Suppression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice by transduced Tat-Annexin protein

We examined that the protective effects of ANX1 on 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced skin inflammation in animal models using a Tat-ANX1 protein. Topical application of the Tat-ANX1 protein markedly inhibited TPAinduced ear edema and expression levels of cyclooxygenase-2 (COX-2) as well as pro-inflammatory cytokines such as interleukin- 1 beta (IL-1 β), IL-6, and tumor necrosis factor-alpha (TNF-α). Also, application of Tat-ANX1 protein significantly inhibited nuclear translocation of nuclear factor-kappa B (NF-κ B) and phosphorylation of p38 and extracellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TPA-treated mice ears. The results indicate that Tat-ANX1 protein inhibits the inflammatory response by blocking NF-κ B and MAPK activation in TPA-induced mice ears. Therefore, the Tat-ANX1 protein may be useful as a therapeutic agent against inflammatory skin diseases.     

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.     

Purification of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase from mouse epidermis

Ornithine decarboxylase was purified at least 1500-fold from mouse epidermis pretreated with five consecutive doses of 12-O-tetradecanoylphorbol-13-acetate and 3-isobutyl-1-methylxanthine at 3- to 4-day intervals. Following DEAE-cellulose chromatography and ammonium sulfate precipitation, ornithine decarboxylase was purified further by affinity chromatography. Ornithine decarboxylase was then radioactively labeled by covalently binding [3H]-alpha-difluromethylornithine to the enzyme following polyacrylamide gel electrophoresis under non-denaturing conditions. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining of protein, a band was identified that corresponded to a molecular weight of approx. 56,000, coincident with a peak of radioactivity. This is the first study to purify ornithine decarboxylase from mouse epidermis.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

TPA(12-O-tetradecanoyl-phorbol-13-acetate) as a carcinogen for mouse skin

Virchows Archiv B Cell Pathology - In order to study a possible dose/response relationship in the tumorigenic effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), groups of hairless mice were...     

Cytochrome P450 induction by phenobarbital (PB) is inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA): evidence that protein kinase C regulates induction

The hepatic microsomal monooxygenase system was studied in hypophysectomized male rats exposed for 24 or 48 h to PB and/or TPA, an activator of kinase C. TPA attenuated basal and PB-induced levels of P450, aniline hydroxylase (ANH), ethylmorphine demethylase (EDM) and cytochrome c reductase. Hence, PB may effect induction via the inhibition of kinase C. Supporting this is spectral evidence that PB and TPA do not bind and the fact that TPA did not decrease P450 when co-incubated with O2 and NADPH. Hemin failed to increase P450 levels previously depressed by TPA indicating that TPA acts by lowering apocytochrome levels. This is consistent with its attenuation of PB-effected increases in hepatic RNA. TPA effects were associated with increased hepatic RNA and were blocked by puromycin.     

Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

Lymphocyte activation by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA).

TPA, a highly active tumor-promoting agent, is an effective mitogen for primate peripheral blood lymphocytes. Optimal stimulation of human lymphocytes was obtained 4 days after the addition of TPA at a concentration of 7.5 ng/ml. Lymphocyte fractionation experiments demonstrated that both T and B cells incorporated 3H-thymidine significantly in response to TPA. Lymphocyte blastogenesis was not due to the reactivation of latent herpesviruses by the tumor promoter, since similar responses to TPA were obtained with virus-genome positive or negative cells. Increased levels of DNA synthesis were observed when TPA was added to marmoset, baboon, rhesus monkey, or chimpanzee peripheral blood lymphocytes. Canine peripheral blood lymphocytes and spleen cells from guinea pigs, rats, and mice were not stimulated by TPA. These observations suggest that TPA-induced lymphocyte blastogenesis may be useful for studies of lymphocyte activation and of the molecular mechanisms of action of tumor-promoting phorbol esters.     

Regulation of protein kinase C and ornithine decarboxylase in the epidermis of juvenile white suckers (Catostomus commersoni) by 12-O-tetradecanoylphorbol-13-acetate, 17 α-ethinylestradiol and testosterone

This study was designed to investigate whether potent regulators of mammalian protein kinase C (PKC) and ornithine decarboxylase (ODC) activity also regulate epidermal PKC and ODC activity in fish. Juvenile white suckers (Catostomus commersoni) were given single or multiple subdermal injections of testosterone, 17α-ethinylestradiol or 12-O-tetradeconylphorbol-13-acetate (TPA) dissolved in sunflower oil. Sequential activation of epidermal PKC and ODC was observed in single injection protocols. Maximal PKC activity occurred at 12–48 hr post-injection, with a corresponding increase in ODC activity in the 12–48 hr immediately following this event. In the multiple injection protocols, PKC activity was almost completely depressed after 1 week of injections, during which ODC activity was stimulated 2- to 5-fold, indicating possible differential activation of these two enzymes. Multiple injections of testosterone, 17α-ethinylestradiol and TPA induced histologically distinct epidermal hyperplasia in suckers, although this did not occur in single injection treatments. The mammalian isozymes of PKC are known to be dependent on Ca²⁺ and phospholipid for optimum activity. This study demonstrated that the fish isozyme of PKC is also Ca²⁺ and phospholipid dependent. Our results indicate that PKC and ODC may be good biochemical markers for neoplasia and hyperplasia in fish.     

Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: Inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

Mouse epidermal cells can be subcultured at 31°C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.     

12-O-tetradecanoylphorbol-13-acetate (TPA)-induced c-Jun N-terminal kinase (JNK) phosphatase renders immortalized or transformed epithelial cells refractory to TPA-inducible JNK activity

c-Jun N-terminal kinase (JNK) regulates gene expression in response to various extracellular stimuli. JNK can be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human oral keratinocytes but not in human keratinocytes that have been immortalized (HOK-16B and HaCaT) or transformed (HOK-16B-Bap-T) nor in a cervical carcinoma cell line (HeLa). The refractory JNK activation response to TPA is not due a defect in the JNK pathway, because JNK can be activated by other stimuli, e.g. UV irradiation and an alkylating agent N-methyl-N'-nitrosoguanidine in these immortalized or transformed cells. More importantly, the refractory JNK and JNKK activation response to TPA can be restored by treatment of the cells with a combination of TPA and a protein-tyrosine phosphatase inhibitor, sodium orthovanadate. Furthermore, pretreatment of cells with TPA partially inhibited UV- or N-methyl-N'-nitrosoguanidine-induced JNK activity. These results suggest that a TPA-inducible, orthovanadate-sensitive protein-tyrosine phosphatase may specifically down-regulate JNK signaling pathway in these immortalized/transformed epithelial cells. In contrast, ERK and p38/Mpk2 are not regulated by this TPA-induced phosphatase. This putative protein-tyrosine phosphatase appears to be JNK pathway-specific.     

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.     

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

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

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.     

Staurosporine, a potent protein kinase C inhibitor, fails to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells

Staurosporine, a most potent protein kinase C inhibitor, actually inhibited protein kinase C activity obtained either from cytosol or particulate fraction of mouse epidermis. Staurosporine at the concentrations which exert protein kinase C inhibition, however, failed to inhibit, but markedly augmented 12-O-tetradecanoylphorbol-13-acetate (TPA)-caused ornithine decarboxylase (ODC) induction in isolated mouse epidermal cells. Staurosporine by itself induced ODC activity as TPA does. Mechanism of ODC induction seems different between these two compounds. Another protein kinase C inhibitor, H-7, inhibited both staurosporine- and TPA-caused ODC induction.     

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide

Growth of Acanthamoeba castellaniiwas inhibited by chitosanoligosaccharide (up to 20 mg ml^–1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.     

Activation of protein kinase C by ganglioside GM3 in the presence of calcium and 12-O-tetradecanoylphorbol-13-acetate

Ganglioside GM3 and 12-O-tetradecanoylphorbol-13-acetate activated protein kinase C as substitutes for phosphatidylserine and diacylglycerol. Hydrophobic gangliosides such as GM4 and GM3 were rather more potent activators of protein kinase C than hydrophilic ones such as GD1a and GT1b. Active tumor promoters such as teleocidin, mezerein, phorbol 12,13-acetate and phorbol 12,13-dibenzoate also activated protein kinase C, but not inactive tumor promoters such as phorbol and 4-alpha-phorbol 12,13-didecanoate.