Expression of HNF4alpha in the human and rat choroid plexus – Implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier

<p>Abstract</p> <p>Background</p> <p>The choroid plexus consists of highly differentiated epithelium and functions as a barrier at the interface of the blood-cerebrospinal-fluid (CSF). This tissue may therefore determine the bioavailability and transport of drugs to the brain. Little is known about the expression of drug and xenobiotic metabolizing enzymes (DME) and of drug transporters in the human choroid plexus. Notably, the transcription factor and zinc finger protein HNF4alpha is a master regulator of DMEs and of drug transporters. As of today its activity in the blood-CSF barrier is unknown. Here we report our efforts in determining HNF4alpha activity in the regulation of ABC transporters in the human and rat choroid plexus.</p> <p>Results</p> <p>We report expression of HNF4alpha by qRT-PCR and by immunohistochemistry and evidence transcript expression of the ATP-binding cassette transporters ABCB1, ABCB4, ABCC1-6 in choroid plexus. Additionally, HNF4alpha DNA binding activity at regulatory sequences of ABCB4 and ABCC1 was determined by EMSA bandshift assays with a specific antibody. We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments.</p> <p>Conclusion</p> <p>Our study evidences activity of HNF4alpha in human and rat choroid plexus. This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier.</p

Transcriptome profiling of human hepatocytes treated with Aroclor 1254 reveals transcription factor regulatory networks and clusters of regulated genes

BACKGROUND: Aroclor 1254 is a well-known hepatotoxin and consists of a complex mixture of polychlorinated biphenyls (PCBs), some of which have the ability to activate the aryl hydrocarbon receptor (AhR) and other transcription factors (TFs). Altered transcription factor expression enables activation of promoters of many genes, thereby inducing a regulatory gene network. In the past, computational approaches were not applied to understand the combinatorial interplay of TFs acting in concert after treatment of human hepatocyte cultures with Aroclor 1254. We were particularly interested in interrogating promoters for transcription factor binding sites of regulated genes. RESULTS: Here, we present a framework for studying a gene regulatory network and the large-scale regulation of transcription on the level of chromatin structure. For that purpose, we employed cDNA and oligomicroarrays to investigate transcript signatures in human hepatocyte cultures treated with Aroclor 1254 and found 910 genes to be regulated, 52 of which code for TFs and 47 of which are involved in cell cycle and apoptosis. We identified regulatory elements proximal to AhR binding sites, and this included recognition sites for the transcription factors ETS, SP1, CREB, EGR, NF-kB, NKXH, and ZBP. Notably, ECAT and TBP binding sites were identified for Aroclor 1254-induced and E2F, MAZ, HOX, and WHZ for Aroclor 1254-repressed genes. We further examined the chromosomal distribution of regulated genes and observed a statistically significant high number of gene pairs within a distance of 200 kb. Genes regulated by Aroclor 1254, are much closer located to each other than genes distributed randomly all over the genome. 37 regulated gene pairs are even found to be directly neighbored. Within these directly neighbored gene pairs, not all genes were bona fide targets for AhR (primary effect). Upon further analyses many were targets for other transcription factors whose expression was regulated by Aroclor 1254 (secondary effect). CONCLUSION: We observed coordinate events in transcript regulation upon treatment of human hepatocytes with Aroclor 1254 and identified a regulatory gene network of different TFs acting in concert. We determined molecular rules for transcriptional regulation to explain, in part, the pleiotropic effect seen in animals and humans upon exposure to Aroclor 1254

Topoisomerase II inhibition involves characteristic chromosomal expression patterns

