sq-23377 and Leukemia--Myeloid

Cross-linking the high affinity IgE receptor on the rat basophil leukemia clone 2H3 (RBL-2H3) cell line, an vitro model for mast cell signaling, results in granule release. A great deal of research has focused on the earliest steps in this signaling cascade resulting in models which include the participation of lyn, syk, phospholipase C (PLC), protein kinase C (PKC) and intracellular calcium mobilization. In an effort to look at pathways downstream of calcium mobilization, ionomycin-mediated granule release was studied. The kinase inhibitors PP1 (src family), GF109203X (PKC), PD98059 (MEK1/2), and U0126 (MEK1/2) substantially inhibited ionomycin-mediated granule release, while the p38 kinase inhibitor SB203580 did not. Both p38 and erk were phosphorylated upon ionomycin treatment, but only extracellular regulated kinase (erk) activation was completely inhibited by PP1 treatment and partially inhibited by the MEK inhibitors, thus, correlating with the granule release data. Interestingly, while GF109203X alone had no affect on erk activation, combining it with U0126 completely blocked this response. This suggests the existence an alternate pathway for erk activation that is MEK independent and PKC dependent. Experiments in which ionomycin and PP1 were titrated (independently) demonstrated a correlation between erk phosphorylation and granule release, implicating erk in a PP1-inhibitable pathway operating downstream of calcium and controlling mast cell degranulation.

Topics: Animals; Basophils; Butadienes; Cell Degranulation; Drug Synergism; Enzyme Inhibitors; Flavonoids; Indoles; Ionomycin; Ionophores; Leukemia, Myeloid; Maleimides; Mitogen-Activated Protein Kinases; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Rats; Tumor Cells, Cultured

New therapeutic approaches are needed to improve the cure rates in acute myeloid leukaemia (AML). The present study was designed to investigate whether: (1) cytotoxic lymphocytes could be expanded from AML patients in complete remission; (2) their signal transduction machinery was preserved; (3) these cells were capable of producing cytokines involved in the cytolytic process; and (4) these cells showed cytotoxic activity against allogeneic and autologous blasts. By co-culturing blood mononuclear cells with feeder cells, we obtained an average 5.3-fold increase in the total cell number and a 35-fold increase in natural killer (NK) cells. Expression of the zeta chain and of tyrosine kinases of the Src and Syk-ZAP families involved in the triggering of NK functions was analysed on expanded cells. The results demonstrated a signal transduction apparatus preserved and quantitatively similar to that of normal donors. After phorbol myristate acetate and ionomicin stimulation, the ability of expanded cells to produce interferon gamma and tumour necrosis factor alpha was documented. Patients' expanded cells showed a cytotoxic activity against target lines and allogeneic blasts which was similar to that of normal donors. Purification experiments indicated that the NK cell fraction was responsible for most of the lytic effect. More significantly, these cells also exerted a lytic effect against autologous blasts that could be further enhanced following incubation with low-dose interleukin 2. These findings document the possibility of expanding cytotoxic effectors with preserved signal transduction machinery and autologous killing capacity from AML patients in remission, and suggest a new potential immunotherapeutic strategy for the management of early disease recurrence or of residual disease.

Topics: Acute Disease; Adult; Aged; Cell Division; Coculture Techniques; Cytokines; Cytotoxicity Tests, Immunologic; Female; Humans; Immunotherapy; Interferon-gamma; Ionomycin; Ionophores; Killer Cells, Natural; Leukemia, Myeloid; Male; Middle Aged; Signal Transduction; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha