==================================BSR16================================== 16. Role of Ca2+/phospholipid-dependent protein kinase (Protein kinase C) in the regulation of cellular functions. Role of tumor-promoting phorbol esters in the regulation of cellular functions. 1 UI - 87100480 AU - Bronner C ; Vall:e A ; Gies JP ; Landry Y TI - Dual effect of phorbol ester on serosal mast cell exocytosis: interactions between ionic gradients and protein kinase C. AB - The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) induces a slow secretion of histamine from rat peritoneal mast cells. This secretion is dose-dependent from 0.3 to 10 ng/ml. Higher doses are less efficient. The absence of magnesium and/or potassium increases mast cell exocytosis induced by TPA. In the absence of both potassium and magnesium, extracellular calcium concentrations above 3 X 10(-5)M inhibit the effect of TPA. The sensitivity of mast cells to other inducers of histamine release is modified by pretreatment with TPA. The ionophore A23187-induced secretion is potentiated or inhibited according to the dose of TPA and the concentration of extracellular calcium. The absence of potassium prevents the potentiating effect of TPA. The secretory effect of compound 48/80 is decreased by pretreatment of mast cells with TPA. This effect is more potent in the absence of potassium. These results suggest that the activation of protein kinase C acts as a bidirectional regulator of mast cell exocytosis and is modulated by transmembrane gradients of monovalent and divalent ions. Its inhibitory effects might be favoured by the highest levels of cytosolic calcium and could be related to the inhibition of a guanine nucleotide regulatory protein involved in the transduction of the receptor signal to phosphatidylinositides turnover. MH - A-23187/PHARMACODYNAMICS ; Animal ; Compound 48-80/PHARMACODYNAMICS ; Exocytosis/*DRUG EFFECTS ; Histamine Liberation/DRUG EFFECTS ; In Vitro ; Ions ; Male ; Mast Cells/*DRUG EFFECTS/IMMUNOLOGY/PHYSIOLOGY ; Protein Kinase C/PHYSIOLOGY ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Ann Inst Pasteur Immunol 1986 Sep-Oct;137D(2):249-61 2 UI - 87100190 AU - Gordon EA ; Carney DH TI - Thrombin receptor occupancy initiates cell proliferation in the presence of phorbol myristic acetate. AB - A combination of DIP-thrombin and either PMA (50 ng/ml) or dioctanoyl glycerol stimulates DNA synthesis in serum free cultures of NIL hamster cells similar to that previously reported for the combinatory effect of DIP-thrombin and gamma-thrombin. Thus, PMA or dioctanoyl glycerol appears to generate signals normally stimulated by gamma-thrombin interaction with cells. This stimulation was not observed when cells were treated with DIP-thrombin and 4-beta-phorbol or 4-alpha-phorbol 12,13-didecanoate. Therefore, it appears that this effect is mediated through activation of protein kinase C and that this activation plays an important role in thrombin mitogenesis. MH - Animal ; Cell Cycle/*DRUG EFFECTS ; Cell Line ; Diglycerides/ PHARMACODYNAMICS ; Drug Synergism ; Hamsters ; Phorbol Esters/ PHARMACODYNAMICS ; Protein Kinase C/*PHYSIOLOGY ; Receptors, Endogenous Substances/*PHYSIOLOGY ; Structure-Activity Relationship ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Thrombin/ *ANALOGS & DERIVATIVES/PHARMACODYNAMICS SO - Biochem Biophys Res Commun 1986 Dec 15;141(2):650-6 3 UI - 87085354 AU - Hall F ; Morita M ; Best JB TI - Neoplastic transformation in the planarian: II. Ultrastructure of malignant reticuloma. AB - Cadmium and phorbol ester induced tumorigenesis in the planarian, Dugesia dorotocephala, develops as a cocarcinogenic process involving initiation and promotion in the progression of neoplastic disease. Treatment of intact planarians with sublethal concentrations of cadmium sulfate and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a type of infiltrating tumor that proved to be potentially lethal. Surgical transplantation of such tumorous tissues into otherwise healthy planarians resulted in the same histopathological progression to lethality, which confirmed the metastatic nature of the neoplasia. Electron microscopic studies revealed that both the chemically-induced and the transplantation-based tumors involved, exclusively, the proliferation and differentiation of abnormal reticular cells, referred to as reticuloma cells. Reticular cells normally are ameboid, phagocytic, and are thought to provide the planarian with a phylogenetic predecessor of an immune surveillance system. A considerable incidence of mitosis was observed within the tumor areas; and the sequence of differentiation, from transformed stem cells to mature but nonfunctional reticuloma cells, was elucidated. This profile of differentiation supports the concept of cellular derivation via stem cell dynamics as opposed to dedifferentiation. A variety of ultrastructural abnormalities were characterized: several of which tend to substantiate the anaplastic quality of the reticuloma, while others are more specifically diagnostic for malignancy. These findings further extend the potential usefulness of the planarian malignant reticuloma as a model system for the study of neoplastic stem cell diseases. MH - Animal ; Cadmium/*PHARMACODYNAMICS ; Carcinogens/*PHARMACODYNAMICS ; Cell Transformation, Neoplastic/*CHEMICALLY INDUCED/ULTRASTRUCTURE ; *Cocarcinogenesis ; Planarians/*DRUG EFFECTS ; Support, U.S. Gov't, Non-P.H.S. ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Turbellaria/ *DRUG EFFECTS SO - J Exp Zool 1986 Nov;240(2):229-44 4 UI - 87085353 AU - Hall F ; Morita M ; Best JB TI - Neoplastic transformation in the planarian: I. Cocarcinogenesis and histopathology. AB - Although several investigators have reported that exposure to mammalian carcinogens induces abnormal tumorlike growths and teratogenic remodeling in planarians, there is no general agreement that these, or comparable responses in any other invertebrates, model mammalian carcinogenesis. To investigate this question, freshwater planarians of the species Dugesia dorotocephala were exposed to culture water containing an initiator and a promoter, either alone or in combination. Cadmium, a potent carcinogen, was used as an initiator in the protocol. Treatment with sublethal concentrations of cadmium sulfate produced a benign, but persistent, tumor in a small percentage of the planarians. The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester and well-known promoter, to the cadmium-containing solutions resulted in the induction of a progressive, potentially lethal, transplantable tumor in a large proportion of the treated flatworms. Light and electron microscopy revealed this particular tumor to be composed both of immature cells and of a single mature cell type: newly differentiated, but transformed, reticular cells. Further examination of the infiltrating tissue formations elucidated the profile of differentiation, from a population of mitotically active transformed stem cells through the transitional stages in the associated reticuloma. These results suggest that 1) the freshwater planarian displays the major phenomenology of mammalian cocarcinogenesis and that 2) the planarian reticuloma models several important features of a neoplastic stem cell disease. MH - Animal ; Cadmium/*PHARMACODYNAMICS ; Carcinogens/*PHARMACODYNAMICS ; Cell Transformation, Neoplastic/*CHEMICALLY INDUCED/PATHOLOGY ; *Cocarcinogenesis ; Planarians/*DRUG EFFECTS ; Support, U.S. Gov't, Non-P.H.S. ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Turbellaria/ *DRUG EFFECTS SO - J Exp Zool 1986 Nov;240(2):211-27 5 UI - 87076680 AU - Haarr L ; Kleppe K ; Lillehaug JR TI - Changes in polypeptide synthesis and glycosylation in mouse embryonic fibroblast C3H/10T1/2 Cl 8 cells caused by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. AB - The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate. MH - Animal ; Cell Line ; Cell Transformation, Neoplastic/*CHEMICALLY INDUCED/ METABOLISM ; Embryo ; Fibroblasts/DRUG EFFECTS/*METABOLISM ; Gene Expression Regulation/DRUG EFFECTS ; Glycoproteins/*BIOSYNTHESIS ; Mice ; Mice, Inbred C3H ; Neoplasm Proteins/*BIOSYNTHESIS ; Peptides/ *BIOSYNTHESIS ; Protein Processing, Post-Translational/DRUG EFFECTS ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Biochim Biophys Acta 1986 Dec 19;889(3):334-45 6 UI - 87064483 AU - Hsiao WL ; Wu T ; Weinstein IB TI - Oncogene-induced transformation of a rat embryo fibroblast cell line is enhanced by tumor promoters. AB - Rat embryo fibroblast cell line 6 was transfected with plasmid pT24, which contains the activated human bladder c-Ha-ras oncogene, and the cells were grown continuously in the absence or presence of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or teleocidin. The presence of TPA or teleocidin led to a 6- to 14-fold increase in the number of morphologically transformed foci. No transformed foci were seen when rat 6 cells were transfected with the normal c-Ha-ras oncogene in the absence or presence of TPA, or in cells simply treated with TPA or teleocidin. Enhancement of pT24-induced foci was seen even when the addition of TPA was delayed until day 16. In transfection studies with the drug resistance genes gpt and neo, TPA and teleocidin did not increase the number of Gpt+ or Neo+ colonies. When rat 6 cells were cotransfected with pT24 and neo genes and grown in the absence or presence of TPA, the presence of TPA did not increase the yield of Neo+ colonies but caused a fivefold increase in the number of Neo+ colonies that displayed a transformed morphology. Southern blot analyses of DNAs obtained from these clones indicated that TPA treatment did not influence the extent of integration of either the pT24 or neo gene. DNA samples from all of the morphologically transformed cells displayed a characteristic 2-kilobase SacI fragment homologous to pT24 DNA and expressed relatively high levels of the corresponding mRNA.(ABSTRACT TRUNCATED AT 250 WORDS) MH - Animal ; Cell Transformation, Neoplastic/*DRUG EFFECTS ; Cells, Cultured ; Fibroblasts ; Lyngbya Toxins/*PHARMACODYNAMICS ; Mice ; *Oncogenes ; Rats ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/ *PHARMACODYNAMICS ; Transfection/DRUG EFFECTS SO - Mol Cell Biol 1986 Jun;6(6):1943-50 7 UI - 87064459 AU - Angel P ; P:oting A ; Mallick U ; Rahmsdorf HJ ; Schorpp M ; Herrlich P TI - Induction of metallothionein and other mRNA species by carcinogens and tumor promoters in primary human skin fibroblasts. AB - We used nucleic acid hybridization and cDNA cloning techniques to isolate human sequences that respond to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). These clones were used as probes to examine changes of gene expression that occurred after the proliferation of exponentially growing primary human fibroblasts was arrested. Transcript levels detected by these probes were increased coordinately by treatment of the cells with UV light, mitomycin C, TPA, or the UV light-induced extracellular protein synthesis-inducing factor EPIF (M. Schorpp, U. Mallick, H. J. Rahmsdorf, and P. Herrlich, Cell 37:861-868, 1984). Proteins coded for by these transcripts were characterized by hybrid-promoted translation and by cDNA sequencing. One of the cDNA clones was homologous to the metallothionein IIa gene, and one set of related clones selected RNA for the secreted TPA-inducible protein XHF1 (U. Mallick, H. J. Rahmsdorf, N. Yamamoto, H. Ponta, R.-D. Wegner, and P. Herrlich, Proc. Natl. Acad. Sci. USA 79:7886-7890, 1982). MH - *Cell Transformation, Neoplastic ; Cells, Cultured ; Cloning, Molecular ; DNA/ISOLATION & PURIFICATION ; Fibroblasts/METABOLISM ; Genes, Structural/ *DRUG EFFECTS ; Human ; Metallothionein/*GENETICS ; Nucleic Acid Hybridization ; RNA, Messenger/*GENETICS ; Skin/*DRUG EFFECTS/METABOLISM ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*TOXICITY ; Transcription, Genetic/*DRUG EFFECTS SO - Mol Cell Biol 1986 May;6(5):1760-6 8 UI - 87057429 AU - Lee SA ; Holz RW TI - Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol. AB - The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells. MH - Adenosine Triphosphate/METABOLISM ; Adrenal Medulla/DRUG EFFECTS/ *METABOLISM/SECRETION ; Animal ; Calcium/PHARMACODYNAMICS ; Cattle ; Cell Membrane Permeability/*DRUG EFFECTS ; Cells, Cultured ; Digitonin/ *PHARMACODYNAMICS ; Diglycerides/*PHARMACODYNAMICS ; Glycerides/ *PHARMACODYNAMICS ; Kinetics ; Norepinephrine/*METABOLISM ; Phosphoproteins/ISOLATION & PURIFICATION ; Phosphorylation ; Proteins/ *METABOLISM/SECRETION ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/ *PHARMACODYNAMICS SO - J Biol Chem 1986 Dec 25;261(36):17089-98 9 UI - 87052004 AU - Knowles MA ; Jani H TI - Multistage transformation of cultured rat urothelium: the effects of N-methyl-N-nitrosourea, sodium saccharin, sodium cyclamate and 12-O-tetradecanoylphorbol-13-acetate. AB - Rat bladder epithelial cell transformation was found to involve at least three distinct phenotypic stages in vitro. The effects on this process of N-methyl-N-nitrosourea (MNU), sodium saccharin, sodium cyclamate and 12-O-tetradecanoylphorbol-13-acetate (TPA) alone or in combination were investigated. MNU was a potent inducer