p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Governing Public Health: EU Law, Regulation and Biopolitics

This book contributes towards EU studies and the growing discourse on law and public health. It uses the EU’s governance of public health as a lens through which to explore questions of legal competence and its development through policy and concrete techniques, processes and practices, risk and security, human rights and bioethics, accountability and legitimacy, democracy and citizenship, and the nature, essence and ‘future trajectory’ of the European integration project. These issues are explored first, by situating the EU's public health strategy within the overarching architecture of governance and subsequently by examining its operationalisation in relation to the key public health problems of cancer, HIV/AIDS and pandemic planning.

The book argues that the centrality and valorisation of scientific and technical knowledge and expertise in the EU's risk-based governance means that citizen participation in decision-making is largely marginalised and underdeveloped – and that this must change if public health and the quality, accountability and legitimacy of EU governance and its regulation are to be improved. Subsequently the book goes on to argue that the legitimating discourses of ethics and human rights, and the developing notion of EU (supra-)stewardship responsibility, can help to highlight the normative dimensions of governance and its interventions in public health. These discourses and dimensions provide openings and possibilities for citizens to power ‘technologies of participation’ and contribute important supplementary knowledge to decision-making.

Role played by BRCA1 in transcriptional regulation in response to therapy

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor, implicated in the hereditary predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of cellular processes including DNA repair and recombination, cell cycle checkpoint control, chromatin remodelling and ubiquitination. In addition, substantial data now exist to suggest a role for BRCA1 in transcriptional regulation; BRCA1 has been shown to interact with the Pol II holoenzyme complex and to interact with multiple transcription factors, such as p53 and c-Myc. We have previously identified a range of BRCA1 transcriptional targets and have linked these to specific cellular pathways, including cell cycle checkpoint activation and apoptosis. Current research is focused on the transcriptional mechanisms that underpin the association of BRCA1 deficiency with increased sensitivity to DNA damage-based chemotherapy and resistance to spindle poisons.

Spatial and temporal regulation of the pesticide dieldrin within industrial catchments

The river catchments of south Yorkshire support a very high density of wool processing industries. Dieldrin was once used as a moth proofing agent, as a sheep dip, and as a pesticide to protect wool fleeces during storage and transport, all of which caused pollution of these catchments due to textile processing. Weekly sampling of four of these rivers revealed two classes of dieldrin contamination: the Aire and Calder (the rivers which support very high concentrations of wool processing industries) had higher concentrations (averaging ~3 ng/l) than the Don and Trent (~1 ng/l). The average flux of dieldrin from these rivers into the Humber estuary was 9.8 g/day, with the Aire (of which the Calder is a tributary) and the Trent contributing almost equally, with a smaller contribution from the Don. The Trent has the highest average flow, explaining its large contribution to dieldrin flux. Less detailed sampling of rivers from the north Humber catchment which drain predominantly rural areas had dieldrin concentrations similar to the heavily industrialized southern catchment rivers. This suggests that dieldrin from agronomic and domestic usage may be more persistent than the pollution caused by textile processing industries. Evidence is presented to suggest that the principle dieldrin sources to the Humber catchments are sewage treatment plants, and that the dieldrin sources are in rapid equilibrium with the water column. (C) 2000 Elsevier Science B.V.

Divergent androgen regulation of unfolded protein response pathways drives prostate cancer

The unfolded protein response (UPR) is a homeostatic mechanism to maintain endoplasmic reticulum (ER) function. The UPR is activated by various physiological conditions as well as in disease states, such as cancer. As androgens regulate secretion and development of the normal prostate and drive prostate cancer (PCa) growth, they may affect UPR pathways. Here, we show that the canonical UPR pathways are directly and divergently regulated by androgens in PCa cells, through the androgen receptor (AR), which is critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1α branch, but simultaneously inhibited PERK signaling. Inhibition of the IRE1α arm profoundly reduced PCa cell growth in vitro as well as tumor formation in preclinical models of PCa in vivo. Consistently, AR and UPR gene expression were correlated in human PCa, and spliced XBP-1 expression was significantly upregulated in cancer compared with normal prostate. These data establish a genetic switch orchestrated by AR that divergently regulates the UPR pathways and suggest that targeting IRE1α signaling may have therapeutic utility in PCa.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

ChIPping away at gene regulation

The coordinated regulation of gene expression in higher eukaryotes is complex and poorly understood. Recent technological advances have allowed the first insights into these networks on a genome-wide scale. These investigations have identified transcription factor target sites in the genome and successfully predicted cooperative interactions with other factors. However, a detailed understanding of the processes that coordinate gene expression remains elusive. Here, we highlight the advances that have been made using current methods, and the need for new technologies to address the gaps in our knowledge and to map these complex pathways further.

Regulation of endosome fusion

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

What is being taught in Religious Education classes in Northern Ireland and to what extent should states be concerned about regulation, quality and control of the subject?

This paper reports on a research project designed to discover what schools are teaching in Religious Education in Northern Ireland and what procedures are in place to maintain standards in the delivery of the subject. A search through literature shows that little research has been carried out to determine what is being taught in Religious Education in Northern Ireland. It also indicates that there are very weak systems of control to measure the effectiveness or quality of what is delivered. A survey of the websites of all Post-Primary schools in the region was used to provide some answers to the basic question of what is being taught in RE. Using content and discourse analysis of these alongside supporting documentary sources (textbooks and exam specifications), it is possible to get a clearer picture of how the Northern Ireland Core Syllabus for Religious Education and any additional curricular elements are delivered in schools. The findings show that a significant minority of schools do not publicly articulate what pupils do in religious education. In situations where the content of religious education is made clear, some definite trends are evident. Despite the existence of a statutory core syllabus, there is significant variation in what is taught in schools. The content is most divergent from the syllabus in relation to the teaching of World Religions at Key Stage 3 and at Key Stage 4 whole elements of the syllabus are neglected due to limited conformity between the syllabus and exam specifications. These results raise important questions about the systems of regulation and control of the subject in the region. In law the subject is exempt from formal inspection by the local inspection authority; instead, a form of inspection is allowed for by the Christian churches who design the syllabus, but it is a process that is either entirely neglected or entirely unreported in situations where it does occur. It is argued that these findings raise questions of more general concern for this and other regions in Europe where the teaching of religious education is largely unregulated. For example, to what extent should states take an interest in what is taught in religious education, how it is delivered, what values it promotes and how standards of teaching and learning in the subject are upheld?