RESUMEN
BACKGROUND AND AIMS: There are epidemiological, morphological, and molecular differences between normal mucosa as well as between adenocarcinomas of the right and left side of the large bowel. The aim of this study was to investigate differences in gene expression. METHODS: Oligonucleotide microarrays (GeneChip) were used to compare gene expression in 45 single samples from normal mucosa and sporadic colorectal carcinomas (Dukes' B and C) of the caecum compared with the sigmoid and rectosigmoid. Findings were validated by real time polymerase chain reaction. RESULTS: Fifty eight genes were found to be differentially expressed between the normal mucosa of the caecum and the sigmoid and rectosigmoid (p<0.01), including pS2, S100P, and a sialyltransferase, all being expressed at higher levels in the caecum. A total of 118 and 186 genes were differentially expressed between normal and right or left sided tumours of the colon, showing more pronounced differences in Dukes' C than B tumours. Thirty genes differentially expressed in tumour tissue were common to adenocarcinomas of both sides, including known tumour markers such as the matrix metalloproteinases. Keratins 8, 19, and 20 as well as carbonic anhydrases (II, IV, VII) showed side specific expression and were downregulated in left sided tumours whereas teratocarcinoma growth factor and cyclooxygenase 2 (COX-2) were upregulated in left sided adenocarcinomas. Immunohistochemical analysis confirmed differences in side specific expression for cytokeratin 20 and COX-2. CONCLUSIONS: Differences in gene expression between normal mucosa as well as between adenocarcinomas of the caecum and sigmoid or rectosigmoid exist and should be taken into account when examining new targeted therapeutic regimens.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/metabolismo , Neoplasias del Ciego/genética , Regulación Neoplásica de la Expresión Génica , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Biomarcadores de Tumor/genética , Neoplasias del Ciego/metabolismo , Neoplasias del Ciego/patología , Perfilación de la Expresión Génica/métodos , Humanos , Mucosa Intestinal/metabolismo , Repeticiones de Microsatélite , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Neoplásico/genética , Neoplasias del Recto/genética , Neoplasias del Recto/metabolismo , Neoplasias del Recto/patología , Neoplasias del Colon Sigmoide/genética , Neoplasias del Colon Sigmoide/metabolismo , Neoplasias del Colon Sigmoide/patologíaRESUMEN
Chromosomal instability is common in bladder cancer and could be caused by mutations of mitotic checkpoint genes. Therefore we screened for mutations of the mitotic checkpoint genes hBUB1, hBUB1B, hBUB3 and TTK in six aneuploid bladder cancer cell lines and 15 human bladder tumours. The screening was performed by sequence analysis of the entire coding regions of the four genes. No mutations were detected in any of the four genes. We detected several sequence variations in hBUB1, hBUB1B and TTK both new and previously published. The genetic stability of the four gene loci were tested by loss of heterozygosity (LOH) analysis in the 15 patient samples, showing one LOH for each of the hBUB1B, hBUB3 and TTK loci (6.7%) of the cases, all in different tumour samples. No LOH was detected at hBUB1. We conclude that both mutational inactivation, and loss of one allele, of the examined mitotic checkpoint genes are relatively uncommon.
Asunto(s)
Genes cdc , Pérdida de Heterocigocidad , Mutación , Neoplasias de la Vejiga Urinaria/genética , HumanosRESUMEN
BACKGROUND: Testing for mutations of the TP53 gene in tumors is a valuable predictor for disease outcome in certain cancers, but the time and cost of conventional sequencing limit its use. The present study compares traditional sequencing with the much faster microarray sequencing on a commercially available chip and describes a method to increase the specificity of the chip. METHODS: DNA from 140 human bladder tumors was extracted and subjected to a multiplex-PCR before loading onto the p53 GeneChip from Affymetrix. The same samples were previously sequenced by manual dideoxy sequencing. In addition, two cell lines with two different homozygous mutations at the TP53 gene locus were analyzed. RESULTS: Of 1464 gene chip positions, each of which corresponded to an analyzed nucleotide in the sequence, 251 had background signals that were not attributable to mutations, causing the specificity of mutation calling without mathematical correction to be low. This problem was solved by regarding each chip position as a separate entity with its own noise and threshold characteristics. The use of background plus 2 SD as the cutoff improved the specificity from 0.34 to 0.86 at the cost of a reduced sensitivity, from 0.92 to 0.84, leading to a much better concordance (92%) with results obtained by traditional sequencing. The chip method detected as little as 1% mutated DNA. CONCLUSIONS: Microarray-based sequencing is a novel option to assess TP53 mutations, representing a fast and inexpensive method compared with conventional sequencing.