calcimycin and Leukemia--Lymphoid

We have tested a panel of recombinant hematopoietic growth factors (HGF) including the interleukins (IL) 1, 2, 3, 4, and 6 and the colony stimulating factors GM-CSF, G-CSF, M-CSF for their ability to induce proliferation of precursor B acute lymphoblastic leukemia cells (ALL) from 19 patients. In Ficoll-Isopaque isolated and T cell-depleted ALL bone marrow samples, IL2 (two cases), IL3 (four cases), and GM-CSF (one case) infrequently stimulated DNA synthesis measured by 3H-thymidine (TdR) uptake, and the other recombinant growth factors completely failed to do so. In repeat experiments with ALL blasts purified by fluorescence activated cell sorting (FACS), IL2, IL3, and GM-CSF responses could not be reproduced, suggesting that nonleukemic contaminant cells, and not the ALL blasts, had been stimulated by these factors. Cocktails containing combinations of IL1-IL4 and IL6 also lacked proliferation inducing potency. Depending on the purity of the incubated ALL cell samples, an impure preparation of B cell growth factors that has been reported to contain a highly effective stimulatory activity for precursor B ALL cells induced proliferation of residual normal cells as well as the ALL cells, as was evident from combined analysis of DNA synthesis and karyotyping. Exposure of the ALL blasts to artificial activators of protein kinase C and Ca2+ mobilization resulted in significant rises in 3H-TdR uptake, suggesting that these intracellular compounds are involved in transducing signals that upregulate proliferation. Although it remains possible that some of the human recombinant growth factors promote the growth of precursor B ALL cells in combination with other stimuli, a dominant role in the regulation of proliferation of these cells cannot be attributed to any of these cytokines at the present time.

Topics: B-Lymphocytes; Calcimycin; Cell Division; Colony-Stimulating Factors; Humans; Interleukin-4; Interleukins; Leukemia, Lymphoid; Neoplastic Stem Cells; Recombinant Proteins; Tetradecanoylphorbol Acetate

Cells from the peripheral blood of 22 patients with chronic B-cell disorders were examined for the expression of surface MHC class II antigens. We made use of well-characterized monoclonal antibodies (McAbs) specific for HLA-DP, -DQ and -DR molecules (B7/21, Tü22, RFDR1 and RFDR2) and of another McAb, RFD1, associated with the class II system. By using indirect immunofluorescence and flow cytometry we found both non-coordination and heterogeneity in expression of MHC class II antigens but generally with a hierarchy of positivity: DR greater than DQ greater than DP. This suggests a sequence of gradual acquisition of HLA-D antigens and indicates distinct differences in maturation arrest of the individual cases. However, after treatment with phorbol ester TPA and calcium ionophore A23187, all cases expressed the previously absent molecules indicating that the structural genes for these products remained intact. TPA and A23187 increased both the number of positive cells in most cases and the fluorescence staining intensities of all class II markers including RFD1. Thus, leukaemic cells may express different combinations of class II antigens reflecting: (i) a predetermined order of gradual acquisition of class II molecules; (ii) differences in the stages of maturation arrest; and (iii) in the cases of disordered expression a desynchronized regulation of these markers.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, Surface; B-Lymphocytes; Binding, Competitive; Calcimycin; Epitopes; Female; Histocompatibility Antigens Class II; Humans; Leukemia, Lymphoid; Major Histocompatibility Complex; Male; Middle Aged; Tetradecanoylphorbol Acetate

A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

Topics: Adenosine Diphosphate; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Arachidonic Acid; Arachidonic Acids; Aspirin; Calcimycin; Cell Aggregation; Citrates; Citric Acid; Dyspnea; Female; Humans; Leukapheresis; Leukemia, Lymphoid; Lymphocytes; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Adrenergic, beta

We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL-2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B-CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.

Topics: Antibodies, Neoplasm; B-Lymphocytes; Calcimycin; Drug Synergism; Enzyme Activation; Humans; Interleukin-2; Interleukin-4; Interleukins; Leukemia, Lymphoid; Neoplasm Proteins; Protein Kinase C; Receptors, Immunologic; Receptors, Interleukin-2; RNA, Neoplasm; Tetradecanoylphorbol Acetate

Solid tumors, particularly those involving the colon, breast, and lung, are the most common tumors in humans. However, many technical difficulties exist in obtaining analyzable chromosomes from these tumors, including the inability to stimulate cell division. Phorbol-12,13-dibutyrate (PDBu) is a tumor promoter that activates a variety of cellular responses, including proliferation. Using flow cytometry, we have demonstrated that PDBu acts as a mitogen in primary cultures of colon tumor cells. Based on these results, we developed a short-term culture technique that greatly improves the yield of analyzable metaphases from colon tumors. Stimulated cultures consistently contained at least ten times more metaphases than unstimulated cultures, and chromosome morphology was improved. By modifying this technique with the addition of the calcium ionophore A23187, we have successfully obtained analyzable chromosomes from the peripheral blood of normal individuals, chronic lymphocytic leukemia patients, and a nodular small cell lymphoma patient. These results demonstrate that mitogenic stimulation by PDBu is a valuable technique in the cytogenetic analysis of colon tumors. By using PDBu alone or in combination with other agents, this technique may also be applicable to many other tumors that are difficult to karyotype because of an inability to obtain mitoses.

Topics: Calcimycin; Chromosome Banding; Colonic Neoplasms; Humans; Karyotyping; Leukemia, Lymphoid; Mitogens; Mitosis; Mitotic Index; Phorbol 12,13-Dibutyrate; Phorbol Esters; Tumor Cells, Cultured

The peripheral blood mononuclear cells from 16 patients with B-chronic lymphocytic leukemia (B-CLL, n = 13), B-prolymphocytic leukemia (B-PLL, n = 2) or hairy cell leukemia (n = 1) were incubated in the presence of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore A23187. A synergy between these inducers was found with respect to morphological changes and B cell proliferation and differentiation. A23187 used alone did not activate the cells. B-CLL cells treated with the double stimulus acquired a plasmacytoid morphology, showed significantly higher incorporation of 3H-thymidine and 3H-uridine, and produced significantly higher amounts of monoclonal immunoglobulin compared with the same cells exposed to either of the inducers alone. These results indicate that phorbol ester and calcium ionophore act synergistically on B-CLL cells to induce proliferation and differentiation. B-PLL cells responded more vigorously to the signals provided by TPA and A23187. Previous studies showed that TPA and A23187 can mimic the two physiological second messengers diacylglycerol and inositol trisphosphate in the transduction of signals leading to cell activation, proliferation, and differentiation in normal B cells. The present findings suggest that the capacity of B-CLL and B-PLL cells to differentiate in response to signals of the second messenger pathway is intact.

Topics: Antibody Formation; B-Lymphocytes; Calcimycin; Cells, Cultured; DNA Replication; Drug Synergism; Humans; Leukemia, Hairy Cell; Leukemia, Lymphoid; Lymphocyte Activation; RNA, Neoplasm; Tetradecanoylphorbol Acetate

The calcium ionophore A23187 synergised with phorbol dibutyrate-induced activation of human chronic lymphatic leukaemia B-cells, as assessed by modulation of the membrane receptor for mouse erythrocytes. Thus A23187 (1 microM), which alone had no effect on expression of the receptor for mouse erythrocytes, reduced the EC50 and shortened the lag period for modulation of this receptor by phorbol dibutyrate. This action of A23187 was shown to be due to enhanced binding of [3H]phorbol dibutyrate to its receptor (phospholipid/Ca++ dependent protein kinase C) whose affinity was altered from a predominantly low affinity state (Kd 83 nM) to high affinity (Kd 9 nM). A23187 had no effect on the total number of phorbol dibutyrate receptors. EDTA abolished these actions of A23187.

Topics: B-Lymphocytes; Caenorhabditis elegans Proteins; Calcimycin; Carrier Proteins; Drug Synergism; Edetic Acid; Humans; Kinetics; Leukemia, Lymphoid; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Drug; Receptors, Immunologic

Peripheral blood lymphocytes consisting mainly of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL cells) showed a markedly reduced response to the human B-cell mitogens anti-beta2 microglobulin, Sepharose-bound protein A and Sepharose-bound anti-human immunoglobulin (anti F(ab')2) in all of nine patients studied. On the other hand, CLL cells from three out of eight patients tested responded well to the calcium ionophore A23187. Sepharose-bound protein A and anti-beta2 microglobulin also failed to induce increased uptake of 86Rubidium (potassium analogue) in CLL cells as compared to B-cell-enriched preparations of normal peripheral blood lymphocytes. The capacity of CLL cells to cap various surface markers including beta2 microglobulin was reduced. On the other hand, surface concentrations of beta2 microglobulin were not reduced as measured by fluorescein-labelled anti-beta2-microglobulin in single-cell cytofluorometry. It is concluded that various membrane-associated events elicited by ligand-receptor interactions are altered or blocked in CLL cells.

Topics: Aged; B-Lymphocytes; beta 2-Microglobulin; Calcimycin; Cell Membrane; DNA; Female; Humans; Immunologic Capping; Leukemia, Lymphoid; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Mitogens

Lymphocytes from a patient with chronic lymphocytic leukaemia, in whom therapy was ineffective, were defined as thymus-derived (T) cells by membrane markers (sheep erythrocyte rosettes, complement rosettes, surface immunoglobulin). The lymphocytes responded weakly to two mitogens, phytohaemagglutinin and the calcium ionophore A23187, but not to concanavalin A. Cytogenetic studies of leukaemic cells from unstimulated and mitogen-stimulated cultures revealed an abnormal karyotype with 45 chromosomes and multiple rearrangements. The T cell variant of classical chronic lymphocytic leukaemia is relatively rare; additional reports are needed to determine if the clinical course is typically less benign than in the common B cell variety, or whether this patient simply represented a late, unresponsive phase of the disease.

Topics: Aged; Calcimycin; Cell Membrane; Chromosome Aberrations; Chromosome Disorders; Concanavalin A; Female; Genetic Variation; Humans; Karyotyping; Lectins; Leukemia, Lymphoid; Lymphocyte Activation; Lymphocytes; Mitogens; T-Lymphocytes

The ionophore A23187 was tested for its effect on the sensitivity of normal and leukemic lymphocytes to colchicine, vincristine, and X-irradiation. The ionophore was used because it transfers divalent cations through biological membranes and causes the transformation of normal lymphocytes. The sensitivity of the cells was measured by incubation of lymphocyte suspensions with and without reagent at 37 degrees C for one to seven days and counting the number of viable lymphocytes before and after incubation. Viability of the cells was judged by cytologic criteria as visualized by phase contrast microscopy. Lymphocytes were obtained from patients with chronic lymphocytic leukemia and from normal persons. Colchicine and vincristine killed in one day non-dividing leukemic lymphocytes while normal lymphocytes were much less sensitive to the two alkaloids. X-irradiation (1000 rad) killed nearly all leukemic lymphocytes in two days and normal lymphocytes in three days. Ionophore A23187 (2 X 10(-5)M) was moderately toxic to normal but not to leukemic lymphocytes and caused the transformation of both types of cells. The ionophore inhibited the cytocidal action of colchicine and vincristine to leukemic but not to normal lymphocytes and inhibited the action of X-irradiation to both types of lymphocytes. The present findings and a review of the literature suggest the hypothesis that calcium or other divalent ions are involved in the cytotoxicity of colchicine, vincristine, and X-irradiation of leukemic lymphocytes and that there may be an abnormality of the metabolism of divalent ions in leukemic lymphocytes.

Topics: Anti-Bacterial Agents; Calcimycin; Calcium; Colchicine; Cytotoxicity Tests, Immunologic; Humans; In Vitro Techniques; Leukemia, Lymphoid; Lymphocyte Activation; Lymphocytes; Vincristine; X-Rays