Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

AbstractGenetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ~2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci and 3 loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ~30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

Download Full-text

Antifungal Activities of Antineoplastic Agents:Saccharomyces cerevisiae as a Model System To Study Drug Action

Clinical Microbiology Reviews ◽

10.1128/cmr.12.4.583 ◽

1999 ◽

Vol 12 (4) ◽

pp. 583-611 ◽

Cited By ~ 75

Author(s):

Maria E. Cardenas ◽

M. Cristina Cruz ◽

Maurizio Del Poeta ◽

Namjin Chung ◽

John R. Perfect ◽

...

Keyword(s):

Mammalian Cells ◽

Kinase Inhibitor ◽

Genetic Model ◽

Fungal Infections ◽

Study Drug ◽

Drug Action ◽

Mechanisms Of Action ◽

Model Systems ◽

Screening Tools ◽

Sphingolipid Metabolism

SUMMARY Recent evolutionary studies reveal that microorganisms including yeasts and fungi are more closely related to mammals than was previously appreciated. Possibly as a consequence, many natural-product toxins that have antimicrobial activity are also toxic to mammalian cells. While this makes it difficult to discover antifungal agents without toxic side effects, it also has enabled detailed studies of drug action in simple genetic model systems. We review here studies on the antifungal actions of antineoplasmic agents. Topics covered include the mechanisms of action of inhibitors of topoisomerases I and II; the immunosuppressants rapamycin, cyclosporin A, and FK506; the phosphatidylinositol 3-kinase inhibitor wortmannin; the angiogenesis inhibitors fumagillin and ovalicin; the HSP90 inhibitor geldanamycin; and agents that inhibit sphingolipid metabolism. In general, these natural products inhibit target proteins conserved from microorganisms to humans. These studies highlight the potential of microorganisms as screening tools to elucidate the mechanisms of action of novel pharmacological agents with unique effects against specific mammalian cell types, including neoplastic cells. In addition, this analysis suggests that antineoplastic agents and derivatives might find novel indications in the treatment of fungal infections, for which few agents are presently available, toxicity remains a serious concern, and drug resistance is emerging.

Download Full-text

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060118 ◽

1967 ◽

Vol 25 ◽

pp. 20-21

Author(s):

Dale E. McClendon ◽

Paul N. Morgan ◽

Bernard L. Soloff

Keyword(s):

Growth Medium ◽

Mammalian Cells ◽

Carcinoma Cells ◽

Venom Protein ◽

Sterile Saline ◽

Carbon Coated ◽

Available Information ◽

Loxosceles Reclusa ◽

Essential Growth ◽

Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

Download Full-text

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Download Full-text

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Download Full-text

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

Download Full-text

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158388 ◽

1990 ◽

Vol 48 (3) ◽

pp. 168-169

Author(s):

M. H. Chestnut ◽

C. E. Catrenich

Keyword(s):

Acridine Orange ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Download Full-text

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015900x ◽

1990 ◽

Vol 48 (3) ◽

pp. 290-291

Author(s):

Gustav Ofosu

Keyword(s):

Mammalian Cells ◽

Antitumor Agent ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Limiting Factor ◽

Sarcoma 180 ◽

Electron Microscopic ◽

Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Download Full-text

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bcj20190359 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3241-3260

Author(s):

Sindhu Wisesa ◽

Yasunori Yamamoto ◽

Toshiaki Sakisaka

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Mammalian Cells ◽

Coiled Coil ◽

Transmembrane Domains ◽

Domain Family ◽

Coiled Coil Domain ◽

U2os Cells ◽

Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

Download Full-text

Early signalling events of autophagy

Essays in Biochemistry ◽

10.1042/bse0550001 ◽

2013 ◽

Vol 55 ◽

pp. 1-15 ◽

Cited By ~ 16

Author(s):

Laura E. Gallagher ◽

Edmond Y.W. Chan

Keyword(s):

Protein Kinase ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Mammalian Cells ◽

Mammalian Target Of Rapamycin ◽

Interacting Protein ◽

The Core ◽

Autophagy Gene ◽

Autophagy Induction ◽

Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

Download Full-text