Decreased Replication Origin Activity in Temporal Transition Regions

In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication. Introduction of a transcription unit into the Igh TTR, activation of gene transcription, and enhancement of local histone modifications characteristic of active chromatin did not lead to origin activation. Moreover, very few replication initiation events were observed when two ectopic replication origin sequences were inserted into the TTR. These findings indicate that the Igh TTR represents a repressive compartment that inhibits replication initiation, thus maintaining the boundaries between early and late replication domains

Constitutively Activated PI3K Accelerates Tumor Initiation and Modifies Histopathology of Breast Cancer

The gene encoding phosphatidylinositol 3-kinase catalytic subunit α-isoform (PIK3CA, p110α) is frequently activated by mutation in human cancers. Based on detection in some breast cancer precursors, PIK3CA mutations have been proposed to have a role in tumor initiation. To investigate this hypothesis, we generated a novel mouse model with a Cre-recombinase regulated allele of p110α (myristoylated-p110α, myr-p110α) along with p53fl/fl deletion and KrasG12D also regulated by Cre-recombinase. After instillation of adenovirus-expressing Cre-recombinase into mammary ducts, we found that myr-p110α accelerated breast tumor initiation in a copy number-dependent manner. Breast tumors induced by p53fl/fl;KrasG12D with no or one copy of myr-p110α had predominantly sarcomatoid features, whereas two copies of myr-p110α resulted in tumors with a carcinoma phenotype. This novel model provides experimental support for importance of active p110α in breast tumor initiation, and shows that the amount of PI3K activity can affect the rate of tumor initiation and modify the histological phenotype of breast cancer

Genomic imprinting variations in the mouse type 3 deiodinase gene between tissues and brain regions.

The Dio3 gene, which encodes for the type 3 deiodinase (D3), controls thyroid hormone (TH) availability. The lack of D3 in mice results in tissue overexposure to TH and a broad neuroendocrine phenotype. Dio3 is an imprinted gene, preferentially expressed from the paternally inherited allele in the mouse fetus. However, heterozygous mice with paternal inheritance of the inactivating Dio3 mutation exhibit an attenuated phenotype when compared with that of Dio3 null mice. To investigate this milder phenotype, the allelic expression of Dio3 was evaluated in different mouse tissues. Preferential allelic expression of Dio3 from the paternal allele was observed in fetal tissues and neonatal brain regions, whereas the biallelic Dio3 expression occurred in the developing eye, testes, and cerebellum and in the postnatal brain neocortex, which expresses a larger Dio3 mRNA transcript. The newborn hypothalamus manifests the highest degree of Dio3 expression from the paternal allele, compared with other brain regions, and preferential allelic expression of Dio3 in the brain relaxed in late neonatal life. A methylation analysis of two regulatory regions of the Dio3 imprinted domain revealed modest but significant differences between tissues, but these did not consistently correlate with the observed patterns of Dio3 allelic expression. Deletion of the Dio3 gene and promoter did not result in significant changes in the tissue-specific patterns of Dio3 allelic expression. These results suggest the existence of unidentified epigenetic determinants of tissue-specific Dio3 imprinting. The resulting variation in the Dio3 allelic expression between tissues likely explains the phenotypic variation that results from paternal Dio3 haploinsufficiency.This is the final version of the article. It is available from the Endocrine Society in Molecular Endocrinology here: http://press.endocrine.org/doi/pdf/10.1210/me.2014-1210

Graded and Binary Responses in Stochastic Gene Expression

Recently, several theoretical and experimental studies have been undertaken to probe the effect of stochasticity on gene expression (GE). In experiments, the GE response to an inducing signal in a cell, measured by the amount of mRNAs/proteins synthesized, is found to be either graded or binary. The latter type of response gives rise to a bimodal distribution in protein levels in an ensemble of cells. One possible origin of binary response is cellular bistability achieved through positive feedback or autoregulation. In this paper, we study a simple, stochastic model of GE and show that the origin of binary response lies exclusively in stochasticity. The transitions between the active and inactive states of the gene are random in nature. Graded and binary responses occur in the model depending on the relative stability of the activated and deactivated gene states with respect to that of mRNAs/proteins.The theoretical results on binary response provide a good description of the ``all-or-none'' phenomenon observed in an eukaryotic system.Comment: to be published in Physical Biolog

Ovarian Cancer Progression is Controlled by Phenotypic Changes in Dendritic Cells

