Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues

The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate pithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour aterial when compared to non-malignant tissue (P< 0.05; Mann–Whitney U -test). Inhibin/activin βA- and βB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of βB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While βB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin α-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin βB-subunit mRNA or by a decrease of ActRIB mRNA levels. © 2000 Cancer Research Campaig

DATAMAN: A global database of methane, nitrous oxide, and ammonia emission factors for livestock housing and outdoor storage of manure

Livestock manure management systems can be significant sources of nitrous oxide (N2O), methane (CH4), and ammonia (NH3) emissions. Many studies have been conducted to improve our understanding of the emission processes and to identify influential variables in order to develop mitigation techniques adapted to each manure management step (animal housing, outdoor storage, and manure spreading to land). The international project DATAMAN (http://www.dataman.co.nz) aims to develop a global database on greenhouse gases (N2O, CH4) and NH3 emissions from the manure management chain to refine emission factors (EFs) for national greenhouse gas and NH3 inventories. This paper describes the housing and outdoor storage components of this database. Relevant information for different animal categories, manure types, livestock buildings, outdoor storage, and climatic conditions was collated from published peer reviewed research, conference papers, and existing databases published between 1995 and 2021. In the housing database, 2024 EFs were collated (63% for NH3, 19.5% for CH4, and 17.5% for N2O). The storage database contains 654 NH3 EFs from 16 countries, 243 CH4 EFs from 13 countries, and 421 N2O EFs from 17 countries. Across all gases, dairy cattle and swine production in temperate climate zones are the most represented animal and climate categories. As for the housing database, the number of EFs for the tropical climate zone is under-represented. The DATAMAN database can be used for the refinement of national inventories and better assessment of the cost-effectiveness of a range of mitigation strategies

Overexpression of Full-Length ETV1 Transcripts in Clinical Prostate Cancer Due to Gene Translocation

ETV1 is overexpressed in a subset of clinical prostate cancers as a fusion transcript with many different partners. However, ETV1 can also be overexpressed as a full-length transcript. Full-length ETV1 protein functions differently from truncated ETV1 produced by fusion genes. In this study we describe the genetic background of full-length ETV1 overexpression and the biological properties of different full-length ETV1 isoforms in prostate cancer. Break-apart FISH showed in five out of six patient samples with overexpression of full-length ETV1 a genomic rearrangement of the gene, indicating frequent translocation. We were able to study the rearrangements in more detail in two tumors. In the first tumor 5′-RACE on cDNA showed linkage of the complete ETV1 transcript to the first exon of a prostate-specific two exon ncRNA gene that maps on chromosome 14 (EST14). This resulted in the expression of both full-length ETV1 transcripts and EST14-ETV1 fusion transcripts. In chromosome spreads of a xenograft derived from the second prostate cancer we observed a complex ETV1 translocation involving a chromosome 7 fragment that harbors ETV1 and fragments of chromosomes 4 and 10. Further studies revealed the overexpression of several different full-length transcripts, giving rise to four protein isoforms with different N-terminal regions. Even the shortest isoform synthesized by full-length ETV1 stimulated in vitro anchorage-independent growth of PNT2C2 prostate cells. This contrasts the lack of activity of even shorter N-truncated ETV1 produced by fusion transcripts. Our findings that in clinical prostate cancer overexpression of full-length ETV1 is due to genomic rearrangements involving different chromosomes and the identification of a shortened biologically active ETV1 isoform are highly relevant for understanding the mechanism of ETV1 function in prostate cancer

Capturing complex tumour biology in vitro: Histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means

Spontaneous development of Epstein-Barr Virus associated human lymphomas in a prostate cancer xenograft program

Prostate cancer research is hampered by the lack of in vivo preclinical models that accurately reflect patient tumour biology and the clinical heterogeneity of human prostate cancer. To overcome these limitations we propagated and characterised a new collection of patient-derived prostate cancer xenografts. Tumour fragments from 147 unsupervised, surgical prostate samples were implanted subcutaneously into immunodeficient Rag2-/-γC-/- mice within 24 hours of surgery. Histologic and molecular characterisation of xenografts was compared with patient characteristics, including androgen-deprivation therapy, and exome sequencing. Xenografts were established from 47 of 147 (32%) implanted primary prostate cancers. Only 14% passaged successfully resulting in 20 stable lines; derived from 20 independent patient samples. Surprisingly, only three of the 20 lines (15%) were confirmed as prostate cancer; one line comprised of mouse stroma, and 16 were verified as human donor-derived lymphoid neoplasms. PCR for Epstein-Barr Virus (EBV) nuclear antigen, together with exome sequencing revealed that the lymphomas were exclusively EBV-associated. Genomic analysis determined that 14 of the 16 EBV+ lines had unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements, confirming their B-cell origin. We conclude that the generation of xenografts from tumour fragments can commonly result in B-cell lymphoma from patients carrying latent EBV. We recommend routine screening, of primary outgrowths, for latent EBV to avoid this phenomenon

Capturing complex tumour biology in vitro : histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1 alpha. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.Peer reviewe

Modulation of Androgen Receptor Signaling in Hormonal Therapy-Resistant Prostate Cancer Cell Lines

Background: Prostate epithelial cells depend on androgens for survival and function. In (early) prostate cancer (PCa) androgens also regulate tumor growth, which is exploited by hormonal therapies in metastatic disease. The aim of the present study was to characterize the androgen receptor (AR) response in hormonal therapy-resistant PC346 cells and identify potential disease markers. Methodology/Principal Findings: Human 19K oligoarrays were used to establish the androgen-regulated expression profile of androgen-responsive PC346C cells and its derivative therapy-resistant sublines: PC346DCC (vestigial AR levels), PC346Flu1 (AR overexpression) and PC346Flu2 (T877A AR mutation). In total, 107 transcripts were differentially-expressed in PC346C and derivatives after R1881 or hydroxyflutamide stimulations. The AR-regulated expression profiles reflected the AR modifications of respective therapy-resistant sublines: AR overexpression resulted in stronger and broader transcriptional response to R1881 stimulation, AR down-regulation correlated with deficient response of AR-target genes and the T877A mutation resulted in transcriptional response to both R1881 and hydroxyflutamide. This AR-target signature was linked to multiple publicly available cell line and tumor derived PCa databases, revealing that distinct functional clusters were differentially modulated during PCa progression. Differentiation and secretory functions were up-regulated in primary PCa but repressed i

Bypass Mechanisms of the Androgen Receptor Pathway in Therapy-Resistant Prostate Cancer Cell Models

Background: Prostate cancer is initially dependent on androgens for survival and growth, making hormonal therapy the cornerstone treatment for late-stage tumors. However, despite initial remission, the cancer will inevitably recur. The present study was designed to investigate how androgen-dependent prostate cancer cells eventually survive and resume growth under androgen-deprived and antiandrogen supplemented conditions. As model system, we used the androgen-responsive PC346C cell line and its therapy-resistant sublines: PC346DCC, PC346Flu1 and PC346Flu2. Methodology/Principal Findings: Microarray technology was used to analyze differences in gene expression between the androgen-responsive and therapy-resistant PC346 cell lines. Microarray analysis revealed 487 transcripts differentiallyexpressed between the androgen-responsive and the therapy-resistant cell lines. Most of these genes were common to all three therapy-resistant sublines and only a minority (,5%) was androgen-regulated. Pathway analysis revealed enrichment in functions involving cellular movement, cell growth and cell death, as well as association with cancer and reproductive system disease. PC346DCC expressed residual levels of androgen receptor (AR) and showed significant down-regulation of androgen-regulated genes (p-value = 10 27). Up-regulation of VAV3 and TWIST1 oncogenes and repression of the DKK3 tumor-suppressor was observed in PC346DCC, suggesting a potential AR bypass mechanism. Subsequent validation of these three genes in patient samples confirmed that expression was deregulated during prostate cancer progression

Four plant defensins from an indigenous South African Brassicaceae species display divergent activities against two test pathogens despite high sequence similarity in the encoding genes

