Abstract
AbstractIn the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77–induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.
Highlights
In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt’s lymphoma (BL) cell lines and a subset of germinal center B lymphocytes
We showed that VT-B, a specific ligand of the Gb3/CD77 antigen, induced apoptosis of BL
We first show that anti-CD77 monoclonal antibodies (MoAb) are able to cause apoptosis of BL cells and demonstrate that various second messenger molecules are produced in response to ligation of the CD77 antigen
Summary
Gregory (Birmingham, UK; Mutu I and Mutu III) These cell lines were cultured in RPMI 1640 medium (GIBCO-BRL, Cergy-Pontoise, France) containing 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 20 mmol/L glucose, and 20 mg/mL gentamicine, and supplemented with 5% heat-inactivated fetal calf serum (FCS). Cells were washed and resuspended at 2 1 106 cells/mL in complete medium; 38.13 MoAb or other reagents were added and incubation was performed at 37ЊC. 3 1 106 cells were incubated for various times at 37ЊC in complete medium containing 38.13 MoAb, and lipids were extracted. Ceramide was quantified by the DAG kinase assay as previously described.[18] Briefly, cell pellets were extracted three times with 1 mL of chloroform:methanol (2:1) and 500 mL of water. Labeled lipids were scraped off the plates and quantitated by liquid scintillation counting
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