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Human Molecular Genetics Advance Access originally published online on July 21, 2004
Human Molecular Genetics 2004 13(18):2155-2163; doi:10.1093/hmg/ddh221
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Human Molecular Genetics, Vol. 13, No. 18 © Oxford University Press 2004; all rights reserved

MRE11 mutations and impaired ATM-dependent responses in an Italian family with ataxia-telangiectasia-like disorder

Domenico Delia1, Maria Piane2, Giacomo Buscemi1, Camilla Savio2, Silvia Palmeri3, Patrizia Lulli2, Luigi Carlessi1, Enrico Fontanella1 and Luciana Chessa2,*

1Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy, 2Department of Experimental Medicine and Pathology, II Faculty of Medicine, University ‘La Sapienza’, Roma, Italy and 3Department of Neurological Sciences, Policlinico Le Scotte, University of Siena, 53100 Siena, Italy

Received June 22, 2004; Accepted July 9, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hypomorphic mutations of the MRE11 gene are the hallmark of the radiosensitive ataxia-telangiectasia-like disorder (ATLD). Here, we describe a new family with two affected siblings, ATLD5 and ATLD6, now aged 37 and 36, respectively. They presented with late onset cerebellar degeneration slowly progressing until puberty and absence of telangiectasias, and were cancer-free. Both patients were wild-type for ATM and NBS1, but compound heterozygotes for MRE11 gene mutations [1422C->A, T481K; 1714C->T, R571X]. The 1422C->A allele was inherited from the mother, whereas the 1714C->T, allele paternally inherited, was apparently null as a result of nonsense-mediated mRNA decay (NMD). Interestingly, the 1714C->T mutation is the same as previously identified in an unrelated English ATLD family (probands ATLD3 and ATLD4), suggesting an important role for NMD in saving potentially lethal mutations. Lymphoblastoid cell lines (LCLs) derived from ATLD5 and ATLD6 were normal for ATM, but defective for Mre11, Rad50 and Nbs1 (the MRN complex) protein expression. Their response to {gamma}-radiation was abnormal, as evidenced by the enhanced radiosensitivity, attenuated autophosphorylation of ATM-S1981 and phosphorylation of the ATM targets p53–S15 and Smc1–S966, failure to form Mre11 nuclear foci and defective G1 checkpoint arrest. The fibroblasts, but not LCLs, from ATLD5 and ATLD6 showed an impaired ATM-dependent Chk2 phosphorylation. These findings further underscore the interconnection between ATM activity and MRN function, which rationalizes the clinical similarity between ataxia-telangiectasia (A-T) and ATLD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ataxia-telangiectasia (A-T) is a pleiotropic autosomal recessive disorder characterized by cerebellar ataxia, teleangiectasias, immunodeficiency, radiosensitivity and predisposition to malignancy. Milder A-T cases, termed A-T variants, present later onset of the disease and/or moderate severity of clinical features and longer life span (1–3).

The common phenotypic features of classical A-T and A-T variants are hypersensitivity to ionizing radiation (IR) and radiomimetic drugs, defective cell-cycle checkpoints and alterations of DNA double-strand breaks (DSBs) repair, altogether contributing to genomic instability and cancer predisposition. Classical A-T patients show homozygous or compound heterozygous mutations of the ATM gene which generally lead to the truncation of the protein product (4–9). ATM mutations were also described in some A-T variants and in one A-T Fresno patient (10).

More recently, four probands belonging to two unrelated English families with A-T variant phenotype, defined as A-T-like disorder (ATLD), have been found to carry mutations in the MRE11 gene (11). Of these, family 1 probands (ATLD1 and ATLD2) are affected by a severe form of the disease and carry homozygous MRE11 mutations that lead to low-level expressions of truncated protein. Family 2 probands (ATLD3 and ATLD4) are affected by a milder form of the disease and carry compound heterozygous MRE11 mutations that lead to the expression of a partially active Mre11 protein (11).

The MRE11 gene encodes a protein with nuclease and intrinsic DNA-binding activity that, through interaction with Rad50 and Nbs1, forms the core of the MRN complex involved in DSB sensing, DNA recombination and multiple cell-cycle checkpoints (12,13). The Mre11/Rad50/Nbs1 (MRN complex) proteins are homogeneously diffuse in the nucleus of normal undamaged cells, but after treatment with genotoxic agents are rapidly recruited at sites of DNA lesions, giving rise to the formation of nuclear foci believed to play a role in DSBs processing and checkpoints signalling (13). In ATLD cells, in which the MRE11 mutations result in reduced expression of Mre11 as well as of Rad50 and Nbs1 proteins, the formation of MRN foci is impaired. The recently generated Mre11ATLD1/ATLD1 mice expressing the ATLD1 allele (1897C->T, R633X) appear to recapitulate the phenotypic features of ATLD, including radiation hypersensitivity, chromosomal instability and defective arrest at G1–S, intra-S and G2–M checkpoints (14). Interestingly, these Mre11ATLD1/ATLD1 mice are not prone to malignancy, indicating that the observed phenotypic defects are per se insufficient to significantly enhance the initiation of tumorigenesis (14).

