Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia‐Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia‐Telangiectasia‐like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild‐type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair‐deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11–NBS1–RAD50 complex, an impaired S‐phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation‐induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair‐defective cells and suggest its impairment may contribute to the development of colorectal cancer.
Ataxia‐Telangiectasia (A‐T) and the Nijmegen Breakage Syndrome (NBS) are rare autosomal recessive diseases that are due to mutation of the ATM and NBS1 genes, respectively. They are characterized by hypersensitivity to ionizing radiation, immunodeficiency and an increased predisposition to cancer (Shiloh, 1997). A milder form of A‐T, the Ataxia‐Telangiectasia‐like disorder (AT‐LD) has been linked recently to mutation of the human MRE11 gene (Stewart et al., 1999). Although clinical differences exist amongst these three syndromes, the striking similarity of the cellular phenotypes of A‐T, NBS and AT‐LD (Shiloh, 1997; Petrini, 2000) has lead to the hypothesis that the gene products ATM, NBS1 and MRE11 work in the same pathway. Indeed, in response to DNA damage induced by ionizing radiation, the ATM kinase phosphorylates NBS1 on serine 343, an event required for the activation of the S‐phase checkpoint (Gatei et al., 2000; Lim et al., 2000) and the relocalization of the MRE11–NBS1–RAD50 (M–N–R) complex to ionizing radiation‐induced nuclear foci (IRIF), which represent sites at which DNA is undergoing repair (Nelms et al., 1998). As is clearly shown in yeast, the M–N–R complex, involving the NBS1 homologue Xrs2, participates in the DNA double‐strand break (DSB) repair via the homologous recombination and the non‐homologous end‐joining pathways (Haber, 1998; Khanna and Jackson, 2001).
Mutations in ATM are firmly implicated in cancer (Shiloh, 1997; Stankovic et al., 1999; Schaffner et al., 2000), and NBS patients display an increased risk for lymphoproliferative disorders (Shiloh, 1997). Although MRE11 mutations are also suspected to predispose to cancer via the inactivation of the M–N–R complex, this conclusion has been hampered by the cell‐lethal phenotype of MRE11 gene knockout (Xiao and Weaver, 1997) and the paucity of AT‐LD families described so far (Petrini, 2000). Therefore, a direct link between MRE11 mutations and cancer has not been established yet. Here we report the identification of a splicing defect of the MRE11 precursor transcript linked to mutations of a poly(T)11 repeat within intervening sequence 4 (IVS‐4). This mutation was found exclusively in mismatch repair (MMR)‐deficient cancer cell lines and primary tumours, and is associated with a defective S‐phase checkpoint and abrogation of MRE11 and NBS1 IRIF formation.
Results and Discussion
To search for MRE11 mutations, we PCR amplified the MRE11 transcript from a panel of cancer cell lines. An exponential phase amplification from SW48, LoVo, HCT116, MIP and LS174T colorectal cancer (CRC) cell lines produced extremely reduced levels of the 5′‐fragment compared with other CRC cell lines (Figure 1A). The appearance of a second product suggested the presence of a second transcript in these cells (Figure 1B). Direct sequencing of the PCR products identified the higher molecular weight band as the wild‐type (WT) MRE11 cDNA fragment (Figure 1C). The faster migrating band (defined as 484del88) contained a complete deletion of exon 5 (Figure 1D). The derived transcript is predicted to generate a truncated protein comprising the first 105 (of the 708) amino acids of the WT MRE11 followed shortly by a stop codon (Figure 1D). The skipping of exon 5 might be due to mutation of the splicing signal sequences. We noticed that the polypyrimidine tract of the 3′‐splice site of the human MRE11 IVS‐4 is composed of 11 Ts (Figure 2A). Intriguingly, we found the aberrant 484del88 MRE11 transcript in MMR‐deficient cell lines selectively; in contrast, the MMR‐proficient cells only expressed high levels of WT MRE11 mRNA (Figure 1A and B; Table 1). Therefore, we tested whether the partial splicing defect leading to the generation of the 484del88 transcript was due to alterations at the IVS‐4 poly(T) tract. All the MMR‐proficient cells were homozygous for the WT poly(T)11 allele (Figure 2B; Table 1; data not shown). In contrast, we found deletions of one or two bases involving both MRE11 alleles in LoVo, SW48, HCT116, LS174T and MIP. In agreement with the observation that shortening the length of a stretch of 11 continuous uridines reduces its strength as a signal sequence in splicing competition assays (Coolidge et al., 1997), these data suggest that the shortening of the MRE11 poly(T) tract strongly reduces, but does not abolish completely, the correct splicing process. The occurrence of alterations in this repeat and the appearance of the 484del88 transcript was not restricted to CRC cells; rather they appeared to be associated with a MMR‐deficient phenotype, since they were also present in MMR‐deficient prostate cancer Du145 and endometrial carcinoma AN3CA cells (Figure 1B; data not shown). To date, we have found no evidence for a mutation of this type in a number of MMR‐proficient cancer cell lines (data not shown).
