bryostatin-1 and Leukemia--Myeloid

Topics: Acute Disease; Animals; Antineoplastic Agents; Bryostatins; Cell Differentiation; HL-60 Cells; Humans; Lactones; Leukemia, Myeloid; Macrolides; Myelodysplastic Syndromes; Tumor Cells, Cultured

Bryostatin-1 (Bryo), a macrocyclic lactone of the sea water bryozoan Bugula neritina, is a potent activator of protein kinase C and was found to exhibit antineoplastic activity in several systems. We studied the effect of Bryo on differentiation and growth modulation of human myeloid leukemia cell lines and freshly explanted blood cells from patients with myeloid leukemia. Alterations at the molecular level and phenotypic changes triggered by Bryo were similar, but not identical, to those induced by phorbol esters. Bryo was able to inhibit cellular proliferation as evidenced by [3H]-thymidine uptake and induced morphological changes associated with monocytic differentiation. In studies using continuous cell lines, the glucocorticoid dexamethasone was unable to prevent the Bryo-induced growth inhibition or the induced phenotypic changes. However, in fresh myeloid blood cells dexamethasone attenuated these Bryo-triggered effects. Our own data taken together with reports from the literature reviewed here suggest the following conclusions: (i) Bryo, while lacking tumor promoting activity, is able to induce differentiation in maturation arrested leukemia cells; (ii) it exhibits selective antiproliferative properties in normal or malignant hematopoietic cells and supports growth of multipotent stem cells. These features might qualify Bryostatin-1 as a potential candidate for promising research and possibly for future clinical applications.

Topics: Antineoplastic Agents; Bryostatins; Cell Differentiation; Cell Division; Dexamethasone; Drug Interactions; Enzyme Activation; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Humans; Lactones; Leukemia, Myeloid; Macrolides; Neoplastic Stem Cells; Protein Kinase C; Proto-Oncogenes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A Phase I trial has been conducted in patients with refractory/relapsed acute leukemia in which escalating doses of the protein kinase C (PKC) activator and down-regulator bryostatin 1 (NSC399555), administered as a 24-h continuous infusion on days 1 and 11, were given immediately before and after a split course of high-dose 1-beta-D-arabinofuranosylcytosine (HiDAC; 1.5 g/m(2) every 12 h x 4) administered on days 2 and 3, and 9 and 10. The bryostatin 1 maximally tolerated dose (MTD) was identified as 50 microg/m(2), with myalgias representing the major dose-limiting toxicity (DLT). Other DLTs included prolonged neutropenia and thrombocytopenia, and hepatotoxicity. Of the 23 patients who completed their course of therapy and were fully evaluable for response, the large majority of whom had unfavorable prognostic characteristics, 4 complete remissions (CRs) were obtained. An additional 3 patients were treated at a 3 g/m(2) ara-C (1-beta-D-arabinofuranosylcytosine) dose level to determine whether this HiDAC dose could be administered in conjunction with bryostatin 1. All 3 of these patients experienced DLT, and this dose was considered above the MTD. However, one of the latter patients, who was heavily pretreated, also achieved a CR that persisted 5+ months without maintenance. Finally, 1 patient post-HiDAC and autologous bone marrow transplantation achieved a 5+ month leukemia-free survival although she did not meet the criteria for a CR because of persistent transfusion requirements. Correlative laboratory studies performed on blasts from 9 patients revealed that in vivo administration of bryostatin 1 resulted in variable effects on total blast PKC activity, including decreases in 4 samples, increases in 2, and no change in 3. Previous in vivo bryostatin 1 exposure also exerted disparate effects on the extent of apoptosis observed in blasts exposed to ara-C ex vivo, although increases were noted in a subset of patient samples. Interestingly, in vivo administration of bryostatin 1 by itself induced lethality in some patient specimens. No clear relationship between the in vivo effects of bryostatin 1 on blast PKC activity and the extent of ara-C-related apoptosis that occurred ex vivo was apparent. Together, these findings demonstrate that bryostatin 1 can be safely administered as a continuous infusion before and after a split course of HiDAC in patients with refractory leukemia, and identify the bryostatin 1 MTD as 50 microg/m(2) when given by this schedul

Topics: Acute Disease; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Cytarabine; Female; Humans; Infusions, Intravenous; Lactones; Leukemia, Myeloid; Macrolides; Male; Middle Aged; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase C; Tumor Cells, Cultured

Natural killer (NK) cells are potent effectors of innate antitumor defense and are currently exploited for immune-based therapy of human leukemia. However, malignant blood cells in acute myeloid leukemia (AML) display low levels of ligands for the activating immunoreceptor NKG2D and can thus evade NK immunosurveillance. We examined the possibility of up-regulating NKG2D-specific UL16-binding protein (ULBP) ligands using anti-neoplastic compounds with myeloid differentiation potential. Combinations of 5-aza-2'-deoxycytidine, trichostatin A, vitamin D3, bryostatin-1, and all-trans-retinoic acid, used together with myeloid growth factors and interferon-gamma, increased cell surface ULBP expression up to 10-fold in the AML cell line HL60 and in primary AML blasts. Up-regulation of ULBP ligands was associated with induction of myelomonocytic differentiation of AML cells. Higher ULBP expression increased NKG2D-dependent sensitivity of HL60 cells to NK-mediated killing. These findings identify NKG2D ligands as targets of leukemia differentiation therapy and suggest a clinical benefit in combining a pharmacological approach with NK cell-based immunotherapy in AML.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Bryostatins; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Cytotoxicity, Immunologic; Decitabine; Flow Cytometry; GPI-Linked Proteins; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Leukemia, Myeloid; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Up-Regulation

We evaluated the synergistic activity of AS101 (ammonium trichloro-(dioxoethylene-0-0')-tellurate) with the protein kinase C (PKC) activators, Bryostatin-1 and phorbol-12-myristate-13-acetate (PMA), on human myelocytic leukemia cell differentiation in vitro, and in a mouse model. Use of AS101 with Bryostatin-1 or with a low concentration of PMA resulted in the differentiation of HL-60 cell line to cells with characteristics of macrophages. A similar synergistic effect was found in vivo. Compared with mice treated with AS101 alone or with Bryostatin-1 alone, the infiltration of leukemic cells into the spleen and the peritoneum of mice treated with both compounds, as well as the number of the HL-60 colonies extracted from those organs, were markedly reduced. The antitumor effects were associated with significantly prolonged survival (100% for 125 days) of the treated mice. Finally, the mechanism of action of this antitumor effect was explored, and was found to involve the Ras/extracellular signal-regulated kinase signaling pathway. Combined treatment with AS101 and Bryostatin-1 synergistically increased p21(waf1) expression levels independently of p53. Upregulation of p21(waf1) was necessary for HL-60 cell differentiation, which was found to be both c-raf-1 and mitogen-activated protein kinase dependent. This study may have implications for the development of strategies to induce differentiation in myeloid leukemias, myelodysplasias and possibly in other malignancies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bryostatins; Cell Differentiation; Drug Synergism; Ethylenes; Extracellular Signal-Regulated MAP Kinases; HL-60 Cells; Humans; Leukemia, Myeloid; Macrolides; Neoplasm Transplantation; ras Proteins

The effects of the PKC activator and down-regulator bryostatin 1 and the PKC and Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) were compared with respect to potentiation of 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis in human myelomonocytic leukemia cells (U937). Whereas bryostatin 1 and UCN-01 both markedly enhanced ara-C-induced mitochondrial injury (e.g., cytochrome c and Smac/DIABLO release, loss of mitochondrial membrane potential), caspase activation, and apoptosis, ectopic expression of an N-terminal loop-deleted Bcl-2 mutant protein protected cells from ara-C/UCN-01- but not ara-C/bryostatin 1-mediated lethality. Conversely, ectopic expression of CrmA or dominant-negative caspase-8 abrogated potentiation of ara-C-mediated apoptosis by bryostatin 1 but not by UCN-01. Exposure of cells to ara-C and bryostatin 1 (but not UCN-01) resulted in sustained release of tumor necrosis factor (TNF) alpha; moreover, potentiation of ara-C lethality by bryostatin 1 (but not by UCN-01) was reversed by coadministration of TNF soluble receptors or the selective PKC inhibitor bisindolylmaleimide (1 microM). Finally, similar events were observed in the human promyelocytic leukemia cell line HL-60. Together, these findings suggest that potentiation of ara-C lethality in human myeloid leukemia cells by bryostatin 1 but not UCN-01 involves activation of the extrinsic, receptor-mediated apoptotic pathway, and represents a consequence of bryostatin 1-mediated release of TNF-alpha. They also argue that the mechanism by which bryostatin 1 promotes ara-C-induced mitochondrial injury, caspase activation, and apoptosis involves factors other than or in addition to PKC down-regulation or modulation of Bcl-2 phosphorylation status.

