Search

Cardiosphere-derived cells (CDCs) ameliorate skeletal and cardiac muscle deterioration in experimental models of Duchenne muscular dystrophy. The HOPE-2 trial examined the safety and efficacy of sequential intravenous infusions of human allogeneic CDCs in late-stage Duchenne muscular dystrophy.In this multicentre, randomised, double-blind, placebo-controlled, phase 2 trial, patients with Duchenne muscular dystrophy, aged 10 years or older with moderate upper limb impairment, were enrolled at seven centres in the USA. Patients were randomly assigned (1:1) using stratified permuted blocks to receive CAP-1002 (1·5 × 10Between March 1, 2018, and March 31, 2020, 26 male patients with Duchenne muscular dystrophy were enrolled, of whom eight were randomly assigned to the CAP-1002 group and 12 to the placebo group (six were not randomised due to screening failure). In patients who had a post-treatment PUL 1.2 assessment (eight in the CAP-1002 group and 11 in the placebo group), the mean 12-month change from baseline in mid-level elbow PUL1.2 favoured CAP-1002 over placebo (percentile difference 36·2, 95% CI 12·7-59·7; difference of 2·6 points; p=0·014). Infusion-related hypersensitivity reactions without long-term sequelae were observed in three patients, with one patient discontinuing therapy due to a severe allergic reaction. No other major adverse reactions were noted, and no deaths occurred.CAP-1002 cell therapy appears to be safe and effective in reducing deterioration of upper limb function in patients with late-stage Duchenne muscular dystrophy. Various measures of cardiac function and structure were also improved in the CAP-1002 group compared with the placebo group. Longer-term extension studies are needed to confirm the therapeutic durability and safety of CAP-1002 beyond 12 months for the treatment of skeletal myopathy and cardiomyopathy in Duchenne muscular dystrophy.Capricor Therapeutics.

WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three–amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)–regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway. Mol Cancer Res; 8(7); 975–85. ©2010 AACR.

MicroRNAs (miRNAs) are crucial post-transcriptional regulators of gene expression in species ranging from plants to mammals. Unraveling the complex networks of miRNA-mediated regulation of gene expression requires the ability to identify the mRNA targets of individual miRNAs in native cellular contexts. Here we describe RIP-SIR, an unbiased genome-wide method for identifying interactions between endogenous miRNAs and their targets in almost any tissue or cell type.

INTRODUCTION: Lung cancer is the leading cause of cancer deaths worldwide and greater than 75% of human lung cancer deaths can be attributed to metastasis. Metastatic disease in most existing mouse models of pulmonary adenocarcinoma is typically sporadic and often requires aging mice for several months. These factors limit the usefulness of most current mouse models for basic and pre-clinical research aimed at identifying mechanisms and effective treatments of metastatic disease. We have developed two novel mouse models of metastatic lung cancer and report here on changes in protein expression and immune cell recruitment in the lungs of these models. METHODS AND RESULTS: Mouse models were generated by adding mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway, to a mouse model of Kras-driven pulmonary adenocarcinoma. For both models, tumorigenesis was induced by conditional expression of an oncogenic allele of Kras (KrasG12D), deletion of both alleles of Trp53 and deletion of one allele of Dicer1 in one cell type and the expression of a truncated Dicer1 allele in a different cell type. In the absence of Dicer1 truncation, mice expressing KrasG12D and deleting Trp53 in club cells have a reported median survival of 28.6 weeks after tumor induction. We detected accelerated development of pulmonary adenocarcinoma and lymph node metastasis (12.1 weeks) only when we induced tumorigenesis in club cells and truncated DICER1 in alveolar type II (ATII) cells. Induction of tumorigenesis in ATII cells and truncation of DICER1 in club cells did not accelerate tumorigenesis or metastasis. To evaluate the molecular and cellular changes in the different phenotypes of these models we have characterized protein expression, using spatial proteomics, and immune cell recruitment, using flow cytometry. CONCLUSIONS: Through cell type specific truncation/deletion of Dicer1 we have generated a new mouse model that rapidly develops pulmonary adenocarcinomas and metastatic disease. Our findings support our hypothesis that tumorigenesis and metastasis are influenced by miRNA regulated communication between different cell types. Preliminary flow cytometry and spatial proteomic analyses have suggested cellular targets underlying phenotypic differences between our two mouse models. These models have potential for both understanding the basic processes of metastasis and for pre-clinical studies aimed at preventing and/or treating metastatic lung cancer. Citation Format: Paige Ramkissoon, Julie Wells, Richard S. Maser, Jiayuan Shi, Zheng Gong, Qing Li, Brian Hoffmann, Anne Marchini, Elaine Bechtel, Rosalinda Doty, Gary Ren, Carol J. Bult. New mouse models of metastatic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1321.

Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse, however there are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. With miRNA-seq analysis we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and EGFR mutated lung cancer cells. Whole transcriptome analysis of single-cell derived clones reveals a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived three-dimensional (3D) structures. This link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.

Tertiary lymphoid structures (TLSs) are a recently-described immune response that occurs when chronic inflammation leads to an organ-like organization of immune cells and specialized blood vessels. In humans, they have been shown to develop in and around many different types of tumors, and often lead to a more promising prognosis when found. In non-small cell lung cancer (NSCLC), they have a highly beneficial effect on patients' survival. What remains to be studied is the heterogeneity of the TLSs and the mechanisms of their development. In order to study these important aspects of the immune response to NSCLC, we developed a protocol to image a time course of TLS development in murine bronchiolo-alveolar adenomas and adenocarcinomas, and further expanded on how to quantify, and therefore characterize, these structures.

INTRODUCTION: The focus of this work is to develop new mouse models for metastatic lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and greater than 75% of human lung cancer deaths can be attributed to metastasis. Metastatic disease in most existing mouse models of pulmonary adenocarcinoma is typically sporadic and often requires aging mice for several months. These factors limit the usefulness of mouse models for basic and pre-clinical research aimed at identifying effective treatments of metastatic disease. METHODS AND RESULTS: We have developed new mouse models of pulmonary adenocarcinoma in which we added mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway to a mouse model of Kras-driven pulmonary adenocarcinoma. For both models, we induced tumorigenesis by conditionally expressing an oncogenic allele of Kras (KrasG12D), deleting of both alleles of Trp53 and deleting one allele of Dicer1 in one cell population then expressing truncated Dicer1 in a different cell population. In the absence of Dicer1 truncation, mice expressing KrasG12D and deleting Trp53 in Club cells have a reported median survival of 28.6 weeks after tumor induction. When we also deleted one allele of Dicer1 in Club cells and truncated Dicer1 in alveolar type II cells of these mice, they exhibited respiratory distress and began dying 12.1 weeks after tumor induction. In our initial pilot study, all animals (12/12) developed pulmonary adenocarcinoma and 30% (3/10) had lymph node metastasis. We did not observe accelerated tumorigenesis, however, when we switched cell types, inducing tumorigenesis in alveolar type II cells and truncating Dicer1 in Club cells, or when induced tumorigenesis and truncating Dicer1 within the same cell populations. CONCLUSIONS: Through cell type specific truncation/deletion of Dicer1 we have generated a new mouse model that rapidly develops pulmonary adenocarcinomas and metastatic disease. Our findings support our hypothesis that tumorigenesis and metastasis are influenced by miRNA regulated communication between different cell types. These models have potential for both understanding the basic processes of metastasis and for pre-clinical studies aimed at preventing and/or treating metastatic lung cancer. Citation Format: Julie Wells, Richard Maser, Ashley Tucker, Teresa McGee, Wendy Memishian, Rosalinda Doty, Carol J. Bult. Developing new mouse models of metastatic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 927.

This chapter explores the history of Australian university governance reform and the drivers for change. It argues that a critical element in achieving institutional diversity and responsiveness will be the capacity for universities to negotiate effectively with government around the tension between autonomy and state control in university governance structures, and advance solutions which support both autonomy and accountability. The reforms led to stronger risk management, and more sophisticated business and strategic planning – aspects of governance which are integral to universities’ capacity to set directions. Yet the focus on accountability and autonomy in discussions of Australian universities’ governance and management continues. The social compact which universities, as institutions established to serve the public good, rely upon to justify their claims to taxpayer support is also under pressure. The internal legislation and regulatory frameworks of many Australian universities were perhaps less rapid to evolve in this developing landscape.

INTRODUCTION: The focus of this work is to develop new mouse models for metastatic lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and greater than 90% of human cancer deaths are due to metastasis. Metastatic disease in most existing mouse models of lung cancer is typically sporadic and often requires aging mice for several months. These factors limit the usefulness of mouse models for basic and pre-clinical research aimed at effective treatment of metastatic disease. METHODS AND RESULTS: We have developed a new mouse model of lung cancer and are in the process of developing two additional models. In the first model, we added mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway to a mouse model of Kras-driven pulmonary adenocarcinoma. When we combined conditional expression of an oncogenic allele of Kras (KrasG12D), deletion of both alleles of Trp53 and one allele of Dicer1 in Club cells with expression of truncated Dicer1 in alveolar type II cells, we generated mice with metastatic pulmonary adenocarcinoma. In 30% of these mice, metastatic tumors were observed in the lymph nodes within 11 weeks of tumor induction. In a second mouse model, we switched the cell types expressing the various mutations to determine the effects of cell of origin on tumor progression and metastasis. Our preliminary results suggest that adenocarcinoma develops even faster when KrasG12D is expressed and Trp53 and one allele of Dicer1 are deleted in alveolar type II cells. Finally, based on an analysis of human lung cancer genomics data from TCGA, we generated a mouse model with mutations in Zfhx4 and are crossing these mice with our models that express oncogenic KrasG12D, delete expression of Trp53 and one allele of Dicer1 and truncate the second allele of Dicer1 to determine the effects of adding Zfhx4 mutations on progression and metastasis of pulmonary adenocarcinoma. CONCLUSIONS: Through cell type specific truncation/deletion of Dicer1 we have generated new mouse models that rapidly develop pulmonary adenocarcinomas and metastatic disease. These models have potential for both understanding the basic processes of metastasis and for pre-clinical studies aimed at preventing and/or treating metastatic lung cancer. Citation Format: Julie Wells, Richard S. Maser, Teresa McGee, Wendy Memishian, Rosalinda Doty, Carol J. Bult. Developing new mouse models of metastatic lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1617.

Author(s): Selleri, Licia; Welsh, Ian; Hansen, Karissa; Osterwalder, Marco; Losa-Llabata, Marta; Wells, Julie; Bult, Carol; Mohun, Timothy; Hu, Diane; Marcucio, Ralph; Visel, Axel; Swigut, Tomek

In Canada in 2015, the pass rates on the National Council Licensure Examination (NCLEX-RN) were considerably lower than pass rates on the Canadian Registered Nurse Examination (CRNE) causing nurse educators to express concern regarding the NCLEX-RN. The purpose of this study was to examine the relationship between candidate variables (e. g. academic performance, demographics) on their NCLEX-RN outcome (pass/fail). A cross-sectional data linkage design was employed using multiple sources of data on nursing graduates who wrote the NCLEX-RN in 2015, 2016 and 2017 (n = 259). Results showed that fewer questions answered on the NCLEX-RN and higher grades in various nursing courses (e. g. Introduction to Nursing, Statistics) predicted higher odds of passing the NCLEX-RN. To improve pass rates, nurse educators must integrate diverse methods of testing into existing curricula that mimic the NCLEX-RN exam, specifically computer adaptive exams. Further research is needed to determine other possible challenges for countries considering adopting the NCLEX-RN.

Congenital diaphragmatic hernia (CDH) is a common and severe birth defect. Despite its clinical significance, the genetic and developmental pathways underlying this disorder are incompletely understood. In this study, we report a catalog of variants detected by a whole exome sequencing study on 275 individuals with CDH. Predicted pathogenic variants in genes previously identified in either humans or mice with diaphragm defects are enriched in our CDH cohort compared with 120 size-matched random gene sets. This enrichment was absent in control populations. Variants in these critical genes can be found in up to 30.9% of individuals with CDH. In addition, we filtered variants by using genes derived from regions of recurrent copy number variations in CDH, expression profiles of the developing diaphragm, protein interaction networks expanded from the known CDH-causing genes, and prioritized genes with ultrarare and highly disruptive variants, in 11.3% of CDH patients. These strategies have identified several high priority genes and developmental pathways that likely contribute to the CDH phenotype. These data are valuable for comparison of candidate genes generated from whole exome sequencing of other CDH cohorts or multiplex kindreds and provide ideal candidates for further functional studies. Furthermore, we propose that these genes and pathways will enhance our understanding of the heterogeneous molecular etiology of CDH.

The Macmillan Values Based Standard aims to achieve a transformation in patients' and families' experience of care. Importantly, the standard provides a framework and methodology for improving both patient and staff experience. Of all health professionals, healthcare assistants (HCAs) spend the most ‘face-time’ with patients and are therefore crucial in the drive to transform patients' experience of care. HCAs have contributed to the implementation of the Values Based Standard in a number of Macmillan pilot sites and they have demonstrated how the framework can help drive improvement. Macmillan believes HCAs are key to changing the way patients and their families experience care. For this reason, Macmillan is investing in testing a new development programme, designed specifically to support HCAs, while also ensuring that the organisations they work within focus on the Values Based Standard and provide workforce support to HCAs. This article introduces the programme.

ADH-1 (Exherin™) is a pentapeptide, which competitively inhibits N-cadherin, resulting in vascular disruptive effect of tumor vasculature in preclinical models. This study was designed to assess the toxicity of ADH-1 and to determine the maximal tolerated dose (MTD). Patients and Methods: Adult patients with advanced measurable solid tumors were stratified according to their tumor N-cadherin status. ADH-1 was administered as a short infusion, every six weeks. Assessment of response was done every 6 weeks. PK parameters included: estimated volume of distribution of the central compartment, the α and β phase half-lives, area under the plasma concentration- time curve (AUC), clearance, and volume of distribution. Target lesions were assessed by dynamic contrast enhancing- magnetic resonance imaging (DCE-MRI). Results: 46 patients were enrolled, 25 (54%) had N-cadherin positive status. The doses administered ranged from 50 mg/m2 to 1000 mg/m2, and the MTD was not reached. The PK analysis of the concentration-time data displayed a biphasic profile. Most of the toxicities were grade 1 and 2 with fatigue, nausea, chest pain and dysgeusia being the most common. Eleven patients had disease control, the single patient who had partial response had N-cadherin positive tumor. Conclusion: ADH-1 is a well tolerated drug with a modest anti tumor effect in tumors which express N-cadherin.

To characterize temporal patterns of transcriptional activity during normal lung development, we generated genome wide gene expression data for 26 pre- and post-natal time points in three common inbred strains of laboratory mice (C57BL/6J, A/J, and C3H/HeJ). Using Principal Component Analysis and least squares regression modeling, we identified both strain-independent and strain-dependent patterns of gene expression. The 4,683 genes contributing to the strain-independent expression patterns were used to define a murine Developing Lung Characteristic Subtranscriptome (mDLCS). Regression modeling of the Principal Components supported the four canonical stages of mammalian embryonic lung development (embryonic, pseudoglandular, canalicular, saccular) defined previously by morphology and histology. For postnatal alveolar development, the regression model was consistent with four stages of alveolarization characterized by episodic transcriptional activity of genes related to pulmonary vascularization. Genes expressed in a strain-dependent manner were enriched for annotations related to neurogenesis, extracellular matrix organization, and Wnt signaling. Finally, a comparison of mouse and human transcriptomics from pre-natal stages of lung development revealed conservation of pathways associated with cell cycle, axon guidance, immune function, and metabolism as well as organism-specific expression of genes associated with extracellular matrix organization and protein modification. The mouse lung development transcriptome data generated for this study serves as a unique reference set to identify genes and pathways essential for normal mammalian lung development and for investigations into the developmental origins of respiratory disease and cancer. The gene expression data are available from the Gene Expression Omnibus (GEO) archive (GSE74243). Temporal expression patterns of mouse genes can be investigated using a study specific web resource (http://lungdevelopment.jax.org).

Background Olaparib is an orally available inhibitor of PARP-1. In pre-clinical studies, olaparib was shown to potentiate anti-tumor effects of irinotecan in colon cancer cell lines. This phase I study was conducted to evaluate the safety and tolerability of olaparib in combination with irinotecan. Patients and Methods Patients with advanced colorectal cancer whose disease progressed after at least one systemic therapy regimen were enrolled. Dose escalation and de-escalation were based on toxicity assessment. Pharmacokinetic samples were collected in Cycle 1 for olaparib, irinotecan and SN-38. Results Twenty-five patients were enrolled, 11 patients on a schedule of continuous olaparib and irinotecan every 3 weeks (Part A) and 14 patients on a schedule of intermittent olaparib and irinotecan every 2 weeks (Part B). Continuous olaparib administration was associated with higher than expected toxicities and was not considered to be tolerable. Intermittent olaparib administration was better tolerated, and the recommended phase 2 doses were olaparib 50 mg p.o twice daily days 1-5 and irinotecan 125 mg/m(2) i.v. every 2 weeks. Common toxicities included fatigue, anorexia, diarrhea, nausea, vomiting, neutropenia, thrombocytopenia and abdominal pain. Nine patients had stable disease as the best response, 2 from Part A (3 and 9 months respectively), and 7 from Part B (median duration: 7.4 months; range: 4 to 13 months). There was no pharmacokinetic interaction between olaparib and irinotecan. Conclusions Olaparib can be combined with irinotecan if administered intermittently. Both olaparib and irinotecan required significant dose reductions. The lack of anti-tumor efficacy observed in this trial makes this combination of little interest for further clinical development. Trial Registration ID NCT00535353.

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre–B-cell leukemia transcription factor 1 ( Pbx1 ), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

The National Institute for Health and Clinical Excellence (NICE) in Clinical Guideline 83: Rehabilitation after critical illness has set challenging recommendations regarding the routine evaluation for, and provision of, rehabilitation within critical care units. There is no published information regarding current practice in the UK. To establish current practice in Scotland we undertook a telephone survey of all 23 Scottish ICU lead clinicians and physiotherapists − 96% of lead clinicians and 100% of lead physiotherapists completed the survey. Routine assessment for physical (median two (IQR 1–3 [range 0–4])) and non-physical sequelae (median zero (IQR 0–2 [range 0–5])) is low. Aproximately half of ICUs (52%) provide an individualised, structured rehabilitation programme, 32% include activities of daily living and all provide low intensity rehabilitation (eg limb stretching and positioning exercises). There are significant differences (all p

WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three–amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)–regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway. Mol Cancer Res; 8(7); 975–85. ©2010 AACR.

Ovol2 belongs to the Ovo family of evolutionarily conserved zinc finger transcription factors that act downstream of key developmental signaling pathways including Wg/Wnt and BMP/TGF-beta. We previously reported Ovol2 expression in the basal layer of epidermis, where epidermal stem/progenitor cells reside. In this work, we use HaCaT human keratinocytes to investigate the cellular and molecular functions of Ovol2. We show that depletion of Ovol2 leads to transient cell expansion but a loss of cells with long term proliferation potential. Mathematical modeling and experimental findings suggest that both faster cycling and precocious withdrawal from the cell cycle underlie this phenotype. Ovol2 depletion also accelerates extracellular signal-induced terminal differentiation in two- and three-dimensional culture models. By chromatin immunoprecipitation, luciferase reporter, and functional rescue assays, we demonstrate that Ovol2 directly represses two critical downstream targets, c-Myc and Notch1, thereby suppressing keratinocyte transient proliferation and terminal differentiation, respectively. These findings shed light on how an epidermal cell maintains a proliferation-competent and differentiation-resistant state.