We also observed mutations at the (T)11 repeat in primary CRCs (Table 2). Shortened (T)9 and/or (T)10 alleles were detected in 93% (14 out of 15) of the MMR‐deficient colon cancers analysed. In >50% (eight out of 15) of the cases, the WT allele was absent or under‐represented compared with the mutant alleles, indicating that the tumour may be homozygous for the alteration (Figure 3; Table 2). In contrast, MRE11 mutations were never found in non‐cancer colonic mucosa surrounding the MMR‐deficient CRCs, in MMR‐proficient CRCs or peripheral blood (Figure 3; Table 2). Therefore, this newly described mutation is highly associated with MMR‐deficient CRC in vivo. Furthermore, while we readily PCR amplified the WT MRE11 transcript in three out of three MMR‐proficient primary CRCs, we failed to detect its expression in three out of three MMR‐deficient CRC cases (Figure 3B). In these samples, the expression of the 484del88 transcript and some WT MRE11 mRNA could only be revealed by a nested RT–PCR (data not shown). This suggests that the ratio between the WT and aberrant 484del88 transcript detected by PCR in MMR‐defective cells could be affected by both impairment of WT splicing and selective degradation of the 484del88 transcript. In fact, an aberrant MRE11 transcript was found recently to be degraded by nonsense‐mediated decay (NMD), a process requiring active translation (Maquat, 2000; Pitts et al., 2001). To test whether 484del88 is also degraded through NMD, we studied the effects of cycloheximide on the expression of MRE11 transcripts. Supporting our hypothesis, cycloheximide induced a consistent reduction in the ratio between the WT and aberrantly spliced transcript in SW48, HCT116 and LoVo cells compared with untreated controls (Figure 3C). To prove finally that the expression of the WT MRE11 is impaired by the described mutation at the MRE11 IVS‐4 splicing site, we analysed MRE11 expression by northern and western blotting. We observed a strongly reduced expression of MRE11 at either the mRNA (Figure 3D) or protein (Figure 4A) level in MMR‐deficient cells compared with the MMR‐proficient CRC cells lines and the normal colonic mucosa cell line Int407.
In AT‐LD cell lines, the expression of MRE11 mutant transcripts is associated with reduced expression of the three proteins of the M–N–R complex, impaired ionizing radiation‐induced S‐phase checkpoint [measured as radio‐resistant DNA synthesis (RDS)] and defective IRIF formation (Stewart et al., 1999). To explore the phenotypic consequences of the newly described MRE11 mutation, we also analysed the levels of NBS1 and RAD50 proteins in all CRC cells. Intriguingly, the expression of the p95NBS1 and RAD50 proteins was reduced severely in all MMR‐deficient cells also (Figure 4A). In contrast, the levels of all three M–N–R proteins were similar to those of the untransformed colonic mucosa Int407 and lymphoblastoid cell lines in all MMR‐proficient CRC cells (Figure 4A; data not shown). Among them, only HT29 provided some evidence of reduced expression of p95NBS1 (Figure 4A). Although the molecular base of this finding is still unclear, the M–N–R complex is functional in these cells (see below). This is consistent with the absence of a detectable phenotype in NBS heterozygous carriers, although their cells show reduced p95NBS1 levels (Shiloh, 1997).
