bryostatin-1 and Leukemia--Lymphocytic--Chronic--B-Cell

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia for which therapeutic options remain unsatisfying as cure has remained elusive. Recent laboratory discoveries and promising results from completed phase III studies with fludarabine provide reason to reassess therapeutic goals in the treatment of patients with symptomatic CLL. Early enrollment of patients on protocols using combination therapies with fludarabine and new agents such as flavopiridol, IDEC-C2B8, Campath-1H, UCN-01, bryostatin, FR 901228, and melarsoprol will hopefully represent the next advance to improved overall survival and ultimately the cure of CLL.

Topics: Alkaloids; Antineoplastic Agents, Alkylating; Bryostatins; Flavonoids; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Piperidines; Staurosporine; Topoisomerase I Inhibitors

Preclinical studies suggested that bryostatin 1 might potentiate the therapeutic effects of fludarabine in the treatment of hematologic malignancies. We undertook a phase I study to identify appropriate schedules and doses of bryostatin 1 and fludarabine to be used in phase II studies.. Patients with chronic lymphocytic leukemia (CLL) or indolent lymphoma received fludarabine daily for 5 days and a single dose of bryostatin 1 via a 24-hour continuous infusion either before or after the fludarabine course. Doses were escalated in successive patients until recommended phase II doses for each sequence were identified on the basis of dose-limiting toxic events.. Bryostatin 1 can be administered safely and tolerably with full dose fludarabine (25 mg/m(2)/d x 5). The recommended bryostatin 1 phase II dose is 50 microg/m(2) for both sequences, bryostatin 1 --> fludarabine and fludarabine --> bryostatin 1. The combination is active against both CLL and indolent lymphomas with responses seen in patients who had been previously treated with fludarabine. Correlative studies do not support the hypothesis that bryostatin 1 potentiates fludarabine activity through down-regulation of protein kinase C in target cells.. Bryostatin 1 can be administered with full dose fludarabine, and the combination is moderately active in patients with persistent disease following prior treatment. In view of the activity of monoclonal antibodies such as the anti-CD20 monoclonal antibody rituximab in the treatment of CLL and indolent lymphomas, the concept of combining bryostatin 1 and fludarabine with rituximab warrants future consideration.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bryostatins; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Macrolides; Male; Maximum Tolerated Dose; Middle Aged; Prognosis; Survival Rate; Vidarabine

Bryostatin 1 activates protein kinase C (PKC) with short-term exposure and results in depletion of PKC with prolonged exposure. Preclinical in vitro and in vivo studies demonstrate synergistic activity and increased tumor apoptosis in B-cell malignancies when a prolonged infusion of bryostatin 1 is followed by vincristine.. We embarked on a Phase I trial of bryostatin 1 as a 24-h continuous infusion followed by bolus vincristine in patients with refractory B-cell malignancies other than acute leukemias. Twenty-four evaluable patients were enrolled.. The dose-limiting toxicity was myalgia. The MTD and recommended Phase II dose of bryostatin 1 was 50 microg/m2/24 h followed by vincristine 1.4 mg/m2 (maximum total dose of 2 mg) repeated every 2 weeks. Significant antitumor activity was observed in this relapsed population, including patients who had failed high-dose chemotherapy. This included 5 durable complete and partial responses and 5 patients with stable disease lasting > or =6 months (range, 6-48+ months). Median time to response was 8 months. Correlative studies demonstrated a progressive increase in serum interleukin-6 with bryostatin 1 infusion followed by an additional increase after vincristine. Flow cytometry for detection of apoptosis in B and T cells showed an initial decrease in apoptotic frequency in CD5+ cells within 6 h of bryostatin 1 infusion compatible with its known increase in PKC activity in the majority of patients followed by a return to baseline or overall increase in apoptotic frequency after completion of infusion. All (5 of 5) patients who had an overall increase in apoptotic frequency in CD5+ cells achieved either a clinical response or prolonged stable disease. Four of these 5 patients did not have the initial decrement in apoptosis at 6 h.. Given the lack of myelosuppression and early evidence of clinical efficacy, additional exploration of this regimen in non-Hodgkin's lymphoma and multiple myeloma is warranted.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Female; Flow Cytometry; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Macrolides; Male; Middle Aged; Multiple Myeloma; Neoplasm Recurrence, Local; Salvage Therapy; Vincristine

Topics: Antineoplastic Agents; Bryostatins; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Macrolides; Vidarabine

Bryostatin 1 is a natural product isolated from the marine bryozoan Bugula neritina in 1982 and is currently undergoing evaluation in a number of malignancies. Twenty-five patients with relapsed, low-grade non-Hodgkin's lymphoma or chronic lyphocytic leukemia (CLL) received bryostatin 1 by 72-h continuous infusion every 2 weeks at a dose of 120 microg/m2 per course. Patients who progressed while receiving bryostatin 1 alone could participate in a feasibility study by receiving vincristine administered by bolus i.v. injection immediately after the completion of the bryostatin 1 infusion. The dose of vincristine was escalated in groups of three patients as follows: level 1, 0.5 mg/m2; level 2, 1.0 mg/m2; and level 3, 1.4 mg/m2 with vincristine doses capped at 2.0 mg for all patients. Bryostatin 1 alone resulted in one complete remission and two partial remissions. Nine patients received sequential treatment with bryostatin 1 and vincristine. The addition of vincristine at a dose of 2 mg was feasible and caused the expected dose-related sensory neuropathy. Phenotypic analysis by flow cytometric analysis on pre- and post-bryostatin 1-treated peripheral blood lymphocytes revealed up-regulation in the coexpression of CD11c/ CD22 on CD20+ B cells in two of four CLL patients studied, which is consistent with in vitro findings of differentiation of CLL cells to a hairy cell phenotype.

Topics: Adult; Aged; Antineoplastic Agents; Bryostatins; Disease Progression; Fatigue; Feasibility Studies; Female; Flow Cytometry; Humans; Immunophenotyping; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Macrolides; Male; Middle Aged; Neoplasm Recurrence, Local; Nervous System Diseases; Pain; Remission Induction; Treatment Outcome; Vincristine

To define, in a phase I study in relapsed non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), the maximum-tolerated dose (MTD), major toxicities, and possible antitumor activity of bryostatin 1, a macrocyclic lactone.. Bryostatin 1 was delivered by 72-hour continuous infusion every 2 weeks to patients with relapsed NHL or CLL, at doses that ranged from 12 microg/m2 to 180 microg/m2 per course. Correlative investigations included evaluations of total protein kinase C (PKC) in peripheral blood and lymphoid differentiation in patient tumor tissue.. Twenty-nine patients were treated, including three patients with CLL and 26 with NHL. Generalized myalgia was the dose-limiting toxicity (DLT) and occurred in two of three patients treated with bryostatin 1 at 180 microg/m2 per course. Myalgias were dose-related and cumulative, and often started in the thighs and calves, improved with activity, were somewhat responsive to analgesics, and often took weeks to resolve once taken off study. Six patients were treated at the MTD of 120 microg/m2 per course. Myalgia, headache, and fatigue were common. Hematologic toxicity was uncommon. Total cumulative doses of bryostatin 1 up to 1,134 microg/m2 have been administered without untoward toxicity. Eleven patients achieved stable disease for 2 to 19 months. An in vitro assay for total PKC evaluation in patient peripheral-blood samples demonstrated activation within the first 2 hours with subsequent downregulation by 24 hours, which was maintained throughout the duration of the 72-hour infusion.. This phase I study defined the MTD and recommended phase II dose of bryostatin 1, when administered over 72 hours every 2 weeks, to be 120 microg/m2 (40 microg/m2/d for 3 days). Generalized myalgia was the DLT. Future studies will define the precise activity of bryostatin 1 in subsets of patients with lymphoproliferative malignancies and its efficacy in combination with other agents.

Topics: Adult; Aged; Antigens, CD; Antineoplastic Agents; Biomarkers, Tumor; Bryostatins; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Macrolides; Male; Middle Aged; Muscular Diseases; Pain; Recurrence

In spite of potent first-line therapies for chronic lymphocytic leukemia, treatment remains palliative and all patients frequently relapse. Treatment options for these patients are more limited. BL22 is a recombinant protein composed of the variable region of a monoclonal antibody that binds to CD22 and of PE38, a truncated Pseudomonas exotoxin. BL22 is a very potent drug already used in patients with hairy cell leukemia, whereas in chronic lymphocytic leukemia its cytotoxicity is limited by a lower expression of CD22. Here we demonstrate that this limitation can be overcome by pre-activation of chronic lymphocytic leukemia cells with bryostatin 1.. Primary malignant B cells from chronic lymphocytic leukemia and mantle cell lymphoma patients were used in vitro to assess the therapeutic impact of drug combinations using BL22 and bryostatin 1.. We demonstrate that bryostatin 1 sensitizes chronic lymphocytic leukemia cells for the cytotoxic effects of BL22 through activation of protein kinase C and subsequently increased CD22 surface expression. Dose and time response analysis reveals that activation of protein kinase C further activates an autocrine feedback loop degrading protein kinase C-βII protein. Depletion of protein kinase C-βII and upregulation of CD22 persist for several days following pre-stimulation with bryostatin 1. Therefore, our data provide a rationale for the sequential administration of BL22 following bryostatin 1 treatment. In addition to primary chronic lymphocytic leukemia cells, bryostatin 1 also sensitizes diffuse large B-cell lymphoma and mantle cell lymphoma cells to BL22 induced apoptosis.. Our data suggest that the combination of bryostatin 1 with antibodies directed against CD22 is a potent drug combination for the treatment of low- and high-grade B-cell lymphoma.

Topics: Antibodies; Antineoplastic Agents; Apoptosis; Bryostatins; Enterotoxins; Humans; Immunotoxins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Mantle-Cell; Protein Kinase C; Protein Kinase C beta; Sialic Acid Binding Ig-like Lectin 2; Tumor Cells, Cultured; Up-Regulation

Signals through the B-cell antigen receptor (BCR) are important for the survival of chronic lymphocytic leukemia (CLL) cells. Therefore, factors that influence these signals have important pathophysiological roles in this disease. One key mediator of BCR signaling is protein kinase C beta (PKCbeta), which regulates the activation of I-kappaB kinases and the deactivation of Bruton tyrosine kinase within the signaling pathways initiated by BCR engagement. The present study demonstrates that overexpression of the PKCbetaII isoform is a feature of CLL cells and that activity of this enzyme strongly correlates with CLL cell response to BCR engagement. Thus, intracellular Ca2+ release and increases in cell survival after BCR cross-linking were significantly greater in CLL patients with low levels than in CLL patients with high levels of active PKCbetaII. Furthermore, BCR-induced Ca2+ fluxes could be restored in CLL patients with high levels of active PKCbetaII by pretreating the cells with the PKCbeta-specific inhibitor LY379196. Conversely, BCR-mediated intracellular Ca2+ release could be inhibited in CLL cells with low levels of active PKCbetaII by pretreatment with the PKC agonist bryostatin. Taken together, these results demonstrate that overexpressed active PKCbetaII plays a role in the regulation and outcome of BCR signals that can be important for the progression of CLL.

