Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells
Andrei Ivanov, … , Tim M. Illidge, Mark S. Cragg
Andrei Ivanov, … , Tim M. Illidge, Mark S. Cragg
Published July 20, 2009
Citation Information: J Clin Invest. 2009;119(8):2143-2159. https://doi.org/10.1172/JCI37884.
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Research Article

Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells

Abstract

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcγR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DR–specific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion–related cell death occurs through a lysosome-dependent pathway.

Authors

Andrei Ivanov, Stephen A. Beers, Claire A. Walshe, Jamie Honeychurch, Waleed Alduaij, Kerry L. Cox, Kathleen N. Potter, Stephen Murray, Claude H.T. Chan, Tetyana Klymenko, Jekaterina Erenpreisa, Martin J. Glennie, Tim M. Illidge, Mark S. Cragg

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Figure 7

Cell death evoked by tositumomab and L243 is nonapoptotic and nonautophagic.

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Cell death evoked by tositumomab and L243 is nonapoptotic and nonautopha...
(A) Raji cells, either WT or overexpressing Bcl-2, were treated with mAbs (10 μg/ml) or mitoxantrone (1 μg/ml) in the presence (black columns) or absence (white columns) of the pan-caspase inhibitor QVD-OPH (20 μM) as indicated for 24 hours and then assessed for cell death as before. (B) Raji cells were treated for 24 hours (left panel) with various mAbs (10 μg/ml) or treated for 2 hours, 4 hours, or 8 hours with various mAbs (right panel) and then assessed by Western blot for the expression of Beclin-1, Atg12, ezrin, or actin (the latter 2 as loading controls). Irr, irrelevant treated; M, M15/8. (C) Raji cells were nucleofected with siRNA to Atg12 or Beclin-1 and then incubated for 24 hours. Subsequently, cells were treated with mAbs (10 μg/ml) and assessed for cell death as previously described. The degree of knockdown was verified 48 hours after nucleofection by Western blot (inset). B2 and B3 refer to 2 different targeting regions of Beclin-1. (D) Raji cells were preincubated with TPCK, trypsin-like serine proteases (TLCK), or Y27632 (20 μM) before being treated with tositumomab or L243; cell death was measured 24 hours later. We performed similar experiments with inhibitors over a 50-fold dilution range from 1 to 50 μM with identical results.