An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo

1994 ◽  
Vol 107 (4) ◽  
pp. 813-825 ◽  
Author(s):  
M.R. Shanks ◽  
D. Cassio ◽  
O. Lecoq ◽  
A.L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Membrane Proteins ◽  
Cell Line ◽  
Hybrid Cell ◽  
The Other ◽  
Hepatocyte Polarity ◽  
Rat Hepatoma

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

scholarly journals Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

scholarly journals Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt

Hepatology ◽  
1998 ◽  
Vol 27 (1) ◽  
pp. 191-199 ◽  
Author(s):  
Elisa M. Konieczko ◽  
Amy K. Ralston ◽  
Aleta R. Crawford ◽  
Saul J. Karpen ◽  
James M. Crawford
Keyword(s):  
B Cells ◽  
Bile Salt ◽  
Cell Line ◽  
Hybrid Cell ◽  
Hybrid Cell Line ◽  
Salt Uptake ◽  
Line Following ◽  
Rat Hepatoma

scholarly journals Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

2021 ◽  
Vol 5 (1) ◽  
pp. e202101162
Author(s):  
Yuta Endo ◽  
Yuko Shimizu ◽  
Hanako Nishikawa ◽  
Katsuhiro Sawasato ◽  
Ken-ichi Nishiyama
Keyword(s):  
Membrane Proteins ◽  
Molecular Basis ◽  
Terminal Region ◽  
Integral Membrane Proteins ◽  
The Other ◽  
Other Hand ◽  
Model Protein

Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo. We analyzed the mechanisms that determine the insertion factor dependency in vitro. Glycolipid MPIase was required for insertion of both proteins because MPIase depletion caused a significant defect in insertion. On the other hand, YidC depletion and PMF dissipation had no effects on Pf3-Lep insertion, whereas V15D insertion was reduced. We reconstituted (proteo)liposomes containing MPIase, YidC, and/or F0F1-ATPase. MPIase was essential for insertion of both proteins. YidC and PMF stimulated Pf3-Lep insertion as the synthesis level increased. V15D insertion was stimulated by both YidC and PMF irrespective of the synthesis level. These results indicate that charges in the N-terminal region and the synthesis level are the determinants of YidC and PMF dependencies with the interplay between MPIase, YidC, and PMF.

scholarly journals Screening for 15 pathogenic viruses in human cell lines registered at the JCRB Cell Bank: characterization of in vitro human cells by viral infection

2018 ◽  
Vol 5 (5) ◽  
pp. 172472 ◽  
Author(s):  
Setsuko Shioda ◽  
Fumio Kasai ◽  
Ken Watanabe ◽  
Kohei Kawakami ◽  
Azusa Ohtani ◽  
...  
Keyword(s):  
Viral Infection ◽  
Cell Line ◽  
Cell Lines ◽  
Human Cell ◽  
The Other ◽  
Pcr Analysis ◽  
Human Cell Lines ◽  
Cell Bank

Human cell lines have been used in a variety of research fields as an in vitro model. These cells are all derived from human tissue samples, thus there is a possibility of virus infection. Virus tests are routinely performed in clinical practice, but are limited in cell lines. In this study, we investigated 15 kinds of viruses in 844 human cell lines registered at the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Our real-time PCR analysis revealed that six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in 43 cell lines. Of them, 20 cell lines were transformed by intentional infection in vitro with EBV or HTLV-1. Viruses in the other 23 cell lines and one EBV transformed cell line are derived from an in vivo infection, including five de novo identifications of EBV, B19V or HHV-7 carriers. Among them, 17 cell lines were established from patients diagnosed with virus-associated diseases. However, the other seven cell lines originated from in vivo cells unrelated to disease or cellular tropism. Our approach to screen for a set of 15 viruses in each cell line has worked efficiently to identify these rare cases. Virus tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral infection which can be a characteristic feature of cell lines.

Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line

Hepatology ◽  
2000 ◽  
Vol 31 (1) ◽  
pp. 124-130 ◽  
Author(s):  
Ekkehard Sturm ◽  
Tracy L. Zimmerman ◽  
Aleta R. Crawford ◽  
Stanislav I. Svetlov ◽  
Pazhani Sundaram ◽  
...  
Keyword(s):  
Bile Acid ◽  
B Cells ◽  
Cell Line ◽  
Hybrid Cell ◽  
Hybrid Cell Line ◽  
Acid Uptake ◽  
Bile Acid Uptake ◽  
Rat Hepatoma

scholarly journals Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line.

1992 ◽  
Vol 12 (4) ◽  
pp. 1864-1871 ◽  
Author(s):  
G Q Daley ◽  
R A Van Etten ◽  
P K Jackson ◽  
A Bernards ◽  
D Baltimore
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
3T3 Fibroblasts ◽  
Nih 3T3

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.

scholarly journals Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in HumansIn Vivo

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Franz Tatzber ◽  
Edith Pursch ◽  
Ulrike Resch ◽  
Roswitha Pfragner ◽  
Sandra Holasek ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Line ◽  
Oxidized Ldl ◽  
Malonic Dialdehyde ◽  
Density Lipoprotein ◽  
Ldl Oxidation ◽  
Human Donor ◽  
Human B Cells

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu++-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodiesin vivo. Furthermore, using these antibodies forin vitroexperiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

scholarly journals Evaluation of the effects of acepromazine and xylazineon viability in an equine dermal cell line

2020 ◽  
Vol 14 (4) ◽  
pp. 259-264
Author(s):  
Jéssica Grace da Silveira ◽  
Bruno Egídio Cappelari ◽  
Ana Paula Muterle Varela ◽  
Thais Fumaco Teixeira ◽  
Giovana Dantas de Araujo
Keyword(s):  
Veterinary Medicine ◽  
Cell Viability ◽  
Cell Line ◽  
Mtt Assay ◽  
Tissue Damage ◽  
The Other ◽  
Drug Concentrations ◽  
Dermal Cell

Xylazine and acepromazine are drugs used exclusively in veterinary medicine. Xylazineis used as a sedative, analgesic, and tranquilizer while acepromazine is used as a sedative, pre-anesthetic, and anesthetic adjuvant. In vitrodrug toxicity experimentation is essential to predict possible damage associated with treatment. This study was carried out to evaluate and compare the in vitroeffects of acepromazine and xylazine on cell viability. Equine Dermis cells lines were used to examine different drug concentrations (0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL). An MTT assay was carried out to reveal cell viability. Both tested drugs reduced the viability of ED cells at 0.02 and 0.01 mg/mL. At 0.005 mg/mL, only acepromazine presented an effect. These results corroborate previous studies with xylazine. On the other hand, this is thefirst report about acepromazine and cell viability. Previous studies suggest that the mechanisms involved in reducing cell viability are apoptosis for xylazine and the activation of the autophagic pathway for acepromazine. Both mechanisms have been seen in other drugs of the same classes. These findings reveal that both acepromazine and xylazine cause concentration-dependent cytotoxicity in vitro. Future experiments could further elucidate the mechanisms by which this effect happens and thus circumvent therisk of potential tissue damage in vivo.

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