|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
From the Leukemia Laboratory, Department of Hematology,
The Finsen Centre, Rigshospitalet, Copenhagen, Denmark.
Antibodies against CD20 can activate complement and induce
antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In
B-cell lines, such antibodies also induce apoptosis. In this study, the
expression and function of CD20 on B-cell chronic lymphocytic leukemia
(B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal
CD5+ and CD5 B-cell chronic lymphocytic leukemia (B-CLL) is the
most common form of leukemia in the Western world, with an annual
incidence exceeding 5 new cases per 100 000 individuals, and the
disease must still be regarded as incurable.1,2 The
pathogenesis of B-CLL is poorly understood, but deregulated control of
apoptosis is believed to be critical for the characteristic
accumulation of B lymphocytes in this disease.1,3 B-CLL
cells are long-lived, small, mature
CD5+CD19+CD23+ B cells that appear
to be arrested early in the G1-phase of the cell
cycle.1-5
CD20 is a 33- to 37-kd membrane protein of unknown function, expressed
on mature B cells and their B-lineage bone marrow
precursors.6-8 Because CD20 neither sheds from the cell
surface nor internalizes upon antibody binding, sustained binding of
anti-CD20 antibodies can activate effector mechanisms, such as
complement-mediated lysis and antibody-dependent cellular
cytotoxicity (ADCC), and immunotherapy with anti-CD20 antibodies has
demonstrated significant efficacy in the treatment of B-cell
neoplasms.9-14 However, in B-cell lines, cultured under
conditions that preclude activation of these effector mechanisms,
anti-CD20 antibodies also induce apoptosis.15-17
CD20-induced apoptosis in these cell lines has been shown to be
partially dependent on protein tyrosine kinase activity,
Ca++ mobilization, and effector caspase
activation.16
In this study, we have used freshly isolated primary leukemia cells
from patients with B-CLL to analyze the functional consequences of
binding of a mouse/human chimeric anti-CD20 antibody (rituximab) to
CD20 expressed in the membrane of the malignant cells. We demonstrate that rituximab activates a CD20-mediated signaling pathway that results in apoptosis and is dependent on p38 mitogen activated protein (MAP)-kinase activation.
Patients
Flow cytometry
Cell culture and reagents Mononuclear cells were cultured at 2 × 106/mL in RPMI 1640 containing 10% heat inactivated fetal calf serum (Harlan Sera-Lab, Sussex, England) and antibiotics, in a humidified atmosphere containing 5% CO2. Cell suspensions were supplemented with 4 µg/mL, unless otherwise indicated, of chimeric anti-CD20 antibody rituximab (kindly provided by IDEC Pharmaceuticals, San Diego, CA) or a control human IgG-preparation (SSI-IVIG, a kind gift from Dr Flemming Balstrup, Statens Seruminstitut, Copenhagen, Denmark), containing greater than 95% IgG with 61% IgG1. For cross-linking, the cultures were further supplemented with anti-human IgG1 F(ab)2 fragment (AO407, DAKO) at concentrations 5 times that of the primary antibody. For inhibition of p38 MAP-kinase activity, cells were preincubated for 60 minutes in the presence of 2.5 to 10 µM SB203580, an inhibitor of the p38 pathway, or 10 to 25 µM UO126, an inhibitor of the extracellular signal-regulated kinase (ERK) pathway (Alexis Biochemicals, San Diego, CA) and subsequently supplemented with rituximab or the control preparation.Detection of apoptosis For morphology studies, cytospin preparations were stained with May-Grünwald-Giemsa. To quantitate the extent of apoptosis, cells were harvested, washed in phosphate-buffered saline (PBS), and gently resuspended in 0.4 mL hypotonic fluorochrome solution (0.05 mg/mL propidium iodide (PI) in 0.1% sodium citrate, 0.37% NP40, and 0.02 mg/mL RNase A), as previously described.18-20 There were 5000 events, at a rate of 100 to 200 events per second acquired on a FACSort flow cytometer, with the use of a threshold of 20 in FL-2 linear mode. For phosphatidylserine exposure and membrane integrity analysis, cells were double-stained with fluorescein isothiocyanate (FITC)-labeled annexin-V and PI with the Apoptosis Detection Kit I (6693KK; PharMingen, San Diego, CA) according to the manufacturer's instructions.21 There were 30 000 ungated events acquired on a FACsort flow cytometer; these were analyzed by means of the CellQuest software.Western blot analysis Cells were washed once in ice-cold PBS and lysed in ice-cold lysis buffer (10 mM Hepes [pH 7.3], 0.1% Triton X-100, 400 mM KCl, 2 mM EDTA, 1 mM ethyleneglycotetraacetic acid, 1 mM dithiothreitol [DTT], 1 mM Na3VO4, 1 mM phenylmethyl sulfonyl fluoride, 5 µg/mL leupeptin, 5 µg/mL aprotinin, 5 µg/mL pepstatin). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with the use of equal amounts of whole cell lysate, followed by transfer to nitrocellulose filters. After blocking with 5% milk and overnight incubation with the primary antibodies (c-Jun NH2-terminal protein kinase [JNK] [sc-571], ERK [sc-94-G], p38 [sc-535] (Santa Cruz Biotechnology, Santa Cruz, CA) and pY-JNK (9255S), pY-ERK (9106S), and pY-p38 (9211S) (New England BioLabs, Beverly, MA), the nitrocellulose sheet was further processed by means of horseradish peroxidase-conjugated secondary antibody (PO260, PO448, or PO160) (DAKO) and developed by means of an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Little Chalfont, England).In vitro kinase assay First, 1 × 108 cells were lysed for 30 minutes on ice in 1 mL lysis buffer (50 mM Tris-base, 0.5% NP-40, 150 mM NaCl, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM EDTA, 10 µg/mL leupeptin, 10 µg/mL aprotinin). Cellular debris was removed by centrifugation at 12 000g for 10 minutes at 4°C. Total MAPKAP K2 was immunoprecipitated by addition of 3 µg primary antibody (06-534) (Upstate Biotechnology, Lake Placid, NY) overnight, followed by 10 µL protein G sepharose for an additional hour. The immunoprecipitates were washed 3 times in wash buffer (50 mM Tris base, 0.1% NP-40, 150 mM NaCl, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM EDTA, 10 µg/mL leupeptin, 10 µg/mL aprotinin) and once in kinase assay buffer (25 mM Hepes [pH 7.4], 25 mM -glycerophosphate, 25 mM
MgCl2, 0.1 mM Na3VO4, 2 mM DTT).
Then, 50 µL beads in assay buffer were left as a 1:1 suspension, and
15 µL MAPKAP K2 substrate (12-240) (Upstate Biotechnology) was added.
In vitro kinase reactions were initiated by the addition of 5 µCi
(1.85 × 105 Bq) [ 32-P]adenosine
triphosphate and incubated at 30°C for 20 minutes. Reactions were
spotted on P81 Whatman paper. The samples were washed 3 times for 5 minutes in 75 mM phosphoric acid and once in acetone and then air
dried; their radioactivity was determined in a -counter.
Statistical analysis CD20 expression was analyzed with the unpaired nonparametrical Mann-Whitney test, and statistical analysis was carried out by means of GraphPad Prism software (San Diego, CA).
CD20 expression on normal and malignant lymphocytes CD5+ B-CLL cells have been reported to express CD20 with weaker fluorescence intensity in their plasma membrane than B cells of CD5 B-cell malignancies.20 We
examined the expression of CD20 on B-CLL cells and compared this
expression with normal CD5+ and CD5 B cells
and CD5 low-grade NHL cells (Figure
1). Using dual-color staining and flow
cytometry, we were able to define 3 separate CD20+
populations in healthy donors: (1)
CD5brightCD20dim, (2)
CD5dimCD20bright, and (3)
CD5CD20bright (Figure 1, upper panel, right
top). All B-CLL samples (n = 44) expressed CD20 but in intensities
varying from CD20dim to CD20bright (Figure 1,
left, bottom). CD20 expression on B-CLL cells was significantly weaker
than on the CD20brightCD5dim or negative
healthy populations (P < .001, Mann-Whitney), and the
CD20bright NHL cells (P < .004,
Mann-Whitney). However, the intensity of CD20 staining on B-CLL cells
was not significantly different from that of the healthy
CD5brightCD20dim population (population 1 in
Figure 1, top). Surprisingly, further analysis of this healthy CLL-like
population demonstrated that these cells did not express B-cell
markers such as CD19, CD22, CD23, or surface membrane immunoglobulin;
they did not express myeloid markers such as CD13, CD14, or CD33;
and they did not express the NK-cell marker CD56 (data not shown)
(n = 6). However, the CD5brightCD20dim cells
stained positive for the pan-T-cell marker CD3; they had an
equal distribution between CD4+ and CD8+ cells;
and they expressed predominantly the  -chain of the T-cell receptor although  -positive cells were also identified in this population. Thus, although normal
CD5brightCD20dim cells express CD5 and CD20
with intensities very comparable to those of B-CLL cells, the healthy
population appears to be of T lineage, not of B lineage (Figure
1).
Rituximab induces apoptosis in freshly isolated B-CLL cells We studied the functional effect of rituximab in primary leukemia cells from patients with B-CLL, using 3 corroborative apoptosis assays: morphology analysis, PI staining of cell DNA, and detection of phospholipid asymmetry and plasma membrane integrity, by double staining with annexin V-FITC and PI. We cultured freshly isolated B-CLL cells in the presence or absence of rituximab. When cross-linked, rituximab induced morphological changes typical of apoptosis and significantly increased the fraction of hypodiploid nuclei (observed in 25 independent experiments) (Figure 2A). This induction of apoptosis was dose and time dependent, with the maximal response at a dose of 4 µg rituximab per milliliter medium (Figure 2B-C).
Annexin V binds the membrane phospholipid phosphatidylserine, which is
externalized from the inner to the outer leaflet of the plasma membrane
in the early stage of apoptosis. When membrane integrity is lost, as
seen in the later stage of cell death resulting from either the
apoptotic or the necrotic processes, PI staining becomes
positive. According to the results of our time-course study,
annexin-positive apoptotic cells, after 24 hours of culture, were
almost equally distributed between PI Rituximab-induced apoptosis in B-CLL cells is related to MAP-kinase phosphorylation and dependent on p38 activity Binding of anti-CD20 antibodies to CD20 induces phosphorylation of protein tyrosine kinases and calcium mobilization in B-cell lines.15-17 These proximal membrane signals could theoretically lead to activation of MAP kinases in a CD20-mediated signaling pathway.22 We analyzed whether this was the case in the CD20-mediated apoptosis signaling pathway. MAP-kinase activation was assessed by Western blot analysis on whole cell lysates with anti-phospho kinase (MAPK) antibodies, which specifically recognize an activated form of MAPK (ie, MAPK phosphorylated at tyrosine and serine residues). We cultured freshly isolated B-CLL cells in the presence or absence of rituximab and the cross-linking F(ab)2 fragment. Rituximab, when added alone, induced weak phosphorylation of JNK and ERK and had little or no impact on the phosphorylation of p38 (Figure 3A, top). However, cross-linking of rituximab resulted in strong and sustained phosphorylation of all 3 MAP kinases (observed in 3 out of 3 independent experiments) (Figure 3A, bottom). To determine if this rituximab-induced MAP-kinase activation was related to the induction of apoptosis, we next analyzed the effect of inhibitors of the MAP-kinase pathways. The MEK-inhibitor UO126 had no inhibitory effect on CD20-mediated apoptosis, even though it completely inhibited rituximab-induced ERK activation (data not shown). In contrast, the p38 inhibitor SB203580 completely abrogated rituximab-induced MAPKAP K2 activity downstream of p38 (observed in 3 out of 3 independent experiments) (Figure 3B, bottom), and decreased rituximab-induced apoptosis by a mean of 41% (range, 16%-67%) (Figure 3B, top). In additional dose-response experiments, SB203580 significantly inhibited CD20-mediated apoptosis, even at doses from 2.5 µM (data not shown).