Topics: Bryostatins; Calcium; Cell Survival; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Mesylates; Protein Kinase C; Protein Kinase C beta; Pyrroles; Receptors, Antigen, B-Cell; Signal Transduction

Pre-treatment with bryostatin 1 (bryo) has been shown to potentiate the efficacy of (2-chloro-2-deoxyadenosine, cladribine, 2-CdA) in B-cell chronic lymphocytic leukemia (B-CLL) by increasing the ratio of deoxycytidine kinase (dCK) to 5'-nucleotidase (5'-NT) activity. The bryo-induced increase in dCK/5'-NT activity alone has not been a conclusive indication of final clinical outcome. Therefore, we used an ex vivo assay to investigate factors which may affect the bryo-induced enhancement of 2-CdA efficacy in B-CLL patient-derived samples. Bryo-induced increase in dCK/5'-NT was inversely associated with Rai stage CLL (r=-0.86). Increased dCK/5'-NT activity was not correlated with increased efficacy (cell death) or percentage of cellular [8-3H]-2-CdA converted to [8-3H]-2-CdATP ex vivo. Bryo pre-treatment increased the cellular uptake of [8-3H]-2-CdA and incorporation of [8-3H]-2-CdA metabolites into the DNA fraction. Cell death from 2-CdA was inversely correlated with bryo-induced activity of the DNA repair enzyme, DNA-PKcs, (r=-0.77). Thus, the ability of B-CLL to repair damaged DNA may be a more important predictor of the response to bryo/2-CdA and eventual clinical outcome than dCK/5'-NT activity. Additional CLL patients under bryo-2-CdA therapy are needed to verify these important observations.

Topics: 2-Chloroadenosine; 5'-Nucleotidase; Antimetabolites, Antineoplastic; Bryostatins; Cell Death; Deoxyadenosines; Deoxycytidine Kinase; DNA-Activated Protein Kinase; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Synergism; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Nuclear Proteins; Protein Serine-Threonine Kinases; ras Proteins; Tumor Cells, Cultured

Bryostatin, a macrocyclic lactone and protein kinase C (PKC) modulator, has been shown to have differentiation and anti-tumor activity against several leukemia cell lines in vitro. In this study, we demonstrated Bryostatin-induced differentiation in B-cell chronic lymphocytic leukemia (B-CLL) cells, characterized by an increase in cell size and a marked up-regulation of CD11c expression. The specific inhibitors of the extracellular signal-regulated kinase (ERK) and protein kinase C pathways, PD98059 and GF 109203X respectively, each completely blocked Bryostatin-induced differentiation of B-CLL cells, suggesting that activation of the ERK pathway plays a direct role in this process in a PKC-dependent manner. Furthermore, Bryostatin reduced both spontaneous and drug-induced apoptosis with chlorambucil, fludarabine and 2-chloro-2'-deoxyadenosine (2-Cda) in B-CLL cells. This resistance was associated with an early up-regulation of the anti-apoptotic protein Mcl-1 and post-translational phosphorylation of Bcl-2 at serine 70. The anti-apoptotic effects of Bryostatin were abrogated by GF 109203X, and to a lesser extent by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002. Interestingly, the ERK inhibitor, PD98059 inhibited Mcl-1 expression but had little effect on Bryostatin-induced survival suggesting that the ERK pathway predominantly affects differentiation. Taken together these results present an explanation for Bryostatin-induced B-CLL cell survival in which modulation of the PKC pathway couples differentiation with an increase in antiapoptotic protein expression and calls into question the rationale for its use in the treatment of B-CLL.

Topics: Apoptosis; Bryostatins; Cell Differentiation; Drug Resistance, Neoplasm; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured; Up-Regulation

Bryostatin 1 is known to exhibit in vitro and in vivo activity against chronic lymphocytic leukemia (CLL) cells by inducing their further maturation into plasma-like cells. Signal transducer and activator of transcription (STAT) proteins play a central role in B-lymphocyte growth and function and are aberrantly phosphorylated on serine residues in CLL cells. To determine whether STAT transcription factors are important in Bryostatin 1-induced differentiation of CLL cells, primary CLL cells were examined for signaling events following exposure to Bryostatin 1 in vitro. Western analysis and electrophoretic mobility shift assays revealed that Bryostatin 1 induced tyrosine phosphorylation and DNA binding of STAT1, yet there was no effect on constitutive serine phosphorylation of STAT1. Bryostatin 1-induced STAT1 activation occurred in a manner that was dependent on protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Janus tyrosine kinase (JAK) activation. Evidence indicates that Bryostatin 1 induces STAT1 activation through an interferon gamma (IFN gamma) autocrine loop. However, STAT1 activation by IFN gamma stimulation alone was not sufficient to induce differentiation. This insufficiency is due to the broader effect on gene expression caused by Bryostatin 1 compared with IFN gamma, as demonstrated by microarray analysis. Both up-regulation of CD22 expression and immunoglobulin M (IgM) production, markers of CLL differentiation, were inhibited by a decoy oligonucleotide for STAT1, indicating that STAT1 is necessary for Bryostatin 1-induced differentiation of CLL cells. This study implicates STAT transcription factors as important mediators of Bryostatin 1-induced differentiation of CLL cells and could possibly lead to improved therapeutic approaches for the treatment of CLL.

Topics: Antineoplastic Agents; Blotting, Western; Bryostatins; Cell Differentiation; Cell Nucleus; Cytokines; DNA-Binding Proteins; Down-Regulation; Humans; Immunoglobulins; Interferon-gamma; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Models, Biological; Oligonucleotide Array Sequence Analysis; Phosphorylation; Precipitin Tests; Protein Kinase C; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; STAT1 Transcription Factor; Trans-Activators; Transfection; Tyrosine; Up-Regulation

Bryostatin 1 (bryo 1) has been shown to potentiate the anti-tumor activity of 2-chloro-2-deoxyadenosine (2-CdA) in chronic lymphocytic leukemia (CLL) and in the WSU-CLL cell line. However, like resistant CLL, WSU-CLL cells lose their sensitivity to bryo 1/2-CdA treatment. We report that 2-CdA-induced IAP expression may be a possible mechanism whereby resistance to apoptosis is acquired in these cells. In WSU-CLL cells, three members of the Inhibitors of Apoptosis (IAP) family were identified. Bryo 1 treatment of WSU-CLL cells leads to initiation of the apoptotic cascade and induced a marginal increase in XIAP protein expression. In contrast, 2-CdA treatment, alone or in combination with bryo 1, induced a substantial increase in survivin and XIAP proteins and phosphorylation of BAD. Bryo 1 alone induced caspase-7 and -9 dependent [poly ADP-ribose] polymerase (PARP) cleavage, while sequential treatment with bryo 1 (72 h) followed by 2-CdA (24 h) induced caspase-3,-7, and -9 dependent PARP cleavage and increased apoptosis. Although exposure to bryo 1 initiated apoptotic events, apoptosis was first enhanced by 2-CdA, and then reversed in a time-dependent manner by 2-CdA-induced expression of survival proteins. Taken together, resistance to bryo 1/2-CdA treatment may be the result of 2-CdA-induced IAP inhibition of the intrinsic apoptotic pathway caspases.