<p>Abstract</p> <p>Background</p> <p>The phenomenon of co-localization of transcriptionally upregulated genes showing similar expression levels is known across all eukaryotic genomes. We recently mapped the Aroclor 1254-regulated transcriptome back onto the genome and provided evidence for the statistically significant co-localization of regulated genes. They did, however, not always show similar expression levels, and many of the regulated genes were, in fact, repressed.</p> <p>Results</p> <p>In this study, we were able to reproduce this observation with microarray data stemming from 1) human hepatocytes treated with the gyrase and potential topoisomerase II inhibitor trovafloxacin, 2) human hepatocytes treated with the topoisomerase II inhibitor doxorubicin and 3) mouse lymphoma cells treated with the topoisomerase II inhibitor etoposide. We found statistically significant co-localization of regulated gene pairs – induced and repressed – within the window size of 0–100 kbp. Notably, by using microarray data stemming from lung tissue of a mouse transgenic line overexpressing the transcription factor c-myc, which served as a negative control, we found regulated genes to be located with regard to each other nearly in the same way as genes distributed randomly all over the genome (0–100 kbp).</p> <p>Conclusion</p> <p>We suggest topoisomerase II inhibition by Aroclor 1254, trovafloxacin, doxorubicin, and etoposide to be responsible for significant co-localization of regulated genes through the inability of the stabilized enzyme complexes to religate DNA. Within the permanently opened chromatin domains, neighbored genes might be allowed to be regulated. Overexpression of c-myc, however, does not inhibit topoisomerase II activity. Consequently, the enzyme is able to perform its normal function of transiently breaking and rejoining the DNA double strand. As a result, exclusively target genes are regulated.</p

An update on the mouse liver proteome

<p>Abstract</p> <p>Background</p> <p>Decoding of the liver proteome is subject of intense research, but hampered by methodological constraints. We recently developed an improved protocol for studying rat liver proteins based on 2-DE-MALDI-TOF-MS peptide mass finger printing.</p> <p>This methodology was now applied to develop a mouse liver protein database.</p> <p>Results</p> <p>Liver proteins were extracted by two different lysis buffers in sequence followed by a liquid-phase IEF pre-fractionation and separation of proteins by 2 DE at two different pH ranges, notably 5-8 and 7-10. Based on 9600 in gel digests a total of 643 mouse liver proteins with high sequence coverage (> 20 peptides per protein) could be identified by MALDI-TOF-MS peptide mass finger printing. Notably, 255 proteins are novel and have not been reported so far by conventional two-dimensional electrophoresis proteome mapping. Additionally, the results of the present findings for mouse liver were compared to published data of the rat proteome to compile as many proteins as possible in a rodent liver database.</p> <p>Conclusion</p> <p>Based on 2-DE MALDI-TOF-MS a significantly improved proteome map of mouse liver was obtained. We discuss some prominent members of newly identified proteins for a better understanding of liver biology.</p

A simple and reliable protocol for mouse serum proteome profiling studies by use of two-dimensional electrophoresis and MALDI TOF/TOF mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Unravelling the serum proteome is the subject of intensified research. In this regard, two-dimensional electrophoresis coupled with MALDI MS analysis is still one of the most commonly used method. Despite some improvements, there is the need for better protocols to enable comprehensive identification of serum proteins.</p> <p>Here we report a combination of two proteomic strategies, zoom in acidic and neutral part of 2-D gels and an application of two optimised matrix preparations for MALDI-MS analyses to simplify serum proteome mapping.</p> <p>Results</p> <p>Mouse serum proteins were separated by 2-D electrophoresis at the pH ranges 3–10 and 4–7, respectively. Then in gel tryptic digests were analysed by MALDI-MS. Notably, sample-matrix preparations consisted of either a thin-layer α-ciano-4-hydroxycinnamic acid (CHCA) matrix deposition or a matrix-layer 2,5-dihydroxybenzoic acid (DHB). This enabled an identification of 90 proteins. The herein reported method enhanced identification of proteins by 32% when compared with previously published studies of mouse serum proteins, using the same approaches. Furthermore, experimental improvements of matrix preparations enabled automatic identification of mouse proteins, even when one of the two matrices failed.</p> <p>Conclusion</p> <p>We report a simple and reliable protocol for serum proteome analysis that combines an optimized resolution of 2-D gels spots and improved sample-matrix preparations for MALDI-MS analysis. The protocol allowed automated data acquisition for both CHCA and DHB and simplified the MS data acquisition therefore avoiding time-consuming procedures. The simplicity and reliability of the developed protocol may be applied universally.</p

Exon Array Analysis to Identify Diethyl-nitrosamine Differentially Regulated and Alternately Spliced Genes in Early Liver Carcinogenesis in the Transgenic Mouse ATT-myc Model