The response to DSBs primarily involves the activation of ATM kinase and phosphorylation of several targets, e.g. p53, Chk2, Brca1, Smc1, Nbs1, critical for cell-cycle checkpoint activation, DNA repair and apoptosis (15). Although the MRN complex is a target of ATM kinase, at least two pieces of evidence have recently underscored the critical role of MRN in stimulating the catalytic activity of ATM. Studies with ATLD have shown that Mre11 deficiency compromises the radiation-induced autophosphorylation of ATM on serine 1981, as well as the phosphorylation of its downstream targets, and this defect can be corrected by wild-type MRE11 (16). In vitro biochemical studies have provided additional mechanistic insights into the functional role and requirement of the individual components of the MRN complex (e.g. Nbs1, Mre11) in the stimulation of ATM activity towards some substrates (17). This interdependent ATM/MRN relationship provides a mechanistic explanation for the phenotypic similarity between A-T and ATLD (16).

Here, we describe two siblings, ATLD5 and ATLD6, presenting with a slowly progressive neurological syndrome initially diagnosed as ataxia without Telangiectasia [A(–T)], who have germline mutations of the MRE11 gene. They were shown to be compound heterozygotes for mutations 1422C->A, resulting in base exchange T481K, and 1714C->T, which generates a stop signal at codon 571X. Interestingly, the paternally inherited mutant allele 1714C->T is subjected to nonsense-mediated mRNA decay (NMD), as detected in an unrelated English ATLD family (patients ATLD3 and ATLD4) with the same mutation (18). NMD is a cellular surveillance mechanism that, in most vertebrates, selectively degrades mRNAs with premature termination codons, reducing the amount of non-functional mRNA that would produce truncated proteins potentially exerting dominant negative effects (19,20). Cells from our patients show a number of phenotypic defects, including moderate chromosomal and clonogenic radiosensitivity, reduced expression and function of the MRN complex, attenuated ATM kinase activation and phosphorylation of some of its target; these features are more similar to those observed in ATLD4 than in ATLD2 cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Description of patients
The affected siblings (ATLD5, a 37-year-old man, and ATLD6, a 36-year-old woman) were born to non-consanguineous parents. Their father died of stroke at 61 years of age, whereas their 59-year-old mother is in good health. The maternal lineage included a grandmother who died of stroke, an aunt affected by deaf-mutism and an uncle affected by mild oligophrenia and parkinsonism. The paternal lineage included a grandmother and her two sisters who died of stroke and a grandfather who died of bladder carcinoma.

ATLD5, the elder son, was normal until the age of 3 years, but then developed progressive unsteadiness, and by 6 years of age showed diffuse cerebellar signs, i.e. ataxic gait, delayed speech and writing difficulties, choreoathetoid arm movements and oculomotor apraxia. The disease progressed slowly till the age of 14 and then stabilized. The latest neurological examination at 36 years of age showed cerebellar dysarthria, oculomotor apraxia, ataxic gait with unaided walk for few steps, choreoathethosis of the superior limbs, jerk nystagmus on horizontal and vertical gaze, dysmetria, dyskinetic movements of mouth and slight dystonia of the hands, diffuse hypotonia, reduced tendon reflexes in the arms, and absent ankle jerks with flexor plantar responses.

ATLD6, the younger sister, had a normal psychomotor development, but by 6 years of age had acquired unsteadiness and writing difficulties. The progression of the disease was similar to her brother and resulted in dystonic movements of the face and hands along with cerebellar ataxia, ocular apraxia and cerebellar dysarthria.

Laboratory findings, including immunoglobulins, {alpha}-fetoprotein, lysosomial enzymes, lipoproteins and vitamin-E levels, were in the normal range for both the patients; electromyography revealed a slight motor sensory neuropathy. Repeated CT scans performed in both the patients at various time intervals showed cerebellar atrophy with a moderate size increase of the fourth ventricle, subsequently confirmed by brain MRIs.

Identification of MRE11 mutations
The search for ATM and NBS1 gene mutations failed to reveal any alterations. However, sequence analysis of the cDNAs revealed an MRE11 missense mutation at codon 1442 (C->A; T481K) (Fig. 1), also detected in the maternal cDNA. As the expression of the inherited paternal allele was undetectable, the search for mutations was performed on the genomic DNA by sequencing each of the MRE11 exons. This led to the identification of a single C->T base change in exon 15, corresponding to nucleotide 1714 in the cDNA sequence, which introduces a premature stop codon (R571X) predicted to encode a prematurely truncated protein of 65 kDa. However, such product was not detected in western blots of cell lysates from both the patients, suggesting that the 1714C->T allele might undergo the NMD. This appeared to be the case, as the cDNA carrying the 1714C->T mutation was detected only after prevention of NMD by treatment of cells with anisomycin (Fig. 1). When examined by protein truncation test (PTT), this transcript encoded the expected truncated Mre11 protein of 65 kDa (data not shown). In summary, ATLD5 and ATLD6 are compound heterozygotes for MRE11 gene mutations (1422C->A, T481K; 1714C->T, R571X), and are null for the expression of the paternal allele because of NMD.



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  		Figure 1. Mutation analysis. (A) Electropherograms of MRE11 exon 15 from ATLD5, identifying the maternal 1442C->A and paternal 1714C->T alleles. The latter mutation can be seen in patient's genomic DNA, as well as in the cDNA derived from anisomycin-treated cells. (B) Schematic sequence of Mre11 protein (12) and position of residues that are affected by the mutations in ATLD cases. The vertical hatched lines indicate the DNA-binding regions a and b.

 
Radiosensitivity findings
Chromosomal breakage tests performed on peripheral blood lymphocytes from ATLD5 and ATLD6 showed an increased rate of radioinduced breaks (27 and 25 cells, respectively, with breaks on 50 cells examined), intermediate between normal (16±2) and A-T (34±5) cells. No clonal chromosomal abnormalities, like the t(7;4) translocation typical of A-T, were detected, and the rate of spontaneous chromosome breakage was within the normal range. Colony survival assays, performed on the LCLs established from ATLD5 and ATLD6, showed an increased sensitivity to 1 Gy of irradiation, relative to normal cells (Fig. 2). Nevertheless, their radiosensitivity was less pronounced compared with ATLD4 and ATLD2, the two previously reported cases with mild and severe clinical features, respectively (11).



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  		Figure 2. Radiosensitivity in ATLD cells. Colony survival assays performed on LCLs derived from a normal donor (LBC-N) and from patients with A-T and ATLD. Cells were irradiated with 1 Gy and cultured for 12 days and analysed for the presence of colonies after MTT staining. The values are means of three experiments.

 
Expression of the MRN complex proteins and ATM-dependent responses to radiation
Immunoblots were performed to determine the level of ATM, Mre11, Nbs1 and Rad50 proteins in our ATLD cases and also in comparison with the English cases. Both ATLD5 and ATLD6, like ATLD2 and ATLD4, expressed normal levels of ATM protein, a finding consistent with the failure to detect ATM mutations in these cases. Conversely, the levels of Mre11, as well as those of Rad50 and Nbs1, were significantly reduced in ATLD5 and ATLD6, as in ATLD4 (Fig. 3). ATLD2 appeared negative for Mre11 and Rad50, whereas it showed residual levels of Nbs1 protein (Fig. 3). Thus, the underlying genetic defect in ATLD5 and ATLD6 compromises the expression of the MRN complex components almost to the same extent as in ATLD4, but less severely than in ATLD2.



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  		Figure 3. Defective expression of Mre11, Rad 50 and Nbs1 proteins in ATLD. Whole cell extracts prepared from LCLs were tested in western blots with antibodies specific for ATM, Mre11, Rad50 and Nbs1 proteins. Blots were reprobed for ß-actin to normalize lanes for protein content.

 
The MRN complex is recruited, along with other recognition and repair proteins, at sites of DSBs, giving rise to nuclear foci (21). As MRE11 mutations can impair the function and localization of MRN complex (11), we determined the capacity of ATLD5 and ATLD6 to form nuclear foci. Compared with normal cells, in which the constitutively diffuse Mre11 nuclear fluorescence became localized in foci after irradiation, ATLD5 and ATLD6, besides showing a reduced Mre11 fluorescence, failed to form foci after irradiation, compatible with a defective MRN complex in these cells (Fig. 4).



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  		Figure 4. Mre11 nuclear foci. The immunofluorescence labelling for Mre11 was carried out 8 h after exposure of LCLs to 0 or 12 Gy of irradiation. Note the formation of radiation-induced Mre11 nuclear foci in LBC-N, but not in ATLD5.

 
As the DNA damage-induced ATM activity, as monitored by the autophosphorylation of S1981, is markedly attenuated in ATLD (16,22), we determined occurrence of this defect in our ATLD cases at 30 min postirradiation. Compared with normal cells, the ATM pS1981 signal detected in ATLD5 and ATLD6 was reduced by 50% after 0.25 Gy and 30–40% after 1 Gy (Fig. 5A), to almost the same extent as in ATLD4. The ATM pS1981 signal was even more attenuated in ATLD2 and undetectable in A-T. Thus, the activation of ATM in our cases is only partially impaired.



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  		Figure 5. ATM activation and phosphorylation of Chk2. Whole cell extracts prepared from the indicated LCLs or fibroblasts were tested on western blots with antibodies specific for ATM pS1981 and for total Chk2. (A) LCLs were harvested 30 min after treatment with 0, 0.25 and 1 Gy of IR. Note the difference in ATM autophosphorylation signal, particularly at 0.25 Gy, between LBC-N and ATLD cases. The lack of pS1981 signal in AT52RM cells verified the specificity of the antibody. Blots were tested with an anti-ATM antibody to verify the total amounts of ATM per lane. Chk2 phosphorylation was evaluated on LCLs (B) or primary fibroblasts (FB-ATLD5 and FB-ATLD6) (C) harvested 30 min and 3 h after 10 Gy of IR. Note the absence of a phosphorylation-related mobility delay in ATLD2, FB-ATLD5 and FB-ATLD6.

 
To determine the effect of this attenuated activity of ATM, we analysed the phosphorylation of its downstream targets Chk2, p53 and Smc1. The initial phosphorylation of Chk2 on Thr68 by ATM triggers additional phosphorylation steps that fully activate Chk2 (23). The phosphorylation of Chk2 is impaired in Nijmegen Breakage Syndrome (NBS) cells with defects of the MRN complex owing to inactivating mutations of NBS1 gene (24). In response to IR doses >1 Gy, ATLD5 and ATLD6 lymphoblastoid cells showed a similar phosphorylation-related Chk2 mobility delay as normal cells, and this delay was also seen in ATLD4, but not in ATLD2 or A-T cells (Fig. 5B). Strikingly, however, the Chk2 mobility delay in ATLD5 and ATLD6 appeared cell-type specific, as it was absent in the irradiated fibroblasts from these patients (Fig. 5C).

The phosphorylation of p53–Ser15 was analysed at 30 min post-IR, a time point when this event mostly reflects the activity of ATM, rather than ataxia-telangiectasia and Rad3 related (ATR) (25,26). To assess Ser15 phosphorylation independently of changes in p53 protein levels, prior to irradiation the cells were treated with the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) to allow the accumulation of basal p53 (26). The results (Fig. 6) showed an 8–9-fold increase in p53–pS15 levels in normal cells, and only 1.2-fold in A-T cells, relative to the untreated cell counterparts. In ATLD5 and ATLD6 cells, the phospho-Ser15 levels increased 5.2–5.8-fold, as in NBS cells, whereas in ATLD4 and ATLD2 it increased 3.1- and 2.3-fold, respectively. ATM phosphorylates Smc1–S966, and this event is required for the radiation-induced S-phase cell-cycle checkpoint arrest (27). Compared with untreated cells, the S966 phosphorylation signal in ATLD5 and ATLD6 at 30 min after irradiation was lower than in LBC-N (1.9-, 1.7- and 5.5-fold increase, respectively), but this difference was not seen at 60 min (Fig. 7A). However, ATLD2, and to a lesser extent ATLD4, showed a defective S966 phosphorylation at both time points (Fig. 7B). Flow cytometric cell-cycle analyses were performed to determine the outcome of the defective ATM-p53 response on G1 checkpoint arrest. At 24 h postirradiation, normal cells showed a slightly reduced G1/G2–M ratio that was compatible with a normal G1 checkpoint, whereas A-T cells showed a marked drop of G1-phase cells (thus causing an inversion of the G1/G2–M ratio), indicative of a failure to arrest in G1 (Fig. 8 and Table 1). Compatible with a defective G1 arrest, ATLD5 and ATLD6 cells showed an inversion of the G1/G2–M ratio (0.86±0.15 and 0.77±0.11, respectively). ATLD2, but not ATLD4, showed a similar defect (Fig. 8).



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  		Figure 6. Radiation-induced p53–S15 phosphorylation levels. Western blot analysis of p53 and p53–pSer15 performed on LCLs collected 30 min after exposure to 0 or 10 Gy of IR (top). The cells were preincubated with the proteasome inhibitor LLnL to allow the accumulation of basal p53. The histogram (bottom) displays the levels of p53–pS15, normalized for total p53, obtained by the densitometric analysis of the western blots.

 


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  		Figure 7. Radiation-induced Smc1–S966 phosphorylation. Western-blot analysis for phosphorylated Smc1–S966 and for total Smc1 performed on normal, A-T and ATLD-LCLs harvested 30 or 60 min after treatment with 0 or 10 Gy of IR.

 


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  		Figure 8. Flow cytofluorimetric analysis of cell-cycle phases changes after irradiation. Lymphoblastoid cells were harvested 24 h after exposure to 0 or 10 Gy IR, stained with propidium iodide and analysed by flow cytofluorimetry. The relatively high increase of the G2 peak in irradiated ATLD5 and ATLD6 is indicative of a G1 checkpoint arrest defect.

 

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  		Table 1. G1/G2–M ratio determined on LCLs by DNA flow cytofluorimetry, 24 h after exposure to 0 or 10 Gy of IR
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Inherited mutations of the MRE11 gene have been recently identified in four patients with ATLD, an A-T variant disorder presenting many of the clinical features of A-T, but often absence of telangiectasias, moderate radiosensitivity, later onset and slow progression of the disease, longer survival and no tumor recurrence (11). Among cases classified as A-T variants, carriers of ATR (28) and LIGASE IV (29) germline mutations have also been identified.

In this study, we describe a new family in which heterozygous compound mutations of the MRE11 gene (1422C->A, T481K; 1714C->T, R571X) have been identified in the affected brother and sister, now aged 37 and 36, who were initially diagnosed as A(–T) patients. Consistent with this diagnosis, these cancer-free patients did not exhibit ocular telangiectasias and presented with mild and slowly progressing neurological dysfunctions that manifested when they were 3 years old and stabilized when 14. Analysis of the samples from their parents showed that the 1422C->A allele, which causes a T->K aminoacid change at codon 481, was inherited from the mother, whereas the 1714C->T allele, which results in a premature stop at codon 571, was paternally inherited. Interestingly, the 1714C->T mutation could be only detected from the sequencing of genomic DNA, but not from cDNA, suggesting that this mutation destabilizes the transcript by NMD. This was indeed the case, as the cDNA with the 1714C->T mutation was recovered from cells treated with anysomycin, a drug that prevents mRNA decay. Worthnoting, the 1714C->T allele is the same identified in an unrelated English family (patients ATLD3 and ATLD4) (11), and also in these cases, the transcript is subjected to NMD (18). NMD is a surveillance mechanism which eliminates the errors in the biogenesis of mRNA (20,30,31). The decay of the transcripts containing premature termination codons prevents the expression of potentially deleterious truncated proteins, mitigating the clinical severity (19). MRE11 is an essential gene for survival and, although the six patients reported to date show variable degrees of clinical severity as well as different amount of Mre11 protein, the milder cases, albeit unrelated, share the same 1714C->T null mutation, suggesting that NMD plays a role in saving potentially lethal mutations.