Topics: 2-Chloroadenosine; Aged; Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Bryostatins; Deoxyadenosines; Enzyme Activators; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Male; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.

Topics: Aged; Antineoplastic Agents; Biological Transport, Active; Bryostatins; Carrier Proteins; Cladribine; Deoxycytidine Kinase; Dipyridamole; Down-Regulation; Drug Resistance, Neoplasm; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Male; Membrane Proteins; Nucleoside Transport Proteins; Phosphorylation; Thioinosine; Tumor Cells, Cultured; Vidarabine

Bryostatin 1 (Bryo-1) has been shown to differentiate chronic lymphocytic leukemia (CLL) cells to the hairy cell leukemia phenotype. The purine analogue 2-chlorodeoxyadenosine (2-CdA) exhibits enhanced activity in patients with hairy cell leukemia compared to those with CLL. Here we present a case report of a patient diagnosed with resistant CLL and treated sequentially with Bryo-1 followed by 2-CdA for three cycles. Molecular and biochemical parameters relative to the sequential treatment with these agents in vivo were comparable to those found in the WSU-CLL cell line in vitro (R. M. Mohammad et al., Clin. Cancer Res., 4: 445-453, 1998; R. M. Mohammad et al., Biol. Chem., 379: 1253-1261, 1998). There was a significant reduction of lymphocyte count from 37.1 x 10(3)/microl before the treatment to 3.4 x 10(3)/microl after treatment, and partial remission was achieved 2 months after the treatment. The percentage of morphologically differentiated lymphocytes was increased from 3% before treatment to 92% with the first cycle of Bryo-1. Similarly, expression of CD22, a marker of differentiation, increased from 38% to 97% and was maintained at a high level for the duration of the treatment. Analysis of the molecular markers of apoptosis in isolated peripheral blood lymphocytes revealed an increase in the Bax:Bcl-2 ratio after treatment with Bryo-1 in cycles 2 and 3, with associated poly(ADP-ribose) polymerase cleavage after Bryo-1 and 2-CdA treatment. The deoxycytidine kinase: cytosolic 5'-nucleotidase activity ratio increased modestly after Bryo-1 treatment, indicating increased sensitivity of the peripheral blood lymphocytes to 2-CdA. In summary, we found that sequential treatment with Bryo-1 and 2-CdA caused a significant reduction in peripheral blood lymphocytes (CLL cells) with simultaneous induction of differentiation and the initiation of the Bax: Bcl-2 apoptotic pathway.

Topics: 5'-Nucleotidase; Aged; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Antineoplastic Combined Chemotherapy Protocols; bcl-2-Associated X Protein; Blotting, Western; Bryostatins; Cell Adhesion Molecules; Cladribine; Clinical Trials, Phase I as Topic; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Integrin alphaXbeta2; Lactones; Lectins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Macrolides; Male; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sialic Acid Binding Ig-like Lectin 2

WSU-CLL is a de novo fludarabine resistant cell line established from a patient with advanced chronic lymphocytic leukemia (CLL) refractory to chemotherapy including fludarabine (Flud). Our previous studies indicate that bryostatin 1 (Bryo 1) induces differentiation of WSU-CLL and increases the ratio of dCK/5'-NT activity and Bax/Bcl-2. This study tests the hypothesis that Bryo 1-differentiated cells are more susceptible to Flud than the parent WSU-CLL cells. Flud, given sequentially after Bryo 1, in vitro and in vivo animal studies resulted in significantly higher rates of growth inhibition and improved animal survival. Flud at 100 to 600 nM exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. The sequential exposure to Bryo 1 (10 nM for 72 h) followed by Flud (100 nM) resulted in significantly higher rates of growth inhibition than either the reverse addition of these two agents or each agent alone, but was not significantly different than the concurrent addition of Bryo 1 + Flud. Using 7-amino-actinomycin D staining and flow cytometry, apoptosis was seen in 40.8% of cells treated with Bryo 1 (10 nM, 72 h) followed by Flud, compared with Flud (100 nM, 72 h) followed by Bryo 1 (18.1%). To demonstrate that Bryo 1 enhancement of Flud efficacy was not restricted to in vitro culture, we used the WSU-CLL xenograft model in mice with severe combined immune deficiency (SCID). Bryo 1 + Flud at the maximum tolerated doses (75 microg/kg i.p. and 200 mg/kg i.v., respectively) were administered to mice in different combinations. The survival in days, the tumor growth inhibition ratio (T/C), the tumor growth delay (T-C) in days, log10 kill, as well as mean tumor weight (mtw) of mice treated with Bryo 1 followed by Flud, were significantly better than control and other groups. T/C%, T-C, log10 kill and mtw were as follows: Bryo 1 (36.8%, 10 days, 0.8, 375 mg); Flud (100%, 0. 0 day, 0.0, 1130 mg); Bryo 1 + Flud (14.3%, 12 days, 0.95, 288 mg); Bryo 1 followed by Flud (4.6%, 17 days, 1.35, 35 mg); Flud followed by Bryo (40.3%, 10 days, 0.80, 175 mg). We conclude that: i) Bryo 1 sensitizes WSU-CLL cells to Flud and enhances apoptosis; ii) the sequential treatment with Bryo 1 followed by Flud resulted in higher anti-tumor activity compared with either agent alone, in combination, or the reverse addition of these agents and iii) these results are comparable to those of Bryo 1 followed by 2-CdA suggesting common pathway(s) of interaction between Bryo

