H-7, a protein kinase C inhibitor, inhibits phorbol ester-caused ornithine decarboxylase induction but fails to inhibit phorbol ester-caused suppression of epidermal growth factor binding in primary cultured mouse epidermal cells

12-O-tetradecanoylphorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC) and suppressed 125I-epidermal growth factor (EGF) binding in primary cultured mouse epidermal cells. TPA (30 nM)-caused ODC induction was almost completely blocked by 30 microM H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], a well known protein kinase C inhibitor, but the same concentration of H-7 failed to restore the 125I-EGF binding suppressed by TPA (10 nM). On the other hand, sphingosine, another protein kinase C inhibitor, blocked not only TPA-caused ODC induction but also TPA-caused suppression of 125I-EGF binding. Concentration-response curves of sphingosine for these two TPA-caused cellular responses were almost identical. 1,2-Diacylglycerols such as 1,2-dioctanoylglycerol (30-300 microM) and 1-oleoyl-2-acetylglycerol (OAG) (30-300 microM) mimicked TPA actions. Similar to the case of TPA, suppression of 125I-EGF binding by OAG was barely inhibited by H-7, whereas sphingosine was more effective in inhibiting the OAG-caused suppression of 125I-EGF binding than was H-7. In TPA (50 nM)-pretreated epidermal cells, TPA (10 nM) failed to suppress 125I-EGF binding. H-7 (30 microM) did not affect TPA (30 nM)-caused translocation of protein kinase C. These results clearly demonstrate the differential inhibition by H-7 of the TPA-caused cellular responses and indicate that TPA-caused suppression of 125I-EGF binding to epidermal cells is mediated through protein kinase C function, which is barely inhibited by H-7.     

Protein kinase C-dependent and -independent actions of a potent protein kinase C inhibitor, staurosporine.

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), induced ornithine decarboxylase (ODC) in primary cultured mouse epidermal cells. Staurosporine, a potent protein kinase C inhibitor, also induced ODC activity. Both TPA- and staurosporine-caused ODC inductions were markedly suppressed in the PKC-down-regulated cells. Another PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), inhibited both TPA- and staurosporine-caused ODC inductions. H-7 by itself never induced ODC activity. Under our experimental conditions, staurosporine induced no detectable phosphorylation of endogenous proteins. TPA induced a translocation of PKC from cytosol to membrane whereas an optimal concentration of staurosporine to induce ODC did not induce an obvious translocation of PKC. Indomethacin, a cyclooxygenase inhibitor, inhibited staurosporine-caused ODC induction, but not TPA-caused ODC induction. Staurosporine induced specific morphological changes of epidermal cells both in normal and in PKC-down-regulated cells. These results indicate that staurosporine induces ODC activity in a PKC-dependent manner and morphological changes possibly through a PKC-independent mechanism. The mechanism of ODC induction caused by staurosporine may be in some way different from that caused by TPA.     

The induction of ornithine decarboxylase caused by 12-O-tetradecanoylphorbol-13-acetate in isolated epidermal cells is inhibited by lipoxygenase inhibitors but not by cyclooxygenase inhibitors

The effects of lipoxygenase and cyclooxygenase inhibitors on ornithine decarboxylase (ODC) induction by a potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined in vitro in isolated mouse epidermal cells. Lipoxygenase inhibitors such as quercetin, nordihydroguaiaretic acid (NDGA), 3,4,2',4'-tetrahydroxychalcone, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C) inhibited the TPA-caused ODC induction. Indomethacin, a selective cyclooxygenase inhibitor, failed to inhibit it. These results suggest that the lipoxygenase inhibitors inhibit TPA-caused epidermal ODC induction in mouse skin at least in part by acting directly on epidermal cells while cyclooxygenase inhibitor inhibits it indirectly by acting on cells other than epidermal cells, e.g. cells which are involved in the prostaglandin-dependent inflammatory process.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and tumor promotion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in mouse skin

N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited epidermal ornithine decarboxylase (ODC) induction caused either by 12-O-tetradecanoylphorbol-13-acetate (TPA) or teleocidin in CD-1 mice. Inhibitory effect of W-7 on TPA-caused ODC induction was also observed in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin and even after repetitive TPA treatment. TPA-induced skin tumor promotion was also suppressed by W-7. Meanwhile, W-7 showed only slight inhibitory effects on calcium-activated, phospholipid-dependent protein kinase (protein kinase C) activity of mouse epidermis stimulated either by Ca2+ or TPA in the presence of phosphatidylserine. Thus, it is unlikely that the anti-ODC-inducing and anti-tumor-promoting actions of W-7 are due to its inhibitory effect on protein kinase C. It may be possible that a calmodulin-mediating process is involved in the mechanism of epidermal ODC induction and tumor promotion caused by tumor promoters such as TPA and teleocidin.     

Comparison of some biochemical properties of epidermis in tumor promotion-susceptible and -resistant strains of mice

It has been reported that CD-1 and SENCAR mice are susceptible and C57BL/6 mice are resistant to skin tumor promotion caused by phorbol esters. Specific binding of a phorbol ester to its epidermal receptor site, epidermal protein kinase C activity, and ornithine decarboxylase (ODC) induction in epidermis were compared between tumor promotion-susceptible and -resistant strains of mice. Specific binding of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to the particulate fraction of the epidermis of C57BL/6 mice gave a similar dissociation constant (Kd) and a maximal number of binding sites (Bmax) to those of CD-1 mice. Protein kinase C activity of the epidermal 105,000 xg supernatant was not significantly different between C57BL/6 and CD-1 mice. Protein kinase C activity of the 105,000 xg pellet, however, was significantly higher in C57BL/6 mice than in CD-1 mice. A topical application of TPA to the skin caused epidermal ODC induction in all of these strains of mice. At any doses of TPA, TPA-induced epidermal ODC activity of C57BL/6 mice was always higher than those of SENCAR and CD-1 mice. Maximal induction of epidermal ODC by TPA was also highest in C57BL/6 mice among these three strains of mice. These results indicate that the mechanism of the difference in susceptibility of C57BL/6, CD-1 and SENCAR mice to the tumor-promoting action TPA resides in a step distal to or other than the protein kinase C activation and ODC induction.     

Effects of activators and inhibitors of protein kinase C on two-stage transformation in BALB/3T3 cells

To determine the relationship between protein kinase C and the promotion of carcinogenesis, we investigated the effects of activators and inhibitors of protein kinase C on two-stage transformation in BALB/3T3 cells. Diacylglycerols, which are activators, and specific inhibitors, such as palmitoyl-DL-carnitine chloride (PC), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and staurosporine (ST) were used. Treatment with diacylglycerols enhanced focus formation in 3-methylcholanthrene (3-MC)-initiated cells, but not as much as 12-O-tetradecanoylphorbol-13-acetate (TPA). PC and H-7 inhibited TPA-enhanced transformation by 76 and 79%, respectively. ST, the most potent inhibitor of protein kinase C, had a low inhibitory effect on transformation at non-toxic doses (33% inhibition). The results suggest that protein kinase C may play an important role in the process by which transformation is promoted in BALB/3T3 cells.     

Dose, time and route dependency of the induction of rat hepatic ornithine decarboxylase by 12-tetradecanoylphorbol 13-acetate.

Ornithine decarboxylase (ODC) activity, the rate limiting enzyme in polyamine biosynthesis, was determined after 12-O-tetradecanoylphorbol 13-acetate (TPA) administration to female Sprague-Dawley rats. The extent of induction depended on the dose, exposure, time and route of administration. The most effective dose for ODC induction by the intraperitoneal route was 40 ug TPA/kg which caused 3-5 fold ODC induction. Maximal ODC induction occurred in a narrow time band 5 hours after TPA administration. TPA had no adverse effects on hepatic DNA (measured by alkaline elution), cytochrome P-450 content and reduced glutathione content or serum alanine aminotransferase (SGPT) activity.     

The effects of the protein kinase C inhibitors staurosporine and H7 on the IgE dependent mediator release from RBL 2H3 cells.

RBL 2H3 cells, a model for mast cell function, sensitized with rat IgE, released histamine and peptidoleukotrienes (LT) in response to rabbit anti-rat IgE in a concentration-dependent manner. The calcium ionophore, A23187 also stimulated the release of both mediators but to a greater extent. The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) failed to influence mediator release when added alone, but when added with either A23187 or anti-IgE, TPA significantly enhanced the release of both histamine and LT. The effects of anti-IgE, TPA and A23187 were completely inhibited by prior addition of the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) but not by N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide dihydrochloride (HA1004), a compound which has similar potency to H7 as an inhibitor of some protein kinases but is less potent as a protein kinase C inhibitor. Although other explanations are possible, these results support the hypothesis that the release of histamine and leukotrienes from RBL 2H3 cells resulting from the cross bridging of the IgE receptors, is dependent on activation of protein kinase C.     

Stimulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by phorbol ester: mediation by protein kinase C

We have investigated the mechanisms by which the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates prostaglandin E2 (PGE2) formation in the rat tracheal epithelial cell line EGV-6aigT, which can be grown in serum-free medium. The addition of TPA to cells that were prelabeled with [3H]arachidonic acid did not enhance the release of [3H]arachidonic acid and/or [3H]PGE2, indicating that TPA does not stimulate phospholipase activity. The addition of exogenous arachidonic acid to cells pretreated with TPA resulted in increased PGE2 formation, compared with basal levels, indicating an elevation in prostaglandin H synthase (PHS) activity. PHS activity was maximal at 4 hr and was dependent upon the concentration of TPA. Actinomycin D and cycloheximide blocked the TPA response. The recovery of PHS activity of cells in which the existing PHS was inhibited by aspirin was enhanced by TPA treatment. TPA treatment enhanced the expression of PHS mRNA, as measured by Northern analysis. The addition of actinomycin D and cycloheximide reduced the TPA enhancement of PHS mRNA, indicating that the increase in PHS activity required de novo RNA and protein synthesis. Furthermore, pretreatment of the cells with protein kinase C inhibitors reduced the TPA-dependent stimulation of PHS activity and the expression of PHS mRNA. The data suggest that TPA-stimulated de novo synthesis of PHS is mediated by protein kinase C.     

Differential inhibition of epidermal growth factor binding by photoactivated psoralens and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in cells overexpressing protein kinase C.

A rat fibroblast cell line, R6PKC3, that stably overexpresses the beta-1 form of protein kinase C was used to analyze sensitivity to inhibitors of epidermal growth factor (EGF) binding. R6PKC3 cells overexpress protein kinase C activity 53-fold relative to non-overexpressing control R6C1 cells. Inhibition of EGF binding by the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) and photo-activated psoralens was compared in these cells. We found that 125I-EGF bound both of the cell lines and was rapidly internalized in a temperature-dependent process and metabolized. Binding of EGF to the R6 cells overexpressing protein kinase C was markedly less than binding to R6C1 control cells. In both of the cell lines, TPA and photoactivated psoralens inhibited 125I-EGF binding but the response of these cells to these inhibitors was distinct. R6PKC3 cells were markedly more sensitive to TPA and were resistant to recovery from TPA-induced inhibition of 125I-EGF binding when compared to control cells. These differences were not observed in other subclones of cells overexpressing protein kinase C, suggesting that they may be unique to R6PKC3 cells. In contrast, no major differences in sensitivity to photoactivated psoralens were observed in R6C1 and R6PKC3 cells. These data indicate that TPA and photoactivated psoralens inhibit 125I-EGF binding to these cell lines by distinct mechanisms.     

Aluminofluoride- and epidermal growth factor-stimulated DNA synthesis in MOB 3-4-F2 cells.

In attempt to study the mechanism of F(-)-induced, osteoblast-mediated bone formation, we tried to show the characteristics of Al-F complex-induced mitogenesis in osteoblastic cells. The MOB 3-4-F2 cell line, an osteoblast-like cell line derived from neonatal mouse calvaria, responded to F- (1-2 mM) combined with Al3+ and epidermal growth factor (EGF, 0.01-100 ng/ml) with increased DNA synthesis. Of the several types of Al-F complexes, AlF4- is thought to act as a mitogenic factor. On the other hand, NaF at high concentrations (greater than 2 mM) markedly decreased cell viability. The AlF(4-)-stimulated DNA synthesis at least with a delay of 48 hr, while EGF stimulated DNA synthesis within a few hours (4-6 hr). Both 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine, inhibitors of protein kinase C (PKC), further enhanced DNA synthesis in AlF(4-)-treated cells, whereas 12-O-tetradecanoyl-13-acetate (TPA), an activator of PKC, decreased the DNA synthesis. In EGF-treated cells, staurosporine and TPA, but not H-7, decreased DNA synthesis. In addition, indomethacin, an inhibitor of cyclooxygenase, partly inhibited the EGF-induced mitogenesis, which, however, was restored by addition of PGE2. AlF4-, as well as EGF, stimulated the release of arachidonic acid and its metabolites. Indomethacin failed to inhibit the AlF(4-)-induced mitogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of prostaglandin E2 production by platycodin D isolated from the root of Platycodon grandiflorum.

Platycodin D, isolated from the root of Platycodon grandiflorum A. DC. (Campanulaceae) suppressed prostaglandin E2 production at 10 and 30 microM in rat peritoneal macrophages stimulated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Platycodin D3 and oleanolic acid showed no effect at these concentrations. Western blot analysis revealed that the induction of COX-2 protein by TPA was inhibited by platycodin D in parallel with the inhibition of prostaglandin E2 production. Platycodin D showed no direct effect on COX-1 and COX-2 activities. TPA-induced release of [3H]arachidonic acid from pre-labeled macrophages was also not inhibited by platycodin D.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on substance P-induced histamine release from rat peritoneal mast cells.

12-O-tetradecanoylphorbol-13-acetate (TPA, 1 to 30 ng/ml) produced a dose-related inhibition of substance P (SP)-induced histamine release from rat peritoneal mast cells. TPA itself induced some histamine release over this concentration range (maximum release about 20% of total). Maximum inhibition of SP-induced release by TPA required preincubation with TPA for at least 10 min. The inhibitory action of TPA was observed in the absence as well as in the presence of extracellular calcium (0.4 mM). Inhibition of diacylglycerol kinase by R 59022 or of diacylglycerol lipase by RHC 80267 reduced SP-induced histamine release. Oleolylacetylglycerol (OAG, 50 microM) inhibited histamine release induced by SP but was less potent than TPA. It is concluded that protein kinase C activation in rat peritoneal mast cells is associated with inhibition of SP-induced histamine release.     

Analysis of the stimulative effect of thapsigargin, a non-TPA-type tumour promoter, on arachidonic acid metabolism in rat peritoneal macrophages.

1. At concentrations above 10 ng ml-1, the tumour promoter thapsigargin stimulates the release of radioactivity from [3H]-arachidonic acid-labelled macrophages harvested from rat peritoneal cavity. 2. The release of radioactivity from prelabelled macrophages was augmented more than additively when the cells were incubated in the medium containing both thapsigargin (10 ng ml-1) and other tumour promoters (10 ng ml-1), such as 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and aplysiatoxin. 3. Thapsigargin required extracellular Ca2+ for the stimulation of arachidonic acid release, while TPA did not. 4. Cytoplasmic free calcium level was increased by thapsigargin treatment but not by TPA treatment. 5. An inhibitor of protein kinases, H-7 inhibited the effect of TPA dose-dependently, whereas H-7 did not inhibit that of thapsigargin. 6. These results suggest that thapsigargin stimulates arachidonic acid release by a mechanism different from that of TPA, viz by acting as a selective Ca2+ mobilizer, but not by activating protein kinase C as TPA does.     

Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: the role of protein kinase C and calcium mobilization

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with calcium ionophores has been shown to bypass the requisite antigen- or lectin-induced signal for lymphocyte mitogenesis. This suggests that protein kinase C activation and calcium mobilization may be early events required for lymphocyte proliferation. Therefore, the relationship(s) of protein kinase C activation and calcium mobilization to ornithine decarboxylase induction and cellular proliferation were examined in a rat node lymphoma cell line (Nb2) which is dependent upon prolactin (PRL) for mitogenesis. TPA enhanced PRL-stimulated Nb2 node lymphoma cell ornithine decarboxylase induction and [3H]thymidine incorporation. Addition of a calcium ionophore (A23187) to cultures containing TPA plus PRL increased ornithine decarboxylase above PRL alone or PRL plus TPA but inhibited proliferation compared to the PRL plus TPA regimen. Exposure of cells to TPA or TPA plus A23187 increased [3H]thymidine incorporation in a similar manner to that demonstrated for low-dose PRL. However, optimal concentrations were only 20-25% as effective as mitogens as was optimal PRL. Protein kinase C and calmodulin antagonists inhibited PRL-stimulated ornithine decarboxylase induction and proliferation. Ca2+ chelation or cation channel antagonism inhibited both PRL-stimulated responses. The cyclic AMP analogue, 8Br-cAMP, inhibited PRL-stimulated ornithine decarboxylase activity as well as cellular proliferation processes assessed by [3H]thymidine incorporation. Finally, tumor-promoting phorbol esters inhibited 125I-rPRL binding. These data strongly suggest that protein kinase C activation and calcium mobilization are requisite events for PRL-stimulated ornithine decarboxylase induction and cellular proliferation in Nb2 node lymphoma cells. An additional component that is linked to alterations in K+ channeling is also implicated. These data support a role for protein kinase C in PRL-coupled mitogenesis. However, other critical Ca2+ and/or ion-induced events are also required.     

Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells

Ornithine decarboxylase (ODC) activity of Ehrlich carcinoma cells was increased more than 36-fold after being maintained for 3.5 hr in vitro in a special chamber which allowed continuous perifusion with 0.5 mM ornithine; if incubated in vitro without perifusion the ODC activity was, of course, only 9-fold by the same concentration of ornithine. Ornithine withdrawal from the perifusion medium resulted in a decay of enzyme activity observed after 90 min; this decay was prevented by addition of 55 microM pyridoxal to the medium. The 1,4-diamines putrescine, spermidine, spermine, agmatine, histamine, serotonin, tryptamine, chlorpheniramine and harmaline at 55 microM strongly suppressed ODC induction by 0.5 mM ornithine in perifused Ehrlich ascites cells. Methyl derivatives also behave as strong inhibitors of ODC induction. On the contrary, N-acetylation paralleled with a decrease in the inhibition capacity: 55 microM N-acetyl putrescine, N-acetyl serotonin or N-omega-acetylhistamine suppressed ODC induction by ornithine in 66, 64 and 19%, respectively. The addition to the perifusion medium of the same concentrations of 1,3-diamines (1,3-diaminopropane, 1,3-diamino-2-propanol or the alkaloid gramine) as well as 1,5-diamines (1,5-diaminopentane and the antihistamic doxylamine or cimetidine) failed to suppress the induction of ODC activity by ornithine. Interestingly, 1,4-benzenediamine, which strongly inhibits ODC activity when the induced enzyme is assayed in its presence, did not suppress the induction of the enzyme when both 0.5 mM ornithine and 55 microM 1,4-benzenediamine were present in the perifusion medium. The inhibitory capacity in down-regulating ODC is not due to differences in the diamine uptake by the cells. The results suggest that the N-N distance (6A) and the charge of one amino group are important chemical characteristics for regulatory effects.