We characterized the initiation and evolution of the immune response against a new inducible p53-dependent model of aggressive ovarian carcinoma that recapitulates the leukocyte infiltrates and cytokine milieu of advanced human tumors. Unlike other models that initiate tumors before the development of a mature immune system, we detect measurable antitumor immunity from very early stages, which is driven by infiltrating dendritic cells (DCs) and prevents steady tumor growth for prolonged periods. Coinciding with a phenotypic switch in expanding DC infiltrates, tumors aggressively progress to terminal disease in a comparatively short time. Notably, tumor cells remain immunogenic at advanced stages, but anti-tumor T cells become less responsive, whereas their enduring activity is abrogated by different microenvironmental immunosuppressive DCs. Correspondingly, depleting DCs early in the disease course accelerates tumor expansion, but DC depletion at advanced stages significantly delays aggressive malignant progression. Our results indicate that phenotypically divergent DCs drive both immunosurveillance and accelerated malignant growth. We provide experimental support for the cancer immunoediting hypothesis, but we also show that aggressive cancer progression after a comparatively long latency period is primarily driven by the mobilization of immunosuppressive microenvironmental leukocytes, rather than loss of tumor immunogenicity

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers

Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation

Nonuniform, mosaic expression patterns of transgenes are often linked to transcriptional silencing, triggered by epigenetic modifications of the exogenous DNA. Such phenotypes are common phenomena in genetically engineered cells and organisms. They are widely attributed to features of transgenic transcription units distinct from endogenous genes, rendering them particularly susceptible to epigenetic downregulation. Contrary to this assumption we show that the method used for the isolation of stably transfected cells has the most profound impact on transgene expression patterns. Standard antibiotic selection was directly compared to cell sorting for the establishment of stable cells. Only the latter procedure could warrant a high degree of uniformity and stability in gene expression. Marker genes useful for the essential cell sorting step encode mostly fluorescent proteins. However, by combining this approach with site-specific recombination, it can be applied to isolate stable cell lines with the desired expression characteristics for any gene of interest

The Emotional and Political Power of Images of Suffering: Discursive Psychology and the Study of Visual Rhetoric

Drawing on insights from discursive and rhetorical approaches in psychology, the chapter examines responses to the publication of the photographs of the body of Alan Kurdi, the three-year-old Syrian refugee who drowned off the coast of Turkey in 2015. The chapter considers how and why the images were constructed as inherently ‘moving’, and as possessing the power to elicit emotions, and affect the audience on a ‘visceral’ level. It also looks at how accounts of (and for) emotional reactions to the images were deployed rhetorically to manage accountability associated with viewing, and sharing, images of a dead child. Through the examination of the Kurdi images the chapter also considers the possibility of a psychologically-informed approach to visual rhetoric, one that offers a better understanding of how and why certain images (but not others) are constituted as topics of humanitarian concern, and a source of emotional and political investment

White paper on microbial anti-cancer therapy and prevention

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting (‘Microbial Based Cancer Therapy’) at the US National Cancer Institute in the summer of 2017. Here, we define ‘Microbial Therapy’ to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care

Inactivation of promoter 1B of APC causes partial gene silencing: evidence for a significant role of the promoter in regulation and causative of familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Two promoters, 1A and 1B, have been recognized in APC, and 1B is thought to have a minor role in the regulation of the gene. We have identified a novel deletion encompassing half of this promoter in the largest family (Family 1) of the Swedish Polyposis Registry. The mutation leads to an imbalance in allele-specific expression of APC, and transcription from promoter 1B was highly impaired in both normal colorectal mucosa and blood from mutation carriers. To establish the significance of promoter 1B in normal colorectal mucosa (from controls), expression levels of specific transcripts from each of the promoters, 1A and 1B, were examined, and the expression from 1B was significantly higher compared with 1A. Significant amounts of transcripts generated from promoter 1B were also determined in a panel of 20 various normal tissues examined. In FAP-related tumors, the APC germline mutation is proposed to dictate the second hit. Mutations leaving two or three out of seven 20-amino-acid repeats in the central domain of APC intact seem to be required for tumorigenesis. We examined adenomas from mutation carriers in Family 1 for second hits in the entire gene without any findings, however, loss of the residual expression of the deleterious allele was observed. Three major conclusions of significant importance in relation to the function of APC can be drawn from this study; (i) germline inactivation of promoter 1B is disease causing in FAP; (ii) expression of transcripts from promoter 1B is generated at considerable higher levels compared with 1A, demonstrating a hitherto unknown importance of 1B; (iii) adenoma formation in FAP, caused by impaired function of promoter 1B, does not require homozygous inactivation of APC allowing for alternative genetic models as basis for adenoma formation