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J F Whitfield

Publications and source records attributed to J F Whitfield.

At least 127 records · Page 7Linked to original sources
PubMed1987

Serum-activated T51B rat liver cells transiently accumulate cyclic AMP-dependent protein kinases on their surfaces during the G1 phase.

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.

Animals↗
PubMed1987

Calcium, cyclic AMP and protein kinase C--partners in mitogenesis.

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.

Animals↗
PubMed1987

The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium.

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.

Animals↗
PubMed1986

Protein kinase C stimulation increases the transforming ability of the polyoma virus middle T antigen.

Exposure of dexamethasone-treated cells of the mT-1 line of F111 rat cells bearing a dexamethasone-inducible polyoma virus middle T (mT) antigen gene to very low concentrations of the protein kinase C-stimulating phorbol ester TPA increased the association of mT antigen with the cellular pp60c-src tyrosine protein kinase, as indicated by an increased phosphorylation of tyrosine residues of mT in mT:pp60c-src complexes precipitated from extracts of the TPA-treated cells by anti-mT antibodies. This TPA (hence probably protein kinase C)-enhanced association of mT with pp60c-src was accompanied by a large increase in the transforming ability of mT as indicated by a much enhanced ability of TPA-treated mT-1 cells producing submaximal levels of mT to proliferate while suspended in semi-solid medium and to form foci on confluent monolayers of normal F111 cells. NRCC NO: 26558.

Antigens, Polyomavirus Transforming↗
PubMed1986

Chromatin structure. Nuclease digestion profiles reflect intermediate stages in the folding of the 30-nm fiber rather than the existence of subunit beads.

Conditions have been found for the isolation of rat liver nuclei which maintain chromatin in its native state and suppresses endogenous nuclease activity. Chromatin prepared in this way can be dispersed into buffers containing various concentrations of monovalent or divalent cations so that the 30-nm fiber is either totally or partially decondensed. When probed with micrococcal nuclease, the digestion profiles show that although the 30-nm fiber can be cleaved periodically to generate superbead-like particles this only occurs under certain ionic conditions when the fiber is partially decondensed. It is likely that this cleavage pattern reflects the transient exposure of specific nuclease sensitive sites as the 30-nm fiber condenses, rather than the existence of a specific subunit of a beaded 30-nm fiber. The periodicity of these nuclease-sensitive sites appear to be related to the asymmetric distribution of histone H1 molecules along the length of the fiber.

Animals↗
PubMed1986

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells.

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.

Animals↗
PubMed1986

The glucocorticoid dexamethasone and the tumor-promoting artificial sweetener saccharin stimulate protein kinase C from T51B rat liver cells.

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).

Animals↗
PubMed1986

Ca2+-dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis.

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.

Adenosine Triphosphate↗
PubMed1986

Characterization of G1 transit induced by the mitogenic-oncogenic viral Ki-ras gene product.

NRK rat kidney cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus (ts371) were transformed at 36 degrees C but were phenotypically nontransformed at 41 degrees C because of the abnormal thermolability of the oncogenic 21-kilodalton product of the viral Ki-ras gene. Thus tsK-NRK cells were rendered quiescent in a G0-G1 state by a 48-h incubation in serum-free medium at the nonpermissive, p21-inactivating temperature of 41 degrees C. The serum-starved cells could then be stimulated to transit G1 either as nontransformed cells by adding serum at 41 degrees C or as transformed cells by lowering the temperature to a p21-activating 36 degrees C. The viral p21 protein was as effective as serum in stimulating tsK-NRK cells to transit G1 and to start replicating DNA. While p21 effectively stimulated cells to transit G1 even in unconditioned, serum-free medium, they still needed cell-derived conditioning factors to subsequently divide. The p21 protein also enabled the cells to transit G1 in spite of an extracellular Ca2+ deficiency that inhibited the G1 transit of serum-stimulated cells. p21 activity was needed to stimulate both early and late G1 events. In contrast to serum, p21 did not stimulate total RNA or protein synthesis, but some RNA and protein synthesis must have been needed for the p21-driven G1 transit because it could be stopped by actinomycin D or cycloheximide.

