Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

Potential detrimental effects of a phytoestrogen-rich diet on male fertility in mice.

Soy and soy-based products are widely consumed by infants and adult individuals. There has been speculation that the presence of isoflavone phytoestrogens in soybean cause adverse effects on the development and function of the male reproductive system. The purpose of this study was to examine the influence of dietary soy and phytoestrogens on testicular and reproductive functions. Male mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Although adult mice fed a soy-rich diet exhibited normal male behaviour and were fertile, we observed a reduced proportion of haploid germ cells in testes correlating with a 25% decrease in epididymal sperm counts and a 21% reduction in litter size. LH and androgens levels were not affected but transcripts coding for androgen-response genes in Sertoli cells and Gapd-s, a germ cell-specific gene involved in sperm glycolysis and mobility were significantly reduced. In addition, we found that dietary soy decreased the size of the seminal vesicle but without affecting its proteolytic activity. Taken together, these studies show that long-term exposure to dietary soy and phytoestrogens may affect male reproductive function resulting in a small decrease in sperm count and fertility.

Combined effect of 25-OH vitamin D plasma levels and genetic vitamin D receptor (NR 1I1) variants on fibrosis progression rate in HCV patients.

BACKGROUND: Decreased vitamin D levels have been described in various forms of chronic liver disease and associated with advanced fibrosis. Whether this association is a cause or consequence of advanced fibrosis remains unclear to date. AIMS: To analyse combined effects of 25-OH vitamin D plasma levels and vitamin D receptor gene (VDR; NR1I1) polymorphisms on fibrosis progression rate in HCV patients. METHODS: 251 HCV patients underwent VDR genotyping (bat-haplotype: BsmI rs1544410 C, ApaI rs7975232 A and TaqI rs731236 A). Plasma 25-OH vitamin D levels were quantified in a subgroup of 97 patients without advanced fibrosis. The VDR haplotype and genotypes as well as plasma 25-OH vitamin D levels were associated with fibrosis progression. RESULTS: The bAt[CCA]-haplotype was significantly associated with fibrosis progression >0.101 U/year (P = 0.007; OR = 2.02) and with cirrhosis (P = 0.022; OR = 1.84). Forty-five percent of bAt[CCA]-haplotype patients were rapid fibrosers, 21.1% were cirrhotic. Likewise, ApaI rs7975232 CC genotype was significantly associated with fibrosis progression and cirrhosis. Lower plasma 25-OH vitamin D levels were significantly associated with fibrosis progression >0.101 U/year in F0-2 patients (P = 0.013). Combined analysis of both variables revealed a highly significant additive effect on fibrosis progression with 45.5% rapid fibrosers for bAt[CCA]-haplotype and 25-OH vitamin D < 20 μg/L compared with only 9.1% for the most favourable combination (P = 0.006). In multivariate analysis, the bAt-haplotype was an independent risk factor for fibrosis progression (P = 0.001; OR = 2.83). CONCLUSION: Low 25-OH vitamin D plasma levels and the unfavourable VDR bAt[CCA]-haplotype are associated with rapid fibrosis progression in chronic HCV patients. In combination, both variables exert significant additive effects on fibrosis progression.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Dual activation of pathways regulated by steroid receptors and peptide growth factors in primary prostate cancer revealed by Factor Analysis of microarray data

Background: We use an approach based on Factor Analysis to analyze datasets generated for transcriptional profiling. The method groups samples into biologically relevant categories, and enables the identification of genes and pathways most significantly associated to each phenotypic group, while allowing for the participation of a given gene in more than one cluster. Genes assigned to each cluster are used for the detection of pathways predominantly activated in that cluster by finding statistically significant associated GO terms. We tested the approach with a published dataset of microarray experiments in yeast. Upon validation with the yeast dataset, we applied the technique to a prostate cancer dataset. Results: Two major pathways are shown to be activated in organ-confined, non-metastatic prostate cancer: those regulated by the androgen receptor and by receptor tyrosine kinases. A number of gene markers (HER3, IQGAP2 and POR1) highlighted by the software and related to the later pathway have been validated experimentally a posteriori on independent samples. Conclusion: Using a new microarray analysis tool followed by a posteriori experimental validation of the results, we have confirmed several putative markers of malignancy associated with peptide growth factor signalling in prostate cancer and revealed others, most notably ERRB3 (HER3). Our study suggest that, in primary prostate cancer, HER3, together or not with HER4, rather than in receptor complexes involving HER2, could play an important role in the biology of these tumors. These results provide new evidence for the role of receptor tyrosine kinases in the establishment and progression of prostate cancer.

