calcimycin and Leukemia--Lymphocytic--Chronic--B-Cell

B-cell chronic lymphocytic leukemia (B-CLL) is a hematologic malignancy characterized by the proliferation and accumulation of mature-looking B lymphocytes. Patients with B-CLL exhibit a number of immune defects including: auto-antibodies, depressed cell-mediated immunity and hypogammaglobulinemia (HG). We investigated the control of Ig production in the malignant CLL B-cell at a transcriptional and translation level. We isolated fresh leukemic B-cells from CLL patients and analyzed for the presence of nuclear factors OCT-1, OCT-2, and NF-KB. Malignant B-cells were purified to greater than 90% B-cells, and total cellular RNA and nuclear proteins were isolated from these cells. Mobility shift assays were probed with 32P-labeled oligonucleotides specific to the immunoglobulin (Ig) enhancer and promotor regions. We detected endogenous OCT-1, OCT-2, and NF-KB in all patients tested (n = 5). We then evaluated whether activation of CLL B cells could augment kappa-mRNA levels. CLL cells (n = 3) exposed to phorbol ester and A23187 were harvested at 0, 2, 4, 8, and 48 min and examined for kappa-mRNA by Northern blot. All CLL patients (n = 3) had easily detectable levels of endogenous kappa-mRNA. However, only one patient had an obvious increase in kappa-mRNA post-induction with TPA/A23187. There was no concomitant increase in this patient's OCT-1, OCT-2, or NF-KB level. This finding prompted us to survey other B-CLL patients (n = 6) for Ig nuclear transcriptional factors pre- and post-induction. In summary, CLL B cells express Ig transcriptional factor OCT-1, OCT-2, and NF-KB constitutively. The endogenous level of NF-KB may account for the basal kappa-mRNA detected in B-CLL cells. However, the inability to augment NF-KB levels may, in part, explain the low levels of Ig synthesis in CLL B-cells.

Topics: Base Sequence; Calcimycin; Cell Nucleus; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Immunoglobulin; Humans; Immunoglobulin kappa-Chains; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Molecular Sequence Data; NF-kappa B; Octamer Transcription Factor-2; Oligodeoxyribonucleotides; RNA, Messenger; RNA, Neoplasm; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.

Topics: Antibodies, Monoclonal; Calcimycin; Calcium; Cell Survival; DNA, Neoplasm; Glucocorticoids; Humans; Immunoglobulin M; Leukemia, Lymphocytic, Chronic, B-Cell; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The role of phorbol myristate acetate (PMA: a protein kinase-C (PKC) activator) and calcium ionophore A23187 in the induction mechanism of the interleukin 2 receptor (IL2R) on B-cell chronic lymphocytic leukemia (B-CLL) cells was studied. B-CLL cells from five patients were cultured with PMA or A23187 for 72 h and used for the following experiments. Interleukin 2 (IL2) cross-linking assays showed that PMA induced the expression of IL2R subunits (p55 and p70/75) in all cases examined, but that A23187 induced neither subunit. Radiolabeled IL2 binding assays also demonstrated that PMA induced both high-affinity IL2R (HA-IL2R) and low-affinity IL2R (LA-IL2R) on B-CLL cells, but that A23187 did not. After treatment with PMA, three of five cases did not respond to IL2 even though they expressed HA-IL2R, suggesting impaired signal transduction. No cases responded to IL2 after treatment with A23187. In conclusion, PMA but not A23187 stimulates B-CLL cells to induce the expression of p55 and p70/75, indicating that the PKC pathway plays a more important role than the calcium pathway in the induction of IL2R subunits in B-CLL cells.

Topics: Antigens, Surface; B-Lymphocytes; Binding Sites; Calcimycin; Calcium; Electrophoresis, Polyacrylamide Gel; Humans; Immunophenotyping; Interleukin-2; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Protein Kinase C; Receptors, Interleukin-2; Tetradecanoylphorbol Acetate

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

Phorbol esters (phorbol 12-myristate 13-acetate; PMA) and alpha-interferon (alpha-IFN) act through divergent signal transduction pathways to induce B chronic lymphocytic leukaemia cells (B-CLL) to undergo plasmacytoid differentiation in vitro. By using a panel of PMA-inducible early response gene probes we show that these two different effectors are coupled to second messenger pathways that do not converge on a common gene regulatory programme in differentiation of B-CLL cells. Moreover, using the calcium ionophore, A23187, four distinct regulatory classes of early response gene could be defined implying that multiple regulatory pathways may mediate the process of terminal differentiation in B lymphocytes.

