Methylation and protein expression of DNA repair genes: association with chemotherapy exposure and survival in sporadic ovarian and peritoneal carcinomas

<p>Abstract</p> <p>Background</p> <p>DNA repair genes critically regulate the cellular response to chemotherapy and epigenetic regulation of these genes may be influenced by chemotherapy exposure. Restoration of BRCA1 and BRCA2 mediates resistance to platinum chemotherapy in recurrent BRCA1 and BRCA2 mutated hereditary ovarian carcinomas. We evaluated BRCA1, BRCA2, and MLH1 protein expression in 115 sporadic primary ovarian carcinomas, of which 31 had paired recurrent neoplasms collected after chemotherapy. Additionally, we assessed whether promoter methylation of BRCA1, MLH1 or FANCF influenced response to chemotherapy or explained alterations in protein expression after chemotherapy exposure.</p> <p>Results</p> <p>Of 115 primary sporadic ovarian carcinomas, 39 (34%) had low BRCA1 protein and 49 (42%) had low BRCA2 expression. BRCA1 and BRCA2 protein expression were highly concordant (p < 0.0001). MLH1 protein loss occurred in 28/115 (24%) primary neoplasms. BRCA1 protein loss in primary neoplasms was associated with better survival (p = 0.02 Log Rank test) and remained significant after accounting for either stage or age in a multivariate model (p = 0.04, Cox proportional hazards). In paired specimens, BRCA1 protein expression increased in 13/21 (62%) and BRCA2 protein expression increased in 15/21 (71%) of recurrent carcinomas with low or intermediate protein in the paired primary. In contrast MLH1 expression was rarely decreased in recurrent carcinomas (1/33, 3%). Similar frequencies of MLH1, BRCA1, and FANCF promoter methylation occurred in primary carcinomas without previous chemotherapy, after neoadjuvant chemotherapy, or in recurrent neoplasms.</p> <p>Conclusion</p> <p>Low BRCA1 expression in primary sporadic ovarian carcinoma is associated with prolonged survival. Recurrent ovarian carcinomas commonly have increased BRCA1 and/or BRCA2 protein expression post chemotherapy exposure which could mediate resistance to platinum based therapies. However, alterations in expression of these proteins after chemotherapy are not commonly mediated by promoter methylation, and other regulatory mechanisms are likely to contribute to these alterations.</p

A Genomewide Screen for Suppressors of Alu-Mediated Rearrangements Reveals a Role for PIF1

Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition

Modulateurs d'actine

The invention provides methods and compositions which find use, $i(inter alia), for modulating the stabilization of actin filaments. The compositions may comprise one or more polypeptide moieties derived from a novel human diaphanous polypeptide and/or one or more nucleic acid moieties derived from a novel human diaphanous gene or gene transcript. The invention also provides agents which specifically modify the binding of a natural human diaphanous gene or gene product with a natural binding target thereof, isolated human diaphanous hybridization probes and primers capable of specifically hybridizing with the disclosed human diaphanous genes, human diaphanous-specific binding agents such as specific antibodies, and methods of making and using the subject composition in diagnosis, therapy and in the biopharmaceutical industry.L'invention concerne des procédés et des compositions permettant, entre autres choses, de moduler la stabilisation des filaments d'actine. Ces compositions peuvent comprendre une ou plusieurs fractions de polypeptide dérivées d'un nouveau polypeptide diaphane de l'homme et/ou une ou plusieurs fractions d'acide nucléique dérivées d'un nouveau gène diaphane de l'homme ou d'un produit de transcription génétique. L'invention concerne également des agents qui modifient, de manière spécifique, la liaison d'un gène diaphane naturel de l'homme ou un produit génétique avec une cible de liaison naturelle de ce dernier. L'invention traite aussi de sondes et d'amorces d'hybridation diaphanes de l'homme pouvant s'hybrider de manière spécifique avec les gènes diaphanes de l'homme selon l'invention, des agents de liaison spécifiques diaphanes de l'homme tels que des anticorps spécifiques. Enfin, l'invention a pour objet des procédés pour fabriquer et utiliser les compositions selon l'invention pour des applications de diagnostique et de thérapie et dans l'industrie biopharmaceutique.UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

<i>pfh1-L430P</i> does not complement wild-type <i>pfh1⁺</i> function.

<p>(<b>A</b>) Complementation assay in <i>pfh1::loxP pfh1⁺ kanMX6 loxP</i> strain with either the L430P mutation (<i>leu::pfh1-L430P</i>), Pfh1 (<i>leu::pfh1⁺</i>), or an empty vector (<i>leu::leu⁺</i>) strain after transformation with a plasmid expressing Cre (<i>cre⁺</i>) or Cre-Y324F (<i>cre</i>⁻). The transformation plates are contrast-inverted pictures. (<b>B</b>) Anti-Pfh1 western blot of wild-type Pfh1GFP (WT) and three independent clones (1–3) expressing Pfh1-L430P. <i>S. pombe</i> cells were grown in the absence (−) or presence (+) of thiamine. 0, 12, and 24 hours indicates the amount of time cells were grown in the presence of thiamine. The loading control (LC) is a non-specific background band. (<b>C</b>) Heterologous expression constructs of <i>pfh1-nuc</i> or <i>pfh1-mt*</i> are indicated in the presence of <i>pfh1::loxP pfh1⁺ kanMX6 loxP</i> and <i>leu::pfh1-L430P</i>. Contrast-inverted pictures of transformation plates with <i>cre⁺</i> on the top and <i>cre⁻</i> on the bottom.</p

<i>Alu</i>-URA-<i>Alu</i> pRS415 (pAUA).

<p>A construct containing a <i>URA3</i> gene flanked by identical <i>AluSp</i> repetitive elements was inserted into the low-copy-number yeast plasmid pRS415.</p