scholarly journals CLONAL GROWTH OF MAMMALIAN CELLS IN VITRO

1956 ◽  
Vol 103 (2) ◽  
pp. 273-284 ◽  
Author(s):  
Theodore T. Puck ◽  
Philip I. Marcus ◽  
Steven J. Cieciura
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Growth Curves ◽  
High Energy ◽  
Mechanical Trauma ◽  
Animal Cells ◽  
Isolated Cells ◽  
Cross Sectional ◽  
Growth Physiology

Two methods for simple and rapid plating of single HeLa cells, human, carcinomatous cells, are described. These result in growth and formation of colonies from each single cell. One of these procedures uses irradiated, non-multiplying "feeder" cells to condition the medium. The second requires more gentle handling of the cells, but otherwise is virtually the same as that used in plating bacteria on semisolid, nutrient media. By extension of these methods, it is possible to isolate single mutant colonies and grow pure clonal stocks of animal cells. These genetically uniform strains are much more homogeneous in their behavior than the parental HeLa cell population. Growth curves obtained from developing colonies are highly reproducible. The most active mutant stocks so far isolated display a generation time of 18 to 20 hours. In pooled human serum HeLa cells assume a highly stretched, ameboid form, with marked motility; whereas growth of the same cells in a variety of non-human sera results in tightly packed, columnar, epithelial-like morphology. The two cell types possess volumes, nuclear cross-sections, plating efficiencies, and generation times which are identical within experimental error, but display widely different cross-sectional areas, suggesting that the basic change occurs in the cell surface. It is conceivable that this change may be related to that which enables the cells of a compact tumor to become invasive. Animal cells subjected to the standard trypsinization procedures which involve mechanical trauma and repeated washings in incomplete media leak large amounts of P and suffer impaired ability to reproduce as isolated cells. Application of the methods described in this paper as a tool for quantitative study of normal mammalian cell growth, physiology, genetics, and biochemistry, and the response of cells to drugs, viruses, high energy radiation, and other agents have been indicated.

scholarly journals p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

2007 ◽  
Vol 18 (10) ◽  
pp. 3741-3751 ◽  
Author(s):  
Kiyoko Ogawa-Goto ◽  
Keiko Tanaka ◽  
Tomonori Ueno ◽  
Keisuke Tanaka ◽  
Takeshi Kurata ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Specific Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

Effects of Asbestos Fibers on Mammalian Cells in Culture

1975 ◽  
Vol 33 ◽  
pp. 420-421
Author(s):  
Brenda E. Barry ◽  
Harold W. Fisher
Keyword(s):  
Mammalian Cells ◽  
Growth Curves ◽  
Ultrastructural Changes ◽  
Asbestos Fibers ◽  
Cells In Culture ◽  
Cell Counts ◽  
Direct Cell ◽  
Amphibole Asbestos ◽  
Organic Pesticides

In previous reports from this laboratory, we examined the correlation of the toxic effects of several organic pesticides, herbicides and trace metals on the plating efficiency to the ultrastructural changes found in the treated cells in culture. Since there has been a long-term interest in the effects of exposure of humans and experimental animals to asbestos, the current study was undertaken to determine if ultrastructural changes would also be found in mammalian cells grown in vitro after exposure to asbestos. In a series of preliminary experiments, suitable fractionation by low-speed differential centrifugation and concentrations of amphibole asbestos were found in which there was at least 70% to 90% viability depending on the cell type. The viability of the cells incubated in the presence of the asbestos was determined both by growth curves established from direct cell counts and from staining with trypan blue which does not penetrate viable cells.

Effects of Lithotripter-Generated High Energy Shock Waves on Mammalian Cells in Vitro

1992 ◽  
Vol 147 (1) ◽  
pp. 215-219 ◽  
Author(s):  
Issac Kaver ◽  
Warren W. Koontz ◽  
John D. Wilson ◽  
John M. Guice ◽  
M.J.V. Smith
Keyword(s):  
Shock Waves ◽  
Mammalian Cells ◽  
High Energy