<p>Abstract</p> <p>Background</p> <p>Plant defensins are an important component of the innate defence system of plants where they form protective antimicrobial barriers between tissue types of plant organs as well as around seeds. These peptides also have other activities that are important for agricultural applications as well as the medical sector. Amongst the numerous plant peptides isolated from a variety of plant species, a significant number of promising defensins have been isolated from Brassicaceae species. Here we report on the isolation and characterization of four defensins from <it>Heliophila coronopifolia</it>, a native South African Brassicaceae species.</p> <p>Results</p> <p>Four defensin genes (<it>Hc-AFP1</it>-<it>4) </it>were isolated with a homology based PCR strategy. Analysis of the deduced amino acid sequences showed that the peptides were 72% similar and grouped closest to defensins isolated from other Brassicaceae species. The Hc-AFP1 and 3 peptides shared high homology (94%) and formed a unique grouping in the Brassicaceae defensins, whereas Hc-AFP2 and 4 formed a second homology grouping with defensins from <it>Arabidopsis </it>and <it>Raphanus</it>. Homology modelling showed that the few amino acids that differed between the four peptides had an effect on the surface properties of the defensins, specifically in the alpha-helix and the loop connecting the second and third beta-strands. These areas are implicated in determining differential activities of defensins. Comparing the activities after recombinant production of the peptides, Hc-AFP2 and 4 had IC₅₀values of 5-20 μg ml^-1against two test pathogens, whereas Hc-AFP1 and 3 were less active. The activity against <it>Botrytis cinerea </it>was associated with membrane permeabilization, hyper-branching, biomass reduction and even lytic activity. In contrast, only Hc-AFP2 and 4 caused membrane permeabilization and severe hyper-branching against the wilting pathogen <it>Fusarium solani</it>, while Hc-AFP1 and 3 had a mild morphogenetic effect on the fungus, without any indication of membrane activity. The peptides have a tissue-specific expression pattern since differential gene expression was observed in the native host. <it>Hc-AFP1 </it>and <it>3 </it>expressed in mature leaves, stems and flowers, whereas <it>Hc-AFP2 </it>and <it>4 </it>exclusively expressed in seedpods and seeds.</p> <p>Conclusions</p> <p>Two novel Brassicaceae defensin sequences were isolated amongst a group of four defensin encoding genes from the indigenous South African plant <it>H. coronopifolia</it>. All four peptides were active against two test pathogens, but displayed differential activities and modes of action. The expression patterns of the peptide encoding genes suggest a role in protecting either vegetative or reproductive structures in the native host against pathogen attack, or roles in unknown developmental and physiological processes in these tissues, as was shown with other defensins.</p

Aqueous and gaseous nitrogen losses induced by fertilizer application

In recent years concern has grown over the contribution of nitrogen (N) fertilizer use to nitrate (NO{sub 3}{sup -}) water pollution and nitrous oxide (N{sub 2}O), nitric oxide (NO), and ammonia (NH{sub 3}) atmospheric pollution. Characterizing soil N effluxes is essential in developing a strategy to mitigate N leaching and emissions to the atmosphere. In this paper, a previously described and tested mechanistic N cycle model (TOUGHREACT-N) was successfully tested against additional observations of soil pH and N{sub 2}O emissions after fertilization and irrigation, and before plant emergence. We used TOUGHREACT-N to explain the significantly different N gas emissions and nitrate leaching rates resulting from the different N fertilizer types, application methods, and soil properties. The N{sub 2}O emissions from NH{sub 4}{sup +}-N fertilizer were higher than from urea and NO{sub 3}{sup -}-N fertilizers in coarse-textured soils. This difference increased with decreases in fertilization application rate and increases in soil buffering capacity. In contrast to methods used to estimate global terrestrial gas emissions, we found strongly non-linear N{sub 2}O emissions as a function of fertilizer application rate and soil calcite content. Speciation of predicted gas N flux into N{sub 2}O and N{sub 2} depended on pH, fertilizer form, and soil properties. Our results highlighted the need to derive emission and leaching factors that account for fertilizer type, application method, and soil properties