In Vivo RNAi Screening Identifies Regulators of Actin Dynamics as Key Determinants of Lymphoma Progression

April 1, 2010Mouse models have markedly improved our understanding of cancer development and tumor biology. However, these models have shown limited efficacy as tractable systems for unbiased genetic experimentation. Here, we report the adaptation of loss-of-function screening to mouse models of cancer. Specifically, we have been able to introduce a library of shRNAs into individual mice using transplantable Eμ-myc lymphoma cells. This approach has allowed us to screen nearly 1,000 genetic alterations in the context of a single tumor-bearing mouse. These experiments have identified a central role for regulators of actin dynamics and cell motility in lymphoma cell homeostasis in vivo. Validation experiments confirmed that these proteins represent bona fide lymphoma drug targets. Additionally, suppression of two of these targets, Rac2 and twinfilin, potentiated the action of the front-line chemotherapeutic vincristine, suggesting a critical relationship between cell motility and tumor relapse in hematopoietic malignancies.National Institutes of Health (U.S.) (RO1 CA128803-01)Massachusetts Institute of Technology. Dept. of Biology (Training Grant)Massachusetts Institute of Technology. Undergraduate Research Opportunities ProgramNational Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1-U54-CA112967

Suffix-specific RNAi Leads to Silencing of F Element in Drosophila melanogaster

Separate conserved copies of suffix, a short interspersed Drosophila retroelement (SINE), and also divergent copies in the 3′ untranslated regions of the three genes, have already been described. Suffix has also been identified on the 3′ end of the Drosophila non-LTR F element, where it forms the last conserved domain of the reverse transcriptase (RT). In our current study, we show that the separate copies of suffix are far more actively transcribed than their counterparts on the F element. Transcripts from both strands of suffix are present in RNA preparations during all stages of Drosophila development, providing the potential for the formation of double-stranded RNA and the initiation of RNA interference (RNAi). Using in situ RNA hybridization analysis, we have detected the expression of both sense and antisense suffix transcripts in germinal cells. These sense and antisense transcripts are colocalized in the primary spermatocytes and in the cytoplasm of the nurse cells, suggesting that they form double-stranded RNA. We performed further analyses of suffix-specific small RNAs using northern blotting and SI nuclease protection assays. Among the total RNA preparations isolated from embryos, larvae, pupae and flies, suffix-specific small interfering RNAs (siRNAs) were detected only in pupae. In wild type ovaries, both the siRNAs and longer suffix-specific Piwi-interacting RNAs (piRNAs) were observed, whereas in ovaries of the Dicer-2 mutant, only piRNAs were detected. We further found by 3′ RACE that in pupae and ovaries, F element transcripts lacking the suffix sequence are also present. Our data provide direct evidence that suffix-specific RNAi leads to the silencing of the relative LINE (long interspersed element), F element, and suggests that SINE-specific RNA interference could potentially downregulate a set of genes possessing SINE stretches in their 5′ or 3′ non-coding regions. These data also suggest that double stranded RNAs possessing suffix are processed by both RNAi and an additional silencing mechanism

Exploring factors affecting undergraduate medical students’ study strategies in the clinical years: a qualitative study

The aim of this study is to explore the effects of clinical supervision, and assessment characteristics on the study strategies used by undergraduate medical students during their clinical rotations. We conducted a qualitative phenomenological study at King Saud Bin Abdulaziz University for Health Sciences, College of Medicine, Riyadh, Saudi Arabia during the period from November 2007 to December 2008. We conducted semi-structured focus groups interviews with students and conducted individual interviews with teachers and students to explore students’ and clinical teachers’ perceptions and interpretations of factors influencing students’ study strategies. Data collection was continued until saturation was reached. We used Atlas-ti Computer Software (Version 5.2) to analyse the data, apply the obtained themes to the whole dataset and rearrange the data according to the themes and sub-themes. Analysis of data from interviews with twenty-eight students and thirteen clinical supervisors yielded three major themes relating to factors affecting students’ study strategies: “clinical supervisors and supervision”, “stress and anxiety” and “assessment”. The three themes we identified played a role in students’ adoption of different study strategies in the “community of clinical practice”. It appeared that teachers played a key role, particularly as assessors, clinical supervisors and as a source of stress to students

