Diverse patterns of genomic targeting by transcriptional regulators in Drosophila melanogaster.

Annotation of regulatory elements and identification of the transcription-related factors (TRFs) targeting these elements are key steps in understanding how cells interpret their genetic blueprint and their environment during development, and how that process goes awry in the case of disease. One goal of the modENCODE (model organism ENCyclopedia of DNA Elements) Project is to survey a diverse sampling of TRFs, both DNA-binding and non-DNA-binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TRFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also highlight the stringency of the Polycomb regulatory network, and show association of the Trithorax-like (Trl) protein with hotspots of DNA binding throughout development. Furthermore, the data identify more than 5800 instances in which TRFs target DNA regions with demonstrated enhancer activity. Regions of high TRF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TRF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. Together these data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development.

Adaptation of a chemosensitivity assay to accurately assess pemetrexed in ex vivo cultures of lung cancer.

PURPOSE: Pemetrexed is the only FDA approved treatment for mesothelioma and is a second line agent for treatment of non-small cell lung carcinoma (NSCLC). Pemetrexed is inhibited by folate and its analogs, which are components of many culture media, making it challenging to study pemetrexed in vitro. In order to accurately evaluate pemetrexed's effects in vitro, the protocol for a standard chemosensitivity assay, the ChemoFx drug response marker, had to be modified. EXPERIMENTAL DESIGN: Novel rinse and media change steps were assessed and then added to the assay protocol in order to observe pemetrexed activity. The intraday and interday stability of pemetrexed were also established under the adapted protocol. Then, the modified protocol was used to examine pemetrexed in 65 ex vivo lung cancer specimens. RESULTS: Substituting 5% RPMI + EGF for BEGM allowed pemetrexed to exert its anticancer activity in the ChemoFx DRM. ChemoFx classified 6.2% of the lung specimens as responsive, 9.2% as intermediate responsive and 84.6% as non-responsive to pemetrexed. CONCLUSIONS: Adapting the ChemoFx protocol allowed for the accurate evaluation of pemetrexed anticancer activity in ex vivo lung specimens. ChemoFx evaluation may provide an indication of a patient's clinical response to the drug prior to pemetrexed treatment. Having this information when treatment options are being considered could avoid wasted time, unnecessary costs and needless side effects that are the result of an inappropriate chemotherapy regimen.

Chemoresponse assay for evaluating response to sunitinib in primary cultures of breast cancer.

PURPOSE: Not all patient tumors respond equally to the same type of therapy. An in vitro chemoresponse assay that can suggest individualized tumor response to therapies, in this case sunitinib, can be a valuable guide for clinical decision-making. RESULTS: The assay was shown to be sensitive and reproducible while differentiating renal cell lines based on sunitinib sensitivity and evaluating vendors' supply of the compound. Of the cultured breast cancer tumor specimens treated with sunitinib, ChemoFx classified 7.6% as responsive (R), 20.5% of specimens as intermediate responsive (IR), and 71.7% as non-responsive (NR). EXPERIMENTAL DESIGN: The ChemoFx(®) drug response marker (DRM) (Precision Therapeutics, Inc.) was carried out on SK-OV-3 cells treated with sunitinib to establish appropriate dose ranges and assay thresholds, and to evaluate vendor supplies of sunitinib. Once reference values were determined, the assay was applied to eight different renal cell lines treated with sunitinib, each of which was subsequently classified into responsive, intermediate responsive, and non-responsive groups. Next, ex vivo tumor samples from 39 clinically diagnosed breast cancer patients were grown in culture and assayed for their response to sunitinib using ChemoFx. CONCLUSIONS: Chemoresponse assay assessment is an effective tool for evaluating sunitinib sensitivity in cultured cell lines as well as ex vivo breast cancer samples. An in vitro assay that may indicate an individual patient's clinical response to a chemotherapeutic agent can be beneficial in time, cost, and clinical outcome when therapeutic options are considered.

A cis-regulatory map of the Drosophila genome.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

Overexpression of Prdx6 and resistance to peroxide-induced death in Hepa1-6 cells: Prdx suppression increases apoptosis.

Peroxiredoxins are thiol-specific antioxidants that catalyze the reduction of cellular peroxides and protect cells from ROS-mediated damage and death. Peroxiredoxin gene expression is up-regulated in a number of cancers, suggesting a possible role in cancer cell maintenance. Prdx6, a cytoplasmic protein elevated in certain cancers, is highly expressed in liver and transcriptionally regulated by various oxidative stresses. In the present study, we found that the cancerous Hepa1-6 hepatoma cell line is significantly more resistant to peroxide-induced cytotoxicity than the non-cancerous H2.35 cell line. We also demonstrated that Hepa1-6 cells express approximately 3-fold more Prdx6 mRNA and 2.5-fold more Prdx6 protein than H2.35 cells. Treatment with mithramycin A resulted in a nearly 20% reduction in Prdx6 mRNA in Hepa1-6 cells, suggesting a possible role for Sp1 in Prdx6 up-regulation. We hypothesized that suppression of Prdx6 in Hepa1-6 cells would increase susceptibility to peroxide-induced cell death. Transient transfection of Hepa1-6 cells with Prdx6 siRNA led to a marked reduction in Prdx6 expression, and an increase in peroxide-induced cytotoxicity by apoptosis. Together, these data demonstrate an important anti-apoptotic function for Prdx6 in cancerous liver cells, and suggest that its up-regulation may be a tumor-supportive adaptation in cancerous states.