WTX encodes a tumor suppressor gene inactivated in Wilms tumor and recently implicated in WNT signaling through enhancement of cytoplasmic β-catenin (CTNNB1) degradation. Here, we report that WTX translocates to the nucleus, a property that is modified by an endogenous splicing variant and is modulated by a nuclear export inhibitor. WTX is present in distinct subnuclear structures and co-localizes with the paraspeckle marker p54NRB/NONO, suggesting a role in transcriptional regulation. Notably, WTX binds WT1, another Wilms tumor suppressor and stem cell marker that encodes a zinc-finger transcription factor, and enhances WT1-mediated transcription of Amphiregulin , an endogenous target gene. Together, these observations suggest a role for WTX in nuclear pathways implicated in the transcriptional regulation of cellular differentiation programs.

A team of practitioners, university researchers, and health care policy makers has been working to develop and apply “design sciences” thinking within the challenging context of a national system aiming to bring about a “revolution in health care.” As members of that team, the authors share that thinking and early findings with those interested in the concept, theory, and practice of design as an approach to large-scale organizational change. The article builds on what to date has been a somewhat abstract debate around the design sciences, its aim being to forge stronger links between the concept and the practice of design. Using empirical data from the English National Health Service as a case study, the article seeks to demonstrate how design sciences may first, expand our thinking around organizational theory and practice and second, offer organization development some new methods, approaches, and processes around the “doing” of large-scale change.

In May 2003, a survey questionnaire was distributed to all licensed primary care physicians in Newfoundland and Labrador. The objective was to examine the attitudes, self-reported practices, and continuing medical education (CME) preferences of primary care physicians as they pertain to prostate cancer screening.Data was obtained from 485 primary care physicians using self-reports of prostate cancer screening practices, attitudes towards prostate cancer screening, and CME preferences. Respondents' characteristics were also collected (eg, gender, years of experience).A majority of respondents screen asymptomatic male patients for prostate cancer. Screening behaviour was related to high volume practice settings, fee-for-service and increased with patient age. Most common reasons for screening were family history, age of patient, and patient request. Majority of physicians agreed that prostate screening should be routinely performed on all men beginning at age 50, however half of physicians believe there is lack of evidence to support digital rectal examination (DRE) and one-third of physicians do not believe the prostate-specific antigen (PSA) nor DRE are accurate screening tests. Areas of greatest interest for CME included topics related to prostate cancer screening effectiveness, strategies for prevention, sexual dysfunction, available treatments and their side effects, and management options.Physicians are supportive of the value of screening, however the reliability of and evidence to support DRE and PSA as prostate cancer screening tests are in question. CME which addresses issues surrounding prostate screening and areas related to patient education and counselling are of greatest need.

SUMO-1 conjugation modulates numerous cellular functions, including the subnuclear localization of its target proteins. The WT1 tumor suppressor encodes a four-zinc finger protein with distinct splicing isoforms. WT1(−KTS), encoding uninterrupted zinc fingers, functions as a transcription factor and has a diffusely nuclear distribution; WT1(+KTS), with an insertion of three amino acids (KTS) between zinc fingers three and four, localizes to discrete nuclear speckles, the function of which is unknown. Because the SUMO-1 E2-conjugating enzyme, Ubc9, interacts with WT1, we tested whether sumoylation modulates the cellular localization of WT1. We find here that both WT1 isoforms are directly sumoylated on lysine residues 73 and 177. Although RNA interference-mediated Ubc9 depletion effectively suppresses WT1 nuclear speckles, a SUMO-1–deficient WT1(+KTS)(K73, 177R) double mutant retains localization to speckles. Thus, direct sumoylation of WT1 is not responsible for its cellular localization, and other sumoylated proteins may target WT1 to these nuclear structures. Identification of other components of WT1-associated speckles is likely to provide clues to their function.

The retinoblastoma (Rb) tumor suppressor protein is an important regulator of cell proliferation and differentiation. Many studies have shown that pRb can negatively regulate the activity of the E2F family of transcription factors during G(0) and G(1) phases of the cell cycle, perhaps by serving as a bridge between the E2Fs and transcriptional repressors such as histone deacetylases and methylases. However, pRb has also been shown to localize to discrete DNA foci during S phase, a time at which pRb is thought to be dissociated from E2F. Numerous other DNA binding proteins have been shown to interact with pRb, suggesting that pRb may control progression through S phase by binding to sites in the genome distinct from E2F target gene promoters. To test this hypothesis, we have identified novel pRb binding sites within the human genome using an unbiased approach which relies upon a combination of chromatin immunoprecipitation and CpG microarray analysis. To provide the greatest opportunity of finding distinct sets of pRb binding sites, we examined pRb binding in chromatin obtained from human Raji cells synchronized in either G(0)/G(1) phase or S phase. These experiments have allowed us to identify a large set of new genomic binding sites for the pRb protein. We found that some sites are occupied by pRb only during G(0)/G(1) phase, as would be predicted from previous models of pRb function. We also identified sites to which pRb bound only during S phase and other sites which were bound constitutively by pRb. Surprisingly, we found that E2F1 was present at most of the CpG islands bound by pRb, independent of the phase of the cell cycle. Thus, although pRb has the potential to interact with numerous transcription factors, our data suggest that the majority of DNA-bound pRb is recruited to E2F target promoters during both G(0)/G(1) and S phases.

