A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

A computational model for cell differentiation

In this paper, we present a word set generating mechanism, called cell-differentiation system, inspired by the tissue process formation in multicellular organisms, which might model some properties of evolving communities of living cells at the syntactical level. The tools utilized to model these biological phenomena belong to the formal language theory. In this context chromosomal mutations are defined as operations on strings and the differentiation according to the control of gene expression is represented by some random-context conditions in formal languages.In the presented formal framework we prove that in a simplified form of this formalism, with only one cell-type which is regular, one single cell and no mitosis involved, the problem of establishing whether or not the set of vectors of integers indicating the number of cells in each population, is finite, linear or semilinear, is recursively undecidable. However, one can algorithmically decide whether or not a cell-differentiation system of finite cell-type can produce a specific generation of cells.     

Studies of alpha-protein in human cell cultures

alpha-Protein growth fraction (AGF) eliminates the 60- to 90-day adaptive phase required to establish actively growing cultures of HeLa (Gey), human heart (Girardi), KB (Eagle) and other established cell lines in serum-free chemically defined medium A3. AGF is effective at less than 0.4 microgram per ml. By using the procedures described in the text, it is possible to culture HeLa cells in very simple media such as Eagle's basal medium. The properties of AGF are such that it may be adsorbed on glass or plastic flasks. Glass flasks treated with AGF retain full activity after washing with acetone, and treatment with ethyl ether and chemically defined medium. Adsorbed AGF is destroyed by trypsin. AGF can detoxify protamines, polylysines or histones. It will reverse the aggregation response induced by adding complexes composed of carboxymethylcellulose (CMC) and basic proteins. The results support the contention that highly adsorptive AGF functions at the cell surface and is capable of modifying the response of the cell to its environment.     

Identification and regulation of a molecular module for bleb-based cell motility

Single-cell migration is a key process in development, homeostasis, and disease. Nevertheless, the control over basic cellular mechanisms directing cells into motile behavior in?vivo is largely unknown. Here, we report on the identification of a minimal set of parameters the regulation of which confers proper morphology and cell motility. Zebrafish primordial germ cells rendered immotile by knockdown of Dead end, a negative regulator of miRNA function, were used as a platform for identifying processes restoring motility. We have defined myosin contractility, cell adhesion, and cortex properties as factors whose proper regulation is sufficient for restoring cell migration of this cell type. Tight control over the level of these cellular features, achieved through a balance between miRNA-430 function and the action of the RNA-binding protein Dead end, effectively transforms immotile primordial germ cells into polarized cells that actively migrate relative to cells in their environment.     

Mast cell granule composition and tissue location--a close correlation

This review provides a survey on mast cell heterogeneity, with aspects differing in humans and rodents or which are subject of conflicting evidence being discussed in greater detail. Mast cell subsets have been first defined in rats by their fixation and dye-binding properties, and detailed studies in humans and pigs reveal very similar observations. The dye-binding properties of rat mast cell subsets are causally related to the absence or presence of heparin in their granules. In humans, this relation has not been shown. Rodent mast cell subsets store different chymase-isoforms. In contrast, just a single chymase has been defined in humans, and mast cells are classified by the presence or relative absence of this chymase. Different investigators find quite different proportions of chymase-positive to chymase-negative mast cells. Tryptase(s) are found in most or every human mast cell, but in rodents, they have hitherto been essentially localised to mast cells in connective tissues. Human mast cell subsets may also be defined by their expression of receptors such as C5aR and possibly the beta-chemokine receptor CCR3; the CCR3 expression seems to be related to the human mast cell chymase expression. Ultrastructural studies are helpful to distinguish human mast cell subsets, and allow to distinguish between chronic and acute activation. The phenotypical characteristics may change in association with inflammation or other disease processes. Studies in humans and pigs show changed dye-binding and fixation properties of the granules. Experimental rodent infection models reveal similar changes of chymase isoform expression. Human lung mast cells have been reported to strongly upregulate their chymase content in pulmonary vascular disease. This line of evidence can explain some inconsistent information on mast cell heterogeneity and may help to understand the physiological role of mast cells.     

