RESUMEN
Numerous single nucleotide polymorphisms (SNPs) have been identified in the human genome, yet the functional significance of most is unknown. CYP3A4 is a key enzyme in the metabolism of numerous compounds. An A-->G substitution 290 bp upstream of the CYP3A4 transcription start site (CYP3A4*1B) has been associated with cancer phenotypes, but its phenotypic effects are unclear. To investigate the functional significance of CYP3A4*1B, we generated two luciferase reporter constructs: 1-kb (denoted L, long) and 0.5-kb (denoted S, short) promoter fragments containing either the variant (V(L),V(S)) or the wild-type (W(L), W(S)) sequences. We evaluated the effect of the variant sequence in the HepG2 and MCF-7 cell lines, and in primary human hepatocytes from three donors. Reporter constructs with the variant sequence had 1.2- to 1.9-fold higher luciferase activity than constructs with wild-type sequence in the cell lines (P < 0.0001) and hepatocytes (P = 0.021, P = 0.027, P = 0.061). The ratio of transcriptional activity for V(S):W(S) was similar to the V(L):W(L) ratio in HepG2 cells, but the V(S):W(S) ratio was consistently less than the V(L):W(L) ratio in MCF-7 cells. This suggests that CYP3A4 expression is higher from the variant promoter and that a repressor sequence may exist in the longer constructs. Electrophoretic mobility shift assays demonstrated specific binding of a component of HepG2 nuclear extract to both wild-type and variant promoters with consistently higher binding affinities to the wild-type sequence. This suggests the existence of a transcriptional repressor responsible for the lower CYP3A4*1A activity. Therefore, the phenotypic effects of the variant CYP3A4*1B may be associated with enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Transcripción Genética , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citocromo P-450 CYP3A , Genes Reporteros , Vectores Genéticos , Genoma Humano , Hepatocitos/metabolismo , Humanos , Luciferasas/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Transfección , beta-Galactosidasa/metabolismoAsunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Mutación de Línea Germinal , Neoplasias Primarias Múltiples/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Análisis Mutacional de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Salud de la Familia , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana Edad , Mutación , Linaje , Polimorfismo Genético , Supresión GenéticaRESUMEN
We identified 17 BRCA1 mutations in 86 Austrian breast and ovarian cancer families (20%) that were screened for mutations by denaturing high-performance liquid chromatography (DHPLC) and the protein truncation test (PTT). Eleven distinct mutations were detected, 4 of them (962del4, 2795del4, 3135del4 and L3376stop) not previously reported in families of non-Austrian origin. In addition, 6 rare missense mutations (allele frequency < 1%) with unknown biological effects were identified. Four mutations occurred more than once in the Austrian population: 2795del4 (3 times), Cys61Gly (3 times) 5382insC (2 times) and Q1806stop (2 times). Haplotype analysis of the 4 recurrent mutations suggested a common ancestor for each of these. Thirty-four breast cancer cases from 17 families with BRCA1 mutations were further analyzed. We observed a low median age of onset (39.5 years). Sixty-eight percent of all BRCA1 breast cancer cases had negative axillary lymph nodes. This group showed a significant prevalence of a negative estrogen and progesterone receptor status and stage I tumors compared with an age-related, node-negative control group. The prevalence of grade III tumors was marginally significant. Survival analysis either with a control group matched for age (within 5 years), grade, histologic subtype and estrogen receptor status, or with an age-related, node-negative comparison group, showed no statistical difference.