Topics: Alkaloids; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bryostatins; Carrier Proteins; Caspases; Cytarabine; Cytochrome c Group; Drug Synergism; Enzyme Activation; Gene Deletion; HL-60 Cells; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Lactones; Leukemia, Myeloid; Macrolides; Maleimides; Mitochondrial Proteins; Mutation; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Staurosporine; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; U937 Cells

Bryostatin-1 inhibits acute myeloid leukemia (AML) in vitroat doses that stimulate the growth of normal hematopoietic progenitors.Although bryostatin-1 has a number of distinct biological activities, those specifically responsible for its antileukemic activity are unclear. We found that bryostatin-1 (10(-8) M) inhibited cell cycling at G(1), induced phenotypic evidence of differentiation, and limited the clonogenic growth of both AML cell lines and patient specimens. This activity was markedly enhanced by granulocyte/macrophage-colony stimulating factor, whereas growth factor-neutralizing antibodies completely inhibited both the differentiating and antileukemic activities of bryostatin-1. Cell cycle inhibition and growth factors were also required for the differentiating activities of two unrelated agents, hydroxyurea and phenylbutyrate. These data suggest that many pharmacological differentiating agents require both cell cycle arrest and lineage-specific growth factors for full activity and may explain why these agents have demonstrated only limited clinical efficacy.

Topics: Acute Disease; Antigens, CD; Antineoplastic Agents; Apoptosis; Bryostatins; Cell Cycle; Cell Differentiation; Cell Lineage; Drug Combinations; Enzyme Activation; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Hydroxyurea; Lactones; Leukemia, Myeloid; Macrolides; Phenylbutyrates; Tumor Cells, Cultured

Primary acute myeloid leukaemia (AML) blasts can be induced to differentiate into dendritic-like leukaemia cells (DLLC) by culture with certain cytokine combinations. DLLC offer potential for use as autologous vaccines based on their ability to present putative leukaemia-specific antigens to T cells. It has been reported, however, that in around 30-50% of AML cases the leukaemia cells are not capable of undergoing DLLC differentiation. The purpose of this study was to identify the features that represent successful DLLC differentiation and, for those cases shown to be resistant to cytokine-induced differentiation, to use differentiating agents in an attempt to overcome the differentiation block. Leukaemia cells derived from 42 patients with AML were cultured in vitro with cytokines GM-CSF, IL-4 and TNFalpha/CD40L. In 22 cases the leukaemic cells underwent DLLC differentiation based on characteristic morphological changes and expression of costimulatory and dendritic cell-associated molecules. Four cases were not evaluable because of poor viability over the culture period. The remaining 16 cases failed to show evidence of DLLC differentiation. Many of these differentiation resistant cases were associated with poor risk karyotypic features. Nine of the resistant cases were selected for further study. Differentiating agents trichostatin (TSA), azacytidine (AZA) and bryostatin (BRYO) were used in combination with cytokines for the first 96 h of the culture period. Bryostatin (BRYO) alone was shown to be capable of overcoming differentiation resistance and allowing DLLC differentiation to proceed.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Azacitidine; Bryostatins; CD40 Ligand; Cell Differentiation; Child; Cytokines; Dendritic Cells; Drug Resistance; Drug Synergism; Female; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-D Antigens; Humans; Hydroxamic Acids; Immunophenotyping; In Situ Hybridization, Fluorescence; Interleukin-12; Interleukin-4; Karyotyping; Lactones; Leukemia, Myeloid; Lymphocyte Culture Test, Mixed; Macrolides; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The effects of the protein kinase C (PKC) activator and down-regulator bryostatin 1 were examined with respect to paclitaxel-induced apoptosis and antiproliferative activity in human myeloid leukemia cells (U937) displaying enforced expression of the anti-apoptotic protein Bcl-xL. Overexpression of Bcl-xL blocked various aspects of paclitaxel-mediated apoptosis, including caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), loss of mitochondrial membrane potential (Delta Psim), and release of cytochrome c. However, subsequent (but not prior) exposure of paclitaxel-treated U937/Bcl-xL cells (500 nM; 6 h) to bryostatin 1 (10 nM; 15 h) restored the extent of apoptosis, caspase activation, and mitochondrial damage to levels approximating those in paclitaxel-treated empty-vector control cells (U937/Neo). Potentiation of paclitaxel-induced apoptosis by bryostatin 1 in U937/Bcl-xL cells occurred primarily in the G2M cell population, and was associated with alterations in Bcl-xL gel mobility and a reduction in paclitaxel-mediated stimulation of CDK1 activity. Enhancement of paclitaxel-induced apoptosis by bryostatin 1 in Bcl-xL overexpressors was accompanied by a corresponding reduction in clonogenic potential. In contrast to its effects on apoptosis, bryostatin 1 failed to restore paclitaxel-mediated increases in free Bax levels in U937/Bcl-xL cells. Lastly, the actions of bryostatin 1 were mimicked by a pharmacologic inhibitor of the MEK1/MAP kinase pathway (PD98059), but not by SB203580, an inhibitor of p 38 MAP kinase. Moreover, sequential exposure of both U937/Neo or/Bcl-xL cells to paclitaxel followed by bryostatin 1 or PD98059 was associated with a net reduction in MAP kinase activity. Collectively, these findings indicate that protection against paclitaxel-mediated mitochondrial dysfunction and apoptosis in human U937 leukemia cells conferred by Bcl-xL overexpression can be substantially overcome by bryostatin 1 and possibly other agents that interrupt the MAP kinase signal transduction pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bryostatins; Choristoma; Humans; Lactones; Leukemia, Myeloid; Macrolides; Mitochondria; Paclitaxel; Proto-Oncogene Proteins; U937 Cells

Bryostatin has shown promise both as a cytotoxic agent and more recently as a modulator of 1-beta-D-arabinofuranosylcytosine (ara-C) resistance. This compound is currently in phase I and II trials as a single agent. We have used the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay as a means of investigating the direct effects of bryostatin and the effects of co-incubating this agent with ara-C on fresh blast cells from 53 patients with acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Additional studies evaluated the levels of accumulation and retention of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) in cells exposed to ara-C with and without bryostatin. Cells were exposed to bryostatin at a range of concentrations (0.1-100 nM) for 48 h and at 1 nM for both modulation studies and assessment of ara-CTP production. We found bryostatin to be cytotoxic in 18/58 (31%) tests whilst potentiation of formazan production in the MTT assay was seen in 21/58 (36%) patients. On co-incubation with bryostatin, 16/58 (27%) tests showed increased cytotoxicity to ara-C. Furthermore, there was a significant increase in the accumulation of ara-CTP on co-incubation with bryostatin (p = 0.0401). We found patients with in vitro resistance were more likely to become sensitised following exposure to bryostatin (p < 0.01). This study has emphasised the need to optimise treatment regimens for individual patients using this approach.

Topics: Acute Disease; Antineoplastic Agents; Arabinofuranosylcytosine Triphosphate; Bryostatins; Cell Death; Cytarabine; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Lactones; Leukemia, Myeloid; Leukocytes, Mononuclear; Macrolides; Tetrazolium Salts; Thiazoles

The functional role of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in leukemic cell G1 arrest, differentiation, and apoptosis induced by two PKC activators (PMA and bryostatin 1) was examined using antisense-expressing lines [U937/p21AS(F4) and U937/p21AS(B8)]. Following incubation with 10 nM PMA (24 h), antisense-expressing cells displayed induction of p27(KIP1) but not of p21, whereas empty vector-containing cells (U937/pREP4) exhibited induction of both p21 and p27. Antisense-expressing cells were impaired in G1 arrest, dephosphorylation of the retinoblastoma protein, dephosphorylation and reduction in activity of cyclin-dependent kinase 2, and acquisition of differentiated features (e.g., plastic adherence). Bryostatin 1 induced p27 but not p21 in control cells and was less effective than PMA in initiating G1 arrest and related events. Nevertheless, disruption of p21 expression abrogated the effects of bryostatin 1 on cell cycle arrest and cellular maturation. Dysregulation of p21 did not, however, modify PMA- or bryostatin 1-mediated down-regulation of c-Myc protein. Unexpectedly, disruption of p21 failed to attenuate the net reduction in viable cell number following PMA or bryostatin 1 treatment inasmuch as impaired differentiation was accompanied by a lowered threshold for PMA- and bryostatin 1-induced apoptosis. Inhibition of p21 expression also promoted PMA- and bryostatin 1-mediated loss of mitochondrial transmembrane potential (DeltaPsim ) and release of cytochrome c into the cytosol. Together, these findings demonstrate a critical functional role for p21 in regulating myelomonocytic leukemic cell G1 arrest and differentiation following exposure to two PKC activators exhibiting disparate patterns of activity. They also suggest that following treatment with these agents, dysregulation of p21 prevents leukemic cells from engaging a normal differentiation program through a c-Myc-independent mechanism, and instead directs cells along an apoptotic pathway.

Topics: Apoptosis; Bryostatins; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; G1 Phase; Humans; Lactones; Leukemia, Myeloid; Macrolides; Protein Kinase C; Tetradecanoylphorbol Acetate; U937 Cells

We have examined the effects of the macrocyclic lactone protein kinase C (PKC) activator bryostatin 1 on taxol-induced apoptosis and inhibition of clonogenicity in the human monocytic leukemia cell line U937. Exposure of cells to bryostatin 1 (10 nM; 15 hr) after (but not before) a 6-hr incubation with 0.5 microM taxol significantly increased apoptosis and resulted in an approximately 3 log reduction in clonogenicity. Cell cycle analysis revealed that the increase in apoptotic cells following bryostatin 1 treatment occurred primarily in the population undergoing taxol-mediated G2M arrest. The actions of bryostatin 1 were not attributable to potentiation of taxol-induced tubulin stabilization or to a reduction in the intracellular retention of taxol. Following exposure of cells to taxol, the Bcl-2 protein displayed an alteration in mobility that was not modified appreciably by bryostatin 1 treatment. The mobility shift in Bcl-2 protein from cells exposed to taxol followed by bryostatin 1 was eliminated by treatment of lysates with the protein phosphatase 2A (PP2A); the latter effect was blocked by okadaic acid. Treatment of cells with taxol followed by bryostatin 1 did not increase the amount of total Bax (compared with treatment with taxol alone), but did increase the amount of free Bax in the supernatant fraction. Finally, the ability of bryostatin 1 to potentiate taxol-induced apoptosis in U937 cells was mimicked closely by 2'-amino-3'-methoxyflavone (PD98059), a specific inhibitor of the mitogen-activated protein kinase (MAPK) kinase (MEK). Collectively, these findings indicate that bryostatin 1 increases the susceptibility of U937 cells to taxol-induced apoptosis and inhibition of clonogenicity. They also raise the possibility that this phenomenon may involve functional alterations in Bcl-2 and/or other proteins involved in regulation of the cell death pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Cell Survival; Drug Synergism; Humans; Lactones; Leukemia, Myeloid; Macrolides; Paclitaxel; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; U937 Cells

The potent anti-neoplastic actions displayed in vitro by bryostatins have led to the introduction of short-term bryostatin-1 infusions in phase I clinical trials. Because bryostatin (bryo) undergoes a rapid clearance in vivo, we were interested in its scheduling effects on acute myeloid leukemia (AML) clonogenicity. Therefore, we assessed the primary plating efficiency (PE1) of AML samples in response to several bryo concentrations after various preincubation periods in a semi-solid colony forming assay. Whereas continuous exposure to 10 nM bryo during the assay period reduced the PE1 in nearly all samples tested, preincubation of eight AML patients' specimens for 1, 2, 3 or 4 days with bryo in a dose range of 0.1-10 nM showed a heterogenous PE1 response. Stimulatory as well as inhibitory effects on leukemic clonogenic growth were seen within individual specimens depending on dose and preincubation time with no single incubation time or concentration that caused unequivocal common overall inhibition of clonogenic growth in most or all of the samples. Higher doses of bryo also failed to result in specific inhibition of leukemic cells: 4/8 samples showed an increased clonogenic response to 250 nM bryo whereas normal bone marrow progenitor cells were consistently inhibited in their clonogenicity at this dose. A marked similarity, i.e. undulatory effects with increasing bryo concentrations, was found for HL60 leukemic cells. In conclusion, the effects of bryo on clonogenic leukemia cell growth are strongly dependent on scheduling and dose varying between and within individual AML samples. These results caution against in vivo bryo pulse therapy, as currently applied, for treatment of AML.

Topics: Acute Disease; Adult; Aged; Antineoplastic Agents; Bryostatins; Cells, Cultured; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Lactones; Leukemia, Myeloid; Macrolides; Male; Middle Aged; Reproducibility of Results; Tumor Stem Cell Assay

Bryostatin 1 is a natural antineoplastic agent that activates protein kinase C. Treatment of U937 human leukemic cells with bryostatin 1 caused a 60% reduction in cell growth, whereas another protein kinase C activator, phorbol myristate acetate (PMA), completely inhibited U937 cell growth. Both bryostatin 1 and PMA induced inhibition of cyclin-dependent kinase 2 (cdk2) activity. The first phase of cdk2 inhibition correlated with the transient induction of p21, a known inhibitor of cdk2. In contrast, the second phase of cdk2 inhibition correlated with the dephosphorylation of cdk2 on threonine-160, which must be phosphorylated for cdk2 activity. The level of growth inhibition induced by these two compounds correlated with the degree of cdk2 dephosphorylation as follows: bryostatin 1, 60%; PMA, 100%.

Topics: Antineoplastic Agents; Base Sequence; Blotting, Western; Bryostatins; CDC2-CDC28 Kinases; Cell Division; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Humans; Lactones; Leukemia, Myeloid; Macrolides; Molecular Sequence Data; Phosphorylation; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride a

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; Cytarabine; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Synergism; Humans; Lactones; Leukemia, Myeloid; Macrolides; Protein Kinase C; Tumor Cells, Cultured

Annexin VIII is a calcium- and phospholipid-binding protein with anticoagulant activity. Annexin VIII mRNA was found to be specifically expressed in acute promyelocytic leukemia (APL) cells; it was not found in other types of acute myeloid leukemia (AML) nor in lymphoid malignancies. Using Northern blot analysis we investigated annexin VIII expression in 142 continuous human leukemia and lymphoma cell lines at the mRNA level. While the only APL cell line, NB-4, was indeed positive, other cell lines also displayed annexin VIII mRNA: 4/22 myeloid cell lines, 8/23 monocytic cell lines, 2/8 megakaryoblastic cell lines, 5/26 lymphoma-derived cell lines, 2/10 myeloma cell lines and 1/44 lymphoid leukemia cell lines. The strongest expression was seen in NB-4 and in the Hodgkin's disease derived cell line HDLM-2. Treatment of NB-4 cells with all-trans retinoic acid (ATRA) or the phorbol ester TPA induced terminal differentiation and down-regulated annexin VIII mRNA expression rapidly within a few hours; vitamin D3 was ineffective in this regard; the protein kinase C activator Bryostatin 1 up-regulated the expression. A panel of initially negative cell lines could not be induced by any of these biomodulators to transcribe annexin VIII. The half-life (T1/2) of annexin VIII mRNA was about 3-4 h using actinomycin D as transcription inhibitor. Treatment with ATRA or TPA prior to exposure to actinomycin shortened the T1/2 to 2 h while Bryostatin 1 extended it to 6h. As 21/141 non-APL cell lines were positive, annexin VIII cannot be used as a marker gene for APL cells; however, it might be associated with myelomonocytic or erythro-megakaryoblastic precursor cells. Annexin VIII gene expression might play a unique role in the proliferation and/or differentiation of leukemic cells and could be associated with the particular abnormal hemostasis of some leukemias.

Topics: Annexins; Blotting, Northern; Bryostatins; Cell Differentiation; Cholecalciferol; Dactinomycin; Gene Expression Regulation, Neoplastic; Half-Life; Humans; Lactones; Leukemia; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Lymphoma; Macrolides; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

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

The AP-1 protein complex binds to specific DNA sequences that regulate transcription of genes responsive to certain growth factors and phorbol esters. This complex is composed of products of the jun and fos gene families. The present studies have examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the regulation of jun-B gene expression during induction of monocytic differentiation. Low levels of jun-B transcripts were present in uninduced HL-60 promyelocytic leukemia cells. In contrast, treatment with TPA was associated with rapid increases in jun-B mRNA levels that were maximal at 3 h and remained elevated at 48 h. The induction of jun-B expression by TPA in these cells preceded that of the c-jun and c-fos genes. Similar increases in jun-B transcripts were detectable in TPA-treated THP-1 and U-937 myeloid leukemia cells, although expression of this gene was transient in the more differentiated THP-1 cells. Run-on assays demonstrated low levels of jun-B gene activation in untreated HL-60 cells, whereas TPA treatment was associated with a 6-fold increase in the transcription rate of this gene. This induction of jun-B expression occurred in the absence of de novo protein synthesis. In contrast, inhibition of protein synthesis was associated with superinduction of TPA-induced jun-B mRNA levels and an increase in stability of this transcript. These findings suggest that jun-B gene expression is regulated at both the transcriptional and posttranscriptional levels during induction of monocytic differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bryostatins; Calcitriol; Cell Differentiation; DNA-Binding Proteins; Gene Expression; Humans; Lactones; Leukemia, Myeloid; Macrolides; Monocytes; Proto-Oncogene Proteins c-jun; Proto-Oncogenes; Tetradecanoylphorbol Acetate; Transcription, Genetic

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