of proliferating preneoplastic epithelial foci. Sodium saccharin alone induced a transient hyperplastic response of the cultured urothelium but did not induce significantly more foci than in controls, though a late toxic effect of saccharin may have masked focus induction. In combination with a single dose of 250 micrograms/ml MNU, sodium saccharin did not increase focus incidence compared with MNU treatment alone but following a single treatment with 25 micrograms/ml MNU which alone induced no foci, saccharin induced a significant number of foci indicating a promoting effect under these conditions. Sodium cyclamate alone induced a marked and prolonged epithelial hyperplasia and a significant increase in focus incidence above controls and above MNU-treated cultures. Following a single treatment with 250 micrograms/ml MNU, cyclamate increased focus incidence still further. TPA, the potent skin tumour promoting agent, alone induced significantly more foci than in controls. Rapidly-proliferating cell lines were established from nine foci induced by treatment with MNU alone or MNU + saccharin. Four of these were tumorigenic. These results indicate that MNU is a potent inducer of preneoplastic foci in rat urothelial cultures. Sodium saccharin and sodium cyclamate can act as promoting agents in this in vitro system as in the in vivo induction of tumours and sodium cyclamate appears to be active as a complete inducer under these conditions. MH - Animal ; Bladder/*DRUG EFFECTS/PATHOLOGY ; Cell Line ; Cell Transformation, Neoplastic/*DRUG EFFECTS ; Cocarcinogenesis ; Cyclamates/ *TOXICITY ; Epithelium/DRUG EFFECTS/PATHOLOGY ; Female ; Hyperplasia ; Methylnitrosourea/*TOXICITY ; Phenotype ; Rats ; Rats, Inbred F344 ; Saccharin/*TOXICITY ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*TOXICITY SO - Carcinogenesis 1986 Dec;7(12):2059-65 10 UI - 87051986 AU - Smith BM ; Gindhart TD ; Colburn NH TI - Possible involvement of a lanthanide-sensitive protein kinase C substrate in lanthanide promotion of neoplastic transformation. AB - The rare earth elements lanthanum and terbium (0.1-1.0 mM), pharmacological analogs of calcium, induced neoplastic transformation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive (P+) and to a lesser extent TPA-resistant (P-) preneoplastic mouse JB6 epidermal cells. A maximum of 2500 anchorage-independent colonies per 10(4) cells were induced in P+ lines, a response comparable to that induced by phorbol esters (1.6-16 nM). The maximum lanthanide-induced colony yield in P- lines was 20% of that in P+ lines (approximately 550 colonies), and was observed under conditions where TPA induced less than 30 colonies per 10(4) cells. Lanthanides and TPA produced a synergistic effect on colony size in P+ cells. Lanthanides are not promoting transformation merely by mimicking high calcium: adding exogenous extracellular calcium (up to 50.0 mM) or using calcium ionophore (up to toxic concentrations) to increase intracellular calcium does not promote transformation. Lanthanum will substitute for calcium in activating partially purified protein kinase C (PKC), the calcium-dependent phorbol ester receptor. However, lanthanides must be promoting transformation by a mechanism other than PKC activation because lanthanides failed to activate PKC in intact JB6 cells. Three independent experiments showed a lack of lanthanum effect on PKC-dependent events in intact cells. First, in contrast to TPA, lanthanum pretreatment of JB6 cells did not produce elevated phosphorylation of an 80-kd substrate. Second lanthanum pretreatment did not cause decreased PKC activity after prolonged exposure. Third, lanthanum and TPA affected epidermal growth factor binding with a different magnitude, time course and calcium dependency. We found, however, a PKC substrate in P+, P- and tumorigenic cell lines that is sensitive to lanthanum and increases its migration in sodium dodecylsulfate-polyacrylamide gels from 23 to 21 kd. The above data suggest that: (i) alterations in cation binding may be sufficient for inducing the transformed phenotype; and (ii) lanthanide promotion of neoplastic transformation may be linked to a lanthanide-sensitive PKC substrate, but is not due to a direct PKC activation. MH - A-23187/PHARMACODYNAMICS ; Calcium Channel Blockers/PHARMACODYNAMICS ; Cell Line ; Cell Transformation, Neoplastic/*DRUG EFFECTS ; Enzyme Activation/DRUG EFFECTS ; Metals, Rare Earth/*TOXICITY ; Phosphorylation ; Protein Kinase C/*PHYSIOLOGY ; Receptors, Epidermal Growth Factor-Urogastrone/METABOLISM ; Tetradecanoylphorbol Acetate SO - Carcinogenesis 1986 Dec;7(12):1949-56 11 UI - 87051806 AU - Romano F ; Marchesini C ; Paccagnella L ; Armato U TI - Antioxidants and blockers of arachidonate metabolism inhibit the mitogenic effects of TPA in hepatocytes: differences in the operative mechanisms according to the cell cycle setting. AB - A single exposure to a low concentration (10(-10) mol/l) of TPA doubled the size of the fraction of neonatal rat hepatocytes flowing into DNA synthesis within 24 hours in 4-day-old primary cultures kept in low-calcium (0.01 mmol/l) HiWoBa2000 synthetic medium, thereby evoking a phenotypically neoplastic feature in normal, i.e. non-initiated cells. Inhibition kinetics studies, in which several antioxidants and blockers of the arachidonate cascade were given, each by itself, simultaneously with or at various time intervals after TPA, showed that the early mitogenic effects of TPA, i.e. the commitment of GO hepatocytes to grow and the reactivation of hepatocytes poised at the G1/S boundary required oxygen radicals and all the main metabolites of arachidonate. Instead, the subsequent flow into S phase of TPA-committed hepatocytes was not controlled by oxygen radicals and prostaglandins but by retinoid-modulable activities and by products of the lipoxygenase and thromboxane synthase pathways of arachidonate metabolism. MH - Animal ; Antioxidants/*PHARMACODYNAMICS ; Arachidonic Acids/*ANTAGONISTS & INHIBITORS ; Ascorbic Acid/PHARMACODYNAMICS ; Cell Cycle/*DRUG EFFECTS ; Glutathione/PHARMACODYNAMICS ; Kinetics ; Lipoxygenases/METABOLISM ; Liver/*CYTOLOGY/DRUG EFFECTS ; Mitosis/*DRUG EFFECTS ; Rats ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Thromboxane Synthetase/ METABOLISM ; Vitamin E/PHARMACODYNAMICS SO - Cell Biol Int Rep 1986 Oct;10(10):797-811 12 UI - 87051356 AU - Chang CC ; Gibson-D'Ambrosio RE ; Trosko JE ; D'Ambrosio SM TI - Growth-promoting effect of 12-O-tetradecanoylphorbol-13-acetate on cultured normal human fetal kidney epithelial cells. AB - Normal human epithelial cells have been