PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

Modeling cell proliferation for simulating three-dimensional tissue morphogenesis based on a reversible network reconnection framework

Tissue morphogenesis in multicellular organisms is accompanied by proliferative cell behaviors: cell division (increase in cell number after each cell cycle) and cell growth (increase in cell volume during each cell cycle). These proliferative cell behaviors can be regulated by multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze multicellular dynamics, a reversible network reconnection (RNR) model has been suggested, in which each cell shape is expressed by a single polyhedron. In this study, to apply the RNR model to simulate tissue morphogenesis involving proliferative cell behaviors, we model cell proliferation based on a RNR model framework. In this model, cell division was expressed by dividing a polyhedron at a planar surface for which cell division behaviors were characterized by three quantities: timing, intracellular position, and normal direction of the dividing plane. In addition, cell growth was expressed by volume growth as a function of individual cell times within their respective cell cycles. Numerical simulations using the proposed model showed that tissues grew during successive cell divisions with several cell cycle times. During these processes, the cell number in tissues increased while maintaining individual cell size and shape. Furthermore, tissue morphology dramatically changed based on different regulations of cell division directions. Thus, the proposed model successfully provided a basis for expressing proliferative cell behaviors during morphogenesis based on a RNR model framework.     

Two distinct mechanisms for inhibition of cell growth in human prostate carcinoma cells with antioxidant enzyme imbalance.

The purpose of the present study was to determine whether manganese superoxide dismutase (MnSOD) overexpression in DU145 human prostate carcinoma cells affected cell reduction-oxidation state (cell redox) and to correlate changes in cell redox status with cell cycle progression and plating efficiency. One MnSOD-overexpressing cell line had no change in other antioxidant enzymes (AEs) (nonadapted clone), whereas a second MnSOD-overexpressing cell line studied had an increase in catalase (CAT) activity (adapted clone). Correlation of biochemical studies with cell cycle studies suggested that heteroploidy observed in the nonadapted MnSOD-overexpressing cell line may be due to increased intracellular peroxides with resultant disruption of the microtubule network, while a decreased mitotic rate was associated with decreased ATP levels in mitosis. In contrast, the decrease in cell growth in the adapted cell line was demonstrated to be due to a decrease in plating efficiency. Our results demonstrate complex effects of AE imbalance on cell growth of DU145 prostate cancer cells.     

Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants

Ascidian early embryonic cells undergo cell differentiation without cell cleavage, thus enabling mixture of cell fate determinants in single cells, which will not be possible in mammalian systems. Either cell in a two-cell embryo (2C cell) has multiple fates and develops into any cell types in a tadpole. To find the condition for controlled induction of a specific cell type, cleavage-arrested cell triplets were prepared in various combinations. They were 2C cells in contact with a pair of anterior neuroectoderm cells from eight-cell embryos (2C-aa triplet), with a pair of presumptive notochordal neural cells (2C-AA triplet), with a pair of presumptive posterior epidermal cells (2C-bb triplet), and with a pair of presumptive muscle cells (2C-BB triplet). The fate of the 2C cell was electrophysiologically identified. When two-cell embryos had been fertilized 3 h later than eight-cell embryos and triplets were formed, the 2C cells became either anterior-neuronal, posterior-neuronal or muscle cells, depending on the cell type of the contacting cell pair. When two-cell embryos had been fertilized earlier than eight-cell embryos, most 2C cells became epidermal. When two- and eight-cell embryos had been simultaneously fertilized, the 2C cells became any one of three cell types described above or the epidermal cell type. Differentiation of the ascidian 2C cell into major cell types was reproducibly induced by selecting the type of contacting cell pair and the developmental time difference between the contacting cell pair and 2C cell. We discuss similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells and propose the ascidian 2C cell as a simple model for toti-potent stem cells.     

Rhizoid differentiation in Spirogyra: position sensing by terminal cells

Some species of Spirogyra anchor themselves to the substrate by differentiating rhizoids. A rhizoid is differentiated only from the terminal cell, suggesting that this cell can recognize its terminal position in a filament. In the present study, we have analyzed the mechanism for position sensing by the terminal cell. When a filament is cut, a new cell occupies the terminal position, and three phenomena are induced: (1) the cell wall of the cut cell detaches from the new terminal cell; (2) adhesive material is secreted by the terminal cell; and (3) the terminal cell begins to differentiate a rhizoid via tip growth. All of these phenomena were inhibited by adding sorbitol to the external medium, suggesting that turgor pressure is involved in position sensing by the terminal cell. The inhibition by sorbitol was reversible. Upon cutting a filament, the distal end of a new terminal cell became convex. However, when a filament was cut in the presence of sorbitol, the distal end of a new terminal cell became less convex. Either treatment with Gd(3+) or decrease in extracellular Ca(2+) resulted in inhibition of all these phenomena, suggesting possible involvement of stretch-activated ion channel in position sensing by terminal cells.     

Cellulose synthesis is coupled to cell cycle progression at G1 in the dinoflagellate Crypthecodinium cohnii

Cellulosic deposition in alveolar vesicles forms the "internal cell wall" in thecated dinoflagellates. The availability of synchronized single cells, the lack of secondary deposition, and the absence of cellulosic cell plates at division facilitate investigation of the possible roles of cellulose synthesis (CS) in the entire cell cycle. Flow cytograms of cellulosic contents revealed a stepwise process of CS in the dinoflagellate cell cycle, with the highest rate occurring at G(1). A cell cycle delay in G(1), but not G(2)/M, was observed after inhibition of CS. A cell cycle inhibitor of G(1)/S, but not G(2)/M, was able to delay cell cycle progression with a corresponding reduction of CS. The increase of cellulose content in the cell cycle corresponded well to the expected increase of surface area. No differences were observed in the cellulose to surface area ratio between normal and fast-growing G(1) cells, implicating the significance of surface area in linking CS to the coupling of cell growth with cell cycle progression. The coupling of CS to G(1) implicates a novel link between CS and cell cycle control, and we postulate that the coupling mechanism might integrate cell wall integrity to the cell size checkpoint.     

Age-related changes in T cell function.

A comparison was made of the abilities of carrier (BGG)-primed T cell populations from young (4-month old), middle-aged (14- and 19-month old) and old (31- and 34-month old) mice to collaborate with hapten (DNP)-primed B cells from young mice in a cell-transfer system. The plaque-forming cell responses to 2,4-dinitrophenol (DNP) were measured by a modification of the Jerne plaque assay. The DNP-specific antibody-forming cell responses of old T cell/young B cell combinations were significantly lower than those of young T cell/young B cell combinations, both in the number of T cells needed for peak response and in the size of that response. These data indicate that the primed T cell populations of old mice are deficient by a factor of 6 in their ability to initiate B cell proliferation and differentiation into antibody-forming cells.     

Best practices in cell culture: an overview

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.     

3D culture model and computer-assisted videomicroscopy to analyze migratory behavior of noninvasive and invasive bronchial epithelial cells

To date, most of the studies in the field of cell migration have been applied to two-dimensional (2D) models. To mimic the three-dimensional (3D) conditions similar to those observed in vivo during tumor invasion, we developed a 3D model of cell migration in which cells were embedded in a collagen I matrix placed in a double-compartment chamber. Using time-lapse videomicroscopy and interactive cell tracking in a four-dimensional data set, we determined the cell trajectories and their migration kinetics. We compared the 2D and 3D migratory behavior of a noninvasive cell line (16HBE) with the migratory behavior of an invasive cell line (BZR). Our results show that the 3D migration kinetics of the noninvasive cell line were lower than the migration kinetics of the invasive cell line. In contrast, in 2D models, no significant difference was observed between the two cell lines. To validate our 3D model, we further investigated the effect of epidermal growth factor (EGF), a promoter of tumor cell motility and invasion on the noninvasive cell line (16HBE). EGF increased significantly the migration kinetics of the noninvasive cell line. Our results show that the 3D model of cell migration allowed us to differentiate the migratory behavior of invasive and noninvasive cells and that such a model can help in the development of molecular targeted therapy as it approaches the in vivo conditions. tumor invasion; metastasis; image analysis; kinetic migration; epidermal growth factor     

MicroRNA-210 regulates cancer cell proliferation through targeting fibroblast growth factor receptor-like 1 (FGFRL1)

The importance of microRNAs (miRNAs) in human malignancies has been well recognized. Here, we report that the expression of microRNA-210 (miR-210) is down-regulated in human esophageal squamous cell carcinoma and derived cell lines. Marked decreases in the level of miR-210 were observed especially in poorly differentiated carcinomas. We found that miR-210 inhibits cancer cell survival and proliferation by inducing cell death and cell cycle arrest in G(1)/G(0) and G(2)/M. Finally, we identified fibroblast growth factor receptor-like 1 (FGFRL1) as a target of miR-210 in esophageal squamous cell carcinoma and demonstrated that FGFRL1 accelerates cancer cell proliferation by preventing cell cycle arrest in G(1)/G(0). Taken together, our findings show an important role for miR-210 as a tumor-suppressive microRNA with effects on cancer cell proliferation.     

COUP-TFI controls Notch regulation of hair cell and support cell differentiation

The orphan nuclear receptor COUP-TFI (Nr2f1) regulates many aspects of mammalian development, but little is known about its role in cochlear hair cell and Deiter's support cell development. The COUP-TFI knockout (COUP-TFI(-/-)) has a significant increase in hair cell (HC) number in the mid-to-apical turns. The total number of hair cells is not increased over wild type, perhaps because of displaced hair cells and a shortened cochlear duct. This implicates a defect of convergent-extension in the COUP-TFI(-/-) duct. In addition, excess proliferation in the COUP-TFI(-/-) sensory epithelium indicates that the origin of the extra HCs in the apex is complex. Because loss-of-function studies of Notch signaling components have similar phenotypes, we investigated Notch regulation of hair cell differentiation in COUP-TFI(-/-) mice and confirmed misregulation of Notch signaling components, including Jag1, Hes5 and in a manner consistent with reduced Notch signaling, and correlated with increases in hair cell and support cell differentiation. The disruption of Notch signaling by a gamma-secretase inhibitor in an in vitro organ culture system of wild-type cochleae resulted in a reduction in expression of the Notch target gene Hes5 and an increase in hair cell differentiation. Importantly, inhibition of Notch activity resulted in a greater increase in hair cell differentiation in COUP-TFI(-/-) cochlear cultures than in wild-type cultures, suggesting a hypersensitivity to Notch inactivation in COUP-TFI(-/-) cochlea, particularly at the apical turn. Thus, we present evidence that reduced Notch signaling contributes to increases in hair cell and support cell differentiation in COUP-TFI(-/-) mice, and suggest that COUP-TFI is required for Notch regulation of hair cell and support cell differentiation.     

Cell poration and cell fusion using an oscillating electric field.

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.     

Ultrastructural and Cytochemical Study on Mature Pollen of Pinus tabulaeformis

The pollen of Pinus tabulaeformis Cart. comprised two prothallial cells, a generative cell and a tube cell which degenerated at pollen maturation. The generative cell had its own cell wall, seperating from the intine of pollen, but with its side wall attached to the infine. Cytoplasmic channels were present on the side of the generative cell wall, which faced to the tube cell cytoplasm. The generative cell differed conspicuously from the tube cell. The main differences include: ( 1 ) The chromatin in the generative cell nucleus was condensed, but was dispersed and had numerous nueleare pores in the tube cell nucleus; (2)There was no microbody in the generative cell but many microbodies were present in the tube cell cytoplasm; (3)More inclusions were present in the tube cell than in the generative cell. Both the generative cell and the tube cells contained lipid bodies and amyloplasts in the cytoplasm, but there were more amyloplasts in the former. The tube cell also contained a few proteins which was absent in the generative cell. In addition, there were numerous mitochondria, polyribosomes, and a few endoplasmic reticulums and dictyosomes in the generative and tube cells. DAPI staining demonstrated numerous cytoplasmic DNA in both generative cell and tube cell. The mode of cytoplasmic inheritance, and the composition, structure and the nature of the pollen wall of P. tabulaefonnis are also discussed in this paper.     

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.     

Antisera inhibiting mammalian cell spreading and possible cell surface antigens involved

Antiserum against a rat neuronal tumor cell line (B103) has been prepared in rabbit by intravenous injection of live cells. This immune serum (anti-B103) precipitates a few cell surface proteins recognizable by two-dimensional gel electrophoresis as common radioiodinatable spots in 15 different rat neural cell lines and in mouse and rat fibroblast cell lines. The apparent molecular weight of one major common protein (II4) is estimated by SDS gel electrophoresis to be somewhere between 80,000 and 90,000 and another protein (I3) to be 120,000. These two proteins are consistently recognized in various cell lines by this antiserum. Furthermore, at a 1:20 dilution, this serum causes monolayer cells to round up usually within 0.5 h and detach from the plate within 3 h. It also inhibits spreading of freshly plated cells. These effects of the antiserum are reversible. Upon absorption of the antiserum with cells (e.g., absorbed with a glial cell line, B27), the serum no longer causes the rounding up of the monolayer cells, it does not inhibit cell spreading, and it does not immune-precipitate the two common proteins from the cell surface of various cell lines. Antisera against several other rat cell lines also precipitate the same common proteins (II4 and I3) from the cell surface and prevent cell spreading. These data suggest that the antibody acts first at the cell surface and then inhibits cell spreading or rounding up of spread cells. The consistent pattern of the immunoprecipitated cell surface proteins on the two- dimensional gel electrophoresis makes these two common surface proteins (II4 or I3 or both) possible candidates for target proteins to which the antibody binds. Thus, they may play a critical role in cell spreading.     

Nitrogen deficiency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates

Nitrogen deficiency severely inhibits leaf growth. This response was analysed at the cellular level by growing Lolium perenne L. under 7.5 mM (high) or 1 mM (low) nitrate supply, and performing a kinematic analysis to assess the effect of nitrogen status on cell proliferation and cell growth in the leaf blade epidermis. Low nitrogen supply reduced leaf elongation rate (LER) by 43% through a similar decrease in the cell production rate and final cell length. The former was entirely because of a decreased average cell division rate (0.023 versus 0.032 h(-1)) and thus longer cell cycle duration (30 versus 22 h). Nitrogen status did not affect the number of division cycles of the initial cell's progeny (5.7), and accordingly the meristematic cell number (53). Meristematic cell length was unaffected by nitrogen deficiency, implying that the division and mitotic growth rates were equally impaired. The shorter mature cell length arose from a considerably reduced post-mitotic growth rate (0.033 versus 0.049 h(-1)). But, nitrogen stress did not affect the position where elongation stopped, and increased cell elongation duration. In conclusion, nitrogen deficiency limited leaf growth by increasing the cell cycle duration and decreasing mitotic and post-mitotic elongation rates, delaying cell maturation.     

Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage

In the Drosophila bristle lineage, five differentiated cells arise from a precursor cell after a rapid sequence of asymmetric cell divisions (one every 2 hours). We show that, in mitotic cells, this rapid cadence of cell divisions is associated with cell cycles essentially devoid of the G1-phase. This feature is due to the expression of Cyclin E that precedes each cell division, and the differential expression of the S-transition negative regulator, Dacapo. Thus, apart from endocycles (G/S), which occurred in two out of five terminal cells, two other cell cycles coexist in this lineage: (1) an atypical cell cycle (S/G2/M), in which the S-phase is initiated during the preceding telophase; and (2) a canonical cell cycle (G1/S/G2/M) with a brief G1 phase. These two types of cell cycle result from either the absence or very transient expression of Dap, respectively. Finally, we show that the fate determinant factor, Tramtrack, downregulates Cyclin E expression and is probably involved in the exit of the cells from the cell cycle.     

The 'Inka cell' and its associated cells: ultrastructure of the epitracheal glands in the gypsy moth,Lymantria dispar

The epitracheal glands in pharate and young pupae of Lymantria dispar are located at the base of ventrolateral tracheal trunks in the prothoracic and first through eighth abdominal segments. Each gland is composed of four cells the ultrastructure of which is described in this paper. One large cell and one smaller cell have an endocrine function, while a third cell is exocrine. A fourth cell forms a canal running from the exocrine cell into the trachea. The large endocrine cell, but not the smaller endocrine cell has released its secretions in freshly moulted pupae. The exocrine cell is assumed to be involved in the pupal moult events as well. The physiological role of the different cell types is discussed: The large endocrine cell (type I endocrine cell) is supposedly homologous with the 'Inka cell', which produces ecdysis triggering hormone (ETH) and was previously described in Manduca sexta; the functions of the smaller endocrine cell (type II endocrine cell) and the exocrine cell remain unknown.     

Phenotypic heterogeneity in cultured skin fibroblasts from patients with disorders of peroxisome biogenesis belonging to the same complementation group

We have studied fibroblast cell lines derived from a control subject (cell line 85AD5035F) and three patients clinically described as having the Zellweger syndrome (cell line W78/515), the infantile form of Refsum disease (cell line BOV84AD) and hyperpipecolic acidaemia (cell line GM3605), respectively. The mutant cell lines belonged to the same complementation group. The fibroblasts were cultured under identical conditions and were harvested at different time intervals after reaching confluece. Several peroxisomal parameters were determined. In agreement with previous reports, a lowered enzymic activity of acyl-CoA:dihydroxyacetonephosphate acyltransferase and a decrease in latent catalase clearly distinguished the patient cell limes from the control cell line. However, the cell lines exhibited a phenotypic heterogeneity. This was most strikingly encountered when cells were processed for indirect immunofluorescence microscopy and stained with anti-(catalase). The control cells exhibited a punctate fluorescence, which is indicative of the presence of catalase in peroxisomes. In the mutant cell line W78.515 a diffuse fluorescence was observed, indicative of the presence of catalase in the cytosol. In the other two mutant cell lines a puncate fluorescence was observed in some of the cells. Moreover, clear differences in the extent of proteolytic processing of acyl-CoA oxidase were detected. The mutant cell line BOV84AD displayed a control-like pattern with all molecular forms of acyl-CoA oxidase (72, 52 and 20 kDa) present, whereas in the W78/515 cell line only the 72 kDa component could be visualised. The GM3605 cell line was intermediate in this respect.