12-O-tetradecanoylphorbol 13-acetate induces DNA cleavage at linker regions in mouse thymocytes

A phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), induced the cleavage of nuclear DNA at linker regions in cultured mouse thymocytes. Similar DNA fragmentation was induced by 1-oleoyl-2-acetyl-glycerol, a synthetic diacylglycerol, but not by 4 alpha-phorbol-12,13 didecanoate. The DNA fragmentation was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine dihydrochloride, an inhibitor of protein kinase C, as well as actinomycin D and cycloheximide. It appears that TPA induces DNA cleavage through activation of protein kinase C and synthesis of yet unidentified protein(s). That the inhibition of DNA fragmentation was accompanied by a reduction in cell lysis suggests a causal relationship between DNA fragmentation and cell death.     

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.     

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.     

Proteomics-based identification of proteins with altered expression induced by 12-O-tetradecanoylphorbol 13-acetate in nasopharyngeal carcinoma CNE2 cells

Nasopharyngeal carcinoma (NPC) is a malignancy with high incidence in Southern China and South-East Asia. Etiology studies indicate that chemical carcinogen promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), are important factors causing NPC development. However, the mechanism of the TPA effect on NPC remains unclear. In the present study, cells from a poorly differentiated squamous cell carcinoma NPC cell line, CNE2, were stimulated by TPA and proteomics technology was carried out to find protein discrepancies between control and TPA-treated cells. Results revealed that TPA treatment in CNE2 cells could upregulate the expression of ““““triosephosphate isomerase““““ and ““““14-3-3 protein sigma““““ and downregulate the expression of ““““reticulocalbin 1 precursor““““, ““““nucleophosmin““““, ““““mitochondrial matrix protein pl precursor““““, and ““““stathmin““““. The changes in the expression of these genes suggested that TPA induced CNE2 cells to antiproliferation and to apoptosis, which was confirmed by subsequent apoptosis detection. Therefore, the effects of TPA on nasopharyngeal carcinoma cells were distinct from the effects on primary epithelial cells and we suggest reasons for these differences.     

Synergism between subthreshold concentrations of thrombin and 12-O-tetradecanoylphorbol 13-acetate in platelet activation

A comparison was made between the time courses and interdependence of platelet aggregation, serotonin release, and cytosolic free Ca2+ concentration in the same sample of platelets loaded with [14C]-serotonin and Ca2+-sensitive photoprotein aequorin. In 100 micrograms/ml aspirin-treated platelets, neither 0.01 U/ml thrombin nor 50nM TPA, an active phorbol ester, induced significant aggregation, serotonin release, or a rise in the intracellular calcium concentration. However, when these two agents were added together, marked aggregation and release were observed without a change in the cytosolic free Ca2+ concentration. No correlation was observed between the extent of the synergistic effects and time of preincubation with TPA. Potentiatory effects of protein kinase C on receptor-mediated agonists need to be considered in platelet activation.     

A mutagenesis-enhancing activity of 12-O-tetradecanoylphorbol 13-acetate detected in Chinese hamster ovary cells

Tumour-promoting agents may bring about the completion of multi-step carcinogenesis by acting as enhancers of mutagenesis, recombinogens or clastogens. We report here that the classical mouse skin tumour promoter TPA, although non-mutagenic per se, can enhance the induction of OuaR CHO-K1 cell mutants by MNNG approximately 2-fold. This observation was made at a concentration approaching the compounds aqueous solubility limit which was non-cytotoxic. Mutagenesis enhancement was dependent on TPA being present throughout mutation expression and mutant selection. It was not accompanied by any modification of cell sensitivity to mutagen killing. In the same treatment protocol TPA did not enhance either EMS- or UV-induced mutagenesis. TPA exposure over 2 rounds of cell replication failed to produce an increase in the frequency of SCE in control or mutagen-treated CHO-K1 cultures. Likewise TPA exposure over 1 round of cell replication failed to produce an increase in the frequency of chromosomal aberrations. Apparently TPA is not a recombinogen or clastogen but in the right exposure regime is capable of acting to enhance mutagenesis by certain genotoxic agents, an action which may contribute to tumour promotion.     

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with ³⁵S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca⁺⁺ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.     

12-O-tetradecanoylphorbol 13-acetate potentiates the action of cAMP in inducing DNA cleavage in thymocytes

12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the action of cAMP in DNA cleavage in thymocytes induced by a low concentration of adenosine receptor-site agonists such as adenosine, 2-chloroadenosine and forskolin. The enhancement of DNA cleavage by TPA was also observed in dibutyryl cAMP-treated thymocytes. On the other hand, TPA suppressed accumulation of cAMP by the adenosine receptor-site agonists. These results suggest that activation of protein kinase C inhibits cAMP production, but stimulates cAMP-triggered process to induce DNA cleavage and death of thymocytes.     

Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate.

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.     

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.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate stimulates simian virus 40 induction by DNA-damaging agents and tumor initiators.

Simian virus 40 (SV40)-transformed Syrian hamster kidney cells produce infectious SV40 virus particles after treatments which damage DNA, such as UV irradiation or mitomycin C treatment. We have found that the induction of SV40 by DNA-damaging agents is greatly stimulated when a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), is present in the medium. Phorbol, which has a molecular structure similar to TPA but does not have any tumor-promoting activity, showed no such stimulatory effect on SV40 induction. This apparent synergistic effect of DNA-damaging agents and tumor promoter (TPA) was more pronounced when a tumor initiator, benzo [a]pyrene or 2-acetamido-fluorene, was combined with TPA. The effect of TPA on UV-triggered SV40 induction was greatly influenced by the timing of TPA addition to the culture medium, which was most efficient when addition of TPA was 5 to 20 h before UV irradiation. The effect of TPA, however, was not observed in SV40 rescue from hamster cells by cell fusion with permissive monkey (C7) cells.     

Nicardipine-sensitive enhancement of high K+-evoked dopamine release in PC12 cells pretreated with 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on stimulus-evoked dopamine release were studied in PC12 cells. Pretreatment of the cells with TPA resulted in an enhancement of dopamine release which could be further stimulated by high concentrations of K⁺, A23187, but not with carbamylcholine. TPA-dependent, high-K⁺-evoked enhancement of dopamine release was studied in detail: a maximum release was observed (169% of control) in response to 50 mM KCl upon treatment with 10⁻⁷ M TPA for 5 min at 37°C. This enhancement of dopamine release was associated with the concomitant reduction of the concentration rise of intracellular Ca²⁺ ([Ca²⁺]_i) induced by a high concentration of K⁺ monitored by a fluorescent indicator, fura2. Thus, these data provide an example for alteration in the efficiency of stimulus-secretion coupling as pointed out in our previous paper. Moreover, we have shown that nicardipine, CdCl₂, and CoCl₂ inhibit high-K⁺-evoked dopamine release more effectively in TPA treated cells than that of untreated cells, and that the TPA-dependent, high-K⁺-evoked dopamine release observed in TPA treated cells is completely abolished by the presence of nicardipine, Cd²⁺ or Co²⁺, but is only partially inhibited in the presence of verapamil. These relevant findings suggest the possible involvement of protein kinase C in regulating the efficiency of a high-K⁺-evoked dopamine release through the modification of nicardipine-sensitive Ca²⁺ channels.     

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.     

Relationship between membrane sterol composition and responsiveness to 12-O-tetradecanoylphorbol 13-acetate in HL-60 human promyelocytic leukaemia cell lines.

We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.     

Ubiquitination and down-regulation of gap junction protein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment

Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces ubiquitination of connexin-43 (Cx43) in IAR20 rat liver epithelial cells. The accelerated ubiquitination of Cx43 in response to TPA occurred concomitantly with Cx43 hyperphosphorylation and inhibition of cell-cell communication via gap junctions. The TPA-induced ubiquitination of Cx43 was mediated via protein kinase C and partly involved the mitogen-activated protein kinase pathway. Following ubiquitination, Cx43 was internalized and degraded. The loss of Cx43 protein was counteracted by ammonium chloride, indicating that acidification of internalized Cx43 gap junctions is a prerequisite for its degradation. Furthermore, the Cx43 degradation was partly counteracted by leupeptin, an inhibitor of cathepsin B, H, and L. Cx43 internalization and subsequent degradation were blocked by inhibitors of the proteasome. Evidence is provided that Cx43 is modified by multiple monoubiquitins rather than a polyubiquitin chain in response to TPA. Moreover, the TPA-induced ubiquitination of Cx43 was blocked by proteasomal inhibitors. Taken together, the data indicate that Cx43 ubiquitination is a highly regulated process. Moreover, the results suggest that the proteasome might play an indirect role in Cx43 degradation by affecting the level of monoubiquitin conjugation and trafficking of Cx43 to endosomal compartments.     

Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells

The effect of mitogens on phospholipase D activity was investigated in NIH-3T3 fibroblasts by measuring the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity when 1-propanol acts as the phosphatidyl group acceptor. Platelet-derived growth factor (PDGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated phosphatidylpropanol production by the cells. The dose-response relationships for activation of phospholipase D and stimulation of thymidine incorporation by PDGF and TPA were comparable. The possibility that activation of phospholipase D is utilized by mitogens as a trans-membrane pathway for signalling cell growth is discussed.