Kristy Brown

Induction of autophagy and inhibition of tumorigenesis by beclin 1

The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1 (ref. 3), a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30 (refs 4, 5), and is mono-allelically deleted in 40–75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.

Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia

Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as γ-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.

XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors

Xenotropic murine leukemia virus–related virus (XMRV) was recently discovered in human prostate cancers and is the first gammaretrovirus known to infect humans. While gammaretroviruses have well-characterized oncogenic effects in animals, they have not been shown to cause human cancers. We provide experimental evidence that XMRV is indeed a gammaretrovirus with protein composition and particle ultrastructure highly similar to Moloney murine leukemia virus (MoMLV), another gammaretrovirus. We analyzed 334 consecutive prostate resection specimens, using a quantitative PCR assay and immunohistochemistry (IHC) with an anti-XMRV specific antiserum. We found XMRV DNA in 6% and XMRV protein expression in 23% of prostate cancers. XMRV proteins were expressed primarily in malignant epithelial cells, suggesting that retroviral infection may be directly linked to tumorigenesis. XMRV infection was associated with prostate cancer, especially higher-grade cancers. We found XMRV infection to be independent of a common polymorphism in the RNASEL gene, unlike results previously reported. This finding increases the population at risk for XMRV infection from only those homozygous for the RNASEL variant to all individuals. Our observations provide evidence for an association of XMRV with malignant cells and with more aggressive tumors.

Defects in Nuclear and Cytoskeletal Morphology and Mitochondrial Localization in Spermatozoa of Mice Lacking Nectin-2, a Component of Cell-Cell Adherens Junctions

ABSTRACT Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily. Nectin-2 is a component of cell-cell adherens junctions and interacts with l-afadin, an F-actin-binding protein. Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile. The production by nectin-2−/−mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa ofnectin-2 −/− mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.

Ontogeny of the neuronal intermediate filament protein, peripherin, in the mouse embryo

The expression of peripherin, a type III neuron-specific intermediate filament protein, and the middle neurofilament subunit were studied in the mouse embryo using immunofluorescence staining. The earliest staining for both proteins is seen at embryonic day 9 in the myelencephalon, initially as fiber staining followed by cell body staining in the developing facial and acoustic nuclei. As the embryo develops, there is rostral as well as caudal extension of peripherin and staining is seen in the trigeminal ganglia, nerve fibers and in the enteric nervous system. As the spinal cord forms there is anti-peripherin staining in developing motoneurons of the anterior horns while little cell body staining is seen for the middle neurofilament subunit. Both antibodies stain the developing dorsal root and its entry zone, but peripherin is found in the secondary sensory and commissural fibers while the middle neurofilament subunit is not. While both proteins are found in the neurons of the dorsal root ganglia, their distribution varies. The larger peripheral cells of the ganglia contain both proteins while the smaller more central cells, constituting over 60% of the cells in the ganglia, contain only peripherin. A similar picture is found in the sympathetic ganglia where there are cells which contain peripherin. middle neurofilament subunit or both, but where the majority of the neurons have only peripherin in their cell bodies. Peripherin is not found in the developing retina or in the adrenal medulla. Peripherin is also completely absent from cell bodies in the cerebral and cerebellar cortices. These results indicate that peripherin is found in development only in regions in which it is found in the adult. It can either co-exist with neurofilaments in the same neuron or the two may be independently expressed.

Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes

The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration.

τ Regulation of Microtubule-Microtubule Spacing and Bundling

Abstract: τ proteins are microtubule‐associated proteins that promote microtubule polymerization in vitro and in vivo. They are a family of neuronal proteins with apparent molecular weights in the range 50,000–68,000 determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Recently, a new member of this family has been described and its cDNA has been cloned. It has an apparent molecular weight of 116,000 and has been called high‐molecular‐weight τ (HMW τ). All the τ proteins are encoded by a single gene, which undergoes complex alternative splicing. In the present study, we have cloned into the baculovirus a cDNA fully encoding HMW τ as well as a truncated cDNA encoding a protein beginning 13 amino acids in front of the τ microtubule‐binding domain. HMW τ‐recombinant‐virus‐infected Sf9 cells overexpressed HMW τ, which induced the polymerization of microtubules and the formation of long cellular processes similar to those induced by low‐molecular‐weight τ (LMW τ) overexpression. Process cross sections revealed a larger spacing (≈35 nm) between microtubules when induced by HMW τ than when induced by LMW τ (≈20 nm). The truncated construct also induces processes, where microtubules were packed far more closely together (≈10 nm). Although branching did not occur in processes induced by intact τs, 10% of the processes induced by the truncated τ protein branched.

