A Cellular Automaton Model for Tumor Dormancy: Emergence of a Proliferative Switch

Malignant cancers that lead to fatal outcomes for patients may remain dormant for very long periods of time. Although individual mechanisms such as cellular dormancy, angiogenic dormancy and immunosurveillance have been proposed, a comprehensive understanding of cancer dormancy and the “switch” from a dormant to a proliferative state still needs to be strengthened from both a basic and clinical point of view. Computational modeling enables one to explore a variety of scenarios for possible but realistic microscopic dormancy mechanisms and their predicted outcomes. The aim of this paper is to devise such a predictive computational model of dormancy with an emergent “switch” behavior. Specifically, we generalize a previous cellular automaton (CA) model for proliferative growth of solid tumor that now incorporates a variety of cell-level tumor-host interactions and different mechanisms for tumor dormancy, for example the effects of the immune system. Our new CA rules induce a natural “competition” between the tumor and tumor suppression factors in the microenvironment. This competition either results in a “stalemate” for a period of time in which the tumor either eventually wins (spontaneously emerges) or is eradicated; or it leads to a situation in which the tumor is eradicated before such a “stalemate” could ever develop. We also predict that if the number of actively dividing cells within the proliferative rim of the tumor reaches a critical, yet low level, the dormant tumor has a high probability to resume rapid growth. Our findings may shed light on the fundamental understanding of cancer dormancy.     

The TATA-Binding Protein as a Regulator of Cellular Transformation

No abstract available.     

Representation of a Three-Dimensional Epithelial Structure as a Regular Cellular Network in a Drosophila Eye Model

Biophysics - Analysis of the spatial arrangement of the drosophila retina has shown that the structure of cell layers can be represented by three-dimensional stack-like cell lattices. These...     

The N-Terminal Methionine of Cellular Proteins as a Degradation Signal

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Characterization of Avian Paramyxoviruses Isolated from Feral Ducks in Northern Japan: The Presence of Three Distinct Viruses in Nature

Seventy-eight strains of avian paramyxoviruses (PMV) were isolated from cloacal and/or tracheal swabs taken from 1,342 feral ducks, comprised of spot-bill ducks, mallards, pintails, teals, falcated teals, wigeons and buffie-heads, in Wakuya-cho, Miyagi Prefecture, Japan, between 1976 and 1979. Five and a half percent of the ducks were positive for virus. Serological and structural characterization indicated that three different avian paramyxoviruses arc prevalent in the Japanese feral duck population. The first group of PMV was Newcastle disease virus (NDV), and in vivo pathogenecity tests in embryonated chicken eggs and 1-day-old chicks revealed that all the NDV strains isolated were avirulent. The second and most prevalent strain was closely related to PMV-4, duck/Hong Kong/D3/75 strain. The viruses of the third group were recovered only from pintails. They cross-reacted antigenically with PMV-3 when antisera to the PMV-3 reference strains, turkey/Wisconsin/68 and parakeet/Netherlands/449/75, were employed. However, no cross-reaction was observed when antiserum to pintail/ Wakuya/20/78, the prototype of this group, was used. The viruses of the third group also differed in viral polypeptide profile from the reference strains of PMV-3.     

The Toxic Effects of Pathogenic Ataxin-3 Variants in a Yeast Cellular Model

Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 variants carrying the expanded polyQ are prone to associate with each other into amyloid toxic aggregates, which are responsible for neuronal death with ensuing neurodegeneration. We employed Saccharomyces cerevisiae as a eukaryotic cellular model to better clarify the mechanism by which AT3 triggers the disease. We expressed three variants: one normal (Q26), one expanded (Q85) and one truncated for a region lying from the beginning of its polyQ stretch to the end of the protein (291Δ). We found that the expression of the expanded form caused reduction in viability, accumulation of reactive oxygen species, imbalance of the antioxidant defense system and loss in cell membrane integrity, leading to necrotic death. The truncated variant also exerted a qualitatively similar, albeit milder, effect on cell growth and cytotoxicity, which points to the involvement of also non-polyQ regions in cytotoxicity. Guanidine hydrochloride, a well-known inhibitor of the chaperone Hsp104, almost completely restored wild-type survival rate of both 291Δ- and Q85-expressing strains. This suggests that AT3 aggregation and toxicity is mediated by prion forms of yeast proteins, as this chaperone plays a key role in their propagation.     

Volvox as a Model System for Studying the Ontogeny and Phylogeny of Multicellularity and Cellular Differentiation

   apd: 1 March 2001     

Aberrant Copper Chemistry as a Major Mediator of Oxidative Stress in a Human Cellular Model of Amyotrophic Lateral Sclerosis

We have investigated the response to oxidative stress in a model system obtained by stable transfection of the human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase or a mutant of this enzyme (H46R) associated with familial amyotrophic lateral sclerosis. We report that expression of mutant H46R Cu,Zn superoxide dismutase induces a selective increase in paraquat sensitivity that is reverted by addition of D-penicillamine. Furthermore, expression of this mutant enzyme affects the activity of the endogenous wild-type enzyme both in basal conditions and in copper overloading experiments. Our data indicate that aberrant metal chemistry of this mutant enzyme is the actual mediator of oxidative stress and that concurrent impairment of the activity of wild-type endogenous enzyme compromises the cell's ability to respond to oxidative stress.     

The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii

Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used Chlamydomonas reinhardtii as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in Chlamydomonas is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed.     

The Carbohydrate Sensitive Rat as a Model of Obesity

Background

Sensitivity to obesity is highly variable in humans, and rats fed a high fat diet (HFD) are used as a model of this inhomogeneity. Energy expenditure components (basal metabolism, thermic effect of feeding, activity) and variations in substrate partitioning are possible factors underlying the variability. Unfortunately, in rats as in humans, results have often been inconclusive and measurements usually made after obesity onset, obscuring if metabolism was a cause or consequence. Additionally, the role of high carbohydrate diet (HCD) has seldom been studied.

Methodology/Findings

Rats (n=24) were fed for 3 weeks on HCD and then 3 weeks on HFD. Body composition was tracked by MRI and compared to energy expenditure components measured prior to obesity. Results: 1) under HFD, as expected, by adiposity rats were variable enough to be separable into relatively fat resistant (FR) and sensitive (FS) groups, 2) under HCD, and again by adiposity, rats were also variable enough to be separable into carbohydrate resistant (CR) and sensitive (CS) groups, the normal body weight of CS rats hiding viscerally-biased fat accumulation, 3) HCD adiposity sensitivity was not related to that under HFD, and both HCD and HFD adiposity sensitivities were not related to energy expenditure components (BMR, TEF, activity cost), and 4) only carbohydrate to fat partitioning in response to an HCD test meal was related to HCD-induced adiposity.

Conclusions/Significance

The rat model of human obesity is based on substantial variance in adiposity gains under HFD (FR/FS model). Here, since we also found this phenomenon under HCD, where it was also linked to an identifiable metabolic difference, we should consider the existence of another model: the carbohydrate resistant (CR) or sensitive (CS) rat. This new model is potentially complementary to the FR/FS model due to relatively greater visceral fat accumulation on a low fat high carbohydrate diet.     

The Protective Role of Fucosylated Chondroitin Sulfate,a Distinct Glycosaminoglycan,in a Murine Model of Streptozotocin-Induced Diabetic Nephropathy

Background

Heparanase-1 activation, albuminuria, and a decrease in glomerular heparan sulfate (HS) have been described in diabetic nephropathy (DN). Glycosaminoglycan (GAG)-based drugs have been shown to have renoprotective effects in this setting, although recent trials have questioned their clinical effectiveness. Here, we describe the effects of fucosylated chondroitin sulfate (FCS), a novel GAG extracted from a marine echinoderm, in experimentally induced DN compared to a widely used GAG, enoxaparin (ENX).

Methods

Diabetes mellitus (DM) was induced by streptozotocin in male Wistar rats divided into three groups: DM (without treatment), FCS (8 mg/kg), and ENX (4 mg/kg), administered subcutaneously. After 12 weeks, we measured blood glucose, blood pressure, albuminuria, and renal function. The kidneys were evaluated for mesangial expansion and collagen content. Immunohistochemical quantifications of macrophages, TGF-β, nestin and immunofluorescence analysis of heparanase-1 and glomerular basement membrane (GBM) HS content was also performed. Gene expression of proteoglycan core proteins and enzymes involved in GAG assembly/degradation were analyzed by TaqMan real-time PCR.

Results

Treatment with GAGs prevented albuminuria and did not affect the glucose level or other functional aspects. The DM group exhibited increased mesangial matrix deposition and tubulointerstitial expansion, and prevention was observed in both GAG groups. TGF-β expression and macrophage infiltration were prevented by the GAG treatments, and podocyte damage was halted. The diabetic milieu resulted in the down-regulation of agrin, perlecan and collagen XVIII mRNAs, along with the expression of enzymes involved in GAG biosynthesis. Treatment with FCS and ENX positively modulated such changes. Heparanase-1 expression was significantly reduced after GAG treatment without affecting the GBM HS content, which was uniformly reduced in all of the diabetic animals.

Conclusions

Our results demonstrate that the administration of FCS prevented several pathological features of ND in rats. This finding should stimulate further research on GAG treatment for this complication of diabetes.     

Ganglioside GM3 Levels Are Altered in a Mouse Model of HIBM: GM3 as a Cellular Marker of the Disease

Objective

HIBM (Hereditary Inclusion Body Myopathy) is a recessive hereditary disease characterized by adult-onset, slowly progressive muscle weakness sparing the quadriceps. It is caused by a single missense mutation of each allele of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, a bifunctional enzyme catalyzing the first two steps of sialic acid synthesis in mammals. However, the mechanisms and cellular pathways affected by the GNE mutation and causing the muscle weakness could not be identified so far. Based on recent evidence in literature, we investigated a new hypothesis, i.e. the involvement in the disease of the GM3 ganglioside, a specific glycolipid implicated in muscle cell proliferation and differentiation.

Methods

qRT-PCR analysis of St3gal5 (GM3 synthase) gene expression and HPLC quantification of GM3 ganglioside were conducted on muscle tissue from a mouse model of HIBM harboring the M712T mutation of GNE (Gne^M712T/M712T mouse) vs control mice (Gne^+/+ mouse).

Results

St3gal5 mRNA levels were significantly lower in Gne^M712T/M712T mouse muscles vs Gne^+/+ mouse muscles (64.41%±10% of Gne^+/+ levels). GM3 ganglioside levels showed also a significant decrease in Gne^M712T/M712T mouse muscle compared to Gne^+/+ mouse muscle (18.09%±5.33% of Gne^+/+ levels). Although these Gne^M712T/M712T mice were described to suffer severe glomerular proteinuria, no GM3 alterations were noted in kidneys, highlighting a tissue specific alteration of gangliosides.

Conclusion

The M712T mutation of GNE hampers the muscle ability to synthesize normal levels of GM3. This is the first time that a mutation of GNE can be related to the molecular pathological mechanism of HIBM.     

Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.     

Learning from Small Fry: The Zebrafish as a Genetic Model Organism for Aquaculture Fish Species

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.     

The Natural Philosophy of Plant Form: Cellular Autoreproduction as a Component of a Structural Explanation of Plant Form

A map-L-system is described which simulates the developmentof the two-dimensional patterns of cell walls displayed at thesurfaces of shoot apices of Psilotum nudum. The simulation ofthese cellular patterns commences with the division of a triangularcell and continues until a complete set of ten different cells,including new triangular cells, is formed amongst the descendantsof each merophyte. The triangular cells generated by means ofthis division pathway, P1, are, in their three-dimensional aspect,four-sided apical cells. In the plant, they have the potentialityto support the development of a shoot apex. The generation ofnew triangular cells by pathway P1 therefore seems to be a preconditionfor the branching of the shoot. Observed variations upon thecellular pattern developed by pathway P1 have also been analysed.Two of these variant pathways, P2 and P3, suggest the typesof controls which are required to bring about all three (P1–P3)patterns of cells. These controls may involve the participationof the plant cytoskeleton and may also require an influencefrom the apical cell itself. The triangular shoot apical cellsof Psilotum are autoreproductive cells: that is, at each division,one of the daughters is a new triangular cell, the other daughterhas some other shape. This example of triangular cell autoreproductionand self-maintenance and its relation to organogenesis is discussedin light of the views on reproduction and self-maintenance expressedby Agnes Arber (1950) in her book The natural philosophy ofplant form(Cambridge: Cambridge University Press). Copyright2001 Annals of Botany Company Agnes Arber, apical cell, cell division patterns, computer simulation, cytoskeleton, L-systems, Living Systems Theory, meristems, Psilotum, shoot apex, stem cell     

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.     

The Cellular Concentration of the Yeast Ure2p Prion Protein Affects Its Propagation as a Prion

The [URE3] yeast prion is a self-propagating inactive form of the Ure2p protein. We show here that Ure2p from the species Saccharomyces paradoxus (Ure2p_Sp) can be efficiently converted into a prion form and propagate [URE3] when expressed in Saccharomyces cerevisiae at physiological level. We found however that Ure2p_Sp overexpression prevents efficient prion propagation. We have compared the aggregation rate and propagon numbers of Ure2p_Sp and of S. cerevisiae Ure2p (Ure2p_Sc) in [URE3] cells both at different expression levels. Overexpression of both Ure2p orthologues accelerates formation of large aggregates but Ure2p_Sp aggregates faster than Ure2p_Sc. Although the yeast cells that contain these large Ure2p aggregates do not transmit [URE3] to daughter cells, the corresponding crude extract retains the ability to induce [URE3] in wild-type [ure3-0] cells. At low expression level, propagon numbers are higher with Ure2p_Sc than with Ure2p_Sp. Overexpression of Ure2p decreases the number of [URE3] propagons with Ure2p_Sc. Together, our results demonstrate that the concentration of a prion protein is a key factor for prion propagation. We propose a model to explain how prion protein overexpression can produce a detrimental effect on prion propagation and why Ure2p_Sp might be more sensitive to such effects than Ure2p_Sc.     

Synthesis of Hydroxy Derivatives of Highly Potent Non-steroidal CYP 17 Inhibitors as Potential Metabolites and Evaluation of their Activity by a Non Cellular Assay using Recombinant Human Enzyme

Inhibition of CYP 17 is a promising strategy for the treatment of prostate cancer. Recently two non-steroidal compounds with high in vitro activity were synthesized in our group (BW19 and BW95). However, after a few hours they showed in vivo a strong decrease in their activity. This might be due to a fast biodegradation. Potential hydroxy and epoxy metabolites were synthesized and their inhibitory activities were tested by a new non-cellular assay using recombinant enzyme. As source, membrane fractions of E. coli pJL17/OR coexpressing human CYP 17 and rat NADPH-P450-reductase were used. Showing a high and constant CYP 17 activity and a fast and easy isolation procedure the new method was advantageous compared with the microsomal assay. Interestingly, all the new synthesized hydroxy and epoxy compounds except one showed a lower inhibition of CYP 17 than the parent compounds. Thus, the loss of in vivo activity may be partly explained.