Studies on the regulation and localization of 5-lipoxygenase in human B-lymphocytes.
Stimulated B-lymphocytes, isolated from patients with chronic lymphocytic leukemia of B-cell type (B-CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B-lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5-lipoxygenase products. Several thiol-reactive compounds such as N-ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bis[dimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)-PCR analysis demonstrated the expression of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase mRNA in B-CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B-CLL cell membrane. Furthermore, MK886, the FLAP-binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5-HETE formation. Immunocytochemistry showed that 5-lipoxygenase was mainly localized in the nuclei of non-activated B-CLL cells, tonsillar B-lymphocytes and monoclonal B-cells. In contrast, neither human peripheral T-lymphocytes nor Jurkat cells were stained. These results suggest that 5-lipoxygenase and its products function in the nucleus of B-lymphocytes.
Jakobsson
P J
PJ
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Shaskin
P
P
Larsson
P
P
Feltenmark
S
S
Odlander
B
B
Aguilar-Santelises
M
M
Jondal
M
M
Biberfeld
P
P
Claesson
H E
HE
D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001402:B-Lymphocytes / Q000201:enzymology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology*; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D002722:Chlorobenzenes
D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase; D002352:Carrier Proteins; D007211:Indoles; D008565:Membrane Proteins; D013439:Sulfhydryl Reagents; C548592:ALOX5AP protein, human; C060893:MK-886; D002722:Chlorobenzenes