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

Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene

<p>Abstract</p> <p>Background</p> <p>Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (<it>IGF2</it>), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the <it>IGF2 </it>gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.</p> <p>Results</p> <p>Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed <it>IGF2BP1 </it>gene. <it>In vitro </it>binding assays using the CTCF target motif at the <it>IGF2BP1 </it>gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic <it>IGF2BP1 </it>expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express <it>IGF2BP1 </it>primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of <it>IGF2BP1 </it>gene expression.</p> <p>Conclusions</p> <p>Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.</p

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers

AbstractDeregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents

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

Identification of a Preneoplastic Gene Expression Profile in Tubal Epithelium of BRCA1 Mutation Carriers123

Microinvasive carcinomas and high-grade intraepithelial neoplasms are commonly discovered within the fallopian tube of BRCA1 mutation carriers at the time of risk-reducing salpingo-oophorectomy, suggesting that many BRCA1-mutated ovarian carcinomas originate in tubal epithelium. We hypothesized that changes in gene expression profiles within the histologically normal fallopian tube epithelium of BRCA1 mutation carriers would overlap with the expression profiles in BRCA1-mutated ovarian carcinomas and represent a BRCA1 preneoplastic signature. Laser capture microdissection of frozen sections was used to isolate neoplastic cells or histologically normal fallopian tube epithelium, and expression profiles were generated on Affymetrix U133 Plus 2.0 gene expression arrays. Normal-risk controls were 11 women wild type for BRCA1 and BRCA2 (WT-FT). WT-FT were compared with histologically normal fallopian tube epithelium from seven women with deleterious BRCA1 mutations who had foci of at least intraepithelial neoplasm within their fallopian tube (B1-FTocc). WT-FT samples were also compared with 12 BRCA1 ovarian carcinomas (B1-CA). The comparison of WT-FT versus B1-FTocc resulted in 152 differentially expressed probe sets, and the comparison of WT-FT versus B1-CA resulted in 4079 differentially expressed probe sets. The BRCA1 preneoplastic signature was composed of the overlap between these two lists, which included 41 concordant probe sets. Genes in the BRCA1 preneoplastic signature included several known tumor suppressor genes such as CDKN1C and EFEMP1 and several thought to be important in invasion and metastasis such as E2F3. The expression of a subset of genes was validated with quantitative reverse transcription-polymerase chain reaction and immunohistochemistry

Allele-specific transcriptional elongation regulates monoallelic expression of the igf2bp1 gene

Background: Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (IGF2), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the IGF2 gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II. Results: Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed IGF2BP1 gene. In vitro binding assays using the CTCF target motif at the IGF2BP1 gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono-or biallelic IGF2BP1 expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express IGF2BP1 primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of IGF2BP1 gene expression. Conclusions: Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation

<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