Control of 3T3 cell proliferation by calcium

Summary When a population of 3T3 mouse cells was subcultured regularly at confluency, the original epitheliodid or stellate cells disappeared and, by the ninth passage, they had been replaced by spindle-shaped cells. The original cells proliferated only when the extracellular calcium concentration exceeded 0.1mm, and their proliferative activity became maximum only when the calcium concentration was 0.5mm. The spindle-shaped cells were much more sensitive to proliferative stimulation by calcium. Although these cells also could not proliferate without extracellular ionic calcium, they proliferated maximally in the presence of as little as 0.05mm calcium. Thus, calcium is a major regulator of the proliferation of 3T3 mouse cells. Moreover, it appears that the sensitivity of the proliferative machinery to the calcium ion can vary greatly within an established cell line.     

Cell growth, cell division and cell size homeostasis in Swiss 3T3 cells

By separating large and small 3T3 cells we show here that cell growth (in volume) after stimulation from quiescence is not 'autocatalytic'. Rather, large cells grow significantly more slowly, in relative terms, than small cells. It follows that 3T3 cells do not require a size control mechanism operating at the level of division timing in order to achieve cell size homeostasis.     

Polycystin-1 induces cell migration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependent cell cell mechanical adhesion

Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1-/- mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and beta-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated beta-catenin through activation of GSK3beta. Expression of a nondegradable form of beta-catenin in PC-1 MDCK cells restores strong cell-cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis.     

Modeling cell migration in 3D

Cell migration is a multi-scale process that integrates signaling, mechanics and biochemical reaction kinetics. Various mathematical models accurately predict cell migration on 2-D surfaces, but are unable to capture the complexities of 3D migration. Additionally, quantitative 3D cell migration models have been few and far between. In this review we look and characterize various mathematical models available in literature to predict cell migration in 3-D matrices and analyze their strengths and possible changes to these models that could improve their predictive capabilities.     

Interleukin 3 and cell cycle progression

Interleukin 3 (IL-3) is a regulatory glycoprotein required for the proliferation and differentiation of cells from many if not all hemopoietic lineages. With the emergence of the competence-progression model of cell proliferation, which predicts that growth factors function at specific stages of the cell cycle, we examined the possibility that IL-3 functions at a specific stage of the cell cycle. C-63 cells were developed as a cell line from normal murine bone marrow. They have a mast cell phenotype and require pokeweed-stimulated spleen cell-conditioned medium (CM), a rich source of IL-3, for their continued growth. Exponentially growing cells were transferred from growth medium, which contains CM, to medium lacking CM or IL-3. After 24 hours, cell viability had decreased 40-50%. The remaining viable cells did not incorporate 3H-thymidine, and displayed a single peak at G1 in a DNA histogram. Restimulation of these cells with CM or IL-3 resulted in a dramatic rise in 3H-thymidine uptake 20-24 hours after restimulation. DNA histograms of restimulated cultures indicated that the cells were progressing in a wave-like fashion throughout the remainder of the cell cycle. The length of time necessary for cells to be in contact with CM or IL-3 before they could progress into the remainder of the cell cycle was also examined. Cells incubated with CM or IL-3 for less than 16 hours could not progress into S phase, whereas cells incubated for 16 hours or longer could progress into S phase and through the remainder of the cell cycle. These data suggest that IL-3 exerts its function at a specific stage of the cell cycle.     

CXCR3 in T cell function

CXCR3 is a chemokine receptor that is highly expressed on effector T cells and plays an important role in T cell trafficking and function. CXCR3 is rapidly induced on naïve cells following activation and preferentially remains highly expressed on Th1-type CD4⁺ T cells and effector CD8⁺ T cells. CXCR3 is activated by three interferon-inducible ligands CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I-TAC). Early studies demonstrated a role for CXCR3 in the trafficking of Th1 and CD8 T cells to peripheral sites of Th1-type inflammation and the establishment of a Th1 amplification loop mediated by IFNγ and the IFNγ-inducible CXCR3 ligands. More recent studies have also suggested that CXCR3 plays a role in the migration of T cells in the microenvironment of the peripheral tissue and lymphoid compartment, facilitating the interaction of T cells with antigen presenting cells leading to the generation of effector and memory cells.     

Multifunctional 14-3-3 proteins of plant cell

The 14-3-3 proteins are a family of highly conserved proteins found in all eukaryotes - from the yeasts to mammals. They regulate several cellular processes recognizing unique conservative, mostly phosphorylated motif of partner proteins. Binding of the 14-3-3 proteins regulates their partners through a variety of mechanisms, such as altering their catalytic activity, subcellular localization, stability or altering their interactions with other protein molecules. The native 14-3-3 proteins are present in form of homo- and hetero-dimers. The most structurally variable N-and C-termini are responsible for isoform specific protein-protein interactions, and cellular localization. In plant cell, 14-3-3 proteins appear to play an important role in regulation of key enzymes of carbon and nitrogen metabolism, modulation ion pumps and channels. They are also involved in signal transduction pathways and even in gene expression.     

Mammary serum factor initiates cell growth in 3T3 cell variants which are non-responsive to EGF

The structural organization of the histone-containing spermatozoa of the goldfish C. auratus is studied. Chemical fractionation shows that the five standard histones are present. Histone H1 subfractions are also similar to those found in other organisms. This is the first organism studied in which no peculiar basic protein is found in spermatozoa, since in all the organisms described up to now, unique protein components are always present, either alone or accompanied by somatic-like histones. Ultrastructural studies show that this chromatin is organized as a bundle of fibers of about 25 nm diameter, which upon spreading give the typical ‘beads-on-a-string’ appearance. Nuclease digestion demonstrates a repeat length of 205 base pairs (bp), slightly longer than in somatic tissues, but shorter than in echinoderm spermatozoa.     

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles’ heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.     

Mouse 3T3 cell filtrability correlating with concanavalin A agglutinability

In a previous study of the dextran gel sphere model system, a possible correlation between cell deformability and agglutinability by concanavalin A was indicated. Cell deformability was evaluated as filtrability, using polycarbonate membrane filtration. With 25-mm diameter filters and 5-ml cell suspensions at (0.8–16) · 10⁵ cells/ml, the filtrability at a given filter pore size was highly reproducible and was not affected by variations in cell population, viability, washings of cells retained on filter, or temperature. The filtrability of EDTA-dissociated 3T3 cells through 12-μm pore size filter was 8%, and a suspension of 10⁶ cells/ml was not agglutinated by 600 μg concanavalin A. The filtrability of trypsin-dissociated 3T3 cells was 95%, and these cells were agglutinated by 200 μg of the lectin. EDTA-dissociated SV-3T3 cells had a filtrability of 73% and were also highly agglutinable. Formalin fixation reduced the high filtrability to 6%, and also abolished the agglutinability. As a further test of the correlation, trypsin-dissociated 3T3 cells were admixed with the fixed cells. The agglutinability varied with the proportions of the two cell components, and the admixtures could be separated according to filtrability into the original components with distinctly different agglutinability. Furthermore, 25% of a random population of EDTA-dissociated SV-3T3 cells retained by the filter were found to be non-agglutinable. The separated SV-3T3 cell fractions could also form admixtures of different agglutinability. It is concluded that the agglutinability of mouse 3T3 and SV-3T3 cells by concanavalin A can be correlated with the predicted by cell filtrability.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Labile intracellular zinc is associated with 3T3 cell growth

Increasing evidence shows that labile intracellular zinc is metabolically important. Depletion of labile intracellular zinc using chelators suppresses DNA synthesis. In this study, we tested the hypothesis that labile intracellular zinc could be modulated via varying zinc nutrition. This could result in an altered availability of labile intracellular zinc, which, in turn, could influence zinc-dependent cellular events involved in cell proliferation and ultimately suppress growth. Labile intracellular zinc was detected by using N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), a membrane-permeable fluorescence probe. After 48 h culture in a zinc-depleted medium, labile intracellular zinc in 3T3 cells was diminished along with a suppressed DNA synthesis and cell proliferation. In contrast, supplementation of zinc to the zinc-depleted medium increased the labile intracellular zinc and promoted DNA synthesis and cell proliferation. Furthermore, growth factor-dependent stimulation of DNA synthesis and cell proliferation was also accompanied by increased labile intracellular zinc. Together, our data showed an association between the labile intracellular zinc, detected using TSQ, and 3T3 cell growth, suggesting that labile intracellular zinc could be an important cellular link between zinc nutrition and growth.     

Expression of Nanog gene promotes NIH3T3 cell proliferation

Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion.     

The red cell 3 phosphoglycerate kinase polymorphism

Summary Blood samples from 778 Burundian, Rwandan, and Zaïran negroes were examined by electrophoresis on cellulose acetate for phosphoglycerate kinase polymorphism. PGK 1 was observed in all but 6 hemolysates; in these cases a new allele was detected, having a frequency of about 0.018 in Rwandans.     

From 3D cell culture to organs-on-chips

3D cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems. We review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These 'organs-on-chips' permit the study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing.     

Interleukin 3-dependent hematopoietic progenitor cell lines

Several biological phenotypes of growth factor-dependent cell lines have been described in recent years, including those with T lymphocyte, neutrophil granulocyte, basophil/mast cell, B lymphocyte, and multipotential stem cell properties. The growth factors for each cell lineage are a subject of intense study. Continuous mouse bone marrow cultures infected with RNA type C viruses (retroviruses) produce nonadherent hematopoietic cells over a longer duration than control cultures. Marrow cultures derived from strains with spontaneously induced ecotropic endogenous retrovirus demonstrate a greater longevity than those from strains with no replicating virus. Cultures infected with murine leukemia virus also generate a greater number, compared with controls, of cloned permanent suspension cell lines dependent for growth on a 41,000-dalton glycoprotein (interleukin 3 [IL 3]). Some are multipotential with capacity for differentiation to erythroid, neutrophil, eosinophil, and basophil/mast cell types. Other cloned IL 3-dependent cell lines are committed to a single pathway. Studies with Friend spleen focus-forming virus indicate that the first effect in the marrow culture is mediated through a subset of adherent hematopoietic stem cells. Bone marrow culture-derived IL 3-dependent cell lines provide a model with which to study the role of viral genes in the control of differentiation and self-renewal capacity of hematopoietic stem cells.