| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Immunology Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Human CD20 and its murine equivalent, Ly-44, have been cloned, and their genetic sequences have been characterized (17, 18, 19, 20). Based on hydrophobicity data and the lack of a signal sequence, CD20 is predicted to have intracellular N- and C-termini, four transmembrane spans (TM1–4), and an extracellular domain between TM3 and TM4 (18, 19). We sought to dissect the cytoplasmic regions of CD20 in search of sequences involved in controlling its redistribution. However, since signal sequences are not always necessary for extracellular localization of N-terminal regions (21, 22, 23, 24, 25, 26), the predicted membrane orientation of CD20 required confirmation.
In this study Abs generated against peptides in the N- and C-terminal regions of CD20 were used to determine its membrane orientation. Then, deletion mutants of intracellular regions were generated and examined to determine their effects on the redistribution of CD20 to Triton-insoluble membrane microdomains.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Raji lymphoblastoid B cells were grown in RPMI/5% FBS. Molt-4 T cells expressing transfected CD20 cDNA (27) were grown in RPMI/10% FBS in the presence of 0.4 mg/ml geneticin (Life Technologies, Gaithersburg, MD). CD20-specific 2H7 mAb was provided by Dr. J. Ledbetter (Bristol-Myers Squibb, Seattle, WA). Antisera against the amino and carboxyl regions of CD20 (herein named anti-CD20N and anti-CD20C) were generated by immunizing rabbits with either OVA-conjugated CD20N peptide (CD20N-P; residues 25–41, SGPKPLFRRMSSLVGPT) or OVA-conjugated CD20C peptide (CD20C-P; residues 231–245, SAEEKKEQTIEIKEE; peptides were provided by James Blake, Bristol-Myers Squibb). The Abs were affinity purified using the Pierce Sulfolink Kit (Rockford, IL).
Mutagenesis and transfections
The generation of the two truncation mutants used in this study,
N
1–49 and C253–297, were described previously (27).
C219–225, C226–252, and the Cys220/Ala
point mutation (C220A) were produced by overlap extension PCR (28)
using the internal primer pairs shown in Table I
and the CD20 cDNA template provided by
Dr. Ivan Stamenkovic (Charleston, MA). Outside primers used were
5'-ATAATGAATTCATTGAGCCTCTTT-3' (5' primer that includes a unique
EcoRI restriction site at position 451 of the CD20 cDNA) and
5'-AATCACTTAAGGAGAGCT-3' (3' primer that includes a unique
AflII site at position 983). The PCR fragments were digested
with EcoRI and AflII, then cloned into
pBluescript containing a CD20 cDNA insert from which the
EcoRI-AflII fragment had been excised. The
sequence of each construct was confirmed before subcloning into the
BCMGSneo mammalian expression vector (29). Transfection into Molt-4 T
cells and selection of geneticin-resistant clones, were performed as
previously described (27).
|
Cells were suspended and incubated in RPMI/5% FBS with anti-CD20 or control Abs, washed once, and resuspended in 100 µl (1/100) dilution of either FITC-conjugated rabbit anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) or FITC-conjugated goat anti-rabbit IgG (ICN, Costa Mesa, CA) as appropriate. For intracellular staining, Raji B cells (1 x 106 cells/sample) were permeabilized with 0.05% saponin in RPMI/5% FBS for 30 min on ice. Subsequent Ab incubation and wash steps were performed as described above, except in the presence of 0.01% saponin. For the peptide inhibition studies, anti-CD20N and anti-CD20C Abs were preincubated with the immunizing peptides CD20N-P and CD20C-P, respectively, for 3 h at room temperature. Immunofluoresence of all samples was measured using a FACScan cytometer (Becton Dickinson, Mountain View, CA).
Trypsin and proteinase K digests
Raji cells (2 x 106/sample) were washed and resuspended in 50 mM Tris-HCl (pH 7.5), then incubated alone or with trypsin (0.2 µg/µl) for 15 min on ice. For proteinase K digests, Raji cells were washed and resuspended in PBS, then incubated alone or with 12.5 µM proteinase K for 15 min on ice. Protease inhibitors (4 mM Pefabloc (Boehringer Mannheim, Laval, Quebec), 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM PMSF) were added to halt digestion. Samples were then rapidly centrifuged, and the supernatants were aspirated. Cell pellets were lysed directly in 2x sample buffer, heated to 100°C for 5 min, and loaded on 12.5% polyacrylamide gels (4 x 105 cell equivalents/lane).
Cell stimulation and sample preparation
Cells (5 x 106/sample) were washed in PBS, not treated or treated with 5 µg of 2H7 mAb at 37°C for 15 min, then pelleted and lysed in 20 mM Tris, pH 7.5, containing 0.5% Triton X-100, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 5 mM EDTA, and 1 mM PMSF. After 15 min on ice, samples were centrifuged at 14,000 x g for 15 min at 4°C to pellet the insoluble material. Lysates were transferred to clean tubes and mixed with 2x SDS sample buffer. The insoluble pellets were washed four times with lysis buffer and then solubilized in 2x SDS sample buffer. Samples were heated to 100°C for 5 min before separation on 10% polyacrylamide gels (1–3 x 105 cell equivalents of lysates/lane; 5 x 105 cell equivalents of pellets/lane). For immunoprecipitation, lysates from 107 cells were transferred to clean tubes, incubated overnight at 4°C with either normal rabbit serum or anti-CD20N antiserum (2 µl), then mixed with 20 µl of protein A-Sepharose (Repligen, Cambridge, MA) for 2 h. The Sepharose beads were washed three times with lysis buffer and once with PBS, and then mixed with 2x SDS sample buffer. Prestained m.w. markers (Life Technologies or New England Biolabs (Beverly, MA)) were run on each gel, and proteins were transferred to Immobilon P (Millipore, Bedford, MA) membranes for immunoblotting.
Immunoblots
Membranes were blocked overnight with 5% BSA, then incubated for 3 h with anti-CD20N or anti-CD20C Abs diluted in 1% BSA. After washing the membranes, bound Abs were detected with protein A-horseradish peroxidase (Bio-Rad, Richmond, CA). Proteins were visualized using chemiluminescence (Pierce) recorded on Kodak X-OMAT film (Eastman Kodak, Rochester, NY) For the peptide inhibition experiments, anti-CD20N and anti-CD20C Abs were incubated alone or with either CD20N-P or CD20C-P for 30 min on ice before immunoblotting.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
To determine the membrane orientation of CD20, Abs against known
epitopes were generated in rabbits by immunization with OVA-conjugated
CD20 N and C region peptides. The resulting antisera, anti-CD20N
and anti-CD20C, were affinity purified and tested for specificity
before use in membrane orientation studies (Fig. 1). Specificity was confirmed in three
ways. First, both antisera detected proteins of 33 to 35 kDa in
immunoblots of whole cell lysates derived from Raji B cells (Fig. 1A). CD20 migrates on SDS-PAGE as a single band, a doublet,
or a triplet depending upon the quantity of protein present and the
resolving power of the gel. The differently migrating species are the
result of differential serine/threonine phosphorylation (30).
Preincubation of the Abs with the immunizing peptides inhibited
binding, while incubation with irrelevant peptides did not (Fig. 1A). Second, immunoprecipitation with anti-CD20N and
detection by immunoblotting with anti-CD20C demonstrated that both
Abs recognized epitopes on the same protein (Fig. 1B).
Third, both Abs specifically recognized CD20 ectopically expressed by
transfection in the Molt-4 T cell line (Fig. 1C).
|
Localization of the N- and C-terminal regions of CD20 was assessed
by indirect immunofluorescence using intact and membrane-permeabilized
Raji B cells. CD20N and CD20C Abs did not recognize CD20 on intact
cells (Fig. 2). However, after
permeabilization there was a specific increase in intracellular
staining by both anti-CD20N and anti-CD20C Abs that was
prevented by preincubation of the Abs with immunizing peptide.
Effective permeabilization of the cells was confirmed by the detection
of the cytoplasmic Src family tyrosine kinase Lyn only after the
permeabilization procedure (data not shown). Since the epitopes
recognized by anti-CD20N and anti-CD20C were accessible only
after permeabilization, these data confirm the intracellular location
of both N- and C-terminal regions of CD20.
|
A). Protease digestion of intact cells was
performed to evaluate the number and the locations of extracellular
loops. Complete digestion of extracellular regions would be expected to
result in either 8- or 15-kDa N-terminal fragments depending on whether
the polypeptide is exposed at the cell surface on the carboxyl side of
TM1. For these studies we used both trypsin, which cuts specifically on
the carboxylic acid side of arginine or lysine, and the nonspecific
protease proteinase K to treat intact Raji B cells as described in
Materials and Methods. There is no trypsin digest site
present in the putative loop between TM1 and TM2, and four sites in the
hydrophilic region between TM3 and TM4. Therefore, the presence of an
extracellular TM1-TM2 loop (Fig. 8A, ii) would be
expected to generate approximately 15- and 8-kDa N-terminal fragments
by trypsin and proteinase K, respectively. Further, there are four
trypsin sites in the short hydrophilic stretch between TM2 and TM3.
These sites would only be exposed in the two transmembrane/one
extracellular loop model (Fig. 8A, i) and would
also generate a significantly larger trypsin fragment compared with
proteinase K. The only topology that would generate 15-kDa fragments
after digestion with either trypsin or proteinase K is shown in Figure 8A, iii.
|
), indicating that the protein
does not exit to the cell surface between TM1 and TM3, and that there
is a single extracellular loop between TM3 and TM4 (Fig. 8
A,
iii). Recognition of more than one band in both the trypsin-
and proteinase K-digested samples by CD20N Ab is probably due to
serine/threonine phosphorylation as seen in the intact protein.
Incomplete digestion by trypsin may account for the third and largest
N-terminal fragment detected. Fragments obtained after proteinase K
digestion were slightly smaller than that obtained after trypsin
digestion, as expected due to clipping of a few extra residues between
the most N-terminal trypsin cut site and the outer leaflet of the
plasma membrane.
|
21 kDa) resulting from
digestion by either trypsin or proteinase K significantly exceeded the
expected size (14 kDa), but is too large to be accounted for by
incomplete digestion of the extracellular loop. The discrepancy in size
may be attributable to post-translational modifications in the
C-terminal region, such as phosphorylation and/or acylation (discussed
further below), or to SDS-resistant protein-protein interactions
occurring after digestion.
Since the epitopes recognized by anti-CD20N and anti-CD20C were
not destroyed by protease digestion, these results, together with those
in Figure 2, confirm their intracellular location. Data from
extracellular protease digestion analyses support a four-TM domain
topology with a single extracellular loop between TM3 and TM4, in
agreement with the hydropathy prediction.
Generation of CD20 deletion mutants
We recently reported that CD20 redistributes from the soluble to
the insoluble fraction of Triton X-100 cell lysates when B cells are
exposed to Abs directed against extracellular epitopes of CD20 before
lysis. The speed with which CD20 can be induced to translocate (6)
indicates that the mechanism does not require de novo transcription or
translation and suggests a conformational and/or post-translational
change in the CD20 molecule. To identify regions that might be involved
in the process of CD20 redistribution, we generated a series of
cytoplasmic domain deletion mutants and tested them for their ability
to translocate to the Triton-insoluble fraction following CD20 mAb
engagement. Generation of a deletion construct that lacked the entire
N-terminal cytoplasmic region (N1–49) was described previously (27)
and was detected in immunoblots using the polyclonal anti-CD20C Ab.
