls or from FA patients. The effect was, however, particularly strong in PD20i cells which show essentially no MRE11 foci after 12 Gy irradiation. Identical results were found for cells from a second FA-D2 patient, PD733 (Figure 1).

Figure 3 IR-induced MRE11 nuclear foci formation in primary fibroblasts and in SV40 T-Ag immortalized fibroblasts. Pairs of log-phase primary and SV40 T-Ag immortalized fibroblasts growing in 8-chamber microscope-slide flasks (BioCoat, Becton Dick- inson) were treated with 12 Gy IR and processed 8 h later for anti-MRE11 staining. For quantification, the slides were coded and 250 or 500 nuclei assessed for the presence of foci using Figure 2 Dose response of MRE11 nuclear foci formation. Con- the DAPI stain to count total nuclei. No threshold for foci num- trol primary fibroblasts growing asynchronously were treated as ber per nucleus was set. Mean percentages of positive cells are gi- described in the legend to Figure 1 with increasing doses of ioniz- ven together with error bars indicating the standard deviation. ing irradiation. After 8 h recovery the cells were processed for White columns: unirradiated cells; black columns: cells irradiated anti-MRE11 staining. Duplicate coded slides were examined mi- with 12 Gy. LN9: primary control fibroblasts; LN9i: SV40 T-Ag croscopically and the number of MRE11 foci in the nuclei of immortalized control fibroblasts; PD20: primary FA-D2 fibro- 100 cells was counted for each treatment. (a) The distribution blasts; PD20i: T-Ag immortalized FA-D2 fibroblasts; FAG326: of foci per nucleus is shown after 0, 4, 8 and 12 Gy irradiation. primary FA-G fibroblasts; FAG326i: T-Ag immortalized FA-G fi- (b) A comparison of the two measurements of foci-formation, foci broblasts; FAE548: primary FA-E fibroblasts; FAE548i: T-Ag per nucleus and percent foci-positive cells immortalized FA-E fibroblasts

Oncogene SV40 large T-antigen and DNA-repair foci M Digweed et al 4876

Figure 4 Phosphorylation of nibrin. Whole cell extracts of irradiated and unirradiated cells were prepared by lysis in RIPA buf- fer and applied to 8% polyacrylamide SDS gels. Proteins were electroblotted to nitrocellulose membranes, blocked in 5% dried milk in TBST and probed with rabbit polyclonal anti-MRE11 and anti-nibrin (p95; Novus Biologicals) for 1 h at room tempera- ture followed by incubation with horseradish peroxidase conjugated secondary antibody. Chemiluminescent detection was performed using the ECL reagents (Amersham). A portion of some irradiated lysates was treated with l-phosphatase (New Eng- land Biolabs) to demonstrate that the shift in nibrin mobility after irradiation is due to phosphorylation. LN9: primary control fibroblasts; LN9i: T-Ag immortalized control fibroblasts; PD20i: T-Ag immortalized FA-D2 fibroblasts; AT7/7: homozygous Ataxia tel- angiectasia primary fibroblasts; AT+/7: heterozygous AT primary fibroblasts; NBS: primary fibroblasts from an NBS patient

Nibrin phosphorylation accompanies nuclear foci formation by the MRE11/RAD50/nibrin complex (Gatei et al., 2000). We therefore examined ATM- mediated phosphorylation of nibrin in T-Ag immortalized cells and primary fibroblasts. As shown in Figure 4, immortalized cells are clearly able to phosphorylate nibrin in a normal fashion after ionizing irradiation. This suggests that nibrin phosphorylation alone is not sufficient for efficient foci formation and that T-Ag reduces MRE11 foci formation independently of nibrin phosphorylation.

Transient SV40 T-antigen expression is sufficient for the attenuation of MRE11 foci formation Figure 5 Transient T-Ag expression and MRE11 foci formation. A reduction in MRE11 foci formation in cells The plasmid vector pPrsTy, which expresses T-Ag from an ori-de- leted SV40 promoter/enhancer, was introduced into 80% conflu- immortalized by T-Ag has been noted previously ent primary fibroblast cultures by lipofection using the (Maser et al., 1997). In order to examine whether the Lipofectamine Plus reagent (Life Technologies). After 48 h for decrease in foci formation was indeed due to the action T-Ag expression, the cells were irradiated with 12 Gy and pro- of T-Ag itself, we transiently transfected two control cessed 8 h later to detect both MRE11 foci as described and T- Ag expression by immunostaining with a monoclonal anti-T-anti- cell strains, two primary FA-G and two primary FA- gen antibody (Oncogene Research Products) and secondary Cy3- D2 cell strains with a T-Ag expression plasmid. The conjugated anti-mouse Ig antibody. (a) Two typical images show- efficiency of transfection varied from 5 to 15% so that ing FA-D2 cells (HND) after pPrsTy-transfection, irradiation and simultaneous staining for MRE11 (Cy2) and T-Ag immunostaining. Ten cells are visible after MRE11 staining (Cy2), (Cy3) after 12 Gy IR (Figure 5a) allowed quantifica- of these, five cells show foci but none of these express T-Antigen (Cy3). (b) Quantification of MRE11 foci-presentation in T-Ag-po- tion of foci formation in T-Ag expressing and non- sitive and T-Ag-negative control (LN8, LN9), FA-D2 (HND, expressing cells within the same population. From the HNK) and FA-G (FA1BER, GNF) cells performed as described quantification shown in Figure 5b it is clear that foci in Figure 3. Data are pooled for the two cell lines in each group formation is indeed attenuated in cells transiently expressing T-Ag before mutational events leading to immortalization can be expected to have occurred. This effect is highly significant (P=0.000017 for control cells influenced not only by T-Ag but also by the FANCD2 with and without T-Ag) and rather more pronounced gene. Retroviral transfer of the FANCD2 cDNA does in the two FA-D2 fibroblast strains in comparison to not rescue the foci-abrogation in T-Ag immortalized the two FA-G strains (P=0.047, FA-G+T-Ag : FA- FA-D2 cells (data not shown) reflecting the dominant D2+T-Ag). These results confirm those with the effect of highly overexpressed T-Ag in these cells which immortalized cell lines described above and indicate cannot be counteracted by retrovirally expressed that the presence of MRE11 in nuclear foci is FANCD2.

Oncogene SV40 large T-antigen and DNA-repair foci M Digweed et al 4877 Table 1 IR-survival of SV40 immortalized ﬁbroblasts Cell line Cell type n 0 Gy 0.5 Gy P 1Gy P

LN9i Control 8 100.0 (+2.9) 98.7 (+3.2) 85.7 (+4.8) PD20i FA-D2 4 100.0 (+2.0) 82.2 (+1.7) 0.004 72.7 (+4.1) ns GM166VA7 NBS 6 100.0 (+2.0) 83.8 (+2.0) 0.003 66.6 (+1.9) 0.004 FAG326i FA-G 4 100.0 (+1.2) 91.0 (+2.4) ns 86.1 (+3.9) ns

Survival is given as a percentage of colony numbers in the untreated cultures together with the standard deviation. n=number of individual data points. P=significance level in the Student’s t-test comparing to the control cells. LN9i: SV40 T-Ag immortalized control fibroblasts; PD20i: T-Ag immortalized FA-D2 fibroblasts; GM1667VA7: SV40 T-Ag immortalized NBS fibroblasts; FAG326i: SV40 T-Ag immortalized FA-G fibroblasts