In their article, A.S. Weinmann and P.J. Farnham (2002, Methods 26) described new techniques for isolating in vivo binding sites for any DNA-binding protein. In this article, we describe complementary methods for detailed in vivo characterizations of such identified protein-DNA interactions. First, we describe how formaldehyde crosslinking and chromatin immunoprecipitation (ChIP), in conjunction with transient transfections or the use of cell lines containing stably integrated constructs or episomes, can be employed to identify which specific nucleotides of a region of DNA are required for recruitment of a particular transcription factor. In contrast to in vivo footprinting, this method not only specifies which nucleotides are bound, but also identifies the protein(s) involved in binding. Next, we discuss the use of the ChIP assay to study how binding of a transcription factor is altered by passage through the cell cycle, by overexpression or deletion of another factor, or during tumorigenesis. Finally, a look toward the future suggests that the ChIP assay may be combined with Western blot analysis or mass spectrometry to identify additional proteins that interact with a transcription factor of interest.

e14637 Background: OVs display oncolytic activity and boost adaptive cell immunity. MG1MA3 is a Maraba virus modified to express tumour antigen MAGE-A3. MG1MA3, both alone and after immune priming with a MAGE-A3 modified adenovirus (AdMA3) may trigger anti-tumour T-cell responses. Methods: N = 41 patients (pts) with MAGE-A3 expressing solid tumours were evaluated in 3 groups (A) Dose escalation of MG1MA3 iv d1+4, q8w (n = 9); (B) Single fixed dose AdMA3 1e10 pfu IM d1 (n = 6); (C) AdMA3 priming d(-14) followed by dose escalated MG1MA3 (d1+4, q8w) (n = 25). Arm A and C had a 3+3 design. Pre + post treatment blood and tissue biopsies were evaluated for viral and immune markers. Endpoints included MTD/MFD, RP2DL, safety, tolerability, pharmacokinetics, viral delivery and replication. Results: Dose limiting toxicities (hypoxia/dyspnea, vomiting, headache) occurred in 4 pts (2 each Arm A +C). RP2DL for arm C was AdMA3 1e10 pfu IM d(-14) then MG1MA3 1e11 pfu iv d1+4. Common treatment related toxicities on Arm C occurring hours to a few days after MG1MA3 included diarrhea, nausea, vomiting, anorexia, chills, fatigue, fever, flu-like symptoms, hypophosphatemia, headache, and hypotension. Preliminary tumour gene expression results reveal induction of pro-inflammatory genes, including chemokines (CCL2, CCL5, CX3CL1, CXCL10), acute phase response proteins (IL-6, TNF), antigen presenting cell (APC) activation markers (CD80, HLA-A, HLA-B), markers of APC and Natural Killer cell infiltration (CD56, CD68, TLR3), as well as a co-incident decrease in the suppressive cytokine TGF-β. MG1MA3 replication was observed in some pts, inferred by detection of circulating genomes on days 4, 8 and 15 after clearance of the input dose. Induction of anti-tumour immune responses (CD8 T cells and antibodies vs MAGE-A3) was demonstrated in 3 of 6 Arm C pts evaluated to date. In one patient, over 1% of circulating CD8 T cells were directed against MAGE-A3. Conclusions: AdMA3 prime followed by MG1MA3 OV boost is feasible with a defined RP2DL, and capable of inducing potent anti-tumour immune response. Alternate schedules will be evaluated. Clinical trial information: NCT02285816.

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

The E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S-phase transition of the mammalian cell cycle (see reference 16 for an extensive review of the E2F regulatory pathway). E2F binding sites are found in the promoters of genes whose products are required for nucleotide synthesis (e.g., dihydrofolate reductase [DHFR] and thymidine kinase [TK]), for DNA replication (e.g., DNA polymerase α and cdc6), and for cell cycle progression (e.g., cyclin E, cyclin D1, c-myc, b-myb, and cdc2). Transcription from each of these promoters increases during late G1 or early S phase, and this regulation is mediated by protein binding to one or more E2F binding sites (3, 11, 22, 30, 34, 43, 44). To date, eight members of the E2F family have been identified: six E2F proteins (E2F1 to -6) and two DP proteins (DP1 and DP2). The E2F and DP proteins bind to DNA as a heterodimer and can function as activators of transcription. Alternatively, E2F-DP heterodimers can also repress transcription when complexed with members of the Rb family of pocket proteins (pRb, p107, and p130) due to the ability of the pocket proteins to bind to and mask the E2F transactivation domain and to recruit histone deacetylases (6, 17, 25). Individual E2Fs preferentially bind to different pocket proteins. E2F1, E2F2, and E2F3, for example, bind to pRb, while E2F4 predominantly binds to p130 and p107 and E2F5 binds to p130. A popular model for how E2F family members regulate G1/S-phase-specific gene expression invokes a complex pattern of protein-protein and protein-DNA interactions that change as cells progress through the cell cycle (Fig. ​(Fig.1A).1A). As depicted, transcription from E2F site-containing promoters is thought to be repressed in G0 phase due to the binding of a trimolecular E2F-DP-pocket protein complex and recruitment of histone deacetylase activity by the pocket protein component (6, 17, 25). As cells progress through the cell cycle, various cyclin–cyclin-dependent kinase (cdk) complexes phosphorylate the pocket proteins, causing release of the hyperphosphorylated pocket protein and associated proteins from the DNA-bound E2F-DP heterodimer (1, 2, 7). Finally, traversal through S phase is thought to be accompanied by cyclin-cdk-mediated phosphorylation of the DP subunit of E2F1-3–DP complexes, resulting in release of the heterodimers from the promoter DNA (15, 21, 42). In some cells, E2F4-containing complexes are thought to be inactivated by relocation to the cytoplasm (28, 38). FIG. 1 E2F-mediated transcriptional regulation. (A) The current model of E2F-mediated transcriptional regulation is thought to involve binding of an E2F-pocket protein complex to promoters in G0 phase of the cell cycle and release of the pocket protein in late ... Although this model is attractive, it is largely based upon circumstantial data, and several important questions remain unanswered. For example, the transcriptional activity of complexes containing E2F4 and E2F5 may be shut off during mid- to late S phase by association with unphosphorylated p107 or p130, rather than by relocation to the cytoplasm (10). Additionally, it is not known if E2F target gene specificity exists or if all six E2Fs bind to and regulate every target gene or if Rb family members display target gene specificity. Determination of target gene specificity has been difficult due to the fact that most cells studied to date contain all of the E2Fs and pocket proteins. Thus, most analyses of E2F and pocket protein binding specificity have been performed using in vitro systems or by overexpression of an individual E2F or pocket protein in cells. In vitro systems, however, cannot recapitulate the complex environment of living cells, and altering the relative amounts of individual proteins through overexpression may abolish important protein-protein interactions. Therefore, we wished to determine which E2Fs and pocket proteins bind to and regulate expression of specific target genes in intact cells under physiological conditions. We felt that the use of an unperturbed in vivo system was of particular importance in the analysis of E2F target gene specificity since regulation occurs in the context of cell cycle progression which cannot be mimicked in vitro and which is often altered when individual E2F proteins are overexpressed. Toward this goal, we have used a formaldehyde cross-linking and immunoprecipitation system to monitor protein-DNA and protein-protein interactions on E2F target genes in living cells. Importantly, this procedure has allowed us to determine the in vivo patterns of E2F and pocket protein binding to specific E2F target genes as cells progress through a cell cycle. Our results indicate that, while some promoters fit the proposed model for E2F-mediated transcriptional regulation, others do not, necessitating individualization of the current model. Furthermore, our data suggest that cell cycle-regulated activity is a stochastic event, as cells have the ability to form several different E2F-pocket protein complexes on a given promoter at each stage of the cell cycle.

Adrenocortical Carcinoma (ACC) is rare with an annual incidence of 0.5-2 cases per million worldwide. Some ACC tumors over express N-cadherin, which correlates with metastatic potential. ADH-1 (Exherin™) is a competitive inhibitor of N-cadherin, resulting in rapid onset of tumor vascular angiolysis and apoptosis in preclinical models. Targeting N-cadherin may cause direct anti-tumor and anti-vascular effects. We report a case of ACC with benefit from ADH-1 therapy. A 24 year old woman with an N-cadherin expressing metastatic ACC was treated on a phase I trial and treated with ADH-1 subsequently received additional doses through a special access program. The patient presented with cushingoid features from cortisol over-secretion and was diagnosed with metastatic ACC in January 2003. Tumor progression followed treatment with a combination of doxorubicin, cisplatin and mitotane. In October 2003, as a part of a phase I clinical trial she was treated with as a single dose of ADH-1 at 150 mg/m(2). This resulted in transient normalization of cortisol, tumor necrosis on CT imaging, and reduction in tumor perfusion on DCE-MRI. Following progression on several additional lines of chemotherapy, she was again treated with ADH-1 under a Special Access Program (SAP). After 33 weekly doses (22 with 150 mg/m(2) and 11 with 300 mg/m(2)) radiographic tumor progression was demonstrated and treatment discontinued. She survived 40 months with metastatic disease, dying 12 months after her last dose of ADH-1. This observation merits consideration for prospectively evaluating the efficacy of ADH-1 in patients with cortisol secreting ACC that over express N-cadherin.

Compared to primary keratinocytes, HaCaT cells are easier to transfect and yet still maintain at least some features of normal epidermal proliferation and differentiation. This chapter describes methods used in our laboratory to maintain HaCaT cells in an undifferentiated state and to use the siRNA technology to efficiently deplete a gene product of interest from these cells. We also provide protocols on how to couple siRNA knockdown with a clonal assay to examine keratinocyte proliferation potential and a luciferase reporter assay to examine promoter regulation.

Compared to primary keratinocytes, HaCaT cells are easier to transfect and yet still maintain at least some features of normal epidermal proliferation and differentiation. This chapter describes methods used in our laboratory to maintain HaCaT cells in an undifferentiated state and to use the siRNA technology to efficiently deplete a gene product of interest from these cells. We also provide protocols on how to couple siRNA knockdown with a clonal assay to examine keratinocyte proliferation potential and a luciferase reporter assay to examine promoter regulation.

MOST research examining the relationship between women's smoking and stress has focused on women living on a low income. The present study looked in detail at comparative groups of women in terms of smoking status (that is, smokers, non-smokers and ex-smokers) and social class, and investigated simi larities and differences in terms of coping strategies. Results suggest that not all women who smoke do so to relieve stress, though women across all social classes suffer from stress. Health promotion should therefore address itself to promoting positive coping mechanisms for all women. Women who do empha sise smoking as a vital coping strategy are likely to live on a low income, in poor housing and often live in isolated conditions with pre-school children. Health promotion must therefore seek to work with welfare agencies in combating the structural factors which cause women to depend on health-damaging coping strategies.

Wilms tumor is a pediatric kidney cancer associated with inactivation of the WT1 tumor-suppressor gene in 5 to 10% of cases. Using a high-resolution screen for DNA copy-number alterations in Wilms tumor, we identified somatic deletions targeting a previously uncharacterized gene on the X chromosome. This gene, which we call WTX , is inactivated in approximately one-third of Wilms tumors (15 of 51 tumors). Tumors with mutations in WTX lack WT1 mutations, and both genes share a restricted temporal and spatial expression pattern in normal renal precursors. In contrast to biallelic inactivation of autosomal tumor-suppressor genes, WTX is inactivated by a monoallelic “single-hit” event targeting the single X chromosome in tumors from males and the active X chromosome in tumors from females.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

3016 Background: Malignant transformation and invasiveness correlate with upregulation of N-cad, a cell adhesion molecule expressed on tumor cells and vascular endothelium. N-cad antagonism by ADH-1, a cyclic pentapeptide (CHAVC), causes rapid tumor vascular disruption and apoptosis in preclinical models. We report the final clinical and PK results from the first human study (AHX-01–001). Methods: 46 subjects with incurable solid tumors stratified according to tumor N-cad expression were enrolled. 99 doses of ADH-1 were given by IV bolus or short infusion (31 in a Special Access Program). Doses ranged from 50–1000 mg/m2, with 3–6 subjects/cohort. Blood and urine were collected for PK on Day 1, assayed by LC/MS/MS, and a 2 compartment analysis was performed. Results: ADH-1 was generally well tolerated and the maximum tolerated dose was not defined. The most commonly reported adverse events were grade 1–2 fatigue (59%), nausea (32%), dysgeusia (25%), and flushing (20%). 4 subjects in the N-cad positive group (n=28, including 3 N-cad unknown) demonstrated anti-tumor activity; a PR of 6 month duration in refractory esophageal carcinoma; a minor response in lung metastases from an unknown primary following a single dose; and >7 months SD in refractory adrenocortical and colorectal carcinoma. No anti-tumor activity was noted in the N-cad negative group (n=18). The mean initial ADH-1 half-life (t½) was 20 min and the mean terminal phase t½ was 2.2 hr. Mean systemic clearance (CL) was 6.5 L/hr/m2. Both t½ and CL were independent of dose. Cmax increased dose proportionally, ranging from 7 to148 μg/mL, and AUC0-∞ tended to increase dose proportionally. The volume of distribution at steady-state (Vss) averaged 14.3 L/m2, indicating modest tissue distribution. Approximately 30% of the dose was excreted unchanged in the urine. Conclusions: ADH-1 was generally well tolerated and demonstrated evidence of anti-tumor activity in subjects with N-cad positive tumors. The PK profile was biphasic and the t½ averaged 2.2 hr. Cmax and AUC0-∞ increased dose proportionally, and CL was independent of dose. No significant financial relationships to disclose.

3038 Background: Malignant transformation and invasive capacity is highly correlated with upregulation of N-cadherin, a cell adhesion molecule expressed on both tumor cells and vascular endothelium. ADH-1 is a cyclic pentapeptide (CHAVC) which competitively inhibits N-cadherin mediated endothelial cell, pericyte and tumor cell interaction resulting in rapid onset of tumor vascular angiolysis and apoptosis in preclinical models. We report safety and pharmacokinetics (PK) from the first human study. Methods: 33 patients with refractory solid tumors received 54 cycles of ADH-1 by bolus injection at 8 dose escalations from 50 to 840 mg/m2to define toxicities (AE) and maximum tolerated dose (MTD) stratified by cadherin status with N-cadherin positive (n=12), unknown (n=3) or negative (n=18) tumors. Dynamic MRI was performed in cohorts 3 to 8 where feasible to assess tumor perfusion changes. Results: MTD has not been reached. The most common AEs were grade 1–2 nausea (27%), fatigue (58%), hot flushes (15%) and ...