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International Agency for Research on Cancer, 69008 Lyon, France [V. D-J., P-O. F., W-M. T., G. S., W. H., G. S., Z. H., Z-Q. W.], and Institute of Human Genetics, Charité-Campus-Virchow, Humboldt Universitaet zu Berlin, D-13353 Berlin, Germany [M. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-irradiation enhanced tumor development in Nbn+/- mice, giving rise to a high frequency of epithelial tumors, mostly in the thyroid and lung, as well as lymphomas. These mice also developed numerous tumors in the ovary and testis. Southern and Western blot analyses showed a remaining wild-type allele and nibrin expression in Nbn+/- tumors. Sequencing analysis confirmed no mutation in the Nbn cDNA derived from these tumors. Cytogenetic analysis revealed that primary Nbn+/- embryonic fibroblasts and tumor cells exhibit increased chromosomal aberrations. These data suggest that haploinsufficiency, not loss of heterozygosity, of Nbn could be the mechanism underlying the tumor development. Taken together, our heterozygous Nbn-knockout mice represent a novel model to study the consequences of NBS1 heterozygosity on tumor development. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The product of the human gene (NBS1), nibrin/p95, forms a complex with hMre11 and hRad50 and functions in DNA DSB repair by homologous recombination or NHEJ, meiotic recombination, DNA damage response, and telomere maintenance (3 , 4) . After DNA damage produced by ionizing radiation, nibrin is phosphorylated by ATM and is responsible for the nuclear focus formation of the Mre11/Rad50 repair complex at the site of DNA damage. This process is apparently independent from H2AX (5) but mediated by MDC1 (mediator of DNA damage checkpoint protein 1; Refs. 6 , 7 ). The Mre11/Rad50/nibrin complex activates downstream molecules, including p53, BRCA1, and Chk2 to control cell cycle progression (see reviews in Refs. 3 , 4 ). In addition, structural maintenance of chromosome protein 1 (SMC1) is phosphorylated in response to DNA damage in an ATM- and NBS1-dependent manner, and the ATM/NBS1/SMC1 pathway is involved in S-phase checkpoint activation (8 , 9) . Finally, it has also been demonstrated that the Mre11/Rad50/nibrin complex interacts with E2F1, indicating that this complex may directly influence S-phase progression (10) . Taken together, these studies suggest an important function for NBS1 in many cellular processes in response to DNA damage.
In an attempt to understand the biological function of NBS1, the mouse homologue of the gene, Nbn, was disrupted in mice. The null mutation of Nbn caused mouse embryos to die in utero between day E3.5 and E7.5 because of stalled growth and expansion of the ICM of mutant blastocysts (11) . Interestingly, mice expressing C-terminal nibrin survive to adulthood and exhibit many phenotypes similar to NBS patients, including growth retardation, lymphoid developmental defects, and development of thymic lymphomas. MEFs derived from these mutants are hypersensitive to ionizing radiation and exhibit defects in the S-phase checkpoint, as well as a high degree of chromosomal breaks (12 , 13) . However, the impact of Nbn mutation in in vivo radiosensitivity and tumorigenesis has not been reported in these animal models.
Epidemiological and clinical studies have proposed that heterozygous NBS patients are prone to malignancy development (14, 15, 16) . Chromosome-painting analysis has demonstrated that NBS heterozygous patients show an increased frequency of chromosomal translocations that can be potentiated by ionizing radiation, which represents an important risk factor in the development of malignancies in heterozygotes with NBS1 gene mutations (17 , 18) . Because the average carrier frequency of the NBS1 founder mutation among newborns is 1 in 177 in the Slav population in the Czech Republic, Poland, and Ukraine (19) , a moderately elevated cancer risk in heterozygous carriers would result in hundreds of new cancer cases in these populations every year. Therefore, experimental investigation into this public health issue would be valuable. In the present study, we examine whether heterozygosity of NBS1 mutation could contribute to tumorigenesis using Nbn-knockout mouse models.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genotyping by PCR and Southern Blot Analysis.
For Southern blot and PCR analyses, genomic DNA isolated from ES cells or from mouse tissues was digested with BamHI, or BamHI and ApaI and hybridized with a 5'-external probe in intron 4. For PCR genotyping, three primers were used: SF1, 5'-CTACTCCCATACACAGACAG-3'; EX6, 5'-CAGGGCGACATGAAAGAAAAC-3'; and neo2, 5'-CCTGCTTGCCGAATATCATG-3'. A 150-bp fragment and a 411-bp fragment were amplified from the wild-type and mutant allele, respectively. For genotyping of blastocysts, embryos or outgrowth were washed twice in PBS and lysed in a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 10 mM NaCl, 0.5% sarcosyl, and 1 mg/ml proteinase K. Samples were then subjected to PCR analysis.
Histopathological Analysis.
