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PubMed1982

Biological responsiveness to the phorbol esters and specific binding of [3H]phorbol 12,13-dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system.

Because of its suitability for genetic studies, the nematode Caenorhabditis elegans was examined for its responsiveness to the phorbol esters. Phorbol 12-myristate 13-acetate had three effects. It inhibited the increase in animal size during growth; it decreased the yield of progeny; and it caused uncoordinated movement of the adult. The effects on nematode size, progeny yield, and movement were quantitated. Concentrations of phorbol 12-myristate 13-acetate yielding half-maximal responses were 440, 460, and 170 nM, respectively. As was expected from the biological responsiveness of the nematodes, specific, saturable binding of phorbol ester to nematode extracts was found. [3H]phorbol 12,13-dibutyrate bound with a dissociation constant of 26.8 +/- 3.9 nM. At saturation, 5.7 +/- 1.4 pmole/mg protein was bound.

Animals↗

PubMed1981

Down regulation of specific binding of [20-3H]phorbol 12,13-dibutyrate and phorbol ester-induced differentiation of human promyelocytic leukemia cells.

Binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDB) to intact human promyelocytic leukemia cells susceptible (HL-60) or resistant (R-35) to phorbol ester-induced differentiation was characterized. Specific binding of [3H]PDB to both HL-60 and R-35 cells at 37 degrees C reached a maximum within 15-20 min. Maximal specific [3H]PDB binding to HL-60 cells was followed by a decline (down regulation) of radioactivity. This down regulation was temperature dependent, because no loss of radiolabel occurred by 1 hr at 4 degrees C. The down regulation of bound [3H]PDB seen in HL-60 cells at 37 degrees C was not observed with R-35 cells. Prior exposure of the HL-60 cells but not of R-35 cells to 1 microM phorbol 12-myristate 13-acetate for 90 min at 37 degrees C caused a marked reduction in the specific binding of [3H]PDB. When [3H]PDB binding was carried out at 4 degrees C, [3H]PDB bound to both cell types in a rapid, specific, and reversible manner. At equilibrium, HL-60 and R-35 cells were found to contain almost the same number of binding sites, which had dissociation constants of about 50 nM, indicating that the failure of R-35 cells to undergo PDB-induced differentiation was not associated with any change in the affinity or in the number of [3H]PDB binding sites. These results indicate that the down regulation of specific [3H]PDB binding may be a crucial early event in the control of phorbol ester-induced terminal differentiation in HL-60 cells. Furthermore, we suggest that such down regulation may be involved in other cellular and biochemical effects of phorbol diester tumor promoters.

Binding Sites↗

PubMed1974

The tumor-promoter phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate), a potent aggregating agent for blood platelets.

The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate, a potent tumor-promoting agent, caused irreversible platelet aggregation when more than 0.02 microM was stirred with human citrated or heparinized platelet-rich plasma (PRP). With washed platelets, 1 nM was effective. The alcohol phorbol, which has little tumor-promoting activity, failed to cause platelet aggregation. With all but low concentrations of phorbol ester, aggregation was succeeded by a rapid phase. The latter was prevented or reduced by enzymes which destroy ADP and by aspirin, was associated with a change in platelet shape, and was presumably due to released ADP. At higher concentrations, only a rapid phase was seen, and these inhibitors were not effective. Low concentrations did not aggregate platelets in PRP containing sufficient EDTA or EGTA to chelate ionized calcium or in PRP from thrombasthenic patients; higher concentrations caused slight aggregation. Both the primary, non-ADP-dependent aggregation and the rapid ADP-dependent aggregation were markedly inhibited by substances which increase cyclic AMP, metabolic inhibitors, and the sulfhydryl inhibitor N-ethylmaleimide. Phorbol ester reduced platelet cyclic AMP only when it had been previously elevated by prostaglandin E(1). 1 microM did not release beta-glucuronidase, lactic dehydrogenase, or inflammatory material from platelets in 4-5 min despite marked aggregation, but liberated all three in 30 min. The possibility is discussed that low phorbol ester concentrations cause primary aggregation by a direct action on platelet actomyosin.

Adenosine Diphosphate↗

PubMed1990

Effect of resiniferatoxin pretreatment on the inflammatory response to phorbol-12-myristate-13-acetate in mouse strains with different susceptibilities to phorbol ester tumor promotion.