As reported previously (27), deletion of the C-terminal region from
either residue 215 or 222 to the end of the molecule at position 297
resulted in no or poor expression, respectively. However, internal
deletions were expressed well (Fig. 4),
and the C-terminal region was therefore analyzed using three
constructs, C219–225, C226–252, and C253–297, that were
detected in immunoblots with the anti-CD20N Ab. All constructs were
stably expressed in Molt-4 T cells, and expression levels were
monitored by indirect immunofluorescence using the 2H7 mAb against an
extracellular epitope (Fig. 4). Clones expressing either high or lower
levels of wild-type CD20, as shown in Figure 4, were selected and used
in different experiments to match the expression of the mutated forms
of CD20 as closely as possible.
|
Cells were incubated with or without 2H7 anti-CD20 mAb and
lysed in Triton X-100-containing buffer, and both soluble and insoluble
fractions were collected as described in Materials and
Methods. The presence of CD20 in the soluble lysates or insoluble
pellets was detected by immunoblot after SDS-PAGE, and the amounts were
estimated by densitometry analysis of quadruplicate samples. In
untreated cells, CD20 was always found entirely in the Triton-soluble
fractions. As reported previously for CD20 on B cells, Ab-mediated
ligation of wild-type CD20 in the transfected Molt-4 T cells induced
virtually all CD20 protein to translocate to the insoluble compartment.
This was observed both by the disappearance of CD20 from the soluble
lysates and by its appearance in the insoluble pellets (Fig. 5). In contrast, a substantial amount of
CD20 remained in the soluble compartment after Ab treatment when seven
amino acids in the membrane-proximal region of the cytoplasmic
C-terminal domain (C219–225) were deleted (Fig. 5). The inhibition
was not complete, however, as most clearly observed by the detection of
some CD20 in the insoluble pellets. Densitometry analysis of
quadruplicate samples estimated the degree of translocation to be about
25% compared with almost 100% for wild-type CD20. Deletion of an
adjacent stretch of 26 residues (C226–252) inhibited translocation
also, albeit to a lesser extent (75% translocated), whereas
deletion of the C-terminal 45 residues (C253–297) had no
discernible effect on Ab-induced translocation. The CD20 mutant lacking
the N-terminal region (N1–49) was detected using the anti-CD20C
polyclonal antiserum. This antiserum exhibited some background
reactivity; nevertheless, it was clear in multiple experiments that the
N-terminal deletion minimally reduced Ab-induced CD20 translocation by
about 10% (Fig. 6).
|
|
The sequence 219 to 225 includes a cysteine residue at position
220 that is conserved in the other two members of the CD20 family of
related proteins, i.e., FcERIß and HTm4 (31, 32). The proximity of
Cys220 to the inner leaflet of the plasma membrane makes it
a potential site of reversible lipid modification by palmitoyl
transferase. Palmitoylation is required for the caveolar localization
of the Src family tyrosine kinase Hck and the endothelial nitric oxide
synthase (33, 34), and is found on a number of other proteins in these
microdomains, including several of the G
signaling proteins and
caveolin, a structural protein of caveolae. To determine whether a
requirement for palmitoylation of Cys220 accounted for the
inhibitory effect of the 219 to 225 deletion, alanine was substituted
for cysteine at this position. Expression of C220A on transfected
Molt-4 cells, as measured by indirect immunofluorescence, is shown in
Figure 4. Mutation of
Cys220 to alanine did not prevent or reduce Ab-induced
translocation of CD20 to the insoluble pellet (Fig. 7), eliminating
palmitoylation, or other potential modifications, of Cys220
as a mediator of CD20 redistribution. A second cytoplasmic cysteine is
present in the loop between the second and third transmembrane regions;
however, this cysteine is not conserved in FcERIß, HTm4, or murine
CD20, making it an unlikely candidate for an essential function. Since
deletion of the internal loop prevented high level expression of CD20
(27), we could not detect the mutated protein in immunoblots and were
unable to assess the impact of this deletion on CD20 translocation
(data not shown).
|
B). Although this region contains
a potential palmitoylation site, this site was not required for the
translocation of CD20. A similar observation was reported for caveolin;
although palmitoylated, this modification is not required for caveolar
localization of caveolin (35). Since multiple potential phosphorylation
sites exist in both 219 to 225 and 226 to 252 sequences, it is possible
that phosphorylation of CD20 is required for its translocation. CD20 is
known to be phosphorylated (36, 37), and the level of phosphorylation
is increased upon activation with phorbol esters (PMA) or Abs directed
against surface IgM (37, 38, 39, 40). The PMA-induced increase in
phosphorylation of CD20 suggests that PKC may phosphorylate CD20 in
vivo, as it does in vitro (39). Interestingly, however, we found that
pretreatment of B cells with PMA does not promote, but rather inhibits,
CD20 translocation (I. Popoff and J. Deans, unpublished observations).
An alternate role for the identified translocation control region in
CD20 may be its involvement in protein-protein interactions induced by
a conformational change in CD20 upon Ab ligation. The putative
interaction motif is likely to include sequence information spanning
the 219 to 225 and 226 to 252 regions, and work is in progress in this
laboratory to further define the boundaries of the region and to
identify interacting proteins. We speculate that the membrane-proximal sequence in the cytoplasmic C-terminal of CD20 may be involved in promoting oligomerization of CD20. Evidence from chemical cross-linking studies has supported the proposal that CD20 may exist as homodimers and -tetramers that could aggregate to form larger complexes (41). Interestingly, a membrane-proximal sequence in the cytoplasmic C-terminal of caveolin-1 has been shown to be involved in homotypic interactions that are thought to mediate the formation of caveolae (42, 43).
Only a few other examples of targeted redistribution of membrane receptors to caveolae or equivalent microdomains have been reported. The muscarinic acetylcholine receptor, the bradykinin receptor, and the protease receptor tissue factor are translocated into caveolae in response to agonists (11, 12, 13, 14, 15, 16). GPI-linked proteins, which have also been implicated in signaling, become enriched in caveolae upon Ab cross-linking (44), and the high affinity receptor for IgE (FcERI), when aggregated, redistributes to detergent-resistant membrane domains (15, 16). There is currently no information, however, on the mechanisms underlying this phenomenon. CD20 shares significant sequence homology with the ß subunit of the FcERI complex, particularly in regions corresponding to the membrane-spanning domains (32). Although Cys220 of CD20 is conserved in FcERIß, the surrounding amino acids are not. The mechanism involved in localizing FcERIß to detergent-insoluble microdomains may in any case be different, since it is released from the insoluble domains in concentrations of Triton X-100 >0.05% (16), whereas activated CD20 remains insoluble in 1% Triton X-100.
CD20 has been reported to mediate a number of biologic activities in B cells upon Ab engagement. It is likely that the redistribution of CD20 to insoluble membrane microdomains is a prerequisite event to the initiation of signaling and/or calcium channel formation. The identification of a cytoplasmic sequence that prevents CD20 redistribution is expected to lead to a full understanding of the underlying mechanism and perhaps to new insights into B cell activation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Julie Deans, Department of Medical Biochemistry, University of Calgary, Health Sciences Center, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1. E-mail address:
3 Abbreviations used in this paper: GPI, glycosylphosphatidylinesitol; TM, transmembrane; CD20N-P, CD20N peptide.
Received for publication March 4, 1998. Accepted for publication May 26, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
subunits Gq and Gi in caveolae in DDT1 MF-2 smooth muscle cells. J. Biol. Chem. 272:17858.
