RESUMEN
PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in several tumor types, and its expression is influenced by the length of a 5'-end microsatellite repeat (CA)n: the longer the repeat, the lower the expression. Dinucleotide repeats accumulate insertion/deletion types of mutations in tumors with microsatellite instability. We designed this study to estimate the occurrence of these mutations in EGFR(CA)n and their relevance in carcinogenesis of microsatellite instability-positive colon and gastric tumors. EXPERIMENTAL DESIGN: We analyzed the frequency of EGFR(CA)n mutations in vivo in 55 colorectal and 14 gastric microsatellite instability-positive cancers, and in vitro in single-cell clone cultures of microsatellite instability-positive colon tumor cell line LS174. Single-cell clone cultures with different repeat lengths were analyzed by fluorescent-activated cell sorter for EGFR cell-surface expression. A correlation analysis was done between EGFR(CA)n mutations and mutations in KRAS, BRAF, and p53. RESULTS: Unlike single-cell clone cultures, which exhibited higher rate of deletions compared with insertions, most of EGFR(CA)n mutations in colon and gastric tumors were insertions. Longer EGFR(CA)n correlated with lower EGFR cell-surface expression in single-cell clone cultures. In colon cancers, the elongation of the repeat was associated negatively with mutations in KRAS and BRAF, but not in p53. CONCLUSIONS: The EGFR(CA)n elongation observed in tumors cannot be explained by an intrinsic property of this repeat favoring insertions versus deletions. Instead, a selection for repeat elongation occurs in microsatellite instability-positive tumors, leading to EGFR down-regulation. These findings suggest that in microsatellite instability-positive tumors current therapies targeting EGFR overexpression may have either no effect or an opposite to the expected effect.
Asunto(s)
Neoplasias del Colon/genética , Repeticiones de Dinucleótido/genética , Receptores ErbB/genética , Inestabilidad de Microsatélites , Regiones no Traducidas 5'/genética , Secuencia de Bases , Línea Celular Tumoral , Neoplasias del Colon/patología , Análisis Mutacional de ADN , Regulación hacia Abajo , Citometría de Flujo , Frecuencia de los Genes , Genes ras/genética , Genotipo , Humanos , Mutagénesis Insercional , Mutación , Poli A/genética , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Proteínas Proto-Oncogénicas B-raf/genética , Eliminación de Secuencia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Genetic or epigenetic inactivation of the DNA mismatch repair genes in tumor precursor cells results in a strong mutator phenotype, known as the microsatellite mutator phenotype (MMP), or microsatellite instability (MSI). This mutator phenotype causes mutations in genes responsible for the regulation of cell growth and survival/death and thus promotes the development and progression of tumors. In the present study, we examined the DNA topoisomerase II genes (topIIalpha and topIIbeta) as mutational targets for MMP. We screened 10 MSI-positive human tumor cell lines and 30 MSI-positive colorectal tumors for mutations within the entire coding region of the topIIalpha gene and two coding poly(A)7 sequences of topIIbeta. Mutations in either the topIIalpha or topIIbeta gene were found with an overall frequency of 18% (in 10% of the primary tumors and in 44% of the cell lines). This indicates that modulation of the DNA topoisomerase II (TOPII) activity may be important for the development of MSI-positive cancer.
Asunto(s)
Neoplasias del Colon/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Neoplasias/genética , Línea Celular Tumoral , Neoplasias del Colon/enzimología , ADN-Topoisomerasas de Tipo II/metabolismo , Femenino , Mutación del Sistema de Lectura , Humanos , Masculino , Repeticiones de Microsatélite/genética , Mutación Missense , Proteínas de Neoplasias/metabolismoRESUMEN
Genetic or epigenetic inactivation of DNA mismatch repair genes results in a strong mutator phenotype, known as the microsatellite mutator phenotype or microsatellite instability (MSI). This mutator phenotype causes mutations in genes responsible for the regulation of cell growth and survival/death and thus promotes the development and progression of tumors. In addition to such tumorigenic lesions, mutations in genes of other types of DNA repair, for example, DNA double-strand break (DNA DSB) repair, are found in tumor cells with MSI. We report here that the majority of MSI-positive tumor cell lines of different tissue origins (endometrial, ovarian, prostate, and colorectal carcinomas) are hypersensitive to bleomycin, a DNA DSB producing chemotherapeutic drug. We suggest that this hypersensitivity may be a result of inactivation of the DNA DSB repair activity by concomitant mutations of different DNA DSB repair genes. To provide experimental support to this hypothesis, we show that the subclones of the MSI-positive colorectal cancer cell line HCT-8 that bear heterozygous frameshift mutations in the DNA DSB repair gene DNA-PK(CS) are more sensitive to a combined treatment with bleomycin and the DNA protein kinase inhibitor LY294002 than the original HCT-8 cells, which are wild type for this gene. These results may be useful in designing therapies for MSI-positive cancer.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Daño del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Línea Celular Tumoral , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteína Quinasa Activada por ADN , Mutación del Sistema de Lectura , Genes p53/genética , Humanos , Repeticiones de Microsatélite/genética , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
BACKGROUND: Matrix metalloproteinases (MMPs) contribute in multiple ways to all stages of tumor development, and a number of DNA polymorphisms in the MMP genes are associated with an increased risk of cancer. We previously identified the MMP-21 gene 572C/T polymorphism leading to Ala191Val substitution within the enzyme's catalytic domain. We performed a case-control study to test association between this polymorphism and the risk of breast cancer. PATIENTS AND METHODS: 572C/T polymorphism was analyzed by RFLP method in 396 unrelated Russian females: 76 breast cancer patients and 320 disease-free blood donors. RESULTS: The frequencies of C/C, T/C and T/T genotypes in patients (69.7%, 25.0% and 5.3%) did not differ significantly from those in controls (61.9%, 34.7% and 3.4%); the polymorphism was not associated with the increased tumor size and the presence of metastases. CONCLUSION: The MMP-21 gene 572C/T polymorphism has no significant effect on the development and progression of breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Metaloproteinasas de la Matriz/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Humanos , Metaloproteinasas de la Matriz Secretadas , Persona de Mediana Edad , Polimorfismo GenéticoRESUMEN
We previously applied arbitrarily primed polymerase chain reaction DNA fingerprinting to identify molecular genetic alterations in primary breast carcinomas. One of the most frequently observed fingerprint alterations was a reduction in the intensity of the MCG1-B2 band in 32% of tumors, indicating recurrent loss of X-chromosome segments. This article reports a mapping analysis of those chromosomal deletions. The subchromosomal origin of MCG1-B2 was determined to be the Xq25 chromosomal region. Loss of heterozygosity (LOH) analysis was carried out on 72 infiltrating ductal carcinomas with a panel of seven microsatellite markers spanning Xq25. The smallest common region of the X-chromosome deletions was mapped to between markers DXS8059 and DXS8009, with the highest LOH frequency of 52.4% at the DXS8098 locus. The LOH at DXS8098 was associated with larger tumor size (> 3 cm) (P = 0.048, Fisher exact test), higher histologic grade (P = 0.036, Fisher exact test), and axillary lymph node metastasis (P = 0.020, Fisher exact test). These results suggest that the Xq25 region harbors a putative tumor suppressor gene whose inactivation in breast cancer is associated with tumor progression and metastasis. LOH at this region, therefore, potentially could be used as a prognostic marker for disease development. One of the two X chromosomes is transcriptionally silent in women. The loss of the Xq25 region detected in this study occurred preferentially on the inactive X chromosome. This suggests that the putative tumor suppressor gene may escape X inactivation.