Immortalization and characterization of Nijmegen Breakage Syndrome fibroblasts

doi:10.1016/S0921-8777(99)00009-9

Mutation Research/DNA Repair

Volume 434, Issue 1, 14 May 1999, Pages 17-27

Mutation Research/DN...

https://doi.org/10.1016/S0921-8777(99)00009-9 Get rights and content

Abstract

Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.

Introduction

Nijmegen Breakage Syndrome (NBS) is an autosomal recessive hereditary disorder characterized by microcephaly, a `bird-like' facial appearance, growth retardation, immunodeficiency, radiosensitivity, chromosomal instability and a very high incidence of lymphoreticular malignancies (reviewed in Refs. 1, 2). The disease appears to be prevalent among the Eastern and Central European population, in particular among the Czech and Polish people. Abnormal response of NBS cells to ionizing radiation (IR) includes: 1) an increased sensitivity in terms of elevated levels of chromosomal aberrations, 2) an increased cell killing, and 3) a diminished inhibition of DNA synthesis called radioresistant DNA synthesis (RDS). This abnormal response to IR closely resembles that of Ataxia telangiectasia (AT) and was therefore thought to be caused by a defect in cell cycle regulation. However, there is still considerable controversy in this respect. Although after IR a reduced and delayed p53 response has been found in NBS cells 3, 4, 5, some investigators have found an abnormal cell cycle arrest at G1-S and a prolonged accumulation of cells in the G2 phase 3, 6, 7, 8, while others have reported normal G1 and G2 block in response to IR 5, 9.

NBS1, the gene defective in NBS has been mapped by linkage studies [10]and microcell-mediated chromosome transfer to 8q21–24 [11], and has recently been cloned 12, 13. The product of this gene, nibrin or NBS1, is a novel protein, which is a member of the hMre11/hRad50 protein complex [14], suggesting that the NBS1 gene is involved in DNA double-strand break (DSB) repair. This hypothesis is supported by the finding that hMre11 and hRad50 co-localize in nuclear foci in response to treatment of human cells with DSB-inducing agents [15]. In NBS cells the formation of hMre11/hRad50 foci in response to IR is completely abolished (although hMre11 can still bind to hRad50), suggesting that nibrin is required for the re-localization of the hMre11/hRad50 protein complex to DSB [14]. It has been suggested that in yeast, Mre11 and Rad50 form together with Xrs2 a protein complex with a nuclease activity 16, 17, 18. Although the human NBS1 and yeast XRS2 genes only share very limited sequence homology 13, 14, they probably exert homologous functions in the Mre11/Rad50 complex. Recently it has been reported that indeed the human Mre11/Rad50 complex together with a protein of 95 kDa, likely to be nibrin, possesses nuclease activity 19, 20. In yeast the Mre11/Rad50/Xrs2 complex is, apart from DSB processing, also involved in telomere replication 21, 22, 23. Therefore, in humans nibrin may also be involved in telomeric length maintenance.

The cellular features of NBS are very similar to AT, suggesting that their gene products function in the same pathway operating in response to IR. However, the clinical features of both disorders are quite distinct. While AT patients suffer from cerebellar ataxia, oculocutanous telangiectasia and have elevated serum levels of α-fetoprotein, NBS patients display microcephaly, are growth retarded and about 60% shows borderline to moderate mental retardation. AT as well as NBS patients show a high incidence of cancer, predominantly lymphoreticular malignancies, but cancer predisposition and genomic instability is presumably more pronounced in NBS than in AT [2]. Strikingly, in blood relatives of NBS patients a significant elevation of various cancers was found [24]. Epidemiological studies also revealed an elevation of certain cancers among relatives of AT patients, especially breast cancer in women (reviewed in Ref. [2]).

The gene defective in AT cells was localized on chromosome 11q23.1 [25]and has been cloned [26]. The ATM protein (ataxia–telangiectasia mutated) is a member of a large protein family, of which all proteins contain a PI 3-kinase domain. Most of these proteins are involved in cellular responses to genotoxic damage or in cell cycle control [27]. In AT cells there is no major defect in the repair kinetics of DNA single-strand breaks (SSB) and DSB, but some studies revealed that a subclass of DSB is not repaired in AT cells (Ref. [28]and references therein). Cell cycle regulation is disturbed in irradiated AT cells, as is evident from their failure to induce p53 and G1-S or G2-M arrest (reviewed in Ref. [2]).

In this paper we describe the cellular characteristics of primary NBS-1LB fibroblasts from a newly diagnosed Polish patient and its derived SV40 transformed fibroblast cell line NBS-1LBI. Both primary and immortal cells show a 2-fold increase in sensitivity towards X-rays, display RDS, and carry a 5-nt deletion in the NBS1 gene. Notwithstanding that NBS cells showed an increased sensitivity to DSB-inducing agents, we found that the overall rates of rejoining of IR-induced DSB were similar to those of wild type cells.

Section snippets

Cells and culture conditions

Primary skin fibroblasts derived from a normal donor (VH25) and primary buccal mucosa fibroblasts derived from a 5 year old Polish NBS patient (NBS-1LB) were cultured in DMEM/F12 medium supplemented with 10% FBS and PS (100 units/ml penicillin, 1 mg/ml streptomycin). The SV40-transformed human fibroblast lines MRC5V1 [29]and 1BR.3GN2 [30]derived from normal donors, the osteosarcoma derived HeLa cells, and the SV40-transformed human fibroblast cell lines AT5BIVA (AT cells) [31]and NBS-1LBI (NBS

Generation of the immortal NBS cell line NBS-1LBI

The primary fibroblast strain, NBS-1LB, was established from a biopsy of buccal mucosa from a NBS patient. Early passage numbered primary cells of this strain were transfected with an SV40 ori-defective early region plasmid. After 6 weeks about 150 colonies of transformed cells appeared and they were mixed and propagated in culture. Approximately 6 months after transfection a decrease in growth rate was observed, but no profound crisis occurred. The transformed cells have been in continuous

Discussion

In the present study we report on the cellular and molecular characteristics and the analysis of DSB-repair in NBS cells. An immortal SV40-transformed NBS fibroblast cell line, NBS-1LBI, derived from NBS-1LB primary fibroblasts from a newly diagnosed NBS patient, was generated. The causative, typical Slavic NBS mutation, 657del5, was found to be present in both immortal and primary cells, confirming the diagnosis of NBS in this patient. The cellular response of NBS-1LBI cells to IR, in terms of

Acknowledgements

This work was supported by grants 9.0.6 and 9.0.14 from the J.A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, The Netherlands and by the European Union grant F14PCT90010.

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Immortalization and characterization of Nijmegen Breakage Syndrome fibroblasts

Abstract

Introduction

Section snippets

Cells and culture conditions

Generation of the immortal NBS cell line NBS-1LBI

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Clin. Immunol. Immunopathol.

Mutation Res.

Am. J. Hum. Genet.

Cell

Cell

Mol. Cell

J. Biol. Chem.

Curr. Biol.

Mutation Res.

Virology

Cell

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The gene for the ataxia–telangiectasia variant, Nijmegen Breakage Syndrome, maps to a 1-cM interval on chromosome 8q21

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