The MRE11 mutations in our ATLD cases impaired not only the expression of Mre11, but also of Rad50 and Nbs1 proteins, to the same extent as in ATLD4, but not as much as in ATLD2, where these proteins were almost undetectable (11). Furthermore, these MRE11 mutations conferred partial radiosensitivity, as demonstrated by colony survival assays, and disrupted the activity of the MRN complex to form nuclear foci in response to radiation.

The MRN complex, besides being a target of ATM, is a direct inducer of ATM kinase. Accordingly, in Mre11-deficient ATLD cells, the autophosphorylation of ATM–S1981 was found markedly attenuated compared with normal cells, and this defect could be corrected by wild-type MRE11 (16). Biochemical studies have further substantiated the role of MRN in ATM activation (17). The attenuated activation of ATM in ATLD cells, resulting in a defective phosphorylation of ATM targets, actually provides an explanation for the phenotypic similarities between A-T and ATLD (16). The autophosphorylation of ATM–S1981 in irradiated ATLD5 and ATLD6 was attenuated, though not as markedly as in A-T or ATLD2 cells. This attenuation was associated with reduced phosphorylation of p53–S15, an event involved in p53 stabilization and transcriptional induction of its target gene p21waf1, a key inhibitor of the G1–S checkpoint (25). This defective phosphorylation of p53 correlated with failure of our ATLD cases to properly enforce a G1 arrest after irradiation. Chk2, a kinase involved in multiple cell-cycle checkpoints arrest after DNA damage (32), is targeted by ATM on T68, and this event triggers the hyperphosphorylation and full activation of Chk2. We have shown a normal hyperphosphorylation of Chk2 in LCLs from ATLD5 and ATLD6, indicating that ATM, despite its reduced activity, is able to activate Chk2. Interestingly, however, the hyperphosphorylation of Chk2 was impaired in the fibroblasts from these cases, suggesting that the efficiency of ATM or access to its substrates may somehow exhibit a tissue specificity.

The phosphorylation of Smc1–S966 by ATM is required for the radiation-induced S-phase cell-cycle checkpoint arrest (27). We have shown that the phosphorylation of this target of ATM is delayed in ATLD5 and ATLD6, suggesting that the attenuated activation of ATM has a limited effect on Smc1.

Chromosomal instability is a common genetic defect of A-T, NBS and ATLD (33), yet neither ATLD5 nor ATLD6 showed chromosomal translocations, in contrast to ATLD1/2 and ATLD3/4 in which these abnormalities were seen in 8 and 1% of blood lymphocytes, respectively (11). This result suggests that the genetic defect of the Italian cases is not so severe to impair chromosomal stability. Although a major outcome of genome instability is cancer, the lack of tumors among the English and Italian ATLD patients would suggest that Mre11 deficiency alone does not increase cancer predisposition. This conclusion, clearly precluded by the limited human cohort number, would be compatible with recent findings showing that Mre11ATLD1/ATLD1 mice exhibit chromosomal instability, but are not cancer prone (14).

In conclusion, we have identified two new ATLD siblings with heterozygous compound mutations of the MRE11 gene causing defective expression and function of the MRN complex, impairment of ATM activity and downstream signalling in response to IR. These findings lend further support to the interconnection between MRN and ATM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell lines and treatments
Lymphoblastoid cell lines (LCLs) were established by Epstien–Barr virus immortalization of peripheral blood lymphocytes from ATLD5 and ATLD6. Normal cells (LBC-N) were obtained from a normal donor, and A-T cells from patient AT52RM (34). The LCLs from ATLD2 and ATLD4 have been described (11). The NBS-derived cell line GM07078 was purchased from Coriell Cell Repository (Camden, NJ, USA). Skin biopsies from ATLD5 and ATLD6 were used to generate fibroblast cell lines. Cells were cultured at 37°C in a CO2 incubator using RPMI 1640 supplemented with 15% of heat inactivated fetal calf serum, 1% of glutamine and antibiotics.

Radiosensitivity analysis
Chromosome breakage analysis was performed on peripheral lymphocytes from patients and normal age- and sex-matched controls. Cells were irradiated in the G2-phase with 0.25 Gy delivered at a rate of 70 cGy/min with a Gilardoni MGL 300/6-D X-ray apparatus. After addition of colcemid and incubation for 90 min, cells were used to prepare chromosome spreads on slides. After Giemsa staining, chromatid and chromosome breaks, rings and dicentric chromosomes on 50 cells were scored by two operators for each treatment. GTG (G-banding with trypsin and Giemsa Staining) banding was performed to evaluate clonal rearrangements involving chromosomes 7 and 14. The colony survival assay and colony efficiency calculations were performed as reported (35), on LCLs seeded into 96-well tissue culture plates (100, 200 and 400 cells/well), irradiated with 0 or 1 Gy, cultured for 12 days and stained with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the colonies containing >32 cells were scored positive by optical microscopy. Experiments were performed in quadruplicate plates.

