Cancer is a progressive genetic disease characterized by progressive accumulation of mutations in both coding and non-coding sequences 1. Simple sequence repeats or microsatellites are highly unstable in some human neoplasms, identifying a class of tumors with the microsatellite mutator phenotype (MMP+) (for review see 2). MMP+ve status is defined by instability in more than 30% of analised microsatellites, including at least one mononucleotide (such as BAT26 or BAT25) 3. Microsatellite instability (MSI) has been described as a frequent genetic alteration in various human solid tumors including sporadic and familial gastric carcinomas (GCs). In almost all patients with hereditary nonpolyposis colorectal cancer (HNPCC), molecular studies have shown that MSI is caused by germline mutations in genes encoding proteins required for DNA mismatch repair (MMR) 4. Genes such as hMSH2, hMLH1, PMS1, PMS2 and hMSH6/GTBP5678, were defined as "caretakers" because, when disrupted, they favor the acquisition of mutations at high frequency in several genes including oncogenes and tumor suppressor genes 9. However, in a significant fraction of sporadic MMP+ tumors, mutation analysis has not allowed the identification of the genes responsible for MSI 210, suggesting that, in such cases, MSI might be caused by epigenetic mechanisms. In fact, gene silencing by promoter hypermethylation was recently shown for the hMLH1 gene 111213.

The significance of MSI in tumorigenesis has been supported by the demonstration that the genetic instability characterizing MMP+ gastrointestinal cancers may target short repetitive tracts within the coding region of genes important for cell survival and proliferation. Insertion/deletion mutations were found in the genes for the transforming growth factor-beta type II receptor (TGFβ1-RII), the insulin-like growth factor type II receptor (IGFIIR), the DNA mismatch-repair genes hMSH3 and hMSH6, the proapoptotic gene BAX and the transcription factor E2F-41415161718. In all the cases, with the exception of the E2F-4, the nucleotides insertion/deletion resulted in frameshift mutations and protein truncation. Although the biological significance of these alterations is not fully established, it was proposed that the absence of TGFβ1-RII receptors from the surface of gastrointestinal epithelial cells could eliminate the negative growth control of TGF-β, leading to uncontrolled cell growth 19. IGFIIR has a role in growth suppression by binding and activating the TGFβ1 receptor complex and by mediating internalization, with subsequent degradation, of the growth factor IGF-II 2021. BAX promotes apoptosis and it was reported that BAX inactivation leads to rapid tumor growth 22. The human genes hMSH3 and hMSH6 are homologous to the bacterial MutS gene whose products bind DNA mismatches to initiate strand-specific repair of DNA replication errors 23. Somatic and germline mutations in hMSH6 were associated with sporadic MMP+ colorectal carcinomas and HNPCC 6.

We have found previously that the BLM gene may also be a target of MSI 24. The frameshifts, found in a short poly-adenine repeat, were predicted to generate a truncated and non-functional BLM protein. Homozygous or compound heterozygous mutations at the BLM gene were shown to cause the Bloom syndrome (BS; OMIM 210900), a pre-malignant condition characterized by chromosomal instability and increased mutational rate. BS patients are predisposed to a wide variety of neoplams diagnosed at early age. Noteworthy, among 100 neoplasms recorded in the BS registry, 19 were gastrointestinal (esophagus, stomach and colon) and only two were diagnosed after the age of 40 25. These data indicate that mutations in the BLM gene can promote tumorigenesis.

To better define the role of BLM in gastrointestinal tumorigenesis we screened a panel of MMP+ and MMP- gastric carcinomas (GCs) for: a) frameshifts in the BLM coding region microsatellite and b) for possible associations with other intragenic frameshifts.

Several studies have documented that alternative pathways may exist in gastrointestinal MMP+ and MMP- cancers characterized by different sets of altered genes 40. A gradual model of mutator mutations ("the mutator that mutates the other mutator") was proposed to define the mutator versus the suppressor pathways in gastrointestinal tumors 41. By analyzing the timing of mutational events in genetically unstable GCs, it was proposed that the first targets of mismatch repair deficiency are the mononucleotide tracts of TGFβ1-RII and BAX. The frameshift mutations of hMSH3 and hMSH6 appear to be secondary mismatch repair lesions, which generate mutations in IGFIIR42. The finding that frameshift mutations at TGFβ1-RII are the most frequent and the IGFIIR are the less frequent in gastric MMP+ tumors supports to the model in which early events should be present in the majority of tumors and late mutations only in a minor fraction 43. This finding is also confirmed in our study (Table 2). BLM frameshift mutations are associated more frequently with first and second step alterations proposed in this model, suggesting that they are secondary to mismatch repair defects (in hMSH3 and/or hMSH6) present in cells which do not undergo apoptosis (possibly in consequence of BAX inactivation). Noteworthy, in case 3P, in addition to BLM, the only frameshifts were found in BAX (see figure 1) and p53 (data not shown) genes, suggesting that, in some instances, BLM alteration may occur without hMSH3 and/or hMSH6 mutations, in cells with abnormal p53-mediated pathway of apoptotic response to DNA mismatch.

The involvement of BLM in tumorigenesis outside BS patients seems conceivable, since this gene appears to act as a caretaker. BLM mutations are known to cause an increased genomic instability characterized by an elevated number of chromosomal breaks, gaps and rearrangements and by an excessive number of mutations in both coding and noncoding regions, likely originated by unequal sister-chromatid exchange, characteristic feature of the Bloom syndrome 4445. Accordingly, BS exhibits a combination of genomic instability and elevated cancer risk, an association found also in other diseases caused by defects in caretaker genes (such as HNPCC, WS, ataxia-telangiectasia and xeroderma pigmentosum). Similar to the mutations found in BS, the frameshifts described in GCs abolish the helicase function of the BLM protein 24. BLM protein has a proven DNA unwinding activity 46 and could be involved in processes that are disturbed in malignant cells, such as DNA replication, recombination, chromosome segregation, DNA repair and transcription. Indeed, the fission yeast BLM gene homologue (radl2⁺/rqhl⁺) was shown to regulate the S-phase checkpoint and was proposed to couple chromosomal integrity with cell cycle progression 4748. However, the finding of BLM mutations in MMP+ tumors generates a paradox: BLM mutations are predicted to generate chromosomal instability while most of the MMP+ tumors are diploid. Nevertheless, some MMP+ tumors are aneuploid and BLM loss-of-function mutations may have pleiotropic consequences, possibly affecting also the microsatellite instability pathway, as suggested by the description of an increased intra-gene mutations in Bloom syndrome 4445. In suppoprt to our suggestion is the finding that we have found that BLM is mutated in LoVo cell line, a colon cancer cell line with both microsatellite and chromosomal inatability 43. Much more, it was recently reported that SGS1 gene, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination and is redundant with DNA mismatch repair (MMR) for suppressing gross chromosomal rearrangements and for suppressing recombination between divergent DNA sequences 49.

Our results indicate that BLM frameshifts are frequent alterations in GCs and are specifically associated with MMP+ tumors displaying at least 2 unstable microsatellites. Mutations in BLM appear to be frequently associated with frameshifts in BAX and in hMSH6 and/or hMSH3, and tumors with BLM alterations present a number of unstable mono- and trinucleotide repeats located in coding regions significantly higher than that observed in MMP+ tumors without BLM frameshifts. We suggest that BLM loss of function by MSI in gastric tumors is an intermediate mutational event, which may increase the instability of a pre-existent unstable genotype.