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Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the bryostatin 1 structure is largely sufficient for bryostatin 1-like activity, provided the molecule also possesses an appropriate PKC binding domain.

As an extension of our earlier structure/activity investigation of resveratrol (1a) cancer cell growth inhibitory activity compared to the structurally related stilbene combretastatin series (e.g., 2a), an efficient synthesis of E-stilstatin 3 (3a) and its phosphate prodrug 3b was completed. The trans-stilbene 3a was obtained using a convergent synthesis employing a Wittig reaction with phosphonium bromide 9 as the key reaction step. Deprotection of the Z-silyl ether 13 gave E-stilstatin 3 (3a) as the exclusive product. The structure and stereochemistry of 3a was confirmed by X-ray crystal structure determination.

Bioassay-guided fractionation of extracts of various plants, marine organisms, and microorganisms has led to the discovery of new natural sources of a number of known compounds that have significant biological activity. The isolation of interesting and valuable cancer cell growth inhibitors including majusculamide C ( 1), axinastatin 5 ( 5), bengazoles A ( 6), B ( 7), and E ( 8), manzamine A ( 10), jaspamide ( 11), and neoechinulin A ( 19) has been summarized.

An investigation of the Phillippine Ampelocissus sp. roots for cancer cell growth inhibitory components led to the isolation of a new acetogenin characterized as 22-epicalamistrin (1) employing primarily 2D NMR and high-resolution mass spectral analysis. Two other antineoplastic constituents proved to be the known acetogenin uvaribonin (2) and chalcone 3. Constituents 1-3 were all found to show significant cancer cell growth inhibitory activity against a panel of human cancer cell lines.

Spongistatin 1 is a macrocyclic lactone polyether from the marine sponge Hyrtios erecta. The aim of this study was to evaluate the in vitro and in vivo antifungal efficacies and mechanism of spongistatin 1. Spongistatin 1 was fungicidal for the majority of 74 reference strains and clinical isolates, including those resistant to flucytosine, ketoconazole or fluconazole, and retained activity in the presence of human serum or at lowered pH. The duration of the postantifungal effect following 1 h exposure to one, four and eight times the minimal inhibitory concentration was strain-dependent. Spongistatin 1 was significantly more efficacious than amphotericin B in reducing kidney infectious burden in a murine model of disseminated candidiasis, and reduced the lung burden in a murine model of pulmonary cryptococcosis. When cryptococcal microtubules were visualized by fluorescence microscopy and iterative deconvolution, spongistatin 1 was shown to disrupt first cytoplasmic and then spindle microtubules in a time- and concentration-dependent manner. Microtubule disruption was accompanied by an abnormal distribution of nuclei in budding cells and an inhibition of cell division, resulting in cells arrested in a large-budded stage. Spongistatin 1 should be pursued as a potential antifungal agent and as a probe to study, major cellular processes.

Bryostatin 1 is a protein kinase C modulator that shows antineoplastic activity in a variety of tumor systems. This study examined the effects of bryostatin 1 administration on papilloma growth in rabbits. Investigations of optimal route, dose, and schedule were performed. Several groups of rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) DNA. Bryostatin 1 was administered i.v., both daily and weekly, and intralesionally both weekly and bi-weekly. Intralesionally dosed papillomas were examined histologically for immune cell infiltration. In weekly and daily i.v. trials, 2.5 and 1.0 microg/kg, respectively, showed the greatest overall reduction in tumor size. Bryostatin 1 administered intralesionally also slowed papilloma growth. Treated lesions had significantly higher numbers of heterophils and eosinophils.

Spongistatin 1, a macrocyclic lactone polyether from the marine sponge Hyrtios erecta, was fungicidal for a variety of opportunistic yeasts and filamentous fungi, including strains resistant to amphotericin B, ketoconazole and flucytosine. In broth macrodilution assays, MICs ranged from 0.195 to 12.5 microg/ml, and minimum fungicidal concentrations ranged from 3.12 to 25 microg/ml. Initial disk diffusion screens with six related macrocyclic lactone polyethers from H. erecta and Spirastrella spinispirulifera, revealed that these polyethers were also antifungal. The fungicidal activity of spongistatin 1 was confirmed in killing kinetics studies, where killing of Candida albicans and Cryptococcus neoformans occurred within 6 and 12 h, respectively. During the killing kinetics experiments, non-treated C. albicans maintained the yeast morphology. However, elongated forms resembling germ tubes were the predominant morphologic form in spongistatin 1-treated C. albicans cultures. The spongistatins show promise as potential antifungal agents and as probes to study fungal morphogenesis and nuclear division.

Separation of two trace cancer cell growth inhibitory (P388 leukemia) fractions from about 1000 kg of wet Gulf of Mexico Bugula neritina (Bryozoa) has led to the isolation of bryostatins 16-18 (2-4). A combination of HRFABMS and high-field (400 MHz) 1H- and 13C-NMR spectral analyses were employed to assign the structures. The three new 20-desoxybryostatins 16 (2), 17 (3), and 18 (4) showed significant growth inhibitory activity (P388 ED50, 2, 9.3 x 10(-3) micrograms/mL, 3, 1.9 x 10(-2) micrograms/mL, and 4, 3.3 x 10(-3) micrograms/mL) against murine P388 lymphocytic leukemia.

Bryostatins have been isolated from diverse Japanese coastal specimens of Bugula neritina guided by inhibitory activity against fertilized sea urchin egg cell division. B. neritina from the Gulf of Aomori, Japan, has been found to contain bryostatin 10 [1b] in high yield (10(-3)%) for this class of compounds. The 1H- and 13C-nmr signals of bryostatin 10 [1b] were reassigned by 2D nmr techniques. The conformation of bryostatin 10 [1b] in solution was revealed by nmr studies. This compound also exhibited activity in a steroidogenesis assay by increasing the production of adrenocortical hormones nearly twofold.

The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. Since dolastatin 10 is itself a noncompetitive inhibitor of vinca alkaloid binding to tubulin, this implies at least three distinct binding sites for the structurally complex and diverse natural products that interfere with each others binding to tubulin and with nucleotide exchange. Spongistatin 1, in contrast to both vinca alkaloids and peptide antimitotic agents like dolastatin 10, does not induce formation of a GTP-independent, morphologically distinctive polymer ("aggregate"). We also examined eight compounds closely related structurally to spongistatin 1 (spongistatins 2-9). The most distinctive in their properties were spongistatins 6 and 8. These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.

A highly cytotoxic macrocyclic lactone polyether has been isolated from a Spongia species and named spongistatin 1. With L1210 murine leukemia cells an IC50 value for cell proliferation of 20 pM was obtained, and an increase in the mitotic index concordant with the decrease in cell number was observed. Kangaroo rat kidney PtK1 cells were examined by indirect immunofluorescence with a spongistatin 1 concentration that caused 50% reduction in cellular protein (0.3 nM) and with a 10-fold higher concentration. These cells displayed mitotic and nuclear aberrations at both concentrations, and intracellular microtubules were reduced in number at the lower concentration and disappeared at the higher. Similar changes in PtK1 cells were observed after treatment with equivalent toxic concentrations of the antimitotic agents colchicine, vinblastine, halichondrin B, and dolastatin 10. Spongistatin 1 inhibited the glutamate-induced polymerization of purified tubulin (IC50 value of 3.6 microM versus 2.1 microM for dolastatin 10 and vinblastine and 5.2 microM for halichondrin B). Spongistatin 1 had no effect on the binding of colchicine to tubulin, but it was a potent inhibitor of the binding of vinblastine and GTP to tubulin. In initial experiments with 5 microM tubulin and 5 microM vinblastine, spongistatin 1 and dolastatin 10 both had IC50 values of 2 microM, whereas halichondrin B had an IC50 value of 5 microM. Spongistatin 1 thus represents a new member of the group of complex natural products that inhibit mitosis by binding in the Vinca alkaloid domain of tubulin.

Data generated in the new National Cancer Institute drug evaluation program, which is based on inhibition of cell growth in 60 human tumor cell lines, were used to compare new compounds with agents of known mechanism of action in terms of their differential cytotoxicity. Two marine natural products, halichondrin B and homohalichondrin B, appeared repeatedly when the data base was probed with known antimitotic agents. We confirmed that both compounds were highly cytotoxic (IC50 values for L1210 murine leukemia cells of 0.3 and 1 nM, respectively), with accumulation of cells arrested in mitosis at toxic concentrations, that both inhibited the polymerization of purified tubulin, and that both inhibited microtubule assembly dependent on microtubule-associated proteins. Limited amounts of homohalichondrin B, the less active agent, were available, so only halichondrin B was studied in detail. Halichondrin B did not interfere with colchicine binding to tubulin, but it was a noncompetitive inhibitor of the binding of vinblastine to tubulin (apparent Ki, 5.0 microM). Halichondrin B was therefore compared with other agents which interfere with the binding of vinca alkaloids to tubulin (vinblastine, maytansine, dolastatin 10, phomopsin A, rhizoxin) in terms of its effects on tubulin polymerization, inhibition of GTP hydrolysis, inhibition of nucleotide exchange, and stabilization of tubulin, as well as the quantitative assessment of its effects on vinca alkaloid binding and inhibition of cell growth. Since halichondrin B was originally isolated from the same organism as the phosphatase inhibitor okadaic acid, and since it is about 50-fold more effective than okadaic acid as an inhibitor of L1210 cell growth, perturbations of cellular microtubules observed following treatment with okadaic acid should be interpreted cautiously.

Phorbol esters inhibit chemically induced differentiation in Friend erythroleukemia cells. This study examines the effect of the macrocyclic lactone bryostatin 1 on phorbol ester responses in a Friend erythroleukemia cell clone, PS 7. In several biological systems, bryostatin 1 was reported to mimic phorbol ester action, including activation of protein kinase C, but in HL-60 cells it blocked phorbol ester-induced differentiation. We report here that bryostatin 1 blocks phorbol ester action in Friend cells (clone PS 7), a second differentiating system. In this system, in contrast to HL-60 cells, the phorbol esters inhibit rather than induce differentiation. Bryostatin 1 restores the differentiation response [50% effective dose, 15 +/- 3.5 nM (SEM)] as well as blocks a second phorbol ester effect, induction of cellular adherence. The inhibition of erythroid differentiation by dexamethasone, a nonphorbol compound whose action presumably is not protein kinase C mediated, is unaffected by bryostatin 1. Although bryostatin 1 inhibits [3H]phorbol 12,13-dibutyrate binding in intact Friend erythroleukemia cell clone PS 7, the mechanism for the antagonism of phorbol ester action by bryostatin 1 in Friend cells cannot be explained by simple competition at the binding site.

The South American Simaba cuspidata Spruce and North Indian Ailanthus grandis Prain were investigated as sources of potentially useful antineoplastic agents. Both of these Simaroubaceae plant species were found to produce 6 alpha-tigloyloxychaparrinone (4a) and the new quassinoid 6 alpha-tigloyloxychaparrin (3b). The latter structure was determined by interpretation of spectral data and oxidation to 6 alpha-tigloyloxychaparrinone (4a). While both glycol 3b and alpha-ketol 4a were found to significantly inhibit growth of the murine P388 lymphocytic leukemia cell line, only the alpha-ketol (4a) inhibited growth of the corresponding in vivo system.

The bryostatins, a group of macrocyclic lactones isolated on the basis of their antineoplastic activity, protein kinase C in vitro and block phorbol ester binding to this enzyme. In some cellular systems, bryostatins mimic phorbol ester action. In other systems, however, the bryostatins display only marginal agonistic action and, instead, inhibit phorbol ester-induced responses. At least in primary mouse epidermal cells, a transient duration of action of bryostatin 1 could rationalize these differences. To determine whether this model of transient activation could explain the dual actions of bryostatin 1 in other cell systems, we have examined the effects of bryostatin 1 on short-term responses in C3H 10T1/2 mouse fibroblasts. Even at very short exposures (30 min), bryostatin 1 blocked phorbol ester-induced arachidonic acid metabolite release and induced only minimal release when assayed alone. In contrast, epidermal growth factor binding was markedly and rapidly decreased in bryostatin 1-treated C3H 10T1/2 cells, and this decrease showed only limited reversal 16 h after initial exposure. Bryostatins 2, 3, 4, 10, and several of their derivatives caused variable arachidonic acid metabolite release (10 to 60% of phorbol ester control) and correspondingly variable inhibition of phorbol ester action. Our findings on arachidonic acid metabolite release argue against transient activation of the protein kinase C pathway as the sole explanation of bryostatin 1 action. They indicate, moreover, differences in the structure-activity relations of the bryostatins for the phorbol ester-mimetic and phorbol ester-inhibitory actions.

The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3H]bryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structure-activity relations for the bryostatin class of compounds provide additional support for the validity of the model.

Bryostatin 1, like the phorbol esters, activates protein kinase C. However, bryostatin 1 induces only some of the effects in cultured cells which result from phorbol ester treatment, whereas it blocks other responses to the phorbol esters. In mouse keratinocytes in particular, bryostatin 1 induces ornithine decarboxylase, a marker of proliferation, but blocks induction of markers of differentiation. Because of the postulated role of induction of differentiation in tumor promotion, we have now examined bryostatin 1 as a tumor promoter and as an inhibitor of phorbol ester tumor promotion in the initiation-promotion model of skin carcinogenesis. After initiation with 7,12-dimethylbenz[a]anthracene, weekly topical treatments of the backs of mice with 1 microgram (1.1 nmol) bryostatin 1 induced epidermal hyperplasia and inflammation, although not to the extent seen after treatment with the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment with bryostatin 15 min before each TPA exposure reduced the phorbol ester-induced hyperplasia. Bryostatin 1 was ineffective as a complete tumor promoter and displayed very weak activity as a second stage promoter upon treatment of initiated mice for 30 weeks. Combined exposure of mice to bryostatin 1 and TPA resulted in a substantial inhibition of promotion by TPA. Our in vivo results extend earlier in vitro findings that bryostatin 1 acts as a partial inhibitor of protein kinase C function.

A fifteen year investigation of marine animal components as sources for new and potentially useful cancer chemotherapeutic drugs has led to our discovery of a number of such valuable substance. The especially productive Indian Ocean sea hare Dolabella auricularia has yielded (100 kg leads to or approximately 1 mg each) a series of very potent cell growth inhibitory substances designated dolastatins 1-9. The first member of this new series, dolastatin 1, may represent the most potent anticancer agent so far uncovered with, e.g., a curative response (33%) using a dose of 11 microgram/kg (T/C 240, to T/C 139 at 1.37 microgram/kg) in the National Cancer Institute's murine B16 melanoma. Structural elucidation of the new antineoplastic agents is underway, and recent progress is illustrated with peptide dolastatin 3 (P388 ED 50 2.7 x 10(-7) microgram/ml).

An x-ray crystal structure determination of a dinostanol from the dinoflagellate Protoceratium reticulatum and zooxanthellae from Orbulina universa was completed. The novel sterol was shown to be 3 beta-hydroxy-4 alpha, 23R,24R-trimethyl-5 alpha-cholestane and may be one of the molecular fossils found in sediment cores from the deepest Black Sea trench.

The desert-marigold Baileya multiradiata Harv. and Gray (Compositae) has been found to produce a series of sesquiterpenes that markedly inhibit growth of the murine lymphocytic leukemia P388 and the corresponding in vitro cell line. Separation guided by bioassay led to the isolation of baileyin (1), fastigilins B and C (3a and 2a), radiatin (3b), pleniradin (4a), and the new pseudoguaianolide multiradiatin (2b). The most significant antitumor activity was displayed by fastigilin C (2a) and radiation (3b).

The macrocyclic lactone bryostatin 1 activates protein kinase C as effectively as the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Nevertheless, there are only certain TPA-effects that can be induced by bryostatin 1. These include stimulation of epidermal DNA synthesis and alkaline phosphatase activity in vivo as well as activation of the Ca2+-independent, phospholipid-requiring phosphorylation of an epidermal protein in a cell-free system. Various other TPA-effects in vivo and in vitro, which are not mimicked by bryostatin 1 can be inhibited by applying bryostatin 1 30 min prior to TPA. TPA-effects suppressible by bryostatin 1 include the Ca2+-dependent stimulation of arachidonic acid and prostaglandin E2 release, of ornithine decarboxylase (ODC) activity and ODC-mRNA expression and of transglutaminase activity in keratinocytes in vivo and/or in vitro and, in addition, Epstein-Barr virus induction in Raji cells. The same is true for the conversion step (first stage of promotion) of multistage carcinogenesis. In contrast to the TPA induction of arachidonic acid and prostaglandin E2 release and of transglutaminase activity, induction by the Ca2+-ionophore and by high Ca2+-shift, respectively, are not significantly inhibited by bryostatin 1. We suggest that bryostatin 1 might inhibit a specific 'Ca2+-component' of TPA action.

An improved procedure based on centrifugation at 37,000 g has been developed for isolating the antineoplastic glycoprotein strongylostatin 1 from Strongylocentrotus droebachiensis (Müller). The green sea urchin has also been found to contain a second protein anticancer constituent designated strongylostatin 2. Treatment of the murine P388 lymphocytic leukemia with strongylostatin 2 resulted in 39-42% life extension at a dose of 4.5 mg/kg.

This study examines the action of phorbol 12-myristate 13-acetate (PMA) on the synthesis of cholesterol sulfate in cultured normal and transformed human epidermal keratinocytes and assesses the antagonistic effects by retinoids and bryostatins on PMA action in relation to the multistep program of squamous differentiation. Treatment of normal human epidermal keratinocytes (NHEK) with PMA induces terminal cell division (irreversible growth-arrest) and causes a time- and dose-dependent increase in the incorporation of Na2(35)SO4 into cholesterol sulfate, a marker for squamous cell differentiation. This stimulation in sulfate incorporation appears specific for cholesterol sulfate and is due to increased levels of cholesterol sulfotransferase activity. The increase in cholesterol sulfate accumulation parallels the increase in transglutaminase type I, another marker for squamous differentiation. Several transformed NHEK cell lines do not exhibit increased levels of cholesterol sulfate and transglutaminase type I activity after PMA treatment, indicating that they acquired defects in the regulation of squamous differentiation. Bryostatins 1 and 2, and several diacylglycerol analogues neither inhibit cell proliferation nor increase cholesterol sulfate synthesis or transglutaminase activity, indicating that these agents do not induce terminal differentiation. In contrast, the bryostatins block the increase in cholesterol sulfate and transglutaminase activity as well as the commitment to terminal cell division by PMA. Bryostatin 1 inhibits the commitment to terminal cell division and the accumulation of cholesterol sulfate significantly even when added 8 h after PMA administration. Retinoids inhibit cholesterol sulfate accumulation and the increase in transglutaminase activity by PMA but do not affect the commitment to terminal cell division. In summary, phorbol esters induce in NHEK cells a program of squamous differentiation. This process of differentiation consists of the commitment to terminal cell division and expression of a squamous phenotype. Expression of this phenotype is accompanied by an accumulation of cholesterol sulfate and increased cholesterol sulfotransferase activity. Bryostatins 1 and 2 and retinoic acid affect this differentiation process at different stages.

The bryostatins, macrocyclic lactones isolated on the basis of their antineoplastic activity, activate protein kinase C in vitro and inhibit phorbol ester binding to the enzyme. In intact cells, the bryostatins induce some phorbol ester responses, such as neutrophil activation, but paradoxically they not only fail to induce other responses, e.g., differentiation in HL-60 promyelocytic leukemia cells, but actually block response to the phorbol esters. We compare here bryostatin I and phorbol 12,13-dibutyrate as inhibitors of cell-cell communication in cultured primary mouse epidermal cells. Like phorbol 12,13-dibutyrate, bryostatin I at nanomolar concentrations markedly inhibited cell coupling. It differed from the phorbol esters, however, in that its action was more transient. By 4 h of incubation bryostatin 1 caused little inhibition of coupling. Moreover, coincubation of bryostatin 1 and phorbol 12,13-dibutyrate gave no greater response at this time than that found for bryostatin 1 alone. Time-dependent inhibition of the protein kinase C pathway could account for many of the observed differences between the actions of the phorbol esters and bryostatin 1.

The antineoplastic constituents of the marine ascidian Aplidium californicum were found to be bryostatins 4 and 5. Bioassay-directed isolation procedures using the National Cancer Institute's P-388 lymphocytic leukemia system led to the bryostatins. The probable symbiotic relationship between A. californicum and the bryozoan Bugula neritina was discussed.

The effect of bryostatin 1 on [26-3H]phorbol 12,13-dibutyrate [( 3H]PDBu) binding to a washed particulate preparation from rat brain was examined. Bryostatin 1 inhibited phorbol ester binding at concentrations considerably lower than previously reported. As would be expected for a ligand of high affinity, the apparent displacing potency of bryostatin 1 was dependent on the concentration of tissue/binding sites included in the assay. Decreasing the concentration of [3H]PDBu binding sites to the picomolar detection limit resulted in apparent bryostatin displacing potencies in the picomolar range with these values representing an upper estimate of the true affinity. When included in saturation studies with [3H]PDBu, bryostatin 1 displayed mixed competitive/non-competitive inhibition. Using either repetitive washing or dialysis of the membrane preparation, it was not possible to reverse the inhibition produced by bryostatin 1. The greater affinity of bryostatin 1 compared to other classes of agents that act directly on protein kinase C and the stability of its association may contribute to the unique biological properties of the bryostatins.

A structure/activity study of selected limonoids from two plant families (Meliaceae and Rutaceae) of the Rutales order against the murine P-388 lymphocytic leukemia system was undertaken. The presence of both a 19 leads to 28 lactol and a 14,15 beta-epoxide group was found especially important for pronounced inhibition of the PS in vitro cell line. Substitution of an A-ring alpha,beta-unsaturated ketone (3-oxo-1-ene) for the lactol led to diminished activity, while reduction of the olefin caused complete loss of activity. At the dose levels employed, even very good PS in vitro inhibition was not translated into PS in vivo antineoplastic effects.

Bryostatin 1, a macrocyclic lactone, functions like the phorbol esters biochemically in binding to and activating protein kinase C. Biologically, however, although it induces some phorbol ester responses such as mitogenesis in Swiss 3T3 cells, it paradoxically blocks the effects of the phorbol esters on differentiation in HL-60 promyelocytic leukemia cells and Friend erythroleukemia cells. Since the phorbol esters induce proliferation and terminal differentiation in distinct subpopulations of epidermal basal cells, we have now examined the action of bryostatin 1 in that system. Bryostatin 1 decreased epidermal growth factor binding and induced ornithine decarboxylase activity, the latter a marker of proliferation. The magnitude of the maximal induction of ornithine decarboxylase was less than for phorbol 12,13-dibutyrate. Bryostatin 1 only transiently caused the morphological change typical of phorbol ester treatment and did not induce transglutaminase or cornified envelope production, markers of the differentiative pathway. Combined treatment with bryostatin 1 and phorbol 12,13-dibutyrate gave similar results to treatment with bryostatin 1 alone, i.e., slight reduction to complete inhibition of phorbol ester action, depending on the response. The mechanism may reflect time dependent block of the protein kinase C pathway by bryostatin 1 in this system; although bryostatin 1 inhibited epidermal growth factor binding at short incubation times (1-2 h), by 4 h of incubation its inhibition was markedly reduced and it correspondingly blocked inhibition of epidermal growth factor binding by phorbol 12,13-dibutyrate. Since induction of terminal differentiation is proposed to be an essential component of phorbol ester mediated tumor promotion in skin, our findings suggest that bryostatin 1 may function as an inhibitor of phorbol ester promotion.