Prototypical Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

High-Throughput Screening for Novel Prostate Cancer Drug Targets –Getting Personal

Prostate cancers form a heterogeneous group of diseases and there is a need for novel biomarkers, and for more efficient and targeted methods of treatment. In this thesis, the potential of microarray data, RNA interference (RNAi) and compound screens were utilized in order to identify novel biomarkers, drug targets and drugs for future personalized prostate cancer therapeutics. First, a bioinformatic mRNA expression analysis covering 9873 human tissue and cell samples, including 349 prostate cancer and 147 normal prostate samples, was used to distinguish in silico prevalidated putative prostate cancer biomarkers and drug targets. Second, RNAi based high-throughput (HT) functional profiling of 295 prostate and prostate cancer tissue specific genes was performed in cultured prostate cancer cells. Third, a HT compound screen approach using a library of 4910 drugs and drug-like molecules was exploited to identify potential drugs inhibiting prostate cancer cell growth. Nine candidate drug targets, with biomarker potential, and one cancer selective compound were validated in vitro and in vivo. In addition to androgen receptor (AR) signaling, endoplasmic reticulum (ER) function, arachidonic acid (AA) pathway, redox homeostasis and mitosis were identified as vital processes in prostate cancer cells. ERG oncogene positive cancer cells exhibited sensitivity to induction of oxidative and ER stress, whereas advanced and castrate-resistant prostate cancer (CRPC) could be potentially targeted through AR signaling and mitosis. In conclusion, this thesis illustrates the power of systems biological data analysis in the discovery of potential vulnerabilities present in prostate cancer cells, as well as novel options for personalized cancer management.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

Review of the Y chromosome and hypertension

The Y chromosome from spontaneously hypertensive rats (SHR) has a locus that raises blood pressure 20-25 mmHg. Associated with the SHR Y chromosome effect is a 4-week earlier pubertal rise of testosterone and dependence upon the androgen receptor for the full blood pressure effect. Several indices of enhanced sympathetic nervous system (SNS) activity are also associated with the SHR Y chromosome. Blockade of SNS outflow reduced the blood pressure effect. Salt sensitivity was increased by the Y chromosome as was salt appetite which was SNS dependent. A strong correlation (r = 0.57, P<0.001) was demonstrable between plasma testosterone and angiotensin II. Coronary collagen increased with blood pressure and the presence of the SHR Y chromosome. A promising candidate gene for the Y effect is the Sry locus (testis determining factor), a transcription factor which may also have other functions.

Allelic frequencies of six polymorphic markers for risk of prostate cancer

The aim of the present study was to evaluate the distribution of polymorphisms for the androgen receptor (AR) (CAG, StuI, GGN), SRD5A2 (Ala49Thr, Val89Leu) and CYP17 (MspA1) genes that are considered to be relevant for risk of prostate cancer. We studied 200 individuals from two cities in the State of São Paulo, by PCR, PCR-RFLP and ASOH techniques. The allelic frequencies of the autosomal markers and the StuI polymorphism of the AR gene were very similar to those described in most North American and European populations. In relation to the CAG and GGN number of repeats, the study subjects had smaller repeat lengths (mean of 20.65 and 22.38, respectively) than those described in North American, European and Chinese populations. In the present study, 30.5% of the individuals had less than 22 CAG repeats and 45.5% had less than 23 GGN repeats. When both repeat lengths are considered jointly, this Brazilian population is remarkably different from the others. Further studies on prostate cancer patients need to be conducted to assess the significance of these markers in the Brazilian population.

Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Transcriptional regulation of bidirectional gene pairs by 17-β-estradiol in MCF-7 breast cancer cells

Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5’flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40% of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17% of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17β-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17β-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs.