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1.
Biochem Cell Biol ; 76(1): 73-82, 1998.
Article in English | MEDLINE | ID: mdl-9666308

ABSTRACT

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -alpha, -beta, -delta, -epsilon, and -zeta are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-beta I has a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-beta I. PKC-beta I is detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-alpha, -delta, and -epsilon were down-regulated by pretreatment of cells with TPA, while PKC-zeta was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-beta I. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-beta I that is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-zeta is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.


Subject(s)
Isoenzymes/analysis , Liver/enzymology , Protein Kinase C/analysis , Animals , Cell Division/drug effects , Cell Line , Epithelial Cells/enzymology , Fluorescent Antibody Technique , Liver/cytology , Rats , Tetradecanoylphorbol Acetate/pharmacology
2.
Cell Signal ; 7(2): 139-50, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7794686

ABSTRACT

We have recently described an in vitro model for liver homeostasis which involves growing T51B rat liver epithelial cells with 1 nM epidermal growth factor (EGF). There is an initial period of hyperplasia, lasting about 3 days, which is followed by an increase in apoptosis. The cell density returns to around the confluent control level 5 days after EGF addition. The dose response of T51B cells to EGF shows three distinct growth patterns. We have carried out EGF binding studies that suggest that the occupancy of the low affinity binding site of the EGF receptor, is responsible for the hyperproliferation seen when the cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems.


Subject(s)
Epidermal Growth Factor/pharmacology , ErbB Receptors/drug effects , ErbB Receptors/metabolism , Liver/cytology , Liver/drug effects , Animals , Apoptosis/drug effects , Binding Sites , Cell Division/drug effects , Cell Line , Dose-Response Relationship, Drug , Epidermal Growth Factor/administration & dosage , Homeostasis , In Vitro Techniques , Kinetics , Liver/metabolism , Models, Biological , Rats
3.
Biochem Cell Biol ; 72(9-10): 429-38, 1994.
Article in English | MEDLINE | ID: mdl-7605615

ABSTRACT

The major objective of this research was the development of an in vitro model for liver homeostasis that would allow the future study of early events in cell proliferation and cell death. The model that was set up involves growing T51B rat liver epithelial cells with a single dose of 1 nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers 2 days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level 5 days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers 3-5 days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, electrophoresis of T51B cell DNA, and quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. This work describes the development and characterization an in vitro model of liver homeostasis that closely parallels in vivo systems where animals are given mitogenic stimuli.


Subject(s)
Apoptosis , Epidermal Growth Factor/pharmacology , Animals , Cell Count , Cell Division , Cell Line , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cell Size , DNA/biosynthesis , Epithelium , Homeostasis , Liver/cytology , Microscopy, Electron , Microscopy, Fluorescence , Rats , Time Factors
4.
Cell Signal ; 6(6): 631-43, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7857767

ABSTRACT

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12-O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.


Subject(s)
Epidermal Growth Factor/pharmacology , Liver/physiology , Protein Kinase C/metabolism , Signal Transduction , Animals , Cell Cycle , Cell Division , Cell Line , Cells, Cultured , DNA/biosynthesis , DNA Replication/drug effects , Down-Regulation , Liver/cytology , Liver/enzymology , Rats , Rats, Inbred F344 , Tetradecanoylphorbol Acetate/pharmacology
5.
Cell Signal ; 1(6): 617-25, 1989.
Article in English | MEDLINE | ID: mdl-2561951

ABSTRACT

Activation of protein kinase C (PKC) by phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat liver cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by beta-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was longer than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.


Subject(s)
Cyclic AMP/metabolism , Liver/metabolism , Protein Kinase C/physiology , Animals , Cell Line , Cholera Toxin/pharmacology , Colforsin/pharmacology , Enzyme Activation/physiology , Liver/drug effects , Rats , Signal Transduction/physiology , Subcellular Fractions/metabolism , Tetradecanoylphorbol Acetate/metabolism , Tetradecanoylphorbol Acetate/pharmacology
6.
J Cell Physiol ; 132(2): 354-8, 1987 Aug.
Article in English | MEDLINE | ID: mdl-3040781

ABSTRACT

Confluent T51B rat liver epithelial cells promptly began accumulating cyclic AMP-binding sites on their surfaces when they were stimulated from quiescence by serum growth factors in medium containing 1.8 mM Ca2+, but they began losing the accumulated binding sites shortly before initiating DNA replication. When the medium contained only 0.02 mM Ca2+, the cells still accumulated surface cyclic AMP-binding sites, but they did not initiate DNA replication and tended to continue accumulating the binding sites. The cyclic AMP-binding sites were eliminated completely by treating intact cells for 5 minutes with 0.005% trypsin (which did not damage the cells), and cyclic AMP caused them to be released from intact, undamaged cells into the medium. The binding sites also comigrated electrophoretically with purified regulatory subunits of type I cyclic AMP-dependent protein kinase, and to a lesser extent the regulatory subunit of type II cyclic AMP-dependent protein kinase. Therefore, it is likely that a transient accumulation of cyclic AMP-dependent protein kinases on the outer surface of the plasma membrane is part of the T51B rat liver cell's prereplicate program.


Subject(s)
Blood , Cyclic AMP/pharmacology , Interphase , Liver/enzymology , Protein Kinases/metabolism , Animals , Azides/metabolism , Calcium/physiology , Cell Line , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , DNA Replication , Electrophoresis, Polyacrylamide Gel , Epithelium/enzymology , Rats , Receptors, Cyclic AMP/metabolism
7.
Cancer Metastasis Rev ; 5(3): 205-50, 1987.
Article in English | MEDLINE | ID: mdl-3030578

ABSTRACT

Evidence is steadily mounting that the proto-oncogenes, whose products organize and start the programs that drive normal eukaryotic cells through their chromosome replication/mitosis cycles, are transiently stimulated by sequential signals from a multi-purpose, receptor-operated mechanism (consisting of internal surges of Ca2+ and bursts of protein kinase C activity resulting from phosphatidylinositol 4,5-bisphosphate breakdown and the opening of membrane Ca2+ channels induced by receptor-associated tyrosine-protein kinase activity) and bursts of cyclic AMP-dependent kinase activity. The bypassing or subversion of the receptor-operated Ca2+/phospholipid breakdown/protein kinase C signalling mechanism is probably the basis of the freeing of cell proliferation from external controls that characterizes all neoplastic transformations.


Subject(s)
Calcium/physiology , Cyclic AMP/physiology , Protein Kinase C/physiology , Proto-Oncogenes , Animals , Cell Cycle , DNA Replication , Humans , Mitosis
8.
J Cell Physiol ; 129(3): 303-9, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3782309

ABSTRACT

Incubating T51B rat liver cells in Ca2+-deficient, serum-rich medium containing only 0.02 mM Ca2+ strikingly decreased the phosphorylation of several trypsin-removable cell surface proteins and arrested the cells in late G1 phase. Raising the Ca2+ concentration in the Ca2+-deficient medium from 0.02 mM to 0.5 mM or adding 80 nM TPA (12-O-tetradecanoyl-phorbol-13-acetate), a protein kinase C activator, stimulated the phosphorylation of a certain set of surface proteins within 5 min and the initiation of DNA replication within the next 2 hr. By contrast, incubation in the same Ca2+-deficient medium, which does not affect the proliferation of neoplastic T51B-261B cells, did not reduce the phosphorylation of cell surface proteins. These observations suggest that the stimulation of a Ca2+-dependent protein kinase (possibly protein kinase C) directly or indirectly phosphorylates certain cell surface proteins that might be part of the mechanism that triggers the Ca2+-dependent G1----S transition of normal cells. They also suggest that an alteration of this Ca2+-dependent protein kinase might be the reason for neoplastic cells being able to proliferate in the face of an external Ca2+ shortage that would stop the proliferation of normal cells.


Subject(s)
Calcium/pharmacology , DNA/biosynthesis , Liver Neoplasms, Experimental/enzymology , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Division , Cell Line , Liver/enzymology , Phosphorylation , Rats , Tetradecanoylphorbol Acetate/pharmacology , Trypsin/metabolism
9.
Biochem Biophys Res Commun ; 135(1): 33-40, 1986 Feb 26.
Article in English | MEDLINE | ID: mdl-3954777

ABSTRACT

Diacylglycerols, such as 1,2-diolein, and tumor-promoting phorbol compounds, such as TPA (12-0-tetradecanoyl phorbol-13-acetate), stimulate the Ca2+/phospholipid-dependent protein kinase C from T51B rat liver cells, probably by sensitizing it to activation by Ca2+, and they reduce the liver cells' content of EDTA-extractable (i.e., soluble) protein kinase C activity. Evidence is presented that indicates that the glucocorticoid, dexamethasone, and the tumor-promoting artificial sweetener, saccharin, also trigger a Ca2+-dependent increase in the activity of the protein kinase C from T51B liver cells and reduce the cells' content of EDTA-extractable protein kinase C activity. However, these novel stimulators do not activate the enzyme by binding to the same site as diacylglycerols and TPA, although they do alter this site as indicated by an increase in the binding of the TPA analogue PDBu (phorbol 12,13-dibutyrate).


