bryostatin-1 and Leukemia--Myeloid--Acute

Acute myeloid leukemia (AML) still is fatal in the majority of patients. Therefore, we evaluated the antileukemic effect of the alkylating agent treosulfan in AML.. Chemosensitivity tests were performed in AML cell lines and primary cells from patients. Because protein kinase C (PKC) is known to play an integral role in the regulation of diverse cellular functions such as apoptosis, several PKC modulators were evaluated in conjunction with treosulfan.. U937, THP-1, HL-60, TUR cells, and primary AML cells obtained from five consecutive patients displayed dose-dependent sensitivity to treosulfan. The LC(90) was approximately 100 microM, which is several fold below clinically achievable plasma levels. Cell death was associated with cellular events indicating apoptosis such as breakdown of the mitochondrial transmembrane potential, proteolytic activation of caspase-3, or appearance of a sub-G(1) DNA peak. Synergistic antileukemic effects were observed in all cell lines and patient samples tested using the PKC activators bryostatin-1 and 12-O-tetradecanoylphorbol-13-acetate (TPA), whereas the PKC inhibitor GF109203X substantially reduced apoptosis. Furthermore, long-term preincubation with bryostatin-1 or TPA, both of which are known to down-regulate PKC protein levels, likewise inhibited treosulfan-induced apoptosis. Immunoblots revealed membrane translocation of PKC-delta, indicating activation of this enzyme upon treosulfan exposure.. Our data provide evidence for a strong antileukemic effect of treosulfan in both cell lines and AML blasts from patients at concentrations below the plasma levels described at standard dose levels. Furthermore, the proapoptotic effect of treosulfan is mediated at least in part by activation of PKC isoforms and can be augmented by coincubation with bryostatin-1.

Topics: Aged; Apoptosis; Bryostatins; Busulfan; Cell Line; Female; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Male; Middle Aged; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Species Specificity

The effects of the protein kinase C activator bryostatin 1, either with or without recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) were examined with respect to the in vitro metabolism of ara-C in leukaemic myeloblasts obtained from 10 patients with acute myelogenous leukaemia (AML). Coincubation of cells with 12.5 x 10(-9) M bryostatin 1 and 10(-5) M ara-C for 4 h resulted in a significant increase in ara-CTP formation (compared to controls) in 6/10 specimens (mean increase 106%; range 38-255%), and no change in the remainder. In contrast, coincubation of cells with 1.25 ng/ml rGM-CSF resulted in a significant increase in only one specimen, and decreases in two. Bryostatin 1 also significantly increased ara-C DNA incorporation in 6/9 evaluable samples, including two which did not display an increase in ara-CTP formation. Coincubation of cells with both bryostatin 1 and rGM-CSF did not lead to a further increase in ara-CTP formation or ara-C DNA incorporation compared to values obtained with either agent alone. Finally, exposure of blasts to bryostatin 1 for 24 h before ara-C led to an increase in ara-CTP formation in 3/8 additional specimens, and a decrease in one sample displaying evidence of bryostatin 1-induced macrophage differentiation. Incubation of cells with both rGM-CSF and bryostatin 1 for this period resulted in ara-CTP levels equivalent to those obtained with bryostatin 1 alone. These studies indicate that while bryostatin 1 exerts a heterogeneous effect on ara-C metabolism in leukaemic myeloblasts, it is capable of potentiating ara-C phosphorylation in a subset of patient samples, including some that do not exhibit an increase in response to rGM-CSF. They also raise the possibility that bryostatin 1-induced potentiation of ara-C metabolism in some leukaemic cells may contribute, at least in part, to the antileukaemic efficacy of this drug combination.

Topics: Antineoplastic Agents; Arabinofuranosylcytosine Triphosphate; Bryostatins; Cytarabine; Drug Interactions; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Recombinant Proteins; Time Factors

We have examined the effect of a combined 24 h exposure to cytosine arabinoside (ara-C) and the protein kinase C activator bryostatin 1, either alone or in conjunction with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), on the clonogenic growth of 14 primary samples from acute myelogenous leukemia (AML) patients, as well as normal human committed and early hematopoietic progenitors. Incubation of blasts with 1 microM ara-C and 12.5 nM bryostatin 1(+/- 1.25 ng/ml rGM-CSF) resulted in a heterogeneous pattern of inhibitory effects toward primary leukemic colonies, ranging from 32-98%, and subadditive to synergistic drug interactions. However, exposure of blasts to ara-C and bryostatin 1, either with or without rGM-CSF, eliminated leukemic cell self-renewal in 80-93% of samples, and very substantially reduced growth in the remainder. Exposure of normal human bone marrow mononuclear cells to identical concentrations of ara-C and byostatin 1 permitted the survival of 23% of committed myeloid progenitors (granulocyte-macrophage colony-forming units), and greater than 50% when rGM-CSF was included. Finally, exposure of bone marrow populations highly enriched for progenitor cells (CD34+, DR-, CD71-) to ara-C and bryostatin 1 +/- rGM-CSF for 24 h led to minimal reductions (e.g. 10-15%) in the survival of early hematopoietic progenitors (high proliferative potential colony-forming cells). Together, these findings indicate that combined exposure in vitro to ara-C and bryostatin 1, both with and without rGM-CSF, effectively inhibits the growth of leukemic cells with self-renewal capacity, while sparing a significant fraction of normal committed and primitive hematopoietic progenitors.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Bryostatins; Clone Cells; Drug Interactions; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Middle Aged; Recombinant Proteins; Vidarabine

Bryostatin 1 is a macrocyclic lactone activator of protein kinase C which has displayed promising antileukemic potential in pre-clinical studies. We have assessed the effect of bryostatin 1 on the in vitro clonogenic response of leukemic myeloblasts obtained from 12 patients with acute non-lymphocytic leukemia to recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and have compared these responses to those of normal human hematopoietic progenitors. Although leukemic blast progenitors responded in a heterogenous manner to bryostatin 1 as a single agent, co-administration of 12.5 or 100 nM bryostatin 1 in conjunction with 1.25 ng/ml rGM-CSF resulted in a significant reduction in colony formation (compared to rGM-CSF alone) in 8/12 specimens, and sub-additive stimulatory effects in all samples. In addition, the exposure of cells to 12.5 nM bryostatin 1, either alone or in conjunction with 1.25 ng/ml rGM-CSF, substantially reduced or eliminated leukemic cell self-renewal capacity in all samples assayed. In contrast to the effects observed in leukemic cells, exposure of adherent and T-cell depleted normal bone marrow mononuclear cells to equivalent concentrations of bryostatin 1 and rGM-CSF consistently produced supra-additive effects on the growth of normal committed myeloid progenitors (day 14 CFU-GM). When normal marrow cells were further enriched for progenitors (MY-10+), concentrations of bryostatin 1 that were unable to support growth when administered alone significantly potentiated the number of GM colonies formed in response to rGM-CSF. These studies suggest that bryostatin 1 may modulate the in vitro response of certain normal and leukemic progenitor cells to rGM-CSF, and that the nature of this response differs between the two cell types. They also indicate that bryostatin 1 may be particularly effective in limiting the self-renewal capacity of leukemic myeloblasts, an in vitro characteristic with potentially important in vivo significance.

Topics: Antigens; Bryostatins; Cell Division; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Neoplastic Stem Cells; Phorbol 12,13-Dibutyrate; Protein Kinase C; Recombinant Proteins; Tumor Cells, Cultured

Protein kinase C (PKC), an enzyme involved in signal transduction, is the receptor for both the tumor-promoting phorbol esters and the anti-neoplastic bryostatins. In many cells, phorbol esters and bryostatins cause similar effects; we have found that both agents increase actin polymerization in neutrophils. In some cells, however, the two agents result in different cell processes; we have found consistently different effects of these agents on actin conformation in myeloblasts obtained from leukemic patients. The patients tested all had increases in F-actin in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) and most had decreases in F-actin in response to bryostatin. The data suggests that leukemic myeloblasts have a different cytoskeletal response to a tumor promoter and an antineoplastic agent despite their common receptor.

Topics: Actins; Adult; Aged; Antineoplastic Agents; Bone Marrow; Bryostatins; Female; Flow Cytometry; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Male; Microscopy, Electron, Scanning; Middle Aged; Protein Conformation; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, has demonstrated both antineoplastic activity against the murine P388 leukemia line in vivo and stimulatory activity against mouse and human hematopoietic progenitors. We studied the effects of bryostatin 1 on the growth of human leukemias in vitro. Bryostatin 1 inhibited 1 to 4 logs of clonogenic leukemia cell growth from three of four leukemia cell lines. Bryostatin 1 also inhibited, by at least 1 log, the proliferation of clonogenic acute nonlymphocytic leukemia (ANLL) cells from 10 to 12 patients with newly diagnosed or relapsed ANLL. Maximal inhibition of leukemic growth occurred at 10(-9) to 10(-7) mol/L bryostatin 1. Interestingly, bryostatin 1 also inhibited the growth of hematopoietic progenitors from eight patients with myelodysplastic syndromes (MDS). Leukemia cells exposed to bryostatin 1 for up to 96 hours and then washed, demonstrated no substantial inhibition of clonogenic growth, indicating that the anti-leukemic effect of bryostatin 1 is cytostatic. The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) produced more potent inhibition of clonogenic leukemia growth, and this inhibition was blocked by bryostatin 1. Thus, the anti-leukemic activity of bryostatin 1 may be mediated through activation of protein kinase C. Bryostatin 1 inhibits clonogenic leukemia cells at concentrations that stimulate normal hematopoietic progenitors. The differential effects of bryostatin 1 on normal and abnormal hematopoiesis suggest that bryostatin 1 may have value in the treatment of leukemias and MDS.

Topics: Antineoplastic Agents; Bryostatins; Cell Line; Hematopoietic Stem Cells; Humans; Lactones; Leukemia; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukemia, Prolymphocytic; Leukemia, Promyelocytic, Acute; Macrolides; Myelodysplastic Syndromes; Phorbol Esters; Tumor Cells, Cultured

Bryostatin 1, a macrocyclic lactone isolated from a marine bryozoan, has significant antineoplastic activity against the murine cell line P388. Like phorbol esters, bryostatin 1 is capable of binding to and activating protein kinase C, but these two compounds differ in the ability of bryostatin 1 to act as a tumor promoter. We have investigated whether bryostatin 1 can modulate the differentiated phenotype of fresh samples of human myeloid leukemia. We find that six of seven samples responded to bryostatin treatment with changes associated with a more differentiated phenotype including increases in macrophage-like morphology and an increase in adherence and OKM1 and alpha-naphthyl acetate esterase activity positivity. The percentage of cells within each sample evidencing these changes varied markedly among the seven patients' cells examined. Because of the effects of bryostatin on fresh samples we examined the ability of bryostatin to differentiate four HL-60 cell sublines obtained from different laboratories. We found that two of the cell lines did not respond either with an inhibition of growth or morphological change, while one was inhibited, and one showed both growth inhibition and some induction of macrophage-like morphology when treated with bryostatin. To test whether other differentiating agents would enhance the effects of bryostatin 1, we added tumor necrosis factor alpha and bryostatin to these four cell lines. The addition of both agents effected an additive inhibition of growth. These data suggest that bryostatin 1 alone or in combination with other biological response modifiers may have a role in the treatment of human leukemia.

Topics: Antineoplastic Agents; Bryostatins; Cell Differentiation; Humans; Lactones; Leukemia; Leukemia, Myeloid, Acute; Macrolides; Protein Kinase C; Proto-Oncogenes; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth.

Topics: Bryostatins; Cell Line; Enzyme Activation; Humans; Lactones; Lamin Type B; Lamins; Leukemia, Myeloid, Acute; Macrolides; Nuclear Envelope; Nuclear Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphorylation; Protein Kinase C

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

The effects of protein kinase C (PKC) inhibitor polymyxin B (PMB) and PKC activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on intact HL60 cells were examined. It was found that each of the three agents exhibited similar effects on phosphorylation of certain endogenous proteins, PKC translocation from cytoplasm to plasma membrane and formation of CDP-choline. TPA, however, was the only agent that stimulated phosphatidylcholine formation. Differentiation of HL60 cells was potently induced by TPA; in comparison bryostatin was a relatively weaker inducer and PMB was without effect. The data indicated that the effects of the PKC inhibitor PMB on intact cells could not be predicted by its in vitro activity, and that certain TPA-dependent but PKC-independent reactions might be crucial in HL60 cell differentiation.

Topics: Bryostatins; Cell Adhesion; Cell Differentiation; Cell Line; Dinoprostone; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phorbol Esters; Phosphatidylcholines; Phospholipids; Phosphorylation; Polymyxin B; Polymyxins; Prostaglandins E; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate

When human promyelocytic leukemia cells (HL-60) are induced by phorbol esters to differentiate to macrophages, the process is accompanied by immediate activation of protein kinase C (PK-C) in the cytoplasm and later changes in DNA and RNA synthesis. Although these events are temporarily related, it remains unclear how activation of this protein kinase leads to changes in nuclear transcription. In this study, we find that bryostatin, a macrocyclic lactone which does not induce differentiation of HL-60 cells but activates PK-C, mimics the effects of phorbol esters on protein phosphorylation and PK-C location. Treatment of HL-60 cells with bryostatin stimulates phosphorylation of the surface transferrin receptor and in the cytoplasm of five proteins having the molecular weights of 17-43 kDa over the same time course as that stimulated by phorbol esters. Similarly, prolonged treatment with bryostatin, like that with phorbol esters, causes the loss of all cellular PK-C activity. Unlike the phosphorylation studies, bryostatin treatment, over a 1-100 nM concentration range and for varying lengths of time, did not affect HL-60 c-myc RNA levels, while phorbol ester treatment rapidly decreased c-myc RNA levels. These data suggest that neither the activation of PK-C and the phosphorylation of specific substrates nor the loss of total cellular PK-C activity from HL-60 cells is sufficient to induce marked decreases in c-myc levels and differentiation of HL-60 cells.

Topics: Bryostatins; Cell Compartmentation; Cell Membrane; Cytoplasm; Gene Expression Regulation; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins; Receptors, Transferrin; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

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

Phorbol esters bind to and activate a calcium phospholipid-dependent protein kinase (C kinase). Some researchers believe that activation of C kinase is necessary for the induction of phorbol ester biologic effects. Our research indicates that bryostatin, a macrocyclic lactone that binds to the phorbol ester receptor in human polymorphonuclear leukocytes, also binds to this receptor in the human promyelocytic leukemia cell line, HL-60. Bryostatin activates partially purified C kinase from HL-60 cells in vitro, and when applied to HL-60 cells in vivo, it decreases measurable cytoplasmic C kinase activity. Unlike the phorbol esters, bryostatin is unable to induce a macrophage-like differentiation of HL-60 cells; however, bryostatin, in a dose-dependent fashion, blocks phorbol ester-induced differentiation of HL-60 cells and, if applied within 48 hr of phorbol esters, halts further differentiation. These results suggest that activation of the C kinase by some agents is not sufficient for induction of HL-60 cell differentiation and imply that some of the biologic effects of phorbol esters may occur through a more complex mechanism than previously thought.

Topics: Binding, Competitive; Bryostatins; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phorbol Esters; Protein Kinase C; Receptors, Drug; Receptors, Immunologic