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Cell Division; Disease Models, Animal; Drug Resistance, Neoplasm; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Mice; Mice, Inbred ICR; Mice, SCID; Neoplasm Transplantation; Transplantation, Heterologous; Tumor Cells, Cultured; Vidarabine

Previous studies on intact cells have shown that bryostatin 1 (Bryo 1) induces significant alterations in the membranes of WSU-CLL cells (a drug-resistant B-CLL cell line), changes which may play an important role in the mechanism of reduced drug resistance of B-CLL cells to 2-chlorodeoxyadenosine (2-CdA). However, it is not clear whether the plasma membranes or the mitochondria, or both are involved; nor is it known which of these two targets is more important for regaining the cells former drug sensitivity. For the present study, we treated WSU-CLL cells with Bryo 1, isolated plasma membranes and mitochondria, and then subjected the purified fractions to infrared (IR) spectroscopic and chromatographic analyses. IR spectroscopy revealed a decreased glycosylation of both plasma membranes and mitochondria in Bryo 1-treated cells compared to untreated cells. The amount of lipid relative to protein was increased in both types of membranes, but considerably more enhanced in the plasma membrane fraction of the Bryo 1-treated cells than in mitochondria. Quantitative lipid analysis by thin layer chromatography also revealed that Bryo 1 treatment significantly increased the phospholipid content in plasma membranes, whereas the lipids in the mitochondria remained essentially unchanged. Changes in lipid composition were quite dramatic for plasma membranes where phosphatidylcholines were decreased by 50%, phosphatidylethanolamines doubled and sphingomyelins increased five-fold compared to the lipid composition in plasma membranes of untreated cells. In addition, the IR spectroscopic analysis provided evidence for an increased plasma membrane fluidity in Bryo 1-treated cells, whereas the fluidity of the mitochondria remained essentially unchanged; marker bands indicating mitochondrial DNA decreased upon Bryo 1 treatment. These results suggest that Bryo 1 increases the sensitivity of WSU-CLL cells to chemotherapeutic agents such as 2-CdA by action on two cell targets: (1) introduction of significant changes in plasma membrane permeability or fluidity through modifications in lipid content and composition as well as by reducing the surface glycosylation; (2) introduction of changes in lipid and DNA content of the mitochondria. Small alterations in the lipid composition of the mitochondria may provide the conditions for an altered proton gradient and transmembrane potential leading to apoptosis and decreased cell survival.

Topics: Antineoplastic Agents; Bryostatins; Cell Membrane; Drug Resistance, Neoplasm; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Membrane Lipids; Membrane Proteins; Mitochondria; Spectroscopy, Fourier Transform Infrared; Tumor Cells, Cultured

Modulating signal transduction pathways represents a promising approach for altering the biological behaviour of haemopoietic malignancies. B-cell chronic lymphocytic leukaemia (B-CLL) cells were treated in vitro with CD40-ligand (CD40L) (CD154) or the protein kinase C modulator Bryostatin-1, exploring the effects on: (a) sensitivity to apoptosis induction by chemotherapeutic drugs (fludarabine, dexamethasone) or anti-Fas antibody; (b) expression of apoptosis-regulatory proteins (Bcl-2, Bcl-X, Mcl-1, Bax, Bak, BAG-1, Flip, XIAP); (c) expression of cell surface co-stimulatory antigens (CD80 [B7.1]; CD54 [ICAM-1]; CD70); and (d) expression of immune modulatory receptors (CD27, CD40, CD95 [Fas]). CD40L and Bryostatin decreased both spontaneous and drug-induced apoptosis in most B-CLL specimens tested. Apoptosis resistance was associated with CD40L- and Bryostatin-induced elevations in the anti-apoptotic Bcl-2 family protein Mcl-1. CD40L also induced striking increases in the levels of the anti-apoptotic protein Bcl-XL in B-CLLs. CD40L stimulated increases in the surface expression of CD40, CD54, CD69, CD70, CD80 and CD95, whereas Bryostatin induced expression of CD40, CD54, CD69 and CD95 but not the co-stimulatory molecules CD70 and CD80. Despite elevations in the expression of CD95 (Fas), anti-Fas antibodies failed to induce apoptosis of CD40L- and Bryostatin-treated B-CLL cells. This Fas-resistance was associated with increased expression of the Fas-antagonist Flip in CD40L-treated, and with elevations in the caspase inhibitor XIAP in Bryostatin-treated B-CLLs. The potential anti-apoptotic properties of CD40L and Bryostatin should be taken into consideration when employing these agents in clinical trials involving patients with B-CLL.

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; CD40 Ligand; Cell Survival; Drug Resistance, Neoplasm; Enzyme Activators; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Membrane Glycoproteins; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

We have previously reported that bryostation 1 (Bryo 1) induces differentiation of chronic lymphocytic leukemia (CLL) in vitro to a hairy cell (HC) stage. This study tests the hypothesis that Bryo 1-differentiated CLL cells are more susceptible to 2-chlorodeoxyadenosine (2-CdA) than parent CLL cells. A recently established EBV-negative CLL line (WSU-CLL) from a patient resistant to chemotherapy including fludarabine was used to test this hypothesis. Both Bryo 1 (10-1000 nM) and 2-CdA (5.6-22.4 microM) exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. In vitro, the sequential exposure to Bryo 1 (100 nM for 72 h) followed by 2-CdA (11.2 microM) resulted in significantly higher rates of growth inhibition than either agent alone. Changes in immunophenotype, enzymes, lipids, proteins, and the DNA of WSU-CLL cells were studied before and after Bryo 1 treatment. Bryo 1 induced a positive tartrate-resistant acid phosphatase reaction and two important markers, CD11c and CD25, after 72 h of culture, confirming the differentiation of CLL to HC. The Fourier transformation infrared spectroscopic analysis showed that the amount of membrane lipids significantly increased in Bryo 1-treated cells compared to controls after 24 h, whereas the protein content, as well as the DNA content, decreased. This finding supports the change of CLL to HC. To evaluate the in vivo efficacy of Bryo 1 and 2-CdA, we used a xenograft model of CLL in WSU-CLL-bearing mice with severe combined immune deficiency. s.c. tumors were developed by injection of 10(7) WSU-CLL cells, and fragments were then transplanted into a new batch of severe combined immunodeficient mice. Bryo 1 and 2-CdA at the maximum tolerated doses (75 micrograms/kg i.p. and 30 mg/kg s.c., respectively) were administered to the mice at different combinations and schedules. The survival in days, the tumor growth inhibition ratio, the tumor growth delay, and the log10 kill of the mice treated with Bryo 1 followed by 2-CdA were significantly better than the control and other groups. We conclude that the sequential treatment with Bryo 1 followed by 2-CdA resulted in higher antitumor activity and improved animal survival.

Topics: Acid Phosphatase; Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Cell Division; Cladribine; DNA, Neoplasm; Drug Administration Schedule; Drug Screening Assays, Antitumor; Humans; Lactones; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lipid Metabolism; Macrolides; Male; Mice; Mice, SCID; Neoplasm Proteins; Neoplasm Transplantation; Spectroscopy, Fourier Transform Infrared; Transplantation, Heterologous; Tumor Cells, Cultured

We tested the activity of dolastatin 10 (a natural product derived from the shell-less marine mollusk, Dolabella auricularia, a sea hare) and its structural modification, auristatin PE, alone and in combination with bryostatin 1 (a protein kinase C activator derived from the marine bryozoan Bugula neritina) on a human B-cell chronic lymphocytic leukemia cell line (WSU-CLL) and in a severe combined immune deficient (SCID) mouse xenograft model bearing this cell line. WSU-CLL cells were cultured in RPMI 1640 at a concentration of 2 x 10(5)/ml using a 24-well plate. Agents were added to triplicate wells, and cell count, viability, mitosis, and apoptosis were assessed after 24 h of incubation at 37 degrees C. Results showed that dolastatin 10 had no apparent inhibition of cell growth at concentrations less than 500 pg/ml. Auristatin PE, on the other hand, showed significant growth inhibition at concentrations as low as 50 pg/ml. Auristatin PE-treated cultures, at this concentration, exhibited 27 and 4.5% mitosis and apoptosis, respectively. Dolastatin 10, at the same concentration, did not exert any effect and was comparable with that of control cultures. In the WSU-CLL-SCID mouse xenograft model, the efficacy of these agents alone and in combination with bryostatin 1 was evaluated. Tumor growth inhibition (T/C), tumor growth delay (T-C), and log10 kill for dolastatin 10, auristatin PE, and bryostatin 1 were 14%, 25 days, and 1.98; 2%, 25 days, and 1.98; 19%, 13 days, and 1.03, respectively. Auristatin-PE produced cure in three of five mice, whereas dolastatin 10 showed activity but no cures. When given in combination, auristatin PE + bryostatin 1-treated animals were all free of tumors (five of five) for 150 days and were considered cured. Dolastatin 10 + bryostatin 1-treated animals produced cure in only two of five mice. We conclude that: (a) auristatin-PE is more effective in this model than dolastatin 10; (b) auristatin PE can be administered at a concentration 10 times greater than dolastatin 10; (c) there is a synergetic effect between these agents and bryostatin 1, which is more apparent in the bryostatin 1 + auristatin PE combination. The use of these agents should be explored clinically in the treatment of CLL.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bryostatins; Cell Count; Depsipeptides; Drug Screening Assays, Antitumor; Female; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Mice; Mice, SCID; Mitosis; Oligopeptides; Subrenal Capsule Assay; Survival Analysis; Treatment Outcome; Tumor Cells, Cultured

Infrared (IR) spectroscopy was used to compare the drug resistance mechanism of cells from chronic lymphocytic leukemia (CLL) patients with that of WSU-CLL cells. Bryostatin 1 (Bryo 1), a macrocyclic lactone and protein kinase C activator, was used to render WSU-CLL cells more susceptible to 2-chlorodeoxyadenosine (2-CdA). The IR spectroscopic analysis revealed some changes in protein and DNA content in Bryo 1-treated WSU-CLL cells, however, the most significant alterations were observed in the membrane lipids, which resemble those found between 2-CdA-sensitive and 2-CdA-resistant cells from CLL patients. In addition, Bryo 1 treatment induced WSU-CLL cells to become CD11c, CD25 and tartrate-resistant acid phosphatase-positive, specific markers for hairy cell leukemia, a disease exquisitely sensitive to 2-CdA. Our results suggest that 2-CdA-sensitive CLL cells have cellular characteristics resembling the hairy cell stage. The similarity between the membrane lipids in 2-CdA-sensitive CLL cells and the Bryo 1-treated WSU-CLL cell line supports the suggestion that membrane lipid alteration might be an important step in the drug resistance mechanism of CLL cells.