Western blot analysis
Cells were washed with phosphate-buffered saline (PBS) and 0.1 mM Na3VO4 (Sigma), pelleted and lysed in Laemli buffer [0.125 M Tris–HCl pH 6.8, 5% sodium dodecyl sulphate (SDS)] containing protease and phosphatase inhibitors. After boiling for 2 min and sonication, lysates were quantitated by Bradford assay. Aliquots containing 40 µg/ml of protein and 5% ß-mercaptoethanol were size-fractionated on 7–10% SDS–ployacrylamide gel electrophoresis (PAGE) and electroblotted onto PVDF membranes. After blocking with 5% non-fat dried milk in PBS and 0.1% Tween-20, membranes were incubated with monoclonal antibodies for ATM (clone 4D2, made in-house) and Chk2 (clone 44D4/21, made in-house) (36), and with rabbit antibodies specific for NBS1 and Mre11 (GeneTex, San Antonio, TX, USA), p53 phospho-S15 (Cell Signaling Technology, Beverly, MA, USA), ATM phospho-S1981 (Rockland Immunochemicals, PA, USA), Smc1 phospho-S966 (Bethyl Laboratories, Inc., Montgomery, TX, USA), total Smc1 (Bethyl Laboratories) and ß-actin (Sigma, Italy). Blots tested for ATM phospho-S1981 were reprobed with the monoclonal antibody MAT3-4G10/8 (37) to normalize for total amounts of ATM. Immunoreactive bands were visualized by ECL Super Signal (Pierce, Rockford, IL, USA) on autoradiographic films, scanned and quantitated by ImageQuant software (Molecular Dynamics).

PTT analysis and DNA sequencing
PTT analysis of Mre11 full-length protein was performed as reported (18), using TNT T7 Coupled Reticulocyte Lysate System (Promega). The cDNA was obtained by reverse transcription–polymerase chain reaction (RT–PCR) from lymphoblastoid cells, treated or untreated for 4 h with 0.1 mM anisomycin. The PCR conditions were 35 cycles of denaturation at 94°C for 10 s, annealing at 57°C for 30 s and extension at 68°C for 2 min using Expand Long template Taq polymerase (Roche). The in vitro translated products were separated on 8% SDS–PAGE and visualized by autoradiography.

The cDNA from anisomycin-treated cells, as well as genomic DNA, were sequenced for MRE11 gene as reported (18). Briefly, PCR products were purified and 50 ng sequenced in 20 µl reactions containing 2 µl ABI Prism Big Dye Terminator (PE biosystems), 6 µl buffer and 1 pmol primer. The PCR conditions were 25 cycles of 96°C, 10 s; 50°C, 5 s; 60°C, 4 min. Sequencing reactions were subsequently ethanol precipitated and then resuspended in formamide/dye (5 : 1) loading buffer. Samples were run on the ABI Prism 377 DNA sequencer according to ABI protocols.

Cell-cycle analysis
Radiation-induced cell-cycle phase modifications were examined by flow cytofluorimetry on propidium iodide stained cells (34) using a FACSCalibur instrument fitted with a Cell Quest software package (Becton Dickinson, Sunnyvale, CA, USA).

IR-induced foci
Unirradiated cells or irradiated with 12 Gy were incubated for 8 h, cytocentrifuged onto glass slides and air-dried. After fixation with 4% formaldehyde for 10 min, slides were washed twice in PBS, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 10% normal goat serum and incubated overnight with 1 : 100 dilution of a rabbit antibody to human Mre11 (GeneTex). Following washing and incubation for 30 min with 1 : 50 dilution of fluorescein-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA), slides were washed again and counterstained with Hoechst 33258. Foci were analysed with an Axioskop 2 Plus fluorescence microscope equipped with an AxioCam digital camera (Zeiss, Germany). At least 100 nuclei were scored for each preparation, and were considered positive if contained at least five distinct foci.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Professor A.M. Taylor, The University of Birmingham, Cancer Research, UK and Institute for Cancer Studies, the Medical School Edgbaston, Birmingham, UK for providing the ATLD2 and ATLD4 cells, and for sharing preliminary results. Professor Yossi Shiloh, Department of Human Genetics and Molecular Medicine Sackler School of Medicine, Tel Aviv University, kindly provided the anti-ATM monoclonal antibody. This work was financed by grants of the Italian Telethon Foundation (grants E764 and GP0205Y01), Italian Association for Cancer Research (AIRC), Consiglio Nazionale Ricerche (CNR grant CU03.00416), the Italian Ministries of Health and of University and Research (FIRB grant RBNE01RNN7).