Loss of MRE11 foci forming ability correlates with p53, 53BP1, may be relevant. 53BP1 is a 217 kD increased radiosensitivity in immortalized FA-D2 cells transcriptional co-activator of p53 and has been reported to accumulate rapidly (within 5 – 15 min) at Abrogation of MRE11 foci formation in FA-D2 cells the sites of DSBs after ionizing irradiation (Schultz et could be expected to be reflected in an increased al., 2000). The presence of 53BP1 in nuclear foci cytotoxicity towards IR. To address this question we precedes MRE11/RAD50/nibrin, suggesting it is examined colony formation in irradiated, T-Ag involved in an earlier step in the pathway (Schultz immortalized, control, NBS, FA-G and FA-D2 cells. et al., 2000). It seems likely, but remains to be In each experiment, cells were plated at low density demonstrated, that the expression of T-Ag in and irradiated 18 h later with 0, 0.5 and 1 Gy as mammalian cells disrupts not only p53 but also described. After 10 days for colony growth the cells 53BP1 and that this may be related to the reduction were fixed, stained and colonies counted. As shown in in MRE11 foci formation after ionizing radiation in Table 1, there is indeed a statistically significant T-Ag expressing cells. decrease in survival of PD20i cells after a low radiation We suggest that disturbance of DNA repair by the dose of 0.5 Gy. After this treatment, approximately MRE11/RAD50/nibrin pathway in the presence of T- 20% of PD20i cells fail to survive, making this cell line Ag may lead to increased mutations and thus as radiosensitive as the NBS cell line, GM166VA7. At contribute to cell immortalization by T-Ag. This may 1 Gy there is also a clear, but statistically no longer reflect yet another activity of p53 as a mediator of significant, reduction in colony formation in PD20i DNA repair, here through a protein complex involved cells in comparison to both T-Ag immortalized control in both homologous recombination and non-homo- and FA-G cells. logous end joining of DSBs. Such an interaction is Whilst there is no evidence for a specific disturbance further supported by the results of p53 co-immuno- in MRE11/RAD50/nibrin foci formation in immorta- precipitation experiments in T-Ag-immortalized cardio- lized or primary fibroblasts from any other FA myocyte cell lines (Lanson et al., 2000). complementation group (Figures 1 and 4 and Digweed et al., 2002), MRE11/RAD50/nibrin foci are uniquely lacking in irradiated T-Ag immortalized cells from the SV40 transformation and FA two FA-D2 patients examined (Figures 1, 3 and 5b). This suggests the presence of a FANCD2-S-dependent Several investigations have reported that FA fibro- pathway for relocation of MRE11 to discrete nuclear blasts are particularly sensitive to transformation by foci. Whether this involvement of FANCD2 in the SV40 T-Ag (Todaro et al., 1966; Dosik et al., 1970; recruitment of MRE11 to foci reflects a ‘rescue Lubiniecki et al., 1980). This phenomenon is directly pathway’ for cells with a compromised p53 response linked to the primary defect in these cells since or, rather, a minor constitutively operative mechanism correction of FA-C cells by transduction with remains to be determined. FANCC has been shown to reduce their transformation potential (Liu et al., 1996). Since cellular transformation requires integration of the virus, the Cell immortalization by SV40 T-antigen and the 3 – 50-fold increase in transformation frequency of FA MRE11/RAD50/nibrin pathway cells suggests that integration is more readily achieved. Viral integration might be promoted in cells The immortalizing capacity of T-Ag is generally repairing DSBs by error prone mechanisms such as attributed to its association with p53 and the impact NHEJ and homology-directed single strand annealing this has on the G1 checkpoint (Zhu et al., 1991). rather than error-free homologous recombination. The However, p53 clearly has further activities within the characteristic chromosome aberrations seen in FA cell, such as the regulation of homologous recombina- suggests that FA cells may differ from other cells in tion triggered by replication arrest, possibly via just such a way. Furthermore, additional interference RAD51 (Saintigny and Lopez, 2002). by T-Ag, through the disturbance of MRE11- The interaction of T-Ag with p53 is well documen- relocation to DSBs, may be even less well tolerated ted and in this connection a further binding partner of by the FA cell.

Oncogene SV40 large T-antigen and DNA-repair foci M Digweed et al 4878 Acknowledgments Excellent technical assistance was provided by Gabriele We are indebted to Dr Monika Gra¨ ssmann, Free Hildebrand and Janina Radszewski, for which we are University Berlin, for the SV40 T-Ag expression plasmid grateful. This work was supported by the Fritz-Thyssen- and to Dr Kenshi Komatsu, Hiroshima University, Japan, Stiftung (Az 2000/01/69) and the Deutsche Forschungsge- for the SV40 immortalized NBS cell line, GM166VA7. meinschaft (SFB 577-B1).

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