The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function

BACKGROUND: The WD motif (also known as the Trp-Asp or WD40 motif) is found in a multitude of eukaryotic proteins involved in a variety of cellular processes. Where studied, repeated WD motifs act as a site for protein-protein interaction, and proteins containing WD repeats (WDRs) are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins. In the model plant Arabidopsis thaliana, members of this superfamily are increasingly being recognized as key regulators of plant-specific developmental events. RESULTS: We analyzed the predicted complement of WDR proteins from Arabidopsis, and compared this to those from budding yeast, fruit fly and human to illustrate both conservation and divergence in structure and function. This analysis identified 237 potential Arabidopsis proteins containing four or more recognizable copies of the motif. These were classified into 143 distinct families, 49 of which contained more than one Arabidopsis member. Approximately 113 of these families or individual proteins showed clear homology with WDR proteins from the other eukaryotes analyzed. Where conservation was found, it often extended across all of these organisms, suggesting that many of these proteins are linked to basic cellular mechanisms. The functional characterization of conserved WDR proteins in Arabidopsis reveals that these proteins help adapt basic mechanisms for plant-specific processes. CONCLUSIONS: Our results show that most Arabidopsis WDR proteins are strongly conserved across eukaryotes, including those that have been found to play key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and /or structure outside of the WDR regions

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation

In aggressive variants of non-Hodgkin lymphomas, Ezh2 is strongly expressed and polycomb repressive complex PRC1.4 dominates over PRC1.2

Polycomb group (PcG) proteins are important for the regulation of hematopoiesis by regulating chromatin compaction and silencing genes related to differentiation and cell cycle. Overexpression of enhancer of zeste homologue 2 (Ezh2) and Bmi-1/PCGF4 has been implicated in solid organ cancers, while Mel-18/PCGF2 has been reported as a tumor suppressor. Detailed expression profiles of PcG proteins and their diagnostic significance in malignant lymphomas are still unknown. In this study, we analyzed the expression levels of Ezh2, Bmi-1, Mel-18, and Ki67 in 197 Hodgkin's and non-Hodgkin's lymphoma patient samples and in lymphoma cell lines using immunohistochemistry, fluorescent immunocytochemistry, and Western blotting. Immunohistochemical staining showed that Ezh2 expression was significantly increased in aggressive compared to indolent subtypes of B cell neoplasms (P = 0.000-0.030), while no significant differences in Bmi-1 expression were found between these subtypes. Compared to the normal counterpart, T cell lymphomas showed significant overexpression of Bmi-1 (P = 0.011) and Ezh2 (P = 0.000). The Ki67 labeling index showed a positive correlation with Ezh2 expression in B cell lymphomas (correlation coefficient (Co) = 0.983, P = 0.000) and T/NK cell lymphomas (Co = 0.629, P = 0.000). Fluorescent immunohistochemical staining showed coexpression of Ezh2 and Ki67 in the same tumor cells, indicating that Ezh2 expression correlates with cell proliferation. Both B and T/NK cell neoplasms showed low expression of Mel-18 and high expression of both Bmi-1 and Ezh2. In conclusion, in aggressive lymphoma variants, Ezh2 is strongly expressed and polycomb repressive complex PRC1.4 dominates over PRC1.2. Coexpression of Bmi-1 and Ezh2 is a characteristic of aggressive lymphomas. Ezh2 correlates with the proliferation and aggressive nature of non-Hodgkin's lymphomas

Estimating and comparing the reliability of a suite of workplace-based assessments: an obstetrics and gynaecology setting

This paper reports on a study that compares estimates of the reliability of a suite of workplace based assessment forms as employed to formatively assess the progress of trainee obstetricians and gynaecologists. The use of such forms of assessment is growing nationally and internationally in many specialisms, but there is little research evidence on comparisons by procedure/competency and form-type across an entire specialty. Generalisability theory combined with a multilevel modelling approach is used to estimate variance components, G-coefficients and standard errors of measurement across 13 procedures and three form-types (mini-CEX, OSATS and CbD). The main findings are that there are wide variations in the estimates of reliability across forms, although there is little evidence that reliability varies systematically by form-type. However, the results suggest that the guidance on assessment within the specialty does not always allow for enough forms per trainee to ensure that the reliability of the assessment process is adequate. Methodologically, the problems of accurately estimating reliability in these contexts through the calculation of variance components and, crucially, their associated standard errors are considered. The importance of the use of appropriate methods in such calculations is emphasised, and the unavoidable limitations of research naturalistic settings are discussed