Subject(s)
Dexamethasone/pharmacology , Liver/enzymology , Protein Kinase C/metabolism , Saccharin/pharmacology , Animals , Cell Compartmentation/drug effects , Cells, Cultured , Diglycerides/pharmacology , Enzyme Activation/drug effects , Epithelium/enzymology , Rats , Tetradecanoylphorbol Acetate/pharmacology
10.
Exp Cell Res ; 160(1): 197-205, 1985 Sep.
Article in English | MEDLINE | ID: mdl-4043243

ABSTRACT

The G0----G1 and G1----S transitions (but not the intervening events) in the G1 phase of T51B rat liver epithelial cells in serum-stimulated confluent cultures required a high concentration of extracellular Ca2+ and were accompanied or immediately preceded by increases in the amount of EDTA-extractable protein kinase C, a Ca2+/phospholipid-dependent enzyme. Involvement of this Ca2+-dependent enzyme in the two Ca2+-dependent transitions was further indicated by the facts that 12-O-tetradecanoyl phorbol-13-acetate (TPA), a compound that stimulated protein kinase C from T51B cells even in the absence of Ca2+, enabled these cells to transit G1 in Ca2+-deficient medium, while a TPA analogue (4 alpha-phorbol-12, 13-didecanoate (4 alpha-PDD) that did not stimulate the enzyme in cell-free preparations did not promote G0----G1 or G1----S transit in Ca2+-deficient medium.


Subject(s)
Interphase , Liver/enzymology , Protein Kinase C/metabolism , Animals , Calcium/pharmacology , Cell Line , Culture Media , DNA Replication , Epithelial Cells , Epithelium/enzymology , Interphase/drug effects , Liver/cytology , Liver/metabolism , Phospholipids/pharmacology , Rats , Tetradecanoylphorbol Acetate/pharmacology
11.
Cancer Lett ; 21(3): 293-302, 1984 Jan.
Article in English | MEDLINE | ID: mdl-6692347

ABSTRACT

T51B rat liver cells and several carcinogen-treated, carcinogen + saccharin-treated, or spontaneously altered clones of T51B cells were tested for their abilities to form colonies in calcium-deficient medium and soft agar and to produce tumors in athymic nude mice. Most (10 out of 11) of the clones which were derived from colonies in calcium-deficient medium were unable to form colonies in soft agar and 8 out of 11 were non-tumorigenic. Conversely, 6 out of 9 clones derived from colonies in soft agar were unable to multiply significantly in calcium-deficient medium and 5 of these 6 clones were also non-tumorigenic. Two of these 9 soft agar-growing clones were tumorigenic, one of which also proliferated in calcium-deficient medium, and the other of which acquired the ability to proliferate in calcium-deficient medium after it became able to form tumors in athymic nude mice. Thus, T51B rat liver cells gain the ability to grow in calcium-deficient medium and soft agar independently during the process of neoplastic transformation and neither characteristic by itself reliably predicts tumorigenicity.


Subject(s)
Calcium/physiology , Cell Transformation, Neoplastic/drug effects , Liver Neoplasms/pathology , Animals , Cell Division/drug effects , Cell Line , Culture Media , Liver/pathology , Rats
12.
Biochem Biophys Res Commun ; 115(1): 383-90, 1983 Aug 30.
Article in English | MEDLINE | ID: mdl-6225434

ABSTRACT

Extracellular Ca2+-deprivation inhibited the proliferation of normal T51B rat liver cells and reduced (2-4 fold) the amount of EDTA/EGTA-extractable protein kinase C activity. By contrast, preneoplastic and neoplastic T51B rat liver cells were able to proliferate in Ca2+-deficient medium and retained all of their extractable protein kinase C activity.


Subject(s)
Aflatoxins/toxicity , Calcium Chloride/pharmacology , Carcinogens/toxicity , Cell Transformation, Neoplastic/drug effects , Liver/physiopathology , Precancerous Conditions/physiopathology , Protein Kinases/metabolism , Aflatoxin B1 , Animals , Cell Division/drug effects , Cell Line , DNA Replication/drug effects , Kinetics , Liver/drug effects , Liver/enzymology , Liver Neoplasms/chemically induced , Liver Neoplasms/physiopathology , Protein Kinase C , Rats
15.
Brain Res Bull ; 7(1): 57-61, 1981 Jul.
Article in English | MEDLINE | ID: mdl-6456038

ABSTRACT

A major phosphoprotein of synaptic membranes, the phosphorylation of which is stimulated by Ca2+ and inhibited by ACTh, appears to be identical with protein B-50 described by Zwiers, Schotman and Gispen [40]. We have investigated its subsynaptic localization by means of a variety of subfractionation techniques and compared it with that of a number of other phosphoproteins found in synaptic membranes. It appears to be predominantly, if not exclusively, associated with presynaptic membranes of low bouyant density. This localization pattern is similar to, but somewhat more extreme than that exhibited by Protein I, as a brain specific phosphoprotein studied by Greengard and his collaborators [11].


Subject(s)
Phosphoproteins/metabolism , Synapses/metabolism , Animals , Cerebral Cortex/metabolism , Electrophoresis, Polyacrylamide Gel , GAP-43 Protein , Nerve Tissue Proteins/metabolism , Rats , Synapsins , Synaptic Membranes/metabolism , Synaptic Vesicles/metabolism , Synaptosomes/metabolism
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