Andrew H. Coles

Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue

Wnt5a is a member of the Wnt family of secreted glycoproteins that play essential organizing roles in development. Similar to other Wnt members, Wnt5a can upregulate cell proliferation and has been proposed to have oncogenic function. Here we report that Wnt5a signals through the noncanonical Wnt/Ca++ pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation in a cell-autonomous manner. Wnt5a hemizygous mice develop myeloid leukemias and B cell lymphomas that are clonal in origin and display loss of Wnt5a function in tumor tissues. Furthermore, analysis of human primary leukemias reveals deletion of the WNT5A gene and/or loss of WNT5A expression in a majority of the patient samples. These results demonstrate that Wnt5a suppresses hematopoietic malignancies.

The ING Gene Family in the Regulation of Cell Growth and Tumorigenesis

The five members of the inhibitor of growth (ING) gene family have garnered significant interest due to their putative roles as tumor suppressors. However, the precise role(s) of these ING proteins in regulating cell growth and tumorigenesis remains uncertain. Biochemical and molecular biological analysis has revealed that all ING members encode a PHD finger motif proposed to bind methylated histones and phosphoinosital, and all ING proteins have been found as components of large chromatin remodeling complexes that also include histone acetyl transferase (HAT) and histone deacetylase (HDAC) enzymes, suggesting a role for ING proteins in regulating gene transcription. Additionally, the results of forced overexpression studies performed in tissue culture have indicated that several of the ING proteins can interact with the p53 tumor suppressor protein and/or the nuclear factor‐kappa B (NF‐κB) protein complex. As these ING‐associated proteins play well‐established roles in numerous cell processes, including DNA repair, cell growth and survival, inflammation, and tumor suppression, several models have been proposed that ING proteins act as key regulators of cell growth not only through their ability to modify gene transcription but also through their ability to alter p53 and NF‐κB activity. However, these models have yet to be substantiated by in vivo experimentation. This review summarizes what is currently known about the biological functions of the five ING genes based upon in vitro experiments and recent mouse modeling efforts, and will highlight the potential impact of INGs on the development of cancer. J. Cell. Physiol. 218: 45–57, 2009.

Deletion of p37Ing1 in Mice Reveals a p53-Independent Role for Ing1 in the Suppression of Cell Proliferation, Apoptosis, and Tumorigenesis

ING proteins have been proposed to alter chromatin structure and gene transcription to regulate numerous aspects of cell physiology, including cell growth, senescence, stress response, apoptosis, and transformation. ING1, the founding member of the inhibitor of growth family, encodes p37(Ing1), a plant homeodomain (PHD) protein that interacts with the p53 tumor suppressor protein and seems to be a critical cofactor in p53-mediated regulation of cell growth and apoptosis. In this study, we have generated and analyzed p37(Ing1)-deficient mice and primary cells to further explore the role of Ing1 in the regulation of cell growth and p53 activity. The results show that endogenous levels of p37(Ing1) inhibit the proliferation of p53-wild-type and p53-deficient fibroblasts, and that p53 functions are unperturbed in p37(Ing1)-deficient cells. In addition, loss of p37(Ing1) induces Bax expression and increases DNA damage-induced apoptosis in primary cells and mice irrespective of p53 status. Finally, p37(Ing1) suppresses the formation of spontaneous follicular B-cell lymphomas in mice. These results indicate that p53 does not require p37(Ing1) to negatively regulate cell growth and offers genetic proof that Ing1 suppresses cell growth and tumorigenesis. Furthermore, these data reveal that p37(Ing1) can negatively regulate cell growth and apoptosis in a p53-independent manner.

Mutation of the SNF2 family member Chd2 affects mouse development and survival.

The chromodomain helicase DNA‐binding domain (Chd) proteins belong to the SNF2‐like family of ATPases that function in chromatin remodeling and assembly. These proteins are characterized by the presence of tandem chromodomains and are further subdivided based on the presence or absence of additional structural motifs. The Chd1–Chd2 subfamily is distinguished by the presence of a DNA‐binding domain that recognizes AT‐rich sequence. Currently, there are no reports addressing the function of the Chd2 family member. Embryonic stem cells containing a retroviral gene‐trap inserted at the Chd2 locus were utilized to generate mice expressing a Chd2 protein lacking the DNA‐binding domain. This mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis and in perinatal lethality. Animals heterozygous for the mutation showed decreased neonatal viability and increased susceptibility to non‐neoplastic lesions affecting most primary organs. In particular, approximately 85% of the heterozygotes showed gross kidney abnormalities. Our results demonstrate that mutation of Chd2 dramatically affects mammalian development and long‐term survival. J. Cell. Physiol. 209: 162–171, 2006.

Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.

Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics for the treatment of neurological disorders, such as Huntingtons disease. Small, endogenous vesicles known as exosomes have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into exosomes are lacking. Here, we show that hydrophobically modified small interfering RNAs (hsiRNAs) efficiently load into exosomes upon co-incubation, without altering vesicle size distribution or integrity. Exosomes loaded with hsiRNAs targeting Huntingtin mRNA were efficiently internalized by mouse primary cortical neurons and promoted dose-dependent silencing of Huntingtin mRNA and protein. Unilateral infusion of hsiRNA-loaded exosomes, but not hsiRNAs alone, into mouse striatum resulted in bilateral oligonucleotide distribution and statistically significant bilateral silencing of up to 35% of Huntingtin mRNA. The broad distribution and efficacy of hsiRNA-loaded exosomes delivered to brain is expected to advance the development of therapies for the treatment of Huntingtons disease and other neurodegenerative disorders.

Noncanonical Wnt signaling promotes apoptosis in thymocyte development

The Wnt–β-catenin signaling pathway has been shown to govern T cell development by regulating the growth and survival of progenitor T cells and immature thymocytes. We explore the role of noncanonical, Wnt–Ca2+ signaling in fetal T cell development by analyzing mice deficient for Wnt5a. Our findings reveal that Wnt5a produced in the thymic stromal epithelium does not alter the development of progenitor thymocytes, but regulates the survival of αβ lineage thymocytes. Loss of Wnt5a down-regulates Bax expression, promotes Bcl-2 expression, and inhibits apoptosis of CD4+CD8+ thymocytes, whereas exogenous Wnt5a increases apoptosis of fetal thymocytes in culture. Furthermore, Wnt5a overexpression increases apoptosis in T cells in vitro and increases protein kinase C (PKC) and calmodulin-dependent kinase II (CamKII) activity while inhibiting β-catenin expression and activity. Conversely, Wnt5a deficiency results in the inhibition of PKC activation, decreased CamKII activity, and elevation of β-catenin amounts in thymocytes. These results indicate that Wnt5a induction of the noncanonical Wnt–Ca2+ pathway alters canonical Wnt signaling and is critical for normal T cell development.

Inhibitor of growth-4 promotes IκB promoter activation to suppress NF-κB signaling and innate immunity

Ing4 is a member of the inhibitor of growth (ING) family of chromatin-modifying proteins. Biochemical experiments indicate that Ing4 is a subunit of the HB01-JADE-hEAF6 histone acetyltransferase complex responsible for most nucleosomal histone H4 acetylation in eukaryotes, and transfection studies suggest that Ing4 may regulate a wide variety of cellular processes, including DNA repair, apoptosis, cell-cycle regulation, metastasis, angiogenesis, and tumor suppression. However, in vivo evidence for a physiological role for Ing4 in cell-growth regulation is lacking. We have generated Ing4-deficient mice to explore the role of Ing4 in development, tumorigenesis, and in NF-κB signaling. Ing4-null mice develop normally and are viable. Although mice deficient for Ing4 fail to form spontaneous tumors, they are hypersensitive to LPS treatment and display elevated cytokine responses. Macrophages isolated from Ing4-null mice have increased levels of nuclear p65/RelA protein, resulting in increased RelA binding to NF-κB target promoters and up-regulation of cytokine gene expression. However, increased promoter occupancy by RelA in LPS-stimulated, Ing4-null cells does not always correlate with increased NF-κB target-gene expression, as RelA activation of a subset of cytokine promoters also requires Ing4 for proper histone H4 acetylation. Furthermore, activation of the IκBα promoter by RelA is also Ing4-dependent, and LPS-stimulated, Ing4-null cells have reduced levels of IκBα promoter H4 acetylation and IκB gene expression. Thus, Ing4 negatively regulates the cytokine-mediated inflammatory response in mice by facilitating NF-κB activation of IκB promoters, thereby suppressing nuclear RelA levels and the activation of select NF-κB target cytokines.

Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntingtons disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

Hdac6 regulates Tip60-p400 function in stem cells

In embryonic stem cells (ESCs), the Tip60 histone acetyltransferase activates genes required for proliferation and silences genes that promote differentiation. Here we show that the class II histone deacetylase Hdac6 co-purifies with Tip60-p400 complex from ESCs. Hdac6 is necessary for regulation of most Tip60-p400 target genes, particularly those repressed by the complex. Unlike differentiated cells, where Hdac6 is mainly cytoplasmic, Hdac6 is largely nuclear in ESCs, neural stem cells (NSCs), and some cancer cell lines, and interacts with Tip60-p400 in each. Hdac6 localizes to promoters bound by Tip60-p400 in ESCs, binding downstream of transcription start sites. Surprisingly, Hdac6 does not appear to deacetylate histones, but rather is required for Tip60-p400 binding to many of its target genes. Finally, we find that, like canonical subunits of Tip60-p400, Hdac6 is necessary for robust ESC differentiation. These data suggest that Hdac6 plays a major role in the modulation of Tip60-p400 function in stem cells. DOI: http://dx.doi.org/10.7554/eLife.01557.001

Recent trends in post-discharge mortality among patients with an initial acute myocardial infarction

The objectives of this study were to describe contemporary postdischarge death rates of patients hospitalized at all Worcester, Massachusetts, hospitals after initial acute myocardial infarctions (AMIs) and to examine factors associated with a poor prognosis. The medical records of patients discharged from 11 central Massachusetts medical centers after initial AMIs during 2001, 2003, 2005, and 2007 were reviewed, identifying 2,452 patients. This population was composed of predominantly older patients, men (58%), and whites. Overall, the 3-month, 1-year, and 2-year all-cause death rates were 8.9%, 16.4%, and 23.4%, respectively. Over time, reductions in postdischarge mortality were observed in crude as well as multivariate-adjusted analyses. In 2001, the 3-month, 1-year, and 2-year all-cause death rates were 11.1%, 17.1%, and 25.6%, respectively, compared to rates of 7.9%, 12.7%, and 18.6% in patients discharged in 2007. Older age, male gender, hospitalization for a non-ST-segment elevation AMI, renal dysfunction, and preexisting heart failure were associated with an increased risk for dying after hospital discharge. These results suggest that the postdischarge prognosis of patients with initial AMIs has improved, likely reflecting enhanced in-hospital and postdischarge management practices. In conclusion, patients with initial AMIs can also be identified who are at increased risk for dying after hospital discharge, in whom increased surveillance and targeted treatment approaches can be directed.