   FOOTNOTES
 
* To whom correspondence should be addressed at: II Faculty of Medicine, A.O.S. Andrea, Via di Grottarossa 1035, I-00189 Roma, Italy. Tel/Fax: +39 0680345258; Email: luciana.chessa{at}uniroma1.it


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Fiorilli, M., Antonelli, A., Russo, G., Crescenzi, M., Carbonari, M. and Petrinelli, P. (1985) Variant of ataxia-telangiectasia with low-level radiosensitivity. Hum. Genet., 70, 274–277.[CrossRef][ISI][Medline]

  2. Taylor, A.M.R., Flude, E., Laher, B., Stacey, M., McKay, E., Watt, J., Green, S.H. and Harding A.E. (1987) Variant forms of ataxia telangiectasia. J. Med. Genet., 24, 669–677.[Abstract]

  3. Chessa, L., Petrinelli, P., Antonelli, A., Fiorilli, M., Elli, R., Marcucci, L., Federico, A. and Gandini E. (1992) Heterogeneity in ataxia telengiectasia: classical phenotype associated with low cellular radiosensitivity. Am. J. Med. Genet., 42, 741–746.[CrossRef][ISI][Medline]

  4. Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, D.A., Smith, S., Uziel, T., Sfez, S. et al. (1995) A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science, 268, 1749–1753.[Abstract/Free Full Text]

  5. Gilad, S., Khosravi, R., Shkedy, D., Uziel, T., Ziv, Y., Savitsky, K., Rotman, G., Smith, S., Chessa, L., Jorgensen, T.J. et al. (1996) Predominance of null mutations in ataxia-telangiectasia. Hum. Mol. Genet., 5, 433–439.[Abstract/Free Full Text]

  6. Vorechovsky, I., Luo, L., Prudente, S., Chessa, L., Russo, G., Kanariou, M., James, M., Negrini, M., Webster, D.B. and Hammarstrom, L. (1996) Exon scanning mutation analysis of the ATM gene in patients with ataxia-telangiectasia. Eur. J. Hum. Genet., 4, 352–355.[ISI][Medline]

  7. Brown, K.D., Ziv, Y., Sadanandan, S.N., Chessa, L., Collins, F.S., Shiloh, Y. and Tagle, D.A. (1997) The ataxia-telangiectasia gene product, a constitutively expressed nuclear protein that is not upregulated following genome damage. Proc. Natl Acad. Sci. USA, 94, 1840–1845.[Abstract/Free Full Text]

  8. Teletar, M., Teraoka, S., Chun, H.H., Wang, Z., Liang, T., Castelvi-Bel, S., Borresen-Dahle, A.-L., Chessa, L., Bernatowska-Matuszkiewicz, E., Porras, O. et al. (1998) Ataxia-telangiectasia: identification and detection of founder-effect mutations in ethnic populations. Am. J. Hum. Genet., 62, 86–97.[CrossRef][ISI][Medline]

  9. Teraoka, S.N., Telatar, M., Becker-Catania, S., Liang, T., Onengut, S., Tolun, A., Chessa, L., Sanal, O., Bernatowska, E., Gatti, R.A. and Concannon, P. (1999) Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and phenotypic consequences. Am. J. Hum. Genet., 64, 1617–1631.[CrossRef][ISI][Medline]

  10. Gilad, S., Chessa, L., Khosravi, R., Russel, P., Galanty, Y., Piane, M., Gatti, R.A., Jorgensen, T.J., Shiloh, Y. and Bar-Shira, A. (1998) Genotype-phenotype relationships in ataxia-telangiectasia (A-T) and A-T variants. Am. J. Hum. Genet., 62, 551–561.[CrossRef][ISI][Medline]

  11. Stewart, G.S., Maser, R.S., Stankovic, T., Bressan, D.A., Kaplan, M.I., Jaspers, N.G.J., Raams, A., Byrd, P.J., Petrini, J.H.J. and Taylor, A.M.R. (1999) The DNA double-strand break gene hMre11 is mutated in individuals with ataxia-telangiectasia-like disorder. Cell, 99, 577–587.[CrossRef][ISI][Medline]

  12. D'Amours, D. and Jackson S.P. (2002) The Mre11 complex: at the crossroads of DNA repair and checkpoint signalling. Nat. Rev. Mol. Cell. Biol., 3, 317–327.[CrossRef][ISI][Medline]

  13. Petrini, J.H. and Stracker, T.H. (2003) The cellular response to DNA double-strand breaks: defining the sensors and mediators. Trends Cell Biol., 13, 458–462.[CrossRef][ISI][Medline]

  14. Theunissen, J.W., Kaplan, M.I., Hunt, P.A., Williams, B.R., Ferguson, D.O., Alt, F.W. and Petrini, J.H. (2003) Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11 (ATLD1/ATLD1) mice. Mol. Cell, 12, 1511–1523.[CrossRef][ISI][Medline]

  15. Shiloh, Y. (2003) ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer, 3, 155–168.[CrossRef][ISI][Medline]

  16. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L. and Shiloh, Y. (2003) Requirement of the MRN complex for ATM activation by DNA damage. EMBO J., 22, 5612–5621.[CrossRef][ISI][Medline]

  17. Lee, J.H. and Paull, T.T. (2004). Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science, 304, 93–96.[Abstract/Free Full Text]