We then assessed the integrity of the S‐phase checkpoint in the CRC cell lines. Following exposure of cells to 10 or 20 Gy γ rays, DNA synthesis was reduced markedly in MMR‐proficient cells (Figure 4B) expressing high levels of M–N–R complex proteins. In sharp contrast, the MMR‐defective cells expressing minimal levels of M–N–R complex proteins, such as the LS174T, LoVo and MIP cell lines (Figure 4A), were significantly more resistant to DNA synthesis inhibition (Figure 4B) and they still showed between 69 and 75% of DNA synthesis after irradiation with 10 Gy, and between 58 and 65% after 20 Gy, compared with unirradiated controls. Thus, they displayed an RDS phenotype similar to AT‐LD cell lines, whose DNA synthesis was reported to be reduced to only 70–80% and 55–65% after exposure to 10 and 20 Gy, respectively (Stewart et al., 1999). Furthermore, following radiation, MRE11 and NBS1 IRIFs were observed in the majority of WIDR, HT29 and Int407 cells (Figure 4C; data not shown). In contrast, there was no significant formation of IRIFs in MIP, LoVo, LS174T and HCT116 cells (Figure 4C; data not shown). These results indicate that at least two known functions of the M–N–R complex are defective in mutated MRE11, but not WT CRC, cell lines.
Mutations of mononucleotide repeats within coding sequences are frequent in MMR‐deficient cells (Aaltonen et al., 1993; Ionov et al., 1993; Thibodeau et al., 1993), and they contribute to carcinogenesis through the inactivation of cancer‐related genes, including TGFβ‐RII, IGF‐IIR and Bax. In this paper, we have shown that mutations of an intronic repeat in the human MRE11 gene result in aberrant splicing, giving rise to the unstable 484del88 transcript and an associated reduced expression of all components of the M–N–R complex. Examples of MRE11 mutations have been reported in breast and hematological malignancies (Fukuda et al., 2001). However, their functional consequences had not been investigated. In contrast, we showed that CRC cells expressing the 484del88 aberrant MRE11 transcript are defective in the radiation‐induced S‐phase checkpoint and IRIF relocalization. The occurrence of a monoallelic inactivation of RAD50 in ∼30% of MMR‐deficient CRC cell lines and primary tumours (Tables 1 and 2; Kim et al., 2001) might add a further contribution to the inactivation of the M–N–R complex, although this aspect needs to be investigated further. However, the MRE11 alteration we described appears to be sufficient to impair both the S‐phase checkpoint and M–N–R complex relocalization to IRIF in the MMR‐defective MIP cell line, where we failed to detect mutations in the RAD50 (A)9 repeat and NBS1 gene (Table 1; data not shown). Inactivation of other components of the S‐phase checkpoint, such as ATM, BRCA1 and CHK2, is associated with cancer (Bell et al., 1999; Falck et al., 2001; Xu et al., 2001). Intriguingly, the M–N–R complex is likely to work in the very early events of the radiation response, since its activity is required for CHK kinase activation and RAD9 phosphorylation in yeast (Grenon et al., 2001). The M–N–R complex also plays a role in DNA repair and M–N–R relocalization to IRIF (the sites of DNA damage) is thought to reflect this (Nelms et al., 1998). It is likely that abrogation of either of these key protective mechanisms will result in further genome instability. Indeed, it is a common observation that in addition to the microsatellite instability causing subtle but diffuse genetic alterations, MMR‐defective tumour cells show an increased degree of chromosomal aberration compared with non‐neoplastic cells, but not the gross aneuploidy observed in ‘chromosomally instable’ tumours bearing mutations in the APC or mitotic checkpoint genes. This mild form of chromosomal instability is compatible with a DNA DSB repair defect. Finally, since the M–N–R complex is also involved in the maintenance of telomere integrity, loss or impairment of additional functions exerted by this complex might provide a further advantage to cancer cells.