scholarly journals Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1291 ◽  
Author(s):  
Pradeep Kumar Singh ◽  
Søren S.-R. Bohr ◽  
Nikos S. Hatzakis
Keyword(s):  
Cancer Cells ◽  
Hela Cells ◽  
Single Particle ◽  
Mammalian Cells ◽  
Model Membranes ◽  
Live Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Sophorolipids (SLs) are naturally produced glycolipids that acts as drug delivery for a spectrum of biomedical applications, including as an antibacterial antifungal and anticancer agent, where they induce apoptosis selectively in cancerous cells. Despite their utility, the mechanisms underlying their membrane interactions, and consequently cell entry, remains unknown. Here, we combined a single liposome assay to observe directly and quantify the kinetics of interaction of SL micelles with model membrane systems, and single particle studies on live cells to record their interaction with cell membranes and their cytotoxicity. Our single particle readouts revealed several repetitive docking events on individual liposomes and quantified how pH and membrane charges, which are known to vary in cancer cells, affect the docking of SL micelles on model membranes. Docking of sophorolipids micelles was found to be optimal at pH 6.5 and for membranes with −5% negatively charge lipids. Single particle studies on mammalian cells reveled a two-fold increased interaction on Hela cells as compared to HEK-293 cells. This is in line with our cell viability readouts recording an approximate two-fold increased cytotoxicity by SLs interactions for Hela cells as compared to HEK-293 cells. The combined in vitro and cell assays thus support the increased cytotoxicity of SLs on cancer cells to originate from optimal charge and pH interactions between membranes and SL assemblies. We anticipate studies combining quantitative single particle studies on model membranes and live cell may reveal hitherto unknown molecular insights on the interactions of sophorolipid and additional nanocarriers mechanism.

scholarly journals ACTION OF X-RAYS ON MAMMALIAN CELLS

1957 ◽  
Vol 106 (4) ◽  
pp. 485-500 ◽  
Author(s):  
Theodore T. Puck ◽  
Dimitry Morkovin ◽  
Philip I. Marcus ◽  
Steven J. Cieciura
Keyword(s):  
Survival Data ◽  
Mammalian Cells ◽  
Radiation Effects ◽  
Lethal Dose ◽  
Human Cells ◽  
X Rays ◽  
Survival Curves ◽  
Isolated Cells ◽  
X Irradiation

Survival curves of normal human cells from a variety of tissues exposed to varying doses of x-irradiation have been constructed, which permit definition of the intrinsic radiation sensitivity of the reproductive power of each cell type. The mean lethal dose of x-irradiation for all the cells employed, including those from normal and cancerous organs, those exhibiting diploid and polyploid chromosome number; those from embryonic and adult tissues, including recently isolated cells and cultures which had been maintained in vitro for many years, and cells exhibiting either epithelioid or fibroblastic morphology, was found to be contained between the limits of 50 to 150 r. Other similarities in the pattern of radiation effects, such as giant formation and abortive colonial growth, in these cells and that of the HeLa S3, previously studied, confirm the hypothesis that the pattern of reaction to x-irradiation previously elucidated, is representatative, at least in over-all outline, for a large variety of human cells. While the radiation survival curves of various human cells are similar in the gross, small but important characterizing differences have been found. All epithelioid cells so far studied are approximately 2-hit, and more radioresistant than the fibroblast-like cells whose survival data correspond to a mean lethal dose of around 60 r, and which so far can be fitted by either 1-hit or 2-hit curves. The earlier prediction that the major radiobiologic damage to mammalian cells is lodged in the genetic apparatus was confirmed by the demonstration of high frequency of mutants among the survivors of doses of 500 to 900 r. All the data on the x-radiosensitivity of these cells can be explained on the basis of a defect resulting from primary damage localized in one or more chromosomes. These considerations afford a convincing explanation of several aspects of the mammalian radiation syndrome.

scholarly journals Integrin and ligand-independent PDGFr signaling synergistically contribute to directional migration of Xenopus mesendoderm

2018 ◽  
Author(s):  
Crystal M. Richardson ◽  
Bette J. Dzamba ◽  
Pooja R. Sonavane ◽  
Douglas W. DeSimone
Keyword(s):  
Mammalian Cells ◽  
Isolated Cells ◽  
Integrin Α5β1 ◽  
Type Iii ◽  
Normal Expression ◽  
Pdgf Signaling ◽  
Tissue Migration ◽  
Cytokeratin Filaments