  18. Pitts, S.A., Kullar, H.S., Stankovic, T., Stewart, G.S., Last, J.I., Bedenham, T., Armstrong, S.J., Piane, M., Chessa, L., Taylor, A.M. and Byrd, P.J. (2001) Hmre11: genomic structure and a null mutation identified in a transcript protected from nonsense-mediated mRNA decay. Hum. Mol. Genet., 10, 1155–1162.[Abstract/Free Full Text]

  19. Bateman, J.F., Freddi, S., Nattrass, G. and Savarirayan, R. (2003) Tissue-specific RNA surveillance? Nonsense-mediated mRNA decay causes collagen X haploinsufficiency in Schmid metaphyseal chondrodysplasia cartilage. Hum. Mol. Genet., 12, 217–225.[Abstract/Free Full Text]

  20. Lewis, B.P., Green, R.E. and Brenner, S.E. (2003) Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl Acad. Sci. USA, 100, 189–192.[Abstract/Free Full Text]

  21. Mirzoeva, O.K. and Petrini, J.H. (2001) DNA damage-dependent nuclear dynamics of the Mre11 complex. Mol. Cell. Biol., 21, 281–288.[Abstract/Free Full Text]

  22. Carson, C.T., Schwartz, R.A., Stracker, T.H., Lilley, C.E., Lee, D.V. and Weitzman, M.D. (2003) The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J., 22, 6610–6620.[CrossRef][ISI][Medline]

  23. Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K. and Elledge, S.J. (2000) Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc. Natl Acad. Sci. USA, 97, 10389–10394.[Abstract/Free Full Text]

  24. Buscemi, G., Savio, C., Zannini, L., Micciche, F., Masnada, D., Nakanishi, M., Tauchi, H., Komatsu, K., Mizutani, S., Khanna, K. et al. (2001) Chk2 activation dependence on Nbs1 after DNA-damage. Mol. Cell. Biol., 21, 5214–5222.[Abstract/Free Full Text]

  25. Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C.W., Chessa, L., Smorodinsky, N.I., Prives, C., Reiss, Y., Shiloh, Y. and Ziv, Y. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science, 281, 1674–1677.[Abstract/Free Full Text]

  26. Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M.B. and Siliciano, J.D. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science, 281, 1677–1679.[Abstract/Free Full Text]

  27. Kim, S.T., Xu, B., Kastan, M.B. (2002) Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev., 16, 560–570.[Abstract/Free Full Text]

  28. O'Driscoll, M., Ruiz-Perez, V.L., Woods, C.G., Jeggo, P.A. and Goodship, J.A. (2003) A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat. Genet., 33, 497–501.[CrossRef][ISI][Medline]

  29. Maraschio, P., Spadoni, E., Tanzarella, C., Antoccia, A., Di Masi, A., Maghnie, M., Varon, R., Demuth, I., Tiepolo, L. and Danesino, C. (2003) Genetic heterogeneity for a Nijmegen breakage-like syndrome. Clin. Genet., 63, 283–290.[CrossRef][ISI][Medline]

  30. Fischmeyer, P.A., van Hoof, A., O'Donnell, K., Guerrerio, A.L., Parker, R. and Dietz, H.C. (2002) An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science, 295, 2258–2261.[Abstract/Free Full Text]

  31. Maquat, L.E. (2002) Skiing toward nonstop mRNA decay. Science, 295, 2221–2222.[Abstract/Free Full Text]

  32. Bartek, J., Falck, J. and Lukas, J. (2001) CHK2 kinase—a busy messenger. Nat. Rev. Mol. Cell. Biol., 2, 877–886.[CrossRef][ISI][Medline]

  33. Taylor, A.M. (2001) Chromosome instability syndromes. Best Pract. Res. Clin. Haematol., 14, 631–644.[Medline]

  34. Delia, D., Mizutani, S., Tagliabue, E., Fontanella, E., Asada, M., Yamada, T., Taya, Y., Prudente, S., Saviozzi, S., Frati, L. et al. (2000) ATM protein and p53 serine 15 phosphorylation in ataxia telangiectasia (AT) patients and AT heterozygotes. Br. J. Cancer, 82, 1938–1945.[CrossRef][ISI][Medline]

  35. Sun, X., Becker-Catania, S., Chun, H.H., Hwang, M.J., Huo, Y., Wang, Z., Mitui, M., Sanal, O., Chessa, L., Crandall, B. and Gatti, R.A. (2002) Early diagnosis of ataxia-telangiectasia using radiosensitivity testing. J. Pediatr., 140, 724–731.[CrossRef][ISI][Medline]

  36. Zannini, L., Lecis, D., Lisanti, S., Benetti, R., Buscemi. G., Schneider, C. and Delia, D. (2003) Karyopherin-alpha2 protein interacts with Chk2 and contributes to its nuclear import. J. Biol. Chem., 278, 42346–42351.[Abstract/Free Full Text]

  37. Andegeko, Y., Moyal, L., Mittelman, L., Tsarfaty, I., Shiloh, Y. and Rotman, G. (2001) Nuclear retention of ATM at sites of DNA double strand breaks. J. Biol. Chem., 276, 38224–38230.[Abstract/Free Full Text]


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