Mimicry of bryostatin 1 induced phosphorylation patterns in HL-60 cells by high-phorbol ester concentrations.

The bryostatins are a group of macrocyclic lactones isolated from the marine bryozoan Bugula neritina. Bryostatin 1, like the phorbol esters, activates protein kinase C; however, it partially inhibits the phorbol ester induced differentiation of the human promyelocytic leukemic cell line HL-60. We compared the phosphorylation response in HL-60 cells treated with phorbol 12,13-dibutyrate or bryostatin 1. Bryostatin 1 enhanced the phosphorylation of the same proteins as did typical concentrations (10(-8)-10(-9) M) of phorbol 12,13-dibutyrate. In addition, bryostatin 1 caused the appearance of 2 phosphorylated protein spots with molecular weights of 70,000 and pIs of 6.3-6.4. These latter phosphorylations were evident after a 30-min exposure to bryostatin 1 at 6 nM. Phorbol 12,13-dibutyrate concentrations of at least 600 nM, approximately 100-fold that necessary to induce differentiation, also induced the appearance of these phosphoprotein spots. The Mr 70,000 phosphoproteins were located in the ionic detergent-soluble cellular fraction which would contain the cytoskeletal proteins. Their phosphorylation was almost totally on serine residues. We speculate that phorbol esters at very high concentrations may more closely resemble bryostatin 1.

Differential effects of bryostatins and phorbol esters on arachidonic acid metabolite release and epidermal growth factor binding in C3H 10T1/2 cells.

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.

Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs.

Synthesis and biological activities of the marine bryozoan alkaloids convolutamines A, C and F, and lutamides A and C.

Synthesis of convolutamines and lutamides, new 2,4,6-tribromo-3-methoxyphenethylamine alkaloids isolated from Floridian marine bryozoan Amathia convoluta, was accomplished by a sequence of reactions starting from 3-hydroxyphenethylamines. Cytotoxities of the synthetic lutamides, convolutamines and their de-O-methyl derivatives were examined using drug-sensitive and -resistant P388 as well as KB cell lines. The bioassay suggests that the 2,4,6-tribromo-3-methoxyphenethylamine is an indispensable unit for detection of the activities. Additionally, a reversal of drug resistance by those alkaloids is recognized.

Isolation and structure of five new cancer cell growth inhibitory bufadienolides from the Chinese traditional drug Ch'an Su.

Five new bufadienolides, 3beta-formyloxyresibufogenin (1), 19-oxobufalin (2), 19-oxodesacetylcinobufagin (3), 6alpha-hydroxycinobufagin (4), and 1beta-hydroxybufalin (5), have been isolated together with the previously known bufadienolides 6-20 from the Chinese traditional drug "Ch'an Su". The structures were elucidated employing spectroscopic methods. Bufadienolides 1-5 provided significant inhibitory activity against the KB and HL-60 cancer cell lines. In addition, bufadienolide 1 was found active against the MH-60 cancer cell line.

Binding of [26-3H]-epi-bryostatin 4 to protein kinase C.

Structure activity analysis of protein kinase C modulators may permit design of selective inhibitors of this important regulatory enzyme. Modeling suggests that the C-26 secondary hydroxyl of bryostatin is homologous to the C-20 primary hydroxyl of phorbol or the C-3 primary hydroxyl of sn-1,2-diacylglycerols (Wender, P. A.; Cribbs, C. M.; Koehler, K. F.; Sharkey, N. A.; Herald, C. L.; Kamano, Y.; Pettit, G. R.; Blumberg, P. M. Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C. Proc. Natl. Acad. Sci. USA 85:7197-7201; 1980). We have characterized the binding activity to protein kinase C of the epimer of bryostatin 4 with the configuration at C-26 inverted from R (natural configuration) to S. The Kd of [26-3H]-epi-bryostatin 4 for protein kinase C reconstituted in the presence of phosphatidylserine was 13 +/- 2 nM. [26-3H]-Epi-bryostatin 4 thus retained moderate absolute affinity. Relatively, however, inversion at the chiral center at C-26 caused a dramatic decrease in binding affinity. Binding of [26-3H]-epi-bryostatin 4 was competitively inhibited by phorbol 12,13-diacetate, as expected for ligands that interact at the same binding site.

Antineoplastic agents, 116. An evaluation of the marine ascidian Aplidium californicum.

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.

An improved source of bryostatin 10, Bugula neritina from the Gulf of Aomori, Japan.

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.

Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C.

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.