Control Of Cell Number Research Articles

Deletion of Bax eliminates sex differences in the mouse forebrain.

Several of the best-studied sex differences in the mammalian brain are ascribed to the hormonal control of cell death. This conclusion is based primarily on correlations between pyknotic cell counts in development and counts of mature neurons in adulthood; the molecular mechanisms of hormone-regulated, sexually dimorphic cell death are unknown. We asked whether Bax, a member of the Bcl-2 family of proteins that is required for cell death in many developing neurons, might be essential for sex differences in neuron number. We compared Bax knockout mice and their WT siblings, focusing on two regions of the mouse forebrain that show opposite patterns of sexual differentiation: the principal nucleus of the bed nucleus of the stria terminalis, in which males have more neurons than do females, and the anteroventral periventricular nucleus (AVPV), where females have more neurons overall and many more dopaminergic neurons than do males. Testosterone, or its metabolites, is responsible for the sex differences in both nuclei. A null mutation of the Bax gene completely eliminated sex differences in overall cell number in both the principal nucleus of the bed nucleus of the stria terminalis and AVPV. Thus, Bax-dependent cell death is required for sexual differentiation of cell number, regardless of whether testosterone decreases or increases cell death. In contrast, the sex difference in AVPV dopaminergic cell number, as measured by tyrosine hydroxylase immunohistochemistry, was not affected by Bax gene deletion, demonstrating heterogeneity of mechanisms controlling cell number within a single nucleus.

Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway.

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK(CS)) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK(CS)-deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PK(CS) gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

Detection of apoptosis during planarian regeneration by the expression of apoptosis-related genes and TUNEL assay

Apoptosis is a tightly organized cell death process that plays a crucial role in metazoan development, but it has not yet been revealed whether apoptotic events are involved in the process of regeneration. Here, we tried to detect apoptotic cells during planarian regeneration using the TdT-mediated dUTP nick-end labeling (TUNEL) assay as well as the expression of apoptosis-related genes. Three novel cDNAs were isolated from a planarian cDNA library and shown to be closely related to other metazoan caspases at the amino acid sequence level. One of these cDNAs, Caspase-like gene 3 ( DjClg3), was expressed primarily in apoptotic cells by double detections with the TUNEL assay. Whole mount in situ studies indicated that DjClg3 was expressed in the cells of the mesenchymal space and also around the pharynx of the intact body. Its expression in the regenerating head piece was seen in the blastema and less significantly in the brain, while in the regenerating tail piece, DjClg3 expression was detected uniformly throughout the entire region. In parallel experiments, we performed in situ TUNEL assays to localize the regions where cell death occurred during regeneration and comparable results to the DjClg3 expression patterns were obtained. This is the first report to show that planarians have apoptosis-related genes and the results suggest that the apoptotic mechanism probably takes place to a large extent in normal intact worms as well as during their regeneration. We hypothesize that the presence of apoptosis in planarians may have a role in controlling cell numbers, eliminating unnecessary tissues or cells and remodeling the old tissues of regenerating body parts.

Viability of Acid-Adapted Escherichia coli O157:H7 in Ground Beef Treated with Acidic Calcium Sulfate

The effects of lactic acid, acetic acid, and acidic calcium sulfate (ACS) on viability and subsequent acid tolerance of three strains of Escherichia coli O157:H7 were determined. Differences in tolerance to acidic environments were observed among strains, but the level of tolerance was not affected by the acidulant to which cells had been exposed. Cells of E. coli O157:H7 adapted to grow on tryptic soy agar acidified to pH 4.5 with ACS were compared to cells grown at pH 7.2 in the absence of ACS for their ability to survive after inoculation into ground beef treated with ACS, as well as untreated beef. The number of ACS-adapted cells recovered from ACS-treated beef was significantly (α = 0.05) higher than the number of control cells recovered from ACS-treated beef during the first 3 days of a 10-day storage period at 4°C, suggesting that ACS-adapted cells might be initially more tolerant than unadapted cells to reduced pH in ACS-treated beef. Regardless of treatment of ground beef with ACS or adaptation of E. coli O157:H7 to ACS before inoculating ground beef, the pathogen survived in high numbers.

Numb and Numblike control cell number during vertebrate neurogenesis

A fundamental problem in nervous system development is how to control progenitor-cell divisions to generate the correct number of cells for the mature brain and spinal cord. A recent report by Petersen and colleagues has identified an essential role for Numb and a related protein, Numblike, in this process. This study demonstrates that Numb and Numblike function to maintain the self-renewal properties of neural progenitor cells in the vertebrate neural tube.

Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development.

During the growth of Drosophila imaginal discs a process called 'cell competition' eliminates slow-proliferating but otherwise viable cells. We report here that cell competition requires the function of the brinker (brk) gene, whose expression is normally repressed by Decapentaplegic (Dpp) signalling but is upregulated in slow-growing Minute/+ cells. Excess brk expression activates the c-Jun amino-terminal kinase pathway, which in turn triggers apoptosis in these cells. We propose that slow-proliferating cells upregulate Brk levels owing to a disadvantage in competing for, or in transducing, the Dpp survival signal. This sequence of events might represent a general mechanism by which weaker cells are eliminated from a growing population, and might serve as a method of controlling cell number and optimizing tissue fitness and hence organ function.

C-Myc regulates mammalian body size by controlling cell number but not cell size.

Overexpression of the proto-oncogene c-myc has been implicated in the genesis of diverse human tumours. c-Myc seems to regulate diverse biological processes, but its role in tumorigenesis and normal physiology remains enigmatic. Here we report the generation of an allelic series of mice in which c-myc expression is incrementally reduced to zero. Fibroblasts from these mice show reduced proliferation and after complete loss of c-Myc function they exit the cell cycle. We show that Myc activity is not needed for cellular growth but does determine the percentage of activated T cells that re-enter the cell cycle. In vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan hypoplasia, in contrast to Drosophila c-myc mutants, which are smaller as a result of hypotrophy. We find that c-myc substitutes for c-myc in fibroblasts, indicating they have similar biological activities. This suggests there may be fundamental differences in the mechanisms by which mammals and insects control body size. We propose that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size.

Apolipoprotein E inhibits serum-stimulated cell proliferation and enhances serum-independent cell proliferation.

Independently of its role in lipid homeostasis, apolipoprotein E (apoE) inhibits cell proliferation. We compared the effects of apoE added to media (exogenous apoE) with the effects of stably expressed apoE (endogenous apoE) on cell proliferation. Exogenous and endogenous apoE increased population doubling times by 30-50% over a period of 14 days by prolonging the G(1) phase of the cell cycle. Exogenous and endogenous apoE also decreased serum-stimulated DNA synthesis by 30-50%. However, apoE did not cause cell cycle arrest; both apoE-treated and control cells achieved equivalent saturation densities at 14 days. Further analyses demonstrated that exogenous and endogenous apoE prevented activation of MAPK but not induction of c-fos expression in response to serum growth factors. Endogenous (but not exogenous) apoE altered serum concentration-dependent effects on proliferation. Whereas control (non-apoE-expressing) cell numbers increased with increasing serum concentrations (1.6-fold for every 2-fold increase in serum), apoE-expressing cell numbers did not differ as serum levels were raised from 2.5 to 10%. In addition, in low serum (0.1%), apoE-expressing cells had elevated DNA synthesis levels compared with control cells. We conclude that apoE does not simply inhibit cell proliferation; rather, the presence of apoE alters the response to and requirement for serum mitogens.

Antagonistic drivers share BuMPy pathway

Seemingly confounding and contradictory, the ability of bone morphogenetic proteins (BMPs) to promote both proliferation and terminal differentiation (and even apoptosis!) of neuronal stem cells—to both expand and limit precursor cell numbers—now makes perfectly good sense. At least it does in light of the conclusions drawn by Ronald McKay, David Panchision, and colleagues (National Institutes of Health, Bethesda, MD), who postulate a mechanism by which BMP ligands control both the production and fate of neural precursor cells. Figure Too much BMPR-IB (right) causes widespread cell death. Panchision attributes this “dynamic process” to the sequential and linked expression and function of two BMP receptors, BMPR-IA and BMPR-IB. In this model, “the BMP-mediated induction of receptor IB accounts for features of stem cell proliferation, identity, differentiation, and death,” says McKay. Expression of mutant BMP receptors in CNS stem cells in vitro and in transgenic mice led to the startling discovery of a “feed-forward” mechanism. Activation of the BMPR-IA receptor early in development promotes proliferation of neural precursor cells and determines their dorsal identity. But BMPR-IA also induces the expression of Bmpr-1b. BMPR-IB activation then mediates mitotic arrest, resulting in either apoptosis (early in development, perhaps to control cell numbers) or terminal differentiation (later in development, for those cells that are left). Additional competence signals must be required to interpret the BMPR-IB signal into an apopototic or differentiation response. An induction–termination sequence could, suggests Panchision, be a general property of signaling in stem cells. It is unclear whether this self-limiting sequential mechanism, which dead-ends at terminal differentiation or cell death, could be reactivated on demand to regenerate tissues, say during wound healing or limb amputation. McKay envisions using knowledge of the linked receptor model to control stem cell proliferation and differentiation, with an eye toward potential applications in cell therapy and cancer therapeutics. ▪ Reference: Panchision, D.M., et al. 2001. Genes Dev. 15:2094–2110. [PubMed]

Apoptosis in squamous cell carcinoma of the lung: correlation with survival and clinicopathological features

Aims—Apoptosis is recognised as a physiological mechanism for controlling cell numbers and its subversion is thought to contribute to carcinogenesis. The aims of this study were to measure the apoptotic...

Apoptotic capability in ageing T cells

The ageing immune system shows a gradual decline in responsiveness to antigens and tumours due to the emergence of immunosenescence. The main functions of T cells are activation, anergy and apoptosis and these are all affected during ageing. Apoptosis is vital in controlling cell numbers, deleting self-reactive T cells and maintaining immune surveillance. One of the principle instigators of death involves the CD95:CD95-ligand interaction and as T cells age both receptor and ligand levels increase. This view will describe the current knowledge of the apoptotic susceptibility of ageing T cells and evaluate the factors that may affect the apoptotic capability of immunosenescent T cells.

Identification and Sequencing of Bcl and Apaf1 Genes in the Common Carp, Cyprinus Carpio.

INTRODUCTION. The role of apoptosis in the immune system is being studied in cultured carp, Cyprinus carpio an economically important farmed fish. In intensive culturing systems, stress is suspected of reducing the immune competence of farm animals. Our initial studies investigated the effect of dexamethasone, a corticosteroid analogue, on the different immunocompetent cells in the pronephros and thymus. This is currently being extended to identify and sequence an apoptotic regulator (Bcl-x) and an apoptotic activator gene (APAF1) from the carp cDNA library. METHOD. Cells from the pronephros and thymus were maintained in vitro, in L-15 medium (Sigma) supplemented with 2mM L- glutamine and containing penicillin (100i.u./ml), streptomycin (100µg/ml), and 5% heat- inactivated foetal calf serum, at 20 o C. They were then exposed to different concentrations of dexamethasone (0.1µM to10.0µM 2 ) for up to 48h in time. Control cells were those maintained in the culture medium lacking in dexamethasone. Cell viability and percentage of apoptotic cell death at times 0h, 24h and 48h were determined using nigrosine dye exclusion and acridine orange stain respectively. DNA from approximately 10 6 cells was isolated from each of the above samples and subjected to agarose gel electrophoresis. In the differential experiment, the different types of immunocompetent cells were labelled with monoclonal antibodies WCI04, WCI09 and WCI12 and their responses to 1.0µM dexamethasone over 6 hours was recorded. Degenerate primers for the Bcl-xL equivalent in carp were designed following a homology study on the sequences of Homo sapiens (human) and Xenopus laevis (South African clawed frog). The homology between human APAF1 and that of Danio rerio (Zebrafish) was examined, and primers constructed, for the comparable sequence in the carp. These were used in PCR to obtain sequences from the carp cDNA library. The sequences obtained will now be used as probes to sequence the rest of the gene(s). RESULTS. Statistical analysis reveals that there is a significant difference between the number of control cells undergoing apoptotic cell death and that of cells exposed to the different concentrations of dexamethasone (p<0.001) (Fig.1). This effect is greatest between the 0h and 24h time periods and appears to decrease after this time period, and is similar in the pronephros and the thymus.

Aspek Molekuler Apoptosis: Peran Keluarga Bcl-2 dan Keluarga Caspase

Apoptosis, known as programmed cell death, is described as a form of cell death characterized by several morphological and biochemical features, such as membrane blebbing, cytoplasmic and nuclear condensation, DNA fragmentation and formation of apoptosis bodies. Genetic studies on Caeneorhabditis elegans have been further extended to apoptosis in mammals. Apoptosis is required for normal tissue homeostasis, which allows the organism to tightly control cell numbers and tissue size, and protect itself from rogue cells. The basic components that play a major role in apoptosis include the Bcl-2 and Caspase families. Bcl-2 family functions as either pro or anti-apoptosis, while the Caspase family includes the central executioner, the basic effectors of apoptosis, leading ultimately to fragmentation of DNA. This review describes the role of Bcl-2 and Caspase families in the process of apoptosis.

Somatic support cells restrict germline stem cell self-renewal and promote differentiation.

Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.

Cellular Death as a Regulative Mechanism in the Control of Cell-Number towards Normal Establishment of Orderly Structure and Function in the Petrous Ganglion during Development and Aging: An Investigation in the Chick

Cellular Death as a Regulative Mechanism in the Control of Cell-Number towards Normal Establishment of Orderly Structure and Function in the Petrous Ganglion during Development and Aging: An Investigation in the Chick

Wnt signaling and cancer

The regulation of cell growth and survival can be subverted by a variety of genetic defects that alter transcriptional programs normally responsible for controlling cell number. High throughput analysis of these gene expression patterns should ultimately lead to the identification of minimal expression profiles that will serve as common denominators in assigning a cancer to a given category. In the course of defining the common denominators, though, we should not be too surprised to find that cancers within a single category may nevertheless exhibit seemingly disparate genetic defects. The wnt pathway has already provided an outstanding example of this. We now know of three regulatory genes in this pathway that are mutated in primary human cancers and several others that promote experimental cancers in rodents (Fig. 1). In all of these cases the common denominator is the activation of gene transcription by -catenin. The resulting gene expression profile should provide us with a signature common to those cancers carrying defects in the wnt pathway. In this review, the wnt pathway will be covered from the perspective of cancer, with emphasis placed on molecular defects known to promote neoplastic transformation in humans and in animal models.

Apoptosis and cancer

Apoptosis, or programmed cell death, is a central mechanism controlling cell number and the deletion of unwanted cells. Recent efforts have lead to the identification of the machinery involved in the control and execution of the apoptotic program Alteration of the normal apoptotic program has been identified as the possible cause of a number of human diseases, including neoplasia. In this review the means by which disruption of the normal apoptotic machinery may lead or contribute to tumor generation and progression will be discussed.

Friend virus-induced erythroblastosis in STAT5a/b mutant mice: Properties of isolated erythroblasts in vitro

