Characterisations of human prostate stem cells reveal deficiency in class I UGT enzymes as a novel mechanism for castration-resistant prostate cancer.

BACKGROUND: Evidence increasingly supports that prostate cancer is initiated by the malignant transformation of stem cells (SCs). Furthermore, many SC-signalling pathways are shown to be shared in prostate cancer. Therefore, we planned transcriptome characterisation of adult prostate SCs as a strategy to consider new targets for cancer treatment. METHODS: Intuitive pathway analysis was used for putative target discovery in 12 matched selections of human prostate SCs, transiently amplifying cells and terminally differentiated cells. These were pooled into three groups according to the stage of differentiation for mRNA microarray analysis. Targets identified were validated using uncultured primary tissue (n=12), functional models of prostate cancer and a tissue microarray consisting of benign (n=42) and malignant prostate (n=223). RESULTS: A deficiency in class 1 UDP glucuronosyltransferase (UGT) enzymes (UGT1A) was identified in prostate SCs, which are involved in androgen catabolism. Class 1 UGT enzyme expression was also downregulated in cancer SCs and during progression to metastatic castration-resistant prostate cancer (CRPC). Reduction of UGT1A expression in vitro was seen to improve cell survival and increase androgen receptor (AR) activity, as shown by upregulation of prostate-specific antigen expression. INTERPRETATION: Inactivation of intracellular androgen catabolism represents a novel mechanism to maintain AR activity during CRPC.

The induction of core pluripotency master regulators in cancers defines poor clinical outcomes and treatment resistance.

Stem cell characteristics have been associated with treatment resistance and poor prognosis across many cancer types. The ability to induce and regulate the pathways that sustain these characteristic hallmarks of lethal cancers in a novel in vitro model would greatly enhance our understanding of cancer progression and treatment resistance. In this work, we present such a model, based simply on applying standard pluripotency/embryonic stem cell media alone. Core pluripotency stem cell master regulators (OCT4, SOX2 and NANOG) along with epithelial-mesenchymal transition (EMT) markers (Snail, Slug, vimentin and N-cadherin) were induced in human prostate, breast, lung, bladder, colorectal, and renal cancer cells. RNA sequencing revealed pathways activated by pluripotency inducing culture that were shared across all cancers examined. These pathways highlight a potential core mechanism of treatment resistance. With a focus on prostate cancer, the culture-based induction of core pluripotent stem cell regulators was shown to promote survival in castrate conditions-mimicking first line treatment resistance with hormonal therapies. This acquired phenotype was shown to be mediated through the upregulation of iodothyronine deiodinase DIO2, a critical modulator of the thyroid hormone signalling pathway. Subsequent inhibition of DIO2 was shown to supress expression of prostate specific antigen, the cardinal clinical biomarker of prostate cancer progression and highlighted a novel target for clinical translation in this otherwise fatal disease. This study identifies a new and widely accessible simple preclinical model to recreate and explore underpinning pathways of lethal disease and treatment resistance.

Correction: The induction of core pluripotency master regulators in cancers defines poor clinical outcomes and treatment resistance.

The original version of this article contained an error in Fig. 5a where the colours of the labels representing the Hinge and LBD of the AR were incorrect and did not match the corresponding exons. The corrected version of this Figure now appears in the article. The conclusions of this paper were not affected. The authors apologise for this error and any confusion caused.

Sequence organization of the soybean genome.

The total complexity of one constituent soybean (Glycine max) genome is estimated to be 1.29 . 10(9) nucleotide pairs, as determined by analysis of the reassociation kinetics of sheared (0.47 kilobase) DNA. Single copy sequences are estimated to represent from 53 to 64% of the genome by analysis of hydroxyapatite binding of repetitive DNA as a function of fragment length. From 65 to 70% of these single copy sequences have a short period interspersion with 1.11--1.36 kilobase lengths alternating with 0.3--0.4 kilobase repetitive sequence elements. The repetitive sequences of soybean DNA are interspersed both among themselves and among single copy regions of the genome.

Diadenosine 5',5'''-P1,P4-tetraphosphate (AP4A) levels under various proliferative and cytotoxic conditions in several mammalian cell types.

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) has been proposed as an intracellular signal for growth. In order to test this hypothesis Ap4A levels were followed in several cell types under various conditions. Quiescent dog thyroid cells in a primary culture were induced to proliferate by addition of a mixture of epidermal growth factor, thyrotropin and foetal calf serum; V79 cells were synchronized by serum depletion then stimulated to proliferate by addition of foetal calf serum. Protein and DNA synthesis increased in both cases, although no significant changes in Ap4A levels per cell could be demonstrated. HeLa D98/AH2 and L929 cells were treated with human recombinant tumour necrosis factor alpha which caused marked cell death. This was measured by a decrease in DNA content and a release into extracellular medium of incorporated radioactive precursor. No concomitant variations in Ap4A concentrations could be observed under these conditions. The data from these various systems do not support the hypothesis that changes in Ap4A levels regulate cellular proliferation.

Rapid phosphorylation of a 27 kDa protein induced by tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to induce the phosphorylation of a 27 kDa protein in a time- and concentration-dependent manner in HeLa D98/AH2, ME 180 and bovine aortic endothelial cells. This phosphorylation could be reproduced by the calcium ionophore, A23187. However, this phosphorylation was not observed in L929 cells, for which TNF is highly cytotoxic, suggesting that it might play a role in actions of TNF other than the induction of cell death.

SIZE: a program to determine the size of nucleic acid and protein molecules from gel mobilities.

The relationship between fragment length and the reciprocal of mobility and the least-squares curve fitting analysis for this relationship have been combined into a user-friendly program for determining the size of unknown DNA fragments by reference to known standards. Protein sizes can be determined in the same way. The program offers an accurate, simple-to-use mechanism for sizing molecules that requires only an IBM-compatible computer.

Effect of adenosine 3',5' monophosphate on the mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine.

The effect of increased cellular concentrations of adenosine 3',5' monophosphate (cAMP) upon mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in V79 Chinese hamster lung cells. Incubation with either forskolin, which increased the accumulation of cAMP, or 8BrcAMP, an analogue of cAMP, resulted in an increase in the mutation frequency which was concentration-dependent, regardless of whether these agents were added before or after mutagen treatment. Increased cAMP concentrations were shown in these cells to inhibit growth; however, this does not seem to be the mechanism responsible for the increase in mutation frequency as low serum concentrations which also retard growth reduced the mutation frequency observed with MNNG.

Small-scale techniques for the analysis of recombinant plasmids.

Using the cloning of part of the T-DNA of pTiC58 from Agrobacterium tumefaciens as an example, techniques are described which enable recombinant plasmids to be mapped and used as hybridization probes. In all cases the starting material is a colony of cells grown on an agar plate which is then subjected to lysis by lysozyme and Triton X-100 in volumes of the order of 300 microliters thus eliminating the need for handling and centrifuging liquid cultures under restrictive containment conditions.

Eosinophilic cholecystitis as a possible late manifestation of the eosinophilia-myalgia syndrome.

We describe a case of acute acalculous cholecystitis occurring in a 43-year-old woman with a history of the eosinophilia-myalgia syndrome, associated with the ingestion of 1-tryptophan. The patient underwent a laparoscopic cholecystectomy and subsequent histological examination of the gallbladder revealed an infiltrate predominantly of eosinophils, suggesting a possible relationship to the underlying condition. This may represent a late complication of the eosinophilia-myalgia syndrome--such an association has not previously been reported in the literature. The gastrointestinal and hepatic complications of this syndrome are discussed.

Long-term remission from gout associated with fenofibrate therapy.

Short-term studies with fenofibrate, an established treatment for hyperlipidaemia, have shown that its unique side effect of urate lowering is mediated through enhanced renal urate clearance. The long-term effects of fenofibrate on hyperuricaemia and gout have not previously been reported. We report two patients with hyperlipidaemia in association with hyperuricaemia in whom long-term fenofibrate therapy was associated with a sustained reduction in serum urate and lipid levels, together with remission from recurrent attacks of acute gout. The mechanisms involved in these effects and the potential role for fenofibrate in the management of gout are discussed.

Power lines: Derrida, discursive psychology and the management of accusations of teacher bullying.

This study connects broad issues of classroom control and the disciplining of pupils by teachers with a detailed examination of the way teachers deal with an implied accusation that they have been bullying. The analysis of interviews develops with reference to discursive psychology and Derrida's development of deconstruction. Billig's (1992) insights into ways that participants' accounts can neutralize threats to established social arrangements are employed in relating detailed analytic points to the broader power relations between teacher and pupil. Interviews were conducted with Scottish secondary school teachers, and subjected to close textual analysis. This resulted in the development of three themes: (1) Subjectivity Construction, in which the functional role of the construction of mental entities is examined; (2) Normalizing Techniques, identifying strategies whereby intimidation can be constructed as normal; and (3) Figuration, examining the utility of figurative language--metaphors, maxims, and so on. These themes display the subtlety and complexity of teachers' strategies for distancing themselves from being held accountable for reported intimidation. To conclude, three broader features of the study are discussed: the contribution to discursive psychology that Derrida's deconstructive philosophy can make; the respecification of psychology and subjectivity as participants' resources for action; and the contribution that this type of detailed study can make to issues of power and social critique.

An atraumatic femoral fracture in a patient with rheumatoid arthritis and osteoporosis treated with denosumab.

Osteoporosis is responsible for a significant burden both individually and socially, but is readily treated with antiresorptive agents and mineral supplementation. However, long-term usage of these agents, notably bisphosphonates, is rarely associated with atypical fractures. Denosumab is a monoclonal antibody that reduces osteoclast activity and thus increases bone mineral density. In this case report, we present a 78-year-old woman with a background of rheumatoid arthritis and osteoporosis who presented with an atypical diaphyseal femoral fracture.

Both Fcgamma and complement receptors mediate transfer of immune complexes from erythrocytes to human macrophages under physiological flow conditions in vitro.

Abnormal clearance by the mononuclear phagocytic system of immune complexes (IC) is important in the pathogenesis of systemic lupus erythematosus (SLE). We have developed an in vitro model to investigate the cellular mechanisms involved in the transfer of soluble IC from erythrocytes to human macrophages under physiological flow conditions. In this assay, erythrocytes bearing fluorescently labelled IC are perfused over monolayers of human monocytes or monocyte-derived macrophages in a parallel-plate flow chamber, and transfer quantified using confocal microscopy and flow cytometry. Using aggregated human IgG as a model IC, we have been able to demonstrate transfer of IC from erythrocytes to macrophages. Blocking studies with specific neutralizing antibodies have shown that both complement and Fcgamma receptors are required for IC transfer. Blockade of CR4 (alpha(x)beta(2) integrin), FcgammaRIIa or FcgammaRIII reduced transfer, while anti-CR3 (alpha(m)beta(2) integrin) had no effect. Blockade of CR3, FcgammaRIIa or FcgammaRIII also reduced the number of adhesive interactions between fluorescently labelled IC-bearing erythrocytes and macrophage monolayers. Taken together with the transfer data, this suggests differing roles for these receptors in the human IC transfer reaction that includes an adhesive function which facilitates IC processing by mononuclear phagocytes. Finally, a functional effect of the FcgammaRIIa R131/H131 polymorphism, important in susceptibility to SLE, has also been demonstrated using this model. Uptake of IgG(2) but not IgG(1)-containing soluble IC was reduced by macrophages from individuals homozygous for the R131 allelic variant of the receptor.

Extra DNA in the T region of crown gall Ti-plasmid pTiA66.

The mutant tumorigenic phenotype of pTiA66, a derivative of the broad host range octopine Ti-plasmid pTiA6, results from a 2.6-Kb insertion into EcoRI fragment 32g of the T region, which has been implicated in the auxin synthesis disruption tumor character. The inserted DNA is closely related to sequences from BamHI fragment 11 of the same or a related plasmid but probably originally derives from a chromosomal sequence.

The effect of right terminal repeat deletion on the oncogenicity of the T-Region of pTiT37.

A modified pTiT37 plasmid was constructed by deleting a 103 base fragment between an AhaIII and a Bc/I site. This fragment, located to the right of the nopaline synthase gene contains the right terminal 25 base pair repeat sequence which defines the right limit of the T-Region. The effect of this deletion was determined on a number of host plants. In contrast to previous reports, the deletion does not destroy tumorigenicity on all plant species. It had no effect on tumorigenicity when Linum usitatissimum was used as the test species and an attenuating effect when Kalanchoë tubiflora was used. Only when Nicotiana tabacum was used did the mutant appear avirulent. We propose from these data and the phenotype of those tumours that form, that a pseudo border located in the 3' untranslated region of the ipt locus has been used to provide the right hand limit of the T-Region in the absence of the normal border.