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Teleocidin, which was isolated from mycelia of Streptomyces, is a potent tumor promoter in mouse skin. The catalytically hydrogenated compound dihydroteleocidin B markedly enhanced malignant cell transformation induced by 3-methylcholanthrene or ultraviolet radiation. Dihydroteleocidin B was at least 100 times more effective in enhancing transformation than 12-O-tetradecanoyl phorbol-13-acetate, the strongest promoter known until now, whereas both promoters showed equal capacities to induce early membrane effects and DNA synthesis.

Teleocidin and 12-O-tetradecanoylphorbol-acetate (TPA), two potent tumor promoters, are also excellent mitogens for 3T3 cells. I now demonstrate that teleocidin is unable to stimulate mitogenesis in TPA non-proliferative 3T3 variants that retain TPA receptors.

(-)-Indolactam-V, which has the partial structure of teleocidins A and B, and has tumor-promoting activity, is a good model for use in studies on the relation between structure and tumor-promoting activity, whereas (+)-indolactam-V has no tumor-promoting activity. In this work, five racemic indolactams differing only in their alkyl group at C-12 of (-)-indolactam-V were synthesized and tested for biological and biochemical activities related to tumor promotion. The activities tested were inductions of ornithine decarboxylase in mouse skin and human promyelocytic leukemia (HL-60) cell adhesion, inhibition of specific [3H]12-O-tetradecanoyl-phorbol-13-acetate binding to a mouse particulate fraction and activation of protein kinase C in vitro. The results showed that (+/-)-indolactam-L and (+/-)-indolactam-F had almost the same activities as (+/-)-indolactam-V, suggesting that (-)-indolactam-L and (-)-indolactam-F are new tumor promoters with as high potency as (-)-indolactam-V. (+/-)-Indolactam-t-L, which has a highly lipophilic group at C-12 of (-)-indolactam-V, showed the highest activities in the above tests. (-)-Indolactam-t-L might have stronger tumor-promoting activity than (-)-indolactam-V. Furthermore, the results with (-)-indolactam-t-L indicated the possibility of designing new tumor promoters with stronger activity than teleocidin.

In the two-stage model of skin carcinogenesis, it is believed that initiators bind to DNA and that tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA) bind noncovalently to membrane-associated high-affinity receptors, probably protein kinase C. Two other types of potent tumor-promoting substances, aplysiatoxin and teleocidin, appear to act also by binding to and activating protein kinase C, even though their chemical structures are quite different. Therefore, we have undertaken computer modeling of the special relationship of various functional groups in these three chemical classes of tumor promoters in an attempt to explain how these diverse structures bind to the same receptor molecule. We propose a stereochemical model in which the oxygens in TPA at C-3, C-4, C-9, and C-20 (O-3, O-4, O-9, and O-20) correspond to the O-11, N-13, N-1, and O-24 positions in teleocidin and the O-27, O-3, O-11, and O-30 oxygens in aplysiatoxin, respectively. In this model all distances with respect to overlap of the corresponding atoms are less than 1 A. In addition, all three types of molecules have their hydrophobic moieties oriented in a similar position. This model is further discussed with respect to other compounds showing various degrees of activity as tumor promoters, including mezerein, ingenol, and 4 alpha-TPA. The model explains how chemically diverse structures can have similar biological activity as tumor promoters and provides a basis for designing both agonists and antagonists of tumor promoters.

We determined the mitogenic and comitogenic properties of tumor promoters in the indole alkaloid series; agents that differ structurally from 12-0-tetradecanoylphorbol-13-acetate (TPA), which is known to be a lymphocyte mitogen. Teleocidin and dihydroteleocidin B were mitogenic for human lymphocytes, and lyngbyatoxin A elicited little or no mitogenesis. Catalase enhanced the mitogenicity of teleocidin and dihydroteleocidin B, and lyngbyatoxin A was also mitogenic in the presence of catalase. The potency of the agents was TPA = teleocidin greater than dihydroteleocidin B greater than lyngbyatoxin A. Dimethylsulfoxide and butyric acid markedly inhibited proliferation induced by these agents in human lymphocytes. The indole alkaloid tumor promoters were all comitogenic for murine thymocytes and induced production of interleukin-2. While the comitogenic effect of teleocidin was similar to that of TPA in its susceptibility to inhibition by dimethylsulfoxide and butyric acid, the comitogenic effect of dihydroteleocidin B and lyngbyatoxin A were less susceptible to these agents. These findings may facilitate the identification of cellular sites responsible for the inhibitory effect of differentiating agents on proliferative responses of lymphocytes.

The new potent tumor promoters teleocidin and debromoaplysiatoxin , which are structurally unrelated to phorbol esters, activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). The concentrations of 12-O-tetradecanoylphorbol-13-acetate, teleocidin and debromoaplysiatoxin for half-maximum activation of protein kinase C were found to be approximately 3 ng/ml, 40 ng/ml and 400 ng/ml, respectively. These three types of tumor promoters bind to protein kinase C, and appear to exhibit their pleiotropic actions through activation of this enzyme.

A tumor promoter teleocidin induced insulin secretion from isolated pancreatic islets in a concentration-dependent manner. The teleocidin-induced secretion was inhibited by p-bromophenacyl bromide, nordihydroguaiaretic acid, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone, but not by indomethacin. Insulinotropic concentrations of teleocidin stimulated 6-keto-prostaglandin F1 alpha release from pancreatic islets. These results suggest that phospholipase A2 activation and lipoxygenase product(s) are involved in the mechanism of teleocidin-induced insulin secretion.

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.

The effects of tumor promoters on the expression of murine leukemia virus (MuLV) were studied in tissue culture. Dihydroteleocidin B, an indole alkaloid, recently found to be a tumor promoter, enhanced not only the production of Moloney MuLV (M-MuLV) by a mouse fibroblast cell line, C3H2K, persistently infected with M-MuLV, but also growth on the C3H2K cells. The production of infectious M-MuLV by M-MuLV-infected C3H2K cells that had been treated with dihydroteleocidin B for 1-7 days was four or five times higher than that of control cells. C3H2K cells grew faster and became stationary at higher cell densities in the presence of dihydroteleocidin B than in its absence. The tumor-promoting phorbol ester phorbol-12, 13-didecanoate and 12-O-tetradecanoylphorbol-13-acetate also enhanced the production of M-MuLV, but their effects were not so strong as that of dihydroteleocidin B. These tumor promoters, however, did not induce production of endogenous MuLV in C3H2K or K-BALB cells.

Phorbol diester tumor promoters are potent cocarcinogens which also possess activity in a variety of biological assays. We have examined the effect of phorbol diesters on secretion of somatostatin-like immunoactivity (SRIF-LI) by dispersed cells of fetal rat brain maintained in long term culture. Phorbol-12-myristate-13-acetate (PMA) and phorbol 12, 13-dibutyrate stimulate SRIF-LI secretion in a dose-dependent fashion. 4-O-Methyl-PMA is approximately 100 times less potent than phorbol 12, 13-dibutyrate. 4-beta-Phorbol was inactive. Treatment with a nonphorbol irritant, teleocidin, also was associated with significantly augmented release of SRIF-LI. Significant stimulation is seen within 7.5 min of treatment and response is linear over 1 h. Administration of phorbol diesters in low calcium buffer (0.1 mM) with or without cobalt or pretreatment with verapamil are associated with significantly diminished secretion. Substitution for sodium ion by choline or pretreatment with tetrodotoxin (10(-7) M) also inhibits response to PMA. gamma-Aminobutyric acid (50 microM) or the gamma-aminobutyric acid agonist muscimol (5 microM) decrease response to PMA as does sodium pentobarbital (IC50 approximately 30 microM). Phenobarbital is less potent as an inhibitor; significant suppression is not seen until approximately 300 microM. The data are consistent with an action for phorbol diesters mediated at least in part by voltage dependent sodium channels and calcium influx into excitable cells. Inhibition by hyperpolarizing agents is compatible with this mechanism. Phorbol diesters may thus mimic endogenous modulator substances active at the nerve cell membrane.

The synthesis of a unique protein with a molecular weight of 32,000 (p32) in BALB/c 3T3 cells has been shown previously to increase after treatment with potent tumor-promoting phorbol esters (Hiwasa et al., Proc. Natl. Acad. Sci. U. S. A., 79: 1800, 1982). In the present study, two new classes of tumor promoters which are structurally different from phorbol esters were investigated for their potencies to enhance p32 synthesis. Teleocidin, dihydroteleocidin B, and lyngbyatoxin A, which are indole alkaloid tumor promoters, enhanced p32 synthesis to the same extent that 12-O-tetradecanoylphorbol-13-acetate did. However, no increase was observed by treatment with the biologically inactive hydrolysate of teleocidin. Polyacetate tumor promoters such as aplysiatoxin and debromoaplysiatoxin also stimulated p32 synthesis, but their effective concentrations were higher than those of 12-O-tetradecanoylphorbol-13-acetate. When 3T3 cells were treated with a combination of two of the three tumor promoters, TPA, teleocidin, and aplysiatoxin, no synergistic effect of p32 synthesis was observed. This implies that these tumor promoters enhance the synthesis of p32 through the same mechanism.

Aplysiatoxin is a new class of potent tumor promoter and debromoaplysiatoxin may be a weak promoter. Aplysiatoxin and debromoaplysiatoxin showed the same potency on irritation of mouse ear and induction of ornithine decarboxylase activity on mouse skin, but debromoaplysiatoxin was much weaker than aplysiatoxin in induction of adhesion of HL-60 cells.

A strong skin irritant, lyngbyatoxin A, isolated from the marine blue-green alga Lyngbya majuscula is structurally related to teleocidin. Since lyngbyatoxin A satisfied our short-term screening tests for possible tumor promoters, viz. irritation of mouse ear, induction of ornithine decarboxylase (ODC) in mouse skin, and adhesion of human promyelocytic leukemia cells (HL-60), a two-stage carcinogenesis experiment was carried out. Tumor incidences in the groups treated with 7,12-dimethylbenz(a)anthracene (DMBA) plus lyngbyatoxin A and with DMBA plus 12-O-tetradecanoylphorbol-13-acetate (TPA) were 86.7% and 93.3% in week 30, respectively. The average number of tumors per mouse was 3.7 in the former group and 10.5 in the latter group. This paper reports for the first time the potent tumor-promoting activity of lyngbyatoxin A and also the histological examination of tumors.

The diterpene diester phorbol 12-myristate 13-acetate and the alkaloid teleocidin B are structurally unrelated natural products that display similar potent irritant and tumor-promoting activities. Computer modeling of these and other structural classes of tumor promoters show a marked similarity in the relative positions of certain heteroatoms and hydrophobic groups. For phorbol this mapping consists of the C-4, C-9, and C-20 hydroxyl groups as well as a hydrophobic region filled by a long-chain acyl functionality attached to either the C-12 or the C-13 positions. Diacylglycerols, thought to be the endogenous activators of the major phorbol ester receptor protein kinase C likewise fit this model in a stereospecific fashion. As an initial test of the utility of the model, members of a new and simplified class of activators were synthesized that possess the predicted essential structural features. These compounds all inhibited specific phorbol ester binding to protein kinase C, albeit with low affinity (10-60 microM); further analysis of one derivative, decylhydroxylindole, confirmed that the inhibition of phorbol ester binding was competitive. This same derivative inhibited epidermal growth factor binding in intact Swiss 3T3 cells and studies with another derivative showed phosphorylation of a 40-kDa protein in platelets. Both of these in vivo responses are characteristic of phorbol esters.

The tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-didecanoate, teleocidin, and dihydroteleocidin, at nM levels, but not the non-tumor-promoting 4 alpha-phorbol-12,13-didecanoate even at microM concentrations, stimulated arachidonic acid metabolism in cultured rat liver cells. These liver cells synthesize primarily prostaglandin I2 [measured as its nonenzymatic hydrolytic product, 6-keto-prostaglandin F1 alpha (PGF1 alpha)]. The production of 6-keto-PGF1 alpha increased with time of incubation with TPA and was essentially complete in 4 hr. Cycloheximide, at nM levels, blocked the TPA-stimulated 6-keto-PGF1 alpha production in a dose-dependent manner; this inhibition was related to inhibition of protein synthesis. Chelation of Ca2+ by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, treatment of the cells with the Ca2+ channel blocker, nifedipine, or inhibition of intracellular Ca2+ mobilization by 8-(diethylamine)octyl-3,4, 5-trimethoxybenzoate hydrochloride also inhibited TPA-stimulated 6-keto-PGF1 alpha production. The steroidal antiinflammatory drug, dexamethasone, a potent in vivo inhibitor of tumor promotion, was an inhibitor of 6-keto-PGF1 alpha stimulation by TPA.

Teleocidin, a new potent tumor promoter, produces biological effects similar to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on human lymphoblastoid cell lines. A wide range of concentrations (4-16 ng/ml) of teleocidin were optimal for induction of replication of latent Epstein-Barr virus (EBV) genomes in P3HR-1 cells; cell growth was significantly inhibited in this dose range. In contrast, treatment of Raji cells with an identical dose range of teleocidin did not induce replication of latent EBV genomes. As with TPA, P3HR-1 cells in the stationary phase were twice as responsive to teleocidin induction as exponentially growing cells. The activation of P3HR-1 cells both by teleocidin and TPA greatly enhanced the synthesis of EBV DNA and EBV-associated polypeptides as determined by cRNA - DNA hybridization and by analysis on polyacrylamide gels. Both drugs also increased production of biologically active virus as monitored by the synthesis of viral DNA in superinfected Raji cells. These effects were completely inhibited by retinoic acid. However, retinoic acid did not inhibit the spontaneous viral DNA replication that occurs in P3HR-1 cells not treated with the inducers. Thus, it appears that retinoic acid antagonizes the inducing effects of TPA and teleocidin, but not viral replication itself.

The potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) and related diterpene phorbol esters have been shown to enhance viral transformation and anchorage-independent growth, inhibit differentiation, and stimulate phosphatidylcholine turnover in various cell culture systems. In the present study, we report that teleocidin, and indole alkaloid isolated from Streptomyces, induces several biological effects similar to those of TPA in cell culture. Both TPA and teleocidin enhanced transformation of a clone of Fischer rat embryo cells (CREF) by a temperature-sensitive mutant of adenovirus type 5 (H5ts125); enhanced the cloning efficiency in agar of E11 cells, a clone of H5ts125-transformed Sprague-Dawley rat embryo cells; inhibited melanogenesis in murine B-16 melanoma cells; inhibited myogenesis in myoblast cultures established from normal human skeletal muscle; and stimulated choline release from prelabeled phospholipids of C3H10T 1/2 mouse cells. In general, TPA and teleocidin were equipotent in inducing these biological effects and were most active in the 3- to 10-ng/ml range, i.e., approximately 10(-8) to 10(-9) M. These studies provide further evidence that teleocidin represents a new class of tumor-promoting agents with properties similar to, if not identical with, those of the phorbol ester tumor promoters. These findings also suggest that cell culture systems can be used to identify new types of tumor-promoting agents in addition to the diterpene phorbol esters.

Phorbol esters and the structurally dissimilar teleocidins and ingenols bind to and activate protein kinase C (PKC) during the course of tumour promotion. These compounds are referred to as TPA-like tumour promoters (from 12-O-tetradencanoyl phorbol-13-acetate, the most active of the class) and are amongst the most potent tumour promoters known. Despite their structural dissimilarity, all three groups of molecules have been shown to bind to the diacylglycerol site of PKC with high affinity. It is thought that this binding to and consequent activation of PKC is the crucial step in tumour promotion by these compounds. The aim of this work was to provide a description of the binding site by comparing structural features (in particular the electrostatic potential) with the activity of numerous derivatives of the three classes. Initially the description was obtained by consideration of the phorbol derivatives, and then refined using the teleocidins and ingenols. The activity data were collected from a variety of sources and the structures calculated using the semi-empirical MNDO approximation embodied in the MOPAC program. Where possible, the crystal structure was obtained from the Cambridge Crystallographic Database, and used as a starting point for the calculation. In other cases, a preliminary calculation was carried out using the molecular mechanics program AMBER. Electrostatic potentials were calculated and displayed using an in-house program 3D2, while superpositions of molecules were carried out using CHEM-X.

In the present paper, recent biochemical studies of retroviruses carried out in our laboratory are summarized. Protein compositions, peptide maps of internal structural proteins, neighborhoods of major structural proteins, and serological properties of reverse transcriptases of type D virus isolates from human cells (including Graffi's isolate termed PMFV and also isolates from HeLa- and HEp-2 cells) were compared with those of type D viruses from Old World (Mason-Pfizer virus of rhesus monkeys, langur virus) and New World (squirrel monkey retrovirus) monkeys. The results provide various new informations on, and further demonstrate the diversity of primate type D viruses. Other studies showed that tumor promoting agents (phorbol ester TPA, indole alkaloid teleocidin) are able to considerably increase, in a transient manner, the production of type C and type D primate retroviruses in persistently infected human cells. From experiments demonstrating a disintegrating activity of chelating agents (EDTA, EGTA) and certain psychoactive drugs (including trifluoperazine) on various primate and nonprimate retroviruses it is concluded that divalent cations, probably Ca2+ ions, and possibly also cation-binding proteins are associated with retroviral membranes and that complexing of these components results in loss of viral infectivity.

Three different diterpen polyols and the teleocidin have antileukemic effects on the human thymic leukemia cell line HPB-ALL by inducing phenotypic differentiation. We tested TPA (phorbol esters), mezerein (daphnane), milliamin (ingenol ester) and teleocidin B (teleocidins) as representative of the chemical structure of each principle and found that these agents have the similar biological activity of inducing phenotypic differentiation in HPB-ALL cells. Moreover, competitive binding of [3H]PDB to cell-surface receptors was significantly inhibited by these agents; n-butyrate and phorbol per se did not inhibit the binding of [3H]PDB. Despite differences in the structure of the principles, these agents may have similarly acting side-chain structures.

Dihydroteleocidin B, which is a derivative of teleocidin from Streptomyces, showed potent tumor-promoting activity in vivo when painted on mouse skin. Although the chemical structure of dihydroteleocidin B is entirely different from those of phorbol esters, the tumor-promoting activity of dihydroteleocidin B was found to be comparable to that of 12-O-tetradecanoylphorbol 13-acetate (TPA) in vivo. Teleocidin from Streptomyces and lyngbyatoxin A and debromoaplysiatoxin from the marine blue-green alga Lyngbya majuscula induced ornithine decarboxylase activity when painted on mouse skin, their effects being similar to those of dihyroteleocidin B and TPA. 13-cis-Retinoic acid inhibited this ornithine decarboxylase induction when painted on the skin 1 hr before these natural products. These three compounds produced adhesion of human promyelocytic leukemia cells (HL-60) to the flasks and inhibited differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. The in vitro biological potencies of teleocidin and lyngbyatoxin A were almost as great as those of dihydroteleocidin B and TPA, but that of debromoaplysiatoxin was much weaker.

Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.

Effects of teleocidin B, 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,7-dichlorodibenzo-p-dioxin (DCDD) on normal human bronchial epithelial cell cultures were assessed by quantitation of cellular morphology, clonal growth (population doublings per day), cross-linked envelope (CLE) formation and the enzymatic activities of aryl hydrocarbon hydroxylase (AHH), ornithine decarboxylase (ODC) and plasminogen activator (PA). Toxicity was assessed by clonal growth assays. Teleocidin B and TPA had similar effects on growth, morphology and enzyme activities. When the cells were incubated with TPA or teleocidin B at concentrations of 1-100 nM for 6 h, RNA synthesis was unaffected, but DNA synthesis decreased and squamous differentiation, marked by an increase in cell surface area and cross-linked envelope formation, was increased. TPA and teleocidin B also increased ODC activity in LHC-0 medium (a maintenance medium without epidermal growth factor) but caused a decrease of ODC activity in LHC-4 (a growth medium containing epidermal growth factor). Finally, TPA and teleocidin B each caused an increase of PA and a decrease of AHH activities in both media. Phorbol, a non-promoting analogue of TPA, had no effect on growth, morphology or biochemical assays. TCDD (100 nM) caused a 15% decrease in cell growth when cells were incubated in LHC-4, and this was accompanied by an increase in cell surface area, PA activity, and CLE formation. TCDD caused an increase in AHH and ODC activities when the cells were incubated in either LHC-0 or LHC-4 medium. DCDD did not alter cell growth, and its morphological and biochemical effects were similar to those of TCDD although less marked. In conclusion, results reported here are consistent with the hypothesis that an important property of some tumor promoters is their ability to induce terminal differentiation in normal, non-initiated epithelial cells.

An indole alkaloid, teleocidin, induced phenotypic differentiation in the thymic ALL cell line HPB-ALL. Within 30 min of seeding in the presence of teleocidin, the cells formed a smooth round shape. Upon 5-day exposure to teleocidin, most of the cells became smaller and reminiscent of large or atypical lymphocytes. A thymic antigen stained with monoclonal antibody OKT6 was remarkably reduced while Leu2a-positive cells were increased in the treated cells. Upon teleocidin-induced phenotypic differentiation, the growth rate of cells was inhibited, their ability to incorporate DNA via [3H]-labelled precursors was reduced, their ability to bind sRBC rosettes was increased. These changes were paralleled to that induced by 12-o-tetradecanoyl-phorbol-13-acetate.

Addition of epidermal growth factor (EGF) to Chang liver cells in a low serum culture causes a long-lasting, two- to three-fold increase in the acetylation of nuclear proteins. This EGF effect is manifested before an increase of cell proliferation in response to EGF. Studies by SDS-polyacrylamide gel electrophoresis and isoelectric focusing show that both histones and acid-insoluble proteins of lower molecular weights are acetylated upon administration of EGF. Tumor promoter teleocidin, which inhibits internalization and nuclear accumulation of EGF, acts antagonistically with EGF in enhancing the acetylation of nuclear proteins. Lysosomal inhibitor chloroquine enhances nuclear accumulation of EGF but has no significant effects on the stimulation of nuclear protein acetylation by EGF. These data appear to suggest that the EGF-enhancement of the acetylation of nuclear proteins is mediated by the steps such as an internalization or nuclear accumulation of EGF or EGF-receptor complex.

Tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA), mezerein, teleocidin, aplysiatoxin, and benzoyl peroxide, although structurally unrelated, induce similar, profound changes in morphology in differentiated epithelial Madin-Darby canine kidney (MDCK) cell colonies. The alteration is evident in the organization of intermediate filaments in intact cells and in whole mounts of the nuclear matrix-intermediate filament (NM-IF) scaffold of the epithelial sheet. This substructure, obtained by salt extraction of the cytoskeletal framework, represents only 5% of the total cell protein but contains all of the intermediate filaments, nuclear matrix, and desmosomal core proteins arranged essentially as in the intact cell. The NM-IF is profoundly reorganized after exposure to TPA and retains the morphological changes observed in intact cells. These include bundling of the intermediate filaments, disruption of cell-cell borders, and marked deformation of the polygonal geometry of epithelia. Thus, TPA and all other complete or second-stage tumor promoters examined have a characteristic morphological signature that is not induced by mitogens, metabolic inhibitors, or agents known to disrupt microtubules or microfilaments. This signature, characteristic of tumor promoters, occurs in the absence of both protein and RNA synthesis. These results suggest that this response is prior to and independent of other biochemical markers for tumor promoters. Of the major filament systems, the cytokeratin network is implicated as an early or possibly primary site of tumor-promoter action because characteristics of the promoted cytoskeletal signature are observed in epithelial colonies after prior exposure to colchicine or cytochalasin D. Despite the massive reorganization of cytoskeletal morphology induced by TPA, the distribution of prelabeled proteins into structural fractions (i.e., cytoskeletal, chromatin, and the NM-IF) remains essentially unchanged. The sensitivity and specificity of the epithelial cell response suggest its possible use as a screen for promoting compounds.

Teleocidin, isolated from Streptomyces mediocidicus ISP 5021, is an indole alkaloid. The teleocidin used was composed of teleocidin A, teleocidin B and their isomers. A hydrogenated derivative of teleocidin B, dihydroteleocidin B, was recently reported to have tumor promoting activity in vivo and various biological activities in vitro, with a specific activity comparable to that of 12-O-tetradecanoylphorbol-13-acetate (TPA). This paper describes the potent tumor promoting activity of the parent compound of dihydroteleocidins, teleocidin, on mouse skin in two-stage carcinogenesis. The tumor promoting activity was evaluated by measuring the incidence and yield of tumors, and by histological examination. Groups of mice were given a single application of 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) and then 2.5 micrograms of teleocidin twice weekly or the same dose of DMBA plus 2.5 micrograms of TPA twice weekly. Both groups showed 100% tumor incidence after 24 weeks, and the tumor yields were 4.0 tumors per mouse in the former group and 9.8 per mouse in the latter group in week 30. We confirmed, through this experiment, that teleocidin is as potent as TPA in in vivo two-stage carcinogenesis in mouse skin. These two structurally unrelated classes, indole alkaloid and phorbol ester, showed tumor promoting activity in almost the same range.

Experiments on the effect of transplacental initiation with 7,12-dimethylbenz(a)anthracene (DMBA) and postnatal promotion with teleocidin were carried out in mice. The percentage of tumor-bearing mice among females treated with DMBA transplacentally on day 17 of gestation and postnatally by topical application of teleocidin to the skin of the back was 73.3% in week 30, whereas that among females treated with DMBA on day 10 of gestation and postnatally by topical application of teleocidin was 20.0%. This indicates that teleocidin shows potent tumor promoting activity on mouse skin in a transplacental initiation and postnatal promotion protocol. Furthermore, in the males treated with DMBA transplacentally on day 17 of gestation and given diet containing 0.01% teleocidin postnatally five tumors of the forestomach were found in 5 of 19 effective mice (26.3%) in week 52. One of these five tumors was a squamous cell carcinoma, and the others were papillomas. This indicates that teleocidin also has tumor promoting activity in the forestomach of mice.

The effect of teleocidin, a new, naturally occurring tumor promoter on induction of Epstein-Barr virus (EBV), was compared with that of a known tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Early antigen (EA) and/or capsid antigen (VCA) of EBV was induced in the EBV genome-carrying cell lines C-6 and P3HR-I cells by teleocidin, its effect being maximal at a concentration of 12.5 ng/ml. The production of infectious EBV from P3-HR-I cells was enhanced by teleocidin maximally at a concentration of 0.5 to 2.5 ng/ml. The outgrowth of EBV-transformed cells from peripheral lymphocytes of seropositive healthy donors was also enhanced by teleocidin at a concentration of 0.02 to 0.5 ng/ml. TPA tested simultaneously in all experiments exhibited the same activities as teleocidin, and was effective at similar concentrations. Teleocidin enhanced both EA and VCA synthesis in P3HR-I cells additively with n-butyrate, but not with TPA. This suggests that teleocidin and TPA have a common mechanism of action, although their chemical structures are different.

Topical application of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3], an active form of vitamin D3, markedly inhibited induction of ornithine decarboxylase caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin B in mouse skin. The degree of inhibition was dependent on the dose and time of application of 1 alpha, 25(OH)2D3. Application of 1 micrograms 1 alpha, 25(OH)2D3 within 30 min before or after treatment with 10 micrograms TPA caused about 72% inhibition of ODC induction at 4 hr. Similar degrees of inhibition were obtained with dose ratios of 1 alpha, 25(OH)2D3 to TPA of 1:3, 1:10, and 1:30. The dose required for 50% inhibition was 0.063 micrograms, or 0.15 nmol, which is about one-half that of retinoic acid, a known inhibitor of induction of ODC activity by TPA. 1 alpha, 25(OH)2D3 had a specific inhibitory effect, in which 100 times higher doses or more of other derivatives of vitamin D3, such as 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, and vitamin D3, were required to inhibit ODC induction by TPA. 1 alpha, 25(OH)2D3 did not inhibit epidermal hyperplasia induced by TPA.

The indole alkaloid tumor promoter dihydroteleocidin B (DHTB) was shown to reduce the natural killer (NK) cell susceptibility of two established cell lines, U937 and K562. The decrease in NK susceptibility correlated with the induction of differentiation as documented by positive benzidine staining in the erythroleukemia K562 and by antibody-dependent cellular cytotoxicity effector function in the histiocytic lymphoma line U937, respectively. In contrast, DHTB treatment did not alter the NK sensitivity of the NK-resistant B-lymphoblastoid-cell line RPMI 8866. Cold target inhibition experiments suggested that both effector-target recognition and post-recognition steps were affected by DHTB. These results lend further support to the notion that NK susceptibility of a given tumor cell may vary with the stage of differentiation.

Twelve aplysiatoxin compounds have been evaluated as possible tumor promoters in vivo by means of three biological tests: viz. irritation of mouse ear, induction of ornithine decarboxylase in dorsal skin of mice, and inhibition of specific binding of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to an epidermal particulate fraction. The potencies of these three biological activities correlate well for each derivative. Bromoaplysiatoxin shows biological activities that are similar to those of the strong tumor promoter, aplysiatoxin. The present studies suggest that the C-3, C-20 and C-30 hydroxyl groups of the aplysiatoxins are involved in binding to the specific receptor of TPA.