Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complementmediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

SUMMARYCD20 is an effective target for therapeutic B-cell depletion with monoclonal antibodies. One proposed mechanism of action is direct cytotoxicity mediated via tyrosine kinase-dependent signalling pathways activated upon CD20 cross-linking. The association of CD20 with membrane microdomains known as lipid rafts, enriched in src-family tyrosine kinases and other signalling effectors, suggests an indirect mechanism of anti-CD20-induced apoptosis in which activation of src-family kinases occurs as a consequence of lipid raft clustering.

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated.

O-linked N-Acetylglucosamine (O-GlcNAc) post-translational modifications originate from the activity of the hexosamine pathway, and are known to affect intracellular signaling processes. As aberrant responses to microenvironmental signals are a feature of chronic lymphocytic leukemia (CLL), O-GlcNAcylated protein levels were measured in primary CLL cells. In contrast to normal circulating and tonsillar B cells, CLL cells expressed high levels of O-GlcNAcylated proteins, including p53, c-myc and Akt. O-GlcNAcylation in CLL cells increased following activation with cytokines and through toll-like receptors (TLRs), or after loading with hexosamine pathway substrates. However, high baseline O-GlcNAc levels were associated with impaired signaling responses to TLR agonists, chemotherapeutic agents, B cell receptor crosslinking and mitogens. Indolent and aggressive clinical behavior of CLL cells were found to correlate with higher and lower O-GlcNAc levels, respectively. These findings suggest that intracellular O-GlcNAcylation is associated with the pathogenesis of CLL, which could potentially have therapeutic implications.

CD20 is a B cell integral membrane protein capable of initiating growth-modulating signals in human B lymphocytes upon its engagement with monoclonal anti-CD20 antibodies. In this report, we demonstrate that treatment of B cells with CD20 antibodies induces rapid redistribution of CD20 into a detergent-insoluble membrane compartment. Redistribution is detected as early as 15 s, following antibody addition, and involves up to 95% of CD20 molecules, depending on the antibody used. All of the detergent-insoluble CD20 was found in the low density fractions of sucrose density gradients, indicating that CD20 redistributes to glycolipid-rich membrane domains, analogous to caveolae in some cell types. As CD20 has previously been shown to associate with Src family tyrosine kinases, their co-existence in these compartments suggests a link to the role of CD20 in signal transduction. This study provides insight into the mechanism by which CD20 commmunicates signals to the cell interior and indicates that the search for membraneproximal intracellular signaling partners should be directed to the Triton-insoluble fraction.

B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through storeoperated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium. The activity of this SOC pathway was abolished by deletion of a cytoplasmic sequence in CD20 essential for its efficient raft localization. Strontium-permeable SOC channels were detected in B cells, and B cell receptorstimulated influx was significantly reduced by downregulation of CD20 expression using short interfering RNA and also by cholesterol depletion. This is the first evidence that raft-associated CD20 constitutes a component of a SOC entry pathway activated by the B cell receptor.The function of CD20, a tetraspan transmembrane protein expressed in B lymphocytes, is not yet fully elucidated, although electrophysiological evidence of calcium channel activity has been reported. Increased Ca 2ϩ conductance, detected by whole cell patch clamp recordings, was induced by membrane hyperpolarization in a variety of cell types expressing CD20 ectopically and was similar to the native conductance found in B cells (1, 2). The type of calcium channel that is formed or regulated by CD20 and the conditions of its activation are not known.Intracellular calcium is an essential regulator of cell function (3, 4). Receptor-mediated activation of phospholipase C and consequent inositol 1,4,5-trisphosphate production leads to rapid release of Ca 2ϩ from intracellular stores in the endoplasmic reticulum. Store depletion activates Ca 2ϩ influx across the plasma membrane, replenishing empty stores and providing a sustained increase in the concentration of cytoplasmic free Ca 2ϩ ([Ca 2ϩ ] c ). The Ca 2ϩ influx has been termed capacitative or store-operated Ca 2ϩ (SOC) 1 entry and is essential for regulating diverse cellular responses to receptor-mediated stimuli, including enzyme activation, gene expression, secretion, and cell division (5-7). Electrophysiological studies distinguish several classes of SOC channels, including the highly Ca 2ϩ -selective Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel described in mast cells and T lymphocytes and others with distinct characteristics (7,8). Despite intensive research efforts, the molecular identity of SOC channel proteins remains unclear. The mammalian homologues of the Drosophila transient receptor potential (TRP) gene products, now forming a TRP superfamily with more than 20 members in 3 subfamilies (9), provide the only candidate SOC channel proteins currently known. All TRPs apparently form cation channels but with variable selectivity and activation properties (10, 11).In B lymphocytes, expression of specific genes requires sustained elevation in [Ca 2ϩ ] c (12). As in other cell types, B lymphocytes activate Ca 2ϩ entry after store-depletion, but the molecular components and the mechanism of acti...

Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.

CD20, a non-glycosylated cell-surface protein expressed exclusively on B lymphocytes, is one of a family of 4-pass transmembrane molecules that also includes the ␤ chain of the high affinity receptor for IgE. The precise function of CD20 is unknown, although in vitro effects of CD20-specific antibodies on resting B cells indicate that it is able to transduce an extracellular signal affecting the G 0 /G 1 cell cycle transition. Previous studies have demonstrated that CD20-initiated intracellular signals involve tyrosine kinase activation and that CD20 is tightly associated with both serine and tyrosine kinases. Here, analysis of CD20-associated molecules has revealed that CD20 is associated with the Src family tyrosine kinases p56/53 lyn , p56 lck , and p59 fyn and with 75/80-kDa proteins phosphorylated in vivo on tyrosine residues. Mutagenesis of CD20 was performed to define regions of CD20 involved in intermolecular interactions. Mutants were analyzed in the human T lymphoblastoid cell line Molt-4, in which ectopically expressed wild-type CD20 associated with p59 fyn , p56lck , and 75/80-kDa phosphoproteins. Deletion of major portions of the cytoplasmic regions of CD20 did not abolish its association with either p75/80 or tyrosine kinases. The interaction between CD20 and the Src-related kinases is therefore likely to be independent of CD20 cytoplasmic domains and may occur indirectly. The interaction may be mediated by the p75/80 phosphoproteins, which were found to be tightly associated with the Src family kinases isolated from the CD20 complex.Growth and differentiation of B lymphocytes occurs primarily in the follicles and germinal centers of the spleen and lymph nodes and depends not only on contact with specific antigen but also upon co-stimulatory signals derived from accessory cell surface molecules. Accessory signals may be mediated by soluble factors such as IL-4 or by intercellular receptor-ligand interactions, such as the interaction between CD40 on B cells and its ligand on the surface of activated T cells (1). Like CD40, CD20 is a B cell surface protein with the capacity to initiate intracellular signals and modify cell growth and differentiation (2-8). CD20 is a non-glycosylated 33-kDa protein expressed on all mature B cells (9 -13). The predicted amino acid sequence of CD20 suggests a structure that has four transmembrane-spanning regions with both amino and carboxyl termini located on the cytoplasmic side of the plasma membrane (14 -16). CD20 is unrelated to a large family of proteins with similar overall structure (CD9, CD37, CD69, TAPA-1, and others) but does possess limited sequence homology, particularly within the transmembrane regions, to a recently identified molecule expressed in hemopoietic cells, HTm4, and to the ␤ chain of the high affinity receptor for IgE (Fc⑀RI) (17, 18). The cytoplasmic region of Fc⑀RI␤ contains an immunoreceptor tyrosine-based activation motif (ITAM) 1 and is thought to be involved in relaying signals for the IgE binding ␣ chain (19). HTm4 and CD20 do not cont...