Mitotic separation of daughter cells in the human lymphoma B cell line Daudi involves L-selectin engagement and shedding

Abstract

BACKGROUND AND OBJECTIVE: A novel role for shedding of the surface molecule L-selectin has been proposed as an adjunctive phenomenon during cell detachment from marrow stroma or vessel endothelium. We wished to examine whether variations in expression of L-selectin on a lymphoma B cell line were linked to shedding. DESIGN AND METHODS: Mapping of L-selectin expression on the surface of Daudi lymphoma cells was performed by flow cytometry, fluorescence microscopy, and electron microscopy. Levels of shed L-selectin were evaluated by Western blotting of culture supernatants. Evaluation of cell cycle and proliferative activity was performed by flow cytometry. RESULTS: Large Daudi cells in S+G(2)/M phases were L-selectin positive, whereas small Daudi cells in G(0)/G(1) phase were L-selectin negative. During mitosis, L-selectin was distributed along the cleavage furrow, and gradually lost. Electron microscopy revealed that separating Daudi cells were negative for L-selectin on the entire surface, except minute aggregates of L-selectin within the cleavage furrow. Addition of agents known to interfere with the ligand-binding portion of L-selectin (sulfatides, MoAbs: Lam1.3 and TQ1) results in loss of L-selectin. Removal of L-selectin by digestion with chymotrypsin inhibits Daudi proliferation. The MoAb FMC46 did not interfere with proliferation. Proliferating Daudi cells produced large quantities of shed L-selectin. Inhibition of Daudi proliferation resulted in levels of shed L-selectin below the limit of detection. INTERPRETATION AND CONCLUSIONS: L-selectin is re-distributed on the cell surface of Daudi cells during the last phase of mitosis, in which plasma membrane invagination occurs between newly formed daughter cells. Shedding of L-selectin is involved in the cytokinesis of Daudi cells.