Animals↗
PubMed1985

The mitogenic/oncogenic p21 Ki-RAS protein stimulates adenylate cyclase activity early in the G1 phase of NRK rat kidney cells.

tsK-NRK rat cells infected with a temperature-sensitive mutant of the Kirsten murine sarcoma virus were arrested in the G0/G1 phase of their cell cycle by incubation in serum-deficient medium at a temperature (41 degrees C) which inactivates the virus' abnormally thermolabile mitogenic/oncogenic 21 kDa (p 21) RAS protein product. Reactivating the viral RAS protein by lowering the temperature to a permissive 36 degrees C rapidly (within 1 hour) stimulated adenylate cyclase, sensitized the enzyme to stimulation by GTP and forskolin and caused the tsK-NRK cells to transit G1 and start replicating their DNA about 10 hours later. The 41 degrees C----36 degrees C shift did not affect adenylate cyclase or stimulate G1 transit in uninfected NRK cells. Thus, an oncogenic viral RAS protein was able to stimulate adenylate cyclase and G1 transit in a mammalian cell just as other RAS proteins appear to do in yeast cells.

Adenylyl Cyclases↗
PubMed1985

The regulatory and catalytic subunits of rat liver cyclic AMP-dependent protein kinases respond differently to thyroparathyroidectomy and 1 alpha,25-dihydroxyvitamin D3.

The regulatory subunits of cytoplasmic and nuclear cyclic AMP-dependent protein kinases increased, while the holoenzyme activities fell in the livers of thyroparathyroidectomized (TPTX) rats. 1 alpha,25-dihydroxyvitamin D3 reversed this TPTX-induced decline in holoenzyme activities. TPTX also prevented the increase in holoenzyme activities, but not the increase in the concentration of regulatory subunits that normally occurs in partially hepatectomized animals. An injection of 1 alpha, 25-dihydroxyvitamin D3 into these TPTX animals, at the time of partial hepatectomy restored the increases in holoenzyme activities, without affecting the changes in regulatory subunits. Thus, the synthesis of regulatory and catalytic subunits can be uncoupled by TPTX and 1 alpha, 25-dihydroxyvitamin D3 appears to selectively control the activity of C subunits.

Animals↗
PubMed1985

Cytoplasmic microtubules are essential for the formation of membrane-bound polyribosomes.

Colchicine, at low intracellular concentrations, causes a rapid depolymerization of membrane-associated polyribosomes. Poly(A+) mRNA is rapidly lost from these polysomes, and 80 S monomers are left attached to the membranes of the endoplasmic reticulum. Binding studies and measurements of intracellular colchicine concentrations indicate that the drug is acting via depolymerization of cytoplasmic microtubules. The vinca alkaloids, vincristine and vinblastine, have the same effect on polyribosomes, whereas lumicolchicine is ineffective. Furthermore, cordycepin and actinomycin D are without effect on polyribosomes indicating that colchicine is not simply inhibiting the production or transport of new mRNA. It appears that disruption of the cytoplasmic microtubule network prevents membrane-associated ribosomes from reinitiating protein synthesis resulting in the rapid loss of mRNA.

Animals↗
PubMed1985

The possible cyclic AMP-dependence of an early prereplicative event that determines mitosis in regenerating rat liver.

