bryostatin-1 and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

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

Bryostatin 1 (bryo 1) is known to induce the differentiation and cell cycle arrest of human lymphoid leukemia cells in vitro. The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/mitogen-activated protein (MAP) kinase pathway in B-lymphoid cell differentiation of the Reh Acute Lymphoblastic Leukemia cell line, the effects of bryo 1 on ERK activation were determined. On bryo 1 treatment, the activity of ERK2 (p42) rapidly increased, with ERK1 (p44) protein levels remaining constant. p44/42 immunoprecipitates from lysates of bryo 1-treated cells had increased their ability to phosphorylate the transcription factor Elk-1. Constitutive AP-1 activity was shown to be potentiated after bryo 1 treatment using electrophoretic mobility shift assays. The protein composition of the AP-1 transcription factor complex activated by bryo 1 was analyzed using supershift analysis with specific antibodies against c-Fos, Fos B, c-Jun, Jun B, and Jun D proteins. Supershift analysis revealed that the bryo 1-induced AP-1 complex was composed predominantly of Fos B and Jun D. Therefore, we evaluated the effects of inhibiting MAP/ERK kinase (MEK) on both DNA binding and cellular differentiation. Treatment of Reh cells with 20 microM PD98059, a specific inhibitor of MEK, inhibited bryo 1-induced ERK activity and DNA binding. Furthermore, PD98059 blocked the bryo 1-induced differentiation of Reh cells, as assessed by a number of features associated with lymphoid differentiation, including changes in morphology, cell growth arrest, attachment, and increased expression of the leukocyte integrin CD11c. Moreover, transient transfection of Reh cells with antisense MAP kinase oligonucleotides blocked bryo 1-induced expression of CD11c. Our analysis also shows that CD11c's gene promoter activity is augmented by bryo 1. Therefore, we conclude that activation of the MEK/ERK signaling pathway is necessary for bryo 1-induced differentiation of the pre-B Acute Lymphoblastic Leukemia cell line Reh.

Topics: Bacterial Proteins; Binding Sites; Blotting, Western; Bryostatins; Cell Differentiation; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Humans; Integrin alphaXbeta2; Lactones; Luciferases; Macrolides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Oligonucleotides, Antisense; Plasmids; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Signal Transduction; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.

Topics: Acetylcysteine; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Bryostatins; Cell Extracts; Cysteine Endopeptidases; Humans; Lactones; Macrolides; Multienzyme Complexes; Precipitin Tests; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteasome Endopeptidase Complex; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Ubiquitins

Previous studies have shown that bryostatin 1 induces a decrease in the expression of the antiapoptotic protooncogene Bcl-2 in the human acute lymphoblastic leukemia (ALL) cell line Reh. This down-regulation has been shown to reduce drug resistance of the Reh cells to anti-tubulin polymerization agents. In the present study we investigated the effect of bryostatin 1 alone and in combination with novel anti-tubulin agents (dolastatin 10 and auristatin PE) and the chemotherapeutic vincristine on the inhibitor of apoptosis protein cIAP-1. Cells were cultured with bryostatin 1 (1 nM), dolastatin 10 (0.1 ng/ml), auristatin PE (0.1 ng/ml), or vincristine (0.5 ng/ml) alone or the combination of these anti-tubulins with bryostatin 1. Western blots were conducted to assess the effects of the above agents on cIAP-1 protein level. Flow-cytometric analysis [7-amino-actinomycin D (7AAD)] was conducted to assess apoptosis as well as staining for morphology using tetrachrome stain. Our results show that cIAP-1 is induced in a time-dependent fashion after bryostatin 1 exposure up to 72 h. However, upon treatment of cells with a combination of bryostatin 1 and dolastatin 10 or auristatin PE, the induction of cIAP-1 was abolished, leading to a significant increase in apoptosis. The initial 24- and 48-h reduction in cIAP-1 protein level recorded in the bryostatin 1 and vincristine combination recovered to control levels by 72 h. We believe that this phenomenon is responsible for the reduced apoptosis recorded in this combination. Results of this study should prove useful in guiding the clinical application of these novel agents in the treatment of ALL.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Depsipeptides; Down-Regulation; Flow Cytometry; Humans; Inhibitor of Apoptosis Proteins; Lactones; Macrolides; Oligopeptides; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Vincristine

We have previously reported that the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces further differentiation of the human acute lymphoblastic leukemia cell line Reh to a monocytoid B lymphocyte stage. In the present study, we investigated the differentiating capacity of another protein kinase C (PKC) activator, bryostatin 1 (bryo). Reh cells were treated in vitro with TPA, bryo, or interferon-alpha (IFN-alpha) for a period of 5 days during which cells were analyzed for changes in growth patterns, morphology, cytochemistry, and surface phenotype. Bryo caused a dose-dependent growth inhibition of Reh cells. Morphologically, the treated cells expressed monocytoid features with development of filopodia and numerous vacuoles indicating phagocytic activity. Bryo induced similar phenotypic changes to TPA, including induction of CD11c, increased expression of CD22 and down-regulation of CD10 and CD19. Enzymatically, bryo, like TPA, induced tartrate-sensitive acid phosphatase expression but failed to induce periodic acid Schiff (PAS) and nonspecific esterase (NSE). Bryo inhibited the TPA action on NSE and CD10. IFN-alpha showed additive growth inhibitory and phenotypic effects to bryo. Collectively, our findings indicate that bryo is capable of inducing further differentiation of the Reh cells along the B cell lineage similar to those of TPA.

Topics: Acid Phosphatase; Antigens, CD; Antineoplastic Agents; B-Lymphocytes; Bryostatins; Carboxylesterase; Carboxylic Ester Hydrolases; Cell Differentiation; Cell Division; Humans; Lactones; Macrolides; Microscopy, Electron; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured