Expression of a functional inducible nitric oxide synthase in hairy cell leukaemia and ESKOL cell line.
The expression of nitric oxide synthase (NOS) isoforms was investigated in the established ESKOL hairy cell line and in leukemic cells of patients with hairy cell leukemia (HCL). By reverse transcription-polymerase chain reaction (RT-PCR), these cells were found to spontaneously express inducible NOS (iNOS)-specific mRNA, but not endothelial constitutive NOS (ecNOS) mRNA. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and ESKOL cells, using different anti-iNOS monoclonal antibodies. A protein of 135 kDa was identified by Western blotting in ESKOL and HCL lysates, confirming the presence of an iNOS in these cells. Cytosolic homogenates displayed NOS catalytic activity, as measured by the conversion of 14C-labelled L-arginine into 14C L-citrulline and by detection in situ using the DAF-2DA (diaminofluorescein diacetate) NO-sensitive fluorescent probe. Ligation of CD23 (low affinity IgE receptor) was found to increase iNOS expression in ESKOL and conversely to decrease the percentage of cells undergoing apoptosis, as measured by the percentage of cells expressing annexin V. These results indicate that, as in chronic B cell lymphocytic leukemia cells (B-CLL) a functional iNOS is expressed constitutively in hairy cells that contributes to protecting these tumoral cells from apoptosis.
Roman
V
V
INSERM U365, Institut Curie, Paris, France.
Zhao
H
H
Fourneau
J M
JM
Marconi
A
A
Dugas
N
N
Dugas
B
B
Sigaux
F
F
Kolb
J P
JP
D000578:Amidines / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis; D001120:Arginine / Q000378:metabolism; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D007943:Leukemia, Hairy Cell / Q000201:enzymology / Q000473:pathology; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000201:enzymology* / Q000473:pathology; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000032:analysis; D017455:Receptors, IgE / Q000276:immunology / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000473:pathology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000578:Amidines; D000911:Antibodies, Monoclonal; D001596:Benzylamines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D009363:Neoplasm Proteins; D009573:Nitrites; D017455:Receptors, IgE; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D001120:Arginine; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III