Immunological detection of somatostatin in a primitive hypothalamic mouse cell line, precursor of a neurophysin cell lineage.

A primitive nerve cell line (F7 clone), obtained by SV 40 transformation of mouse fetal hypothalamic cells, has been previously shown to share some properties with a progenitor cell for neurophysin synthesizing neuron. This paper describes the immunological detection, at the light microscope level, of somatostatin in this cell line. A SV 40 transformed neurosecretory clone, synthesizing neurophysin and vasopressin, was used as control.     

Relations among variations in human red cell volume,density, membrane area,hemoglobin content and cation content

It is shown how variations in different properties of red cells can be inter-related provided relations exist among these properties at the single cell level. On the basis of the cell density dependence on cell volume and hemoglobin content, and the assumed volume dependence on red cell cation and hemoglobin content, nine relations among the variations in red cell volume, density, membrane area, hemoglobin content and cation content, and their correlations are derived. Values of seven correlation coefficients are theoretically predicted and are shown to be consistent with the experiments performed by density fractionated red blood cells. The cell volume dependence on cation and hemoglobin content obtained from relations among variations is compared with the predictions obtained by the existing model about the osmotic behavior of the red blood cell. Furthermore, it is shown that data on the variations of the red cell properties indicate the existence of the relation among cation content, hemoglobin content, and membrane area at the level of a single cell.     

The lin-12 locus specifies cell fates in Caenorhabditis elegans

We describe two classes of mutations in the lin-12 locus of the nematode Caenorhabditis elegans. Ten semidominant mutations (lin-12[d]) appear to elevate the level of lin-12 activity. Thirty-two recessive alleles (lin-12[0]), including two amber mutations, appear to eliminate gene activity. The lin-12(d) and lin-12(0) mutations result in reciprocal homeotic transformations in the fates of defined cells in several different tissues. Gene dosage studies suggest that a high level of lin-12 activity specifies one cell fate and a low level specifies an alternative fate. Temperature-shift experiments indicate that lin-12 acts at the time cell fate is determined in wild type. We propose that lin-12 functions as a binary switch to control decisions between alternative cell fates during C. elegans development.     

Effects of substratum morphology on cell physiology

Among the host of substratum properties that affect animal cell behavior, surface morphology has received relatively little attention. The earliest effect of surface morphology on animal cells was discovered almost a century ago when it was found that cells became oriented in response to the underlying topography. This phenomenon is now commonly known as contact guidance. From then until very recentrly, little progress has been made in understanding the role of surface morphology on cell behavior, primarily due to a lack of defined surfaces with uniform morphologies. This problem has been solved recently with the development of photolithographic techniques to prepare substrata with well defined and uniform surface morphologies. Availability of such surfaces has facilitated systematic in vitro experiments to study influence of surface morphology on diverse cell physiological aspects such as adhesion, growth, and function. For example, these studies have shown that surfaces with uniform multipls parallel grooves can enhance cell adhesion by confining cells in grooves and by mechanically interlocking them. Several independent studies have demosterated that cell shape is a major determinant of cell growth and function. Because surface morphology has been shown to modulate the extent of cell spreading and cell shape, its effects on cell growth and function appear to be mediated via this biological coupling between cell shape and function. New evidence in the cell biology literature is emerging to suggest that surface morphology could affect other cell behavioral properties such as post-translational modifications. Further elucidation of such effects will enable better designs for implant and cell culture substrata.     

The cellular basis of cell sorting kinetics

Cell sorting is a dynamical cooperative phenomenon that is fundamental for tissue morphogenesis and tissue homeostasis. According to Steinberg's differential adhesion hypothesis, the structure of sorted cell aggregates is determined by physical characteristics of the respective tissues, the tissue surface tensions. Steinberg postulated that tissue surface tensions result from quantitative differences in intercellular adhesion. Several experiments in cell cultures as well as in developing organisms support this hypothesis.The question of how tissue surface tension might result from differential adhesion was addressed in some theoretical models. These models describe the cellular interdependence structure once the temporal evolution has stabilized. In general, these models are capable of reproducing sorted patterns. However, the model dynamics at the cellular scale are defined implicitly and are not well-justified. The precise mechanism describing how differential adhesion generates the observed sorting kinetics at the tissue level is still unclear.It is necessary to formulate the concepts of cell level kinetics explicitly. Only then it is possible to understand the temporal development at the cellular and tissue scales. Here we argue that individual cell mobility is reduced the more the cells stick to their neighbors. We translate this assumption into a precise mathematical model which belongs to the class of stochastic interacting particle systems. Analyzing this model, we are able to predict the emergent sorting behavior at the population level. We describe qualitatively the geometry of cell segregation depending on the intercellular adhesion parameters. Furthermore, we derive a functional relationship between intercellular adhesion and surface tension and highlight the role of cell mobility in the process of sorting. We show that the interaction between the cells and the boundary of a confining vessel has a major impact on the sorting geometry.     

Water in the orchestration of the cell machinery. Some misunderstandings: a short review

Nowadays, biologists can explore the cell at the nanometre level. They discover an unsuspected world, amazingly overcrowded, complex and heterogeneous, in which water, also, is complex and heterogeneous. In the cell, statistical phenomena, such as diffusion, long considered as the main transport for water soluble substances, must be henceforth considered as inoperative to orchestrate cell activity. Results at this level are not yet numerous enough to give an exact representation of the cell machinery; however, they are sufficient to cease reasoning in terms of statistics (diffusion, law of mass action, pH, etc.) and encourage cytologists and biochemists to prospect thoroughly the huge panoply of the biophysical properties of macromolecule-water associations at the nanometre level. Our main purpose, here, is to discuss some of the more common misinterpretations due to the ignorance of these properties, and expose briefly the bases for a better approach of the cell machinery. Giorgio Careri, who demonstrated the correlation between proton currents at the surface of lysozyme and activity of this enzyme was one of the pioneers of this approach.     

Micromechanical architecture of the endothelial cell cortex

Mechanical properties of living cells are important for cell shape, motility, and cellular responses to biochemical and biophysical signals. Although these properties are predominantly determined by the cytoskeleton, relatively little is known about the mechanical organization of cells at a subcellular level. We have studied the cell cortex of bovine pulmonary artery endothelial cells (BPAECs) using atomic force microscopy (AFM) and confocal fluorescence microscopy (CFM). We show that the contrast in AFM imaging of these cells derives in large part from differences in local mechanical properties, and AFM images of BPAEC reveal the local micromechanical architecture of their apical cortex at approximately 125 nm resolution. Mechanically the cortex in these cells is organized as a polygonal mesh at two length scales: a coarse mesh with mesh element areas approximately 0.5-10 microm2, and a finer mesh with areas <0.5 microm2. These meshes appear to be intertwined, which may have interesting implications for the mechanical properties of the cell. Correlated AFM-CFM experiments and pharmacological treatments reveal that actin and vimentin are components of the coarse mesh, but microtubules are not mechanical components of the BPAEC apical cortex.     

Molecular basis of B cell activation: mitogenicity of native and modified digitalis glycosides.

Lanatoside C, a digitalis glycoside, has previously been shown to be a potent polyclonal B cell activator for mouse lymphocytes. Since the chemical structure of lanatoside C is well defined we investigated the effect of different chemical modifications on the mitogenic properties of the glycoside in order to determine the molecular basis of B cell activation. The presence of an OH- group at C12 in the steroid nucleus proved to be essential for activation to occur in serum-free medium. Likewise, an intact carbohydrate side chain at C3 appeared to be necessary for retaining mitogenic properties. In serum-supplemented cultures, however, a much more complex response pattern was observed where the prerequisites for activation were not as distinct as in serum-free medium.     

