La inestabilidad genómica en cáncer.

Abstract

Microsatellite instability (MSI) characterizes tumors arising in patients with HNPCC syndrome. In these tumors, the loss of MMR compromises the genome integrity, allowing the progressive accumulation of mutations and the establishment of a mutator phenotype in a recessive manner. It is not clear, however, whether MSI can be detected in HNPCC carriers before tumor diagnosis. The aim of this study was to evaluate the presence of genetic instability in MMR gene carriers in peripheral blood lymphocytes of carriers and non-carriers members of two HNPCC families harboring a germline MLH1 and MSH2 mutation, respectively. An extensive analysis of the allelic distribution of single molecules of the polyA tract bat26 was performed using a highly sensitive PCR-cloning approach. In non-carriers, the allelic distribution of single bat26 molecules followed a gaussian distribution with no bat26 alleles shorter than (A)21. All mutation carriers showed unstable alleles with an overall frequency of 5.6%. We therefore suggest that low levels of genomic instability characterize MMR mutation carriers. These observations suggest that somatic mutations accumulate well before tumor diagnosis. Even though it is not clear whether this is due to the presence of a small percentage of cells with lost MMR or due to MMR haploinsufficiency, detection of these short unstable alleles might help in the identification of asymptomatic carriers belonging to families with no detectable MMR gene mutations. In addition to the defect in MMR, alterations of microsatellites in no- coding regions may have other causes. For this reason we have used an alternative model (mitocondrial DNA) to evaluate if these alterations depend only on the defects of the MMR, as previously thought. Somatic mutations at a mitochondrial noncoding polycytidine D310 repeat have been associated with tumor progression. We analyzed whether these alterations are due to the inherent mutability of repeated sequences. Insertion and deletion mutations were found in colon, stomach, endometrium, breast, lung, and prostate tumors. The mutation frequency in colon, gastric, and endometrial tumors was 23, 17, and 11% respectively, which paralleled the relative extent of microsatellite instability in long mononucleotide repeats observed in tumors with mismatch repair deficiency (colon > stomach > endometrium, relative ratio 10:8:4). Colon tumors with mutations of more than one nucleotide were more advanced in tumor progression. Further, two tumors showing a T > C mutation that restored the homopolymeric repeat, harbored sequential deletion mutations of 4 and 6 nucleotides. These results illustrate that the increased mutability of repeated mitochondrial sequences is dependent on the repetitive structure of the DNA molecule and suggest that mutations in the D310, whether homoplasmic or not, and by extrapolation, mitochondrial mutations in general, are not the result of selective pressure during tumorigenesis. We also suggest that the D310 may be used as an universal molecular clock to estimate the relative mitotic history of tumors. The last part of this thesis concentrates in studying the alterations of microsatellites in coding regions and the effect that these will have in tumorogenesis process. Alteration in these genes sequences may be change the behaviour comportment of several pathways.. Maintenance of genomic stability depends on the appropriate cellular responses to DNA damage and the integrity of the DNA repair systems. We analyzed stomach tumors with MSI for frameshift mutations in several potential targets of the mutator phenotype involved in DNA damage-response pathways, and DNA repair system. High frequency of mutations was found within ATR (21%), MED1 (43%), hMSH3 (56%) and hMSH6 (43%) genes. Also, a low frequency of mutations within the CHK1 gene was detected in 9% of tumors. These results confirm ATR, MED1 and CHK1 as targets of the mutator pathway in stomach tumorigenesis, and also suggest a potential role of MED1 increasing, together with hMSH3 and hMSH6, the genomic instability in the mutator pathway as a secondary mutator. Furthermore, these results suggest that the inhibition of the ATR-CHK1 DNA damage-response pathway might be involved in the tumorigenesis of gastric cancer with MSI.

Date of Award	21 Sept 2006
Original language	Undefined/Unknown
Supervisor	Simon Schwartz Riera (Tutor) & Simon Schwartz Navarro (Director)