Polyglycine expansions in eRF3/GSPT1 are associated with gastric cancer susceptibility.

Gastric cancer remains a major cause of death in the developed countries, and a large percentage is still genetically unexplained. Because of their major role in cell survival, mutations in translation factors and altered expression of these genes have been associated with cancer development. Apart from its role in translation termination, the eukaryotic translation release factor 3 (eRF3) is involved in several critical cellular processes, such as cell cycle regulation, cytoskeleton organization and apoptosis. The aim of this study was to evaluate eRF3/GSPT1 gene as a potential genetic susceptibility associated locus for gastric cancer, analysing a stable GGC expansion in exon 1 encoding a polyglycine tract in the N-terminal domain of the protein. DNA was obtained from 139 patients with gastric cancer and from 100 individuals of a healthy control population. The GGC expansion was amplified by PCR and the number of repeats determined by genotyping in an automatic sequencer. There are five known alleles encoding from 8 to 12 glycines. The most common allele encodes 10 glycines. The 12-Gly allele was detected exclusively in the cancer patients (allelic frequency = 5%). Regardless of the genotype, patients with the 12-Gly allele had a 20-fold increased risk for gastric cancer. We also detected a single-base alteration in the gene (G274T) although no correlation with cancer development has been found. Thus, our results show that the GGC expansion may have a potential role in regulating eRF3/GSPT1 expression and/or changing the protein function that can lead to gastric cancer development.

Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological types.

BACKGROUND: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. AIMS: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. METHODS: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. RESULTS: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p < 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. CONCLUSIONS: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.

Molecular methods for the detection of mutations.

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Association of p53 genomic instability with the glutathione S-transferase null genotype in gastric cancer in the Portuguese population.

AIMS: p53 gene mutations are the most common genetic changes known to occur in human cancer. In previous studies, the presence of alterations to the p53 gene has been linked to the null phenotype of the glutathione S-transferase mu gene (GSTM1). GSTM1 appears to be part of a protective mechanism against the development of cancers in which environmental chemical carcinogens are involved. To screen for such an association in stomach cancer, p53 allelic loss and genomic instability and GSTM1 genotypes were investigated in gastric tumour DNA samples from 113 patients. METHODS: The polymerase chain (PCR) reaction was used to amplify a (CA) repeat array in the p53 locus; electrophoresis, genotyping, and allele quantification were performed using an automated DNA sequencer and Genescan software. The presence of the GSTM1 gene was determined by means of a differential PCR in which multiple genes were co-amplified in the same reaction tube. RESULTS: Loss of heterozygosity (LOH) of the p53 gene was found in 36 of 87 informative cases and genomic instability was present in eight of 113 cases. Further analysis into histological subtypes and sites of tumours did not show any positive association with p53 loss. An association between the presence of LOH and the GSTM1 null genotype was not seen; however, all the samples with genomic instability of the p53 gene (eight of 113) also showed a GSTM1 null genotype. CONCLUSION: This study does not support the hypothesis of an association between LOH in the p53 gene and the GSTM1 null genotype, but suggests that the GSTM1 null genotype might influence p53 genomic instability.