reported to be sensitive to growth inhibition by 12-O-tetradecanoylphorbol-13-acetate in contrast to neoplastic counterparts. Our studies with normal human fetal kidney epithelial cells, however, show that 12-O-tetradecanoylphorbol-13-acetate may increase cell density and promote the growth of these cells at early passages. The result, together with three previous reports showing similar effects in normal human melanocytes, prostatic epithelial cells, and an unidentified cell type in human epidermal cell culture, indicates that human cells may exhibit divergent responses to 12-O-tetradecanoyl-phorbol-13-acetate for induction of cellular differentiation or proliferation. MH - Cell Communication/DRUG EFFECTS ; Cell Count ; Cell Division/*DRUG EFFECTS ; Cells, Cultured ; Dose-Response Relationship, Drug ; Epithelium/ CYTOLOGY/DRUG EFFECTS ; Human ; Kidney/DRUG EFFECTS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/ *PHARMACODYNAMICS SO - Cancer Res 1986 Dec;46(12 Pt 1):6360-3 13 UI - 87028548 AU - Sanchez JH ; Abernethy DJ ; Boreiko CJ TI - Reversible expression of morphological transformation in C3H/10T1/2 mouse embryo cultures exposed to 12-O-tetradecanoylphorbol-13-acetate. AB - Treatment of C3H/10T1/2 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine and then 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the production of numerous foci of morphologically transformed cells. When dishes containing foci were provided medium which did not contain TPA, up to 84% of the foci were found to regress. Promotion of morphological transformation by TPA in C3H/10T1/2 cells may thus be a reversible process. MH - Animal ; Cell Transformation, Neoplastic/*DRUG EFFECTS/PATHOLOGY ; Cells, Cultured ; Methylnitronitrosoguanidine ; Mice ; Phenotype ; Tetradecanoylphorbol Acetate/*TOXICITY SO - Carcinogenesis 1986 Nov;7(11):1793-6 14 UI - 87013367 AU - Luikart SD ; Sackrison JL TI - Glycosaminoglycan metabolism of HL-60 cells during differentiation induction by tetradecanoylphorbol-13-acetate. AB - Many biochemical responses to phorbol ester differentiation inducers have been reported, including alterations in synthesis of specific gene products such as glycoproteins. Stage-specific glycosaminoglycan changes have previously been associated with the differentiation process, including a dramatic reduction in cellular chondroitin 4-sulfate during human myeloid leukemia cell maturation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). We have demonstrated that treatment of HL-60 human promyelocytic leukemia cells with 4-methyl-umbelliferyl-beta-D-xyloside increases precursor incorporation into glycosaminoglycans linked to beta-D-xyloside, rather than core protein, eliminating the need for core protein and xylosyltransferase. Therefore, these beta-D-xyloside-treated cells were used to study the decreased glycosaminoglycan production during TPA-induced HL-60 differentiation. Exposure of these pretreated HL-60 cells to TPA, which induces macrophage-like maturation, resulted in a 70% reduction of incorporation of [35S]sulfate into cell-associated glycosaminoglycans. Thus, even in HL-60 cells in which glycosaminoglycan production is maximally stimulated by beta-D-xyloside, TPA is a strong inhibitor of free glycosaminoglycan chain production, and this biochemical effect is associated with other features of leukocyte maturation. MH - *Cell Differentiation/DRUG EFFECTS ; Cell Line ; Glycosaminoglycans/ *METABOLISM ; Human ; Hymecromone/ANALOGS & DERIVATIVES/*PHARMACODYNAMICS ; Macrophages/*CYTOLOGY ; Muramidase/METABOLISM ; Proteoglycans/ BIOSYNTHESIS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Umbelliferones/ *PHARMACODYNAMICS SO - Leuk Res 1986;10(9):1083-90 15 UI - 87002115 AU - Sagara J ; Yamada KM ; Kakunaga T TI - Induction of an unusual type of shared phosphorylation in human and avian cells by tumor-promoting phorbol esters or transformation. AB - Alterations in patterns of protein phosphorylation after exposure to phorbol esters were compared in chicken embryo fibroblasts and KD cells (a human fibroblast line) by two-dimensional gel electrophoresis. A substantial increase in phosphorylation was observed of a major, markedly acidic protein of pI = 4.5 in two-dimensional gels of each cell type. However, the apparent molecular weights of these phosphoproteins differed substantially in the two species with a molecular weight of 67,000 in chicken fibroblasts and one of 80,000 in human KD cells. Both phosphoproteins, termed 67K and 80K respectively, contained phosphoserine and a small amount of phosphothreonine, but no detectable level of phosphotyrosine. Tryptic and chymotryptic phosphopeptide maps of 67K were nearly identical to those of 80K. These results indicate that they are related molecules, even though considerably different in apparent molecular weight, and that the induction of phosphorylation of these closely related, major, acidic phosphoproteins by phorbol esters is conserved from avian to human cells. In chicken embryo fibroblasts infected by a temperature-sensitive mutant of Rous sarcoma virus, phosphorylation of 67K was found to be elevated at 36 degrees C (transformed phenotype) compared to 41.5 degrees C (normal). Although the function of these closely related 67K and 80K phosphoproteins is unknown, the elevated level of phosphorylation could be involved in some aspects of transformation, and the increase is mimicked by treatment with phorbol esters. MH - Amino Acids/ANALYSIS ; Animal ; Avian Sarcoma Viruses ; Cell Transformation, Neoplastic/*METABOLISM ; Cells, Cultured ; Chick Embryo ; Diglycerides/PHARMACODYNAMICS ; Fibroblasts/METABOLISM ; Human ; Molecular Weight ; Peptide Mapping ; Phosphorylation ; Protein Kinase C/ ANALYSIS ; Proteins/*METABOLISM ; Tetradecanoylphorbol Acetate/ *PHARMACODYNAMICS SO - Cancer Res 1986 Oct;46(10):5291-6 16 UI - 87000724 AU - Warren L TI - Sialic acid lyase in human promyelocytic leukemic cells (HL-60) during phorbol-ester-induced differentiation. AB - There is a marked increase in the activity of sialic acid lyase (N-acetylneuraminate lyase; EC 4.1.3.3; also known as sialic acid aldolase) in HL-60 cells induced to differentiate into macrophages by the phorbol ester, tetradecanoylphorbol 12-myristate 13 acetate (TPA). Exposure of HL-60 cells to retinoic acid, butyric acid or dimethyl sulfoxide has little or no effect. The level of the enzyme remains unaltered in HL-60 cells grown in the presence of an inactive analog of TPA, nor does it change in variants of HL-60 cells resistant to TPA. MH - Butyric Acids/PHARMACODYNAMICS ; Cell Differentiation/*DRUG EFFECTS ; Cell Line ; Dimethyl Sulfoxide/PHARMACODYNAMICS ; Human ; Ketoacid-Lyases/ *METABOLISM ; Leukemia, Lymphoblastic/*ENZYMOLOGY ; Phorbol Esters/ PHARMACODYNAMICS ; Sialyltransferases/METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/ *PHARMACODYNAMICS ; Tretinoin/PHARMACODYNAMICS SO - Biochim Biophys Acta 1986 Oct 10;888(3):278-81 17 UI - 86304563 AU - Iacopetta B ; Carpentier JL ; Pozzan T ; Lew DP ; Gorden P ; Orci L TI - Role of intracellular calcium and protein kinase C in the endocytosis of transferrin and insulin by HL60 cells. AB - The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin. Similarly, a 10-fold elevation of the [Ca2+]i using the calcium ionophore ionomycin caused little or no change in the endocytosis of the two ligands. In contrast, activation of protein kinase C by phorbol myristate acetate markedly stimulated the internalization of both occupied and unoccupied transferrin receptors, even in cells with very low [Ca2+]i. The insulin receptor was found to behave differently in response to phorbol myristate acetate, however, in that only the occupied receptors were stimulated to internalize. We conclude that the [Ca2+]i plays only a minor role in regulating receptor-mediated endocytosis, whereas protein kinase C can selectively modulate receptor internalization depending on receptor type and occupancy. MH - Calcium/*PHYSIOLOGY ; Cell Line ; *Endocytosis ; Human ; Insulin/ *METABOLISM ; Leukemia, Myeloblastic ; Protein Kinase C/*PHYSIOLOGY ; Receptors, Endogenous Substances/METABOLISM ; Receptors, Insulin/ METABOLISM ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/ PHARMACODYNAMICS ; Transferrin/*METABOLISM SO - J Cell Biol 1986 Sep;103(3):851-6 18 UI - 86300896 AU - Neckers LM ; Vidal C ; McGlennen R ; Colamonici O TI - Phorbol ester-induced surface transferrin receptor modulation. No correlation with decreased cell proliferation. AB - Both phorbol ester or diacylglycerol (DAG) reduce cell surface transferrin receptor (TFR) number in CEM cells (a human T-cell acute lymphoblastic leukemia line) and HL-60 cells (a human promyelocytic leukemia cell line). This effect occurs with a t1/2 of approx. 30 min and is mimicked by addition of phospholipase C to cell cultures. Although cell surface TFR number is reduced to 25-30% of the control level 5 h after phorbol ester administration, apparent cell proliferation (as measured by [3H]thymidine incorporation) remains unaffected. Although independent of extracellular calcium (EGTA is slightly enhancing), the phenomenon is completely blocked by 30-min pretreatment with the calcium channel blocker diltiazem. Dilitazem pretreatment, while preventing receptor redistribution, does not completely block the phorbol ester-induced increase in TFR phosphorylation thought to be associated with receptor redistribution. Thus, calcium channel blockade effectively dissociates the effects of tetradecanoylphorbol acetate (TPA) on TFR internalization and phosphorylation. Our results also demonstrate that phorbol ester-induced effects on the TFR can be mimicked by the endogenous stimulator of protein kinase C, DAG, whether added directly to cultures or produced by the cells in response to exogenous phospholipase C. Furthermore, the phenomenon of TFR redistribution here described is not associated with a decreased proliferative capacity. MH - *Cell Division ; Cell Line ; Cell Membrane/METABOLISM ; Diglycerides/ PHARMACODYNAMICS ; Diltiazem/PHARMACODYNAMICS ; DNA/BIOSYNTHESIS ; EGTA/ PHARMACODYNAMICS ; Human ; Phorbols/*PHARMACODYNAMICS ; Phospholipase C/ PHARMACODYNAMICS ; Phosphorylation ; Receptors, Endogenous Substances/ DRUG EFFECTS/*METABOLISM ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Exp Cell Res 1986 Sep;166(1):151-60 19 UI - 86305779 AU - Haskard D ; Cavender D ; Ziff M TI - Phorbol ester-stimulated T lymphocytes show enhanced adhesion to human endothelial cell monolayers. AB - Phorbol esters have been used to study changes in the adhesiveness of T cells to endothelial cells (EC) after activation. The phorbol esters 12-O-tetra-decanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (P(Bu)2), but not the biologically inert 4-alpha-phorbol-12,13-didecanoate, strongly increased the binding of 51Cr-labeled T cells to human umbilical vein EC monolayers in microtiter wells. Binding to fibroblasts and gelatin-coated plastic was also increased, but to a lesser extent. Increased binding was observed at 0.3 ng/ml, with maximal enhancement at 33 to 100 ng/ml. Enhancement occurred within 1 min, with maximal increase after 15 min. Preincubation studies with P(Bu)2 showed that binding enhancement was entirely attributable to an effect on T cells, with no action on EC. Additive binding enhancement was seen when phorbol esters and agents that alter adhesion by acting on EC (LPS, IL 1, or IFN-gamma) were used together. The increase in T cell adhesion to EC after T cell activation may contribute to the selective emigration of activated T cells from the blood into developing inflammatory lesions. The rapid increase in binding suggests that this may be an important mechanism for immediate localization of circulating T cells, particularly sensitized T cells, in the cellular immune response, perhaps involving the activation of these cells at the endothelial blood-tissue interface. MH - Cell Adhesion/*DRUG EFFECTS ; Cells, Cultured ; Comparative Study ; Endothelium/*PHYSIOLOGY ; Fibroblasts/PHYSIOLOGY ; Gelatin ; Human ; Interferon Type II/PHARMACODYNAMICS ; Interleukin 1/PHARMACODYNAMICS ; Lipopolysaccharides/PHARMACODYNAMICS ; Phorbol Esters/*PHARMACODYNAMICS ; Phorbols/*PHARMACODYNAMICS ; Support, U.S. Gov't, P.H.S. ; T Lymphocytes/ DRUG EFFECTS/*PHYSIOLOGY ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Umbilical Veins SO - J Immunol 1986 Sep 1;137(5):1429-34 20 UI - 86247909 AU - Mesnil M ; Montesano R ; Yamasaki H TI - Intercellular communication of transformed and non-transformed rat liver epithelial cells. Modulation by TPA. AB - Gap-junctional intercellular communication of transformed and non-transformed rat liver epithelial cell lines was compared using a dye transfer method in the presence and absence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Whereas non-transformed cells (IAR 20, non-tumorigenic in newborn rats and in nude mice) showed very high communication capacity throughout a culture period of 3 weeks, transformed cells (IAR 6-1, tumorigenic in newborn rats and in nude mice) were less able to communicate. Similar correlation between intercellular communication and expression of transformed phenotypes were also found in newly cloned epithelial cell lines, IAR 27E and IAR 27F. When TPA was added to culture medium at 100 ng/ml, intercellular communication in all lines tested was reduced within 60 min. However, communications recovered completely from the effect within 10 h after addition of TPA. Further addition of TPA to the cultures every 24 h for 3 weeks had no effect on intercellular communication (measured 30 min after each TPA addition), suggesting that a single application of TPA made these cells refractory to further doses. A known stimulator of gap-junctional communication, db-cAMP, also increased dye transfer in IAR 20 and IAR 6-1 cells. TPA added to db-cAMP-treated cultures of IAR 20 and IAR 6-1 cells inhibited intercellular communication, suggesting that cAMP is not an antagonist of the effect of TPA on intercellular communication in these cell lines. These results are in sharp contrast to those obtained with the fibroblast cell line BALB/c 3T3, in which db-cAMP antagonized TPA effect and inhibition by TPA of intercellular communication was transient only when administered during their growth phase, and was stable and continuous when TPA was applied at confluence, and suggest that TPA may not be an effective tumour promoter in rat liver. MH - Adenosine Cyclic Monophosphate/PHARMACODYNAMICS ; Animal ; Cell Communication/*DRUG EFFECTS ; Cell Line ; Dose-Response Relationship, Drug ; Epithelium/CYTOLOGY ; Intercellular Junctions/*PHYSIOLOGY ; Liver/ *CYTOLOGY ; Liver Neoplasms, Experimental/*PATHOLOGY ; Phorbols/ *PHARMACODYNAMICS ; Rats ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Time Factors SO - Exp Cell Res 1986 Aug;165(2):391-402 21 UI - 86189658 AU - Kiguchi K ; Henning-Chubb C ; Huberman E TI - Alteration in glycosphingolipid pattern during phorbol-12-myristate-13-acetate-induced cell differentiation in human T-lymphoid leukemia cells. AB - We analyzed the patterns of glycosphingolipids (GSLs) from a line of cells derived from a clone of the human T-cell leukemia cells (CEM) that had been induced to differentiate by phorbol-12-myristate-13-acetate (PMA) into cells with a suppressor-like phenotype. We characterized the differentiation state of the cells by immunofluorescence by using anti-cell surface differentiation-specific monoclonal antibodies (OKT3, OKT4, OKT6, and OKT8). The GSLs were extracted and separated by thin-layer chromatography and the individual bands were quantitated by a dual-wavelength densitometer or by autoradiography of GSLs labeled with [14C]glucosamine and [14C]galactose. Treatment of the CEM cells with 0.16-16 nM PMA for 6 h to 6 days resulted in a dose- and time-dependent increase in the amount of two neutral GSLs [ceramide monohexoside and ceramide dihexoside] and three gangliosides [monosialoganglioside (GM3), sialosylparagloboside, and disialoganglioside (GD3)]. The increase in the neutral GSLs after PMA treatment reached its maximum at 30 h while GM3 peaked at 96 h. The increases in GM3 and sialosylparagloboside are presumably due to an increase in their synthesis levels because PMA promoted an elevated incorporation of glucosamine and galactose into these GSLs. The increase in the amount of GD3, on the other hand, is due to either a decrease in its degradation or use in other metabolic pathways because no detectable increase in glucosamine and galactose incorporation into this ganglioside could be found. Incubation of control or PMA-induced CEM cells with GM3 fractions purified from either CEM cells, human brain, or dog erythrocytes caused a reduction in cell growth and prevented the increase in reactivity of the induced cells with the OKT3 antibody. Incubation with semisynthetic ceramide dihexoside, however, prevented the decrease in reactivity with the OKT4 antibody. The observed changes in GSL patterns during PMA-induced differentiation of the CEM cells into suppressor-like cells and the inhibition of CEM cell growth by GM3 fractions suggest that the GSLs play a role in the control of cell growth and differentiation in the PMA-treated CEM cells. MH - Antibodies, Monoclonal/IMMUNOLOGY ; Cell Differentiation/*DRUG EFFECTS ; G(M3) Ganglioside/PHARMACODYNAMICS ; Gangliosides/ANALYSIS ; Glycosphingolipids/*ANALYSIS/BIOSYNTHESIS/PHARMACODYNAMICS/PHYSIOLOGY ; Human ; Leukemia, Lymphocytic/IMMUNOLOGY/*PATHOLOGY ; Phorbols/ *PHARMACODYNAMICS ; Support, U.S. Gov't, Non-P.H.S. ; T Lymphocytes ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Cancer Res 1986 Jun;46(6):3027-33 22 UI - 86105953 AU - Kokunai T ; Korosue K ; Tamaki N ; Matsumoto S TI - Promoting effect of 12-O-tetradecanoylphorbol-13-acetate on the in vitro malignant transformation of fetal rat brain cells exposed in utero to ethylnitrosourea. AB - In order to investigate the possibility that the theory of two-stage carcinogenesis can be applied to neurogenic carcinogenesis, we analyzed the promoting effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the in vitro malignant transformation of fetal rat brain cells exposed in utero to ethylnitrosourea (ENU). Rat brain cells were transferred to a cultured system at 72 h after a single pulse of ENU (50 mg/kg body weight) to pregnant SD-JCL rats on the 18th day of gestation. The positive findings of glial fibrillary acidic protein and S-100 protein in primary cultured cells by the analysis of immunohistochemistry revealed the neuroglial origin of transformed cells. These cells were divided into 12 groups and were treated twice per week with or without TPA at concentrations from 0.1 to 50.0 ng/ml. From the results of cellular morphology, Concanavalin A agglutinability, colony forming capacity in semisolid soft agar, and tumorigenicity in vivo, malignant transformation of fetal rat brain cells appeared earlier in the ENU group treated with TPA than in the untreated ENU group. On the basis of these observations, it is suggested that TPA might be effective as a tumor promoter on ENU-induced neurogenic carcinogenesis. MH - Animal ; Brain Neoplasms/*CHEMICALLY INDUCED/PATHOLOGY ; Cell Aggregation ; Cell Division ; Cell Transformation, Neoplastic/*DRUG EFFECTS ; Cells, Cultured ; Cocarcinogenesis ; Concanavalin A/DIAGNOSTIC USE ; Ethylnitrosourea/*PHARMACODYNAMICS ; Female ; Glial Fibrillary Acidic Protein/METABOLISM ; Maternal-Fetal Exchange ; Neoplasm Transplantation ; Phorbols/*PHARMACODYNAMICS ; Pregnancy ; Rats ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS ; Tumor Stem Cells/PATHOLOGY SO - Cancer Res 1986 Mar;46(3):1377-81 23 UI - 86206064 AU - Weissmann G ; Azaroff L ; Davidson S ; Dunham P TI - Synergy between phorbol esters, 1-oleyl-2-acetylglycerol, urushiol, and calcium ionophore in eliciting aggregation of marine sponge cells. AB - Aggregation of marine sponge cells (Microciona prolifera) resembles stimulus-response coupling of higher organisms in which activation of protein kinase C and movements of intracellular Ca provide twin signals. We now report that activators of protein kinase C (phorbol esters) and ionomycin act synergistically to aggregate sponge cells. Surprisingly--since extracellular Ca is required for integrity of the species-specific aggregation factor--synergistic aggregation proceeded in the complete absence of added extracellular Ca (2.5-20 mM EDTA). The order of activity of phorbol esters and related compounds was that of their effect on protein kinase C (phorbol myristate acetate, phorbol dibutyrate greater than phorbol diacetate much greater than phorbol, 4 alpha-phorbol). 1-Oleyl, 2-acetylglycerol a synthetic activator of protein kinase C, also showed synergy with ionomycin. Phorbol esters and 1-oleyl, 2-acetylglycerol acted in synergy with ionomycin to liberate membrane Ca as detected by decreased fluorescence of chlortetracycline in prelabeled cells. Moreover, urushiol, the toxic principle of poison ivy, but not pentadecanylcatechol, its inert analogue, showed synergy with ionomycin. Synergistic aggregation was inhibited by calmidazolium (10 microM), piroxicam (20-100 microM), and pertussis toxin (20 micrograms/ml). The data not only confirm that marine sponge cell aggregation follows the general sequence of stimulus-response coupling in the cells of higher organisms but also support, in this most ancient of multicellular creatures, the hypothesis that mobilization of intracellular Ca and activation of protein kinase C provide the twin signals for cell activation in the absence of added extracellular Ca. MH - Calcium/*PHARMACODYNAMICS ; Catechols/*PHARMACODYNAMICS ; Cell Aggregation/*DRUG EFFECTS ; Chlortetracycline/DIAGNOSTIC USE ; Diglycerides/*PHARMACODYNAMICS ; Dose-Response Relationship, Drug ; Drug Synergism ; Enzyme Activation ; Ethers/PHARMACODYNAMICS ; Fluorescence ; Glycerides/*PHARMACODYNAMICS ; Ionophores/*PHARMACODYNAMICS ; Phorbol Esters/*PHARMACODYNAMICS ; Porifera/DRUG EFFECTS ; Protein Kinase C/ PHYSIOLOGY ; Proteins/PHARMACODYNAMICS ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/PHARMACODYNAMICS ; Thiazines/ PHARMACODYNAMICS SO - Proc Natl Acad Sci USA 1986 May;83(9):2914-8 24 UI - 86192858 AU - Lad PM ; Olson CV ; Grewal IS TI - A step sensitive to pertussis toxin and phorbol ester in human neutrophils regulates chemotaxis and capping but not phagocytosis. AB - Treatment of human neutrophils with pertussis toxin (PT) abolishes chemotaxis in response to either platelet-activating factor (PAF) or f-Met-Leu-Phe (FMLP), and capping induced via the concanavalin A (Con A) receptor. These functional effects are accompanied by the inhibition of calcium mobilization by PAF, FMLP and Con A. The agent phorbol 12-myristate-13-acetate (PMA) also inhibits chemotaxis and capping as well as calcium mobilization by these receptors. In sharp contrast, neither PT, cholera toxin (CT), nor PMA, inhibits the phagocytosis of non-opsonized and opsonized Candida albicans, sheep erythrocytes or fluorescent latex beads. Our results suggest that receptor-initiated chemotaxis and capping involve a step that is sensitive to PT and PMA, and that phagocytosis is not regulated in a similar fashion. MH - Chemotaxis, Leukocyte/*DRUG EFFECTS ; Comparative Study ; Concanavalin A/ ANALOGS & DERIVATIVES ; Fluoresceins ; Fluorescent Dyes ; Human ; Kinetics ; N-Formylmethionine Leucyl-Phenylalanine/PHARMACODYNAMICS ; Neutrophils/CYTOLOGY/DRUG EFFECTS/*PHYSIOLOGY ; Pertussis Toxins/ *PHARMACODYNAMICS ; Phagocytosis/*DRUG EFFECTS ; Phorbols/ *PHARMACODYNAMICS ; Platelet Activating Factor/PHARMACODYNAMICS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - FEBS Lett 1986 May 5;200(1):91-6 25 UI - 86145353 AU - Yun K ; Sugihara H TI - Cell differentiation and cell cycle effects on human promyelocytic leukemia cells induced by 12-O-tetradecanoylphorbol-13-acetate. AB - As has been reported, doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) as small as 1 to 100 ng/ml induced human promyelocytic leukemia HL-60 cells to differentiate terminally into macrophage-like cells rather than toward cells of the granulocytic series. This differentiation was accompanied by the appearance of monocyte/macrophage markers and by the disappearance of myeloid markers from the view point of enzyme cytochemistry. Contrasted to untreated HL-60 cells, TPA-treated cells increased in cell size and showed increased phagocytotic activities against opsonized sheep blood red cells and activated yeast. Nitroblue tetrazolium-positive cells increased rapidly after TPA exposure. The alterations of the cell cycle traverse of HL-60 cells by TPA were analyzed by [3H]thymidine autoradiography, flow microfluorimetry, and mitotic cell counting. TPA sequentially caused (a) inhibition of cells to move from G1 to S near G1/S boundary in G1; (b) temporary inhibition in G2; (c) growth arrest of most cells in G1 within 2 to 3 days after TPA exposure. MH - Cell Cycle/*DRUG EFFECTS ; Cell Differentiation/*DRUG EFFECTS ; Cell Line ; Flow Cytometry ; Histocytochemistry ; Human ; Lactate Dehydrogenase/ METABOLISM ; Lactate Dehydrogenase Isoenzymes/METABOLISM ; Leukemia, Myeloblastic ; Mitosis/DRUG EFFECTS ; Nitroblue Tetrazolium/METABOLISM ; Oxidation-Reduction ; Phagocytosis/DRUG EFFECTS ; Phenotype ; Phorbols/ *PHARMACODYNAMICS ; Receptors, Fc/DRUG EFFECTS ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Lab Invest 1986 Mar;54(3):336-44 26 UI - 86131793 AU - Muir JG ; Murray AW TI - Mimicry of phorbol ester responses by diacylglycerols. Differential effects on phosphatidylcholine biosynthesis, cell-cell communication and epidermal growth factor binding. AB - The biosynthesis of phosphatidylcholine (PC) in HEL-37 cells was followed by measuring the incorporation of [32P]Pi into PC. Incorporation was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), but not by sn-1-oleoyl-2-acetylglycerol or sn-1,2-dihexanoylglycerol (diC6). DiC8 was rapidly metabolised by HEL-37 cells to the corresponding PC and phosphatidic acid derivatives. diC8, diC6 and oleoylacetylglycerol effectively displaced [3H]phorbol-12,13-dibutyrate bound to a soluble cell extract from HEL-37 cells, but only diC8 was able to displace the labelled phorbol ester from prelabelled cells. TPA, diC8, diC6 and oleoylacetylglycerol were all effective inhibitors of 125I-labelled epidermal growth factor binding to, and gap junctional communication between, HEL-37 cells. It is concluded that only cell-permeable diacylglycerols stimulate PC biosynthesis which may therefore require interaction with membranes other than the plasma membrane. MH - Animal ; Cell Communication/*DRUG EFFECTS ; Cell Line ; Diglycerides/ *PHARMACODYNAMICS ; Epidermal Growth Factor-Urogastrone/*METABOLISM ; Epidermis ; Glycerides/*PHARMACODYNAMICS ; Intercellular Junctions/DRUG EFFECTS ; Mice ; Phorbols/*PHARMACODYNAMICS ; Phosphatidylcholines/ *BIOSYNTHESIS ; Protein Kinase C/METABOLISM ; Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Biochim Biophys Acta 1986 Feb 21;885(2):176-84 27 UI - 86105950 AU - Kavanagh TJ ; Chang CC ; Trosko JE TI - Characterization of a human teratocarcinoma cell assay for inhibitors of metabolic cooperation. AB - Although a Chinese hamster V79 cell-based assay for inhibitors of metabolic cooperation is currently available, the development of a human cell-based assay is desirable in order to avoid inappropriate extrapolation from animal cells to human cells. Cells derived from a human teratocarcinoma cell line (designated PA-1), which has a stable pseudodiploid karyotype and excellent in vitro growth properties, were used in a metabolic cooperation assay. The assay was based on the metabolic isolation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient variants in the presence of HGPRT-proficient cells and 6-thioguanine. Chemicals which inhibit the transfer of the lethal metabolite of 6-thioguanine from HGPRT-proficient to HGPRT-deficient cells will allow for recovery of the 6-thioguanine-resistant (HGPRT-deficient) cells. Chemicals tested included 12-O-tetradecanoylphorbol-13-acetate and related analogues phorbol-12,13-didecanoate, mezerein, and 4-phorbol-12,13-didecanoate. Concurring with results previously obtained in V79 cells, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-didecanoate strongly inhibited metabolic cooperation, whereas mezerein was moderately inhibitory and 4 alpha-phorbol-12,13-didecanoate was inactive. These cells thus hold promise as a human cell-based assay for inhibitors of metabolic cooperation. MH - Biological Assay ; Cell Communication/*DRUG EFFECTS ; Human ; Hypoxanthine Phosphoribosyltransferase/METABOLISM ; Intercellular Junctions/*PHYSIOLOGY ; Karyotyping ; Phorbol Esters/*PHARMACODYNAMICS ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Teratoma/ *METABOLISM/PATHOLOGY ; Terpenes/PHARMACODYNAMICS ; Tetradecanoylphorbol Acetate/PHARMACODYNAMICS SO - Cancer Res 1986 Mar;46(3):1359-66 28 UI - 86079162 AU - Smith BM ; Gindhart TD ; Colburn NH TI - Extracellular calcium requirement for promotion of transformation in JB6 cells. AB - Extracellular calcium is required in the induction of neoplastic transformation of preneoplastic mouse JB6 epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Depleting extra-cellular calcium by chelation or by use of commercial calcium-depleted medium inhibited TPA-promoted transformation of promotion-sensitive JB6 cells with a half-maximal inhibition at 1.2 mM calcium. Inhibition was reversible by the addition of calcium. The calcium channel blockers lanthanum and nifedipine inhibited promotion of anchorage-independent transformation by TPA maximally at 10.0 microM and 1.0 nM, respectively, suggesting that calcium entry occurs partially via cell channels. None of the above treatments altered expression of transformation in anchorage-independent tumorigenic JB6 cell lines, indicating that the extracellular calcium requirement was at the level of induction, not expression of transformation. The calcium requirement was not merely a requirement for proliferation; calcium concentrations from 0.2 to 1.8 mM had no effect on JB6 cell monolayer growth. Extracellular calcium was required for 7 days for maximal colony induction. The calcium ionophore A23187 was not a promoter and moderately inhibited TPA-promoted transformation, indicating that increases in free cytosolic calcium concentrations are not sufficient for promotion of transformation and may even activate calcium-dependent antipromoting events. The results suggest that a cellular calcium pool supplied by extracellular calcium, but not distinguishable by ionophoretic increases in free cytosolic calcium, is essential in TPA-promoted neoplastic transformation. MH - A-23187/PHARMACODYNAMICS ; Animal ; Calcium/*PHYSIOLOGY ; Cell Line ; Cell Transformation, Neoplastic/*DRUG EFFECTS ; Extracellular Space/ PHYSIOLOGY ; EGTA/PHARMACODYNAMICS ; Lanthanum/PHARMACODYNAMICS ; Mice ; Nifedipine/PHARMACODYNAMICS ; Phorbols/*PHARMACODYNAMICS ; Support, U.S. Gov't, P.H.S. ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Cancer Res 1986 Feb;46(2):701-6 29 UI - 86053246 AU - Takenaga K ; Takahashi K TI - Effects of 12-O-tetradecanoylphorbol-13-acetate on adhesiveness and lung-colonizing ability of Lewis lung carcinoma cells. AB - The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the adherence of low-metastatic Lewis lung carcinoma cells (P-29) to the surface of plastic culture dishes and to monolayers of endothelial cells. This effect was transient, being apparent within 15 min and maximal within 1 h after treatment with TPA. Biologically active analogues of TPA and mezerein also enhanced attachment of P-29 cells, whereas inactive analogues of TPA did not. TPA-treated P-29 cells formed many more pulmonary nodules than did untreated P-29 cells when injected i.v. into C57BL/6 mice. The kinetics of enhancement of attachment of P-29 cells after TPA treatment coincided well with that of enhancement of their lung-colonizing ability. Addition of TPA to P-29 cells stimulated phosphorylation of a cellular protein with a molecular weight of 54,000. The possibility that this phosphorylation was related to activation of Ca2+ phospholipid-dependent protein kinase was suggested by the fact that phospholipid breakdown induced by exogenous treatment of the cells with Clostridium perfringens phospholipase C and 1-oleoyl-2-acetylglycerol also enhanced Mr 54,000 cellular protein phosphorylation. However, neither phospholipase C nor 1-oleoyl-2-acetylglycerol enhanced attachment of P-29 cells or their lung-colonizing ability. MH - Animal ; Cell Adhesion/*DRUG EFFECTS ; Diglycerides/PHARMACODYNAMICS ; Lung Neoplasms/*PATHOLOGY ; Male ; Mice ; Mitogens/PHARMACODYNAMICS ; *Neoplasm Metastasis ; Phorbol Esters/PHARMACODYNAMICS ; Phorbols/ *PHARMACODYNAMICS ; Phospholipase C/PHARMACODYNAMICS ; Protein Kinases/ METABOLISM ; Structure-Activity Relationship ; Terpenes/PHARMACODYNAMICS ; Tetradecanoylphorbol Acetate/*PHARMACODYNAMICS SO - Cancer Res 1986 Jan;46(1):375-80