Animals in a mixed genetic background (C57BL/6 x 129/Sv) were used for spontaneous tumor development, as well as for irradiation treatment. For -radiation, Nbn+/- and wild-type mice were treated at 4–7 weeks old with 8 Gy with a 137 Ce source (IBL-67C irradiator; C1S Biointernational, Gif-Sur-Yvette, France). Animals were sacrificed by ether anesthesia upon decline in health (i.e., weight loss, paralysis, ruffling of fur, or inactivity). A full autopsy was performed and organs were fixed in 4% neutral-buffered formaldehyde, followed by dehydration and paraffin embedding. Histopathological analysis was carried out on 3-µm thick sections stained with H&E or immunostaining as described previously (20)
. Antibodies were: rat monoclonal antimouse CD3 (Serotec, Oxford, United Kingdom), rat monoclonal antimouse CD45R/B220 (PharMingen International, San Diego, CA), and rabbit polyclonal anti-3ß-HSD antibody (a gift from Dr. Mohamed Benahmed, Lyon, France).
Western Blot Analysis.
For protein extraction, tumor and normal tissues were frozen in liquid nitrogen and incubated with an ice-cold radioimmunoprecipitation assay-like buffer [50 mM Tris-HCl (pH 7.4), 250 mM NaCl, 0.5% NP40, 0.1% SDS, 2 mM DTT, and 1 mM phenylmethylsulfonyl fluoride] for 30 min at 4°C, followed by sonication. Lysates were then subjected to Western blot analysis (10% SDS gel). The membrane was probed with a polyclonal antibody raised against the C-terminal part of murine nibrin (nibrin 15CR). The band was visualized by incubation with an enhanced chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, United Kingdom). To illustrate protein loading, the blots were stripped and incubated with an antiactin antibody (clone C4; ICN, Hampshire, United Kingdom).
In Vitro Culture of Blastocysts.
Blastocysts were flushed from the uteri of pregnant female mice at day 3.5 post coitum and cultured in M15 medium (DMEM, 15% FCS, 2 mM glutamine, 5 x 10-5 M ß-mercaptoethanol, 1000 units/ml leukemia inhibitory factor, 100 units/ml penicillin, 100 mg/ml streptomycin, 1% nonessential amino acid, and 1% sodium pyruvate) at 37°C in a humidified incubator (5% CO2). For TUNEL analysis, embryos grown in 96-well plates were washed twice in PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate for 5 min on ice. After two PBS washes, a TUNEL-labeling reagent (Roche, Meylan, France) was applied for 2 h at 37°C and then cells were counterstained with propidium iodide. Photographs were taken using an inverted fluorescent microscope.
Cytogenetic Analysis.
Primary MEFs were isolated from E13.5 embryos derived by intercrossing Nbn+/- mice, according to protocols described previously (21)
. Primary lymphoma cells were isolated and cultured in an M10 medium (DMEM, 10% FCS, 2 mM glutamine, 5 x 10-5 M ß-mercaptoethanol, 100 units/ml penicillin, 100 mg/ml streptomycin, and 1% sodium pyruvate) and stimulated for proliferation by 1.5 µg/ml ConA (Sigma, Saint Quentin Fallavier, France). Preparation of metaphase spreads, telomere staining, and chromosome analysis was performed as described previously (20)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. After electroporation of the targeting vector into D3 ES cells, 7 of 650 clones screened by Southern blot analysis were identified as containing the targeted allele (Fig. 1B)
. Four independent ES clones (Nbn+/-) were injected into blastocysts to generate chimeric mice and two clones produced germ-line offspring.
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. To determine the possible effects of nibrin deficiency, blastocysts were isolated from uteri of Nbn+/- females that had been bred with Nbn+/- males. During the first 4 days of culture, the ICM formed and the trophoectoderm of blastocysts differentiated into giant trophoblast cells, regardless of their genotypes. Although Nbn+/+ and Nbn+/- blastocysts displayed a normal growth pattern, degeneration of the ICM was evident in homozygous Nbn mutants after 4 days in culture. TUNEL analysis revealed enhanced apoptosis in the ICM, as well as in the giant trophoblast cells of Nbn-/- blastocyst outgrowths (Fig. 1D)
. These in vitro culture results indicate that Nbn deficiency results in enhanced cell death and degeneration of the ICM, most likely causing peri-implantation lethality. This finding is consistent with the results reported by Zhu et al. (11)
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Nbn Heterozygous Mice Are Susceptible to Tumor Development.
To test whether Nbn heterozygosity predisposes mice to malignancy, we monitored the tumor development in Nbn+/- mice in a large cohort of Nbn+/- (n = 63) and Nbn+/+ (n = 33) animals over a period of 100 weeks. Statistical analysis showed a significant survival difference between Nbn+/- and wild-type control mice (P = 0.027, log-rank test; Fig. 2A
). Systematic histopathological analysis of 58 Nbn+/- mice revealed that tumor onset and development were significantly enhanced compared with wild-type controls (Fig. 2A)
. It was also noted that the tumor incidence during the observation period was at 78% (45 of 58) in the Nbn+/- group and at 53% (17 of 32) in the wild-type control group. Moreover, the number of tumors per mouse was significantly higher in the Nbn+/- group (1.09 tumors per mouse) compared with the wild-type group (0.59 tumors per mouse; Fig. 2B
).
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). Interestingly, Nbn+/- mice developed a high frequency of epithelial tumors (52%), gonad tumors (12%), and lymphomas (10%; Fig. 2B
). Immunostaining using T- and B-cell-specific markers revealed that lymphomas could infiltrate other organs such as the liver or small intestine and expressed exclusively either the B-cell marker CD45R/B220, or the T-cell marker CD3 (Fig. 3A
, data not shown). The presence of T- or B-cell lymphomas in these Nbn mutant mice are reminiscent of human NBS cases (1)
. Interestingly, many epithelial tumors were also found in a wide array of organs (Fig. 2B)
, including liver adenomas and carcinomas (Fig. 3B)
, mammary gland carcinomas (Fig. 3C)
, and low-grade prostate intraepithelial neoplasia (Fig. 3D)
. Finally, these Nbn+/- mice developed gonad tumors (7 of 58 mice examined), including sex-cord stromal cell tumors and germ cell tumors (Fig. 2B)
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-rays at a dosage of 8 Gy and monitored for tumor formation over a period of 64 weeks. As shown in Fig. 4A
, -irradiation significantly reduced the life span of Nbn+/- mice compared with wild-type controls (P = 0.0035, log-rank text). Histopathological analysis revealed that tumor incidence was dramatically increased after -irradiation in the Nbn+/- group (85%) compared with that in Nbn+/+ control mice (41%; Fig. 4B
). Similar to spontaneous tumor development in Nbn+/- aged mice, more lymphomas and epithelial tumors were observed in radiation-treated Nbn+/- mice when compared with wild-type controls or to unirradiated groups (Figs. 2B
and 4B
). Strikingly, a high frequency of thyroid gland atypical hyperplasia and intraepithelial adenomas was observed in Nbn+/- mice after -irradiation (Figs. 3E
and 4B
). In addition, -irradiation increased the formation of lung cancers (Fig. 3F)
, germ cell tumors and stromal cell tumors in the testis (Leydig cell tumors; Fig. 3, G and H
) and ovary (Fig. 3, I and J)
in Nbn+/- mice. Compared to spontaneously developed tumor types in Nbn+/- mice, we noted that -radiation induced new types of tumors (e.g., thyroid tumors) while it accelerated lymphomas and gonad tumors. These data suggest that nibrin is important in protecting these particular organs against -irradiation-induced damage.
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; data not shown). We further confirmed this result by Western blot analysis, which showed no obvious change in the amount of nibrin in tumors when compared with nontumorigenic tissues (Fig. 5B)
. Immunohistochemical analysis of 18 additional tumors using the antinibrin antibody showed that nibrin expression in all tumors is indistinguishable from that of the nontumoric areas of the affected tissues or normal counterparts (data not shown). To test whether nibrin is mutated in these Nbn+/- tumors, we sequenced reverse transcription (RT)-PCR products derived from the Nbn cDNA of two tumors and found that there was no apparent mutation in these samples when compared with sequences derived from wild-type and heterozygous nontumoric tissues and Nbn+/- ES cells (data not shown). Thus, it seems that the tumor development in Nbn+/- mice is not due to LOH.
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, suggesting that haploinsufficiency of Nbn leads to an increased rate of chromosome aberrations.
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. Notably, one tumor showed that 73% of metaphases contain fusions, including Robertsonian-like configurations and another showed a high number of fragmentations and breaks (14%; Fig. 6A, d and e, B
). These data suggest that Nbn+/- tumors may arise from clonal or polyclonal expansion of cells associated with chromosomal aberrations. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to lymphomas that frequently appeared in Nbn+/- mice, other major tumor types included epithelial tumors affecting the liver, prostate, and mammary glands, as well as gonad malignancy. The tumors reported in NBS patients (homozygous 657del5) are predominantly lymphomas arising in the first two decades of life, but leukemia, gliomas, medulloblastomas, and rhabdomyosarcomas have also been reported (Ref. 27 ; see also reviews Refs. 1 , 2 ). NBS heterozygous patients also develop other tumor types, including melanomas, mammary gland cancers, colon cancers, brain tumors, as well as lymphomas (15 , 16) . The discrepancy in the tumor spectrum between NBS patients (homozygous or heterozygous 657del5) and our Nbn heterozygous mice may be explained by the nature of the Mre11/Rad50/nibrin complex that might be affected by different mutations of Nbn imposed in the cells or different modifiers in humans and mice. It is also possible that Nbn heterozygosity causes progressive defects in chromosomal stability or in other cellular functions in adult tissues. These observations strongly suggest that nibrin plays a general role in suppressing tumorigenesis, not only in lymphoid organs but also in other cell types.
Another interesting observation is that ionizing radiation dramatically enhanced tumor formation in Nbn heterozygous mice, consistent with the notion that NBS patients are sensitive to -irradiation. Moreover, a high frequency of thyroid tumors arose after ionizing radiation. This phenomenon is reminiscent of studies showing that exposure to radiation selectively predisposes individuals to thyroid cancers in late their life (28
, 29)
. The ionizing radiation-induced thyroid cancer is believed to be a consequence of the rearrangement or activation of the Ret/PTC oncogene (30)
. However, whether mutation of Nbn is another genetic factor involved in radiation-induced thyroid pathology or whether nibrin sits in any pathways similar to Ret/PTC requires additional investigation. It is plausible that the function of nibrin in thyroid epithelial cells may be critical in DNA damage response. Taken together, this study establishes a relationship between Nbn heterozygosity, radiation sensitivity in vivo, and increased cancer risk.
It is interesting to note that these Nbn+/- tumors retained the remaining Nbn wild-type allele, suggesting that the mechanism underlying tumor development in Nbn heterozygous mice is most likely not LOH, but rather haploinsufficiency. In support of this hypothesis, our cytogenetic analysis has shown that primary Nbn+/- fibroblasts and tumor cells associate with chromosomal aberrations, including fragmentations/breaks and end-to-end fusions, consistent with the role of nibrin in DSB repair and genomic stability. In addition, loss of telomere signals at fusion sites further supports the function of nibrin in telomere maintenance (31, 32, 33) . This finding is consistent with studies showing that cells isolated from NBS patients exhibit high levels of chromosome breakage and nonspecific translocations (17 , 18) , and NBS1 heterozygous cells show an increased sensitivity to radiation-induced chromosomal aberrations (17) . A plausible explanation for this chromosome phenotype is that the persistence or aberrant repair of DSBs in Nbn+/- cells provide substrates for chromosome end-joining, the quality of which is otherwise monitored by the homologous recombination or NHEJ machinery involving appropriate Mre11/Rad50/nibrin activity (4) . Therefore, Nbn haploinsufficiency may represent a mechanism of tumor development in these Nbn+/- mice. Our observation is consistent with the general notion that reduced NHEJ activity, not LOH, is sufficient to create a predisposition to tumor development in various models, including mice and humans (see reviews Refs. 34 , 35 ). For example, Lig4 heterozygosity induces soft tissue sarcoma development in Ink4a/arf-/- mice (36) , and Ku80 heterozygosity accelerates liver tumor formation in the PARP-1 null background (37) . Moreover, it is interesting to note that mice carrying a heterozygous mutation in H2AX, a molecule that modulates DSB repair by both homologous recombination and NHEJ, are susceptible to tumorigenesis (38 , 39) . Finally, decreased expression of Ku70 and Ku80 was found in some human neoplasms (40 , 41) .
Accumulating evidence shows that heterozygous NBS patients are prone to malignancy development (14, 15, 16)
. Given the high frequency of NBS1 mutation carriers (1 in 177 individuals) in the Slav population in Eastern and Central Europe (19)
, the genetic instability of these heterozygous NBS patients may represent a risk factor for malignancy. Indeed, the present study provides experimental evidence that Nbn heterozygosity predisposes cells to malignancy most likely because of chromosomal instability and defects in DNA repair. Moreover, the high susceptibility of Nbn heterozygous mice to malignancy after exposure to -irradiation may implicate a necessary precaution regarding any radiotherapy applied to these NBS1 carriers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 P-O. F. is a recipient of fellowship from the Comité départmental de la Ligue Nationale contre le Cancer de Haute-Savoie (2000–2002) and the Association de la Recherche pour le Cancer (2002–2003). 
2 To whom requests for reprints should be addressed, at International Agency for Research on Cancer, 150 cours Albert Thomas, 69008 Lyon, France. Phone: 33-4-72-73-85-10; Fax: 33-4-72-73-83-29; E-mail: zqwang{at}iarc.fr
3 The abbreviations used are: NBS, Nijmegen Breakage Syndrome; DSB, double strand break; NHEJ, nonhomologous end-joining; ATM, ataxia telangiectasia mutated; ICM, inner cell mass; MEF, mouse embryonic fibroblast; 3ß-HSD, 3ß-hydroexsteriod dehydrogenase; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling; LOH, loss of heterozygosity.
Received 7/16/03. Revised 9/ 5/03. Accepted 9/12/03.
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