2--7--bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl-ester and Leukemia--Erythroblastic--Acute

Decreased accumulation of the fluorescent dye BCECF [2', 7'-bis-(2-carboxyethyl)-5-(6)- carboxyfluorescein] characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 microM), a known cyclo-oxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Drug Resistance, Multiple; Fluoresceins; Fluorescent Dyes; HL-60 Cells; Humans; Indomethacin; Leukemia, Erythroblastic, Acute; Mice; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Rhodamines; Tumor Cells, Cultured; Vincristine

This study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura-2-AM) and [pH]in (BCECF-AM). Results demonstrate that two types of target cell response to the attack by an NK cell can be distinguished. The target cell either dies immediately, due to the complete breakdown of the membrane impermeability, or the initial membrane damage (i.e., increased membrane permeability) is repaired and the cell "escapes" immediate death. During both responses an increase of [Ca2+]in takes place in the target cells. In the cells that die immediately, however, [Ca2+]in reaches higher levels (approximately 1,400 nM) than in the cells that restore the initial damage (approximately 700 nM). Changes in target cell [pH]in are also detected during both responses. The direction of the change (acidification or alkalinization) as well as the level of the change depend on extracellular pH ([pH]ex). Also, [pH]in remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pH]ex 7.3-7.6 (approximately 3 min).

Topics: Calcium; Calibration; Cell Membrane Permeability; Cytotoxicity, Immunologic; Fluoresceins; Fluorescent Dyes; Fura-2; Homeostasis; Humans; Hydrogen-Ion Concentration; Intracellular Fluid; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Membrane Glycoproteins; Microscopy, Fluorescence; Neoplastic Stem Cells; Perforin; Pore Forming Cytotoxic Proteins; Tumor Cells, Cultured