Our data implicate human MRE11 in cancer development and indicate that it is a major target for inactivation in MMR‐deficient CRC and other cells. Together with the identification of other mutations affecting this complex (Fukuda et al., 2001; Kim et al., 2001; Varon et al., 2001; and the present paper), they also suggest that inactivation of the M–N–R complex is likely to be a frequent event in sporadic cancer.
RT–PCR amplification of the 1235 bp 5′‐fragment of MRE11 from CRC cell lines was performed on total RNA (1 μg) using the Gene Amp Gold RNA PCR Core kit (PE‐Applied Biosystems, Warrington, UK). An aliquot (5 μl) of the RT reaction was subjected to 20–35 PCR cycles, with the MRE11‐specific primers MRE11‐19233 (5′‐GAGTGCATTTTCTGACATTTGAGTAC‐3′) and MRE11‐H (5′‐CTGGCTAAAGCGAAGAACAC‐3′). The DNA fragment for the analysis of the IVS‐4 (T)11 repeat was PCR amplified from genomic DNA (200 ng) through 35 PCR cycles with the primers MRE11‐100F (5′‐AATATTTTGGAGGAGAATCTTAGGG‐3′) and MRE11‐L (5′‐AATTGAAATGTTGAGGTTGCC‐3′). Purified fragments were directly sequenced by an ABI PRISM 377 DNA Sequencer (PE‐Applied Biosystems).
Northern blot analysis was performed on total RNA (20 μg) blotted to nylon membranes (Gene Screen Plus, NEN Life Science Product, Boston, MA) and hybridized to randomly primed cDNA probes.
The expression of MRE11, NBS1 and RAD50 proteins was evaluated by western blotting. Each protein lysate (40 μg) was separated on 8% SDS–PAGE gels, blotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) and probed with rabbit anti‐p95NBS1, anti‐MRE11, anti‐RAD50 and anti‐tubulin (Novus Biologicals, Littleton, CO) polyclonal antibodies. Immunoreactive bands were visualized by enhanced chemoluminescence (Pierce Chemical Co., Rockford, IL).
For thymidine incorporation assays, cells were seeded at 2 × 104 cells/well in 96‐well plates and, after 18–24 h, were γ‐irradiated at 3 Gy/min using a 60Co source at room temperature, labelled with 1 μCi/well [3H]thymidine and incubated for 4 h under standard conditions. Cells were then harvested and counted in a Top Count NST scintillation counter (Packard Instruments, Downers Grove, IL).
For estimation of IRIF formation, after γ‐irradiation with 12 Gy, cells were recovered in fresh complete medium for 8 h followed by fixation and permeabilization in 4% paraformaldehyde with 0.25 Triton X‐100 or prepared as described recently (Mirzoeva and Petrini, 2001). After blocking in 10% normal goat serum (NGS), cells were then washed and incubated with rabbit anti‐p95NBS1 or anti‐MRE11 anti‐sera, and diluted 1/600 and 1/300 in 3% NGS, respectively. After washing, dishes were incubated with FITC‐conjugated goat anti‐rabbit secondary antibody (Cappel, distributed by ICN, Costa Mesa, CA) at 1/50 dilution in 3% NGS for 30 min.
This work was partially supported by grants from the Italian Association for Cancer Research, the Ministries of Health and Research and University, the National Research Council Biotechnology and Oncology Projects, the Neuroblastoma Association, the MURST‐CNR ‘Biomolecole per la Salute Umana’ Program and the Pasteur Institute Cenci‐Bolognetti Foundation.
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