Abnormal microtubule packing in processes of SF9 cells expressing the FTDP-17 V337M tau mutation

Mutations in the gene for the microtubule associated protein, tau have been identified for fronto‐temporal dementia with Parkinsonism linked to chromosome 17 (FTDP‐17). In vitro data have shown that FTDP‐17 mutant tau proteins have a reduced ability to bind microtubules and to promote microtubule assembly. Using the baculovirus system we have examined the effect of the V337M mutation on the organization of the microtubules at the ultrastructural level. Our results show that the organization of the microtubules is disrupted in the presence of V337M tau with greater distances between the microtubules and fewer microtubules per process.

Neurofilament light polypeptide gene N98S mutation in mice leads to neurofilament network abnormalities and a Charcot-Marie-Tooth Type 2E phenotype

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited neurological disorder with a prevalence of 1 in 2500 people worldwide. Patients suffer from degeneration of the peripheral nerves that control sensory information of the foot/leg and hand/arm. Multiple mutations in the neurofilament light polypeptide gene, NEFL, cause CMT2E. Previous studies in transfected cells showed that expression of disease-associated neurofilament light chain variants results in abnormal intermediate filament networks associated with defects in axonal transport. We have now generated knock-in mice with two different point mutations in Nefl: P8R that has been reported in multiple families with variable age of onset and N98S that has been described as an early-onset, sporadic mutation in multiple individuals. Nefl(P8R/+) and Nefl(P8R/P8R) mice were indistinguishable from Nefl(+/+) in terms of behavioral phenotype. In contrast, Nefl(N98S/+) mice had a noticeable tremor, and most animals showed a hindlimb clasping phenotype. Immunohistochemical analysis revealed multiple inclusions in the cell bodies and proximal axons of spinal cord neurons, disorganized processes in the cerebellum and abnormal processes in the cerebral cortex and pons. Abnormal processes were observed as early as post-natal day 7. Electron microscopic analysis of sciatic nerves showed a reduction in the number of neurofilaments, an increase in the number of microtubules and a decrease in the axonal diameters. The Nefl(N98S/+) mice provide an excellent model to study the pathogenesis of CMT2E and should prove useful for testing potential therapies.

A novel melano-lysosome in the retinal epithelium of rhesus monkeys.

The large phagocytic load that confronts the retinal pigment epithelium (RPE) is thought to play a possible role in the pathogenesis of age related macular degeneration (AMD) that afflicts both humans and monkeys. Our knowledge of how RPE degrades phagosomes and other intra-cellular material by lysosomal action is still rudimentary. In this paper we examine organelles that play a role in this process, melanosome, lysosomes and phagosomes, in the RPE of young and old rhesus monkeys in order to better understand lysosomal autophagy and heterophagy in the RPE and its possible role in AMD. We used electron microscopy to detect and describe the characteristics of melanosomes and lysosome-like organelles in the macular RPE of rhesus monkeys (Macaca mulatta) that were 1, 6, 24, 24, 26 and 35 years of age. The measurements include the number, shape and size of these organelles located in the basal, middle and apical regions of RPE cells. Phaagosomes were also examined but not counted or measured for size or shape because of their rarity. Melanosomes were homogeneously dark with a circular or elliptical shape and decreased in number with age. Smaller melanosomes were more common at the basal side of the RPE. Among the small melanosomes, we found an organelle that was losing melanin in varying degrees; in some cases was nearly devoid of melanin. Because of the melanin loss, we considered this organelle to be a unique type of autophagic melano-lysosome, which we called a Type 1 lysosome. We found another organelle, more canonically lysosomal, which we called a Type 2 lysosome. This organelle was composed of a light matrix containing melanosomes in various stages of degradation. Type 2 lysosomes without melanosomes were rare. Type 2 lysosomes increased while Type 1 decreased in number with age. Phagosomes were rare in both young and old monkeys. They made close contact with Type 2 lysosomes which we considered responsible for their degradation. Melanosomes are being lost from monkey RPE with age. Much of this loss is carried out by two types of lysosomes. One, not defined as unique before, appears to be autophagic in digesting its own melanin; it has been called a Type 1 lysosome. The other, a more canonical lysosome, is both heterophagic in digesting phagosomes and autophagic in digesting local melanosomes; it has been called a Type 2 lysosome. Type 1 lysosomes decrease while type 2 lysosomes increase with age. The loss of melanin is considered to be detrimental to the RPE since it reduces melanins protective action against light toxicity and oxidative stress. Phagosomes appear to be degraded by membrane contacts with Type 2 lysosomes. The loss of melanin and the buildup of Type 2 lysosomes occur at an earlier age in monkeys than humans implying that a greater vulnerability to senescence accelerates the rate of AMD in monkeys.