Microsatellite Instability

The influence of DNA repair capacity on genomic integrity is suggested by the positive correlation between life span and DNA repair capacity and evidenced by progeroid syndromes in which DNA repair defects can cause phenotypes resembling premature aging (Lombard et al., 2005). The accurate maintenance of nuclear DNA is critical to cell and organism functions, and therefore numerous DNA repair pathways have evolved for the different types of DNA lesions. Among these pathways, the nucleotide excision repair mechanism (because of its role in repair of UV-induced damage) and the nonhomologous end joining pathway (the predominant double-strand break repair system) are the most intensely studied, and together with the base excision repair pathway do appear to be altered with age (Lombard et al., 2005). The function of the mismatch repair (MMR) system during aging is to scan newly replicated DNA and to deal with replicative and recombinational errors leading to mispaired bases (mismatches) by excising the mutated strand in either direction to the mismatch, thus contributing to genomic protection against replication-induced mutations (Loeb, 1994). This would be most important in rapidly replicating cells, such as TCC.

An experimental approach to the study of defects in this pathway is the evaluation of microsatellite instability (MSI), evidenced by expansions or contractions of microsatellites, highly polymorphic tandemly-repeated sequences from one to six bases, interspersed in the genome and particularly prone to replication slippages that change strand lengths, either by insertion or deletion of repeated units.

Mutations occur both in repeated sequences and randomly throughout the genome. Therefore, MSI indicates a higher susceptibility to mutations. Despite the trend to errors occurring in all dividing cells, microsatellite lengths remain stable due to the intrinsic activity and fidelity of the MMR system. Hence, MSI serves as a marker for a malfunctioning MMR system. There is an increased instability both in the mucosa of aged patients with gastric lymphomas (Starostik et al., 2000) and also in microsatellite sequences from total peripheral blood DNA of old subjects obtained at a 10-year interval (Ben Yehuda et al., 2000; Krichevsky et al., 2004; Neri et al., 2005).

One possible cause of MMR deficiency could be the inactivation or the altered expression of MMR genes due to epigenetic mutations, as suggested by the finding of hypermethylation-mediated MMR gene inactivation in sporadic cancers showing microsatellite instability and by the age-related inactivation of hMLH1 by promoter methylation found in normal colon cells. Data on in vitro aging of TCC suggest that the MMR system could differently change its efficiency during in vitro proliferation, depending upon the cell type and/or that repeated doublings could contribute to the accumulation of genetic alterations not repaired by the MMR system (Neri et al., 2004).

Protocol for MSI analysis

1. Extract genomic DNA from cell pellets using the QIAamp DNA Mini Kit (Qiagen GmbH, Germany) following the manufacturer's instructions; quantify DNA by spectrophotometry determination at 260 nm.

2. For MSI analysis, at least five repeated sequences should be tested; for example, the following loci containing tetra- and pentanucleotide polymorphic tandem repeat sequences: CD4 (12p13), a (TTTTC) repeat located in the 5' nontranscribed region of the T cell surface antigen gene; VWA31 (12p12-pter), an (AGAT) repeat in intron 40 of the von Willebrand factor gene; Fes/FPS (15q25-qter), an (ATTT) repeat in intron 5 of the c-fes proto-oncogene; TPOX (2p23-pter), an (AATG) repeat in intron 10 of the thyroid peroxidase gene; p53 (17p13), an (AAAAT) repeat in the first intron of the TP53 gene.

Carry out the PCR amplifications using the following specific primers:










p53-pF: 5'-ACTCCAGCCTGGGCAATAA-GAGCT-3', p53-pR: 5'-ACAAAACATCCCCTACAACAGC-3'. Combine template DNA (50 ng), 1 ^M each primer, 200 ^M each deoxynucleotide triphosphate, 1.5 mM (for CD4, FesTPOX and p53) or 2.5 mM (for VWA31) MgCl2, 1X PCR buffer II (50 mM KCl, 10 mM Tris-HCl, pH 8.3) and 1.25 U AmpliTaq DNA Polymerase (Perkin Elmer, Roche Molecular Systems, USA) in 25-^1 reactions. Amplification profiles are, respectively: 10 cycles (1' 94° C, 45'' 62°C) followed by 20 cycles (1' 90°C, 45'' 62°C) for CD4; 30 cycles (1' 94°C, 1' 57°C, 1' 72°C) for VWA31; 30 cycles (1' 94°C, 1' 54°C, 1' 72°C) for Fes; 30 cycles (1' 94°C, 1' 64°C, 1' 72°C) for TPOX; and 30 cycles (1' 94°C, 45'' 65°C, 45'' 72°C) for p53. Include in all reactions a negative control (sample without template).

3. Analyze 10 ^l of each PCR product on 2% agarose gels in order to verify amplification and then perform electrophoresis with product-adjusted amounts on 10% nondenaturing polyacrylamide vertical gels (20 cm long, 0.75 mm thick) containing 5% glycerol, in TBE buffer, for 16 to 18 hrs at 100 to 150 V, together with the DNA Molecular Weight

Marker VIII (Roche Molecular Systems, Branchburg, USA) and allelic ladders prepared by mixing known alleles.

4. Silver stain gels at room temperature with continuous shaking. After 10' fixation in 10% EtOH and 2' oxidation in 1% nitric acid, stain the gels for 20' in 0.02% AgNO2, rinse in distilled water, and then reduce in developing solution (3% sodium carbonate and 0.1% formaldehyde) until an optimal band intensity is observed. Stop the development by adding 5% acetic acid for 3' and finally place the gels in distilled water.

5. Conclude genotyping by side-by-side comparison with allelic ladders. Results showing allele modifications in DNA samples derived from the same donor must be confirmed by repeating the analysis.

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