The Phorbol Ester 12-0-tetradecanoylphorbol-13-acetate Research Articles

Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin–proteasome proteolytic pathway

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose–response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 μM), suggesting that it was mediated through the ubiquitin–proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin–proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome α-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E2 14k, also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin–proteasome pathway. TPA also induced degradation of the inhibitory protein, I-κBα, and increased nuclear accumulation of nuclear factor-κB (NF-κB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-κB activation by resveratrol (30 μM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-κB.

The Novel Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) Potently Enhances Apoptosis Induced by Tumor Necrosis Factor in Human Leukemia Cells

Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-kappaB (NF-kappaB) pathway that leads to up-regulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-kappaB-mediated gene expression at a step after translocation of activated NF-kappaB to the nucleus. This effect appears specific for the NF-kappaB pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome c release, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochrome c release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-x(L), suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-kappaB-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia.

Role of ubiquitin carboxyl terminal hydrolase in the differentiation of human acute lymphoblastic leukemia cell line, Reh

We have previously demonstrated that the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), induces differentiation of the acute lymphoblastic leukemia cell line, Reh, to a mature non-dividing state. Associated with this differentiation is the expression of ubiquitin carboxyl terminal hydrolase (UCH-L1). To investigate the role of UCH-L1 in TPA-induced Reh differentiation and apoptosis, molecular and chemical inhibition was used. Molecularly, a sequence-specific antisense oligodeoxynucleotide (AODN) directed against UCH-L1 transcript was used to inhibit the expression of the gene. In addition, its complementary sense oligodeoxynucleotide (SODN) was used to indicate the specificity of AODN action. Chemically, sodium borohydride (NaBH 4), an inhibitor of UCH-L, was used to block the transcript product. TPA-induced changes in Reh cell growth and morphology, UCH-L1 protein expression, apoptosis contour, surface phenotype, and enzymatic profile were assessed in the presence or absence of NaBH 4, AODN or SODN. As previously reported, TPA induced Reh cells to differentiate into monocytoid B lymphocytes and stimulated the apoptotic pathway. However, adding NaBH 4 or AODN inhibited the TPA effect on all parameters measured except apoptosis. The sequence in which NaBH 4 or AODN were added in relation to TPA did not affect any of the response variables measured. The use of SODN did not influence any of the parameters measured, indicating the specificity of the action. Thus, we conclude that UCH-L1 is involved in the differentiation process of the lymphoblastic leukemia cell line, Reh. Our data suggest that TPA-induced apoptosis of Reh cells has a separate pathway from that of differentiation or that UCH-L1 expression is independent of the apoptotic pathway.

Post-Transcriptional Suppression of Human Ornithine Decarboxylase Gene Expression by Phorbol Esters in Human Keratinocytes

The induction of ornithine decarboxylase levels by the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in mouse skin has been shown to be integral to tumor promotion by TPA, and changes in ornithine decarboxylase activity indicate the proliferative state of many different cell types. However, in cultured human epidermal cells, TPA has been reported to be antiproliferative. Therefore, to elucidate pathways that TPA activates in cultured human skin cells, we have examined the levels at which TPA regulates ornithine decarboxylase gene expression in two immortalized human epidermal keratinocyte cell lines, and in normal neonatal keratinocytes. We have found that in cultured human keratinocytes, TPA cases a marked decrease in ornithine decarboxylase enzyme activity (50-90%), with no detectable effect on ornithine decarboxylase mRNA levels. TPA decreased steady-state levels of ornithine decarboxylase immunoreactive protein (approximately 50-67%), accounting for the 50-90% suppression of ornithine decarboxylase activity levels, as well as decreasing new synthesis of ornithine decarboxylase protein (48-50%). However, measurement of ornithine decarboxylase protein half-life showed no significant effect of TPA. Also, prolonged treatment of keratinocytes with phorbol esters abolished the suppression of ornithine decarboxylase activity by TPA. Our data, therefore, suggest that phorbol esters suppress ornithine decarboxylase gene expression predominantly by decreasing ornithine decarboxylase mRNA translatability.

1,25-Dihydroxyvitamin D3 translocates protein kinase C beta to nucleus and enhances plasma membrane association of protein kinase C alpha in renal epithelial cells.

1,25-Dihydroxycholecalciferol (1,25-(OH)2-D3) increases membrane-associated protein kinase C (PKC) activity and immunoreactivity in renal epithelial (Madin Darby bovine kidney, MDBK) cells (Simboli-Campbell, M., Franks, D. J., and Welsh, J. E. (1992) Cell Signalling 4, 99-109). We have now characterized the effects of 1,25-(OH)2-D3 on the subcellular localization of three individual isozymes by immunofluorescence and immunoblotting. Although the total amount of PKC alpha, PKC beta, and PKC zeta are unaffected by 1,25-(OH)2-D3, this steroid hormone induces subcellular redistribution of both PKC alpha and PKC beta. Treatment with 1,25-(OH)2-D3 (100 nM, 24 h) enhances plasma membrane association of PKC alpha and induces translocation of PKC beta to the nuclear membrane. The effects of 1,25-(OH)2-D3 appear to be limited to the calcium-dependent PKC isozymes, since 1,25-(OH)2-D3 has no effect on the calcium independent isozyme, PKC zeta. In contrast to rapid transient PKC translocation seen in response to agents which interact with membrane receptors to induce phospholipid hydrolysis, modulation of PKC alpha and PKC beta is observed after 24 h treatment with 1,25-(OH)2-D3. In MDBK cells, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) (100 nM, 24 h) down-regulates PKC alpha and, to a lesser extent, PKC zeta, without altering their subcellular distribution. TPA also induces translocation of PKC beta to the nuclear membrane. MDBK cells treated with 1,25-(OH)2-D3, but not TPA, exhibit enhanced phosphorylation of endogenous nuclear proteins. In addition to the distinct effects of 1,25-(OH)2-D3 and TPA on PKC isozyme patterns, 1,25-(OH)2-D3 up-regulates both the vitamin D receptor and calbindin D-28K, whereas TPA down-regulates the expression of both proteins. These data support the involvement of PKC in the mechanism of action of 1,25-(OH)2-D3 and specifically implicate PKC beta in 1,25-(OH)2-D3-mediated nuclear events.

IL-2 mRNA levels and degradation rates change with mode of stimulation and phorbol ester treatment of lymphocytes

Transient expression of interleukin 2 (IL-2) in activated T lymphocytes may be due to transcriptional and post-transcriptional regulation. As incubation of lymphocytes with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) prior to mitogenic stimulation results in decreased levels of IL-2 mRNA, we asked if IL-2 mRNA stability was affected. We found that in TPA-treated cells, IL-2 mRNA was degraded more rapidly than in untreated ones whether the mitogenic stimulus was Concanavalin A (Con A), Con A plus TPA, or TPA plus ionomycin. The degradation was blocked if the TPA pre-incubation included cycloheximide. In contrast, when TPA was included as a co-mitogen, i.e. added at the same time as the mitogen, the IL-2 mRNA levels and stability significantly increased. Compared to the levels found in Con A stimulated cells, TPA plus Con A increased IL-2 mRNA levels by as much as 20-fold and the half-life by 5-fold. TPA plus ionomycin increased the message levels at least 100-fold and half-life by nearly 10-fold. These effects on IL-2 mRNA were not general because IL-2 receptor mRNA stability was not changed even though it also is transiently expressed during the course of lymphocyte activation.

Mechanisms of action for an androgen-mediated autoregulatory process in rat thecal-interstitial cells.

In rat thecal-interstitial cells (TIC), treatment with the synthetic androgen mibolerone has led to the documentation of an autoregulatory process for androgen production. In the present study, accumulated evidence has provided insight into the mechanisms of mibolerone action that control this process. Investigations using the nonsteroidal antiandrogen hydroxyflutamide were conducted to characterize mibolerone's mode of action. Hydroxyflutamide had differential effects on hCG action, the 1-microM dose stimulating hCG-induced androsterone synthesis by 27% and the 10-microM concentration decreasing the androgen levels by 84%. In addition, treatment with 1 microM hydroxyflutamide was effective in partially reversing the inhibitory action of mibolerone on hCG-stimulated androsterone production. Thus, the data indicated that mibolerone's mode of action may be mediated, at least in part, via the androgen receptor. The possibility that mibolerone had multiple sites of action prompted studies on the effectiveness of this androgen to alter various signaling pathways. Treatment with increasing concentrations (0.01-100 nM) of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C, resulted in a 75% decrease in hCG-stimulated androgen production at a dose of 100 nM TPA. Treatment with mibolerone (100 nM) was unable to alter the action of TPA on androgen synthesis when doses of 1 and 10 nM TPA were employed. It was also found that Ca2+ can serve as a mediator of mibolerone action. Treatment with a 0.01-microM dose of A23187, a Ca2+ ionophore known to increase intracellular Ca2+, was ineffective in altering hCG-stimulated androsterone synthesis. The concurrent treatment of mibolerone (100 nM) and A23187 (0.01 microM) resulted in the potentiation of mibolerone's inhibitory effects on hCG-stimulated androgen production, thereby suggesting that mibolerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a 1-microM dose of the L-type Ca2+ channel blocker verapamil to TIC cultures was able to partially block the inhibitory effect of mibolerone on androgen synthesis. Evidence for an additional site of mibolerone action was revealed through an analysis of the mRNA levels of P450scc and P450(17) alpha enzymes. Although hCG and insulin-like growth factor I treatment resulted in 20- and 32-fold increases in the amount of P450scc and P450(17) alpha mRNA, respectively, the addition of mibolerone (100 nM) reduced only P450(17) alpha mRNA levels by 91%. Overall, the evidence indicates that mibolerone has multiple sites of action in exerting its regulatory effect on androgen synthesis.

Effect of phorbol ester on vagal stimulation and acetylcholine-evoked exocrine pancreatic secretion and cytosolic free calcium in the rat.

In anaesthetized rats, interaction between either vagal stimulation or acetylcholine (ACh) and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) and staurosporine on pancreatic juice secretion have been investigated in vivo. In vitro, rat cytosolic free calcium concentration (Ca2+)i in pancreatic acini loaded with the fluorescent dye, Fura-2 acetoxymethyl ester (AM) have been analysed after the same modifications. In vivo, vagotomy caused a marked reduction in the rate of pancreatic juice flow, total protein output and amylase secretion compared to basal secretory parameters. Furthermore, bolus injection of either saline, TPA (10(-8) mol/kg b wt), staurosporine (10(-8) mol/kg b wt) or a combination of TPA and staurosporine (all 10(-8) mol/kg b wt) had no statistically significant effect on pancreatic juice flow, protein output and amylase secretion compared to vagotomy values. Electrical stimulation (E.S.) of vagues nerves resulted in marked and statistically significant (P < 0.05) increases in the rate of pancreatic juice flow, total protein output and amylase secretion in rats injected with either saline or staurosporine, compared to control values. In contrast, E.S. of the vagus nerves failed to enhance all secretory parameters in the presence of either TPA or a combination of TPA and staurosporine. In vitro, on isolated acini, ACh evoked dose dependent increases in (Ca2+)i. Pretreatment these acini with either TPA, staurosporine or a combination of TPA and staurosporine had no significant effect on the ACh-induced (Ca2+)i. These results indicate that TPA can decrease the secretory responses evoked by E.S. of the vagus nerves in the anaesthetized rat. This attenuation is not associated with either protein kinase C inhibition or the mobilization of the second messenger Ca2+ but possibly through activation of protein kinase C by TPA.

Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids

Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.

Effects of a fecapentaene on protein kinase C

Protein kinase C (PKC) is a Ca 2+- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). PKC can be activated in vitro by phosphatidylserine (PS) plus Ca 2+. We report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca 2+. In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by Ca 2+ and PS. It can also either enhance or inhibit activation of PKC by the tumor promoter teleocidin, depending on the assay conditions. These results are of interest since fecapentaene is known to be a potent mutagen that is produced by Bacteroides species present in the lumen of the human colon. The present studies raise the possibility that this compound might also play a role in colon cancer by altering the activity of PKC.

Successful culture of adult human melanocytes obtained from normal and vitiligo donors.

The development of growth conditions for human melanocytes from uninvolved skin from vitiligo patients and age-matched normal adults is a prerequisite to understanding the etiology of this pigmentary disorder. By using new growth conditions, pure melanocyte cultures were prepared from normal adults and the pigmented skin of vitiligo donors. Both cell types grew without a lag period and were maintained for more than six months (4-8 passages). The cultures could be expanded from several thousand melanocytes in the original cell suspension to several million cells (2-7 x 10(6] in pure culture. To obtain these results, the current standard conditions for the culture of fetal melanocytes were substantially modified. MEM-S medium was less satisfactory than MCDB-153. Adult normal and vitiligo cells also required the presence of exogenous catalase (20 micrograms/ml) during isolation and primary seeding. Thereafter, this enzyme was not necessary. Melanocytes grew best and gave the highest yields if the concentrations of both calcium (200 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) were low (4-8 nM). Other growth factors included in the MCDB-153 media were bFGF, crude bovine pituitary extract, insulin, transferrin, hydrocortisone, 5% FCS, and the antioxidant alpha-tocopherol. Cholera toxin and isobutylmethylxanthine (IBMX) were omitted from the MCDB-153 growth medium because they slowed down growth even at very low concentrations. The results indicate adult and vitiligo melanocytes can be cultured. Preliminary studies of the normal and vitiligo cells indicate that vitiligo melanocytes retain some of the ultrastructural abnormalities observed in skin even when grown in culture.

Interactions of protein kinase C with receptor- and non-receptor-mediated cyclic AMP generation in swine granulosa cells

Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The swine granulosa cell provides a model to study the interactions between the cAMP and calcium-lipid-dependent signaling pathways. To this end, porcine granulosa cells were incubated in monolayer culture for 1–4 days in the presence of FSH (200 ng/ml), forskolin (85 μM), or cholera toxin (3 μg/ml) with or without an activator of protein kinase C, the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) (30 ng/ml). TPA had little effect on basal cAMP generation (1–4 days) or on follicle-stimulating hormone (FSH)-stimulated cAMP formation during the first 24 h. Phorbol ester did inhibit cAMP formation on day 2 (by ~ 25%), on day 3 (by ~ 70%) and on day 4 (by > 80%). Forskolin-mediated cAMP generation was inhibited (33–56%) on days 1–4, respectively. TPA suppressed dose-dependent FSH (3–300 ng/ml)-stimulated cAMP production on day 2, virtually abolished FSH-provoked cAMP formation on day 4 and inhibited dose-dependent forskolin-stimulated cAMP production on both days. TPA had no effect on the half-maximally effective dose, ED 50, of FSH-stimulated cAMP production but did decrease the ED 50 of forskolin and the maximal stimulatory effect of FSH and forskolin on days 2 and 4. Similar effects were observed with the synthetic diacylglycerols DOG (1,2-dioctanoylglycerol) and OAG (1-oleoyl-2-acetylglycerol). The TPA effect was limited to the mammalian adenylate cyclase as it had no effect on bacterially derived adenylate cyclase from Bordetella pertussis. In contrast, although phorbol ester treatment inhibited the dose-dependent (30–1000 ng/ml) stimulation of cAMP accumulation by cholera toxin on day 2, unexpectedly, the action of TPA on day 4 was reversed in the presence of cholera toxin becoming stimulatory instead of inhibitory. Pretreatment of granulosa cells with pertussis toxin (100 ng/ml for 18 h) did not override the inhibitory action of TPA on either receptor- or non-receptor-mediated cAMP accumulation after 2 or 4 days of incubation. We conclude that the protein kinase C effector pathway is effectively coupled in an inhibitory fashion to both receptor-mediated and non-receptor-mediated activation of adenylate cyclase in porcine ovarian cells. Moreover, the present observations are consistent with the hypothesis that this inhibitory coupling is enacted at least in part at the level of the catalytic subunit of adenylate cyclase.

Phylogeny of lymphocyte heterogeneity: The thymus of the channel catfish

The number of thymocytes (approximately 3 × 107) that were recoverable from fingerling channel catfish remained constant from about 3 to 10 months of age, i.e. from September to April following hatching the previous June. Between 11 and 12 months, i.e. May and June, the thymus dramatically increased in size with 3 × 109 thymocytes being recoverable from the tissue of individual fish. The thymus remained enlarged for several months (throughout the summer) but at about 15 months (in September) began to involute such that by 17 months (November) no thymus tissue could be seen macroscopically. This natural involution could be accelerated by subjecting the fish to handling and transport stress. Thymocytes of channel catfish aged 4 to 16 months exhibited reactivity with monoclonal antibodies against peripheral T cells but not B cells. Thymocytes responded to the mitogen Concanavalin A only in the presence of added accessory cells (peripheral blood monocytes) or a monocyte-derived supernatant (presumably containing IL-1) at permissive temperatures (27°C). Thymocytes could also be induced to divide at nonpermissive temperatures (17°C) when incubated in the presence of the following combinations of stimulants, a) the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and the calcium ionophore A23187, b) TPA and ConA, or c) A23187 and ConA. In those cases where TPA or A23187 were used, accessory cells or their products were not needed. Collectively, these results support the notion that channel catfish thymocytes functionally mimic those lymphocytes in the peripheral blood previously designated as T cells.

Stimulated T cell and natural killer (NK) cell lines fail to synthesize leukotriene B 4

The ability of leukotriene B 4 (LTB 4) to influence T cell and natural killer (NK) cell functions makes the question of LTB 4 generation by these cells important to address. Consequentlym LTB 4 generation was evaluated in a human (Jurkat), and in a murine (EL-4) T cell line as well as in a rat NK cell line (RNK-16). Incubation of each of the 3 cell lines with [1- 14C]arachidonic acid alone or in the presence of phytochemagglutinin (PHA), of calcium ionophore A23187, or concanavalin A (Con A) plus the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) failed to generate radiolabelled LTB 4 or other eicosanoids as determined by thin layer radiochromatography. Using two different radioimmunoassays for LTB 4 also failed to demonstrate the generation of LTB 4 under basal or stimulated conditions. These results support earlier studies that demonstrate that T cells are not capable of de novo synthesis of prostaglandins, thromboxanes, or leukotrienes and also provide evidence that NK cells also do not have the capacity to generate LTB 4 or other eicosanoids. Our findings are also critically discussed in relation to studies claiming eicosanoid synthesis by T cells.

Studies on the subcellular mechanisms mediating the negative estradiol effect of GnRH-induced LH-release

The gonadotropin releasing hormone(GnRH)-induced luteinizing hormone(LH)-secretion by rat pituitary cells in culture is significantly reduced by short-term estrogen treatment. This negative estrogen effect occurs already after approx. 35 min [1] and might thus be mediated via a non-genomic mechanism. In the present study, the effects of short term E2-treatment on selected steps of the GnRH-second messenger cascade were checked to elucidate a possible non-genomic subcellular mechanism, by which the negative E2-effect might be mediated. The following mechanisms were studied: 1. Ca+ + influx (blockade of Ca+ +-channels by verapamil, activation of Ca+ +-channels by K + or veratridine; Ca+ + influx via the ionophores A23187 or ionomycin [2]. 2. Polyphosphoinositide breakdown by monitoring the accumulation of inositol phosphates [3]. 3. The protein kinase C-system by stimulation with the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) [4].

Phorbol esters, but not the hormonal form of vitamin D, induce changes in protein kinase C during differentiation of human histiocytic lymphoma cell line (U-937)

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1, 25-dihydroxy-cholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possibly through a different mechanism of that of phorbol ester.

T-leukemia cell lines CCRF-CEM, HPB-ALL, JM and MOLT-4: changes in isoenzyme profiles during induction of differentiation.

Biochemical analysis has been used to monitor the induction of differentiation in cultured human T-leukemia cell lines (CCRF-CEM, HPB-ALL, JM and MOLT-4) by the phorbolester 12-0-tetradecanoylphorbol 13-acetate (TPA). The isoenzymes of carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase were separated by isoelectric focusing on horizontal thin-layer polyacrylamide gels and stained by histo-cytochemical methods. TPA inhibited the proliferative activity in all four cell lines and led to aggregation of cells seen as floating clusters. TPA induced an increase in number and staining intensity of isoenzymes of all four enzymes in the cell lines studied. This corresponds to an induced isoenzymatic maturation as the progressive increase in number and staining intensity of the isoenzymes parallels the differentiation along the T-cell pathway. However, regardless of the initial stage of arrested differentiation, the cell lines could be induced only to differentiate to a certain more mature stage, but could not be triggered to differentiate terminally with regard to expression of isoenzyme patterns.

Induction of differentiation in the human leukemia cell line SPI-802: morphological, immunological, and isoenzymatic changes.

The phorbolester 12-0-tetradecanoylphorbol 13-acetate (TPA) was used for the induction of differentiation in cells of the human leukemia cell line SPI-802. The cellular morphology, surface marker antigen expression, and isoenzyme profiles of four enzymes (carboxylic esterase, acid phosphatase, hexosaminidase, and lactate dehydrogenase) served as parameters for monitoring the induced phenotypical changes. TPA led to distinct alterations of the morphology and significantly affected the growth rate with cessation of cell proliferation. No major increase in the number of nitro blue tetrazolium-positive cells or aggregation of cells, phagocytosis of latex beads, adherence to plastic surface, or development of pseudopodia were observed. As TPA-treated SPI-802 cells remained negative for these markers of the monocyte-macrophage complex, it can be concluded that the cells did not differentiate into monocytes and macrophages. The immunological marker profile based on testing of 55 monoclonal antibodies, terminal deoxynucleotidyl transferase and two erythrocyte rosette tests indicated a differentiation of SPI-802 cells along the granulocytic cell lineage. This was confirmed by isoenzyme analysis, especially that of carboxylic esterase. An isoenzyme specific for monocytes and macrophages was not detected. In earlier studies it was found that SPI-802 cells produce hemoglobin upon exposure to TPA or hemin. This latter observation and the present results suggested a comparison with the two erythroleukemia cell lines K-562 and HEL. SPI-802 cells appear to have the potential to differentiate along several cell lineages.

Detection of melanogenic proteins in cultured chick-embryo melanocytes

The phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), was used as a reversible inhibitor of melanogenesis. Chick-melanocyte cultures of the black genotype, EIE, were grown in conditioned medium plus TPA. After growth in TPA and after its removal, the cells were pulse labeled with 3H-leucine. The membrane fraction, which included all tyrosinase activity as well as both mature and immature melanosomes, was solubilized with Triton X-100. The proteins were separated using two-dimensional electrophoresis and visualized by fluorography. One defined melanogenic protein, tyrosinase, was isolated, and its location was determined in the two-dimensional protein pattern. The protein patterns for both the TPA-inhibited cells and the cells in which the TPA effects were reversed after removal were compared. in addition to tyrosinase, at least nine TPA-sensitive proteins were found. These were designated as being putative melanogenic proteins which, along with tyrosinase, may be responsible for melanin-granule synthesis.

Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.