Objectives: To identify the regulated genes or the spliced genes of diethylnitorsamine (NDEA) in ATT-myc mice versus control group. Methods: We analysed the 9 hybridizations on the MouseExon10ST array of NDEA treatments and control non- transgenic by application of a mixed model analysis of variance. Results: The 907 genes had regulated significantly between the groups and 916 genes had regulated with a significant exon-group interaction among of them 150 genes had regulated with both gene and possible splicing differences (p<0.01). The 7,618 genes had tested for the alternative gene up-regulation and splicing and compared to the gene-classifications. The genes functions, pathways and gene-classifications in the current study had presented in the contingency table analysis of the set of the regulated genes and alternatively spliced that regulated significantly in the ATT-myc mice treated by diethylnitorsamine versus control non-transgenic. The GOMolFn of gene-classification had 321 groups that had significantly regulated in the set of the regulated genes or differentially spliced. While the GOProcess of gene-classification had 330 groups that had significantly regulated in the set of differentially regulated genes or spliced. Additionally, the CELlLoc of gene-classification had 70 groups that had significantly regulated in the set of differentially regulated genes spliced. Finally, the Pathway gene-classification had 8 groups that had significantly regulated in the set of differentially regulated genes or spliced (p<0.01) in diethylnitorsamine when compared to control group. Conclusion: we summarized the toxicogenomics induced by diethylnitrosamine in early liver carcinogenesis in ATT-myc transgenic mice of liver cancer.   Doi: 10.28991/SciMedJ-2021-0302-6 Full Text: PD

Molecular Characterization of Lung Dysplasia Induced by c-Raf-1

BACKGROUND: Lung cancer is a multistage process with poor prognosis and high morbidity. Importantly, the genetics of dysplasia, a facultative cancer, at the edge of malignant transformation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We employed laser microdissection to harvest c-Raf1- induced dysplastic as opposed to transgenic but otherwise morphologically unaltered epithelium and compared findings to non-transgenic lung. We then employed microarrays to search genome wide for gene regulatory networks. A total of 120 and 287 genes were significantly regulated, respectively. Dysplasia was exclusive associated with up-regulation of genes coding for cell growth and proliferation, cell-to-cell signalling and interaction, lipid metabolism, development, and cancer. Likewise, when dysplasia was compared with non-transgenic cells up-regulation of cancer associated genes, tight junction proteins, xenobiotic defence and developmental regulators was observed. Further, in a comparison of the data sets of dysplasia vs transgenic and dysplasia vs non-transgenic 114 genes were regulated in common. We additionally confirmed regulation of some genes by immunohistochemistry and therefore demonstrate good concordance between gene regulation and coded protein. CONCLUSION: Our study identified transcriptional networks at successive stages of tumor-development, i.e. from histological unaltered but transgenic lungs to nuclear atypia. Our SP-C/c-raf transgenic mouse model revealed interesting and novel candidate genes and pathways that provide clues on the mechanism forcing respiratory epithelium into dysplasia and subsequently cancer, some of which might also be useful in the molecular imaging and flagging of early stages of disease

Genome wide prediction of HNF4α functional binding sites by the use of local and global sequence context

An application of machine learning algorithms enables prediction of the functional context of transcription factor binding sites in the human genome

Toward predictive models for drug-induced liver injury in humans: are we there yet?

Drug-induced liver injury (DILI) is a frequent cause for the termination of drug development programs and a leading reason of drug withdrawal from the marketplace. Unfortunately, the current preclinical testing strategies, including the regulatory-required animal toxicity studies or simple in vitro tests, are insufficiently powered to predict DILI in patients reliably. Notably, the limited predictive power of such testing strategies is mostly attributed to the complex nature of DILI, a poor understanding of its mechanism, a scarcity of human hepatotoxicity data and inadequate bioinformatics capabilities. With the advent of high-content screening assays, toxicogenomics and bioinformatics, multiple end points can be studied simultaneously to improve prediction of clinically relevant DILIs. This review focuses on the current state of efforts in developing predictive models from diverse data sources for potential use in detecting human hepatotoxicity, and also aims to provide perspectives on how to further improve DILI prediction