Phorbol 12-myristate 13-acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium

The addition of low concentrations of phorbol 12-myristate 13-acetate to rabbit neutrophils induces cell aggregation, degranulation, increased oxygen consumption and an increase in the amount of actin associated with the cytoskeleton without a rise in the level of intracellular free calcium as measured using the fluorescent probe quin-2. The ability of phorbol 12-myristate 13-acetate to initiate neutrophil responses similar to those produced by the chemotactic factor without causing a rise in the level of intracellular free calcium suggests two possibilities; that there is a second messenger in addition to calcium or that it activates the cells at a point distal to calcium mobilization. The possible role of diacylglycerol in neutrophil activation is discussed.     

Phorbol 12-myristate 13-acetate inhibits apoptosis in erythroleukemia K562 cells induced by some nucleosides

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Specific stimulation of histone H2B and H4 phosphorylation in mouse lymphocytes by 12-O-tetradecanoylphorbol 13-acetate

This report demonstrates that the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate rapidly stimulates the phosphorylation of histones H2B and H4 in a cell cycle-independent manner. This effect was observed in primary cultures of BALB/c mouse splenocytes, a population of noncycling, G0 cells which are not stimulated to divide by 12-O-tetradecanoylphorbol 13-acetate treatment alone. The biological nature of this cell system allowed the analysis of histone phosphorylation in the absence of a background of cell cycle-dependent changes and in response to a nonmitogenic agent. The phosphorylation of H2B was determined with high resolution through the use of two-dimensional gel electrophoresis. In contrast to 12-O-tetradecanoylphorbol 13-acetate, the mitogen from pokeweed did not induce stimulation of H2B and H4 phosphorylation, but did, however, elicit increases in the phosphorylation of histones H1, H2A, and H3, in parallel with changes in rate of DNA synthesis.     

Ca2+ channel ligand sensitive responses to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in vascular smooth muscle

The action of a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), on isolated rat aortic and tail artery strips has been characterized. TPA (10(-9)-10(-7) M) produced a graded contraction developing maximum tension over 30-40 min. The contraction was irreversible and was not relaxed by prolonged washing with physiologic saline. Relaxation occurred upon washing with Ca2+-free saline but readdition of Ca2+ restored response. TPA was without significant effect in rat tail arteries in physiologic saline but produced responses in saline containing elevated K+ (15 mM). The protein kinase C inhibitor, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)n-propyl]-4-phenylpiperidine dihydrochloride) (5 X 10(-5) M), blocked the response to TPA but was without effect on responses to Bay K 8644 (2,6-dimethyl-3-carbomethoxy-5-nitro-4-(2-trifluoromethylphenyl) 1,4-dihydropyridine), KCl, phenylephrine, and B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). The calcium channel antagonist nifedipine and its analogue, 2,6-dimethyl-3,5-dicarbomethoxy-4-(3-cyanophenyl)-1,4-dihydr opyridine, inhibited TPA responses with IC50 values of 9.28 X 10(-9) and 1.96 X 10(-7) M, respectively. Responses to Bay K 8644 in rat aorta were maximum in the presence of elevated KCl (10 mM), but TPA at concentrations of 10(-9) and 3 X 10(-9) M potentiated responses to Bay K 8644 in physiologic saline to levels approximating those in elevated K+ saline. TPA similarly potentiated responses to Ca2+ in Ca2+-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL)

The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c(PKC), and to the Ca2+ ionophores A23817 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A23817. TPA alone (1-20 ng/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 ng/ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fc gamma receptors (CD16) on LGL and the induction of the expression of IL-2 (CD25) and transferrin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca2+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tac antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum.     

Stimulation and inhibition of Leydig cell steroidogenesis by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate: similarity to the effects of gonadotropin-releasing hormone

To explore the mechanism of gonadotropin-releasing hormone (GnRH) action on Leydig cell steroidogenesis the effects of a GnRH analog (GnRHa) were compared to those of 12-O-tetradecanoylphorbol 13-acetate (TPA). Both compounds acutely stimulated androgen production 2-4 fold with EC50's of 9 nM (TPA) and 0.2 nM (GnRHa). The effects of TPA and GnRHa were not additive and neither compound acutely altered the luteinizing hormone (LH) concentration-response relationship. After 24 h of exposure to TPA or GnRHa the ability of LH to stimulate androgen production was impaired. The parallel effects of TPA and GnRHa on Leydig cell steroidogenesis suggest that they are acting via similar mechanisms; presumably the activation protein kinase C.     

Increased sensitivity to cis-diamminedichloroplatinum(II) in human ovarian carcinoma cells in response to treatment with 12-O-tetradecanoylphorbol 13-acetate

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) enhanced sensitivity to cis-diamminedichloroplatinum(II) (DPP) in human ovarina carcinoma 2008 cells by a factor of 2.53 +/- 0.74 fold (S.D.). Sensitization was maximum 3 h after a 1-h exposure to TPA and had disappeared completely by 7 h after treatment. An equivalent degree of sensitization was produced in a 2008 variant selected for 10-fold resistance to DDP. TPA neither increased nor decreased cellular accumulation of DDP. Phorbol, a TPA analog which does not activate protein kinase C, did not cause sensitization. This synergistic interaction between TPA and DDP was completely inhibited by pretreatment with staurosporine, a protein kinase C inhibitor. Cellular cAMP was not altered by TPA stimulation. Furthermore, cycloheximide, a potent protein synthesis inhibitor, did not block the TPA-induced enhancement of drug sensitivity. These results strongly suggest that DDP sensitivity can be modulated by protein kinase C and regulated by phosphorylation of a protein kinase C substrate in both intrinsically sensitive and DDP-resistant cells.     

Stimulation of insulin release by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the clonal cell line RINm5F despite a lowering of the free cytoplasmic Ca2+ concentration

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the handling of Ca2+ and insulin release were investigated in the clonal insulin-producing cell line RINm5F. The presence of the phorbol ester lowered the free cytoplasmic Ca2+ and suppressed the increase obtained by depolarization with high concentrations of K+. Despite the lowering in cytoplasmic Ca2+ by TPA, there was a concomitant stimulation of insulin release indicating that one feature of protein kinase C activation is to make the secretory system more sensitive to Ca2+. Furthermore, there was no interaction of TPA with the mechanisms responsible for inositol 1,4,5-tris(phosphate) induced Ca2+ release or Ca2+ uptake in permeabilized cells. Although TPA slightly depolarized the RINm5F cells there was no interference with K+-induced depolarization. It is suggested that an additional effect of protein kinase C activation in these cells, is to stimulate the extrusion of Ca2+ over the plasma membrane.     

Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain.

Regulation of death receptor-mediated apoptosis is incompletely understood. Previous studies have demonstrated that phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, inhibits Fas (CD95)-mediated apoptosis in Jurkat (type II) cells but not SKW6.4 (type I) cells. In this study, we demonstrated that PMA also protects Jurkat cells from apoptosis induced by tumor necrosis factor-alpha and the tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). Interestingly, PMA failed to protect Jurkat cells from apoptosis induced by other agents, including etoposide, camptothecin, and gamma-irradiation. Analysis of the initial events induced by agonistic anti-Fas antibodies revealed that PMA inhibited Fas binding to Fas-associated polypeptide with death domain (FADD) in Jurkat cells but not in SKW6.4 cells. Although the protein kinase inhibitor bisindoylmaleimide VIII increased apoptosis induced by agonistic anti-Fas antibody, tumor necrosis factor-alpha, and TRAIL, these effects were not observed with the protein kinase C inhibitor H7 and were not associated with increased FADD recruitment to Fas. These results indicate that PMA inhibits death signaling induced by a number of discrete receptors and suggest that the effects are mediated at the level of receptor-mediated adaptor molecule recruitment.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate inhibits mas/angiotensin receptor-stimulated inositol phosphate production and intracellular Ca2+ elevation in the 401L-C3 neuronal cell line

Stimulation of mas-oncogene transfected 401L-C3 cells by angiotensins leads to the production of inositol phosphates. This response shows dose dependence, and has an apparent rank order of potency angiotensin III greater than or equal to angiotensin II much greater than angiotensin I. Preincubation with 12-O-tetradecanoylphorbol 13-acetate, for 5 min, significantly diminishes both inositol phosphate and intracellular [Ca2+] responses to angiotensins, without affecting those stimulated by the endogenous bradykinin receptor. Incubation of 401L-C3 cells with either phorbol ester or angiotensins leads to elevation of intracellular pH, implying that mas/angiotensin receptor stimulation itself leads to protein kinase C activation. These results suggest the operation of a negative feedback loop specific for the mas/angiotensin receptor signalling pathway, and which may be essential in defining the final biological output response to this receptor stimulation.     

Phorbol 12-myristate 13-acetate prevents apoptosis in erythroleukemia K562 cells induced by some nucleosides

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 18–25.Original Russian Text Copyright © 2005 by Volkova, Malysheva, Nemova.     

Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol 13-acetate in mouse epidermis

A murine mRNA (provisionally called 2ar) is described whose abundance is greatly increased by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate both in JB6 epidermal cells in vitro and in epidermis in vivo. We have previously shown induction of 2ar in epidermal or fibroblast cell lines by tumor promoters, growth factors, and transformation with H-ras. The 2ar mRNA appears to be derived from a single copy gene. It encodes the mouse homolog of rat osteopontin, a 41.5-kDa glycosylated bone phosphoprotein that binds to fibroblasts and osteosarcoma cells and to hydroxylapatite (bone matrix). The rat and mouse sequences are 84% identical at the amino acid level and 87% identical at the nucleotide level. Many of the primary structural features are conserved, including a run of 9-10 aspartic residues and a Gly-Arg-Gly-Asp-Ser cell adhesion sequence. Antiserum raised against portions of the predicted polypeptide immunoprecipitated proteins of apparent Mr 55,000-70,000 both from reticulocyte lysates containing the translation products of hybrid-selected mRNA and from cell culture medium containing metabolically labeled proteins secreted by JB6 cells. The results presented here demonstrate that osteopontin is identical to a transformation-associated phosphoprotein whose level of expression by cultured cells and abundance in human sera has been correlated with tumorigenicity. These results suggest a role for osteopontin in carcinogenesis. The murine version of osteopontin has been given the formal name "secreted phosphoprotein 1" and the designation spp.     

Synergistic enhancement of histamine release from rat peritoneal mast cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate is not reflected by corresponding changes in phospholipid turnover

In an attempt to elucidate further the relationship between changes in phospholipid metabolism in, and histamine secretion from, purified rat peritoneal mast cells, the effects of the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) on these responses in stimulated and unstimulated cells was investigated. TPA caused a dose-dependent increase in the incorporation of 32PO4(3-) into the mast cell phospholipids; phosphatidic acid (PA) and phosphatidylcholine (PC), but not phosphatidylinositol (PI). TPA synergistically enhanced histamine release from cells stimulated by anti-immunoglobulin E (IgE) and the calcium ionophore A23187, reducing its ED50 from 150 nM to 40 nM, but did not alter histamine release from cells stimulated by compound 48/80. The effect of TPA on the changes in 32PO4(3-) incorporation into phospholipids associated with the above secretagogues did not, however, correlate well with the observed effects on histamine secretion induced by the same secretagogues. These observations are discussed in relation to the known effects of phorbol esters upon both secretory processes and phospholipid metabolism in other tissues.     

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.     

The inhibitory GTP-binding protein (Gi) occurs in rat Leydig cells and is differentially modified by lutropin and 12-O-tetradecanoylphorbol 13-acetate.

Luteinizing-hormone (LH)-stimulated cyclic AMP production in rat testis Leydig cells was desensitized by both LH and 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA, but not LH, enhanced the subsequent response to cholera toxin. Treatment of the cells with pertussis toxin potentiated cyclic AMP production in both control and LH-desensitized cells, but did not potentiate further the responses obtained by TPA pretreatment. The results implicate the presence of an inhibitory GTP-binding protein (Gi), which may be inhibited by TPA. The presence of a Gi-like protein within the plasma membrane of Leydig cells was demonstrated by pertussis-toxin-catalysed [32P]ADP-ribosylation of a Mr-40000-41000 protein.     

Stimulation of hepatic glycogenolysis by 12-O-tetradecanoylphorbol-13-acetate (TPA) via a calcium requiring process

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated glycogenolysis in perfused rat liver which was perfused with Krebs-Ringer-bicarbonate buffer containing 1 mM CaCl2 but no substrate. Verapamil (100 microM), diltiazem (100 microM) and trifluoperazin (100 microM), all inhibited the effect of TPA in the presence of CaCl2. Omission of CaCl2 from the perfusate or the addition of EGTA markedly attenuated the effect of TPA. TPA decreased net release of 45Ca from 45Ca-preloaded liver. The effect of maximal concentration of TPA (20 ng/ml) was not additive to that of 0.6 microM A23187. These data suggest that TPA increases calcium influx into hepatocytes and stimulates glycogenolysis through a calcium-calmodulin dependent mechanism.     

Tumour promotor 12-O-tetradecanoylphorbol 13-acetate inhibits angiotensin II-induced inositol phosphate production and cytosolic Ca2+ rise in rat renal mesangial cells

Preincubation of rat renal mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) strongly inhibited the increases of inositol phosphates and of free cytosolic Ca2+ induced by angiotensin II (10(-7) M). TPA had no significant effect on the basal values of inositol phosphates and of free cytosolic Ca2+. Inhibition appeared already after 1 min and was maximal after 5 min. These effects occur without significant changes on angiotensin II binding in intact cells. The concentration of TPA needed (10(-9)-10(-7) M) was in the range believed to cause specifically an activation of protein kinase C. Furthermore the biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. From the entirety of these results it is likely that protein kinase C inhibits angiotensin II activation of phospholipase C at a stage distal to receptor occupancy.     

Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes

Activation of Na⁺-H⁺ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). We determined whether the change in [Ca²⁺]_i was secondary to the uptake of Na⁺, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na⁺] by treating the cells with ouabain or gramicidin failed to affect [Ca²⁺]_i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH₃, significantly elevated [Ca²⁺]_i. These results suggest an important role of cytoplasmic pH in the control of [Ca²⁺]_i in lymphocytes.