AbstractBoth PDGF signaling and adhesion to fibronectin (FN) matrix have been implicated in the directional collective migration of Xenopus mesendoderm cells at gastrulation. However, mesendoderm explants cultured on FN-coated substrates migrate directionally even in the absence of a source of PDGF. Integrin adhesion has been reported to up-regulate PDGF ligand-independent signaling through the PDGF receptor (PDGFr) in cultured mammalian cells. In order to address whether a similar mechanism stimulates PDGFr signaling in the absence of PDGF-A ligand in amphibian mesendoderm, isolated cells were cultured on bacterial fusion proteins containing the Type-III repeats 9-11 of FN (GST-9.11). Type III9-11 contains the RGD and “synergy” (PPSRN) sites required for integrin α5β1 adhesion and activation but lacks the PDGF-A ligand-binding site present in the full-length FN protein. In order to ensure mesendoderm was not exposed to PDGF in vivo prior to removal and culture in vitro, antisense morpholinos were used to inhibit normal expression of PDGF-A ligand in embryos. P-Akt levels were reduced two-fold when either the PDGFr-α was knocked down or when cells were plated on GST-9.11a, which contains a point mutation (PPSRN>PPSAN) that prevents both full activation of integrin α5β1 and cell spreading. Reduced expression of PDGFr-α was accompanied by perturbations in tissue migration, cytoskeletal organization, polarity of cell protrusions, and focal adhesion area. Mesendoderm cells became rounded, and the actin and cytokeratin filaments appeared collapsed and often colocalized near the cell center. Taken together, these findings suggest that integrin adhesion to FN, acting in synergy with PDGFr-α, is sufficient to elevate PI3K-Akt signaling in the mesendoderm even in the absence of the PDGF-A ligand, and to promote forward-directed protrusions and directional tissue migration.

scholarly journals Essential Role of Phosphorylation of MCM2 by Cdc7/Dbf4 in the Initiation of DNA Replication in Mammalian Cells

2006 ◽  
Vol 17 (10) ◽  
pp. 4459-4472 ◽  
Author(s):  
Toshiya Tsuji ◽  
Scott B. Ficarro ◽  
Wei Jiang
Keyword(s):  
Dna Replication ◽  
Hela Cells ◽  
Mammalian Cells ◽  
High Speed ◽  
Wild Type ◽  
Initiation Of Dna Replication ◽  
Complex Display

We report the identification of Cdc7/Dbf4 phosphorylation sites in human MCM2 and the determination of the role of Cdc7/Dbf4 phosphorylation of MCM2 in the initiation of DNA replication. Using immunoblotting, immunofluorescence, and high-speed automated cell-imaging analyses with antibodies specific against MCM2 and Cdc7/Dbf4 phosphorylated MCM2, we show that the chromatin recruitment and phosphorylation of MCM2 are regulated during the cell cycle in HeLa cells. Chromatin-bound MCM2 is phosphorylated by Cdc7/Dbf4 during G1/S, which coincides with the initiation of DNA replication. Moreover, we show that baculovirus-expressed purified MCM2-7 complex and its phosphomimetic MCM2E-7 complex display higher ATPase activity when compared with the nonphosphorylatable MCM2A-7 complex in vitro. Furthermore, suppression of MCM2 expression in HeLa cells by siRNA results in the inhibition of DNA replication. The inhibition can be rescued by the coexpression of wild type MCM2 or MCM2E but not MCM2A. Taken together, these results indicate that Cdc7/Dbf4 phosphorylation of MCM2 is essential for the initiation of DNA replication in mammalian cells.

scholarly journals GENETICS OF SOMATIC MAMMALIAN CELLS

1958 ◽  
Vol 108 (2) ◽  
pp. 259-268 ◽  
Author(s):  
J. H. Tjio ◽  
Theodore T. Puck
Keyword(s):  
Chromosome Number ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Human Subjects ◽  
Chinese Hamster ◽  
Animal Cells ◽  
Active Growth ◽  
Metabolic Characteristics ◽  
American Opossum

A convenient, reliable method for chromosome delineation of animal cells grown as monolayers on glass has been applied to human, opossum, and Chinese hamster cells. Tissue cultured cells from 5 different, normal organs of 7 different human subjects uniformly displayed the expected chromosome number of 46 and showed no variations in morphology or number other than the expected sex differences and a small incidence of polyploidy. The chromosomes of normal cells from the American opossum were as uniform as those of human cells. Cells of the inbred Chinese hamster demonstrated appreciable karyotype variability, the cause of which is under investigation. The chromosome number and morphology of cells from normal human tissues have remained constant after more than 5 months of continuous, rapid growth in tissue culture involving scores of vessel transfers and a number of generations equivalent to many billions of progeny. By the use of routine recloning, even cells of malignant, aneuploid constitution have been maintained in active growth for 3 years and hundreds of generations, with stable chromosomal and metabolic characteristics. The cells of the American opossum and Chinese hamster which possess only 22 chromosomes have been established in vitro and are especially suitable for genetic studies. The readily recognizeable Y and X chromosomes of the male opossum are particularly favorable as cytological markers. Photomicrographs of the chromosomes of the various cells employed are presented.

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