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.     

12-O-tetradecanoylphorbol-13-acetate enhances glycolysis in rat thymocytes

• 1.1. The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a rapid increase in glycolysis in rat thymocytes.
• 2.2. The increase in the glycolytic flux was also reflected by elevated fructose 1,6-diphosphate levels.
• 3.3. TPA treatment did not result in an increase of hexokinase, phosphofructokinase or pyruvate kinase when measured in cell homogenates.
• 4.4. It is suggested that the early increase in glycolysis in TPA treated lymphocytes may result from TPA-mediated increase in glucose transport.

     

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.     

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.     

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.     

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.     

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.     

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.     

Proteome analysis of antiproliferative mechanism of 12-O-tetradecanoylphorbol 13-acetate on cultured nasopharyngeal carcinoma CNE2 cells

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a plant derivative with multiple function as tumor promoter, differentiation revulsant or leukemia therapy drug. The molecular mechanism of its function is perplexing. Many studies have focused on the mechanism of TPA stimulation in tumor promotion of mouse models or terminal differentiation of leukemia cells, but the effect of TPA on nasopharyngeal carcinoma (NPC) remains unclear, while TPA was considered to be associated with NPC development. In the present study, we employed proteomics techniques to study protein changes of a poorly differentiated squamous carcinoma cell line-CNE2 of human NPCs cells induced by TPA. Six significantly and reproducibly changed proteins were identified and their functional implications were discussed in some details.     

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.     

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a protein in human lymphocytes binding satellite DNA

Interactions between a satellite DNA fragment (SH3, 1.8 kb) cloned in pBR325 plasmid and DEAE-protein fractions from human lymphocytes treated with phorbol-12-myristate-13-acetate (TPA) have been studied by filtration technique through nitrocellulose filters. The 0.3 M fraction was found to have a protein which binds SH3 DNA specifically. Cytogenetic studies have shown an increased frequency of chromosome endoreduplications which may be due to the binding of TPA-induced protein to centromeres.     

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)     

Adenosine, deoxyadenosine, and deoxyguanosine induce DNA cleavage in mouse thymocytes

When thymocytes were cultured with adenosine, deoxyadenosine, or deoxyguanosine at 1 mM for 24 h, DNA cleavage at internucleosomal sites with multiples of approximately 180 bp was induced, followed by lactate dehydrogenase release into the medium. In the presence of coformycin, an adenosine deaminase inhibitor, or formycin B, a purine nucleoside phosphorylase inhibitor, DNA cleavage was induced by these nucleosides at concentrations of less than 50 microM. Other purine and pyrimidine ribo- and deoxyribonucleosides did not induce DNA cleavage or LDH release. Because thymocyte nuclei contain a Ca2+,Mg2+-dependent endonuclease, which preferentially cuts DNA in its linker regions, DNA fragmentation induced by the three purine nucleosides was suggested to occur through increased activity of the endonuclease. The DNA cleavage induced by the nucleosides required protein phosphorylation and synthesis, inasmuch as it was inhibited by an inhibitor of protein kinases, H-7, and by an inhibitor of protein synthesis, cycloheximide. The inhibition of DNA cleavage was accompanied by a reduction in lactate dehydrogenase release, suggesting a causal relationship between DNA cleavage and cell death. The DNA cleavage and subsequent cell lysis might be related to the selective thymocyte deletion observed in patients with adenosine deaminase or purine nucleoside phosphorylase deficiency.     

Tumor promoter phorbol-12-myristate-13-acetate induces poly ADP-ribosylation in human monocytes

The tumor promoter phorbol-12-myristate-13-acetate (PMA) induces rapid poly ADP-ribosylation and a drop in cellular NAD concentration in human monocytes. The antioxidants CuZn-superoxide dismutase, catalase, glutathione peroxidase and butylated-hydroxytoluene inhibit the reaction indicating that active oxygen species produced in the PMA-induced oxidative burst represent intermediates. The inhibitor of ADP-ribosyl-transferase, 3-amino-benzamide, inhibited poly ADP-ribosylation but did not prevent the drop in NAD-levels. PMA also causes the slow accumulation of DNA strand breaks in monocytes. The difference in the kinetics of poly ADP-ribosylation and DNA breakage argues against a simple relationship between the two reactions.     

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.     

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.     

Na+/H+ exchange in Ehrlich ascites tumor cells. Regulation by extracellular ATP and 12-O-tetradecanoylphorbol 13-acetate

The effects of extracellular ATP and/or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the intracellular pH of Ehrlich ascites tumor cells were measured using both distribution of [14C]5,5-dimethyloxazolidine-2,4-dione, and the fluorescent indicator 5(6)-carboxyfluorescein. Micromolar concentrations of extracellular ATP induce a biphasic change in the intracellular pH characterized by a rapid acidification of 0.04 pH units followed by an alkalinization of 0.11 pH units. Concurrently with the alkalinization, an increase in the total cellular [Na+] from 37.5 to 45.0 mM is observed. The pH change is half-maximally activated by 0.5-2.5 microM extracellular ATP. The intracellular alkalinization, but not the initial acidification, phase requires extracellular Na+, with half-maximal alkalinization in the presence of 24-32 mM Na+, and is inhibited by amiloride. Exposure of Ehrlich ascites tumor cells to TPA alone produces a slight alkalinization of approximately 0.04 pH units. Conversely, preincubation of the cells with TPA partially inhibits the ATP-induced changes in intracellular pH. Under identical conditions TPA also inhibits the ATP-induced increase in the cytosolic [Ca2+]. The half-maximal dose for both effects is produced by 3-10 nM TPA. These data indicate that extracellular ATP triggers the activation of Na+/H+ exchange. Furthermore, activation of protein kinase C mediates at least part of the Na+/H+ exchange, although a second mechanism may also exist.