All tumor-promoting phorbol esters induce inflammation in mouse skin. The correlation between promoting and inflammatory activities is only partial, however, indicating that only some events in inflammation may be closely coupled to the process of tumor promotion. Resiniferatoxin (RTX), an extremely inflammatory phorbol-related diterpene, acts as an ultrapotent analog of capsaicin to stimulate and then to block the neurogenic inflammatory pathway. In CD-1 mice, we have used pretreatment with RTX to show that the erythema and edema responses to phorbol and 12-deoxyphorbol esters in significant part involve this neurogenic inflammatory pathway. We report here that mouse strains with differing sensitivities to phorbol-ester-induced promotion displayed marked differences in the effect of pretreating with RTX on the edema response following phorbol-12-myristate-13-acetate (PMA) application. In the highly promotion-sensitive SENCAR mouse, RTX pretreatment had little inhibitory effect; the edema response to PMA was similar with or without RTX pretreatment 6 h before PMA application. On the other hand, in C57BL/6J mice, which are resistant to promotion by phorbol esters under the usual protocols, the edema response to PMA was totally eliminated by RTX pretreatment during the first 8 h after PMA administration. DBA/2J mice, which are similar to CD-1 mice in their susceptibility to PMA promotion, responded similarly to CD-1: the edema response was blocked partially by RTX pretreatment during the early phase (up to 8 h) of inflammation. Our results suggest that the RTX-resistant component of PMA-induced edema may correlate better with the sensitivity to promoting action than does the overall inflammatory response.

Animals↗

PubMed1989

In vitro effects of phorbol and phorbol ester on the proliferation and enzyme activities of bone marrow stromal cells in rats.

Stromal cells obtained from bone marrow of Wistar rats were cultured in liquid medium. The cultures were divided into control and experimental groups. After 24 h of culture, the medium was removed and replaced by a new one containing 10(-6) and 10(-9) M 4 beta-phorbol or phorbol 12,13-diacetate ester in the experimental cultures, or phorbol solvent alone in control cultures. After 7 days, the cultures were stained with Wright's stain or were subjected to cytochemical procedures for demonstration of nonspecific esterase, alkaline and acid phosphatases and diaphorase activities. The obtained results showed that phorbols stimulated proliferation: fibroblast colonies, fibroblasts, macrophages, immature reticular cells. The number of adipocytes was markedly lower in the experimental cultures. Phorbols stimulated activity of the investigated enzymes.

Animals↗

PubMed1984

Effect of phorbol 12-myristate 13-acetate and its analogue 4 alpha-phorbol 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils.

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.

Animals↗

PubMed1987

A phorbol ester, phorbol 12-myristate 13-acetate, and a calcium ionophore, A23187, can mimic the luteolytic effect of prostaglandin F2 alpha in isolated rat luteal cells.

To explore the possible role of protein kinase C and calcium in the luteolytic process, we treated luteal cells with a protein kinase C activator, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and with the calcium ionophore, A23187. Lower concentrations of PMA could clearly mimic the inhibitory, luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) on LH-induced cAMP and progesterone production. A nontumor promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no inhibitory effect, indicating a specific PMA effect. The calcium ionophore, A23187, also gave a marked inhibition of LH-induced cAMP and progesterone production. Hormone-stimulated adenylate cyclase activity was markedly impaired after preincubation of the cells with PGF2 alpha, PMA, or A23187, but no effect was seen when the substances were added to isolated membranes. In addition, the stimulation of progesterone production in the luteal cells with the cAMP analogs, 8-bromo-cAMP and (Bu)2cAMP, was almost totally abolished when PMA or A23187 was present. We conclude that PMA and A23187 in many ways mimic the effect of PGF2 alpha in luteal cells. The inhibition of steroidogenesis is partly dependent on depressed activity of the hormone-sensitive adenylate cyclase, but also obtained by inhibiting steps distal to cAMP formation. Both points of action seem to be calcium and/or protein kinase C dependent. In contrast, higher concentrations of PMA markedly stimulated steroidogenesis without affecting the cAMP level, a stimulation not seen after incubation with 4 alpha-phorbol 12,13-didecanoate, suggesting again a specific PMA effect. The stimulation of steroidogenesis by higher concentrations of PMA seems to be specific, but the interpretation of this finding is unclear at present. In conclusion, PMA and A23187 mimic some of the luteolytic properties of PGF2 alpha, not only inhibiting the luteal cAMP system, but also by inducing lesions in the steroidogenic steps beyond the cAMP system.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Interactive effects of pertussis toxin and the phorbol ester tumour promotor, phorbol dibutyrate, on T-lymphocyte mitogenesis and the expression of phenotypic determinants.

The B oligomer of pertussis toxin serves as a weak mitogen in the T lymphocyte, an effect which is associated with an early rise in cytosolic free calcium concentrations, as monitored by Fura-2 fluorescence. Upon co-administration of phorbol dibutyrate, a phorbol ester tumour promotor which activates protein kinase C, pertussis toxin-induced proliferation was synergistically enhanced, as measured by the increased uptake of [3H]thymidine, into cellular DNA. Although phorbol ester co-administration has often been associated with an inhibition of Ca2+-mobilizing pathways, phorbol dibutyrate pretreatment had no inhibitory effect on the pertussis toxin-induced calcium flux and may actually have enhanced this response slightly. Flow cytometric analysis of cell populations expanded by the combined regimen did not provide evidence for the preferential expansion of cells bearing either CD4 or CD8, the T-cell determinants representative of the helper-inducer and cytotoxic-suppressor subsets, respectively. Pertussis toxin and phorbol dibutyrate appear, therefore, to elicit polyclonal stimulation, rather than the selective activation of a given lymphocyte subset. Expression of the transferrin receptor, a marker for nutrient uptake, and CD25, the Tac component of the interleukin-2 (IL-2) receptor, was, however, synergistically enhanced in cells activated by the co-treatment procedure.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Inhibition of the chemotactic peptide-induced elevation of intracellular calcium in differentiated human leukemic (HL-60) cells by the phorbol ester phorbol 12-myristate 13-acetate.

The chemotactic peptide formylmethionylleucylphenylalanine rapidly elevated the intracellular calcium concentration, in a concentration-dependent manner, of human leukemic (HL-60) cells which had been differentiated to polymorphonuclear leukocyte-like cells by pretreatment with dimethyl sulfoxide (1.3%). Preincubation of the cells with phorbol 12-myristate 13-acetate, a protein kinase C-activating phorbol ester, inhibited the formylmethionylleucylphenylalanine-induced rise in intracellular calcium in a time- and concentration-dependent manner. 4 alpha-Phorbol 12,13-didecanoate, which does not activate protein kinase C, was inactive in this capacity. The use of calcium-free medium suggested that the elevation of intracellular calcium by formylmethionylleucylphenylalanine consisted of rapid intracellular release followed by calcium influx. Phorbol 12-myristate 13-acetate (10(-7) M) inhibited both components of the elevation of intracellular calcium, whereas 10(-8) M phorbol 12-myristate 13-acetate inhibited only the calcium influx. The influx of calcium was not prevented by verapamil, which blocks the voltage-dependent calcium channels. These data suggest that, in differentiated HL-60 cells, 12-O-tetradecanoylphorbol-13-acetate rapidly inhibits both the intracellular release of calcium and calcium influx through non-voltage-dependent calcium channels in response to formylmethionylleucylphenylalanine.

Calcium↗

PubMed1986

Calcium ionophore A23187 primes human B-cells for activation by phorbol dibutyrate by converting receptors for phorbol dibutyrate from a low to high affinity state.

The calcium ionophore A23187 synergised with phorbol dibutyrate-induced activation of human chronic lymphatic leukaemia B-cells, as assessed by modulation of the membrane receptor for mouse erythrocytes. Thus A23187 (1 microM), which alone had no effect on expression of the receptor for mouse erythrocytes, reduced the EC50 and shortened the lag period for modulation of this receptor by phorbol dibutyrate. This action of A23187 was shown to be due to enhanced binding of [3H]phorbol dibutyrate to its receptor (phospholipid/Ca++ dependent protein kinase C) whose affinity was altered from a predominantly low affinity state (Kd 83 nM) to high affinity (Kd 9 nM). A23187 had no effect on the total number of phorbol dibutyrate receptors. EDTA abolished these actions of A23187.

B-Lymphocytes↗

PubMed1983

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

As part of our effort to characterize receptors for the phorbol ester tumor promoters, a phorbol ester photoaffinity probe, [20-3H]phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz), was synthesized. In the dark, PaBzBz bound reversibly to brain particulate fractions with a dissociation constant (Kd) of 0.81 +/- 0.09 x 10(-9) M. Specific binding of PaBzBz, at a concentration equal to its Kd, represented 85% of the total bound. At saturation, 24 +/- 5 pmol of PaBzBz were bound per mg of brain protein, a level similar to that observed with [20-3H]phorbol 12,13-dibutyrate. Under the conditions used (concentrations greater than the Kd for PaBzBz), irradiation caused 45% of the PaBzBz binding to become irreversible. Most of the binding (approximately equal to 60%), including most of the specific irreversible binding, was to phospholipid rather than to protein. Based on susceptibility to enzymatic digestion and on chromatographic mobility, the specifically labeled phospholipids were identified as phosphatidylserine, phosphatidylethanolamine, and phosphatidylethanolamine plasmalogen. Although the PaBzBz specifically labeled lipid, labeling was blocked by pretreatment of membranes at 100 degrees C for 5 min or by papain digestion. Therefore, it seems likely that the identified lipids are specifically associated with a protein receptor and are preferentially labeled either because of the location or reactivity of the nitrene generated on the photoaffinity probe.

Affinity Labels↗

PubMed2003

Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters.

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.

Amino Acid Sequence↗

PubMed1985

Effects of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation between Chinese hamster V79 lung fibroblasts.

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT-) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol dibutyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generally support the hypothesis that tumor promoters inhibit metabolic cooperation and illustrate the importance of considering metabolites when testing this hypothesis. The weak capacity of five metabolites of phenol to inhibit metabolic cooperation correlates with the weakness of phenol as a tumor promoter. Interpretation of these results is complicated because two metabolic cooperation-inhibiting metabolites (catechol and quinol) are nonpromoting when tested individually in the same assay where phenol shows promoting activity. Such metabolites may be incomplete (stage) promoters, and exposure to two or more may be required for a promoting effect. The significance of enhanced metabolic cooperation requires further investigation, particularly in relation to antipromoting effects.

Animals↗

PubMed1990

Contrasting effects of phorbol dibutyrate and phorbol myristate acetate in rabbit aorta.

In rat hepatocytes, active phorbol esters inhibited the alpha 1-adrenergic stimulation of phosphatidylinositol labeling with the expected potency order: phorbol myristate acetate (PMA) greater than phorbol dibutyrate (PDB). In contrast, in rabbit aorta the alpha 1-adrenergic action was inhibited dose-dependently by PDB but not by PMA. Similarly PDB (but not PMA) induced a strong contraction in rabbit aorta. The phorbol ester-induced contraction developed slowly, was dose-dependent and independent of extracellular calcium. These effects of PDB in rabbit aorta were neither inhibited by the protein kinase inhibitor H-7 nor mimicked by the synthetic diacylglycerol, OAG. Our results raise some doubts on the mechanism(s) through which the actions of PDB take place in rabbit aorta.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMedDate not supplied

A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils.

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2-) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2(+)-containing and Ca2(+)-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2- production in a dose-dependent manner. In cytochalasin B-treated cells and in the presence of Ca2+, priming with PDBU or PMA did not alter the enhancing effect of cytochalasin B on fMet-Leu-Phe-induced O2- production. In Ca2(+)-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimental conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.

Cells, Cultured↗

PubMed1987

The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, enhances the evoked quanta release of acetylcholine at the frog neuromuscular junction.

The possible role of protein kinase C in the regulation of quantal transmitter release was studied at the frog neuromuscular junction by using the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a compound known to mimic the effects of the physiological activator of the enzyme, endogenous diacylglycerol. The main effect of the phorbol ester was to increase the quantal content, m, of the endplate potential. The initial values of m were adjusted over a wide range by changing the Ca2+ concentration of the extracellular medium, and the TPA-induced fractional increase in m was significantly greater at junctions with a lower initial quantal content. On the other hand, the absolute increases in m induced by the phorbol ester were positively correlated with the square root of the initial quantal content. The possible physiological significance of this correlation is discussed in view of the well known relationship between extracellular Ca2+ concentration and the quantal content of the end plate potential.

Acetylcholine↗

PubMed1989

Absence of phorbol ester-induced down-regulation of myc protein in the phorbol ester-tolerant mutant of HL-60 promyelocytes.

The human promyelocytic leukemia cell line HL-60 has an amplified number of copies of the protooncogene c-myc. It is induced to differentiate by exposure to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have developed a mutant phorbol ester-tolerant (PET) line of HL-60 which undergoes a transient growth arrest but does not differentiate when exposed to TPA (Macfarlane et al., Br. J. Haematol., 68: 291-302, 1988). The defect is not due to a general failure of TPA-induced phosphorylation. In this paper, we show that exposing phorbol ester-sensitive (S) HL-60 cells to TPA caused the disappearance of the c-myc protein antigen (detected on Western blots) in 4 h, whereas TPA had no effect on the c-myc protein content of PET cells. Dimethyl sulfoxide caused the rapid disappearance of the myc antigen in both cells. PET cells had slightly more copies of the c-myc gene detected on Southern blots than S cells. c-myc mRNA was equally unstable in both cells, as determined by Northern blots following actinomycin D. TPA induced the down-regulation of c-myc mRNA in S cells to a greater extent than in PET cells. Dimethyl sulfoxide caused a rapid down-regulation of c-myc mRNA in both cell lines. This shows that PET cells have a defect in the mechanism by which protein kinase C regulates c-myc transcription. Our results provide further evidence that reduction in c-myc expression is necessary for differentiation to occur in HL-60 cells.

Blotting, Southern↗

PubMed1986

Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C.

One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.

Animals↗