The beta-adrenergic blocker dl-propranolol prevented a large proportion of regenerating rat liver cells from entering the mitotic phase of their first cell division cycle without affecting their ability to initiate or complete DNA replication. The drug, at a dose of 20 or 50 mg/kg of body weight, was most effective in reducing mitosis when injected between 1 and 2 hours after the proliferatively activating partial hepatectomy, which was 22 to 23 hours before the peak of DNA-synthetic activity. Propranolol also inhibited the early prereplicative surge of total liver cyclic AMP, which occurs shortly after partial hepatectomy, but this effect was not correlated to the mitosis-inhibiting activity. However, cyclic AMP or dibutyryl cyclic AMP completely reversed propranolol's mitosis-inhibiting action when injected between 1.5 and 2 hours (but not sooner or later) after partial hepatectomy, which was just before the total liver cyclic AMP content began to rise. Thus, there appears to be a transient, propranolol-inhibitable, probably cyclic AMP-initiated event in the early prereplicative development of rat hepatocytes that determines entry into mitosis rather than the initiation of DNA replication.

Adenine Nucleotides↗
PubMed1985

The selective induction of a small number of proteins during G1 transit results from the mitogenic action of pp60v-src in tsASV-infected rat cells.

Since the mitogenic/oncogenic pp60v-src product of the avian sarcoma virus (ASV) mutant, tsLA23, is abnormally thermolabile, tsLA23-NRK cells were phenotypically nontransformed at 40 degrees C and were consequently rendered quiescent by serum deprivation at this temperature. These serum-deprived cells were stimulated to transit G1 either as transformed cells by simply dropping the temperature to a pp60v-src -activating 36 degrees C, or as nontransformed cells by adding serum at 40 degrees C. Serum stimulation rapidly increased total protein synthesis in these cells and over 100 changes in cellular proteins (resolved by two-dimensional gel electrophoresis) occurred during G1 transit. By contrast, pp60v-src-activation did not increase total protein synthesis and only six proteins (18.5-44 kD) were clearly seen to appear or increase when quiescent cells were stimulated to transit G1 by activating pp60v-src. Three of these six pp60v-src- induced proteins also appeared or accumulated during the G1 transit of serum-stimulated cells. The appearance and/or accumulation of the six proteins and the subsequent initiation of DNA replication may have resulted from pp60v-src stimulating only a small number of critical cellular genes because both the protein changes and DNA replication were completely suppressed by the transcription inhibitor actinomycin D.

Animals↗
PubMed1985

Transforming NRK cells with avian sarcoma virus reduces the extracellular Ca2+ requirement without affecting the calcicalmodulin requirement for the G1/S transition.

NRK rat cells infected with a transformation-defective, temperature-sensitive (ts) mutant of the avian sarcoma virus could not proliferate in Ca2+-deficient medium at a nonpermissive temperature (40 degrees C) that inactivated the viral pp60v-src-transforming product and rendered the cells phenotypically untransformed. However, these arrested cells were stimulated to initiate DNA replication with little or no delay while still in the Ca2+-deficient medium, either by adding Ca2+ or calmodulin at 40 degrees C or by reducing the temperature to 36 degrees C which restored the transformed phenotype by rapidly reactivating pp60v-src. The G1/S transition triggered by restoring the transformed phenotype was suppressed by three different anticalmodulin drugs (R24571, trifluoperazine, W7). The suppression by one of these drugs, trifluoperazine, was overcome by adding calmodulin. Thus, neoplastic transformation by the avian sarcoma virus sharply reduces the extracellular Ca2+ requirement for the initiation of DNA replication without bypassing a calcicalmodulin-dependent mechanism also needed for the G1/S transition.

Animals↗
PubMed1985

Involvement of the Ca2+/phospholipid-dependent protein kinase in the G1 transit of T51B rat liver epithelial cells.

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.

Animals↗
PubMed1985

The influence of extracellular calcium on the distribution of protein kinase C in non-neoplastic and neoplastic rat liver cells.

Non-neoplastic T51B rat liver epithelial cells cannot proliferate in Ca2+-deficient medium. This proliferative inhibition in Ca2+-deficient medium is accompanied by a large reduction in the amount of cellular EDTA-extractable Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity. By contrast, tumorigenic epithelial cells from several Morris hepatomas proliferate in Ca2+-deficient medium and either maintain or greatly increase their content of EDTA-extractable protein kinase C.

Animals↗