Topics: B-Lymphocytes; Blotting, Northern; Calcimycin; Cell Differentiation; Gene Expression Regulation, Leukemic; Genes, Regulator; Humans; Interferon Type I; Leukemia, Lymphocytic, Chronic, B-Cell; Phorbol Esters; Plasma Cells; RNA, Messenger; Second Messenger Systems; Signal Transduction; Tumor Cells, Cultured

In view of the changes reported in expression of the different members of the leukocyte-common family of antigens by T cells as they are activated, B cells were examined with monoclonal antibodies against p220-180 (CD45), p220,205 (CD45R), and p180 (UCHL1). Resting tonsil B cells reacted with CD45 and CD45R antibodies, but not with UCHL1. Activation of B cells by anti-IgM and IL-4 and culture of activated B cells with factors which induce proliferation (B-cell growth factor) and differentiation (IL-6) led to only minor changes in the expression of CD45, CD45R, and p180 antigens. Activation with phorbol ester led to minor increases in reactivity with CD45 and CD45R antibodies and led to a weak reactivity with UCHL1. The results suggest that the functional relationship previously described in T cells between p180 and p220,205 is not seen in B cells. p180 is induced, but only at low levels and only with nonphysiologic activation. CD45R is not lost under the same conditions. However, in terminally differentiated B cells, and in some chronic lymphocytic leukemias, p180 expression was seen, often coexpressed with CD45R.

Topics: Antibodies, Anti-Idiotypic; Antigens, CD; Antigens, Differentiation; B-Lymphocytes; Calcimycin; Histocompatibility Antigens; Humans; Immunoglobulin M; Interleukin-4; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocyte Common Antigens; Lymphocyte Activation; Molecular Weight; Palatine Tonsil; Tetradecanoylphorbol Acetate

When citrate export from mitochondria is blocked with 1,2,3-benzenetricarboxylate (BTC) during the G1/S phase of the cell cycle, both DNA synthesis and cell growth are dramatically inhibited in suspension-grown 70Z/3 murine lymphoma cell cultures sustained under otherwise optimal conditions. Synchronized (G0/G1 or G1/S) and unsynchronized cultures are susceptible to this phenomenon. BTC prevents two requirements from being met. (1) It deprives the cytosol of the acetyl CoA necessary for operation of the cholesterogenesis pathway, thereby depleting the supply of mevalonate (MVA) implicated as a requirement for triggering DNA synthesis. (2) It behaves as a nonmetabolizable divalent cation chelator, reducing the availability of Ca2+ and Mg2+, which, in whole cells are both required for DNA synthesis. Such inhibitions are reversible. In whole cells, removal of the inhibitor yields rapid and complete recovery of DNA synthesis. During the prolonged presence of BTC, the addition of MVA plus the Ca2+ ionophore A23187 allows partial recovery of DNA synthesis. In isolated, DNA synthesizing nuclei, on the other hand, the slight inhibition of DNA synthesis by BTC is reversed merely by addition of Mg2+. We conclude that the uninterrupted production of citrate-derived MVA via the mitochondria, at the G1/S boundary of the cell cycle (i.e., subsequent to peak cholesterol synthesis), is mandatory for initiating the duplication of the cell genome. Consequently, by its mitochondrial site of action, BTC can severely limit the otherwise continuous supply of MVA during late G1, which in turn, prevents entry into the S phase, and thereby cell proliferation.

Topics: Animals; Benzene Derivatives; Biological Transport; Calcimycin; Cell Line; Cell Nucleus; Citrates; DNA Replication; Growth Inhibitors; Interphase; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mitochondria; Thymidine; Tricarboxylic Acids

The peripheral blood mononuclear cells from patients with B-chronic lymphocytic leukemia (B-CLL) were incubated for 0.5 h to 72 h in the presence of the phorbol ester TPA, the calcium ionophore A23187, or a combination of these reagents. Using Northern blot analysis, total cellular RNA was prepared from cells harvested at different time points and hybridized with DNA clones specific for the protooncogenes c-fos and c-myc. While untreated control cells lacked detectable amounts of messenger RNA (mRNA), increase in the level of c-fos mRNA was noted as early as 0.5 h after exposure to the inducers. Peaks of c-fos and c-myc transcript accumulation were seen at 1 h and 4 h after induction, respectively. The most effective inducer was double stimulation with TPA plus A23187. The kinetics of c-fos and c-myc mRNA accumulation in B-CLL appear to be similar to those reported for normal lymphocytes that have been either activated by physiologic external stimuli or by direct activators of protein kinase C and calcium flux (such as TPA and A23187). No direct link between oncogene expression and proliferation or differentiation parameters could be established. These results document that expression of c-fos and c-myc genes, which are among the earliest events following stimulation of the protein kinase signal transduction pathway, can be successfully induced in B-CLL cells. The data provide further evidence for the hypothesis that signal transmission downstream of protein kinase C is intact in B-CLL.

Topics: Blotting, Northern; Calcimycin; Cell Differentiation; Cell Division; DNA; Drug Synergism; Gene Expression Regulation; Humans; Immunoglobulin M; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Individual leukaemic B cells of chronic lymphocytic leukaemia (CLL) do not proliferate to B cell growth factor (BCGF) or interleukin 2 (IL-2) when co-stimulated with immunoglobulin (Ig) ligands. To exclude possible defective signalling via surface Ig (sIg), phorbol myristate acetate (PMA) plus calcium ionophore (A23187) were used to activate purified CLL B cells and compared with staphylococcal protein A coupled to sepharose beads (Seph-PA). RNA synthesis and phenotypic changes after PMA plus A23187 stimulation indicate that CLL B cells from (10) different individuals are similarly able to undergo the G0 to the G1 phase transition and express surface activation antigens. In contrast, they are variable in the capacity to show DNA synthesis, which occurred in only six out of 10 cases. Even in the presence of BCGF (10%, v/v) or IL-2 (50 U/ml) four out of nine CLL B cells activated with PMA plus A23187 or PMA alone were still unable to proliferate although they were induced to express CD23, 4F2, CD25 and OKT9 antigens by PMA plus A23187. However, PMA plus A23187 induced IgM secretion which increased further in response to IL-2 even in the absence of DNA synthesis. Moreover, in other CLL B cell populations, the unresponsiveness to growth factors upon co-stimulation with Ig ligands (Seph-PA) may simply reflect a defective signalling via sIg cross-linking which can be circumvented by PMA plus A23187 stimulation. Recombinant Interferon-gamma (50 U/ml) failed to affect DNA synthesis and IgM secretion.

Topics: B-Lymphocytes; Calcimycin; Cell Extracts; Cells, Cultured; Humans; Interleukin-2; Interleukin-4; Interleukins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Tetradecanoylphorbol Acetate

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

Cells from 3 patients with B-chronic lymphocytic leukaemia (B-CLL) and 1 with B-prolymphocytic leukaemia (B-PLL) were treated in vitro with the phorbol ester 12-0-tetradecanoylphorbol (TPA), the calcium ionophore A23187, or a combination of TPA and A23187. TPA induced the cells to adhere to the culture flask or to clump in dense clusters; single cells became enlarged, often with cytoplasmic elongations. Cells treated with TPA plus A23187 acquired a plasmacytoid morphology and formed regular aggregates in culture. Only TPA alone induced the expression of tartrate-resistant acid phosphatase (TRAP) as documented by isoelectric focusing on horizontal thin-layer gels. The TRAP isoenzyme was first detected after 24 h of TPA treatment; its intensity increased during further TPA exposure, being maximally expressed at 72/96 h. The results suggest that, while TPA triggers B-CLL cells to convert to hairy cell leukaemia (HCL)-type cells, the double stimulus which more closely imitates physiological activation initiates a 'normal' differentiation programme which leads to plasma cells.

Topics: Acid Phosphatase; Aged; B-Lymphocytes; Calcimycin; Drug Synergism; Female; Humans; Isoenzymes; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic; Male; Middle Aged; Tartrates; Tetradecanoylphorbol Acetate