This study examined the roles of the Stat5a and Stat5b gene products in growth and differentiation of mouse erythroid cells. BALB/c mice bearing inactivating mutations in both Stat5a and Stat5b were infected with the anemia-inducing strain of Friend virus. The infected mice developed erythroblastosis in the spleen typical of the early phase of Friend disease. Early erythroblasts purified from the spleens of infected mice (Stat5a/b mutants and BALB/c wild type controls) were cultured in medium containing 2 U/mL of erythropoietin (EPO). During the culture period of 60 h, a significant fraction of the Stat5a/b(−/−) erythroblasts differentiated into reticulocytes. However, the population exhibited several properties that were different from controls. Control cell numbers increased 6-fold during the 60 h culture period and about 90% of the resultant population formed reticulocytes. The Stat5a/b(−/−) cells increased in number only 3.5-fold. About 30% of the cells never initiated hemoglobin synthesis, and about one-half of the remainder did not mature into reticulocytes. After 36 h of culture, the Stat5a/b(−/−) erythroblast population contained a higher fraction than normal of cells which were positive for DNA degradation by a flow cytometric TUNEL assay although morphological apoptosis was not pronounced. This pleotropic phenotype does not resemble the type or timing of apoptosis induced by EPO deprivation. The combined result of the above factors was inefficient erythropoiesis by Stat5a/b(−/−) cells in vitro. Several molecular regulators of apoptosis and of cell cycle were examined. Normal levels of Bcl-x production were observed and no activation of caspase 3 or cleavage of PARP was detected. p27 and p21 were significantly elevated in Stat5a/b(−/−) cells. The fractions of cells in G1 and S phases of the cell cycle were different in Stat5a/b (−/−) and control cells throughout the culture period.

Programmed cell death in the developing heart.

In humans every minute millions of cells die by programmed cell death and by the end of their life almost 99.9% of all cells once made have undergone this fate [1]. The functionality of cell death is different during development and adult life. During development apoptosis serves three major functions: deleting vestigial structures, i.e. phylogenetic cell death; controlling cell numbers, i.e. histogenetic cell death, and remodeling structures, i.e. morphogenetic cell death [2]. In adult life, apoptosis mainly serves to maintain homeostasis by counterbalancing mitosis and deleting cells, which are potentially autoimmunoreactive, malignant, or virus infected [3,4]. In contrast to adult life cell death, where cell death is generally considered a pathological event, during cardiac development cell death is a physiological event that is highly regulated in space and time. It took till 1968 when cell death was first recognized in the chicken ventricular myocardium [5]. In the next decade, the spatiotemporal pattern of cell death in the developing heart was extensively studied, in particular by the groups of Pexieder [6–10], Okamoto [11–14] and Ojeda [15–18]. These studies accurately described cell death patterns, but the underlying mechanisms and functions were not addressed. Despite the large body of data regarding the spatiotemporal patterns of cell death in the developing heart, a comprehensive overview of cell death in this developing organ is difficult to obtain. This is mainly because the original data were not published in regular journals and/or in the German language. Moreover, the nomenclature of the various structures of the developing heart has changed significantly during the last decades, which adds to the imperviousness of the data. The objective of this review is to bring together data derived from the earlier as well as from recent studies, and to generate a map of cardiac cell … * Corresponding author. Tel.: +31-20-5669-111; fax: +31-20-6976-177 m.j.vandenhoff{at}amc.uva.nl a.f.moorman{at}amc.uva.nl

Stem cells and the cellular organization of the brain.

The modern field of neuroscience emerged in the hands of neuroanatomists at the end of the last century. They recognized that information flows through arrays of cells and that the structure of the cells shapes the passage of information through the brain. In the middle of this century, the role of ion flux in the movement of information along axons was established. The electron microscope and the microelectrode helped to explain information transfer at the synapse, and study of the modulation of synaptic strength is currently a major area of neuroscience research. The origin of the many types of neuron seen by the early anatomists remains enigmatic, but the solution to this mystery is now emerging. The identification of stem cells and of the mechanisms that control their differentiation into distinct neuron types will contribute to a new understanding of both the origin of neuronal types and neuronal circuits. The current enthusiasm for stem cells is stimulated by interest from both the academic and nonacademic communities. These enthusiasts recognize a simple truth, that the cells are smart; they are the agents that control cell number and type in the brain. They create the structure that generates the higher order brain function. If these premises are true, then in the new century our understanding of the nervous system will be transformed by the facts of stem cell biology. J. Neurosci. Res. 59:298-300, 2000. Published 2000 Wiley-Liss, Inc.