5 pre-B cell-specific genes can associate with each other and with mu heavy chain. J. Exp. Med. 172:969.This article has been cited by other articles:
|
|
 |
|
  Y. Terui, Y. Mishima, N. Sugimura, K. Kojima, T. Sakurai, Y. Mishima, R. Kuniyoshi, A. Taniyama, M. Yokoyama, S. Sakajiri, et al. Identification of CD20 C-Terminal Deletion Mutations Associated with Loss of CD20 Expression in Non-Hodgkin's Lymphoma Clin. Cancer Res., April 1, 2009; 15(7): 2523 - 2530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Johnson, S. Leach, B. Woolcock, R. J. deLeeuw, A. Bashashati, L. H. Sehn, J. M. Connors, M. Chhanabhai, A. Brooks-Wilson, and R. D. Gascoyne CD20 mutations involving the rituximab epitope are rare in diffuse large B-cell lymphomas and are not a significant cause of R-CHOP failure Haematologica, March 1, 2009; 94(3): 423 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Goldenberg, E. A. Rossi, R. Stein, T. M. Cardillo, M. S. Czuczman, F. J. Hernandez-Ilizaliturri, H. J. Hansen, and C.-H. Chang Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody Blood, January 29, 2009; 113(5): 1062 - 1070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Perosa, E. Favoino, C. Vicenti, A. Guarnera, V. Racanelli, V. De Pinto, and F. Dammacco Two Structurally Different Rituximab-Specific CD20 Mimotope Peptides Reveal That Rituximab Recognizes Two Different CD20-Associated Epitopes J. Immunol., January 1, 2009; 182(1): 416 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Polyak, H. Li, N. Shariat, and J. P. Deans CD20 Homo-oligomers Physically Associate with the B Cell Antigen Receptor: DISSOCIATION UPON RECEPTOR ENGAGEMENT AND RECRUITMENT OF PHOSPHOPROTEINS AND CALMODULIN-BINDING PROTEINS J. Biol. Chem., July 4, 2008; 283(27): 18545 - 18552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. James, P. D. Greenberg, M. C. Jensen, Y. Lin, J. Wang, B. G. Till, A. A. Raubitschek, S. J. Forman, and O. W. Press Antigen Sensitivity of CD22-Specific Chimeric TCR Is Modulated by Target Epitope Distance from the Cell Membrane J. Immunol., May 15, 2008; 180(10): 7028 - 7038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Li, S. Shi, W. Qian, L. Zhao, D. Zhang, S. Hou, L. Zheng, J. Dai, J. Zhao, H. Wang, et al. Development of Novel Tetravalent Anti-CD20 Antibodies with Potent Antitumor Activity Cancer Res., April 1, 2008; 68(7): 2400 - 2408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Czuczman, S. Olejniczak, A. Gowda, A. Kotowski, A. Binder, H. Kaur, J. Knight, P. Starostik, J. Deans, and F. J. Hernandez-Ilizaliturri Acquirement of Rituximab Resistance in Lymphoma Cell Lines Is Associated with Both Global CD20 Gene and Protein Down-Regulation Regulated at the Pretranscriptional and Posttranscriptional Levels Clin. Cancer Res., March 1, 2008; 14(5): 1561 - 1570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Garcia, E. J. Brown, and C. Rosales Transmembrane Mutations to Fc{gamma}RIIA Alter Its Association with Lipid Rafts: Implications for Receptor Signaling J. Immunol., March 1, 2007; 178(5): 3048 - 3058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Stel, B. ten Cate, S. Jacobs, J. W. Kok, D. C. J. Spierings, M. Dondorff, W. Helfrich, H. C. Kluin-Nelemans, L. F. M. H. de Leij, S. Withoff, et al. Fas Receptor Clustering and Involvement of the Death Receptor Pathway in Rituximab-Mediated Apoptosis with Concomitant Sensitization of Lymphoma B Cells to Fas-Induced Apoptosis J. Immunol., February 15, 2007; 178(4): 2287 - 2295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Teeling, W. J. M. Mackus, L. J. J. M. Wiegman, J. H. N. van den Brakel, S. A. Beers, R. R. French, T. van Meerten, S. Ebeling, T. Vink, J. W. Slootstra, et al. The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20 J. Immunol., July 1, 2006; 177(1): 362 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xu, M. S. Williams, and L. M. Spain Patterns of expression, membrane localization, and effects of ectopic expression suggest a function for MS4a4B, a CD20 homolog in Th1 T cells Blood, March 15, 2006; 107(6): 2400 - 2408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Zhang, L. A. Khawli, P. Hu, and A. L. Epstein Generation of Rituximab Polymer May Cause Hyper-Cross-linking-Induced Apoptosis in Non-Hodgkin's Lymphomas Clin. Cancer Res., August 15, 2005; 11(16): 5971 - 5980. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Niwa, M. Sakurada, Y. Kobayashi, A. Uehara, K. Matsushima, R. Ueda, K. Nakamura, and K. Shitara Enhanced Natural Killer Cell Binding and Activation by Low-Fucose IgG1 Antibody Results in Potent Antibody-Dependent Cellular Cytotoxicity Induction at Lower Antigen Density Clin. Cancer Res., March 15, 2005; 11(6): 2327 - 2336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Cartron, H. Watier, J. Golay, and P. Solal-Celigny From the bench to the bedside: ways to improve rituximab efficacy Blood, November 1, 2004; 104(9): 2635 - 2642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Li, L. M. Ayer, M. J. Polyak, C. M. Mutch, R. J. Petrie, L. Gauthier, N. Shariat, M. J. Hendzel, A. R. Shaw, K. D. Patel, et al. The CD20 Calcium Channel Is Localized to Microvilli and Constitutively Associated with Membrane Rafts: ANTIBODY BINDING INCREASES THE AFFINITY OF THE ASSOCIATION THROUGH AN EPITOPE-DEPENDENT CROSS-LINKING-INDEPENDENT MECHANISM J. Biol. Chem., May 7, 2004; 279(19): 19893 - 19901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Li, L. M. Ayer, J. Lytton, and J. P. Deans Store-operated Cation Entry Mediated by CD20 in Membrane Rafts J. Biol. Chem., October 24, 2003; 278(43): 42427 - 42434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. T. C. Chan, D. Hughes, R. R. French, A. L. Tutt, C. A. Walshe, J. L. Teeling, M. J. Glennie, and M. S. Cragg CD20-induced Lymphoma Cell Death Is Independent of Both Caspases and Its Redistribution into Triton X-100 Insoluble Membrane Rafts Cancer Res., September 1, 2003; 63(17): 5480 - 5489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Cragg, S. M. Morgan, H. T. C. Chan, B. P. Morgan, A. V. Filatov, P. W. M. Johnson, R. R. French, and M. J. Glennie Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts Blood, February 1, 2003; 101(3): 1045 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Rose, B. E. Smith, and D. G. Maloney Glucocorticoids and rituximab in vitro: synergistic direct antiproliferative and apoptotic effects Blood, August 13, 2002; 100(5): 1765 - 1773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. B. Michel and M. J. Mattes Intracellular Accumulation of the Anti-CD20 Antibody 1F5 in B-Lymphoma Cells Clin. Cancer Res., August 1, 2002; 8(8): 2701 - 2713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Polyak and J. P. Deans Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure Blood, May 1, 2002; 99(9): 3256 - 3262. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Alas, C. Emmanouilides, and B. Bonavida Inhibition of Interleukin 10 by Rituximab Results in Down-Regulation of Bcl-2 and Sensitization of B-cell Non-Hodgkin's Lymphoma to Apoptosis Clin. Cancer Res., March 1, 2001; 7(3): 709 - 723. [Abstract] [Full Text]
|
|
|
|
|
|
Y.-G. Ko, J.-S. Lee, Y.-S. Kang, J.-H. Ahn, and J.-S. Seo TNF-{alpha}-Mediated Apoptosis Is Initiated in Caveolae-Like Domains J. Immunol., June 15, 1999; 162(12): 7217 - 7223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Brown and E. London Structure and Function of Sphingolipid- and Cholesterol-rich Membrane Rafts J. Biol. Chem., June 2, 2000; 275(23): 17221 - 17224. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