Topics: Adult; Aged; Antineoplastic Agents; Bryostatins; Cladribine; Drug Resistance; Female; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Macrolides; Male; Middle Aged; Spectrophotometry, Infrared; Tumor Cells, Cultured

The activities of 2-chlorodeoxyadenosine (2-CdA) metabolizing enzymes, deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase (5'-NT) were measured in control and bryostatin 1 treated CLL cells using an EBV-negative WSU-CLL cell line. This cell line was established from a patient with CLL resistant to fludarabine. The results revealed a significant increase in dCK activity in bryostatin 1 treated cells at 48 and 72 h compared with the control. 5'-NT activity decreased significantly at 48 h. The ratio of dCK to 5'-NT activity was significantly increased in bryostatin 1 treated WSU-CLL cells after 48 h. WSU-CLL cells treated with bryostatin 1 exhibited an increase in the percentage of apoptotic and dead cells from control levels of 16% to 40%. This percentage was further increased to 67% following the addition of 11.2 microM 2-CdA to WSU-CLL cells pretreated with bryostatin 1. Results from Western blot analysis indicate that WSU-CLL cells express high levels of Bcl-2, Bcl-xL and c-myc, and a low level of Bax. p53 in untreated WSU-CLL cells is undetectable. WSU-CLL cells treated with bryostatin 1 showed a significant increase in the ratio of Bax to Bcl-2. To demonstrate that the bryostatin 1 mediated enhancement of 2-CdA efficacy was not restricted to in vitro cell culture, we have studied the tumor growth delay of WSU-CLL xenografts treated with placebo, bryostatin 1, 2-CdA, and bryostatin 1 followed by 2-CdA. SCID mice given bryostatin 1 at 75 microg x kg(-1) x d(-1) for 5 days followed by 30 mg x kg(-1) x d(-1) 2-CdA for 5 days in two cycles, had significantly improved tumor growth delay (P = 0.05). We conclude that bryostatin 1 is not only capable of inducing apoptosis by itself, but also sensitizes de novo resistant WSU-CLL cells to the chemo-therapeutic effects of 2-CdA. The bryostatin 1-induced increased ratio of dCK/5'-NT activity and an increased ratio of Bax/Bcl-2 are at least two mechanisms through which this natural compound is able to potentiate the anti-tumor activity of 2-CdA in otherwise resistant CLL cells.

Topics: 5'-Nucleotidase; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Bryostatins; Cell Division; Cladribine; Deoxycytidine Kinase; Drug Synergism; Female; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Mice; Mice, Inbred ICR; Mice, SCID; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Tumor Cells, Cultured; Tumor Suppressor Protein p53

B-Chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) are both differentiated B-cell lymphoproliferative disorders. Prior studies have suggested that phorbol esters and the macrocyclic lactone Bryostatin-1, which are both protein kinase-C activators, can induce the differentiation of B-CLL cells into HCL cells in vitro, as evidenced by morphology, phenotype and TRAP activity. The differentiating effect of all-trans retinoic acid on B-CLL cells has been less extensively studied. We studied the effects of incubating adherence purified B-CLL cells with phorbol myristic acetate (PMA), all-trans retinoic acid (ATRA), and Bryostatin-1. None of these agents induced a true HCL phenotype (CD5-, CD11c/CD25 coexpression) under the conditions studied.

Topics: Acid Phosphatase; Antigens, CD; Antigens, Neoplasm; B-Lymphocytes; Biomarkers, Tumor; Bryostatins; Cell Differentiation; Enzyme Activation; Humans; Immunophenotyping; Isoenzymes; Lactones; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Neoplastic Stem Cells; Protein Kinase C; Tartrate-Resistant Acid Phosphatase; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The protein kinase C activator bryostatin induces differentiation and antagonizes the effects of tumour-promoting phorbol esters in a number of different cell types. We show here that bryostatin preferentially inhibits phorbol 12-myristate 13-acetate (PMA)-induced proliferation compared with differentiation in a number of different B chronic lymphocytic leukaemia (BCLL) cell populations examined. By using a panel of 11 early-response gene probes in Northern hybridization analysis, we found that the profile of genes induced in response to bryostatin and PMA was qualitatively similar and displayed comparable sensitivities to inhibition with the serine-threonine kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine hydrochloride (H7), consistent with common signalling through protein kinase C. However, the nuclear oncogene. c-myc, which was induced strongly in response to PMA treatment, was only marginally up-regulated by bryostatin. In addition, bryostatin selectively inhibited the magnitude of PMA-responsive induction of c-myc, to a degree commensurate with its antagonistic effects seen at the biological level. Finally, an anti-sense oligonucleotide blockade of c-myc inhibited PMA-induced proliferation but not the differentiation of BCLL cells, implicating this nuclear oncogene as an important determinant distinguishing PMA from bryostatin-coupled biological responses and also as a candidate third-messenger effector target for the anti-tumour effects of bryostatin.

Topics: Bryostatins; DNA Replication; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Neoplastic; Genes, Immediate-Early; Genes, myc; Humans; Immunoglobulin M; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Macrolides; Oligonucleotides, Antisense; RNA; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation

The phorbol esters induce differentiation of chronic lymphocytic leukemia (CLL) cells. Clinical use of this observation has been hampered by the fact that phorbol esters are also tumor promoters. In this study we demonstrate that another protein kinase C activator, without tumor promoting activity, has similar effects on CLL cells. Fresh leukemic cells from the peripheral blood of 13 patients with CLL were isolated and cultured in the absence (control) or presence of Bryostatin 1 or 12-0-tetradecanoylphorbol 13-acetate (TPA). Aliquots of cells were then analyzed after 24, 72 and 120 hours for morphological changes, acid phosphatase (ACP) and the co-expression of two hairy cell-associated surface antigens, CD22 and CD11c, by flow cytometry. Bryostatin 1 induced changes in shape and morphology similar to TPA, with adherence and increase in cell size, abundant cytoplasm and irregular cytoplasmic membrane. Both agents induced a statistically significant increase in the expression of CD22 and CD11c compared with control (p < 0.0008). There was no significant difference between the two agents in the degree of expression of these two markers. Both agents also induced ACP that was tartrate resistant (TRAP). These changes indicate that Bryostatin is as effective as TPA in inducing further differentiation of CLL cells to a hairy cell stage.

Topics: Acid Phosphatase; Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Bryostatins; CD11 Antigens; Cell Adhesion Molecules; Cell Differentiation; Enzyme Activation; Female; Humans; Immunophenotyping; Lactones; Lectins; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Male; Middle Aged; Protein Kinase C; Sialic Acid Binding Ig-like Lectin 2; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The effects of bryostatin 1 (Bryo 1), a protein kinase C (PKC) activator, on proliferation, differentiation and macromolecular synthesis were investigated in the two cell lines EHEB and JVM-2, established from patients with chronic B-cell leukemia. Treatment with Bryo 1 inhibited the proliferation, DNA and RNA synthesis in a time- and dose-dependent fashion. The cells differentiated along the B-cell pathway to plasmacytoid cells as judged by morphological examination and increased their production and secretion of immunoglobulins. c-myc mRNA expression was induced in both cell lines. The phorbol ester TPA, a pharmacological PKC activator, had similar differentiation-inducing effects. The biomodulators failed to induce significant alterations in the cell surface marker profile. Except for their surface markers, all parameters studied were more strongly altered in JVM-2 than in EHEB cells. JVM-2 was established from a patient with B-prolymphocytic leukemia (PLL), whereas EHEB originated from a case of B-chronic lymphocytic leukemia (CLL). These data support the notion that PLL cells appear to be activated B-cells, in contrast to the rather quiescent CLL cells. Since Bryo 1 lacks tumor-promoting activity, this naturally occurring compound, extracted from marine animals, has a potential role in the therapy of B-cell neoplasms as a differentiating agent.

Topics: Antigens, Neoplasm; Antigens, Surface; Antineoplastic Agents; Bryostatins; Cell Differentiation; Cell Division; Humans; Immunoglobulins; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Proto-Oncogenes; Tumor Cells, Cultured

Most B chronic lymphocytic leukemia (CLL) cells express on their surface the CD5 antigen which is an activation marker on normal B cells. To investigate the control of CD5 expression in B-CLL cells, we examined several inducing agents for their effects on CD5 mRNA expression. Northern blot analysis demonstrated that the expression of CD5 mRNA could be up- or down-regulated depending on the inducers used. Treatment with direct activators of protein kinase C (PKC), the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the natural agent bryostatin 1 (Bryo), caused increased CD5 mRNA expression after 8-16 h of incubation. In contrast, exposure to the dual signals of a PKC activator (PMA or Bryo) plus the calcium ionophore A23187 led to down-regulation of CD5 mRNA expression. The molecular alterations at the RNA level were accompanied by morphological changes: PMA and/or Bryo induced cellular features of activation while PMA plus A23187 or Bryo plus A23187 mediated morphological changes indicative of differentiation to plasmacytoid cells. The data suggest that as a consequence of maturation differentiated B-CLL cells down-regulate CD5 expression by analogy with the normal ontogenic process in which plasma cells, the end-stage cells of normal B cell differentiation, are CD5-.

Topics: Aged; Antigens, CD; Antigens, Differentiation; Bryostatins; Calcimycin; CD5 Antigens; Cell Differentiation; Female; Gene Expression Regulation, Neoplastic; Humans; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Male; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We describe in vitro studies undertaken to characterize the expression of the proto-oncogene c-jun during differentiation of B-CLL cells. The phorbol ester TPA and the natural compound Bryostatin 1 (Bryo) were used to directly stimulate protein kinase C (PKC) while the calcium ionophone A23187 was employed to increase intracellular Ca2+. In quiescent cells c-jun mRNA expression was undetectable or at low levels. Upon treatment with TPA or Bryo, the steady-state levels of c-jun mRNA increased rapidly, reached a maximum at 0.5 or 1 hr, and then decreased in the B-CLL cells from all five patients analyzed; this reaction was augmented by the addition of A23187. Induction of c-jun mRNA by direct stimulation of PKC could be blocked by the PKC inhibitor H7. The present observations, along with other results on the induction of long-term phenotypical cellular changes, such as alteration of morphology and other features of differentiation, support the notion that the second messenger (via PKC) and the third messenger (via proto-oncogene products) pathways are intact in B-chronic lymphocytic leukemia cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Bryostatins; Calcimycin; Cell Differentiation; Enzyme Activation; Gene Expression; Humans; Isoquinolines; Kinetics; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Piperazines; Protein Kinase C; Proto-Oncogene Mas; Proto-Oncogenes; RNA, Messenger; Second Messenger Systems; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

Peripheral blood cells from nine patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate). Like the phorbol ester 12-0-tetradecanoyl-phorbol 13-acetate (TPA), bryostatin 1 activates protein kinase C (PKC), which plays a central role in the phosphatidylinositol signal transduction pathway. The effects of bryostatin 1 alone and in combination with TPA or with the calcium mobilizing ionophore A23187 were assessed by morphological appearance, cell adherence and aggregation, RNA and DNA synthesis, and immunoglobulin (Ig) production. While eight of nine B-CLL cultures remained proliferatively inert, bryostatin 1 could effectively trigger activation and differentiation of B-CLL cells in all cases as inferred by the induction of morphological changes, RNA synthesis, and monotypic Ig production. Addition of calcium ionophore A23187 to bryostatin 1-exposed cells resulted in significantly increased values for RNA synthesis and Ig production and in the acquisition of plasmacytoid morphology. Bryostatin 1 and the dual signal of bryostatin 1 plus A23187 mimicked the stimulatory action of TPA and the combination of TPA plus A23187, respectively. Overall, bryostatin 1 was less active than equivalent concentrations of TPA. This lesser efficacy may, however, reflect a quantitative rather than qualitative difference. Bryostatin 1 partially antagonized TPA-mediated effects on B-CLL cells suggesting different modes of action by the two activators. These studies indicate that bryostatin 1 has effective differentiation-inducing properties on B-CLL cells that can differentiation-inducing properties on B-CLL cells that can be accentuated by a calcium ionophore.

Topics: Aged; Antineoplastic Agents; B-Lymphocytes; Biomarkers, Tumor; Bryostatins; Calcimycin; Cell Adhesion; Cell Aggregation; Cell Differentiation; DNA, Neoplasm; Female; Humans; Immunoglobulins; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Male; Middle Aged; Receptors, Antigen, B-Cell; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured