Evidence that arginine vasotocin inhibits human chorionic gonadotropin and cyclic adenosine 3',5' monophosphate stimulated ovarian steroidogenesis

Lyngbyatoxin A, isolated from a marine blue-green alga, and dihydroteleocidin B, a hydrogenated derivative of teleocidin, induce ornithine decarboxylase in mouse skin. In addition, dihydroteleocidin B was recently shown to be a potent tumor promoter in mouse skin. The present studies demonstrate that both lyngbyatoxin A and dihydroteleocidin B induce increased prostaglandin release and choline turnover in HeLa cells at concentrations of 6–20 ng/ml, with a time course similar to that of the potent phorbol ester tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. Thus these indole alkaloids, although structurally different from phorbol ester tumor promoters, share several properties with the latter compounds.     

Induction of histidine decarboxylase activity in mouse skin after application of indole alkaloids,a new class of tumor promoter

The effects of various promoters in two-step carcinogenesis on the induction of histidine decarboxylase in the skin of mice was investigated. The potencies of various phorbol esters in inducing histidine decarboxylase activity were parallel with their tumor-promoting activities. Indole alkaloids such as dihydroteleocidin B and lyngbyatoxin A, which induced ornithine decarboxylase and promoted tumor development in the skin of mice with the same potency as 12-O-tetradecanoylphorbol-13-acetate (TPA), also induced histidine decarboxylase activity. These results suggest that histamine produced by this inducible histidine decarboxylase may play some role in tumor promotion.     

Stimulation of prostaglandin E2 production by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters in macrophages and its inhibition by cycloheximide

The effects of TPA (12-O-tetradecanoylphorbol 13-acetate)-type and non-TPA-type tumor promoters on prostaglandin E2 production by peritoneal macrophages of rats were examined. Among the TPA-type tumor promoters, aplysiatoxin was most potent in stimulating prostaglandin E2 production followed by dihydroteleocidin B, teleocidin, TPA and debromoaplysiatoxin. Prostaglandin E2 production by aplysiatoxin treatment was stimulated at doses up to 0.1 ng/ml. Palytoxin, a non-TPA-type tumor promoter, also stimulated both prostaglandin E2 production and the release of radioactivity from [3H]arachidonic acid-labeled macrophages. However, the dose required for the expression of these effects by palytoxin was up to 3 pg/ml. It was suggested that the tumor promoters are associated with the activity to stimulate arachidonic acid metabolism, irrespective of their type. Cycloheximide, a protein synthesis inhibitor, inhibited both prostaglandin E2 production and the release of radioactivity from prelabeled macrophages stimulated either by the TPA-type tumor promoters or by the non-TPA-type tumor promoter. It is possible that the tumor promoters may induce the synthesis of some proteins responsible for the stimulation of arachidonate metabolism.     

Effects of various tumor promoters on expression of cartilage phenotypes in rabbit costal chondrocytes in culture

12-O-Tetradecanoylphorbol-13-acetate (TPA), a skin tumor-promoting phorbol ester, and teleocidin and aplysiatoxin, which are potent tumor promoters in mouse skin but are chemically unrelated to phorbol esters, induced change of cultured rabbit costal chondrocytes from a polygonal to a fibroblastic shape and inhibited glycosaminoglycan (GAG) synthesis and metachromatic matrix formation in these cells. The potencies of teleocidin and aplysiatoxin to inhibit GAG synthesis were almost the same as that of TPA. On the other hand, Tween 60 and cantharidin, weak mouse skin tumor promoters, phenobarbital, a liver tumor promoter, and saccharin, a bladder tumor promoter, had no effect on the morphology or GAG synthesis of cultured chondrocytes. Like TPA, teleocidin and aplysiatoxin increased DNA and RNA syntheses of chondrocytes. Parathyroid hormone (PTH) and dibutyryl cyclic AMP reversed the morphological and histochemical changes caused by a 4-day treatment with teleocidin or aplysiatoxin as well as with TPA, reversal being apparent after 2 days. PTH increased intracellular cyclic AMP after 2 min in chondrocytes pretreated with teleocidin or aplysiatoxin as well as with TPA. PTH also increased ornithine decarboxylase [ODC; EC 4.1.1.17] activity in these chondrocytes after 4 h. These results show that retention of responsiveness to PTH is a typical characteristic of chondrocytes dedifferentiated by treatment with TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin. The results also suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of teleocidin- and aplysiatoxin-treated chondrocytes.     

A possible naturally occurring tumor promoter, teleocidin B from Streptomyces.

Dihydroteleocidin B, a derivative of teleocidin B, when painted on mouse skin, caused marked induction of ornithine decarboxylase within 4 hrs. This induction of ornithine decarboxylase was inhibited by painting the skin with 13-cis-retinoic acid one hour before dihydroteleocidin B. Dihydroteleocidin B induced cell adhesion of human promyelocytic leukemia cells (HL-60) to the surface of culture flasks, and inhibited terminal differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. Its effective dose for these actions was comparable to that of the potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate. Teleocidin B seems to be a new type of promoter of carcinogenesis.     

Calcium-dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter

The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two-phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase. Ionophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.     

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.     

Effects of tumor promoters (mezerein, teleocidin and palytoxin) on growth hormone secretion from rat anterior pituitary cells cultured in monolayer

The effects of compounds with tumor promoting activity (mezerein, teleocidin and palytoxin) on rat growth hormone (rGH) release was compared to that of TPA (12-O-tetradecanoyl phorbol-13-acetate). Mezerein and teleocidin both of which are activators of protein kinase C (TPA type tumor promoter), elicited rGH release about 3.5 to 4 fold above control values. The ED 50 was 16 nM for mezerein, 1.1 nM for teleocidin and 1.5 nM for TPA. In contrast to mezerein or teleocidin, a non-TPA type tumor promoter (palytoxin) which does not activate protein kinase C failed to stimulate rGH release. These observations suggest that the activation of protein kinase C is essential in the release of rGH induced by the tumor promoters.     

Distribution of tumor promoters in lipid membranes and changes in membrane structure

The interaction of tumor promoters differing in molecular structure, namely, 12-O-tetradecanoylphorbol 13-acetate (TPA) and teleocidin, with dipalmitoylphosphatidylcholine (DPPC) vesicles was studied. Investigation by Fourier transform infrared spectroscopy clarified the differences between the tumor promoters in the mode of interaction with lipid bilayer membranes. The temperature dependence of the bandwidth of the C-H or C = O stretching absorption of lipid molecules in the presence of tumor promoters relative to that in pure DPPC vesicles indicated that TPA is incorporated into the hydrophobic core of the lipid bilayer membrane whilst teleocidin binds predominantly to the membrane surface. However, both tumor promoters tend to restrict the motion of lipid molecules in membranes. The same conclusion was derived from measurements of steady-state fluorescence polarization, which showed that tumor promoters decreased the membrane fluidity. On the other hand, carboxyfluorescein (CF) leakage from vesicles was enhanced by the addition of TPA below the phase-transition temperature, whereas the effect of teleocidin on steady-state CF leakage was not as significant. It is considered that the difference in the profile of the TPA-induced increase in CF leakage compared to that of teleocidin might be ascribable to a different binding site for each tumor promoter in the membranes.     

Teleocidin and phorbol ester tumor promoters exert similar mitogenic effects on human lymphocytes

The tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) affects a wide variety of cellular functions via its binding to protein kinase C (PKC). The TPA molecule contains a diacylglycerol (DAG)-like structure, which may explain its ability to mimic DAG in PKC activation. Teleocidin (TCD) is a different tumor promoter which can compete with TPA in binding to its cell surface receptors even though structurally unrelated to TPA or DAG. Since TCD may use an additional receptor system and/or be distinguished from TPA in its effect on cells, we compared the effects of TPA and TCD on human peripheral blood lymphocytes (PBL). Both tumor promoters preferentially enhanced cell proliferation of sheep erythrocyte-rosetted lymphocytes, which were enriched for T cells. Additionally, TPA and TCD both induced a high density of cell surface receptors for interleukin 2 (IL2) and transferrin, but not synthesis or production of IL2. However, either of the tumor promoters synergized with T cell mitogens to induce high level IL2 production by PBL. In dose response and kinetic studies, matching concentrations of TPA and TCD induced similar effects in PBL. The results thus demonstrate that TPA and TCD are alike in mitogenic capacity, and suggest that structural similarity between the tumor promoter and DAG, the physiological activator of PKC, is not an essential property for promoting tumors or affecting a wide variety of cellular functions.     

Oncogene-induced transformation of a rat embryo fibroblast cell line is enhanced by tumor promoters.

Rat embryo fibroblast cell line 6 was transfected with plasmid pT24, which contains the activated human bladder c-Ha-ras oncogene, and the cells were grown continuously in the absence or presence of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or teleocidin. The presence of TPA or teleocidin led to a 6- to 14-fold increase in the number of morphologically transformed foci. No transformed foci were seen when rat 6 cells were transfected with the normal c-Ha-ras oncogene in the absence or presence of TPA, or in cells simply treated with TPA or teleocidin. Enhancement of pT24-induced foci was seen even when the addition of TPA was delayed until day 16. In transfection studies with the drug resistance genes gpt and neo, TPA and teleocidin did not increase the number of Gpt+ or Neo+ colonies. When rat 6 cells were cotransfected with pT24 and neo genes and grown in the absence or presence of TPA, the presence of TPA did not increase the yield of Neo+ colonies but caused a fivefold increase in the number of Neo+ colonies that displayed a transformed morphology. Southern blot analyses of DNAs obtained from these clones indicated that TPA treatment did not influence the extent of integration of either the pT24 or neo gene. DNA samples from all of the morphologically transformed cells displayed a characteristic 2-kilobase SacI fragment homologous to pT24 DNA and expressed relatively high levels of the corresponding mRNA. Our findings indicate that in this system tumor promoters do not simply enhanced the process of DNA transfection per se. Thus, this model system may be useful for analyzing synergistic interactions between tumor promoters and activated oncogenes during multistage carcinogenesis. It may also serve as a simple screening test for detecting new tumor promoters.     

Identification of tumor promoters by their inhibitory effect on intercellular transfer of lucifer yellow

The effect of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein, teleocidin, anthralin, the Ca²⁺-ionophore A23187, butylated hydroxytoluene (BHT), dichlordiphenyltrichloroethane (DDT) and phenobarbital (PB) on lucifer yellow transfer in cultures of SV-40-transformed Djungarian hamster fibroblasts was studied. TPA, mezerein, teleocidin, A23187, DDT and BHT exerted a strong inhibitory effect on cell-to-cell dye transfer. Anthralin uncoupled cells in 3 experiments out of 6. PB appeared to enhance lucifer yellow transfer. Sodium nitrite, a substance with unknown promoting activity, effectively uncoupled cells. All the promoters investigated had a reversible effect on the dye transfer. The value of the dye transfer method for promoter screening is discussed.Abbreviations BHT butylated hydroxytoluene - DDT dichlordiphenyltrichloroethane - LY Lucifer Yellow - PB phenobarbital - TPA 12-O-tetradecanoylphorbol-13-acetate     

Cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) in human promyelocytic leukemia cells HL-60

12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumor promoter and is known to induce terminal differentiation of human promyelocytic leukemia cells HL-60 to mature monocytes. To investigate the molecular mechanism of TPA actions, TPA-specific binding proteins in HL-60 were analyzed. Anion exchange high-performance liquid chromatography revealed that HL-60 cells possess TPA-specific binding proteins other than protein kinase C (PKC). One of these TPA-specific binding proteins exists in the cytosolic fraction of HL-60 cells, but translocates into the nuclear fraction of HL-60 cells after the treatment of the cells with TPA. The results suggest that HL-60 cells take up TPA into the nuclei via the TPA-specific binding protein. The TPA-specific binding protein binds TPA, phorbol 12,13-di-butylate, teleocidin B-2, teleocidin B-3, and debromoaplysiatoxin in a mutually competitive manner. However, the protein does not bind to okadaic acid, olivoretin C, retinoic acid, or dioxin. This cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) might play an essential role in the action of tumor promoters.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

Involvement of sulfatide in activation of protein kinase C by tumor promoters

Sulfatide (cerebroside sulfate) activated protein kinase C to the same extent as phosphatidylserine did with the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and debromoaplysiatoxin. Sulfatide and phosphatidylserine both induced specific binding of [3H]TPA to protein kinase C, although the ratios of specific to non-specific [3H]TPA binding to protein kinase C with the two were not the same. It is concluded that sulfatide is involved in activation of protein kinase C by tumor promoters in a slightly different way from phosphatidylserine.     

Analysis of tumor-promoter-induced inflammation in rats: participation of histamine and prostaglandin E2

Inflammatory reactions induced by TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoters, including TPA, teleocidin and aplysiatoxin, and chemical mediators responsible for such inflammatory reactions were analyzed. The tumor promoter dissolved in a 0.8% sodium carboxymethyl cellulose solution was injected into a subcutaneous air pouch preformed on the dorsum of rats. Within 30 min after the injection, vascular permeability as measured by the leakage of labeled albumin into the pouch fluid was increased, with a concomitant increase in histamine level. This increase in vascular permeability was inhibited by a histamine antagonist, pyrilamine, and a serotonin antagonist, methysergide. Vascular permeability at 4 h was not inhibited by pyrilamine or methysergide but was inhibited by a cyclooxygenase inhibitor, indomethacin, with a parallel decrease in the prostaglandin E2 level in the pouch fluid. These results suggest that the TPA-type tumor promoters induce inflammation by the mechanism of mast cell degranulation within a short period, this being followed by the stimulation of arachidonic acid metabolism. The mechanism of the in vivo effect of the TPA-type tumor promoters is discussed and compared with in vitro effects that we have previously reported.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.     

Tumor promoters and diacylglycerol activate the Na+/H+ antiporter of sea urchin eggs

Various tumor promoters (TPA, lyngbyatoxin and aplysiatoxin) and diacylglycerol induced cytoplasmic alkalinization of sea urchin eggs independently of intracellular Ca2+ release. This response stimulated protein synthesis and was blocked by amiloride or a lack of extracellular Na+, procedures which inhibit the Na+/H+ antiporter. These results suggest that the antiporter which is responsible for cytoplasmic alkalinization in sea urchin eggs is activated directly or indirectly by protein kinase C in a Ca2+-independent manner.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.