A morphologic and immunofluorescent analysis of primary guinea pig glomerular cell types grown in chemically defined media. Evidence for clonal growth and cell differentiation

Primary kidney guinea pig glomerular cells were successfully grown in chemically defined media containing insulin, transferrin, and fibronectin or glycylhistidyllysine and fibronectin. Morphologic analysis of glomerular cells grown in either of these chemically defined media provided identical results with respect to cell growth properties and cell types involved. Electron microscopic studies of glomeruli early after they had been placed in culture showed definite evidence of "dedifferentiation" of some glomerular cells. Most glomerular cells in later cultures were undifferentiated. However, since electron microscopic analyses of glomeruli in confluent cultures demonstrated that the majority of cells in culture grow from the epithelial side of the glomerular basement membrane, we suggest that these cells were some form of epithelial cell. This conclusion was further strengthened by the fact that cells resembling well differentiated glomerular epithelial cells were seen in cultures of glomeruli grown in chemically defined media; these cells have never been observed in glomeruli grown in calf serum. Fluorescent microscopy of cell stained with the mitochondrial stain rhodamine 123 allowed identification of several glomerular cell types according to distribution, number, and morphology of mitochondria. Similarly, indirect immunofluorescent microscopy studies using antibodies to fibronectin or laminin provided evidence that glomerular cells separated into cell types according to mitochondrial staining properties were unique biochemically. Using these histochemical criteria it was possible to demonstrate that certain of the glomerular cell types could be selectively grown by addition of the enzyme galactose oxidase to the media. Analysis of our morphologic and histochemical results suggests the possibility that clonal growth and differentiation of glomerular epithelial cells occurs when glomeruli are placed in chemically defined media, and our results are compatible with the hypothesis that either "stem cells" or "dedifferentiated" cells are the primary cells dividing in culture.     

Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT29-D4 in serum-free medium

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.     

Advances in plant cell type-specific genome-wide studies of gene expression

Cell is the functional unit of life.To study the complex interactions of systems of biological molecules,it is crucial to dissect these molecules at the cell level.In recent years,major progresses have been made by plant biologists to profile gene expression in specific cell types at the genome-wide level.Approaches based on the isolation of cells,polysomes or nuclei have been developed and successfully used for studying the cell types from distinct organs of several plant species.These cell-level data sets revealed previously unrecognized cellular properties,such as cell-specific gene expression modules and hormone response centers,and should serve as essential resources for functional genomic analyses.Newly developed technologies are more affordable to many laboratories and should help to provide new insights at the cellular resolution in the near future.     

Ion channels in human macrophages compared with the U-937 cell line

Summary The human cell line U-937 has been used extensively to model many macrophage functions. We have examined the cell membranes of human monocyte-derived macrophages (HMDM) and U-937 cells to compare membrane properties as expressed by single ion channel currents. The patch-clamp technique was applied to isolated, nonactivated, inside-out patches of cell membranes obtained from HMDM and from the U-937 cell line. Voltage-gated potassium channels of similar conductance but different kinetics are present in both types of cells, and a calcium-activated potassium channel is present only in the HMDM. These differences in ion channel properties suggest fundamentally different behavior between these two cell types at the level of the cell membrane.     

Extracting cancer cell line electrochemical parameters at the single cell level using a microfabricated device

Cancer cells have distinctive electrochemical properties. This work sheds light on the system design aspects and key challenges that should be considered when experimentally analyzing and extracting the electrical characteristics of a tumor cell line. In this study, we developed a cellularbased functional microfabricated device using lithography technology. This device was used to investigate the electrochemical parameters of cultured cancer cells at the single-cell level. Using impedance spectroscopy analyses, we determined the average specific capacitance and resistance of the membrane of the cancer cell line B16-F10 to be 1.154 ± 0.29 μF/cm², and 3.9 ± 1.15 KΩ.cm² (mean ± SEM, n =14 cells), respectively. The consistency of our findings via different trails manifests the legitimacy of our experimental procedure. Furthermore, the data were compared with a proposed constructed analytical-circuit model. The results of this work may greatly assist researchers in defining an optimal procedure while extracting electrical properties of cancer cells. Detecting electrical signals at the single cell level could lead to the development of novel approaches for analysis of malignant cells in human tissues and biopsies.