mezerein and bryostatin-1

Cotreatment with a minimally toxic concentration of the protein kinase C (PKC) activator (and down-regulator) bryostatin 1 (BRY) induced a marked increase in mitochondrial dysfunction and apoptosis in U937 monocytic leukemia cells exposed to the proteasome inhibitor lactacystin (LC). This effect was blocked by cycloheximide, but not by alpha-amanitin or actinomycin D. Qualitatively similar interactions were observed with other PKC activators (eg, phorbol 12-myristate 13-acetate and mezerein), but not phospholipase C, which does not down-regulate the enzyme. These events were examined in relationship to functional alterations in stress (eg, SAPK, JNK) and survival (eg, MAPK, ERK) signaling pathways. The observations that LC/BRY treatment failed to trigger JNK activation and that cell death was unaffected by a dominant-interfering form of c-JUN (TAM67) or by pretreatment with either curcumin or the p38/RK inhibitor, SB203580, suggested that the SAPK pathway was not involved in potentiation of apoptosis. In marked contrast, perturbations in the PKC/Raf/MAPK pathway played an integral role in LC/BRY-mediated cell death based on evidence that pretreatment of cells with bisindolylmaleimide I, a selective PKC inhibitor, or geldanamycin, a benzoquinone ansamycin, which destabilizes and depletes Raf-1, markedly suppressed apoptosis. Furthermore, ERK phosphorylation was substantially prolonged in LC/BRY-treated cells compared to those exposed to BRY alone, and pretreatment with the highly specific MEK inhibitors, PD98059, U0126, and SL327, opposed ERK activation while protecting cells from LC/BRY-induced lethality. Together, these findings suggest a role for activation and/or dysregulation of the PKC/MAPK cascade in modulation of leukemic cell apoptosis following exposure to the proteasome inhibitor LC. (Blood. 2001;97:2105-2114)

Topics: Acetylcysteine; Amanitins; Aminoacetonitrile; Apoptosis; Benzoquinones; Bryostatins; Butadienes; Curcumin; Cysteine Endopeptidases; Dactinomycin; Diterpenes; Drug Synergism; Enzyme Activation; Flavonoids; Humans; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Lactones; Macrolides; Maleimides; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neoplasm Proteins; Nitriles; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Kinase C; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-raf; Pyridines; Quinones; Terpenes; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Type C Phospholipases; U937 Cells; Ubiquitins

The role of the nuclear phosphoprotein c-Myc has been examined with respect to the regulation of 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis in human leukemia cells exposed to bryostatin 1 and other pharmacologic protein kinase C (PKC) activators. Pretreatment of HL-60 cells for 24 hr with 10 nM bryostatin 1 significantly potentiated the ability of ara-C (10 microM; 6 hr) to induce apoptosis without reducing the expression of c-Myc protein. In contrast, equivalent exposure to the stage 2 tumor-promoting PKC activator mezerein (10 nM) in conjunction with ara-C reduced c-Myc levels by 87% and failed to potentiate apoptosis. Co-administration of bryostatin 1 with mezerein before ara-C prevented down-regulation of c-Myc and augmented cell death, whereas co-treatment with the calcium ionophore A23187 (250 nM) and bryostatin 1 reduced c-Myc levels by 80% and abrogated the increase in ara-C-induced apoptosis. When cells were exposed for 24 hr to a c-myc antisense oligonucleotide (AS-ODN;10 microM) but not to a scrambled sequence ODN (SS-ODN) prior to ara-C, c-Myc expression was reduced by 81%, and apoptosis and cell viability were unperturbed. However, AS-ODN (but not SS-ODN) reduced c-Myc protein in cells pre-exposed to bryostatin 1 by 74% and abrogated potentiation of ara-C-induced apoptosis. The actions of c-myc AS-ODN did not stem from proximal G1 arrest/differentiation or biochemical events, since they were not associated with a reduction in the S-phase cell fraction, p21(WAF1/CIP1) induction, pRb hypophosphorylation, or alterations in ara-C metabolism. Together, these findings indicate that HL-60 cell apoptosis proceeds by both c-Myc-dependent and -independent pathways, and that only the former are involved in the potentiation of ara-C-mediated cell death by bryostatin 1.

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; Cell Cycle; Cytarabine; Diterpenes; DNA Fragmentation; Drug Synergism; Enzyme Activation; HL-60 Cells; Humans; Kinetics; Lactones; Macrolides; Oligonucleotides, Antisense; Phorbol 12,13-Dibutyrate; Protein Kinase C; Proto-Oncogene Proteins c-myc; Terpenes; Tetradecanoylphorbol Acetate; Thionucleotides

Previous studies have shown that pretreatment of human myeloid leukemia cells (HL-60) with the protein kinase C (PKC) activator bryostatin 1 potentiates ara-C-induced apoptosis. To test the hypothesis that this capacity stems from down-regulation of PKC activity and/or Ca2+-dependent (group-I; cPKC) isoform expression, comparisons were made between the effects of this agent and the stage-2 tumor promoter mezerein under conditions favoring either cellular differentiation or drug-induced apoptosis. Twenty-four-hour pretreatment of HL-60 cells with 10 nM bryostatin 1, which does not induce differentiation in this cell line, led to a profound reduction in membrane and cytosolic PKC activity, decreased expression of cPKC isoforms (alpha, betaI, betaII, gamma), and a marked increase in ara-C induced apoptosis. In contrast, 10 nM mezerein, which induces HL-60 cell differentiation, was less effective in down-regulating membrane and cytosolic PKC activity as well as alpha, betaI, and gamma cPKC isoform expression, and failed to potentiate ara-C-related apoptosis. The effects of bryostatin 1 were dominant to those of mezerein, in that the combination resulted in down-regulation of PKC activity and expression and potentiation of ara-C-induced apoptosis, but not cellular maturation. However, coadministration of the Ca2+ ionophore A23187 (250 nM) restored bryostatin 1's differentiating ability while antagonizing its capacity to augment apoptosis, despite failing to reverse bryostatin 1-induced down-regulation of PKC activity and cPKC isoform expression. Furthermore, pretreatment of differentiation-responsive monocytic leukemia cells (U937) with bryostatin 1 substantially reduced PKC activity and cPKC isoform expression, but exerted minimal effects on ara-C-related apoptosis. In contrast, exposure of U937 cells to bryostatin 1 after ara-C dramatically increased apoptosis, a phenomenon that did not occur in differentiation-unresponsive HL-60 cells. Collectively, these observations indicate that down-regulation of total assayable PKC activity and cPKC expression by bryostatin 1 are insufficient, by themselves, to account for potentiation of leukemic cell apoptosis, at least under conditions in which differentiation occurs. They also provide further evidence that a reciprocal and highly schedule-dependent relationship exists between leukemic cell differentiation and drug-induced apoptosis.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Bryostatins; Calcium; Carcinogens; Cell Differentiation; Cytarabine; Diterpenes; Enzyme Activation; HL-60 Cells; Humans; Isoenzymes; Lactones; Macrolides; Protein Kinase C; Signal Transduction; Terpenes; Tumor Cells, Cultured

The activity of protein kinase C (PK-C) has been implicated in the regulation of the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C-activating agents phorbol 12,13-dibutyrate (PDBu), mezerein, and bryostatin 1 on the proliferation and lineage commitment of CD34+ human myeloid progenitor cells stimulated by recombinant interleukin-3 (rIL-3) and/or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Although each of the PK-C activators administered alone induced no colony formation, coadministration of these agents with plateau concentrations of each cytokine (eg, 50 ng/mL) increased the number of day 14 granulocyte-macrophage colony-forming units by 100% to 150%. The number of pure and mixed neutrophil and macrophage colonies was substantially enhanced in the presence of PK-C activators, whereas the percentage and, in most cases, the absolute number of eosinophilic colonies was significantly reduced. The inhibition of eosinophilic colony formation was not overcome by the addition of rIL-5. Although addition of bryostatin 1 24 hours before rIL-3 abrogated the increase in total colony formation observed with simultaneous administration of factors, the inhibition of eosinophilic colonies and the increase in neutrophil/macrophage colonies persisted under these conditions. The addition of bryostatin 1 for up to 144 hours after rIL-3 continued to potentiate total colony formation, whereas the inhibition of eosinophilic commitment was lost after 120 hours. Together, these results suggest that pharmacologic interventions at the level of PK-C may regulate both the proliferation as well as the lineage commitment of human hematopoietic progenitors exposed to rGM-CSF and rIL-3.

Topics: Bryostatins; Cell Differentiation; Cell Division; Diterpenes; Enzyme Activation; Eosinophils; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Hematopoietic Stem Cells; Humans; Interleukin-3; Lactones; Macrolides; Phorbol 12,13-Dibutyrate; Protein Kinase C; Recombinant Proteins; Terpenes

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 microM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 x 10(6) cells/mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 microM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 pmol/10(6)), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10-100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 microM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resul

Topics: Antineoplastic Agents; Arabinofuranosylcytosine Triphosphate; Bryostatins; Cell Count; Cytarabine; Diterpenes; DNA, Neoplasm; Humans; Kinetics; Lactones; Leukemia, Experimental; Leukemia, Myeloid; Macrolides; Phorbol 12,13-Dibutyrate; Phosphorylation; Terpenes; Time Factors; Tumor Cells, Cultured

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.

Topics: Animals; Antibodies, Anti-Idiotypic; B-Lymphocytes; Bryostatins; Calcium; Diterpenes; Enzyme Activation; Immunoglobulin delta-Chains; In Vitro Techniques; Indoles; Ionomycin; Lactams; Lactones; Lymphocyte Activation; Macrolides; Mice; Mice, Inbred DBA; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Protein Kinase C; Receptors, Antigen, B-Cell; Signal Transduction; Terpenes

Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit the growth of A549 human lung carcinoma cells at non-toxic concentrations, whereas 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol, synthetic analogues of the physiological ligands of protein kinase C (PKC), do not. Experiments were conducted to test the hypothesis that other activators of PKC are capable of interfering with A549 cell growth. The non-phorboid tumour promotor mezerein mimicked the growth-inhibitory effect of TPA in that it arrested growth for 5 days, after which cells proliferated again in the continued presence of the agent. TPA was 20 times more potent as a growth inhibitor than was mezerein. Bryostatin 1 at 10 nM and bryostatin 2 at 100 nM also arrested A549 cell growth and inhibited DNA replication as measured by incorporation of [methyl-3H]-thymidine into cells. Inhibition of DNA synthesis to between 90 and 75% of control values developed during the first hour of incubation of the cells with TPA, mezerein or the bryostatins. The extent of inhibition changed little during the subsequent 5 hr of incubation, after which it increased further to reach maximal values within 12 hr. At concentrations above those which caused maximal growth inhibition, the bryostatins abolished both their own inhibition of DNA synthesis and the anti-replicative effect of TPA and mezerein. The results show that activators of PKC other than phorbol esters are capable of inhibiting the growth of A549 cells. The bryostatins not only interfere with A549 cell growth but can also counter the growth-inhibitory effect of PKC activators, presumably via interaction with a target separate from the phorbol ester receptor site.

Topics: Animals; Bryostatins; Cell Division; Diterpenes; DNA Replication; Enzyme Activation; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate; Time Factors

Pulse-chase experiments, performed with 14C-labeled choline, were used to study the possible effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the terminal step of sphingomyelin (CerPCho) synthesis from phosphatidylcholine in intact human promyelocytic leukemic HL60 cells. Addition of TPA for the chase period significantly increased the rate of CerPCho synthesis; maximal stimulation (104%) required only 3 nM TPA. Treatment of cells with TPA for 6 h also increased the mass of CerPCho by 35%. Sphingosine (25 microM) or H7 (100 microM), inhibitors of protein kinase C (PKC) in vitro, inhibited some, but not all effects of TPA on endogenous protein phosphorylation in intact cells, and failed to inhibit TPA-stimulated synthesis of CerPCho. However, bryostatin, mezerein, 1-oleoyl-2-acetylglycerol, and polymyxin B, previously all shown to stimulate PKC in vivo, also stimulated the synthesis of CerPCho. It is suggested that the effect of phorbol ester on CerPCho synthesis is mediated by a subtype of PKC which responds to known activators of enzyme but is not inhibited by H7 or sphingosine.

Topics: Bryostatins; Cell Line; Choline; Diterpenes; Enzyme Activation; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phosphatidylcholines; Phosphorylation; Protein Kinase C; Sphingomyelins; Terpenes; Tetradecanoylphorbol Acetate

Human myeloid leukemia KG-1 cells are induced to differentiate to macrophage-like cells by tumor-promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Cells from the cloned subline, KG-1a, unlike the parental line, are resistant to the differentiating effect of TPA. In the present studies, we investigated in these cells protein phosphorylation stimulated by various protein kinase C activators, including 1-oleoyl-2-acetylglycerol in the presence of the diacylglycerol kinase inhibitor R59022, TPA, mezerein, and bryostatin. All the agents stimulated, to a greater extent and with a higher potency, phosphorylation of several proteins in KG-1 cells than in KG-1a cells. On the other hand, these agents markedly stimulated phosphorylation of other proteins in KG-1a cells compared to that in KG-1 cells. The findings indicated that the actions of the diacylglycerol, 1-oleoyl-2-acetylglycerol, and the non-metabolizable activators (TPA, mezerein, and bryostatin) were very similar but not fully equivalent; and that KG-1a cells exhibited altered (increased or decreased) phosphorylation patterns, perhaps related to the TPA resistance characteristic of this subline of cells.

Topics: Bryostatins; Cell Differentiation; Cell Line; Diglycerides; Diterpenes; Drug Resistance; Enzyme Activation; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phosphorylation; Protein Kinase C; Proteins; Pyrimidinones; Terpenes; Tetradecanoylphorbol Acetate; Thiazoles