Anti-CD20-based immunotherapy of B-cell malignancies has been developed in an attempt to activate immune-effector mechanisms, such as ADCC and complement-mediated lysis, and thereby induce cell death in the malignant clone.10 Activation of both effector mechanisms have been demonstrated in vitro and in vivo, but these observations do not preclude the possibility that the clinical efficacy of rituximab may involve additional effector mechanisms.13,14,23 Because CD5+ B-CLL cells have been reported to express less
CD20 than other B cells,24 we first examined the phenotype
of B-CLL cells in terms of CD5 and CD20 coexpression. We confirmed that
CD20 expression is weaker on B-CLL cells compared with normal CD5+ and CD5 We next demonstrated that, despite the dim CD20 expression, rituximab induced apoptosis in freshly isolated B-CLL cells under conditions in which the contributions from the above-mentioned effector mechanisms are negligible. First, complement is not active in heat-inactivated serum. Second, our samples contained a median of 96.1% B-CLL cells, and the vast majority of the residual cells are CD5+ T-cells. Therefore, the ratio between B-CLL cells and cells capable of mediating the cytotoxic response, ie, NK cells and monocytes, is at best 1:100 or lower, making it unlikely that cytotoxicity has any significant impact on the apoptotic response. The induction of apoptosis requires cross-linking or immobilization of the antibody and appears to be specific for rituximab. First, although the cross-linking antibody by itself, or together with an IgG1 control preparation, can induce a small degree of apoptosis, this response is not observed in all cases in which rituximab induces apoptosis. The mechanism for this unspecific induction of apoptosis is not clear, but may be attributed to interaction through Fc receptors or immunoglobulin on the surface of the B-CLL cell. Second, antibodies against CD19, both alone and cross-linked, failed to induce apoptosis in B-CLL cells. Third, the rituximab preparation did not induce a measurable apoptotic response in normal T lymphocytes. Therefore, we conclude that the major mechanism by which rituximab works in our system is induction of apoptosis. In cell lines, rituximab-induced apoptosis has been reported to involve calcium mobilization and protein tyrosine kinase phosphorylation.16 In many instances, such signals result in activation of serine/threonine MAP kinases. The dynamic balance of activation of the MAP-kinase pathways is thought to be important in determining whether a cell survives or undergoes apoptosis.27 An important finding in our study is the association between activation of MAP kinases and rituximab-induced apoptosis. Specifically, our results indicate that CD20-mediated apoptosis is dependent on the activity of the p38 MAP-kinase in B-CLL cells. The data presented in this study demonstrate that only cross-linking of rituximab induces a strong, sustained activation of p38 and a significant degree of apoptosis. When the activity of p38 was completely blocked, as evidenced by the absence of MAPKAP K2 activity in SB203580-treated cultures, the response to rituximab in terms of apoptosis induction was significantly decreased in 7 out of 7 experiments. The ERK inhibitor UO126 did not prevent rituximab-induced apoptosis. Although the unavailability of a specific JNK inhibitor precludes any conclusions about the role of JNK activation in rituximab-induced apoptosis, our results strongly indicate that rituximab-induced apoptosis is dependent on p38 activation in B-CLL cells. The specificity of the p38 inhibitor we applied has recently been questioned, but the data on this issue are not equivocal.28-30 Furthermore, we observed the inhibitory effect even at low doses at which the specificity of the inhibitor is not questioned (data not shown). The p38 MAP kinase has been found to be important in many cellular apoptosis systems. A dependence of p38 MAPK activity in B-cell-mediated apoptosis has, for example, been demonstrated in B-cell receptor (BCR)-mediated apoptosis in the B104 B-cell line.31 Recently, both rituximab and anti-BCR antibodies were shown to induce apoptosis and lead to ERK activation in B-cell lines at various maturational stages.32 In contrast, analysis of Fas-mediated apoptosis in the BJAB B-cell line showed no dependence on p38 activity.31 These data suggests that the p38 MAPK pathway plays a complex role in different apoptosis pathways in B cells. Although our results demonstrate dependence on p38 MAP-kinase activitation for the induction of CD20-mediated apoptosis, complete inhibition of p38 activity resulted in only partial inhibition of apoptosis. This observation is consistent with previous reports, in which CD20-mediated apoptosis was shown to be partially dependent on calcium mobilization as well as on protein tyrosine kinase activation.16 These studies, along with the data presented here, suggest that the ability of rituximab to induce apoptosis involves a specific receptor-ligand-like interaction between the antibody and CD20. Specific components of the resulting CD20-mediated signaling cascade appear to be important for the ability of the antibody to induce apoptosis in B lymphocytes, and our results indicate that such molecules are to be defined downstream of p38. Identification of these molecules, and dissection of the pathway(s) that link CD20 to the apoptotic machinery, may provide new therapeutic targets in B-CLL and possibly other B-cell neoplasms.
Submitted November 27, 2000; accepted September 27, 2001.
Supported in part by Danish Cancer Society grants 96-00-009 (J.J.) and 99-100-39 (A.M.B.), the A. L. Vengs Foundation, and The John and Birthe Meyer Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jesper Jurlander, The Leukemia Laboratory, Department of Hematology L4041, The Finsen Centre, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark; e-mail: jjurland{at}rh.dk.
1. Jurlander J. The cellular biology of B-cell chronic lymphocytic leukemia. Crit Rev Oncol Hematol. 1998;27:29-52[Medline] [Order article via Infotrieve].
2.
Dighiero G, Travada P, Chevret S, et al.
B-cell chronic lymphocytic leukemia: present status and future directions. French cooperative group on CLL.
Blood.
1991;78:1901-1914
3.
Caligaris-Cappio F.
B-chronic lymphocytic leukemia: a malignancy of anti-self B-cells.
Blood.
1996;87:2615-2620
4.
Geisler CH, Larsen JK, Hansen NE, et al.
Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia.
Blood.
1991;78:1795-1802 5. Matutes E, Owusu-Ankomah K, Morilla R, et al. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. 1994;8:1640-1645[Medline] [Order article via Infotrieve]. 6. Stashenko P, Nadler LM, Hardy R, Schlossman SF. Characterization of a human B-lymphocyte-specific antigen. J Immunol. 1980;125:1678-1685[Abstract]. 7. Rosenthal P, Rimm IJ, Umiel T, et al. Ontogeny of human hematopoietic cells: analysis utilizes monoclonal antibodies. J Immunol. 1983;131:232-237[Abstract]. 8. Tedder TF, McIntyre G, Schlossman SF. Heterogeneity in the B1 (CD20) cell surface molecule expressed by human B-lymphocytes. Mol Immunol. 1988;25:1321-1330[CrossRef][Medline] [Order article via Infotrieve]. 9. Einfeld DA, Brown JP, Valentine MA, et al. Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J. 1988;7:711-717[Medline] [Order article via Infotrieve].
10.
Reff ME, Carner K, Chambers KS, et al.
Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20.
Blood.
1994;83:435-445
11.
Maloney DG, Liles TM, Czerwinski DK, et al.
Phase I clinical trial using escalating single-dose infusions of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma.
Blood.
1994;84:2457-2466 12. Liu AY, Robinson RR, Murray ED, et al. Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biological activity. J Immunol. 1987;139:3521-3526[Abstract].
13.
Golay J, Zaffaroni L, Vaccari T, et al.
Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement mediated lysis.
Blood.
2000;95:3900-3908
14.
Winkler U, Jensen M, Manzke O, et al.
Cytokine-release syndrome in patients with B-cell chronic lymphocytic leukemia and high lymphocyte counts after treatment with an anti-CD20 minoclonal antibody (rituximab, IDEC-C2B8).
Blood.
1999;94:2217-2224
15.
Shan D, Ledbetter JA, Press OW.
Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies.
Blood.
1998;91:1644-1652 16. Shan D, Ledbetter JA, Press OW. Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother. 2000;48:673-683[CrossRef][Medline] [Order article via Infotrieve]. 17. Hofmeister JK, Cooney D, Coggeshall KM. Clustered CD20 induced apoptosis: src-family kinase, proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis. Blood Cells Mol Dis. 2000;26:133-143[CrossRef][Medline] [Order article via Infotrieve].
18.
Jurlander J, Lai CF, Tan J, et al.
Characterization of interleukin-10 receptor expression on B-cell chronic lymphocytic leukemia cells.
Blood.
1997;89:4146-4152 19. Freid J, Perez AG, Clarkson BD. Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures: some pitfalls in staining and data analysis. J Histochem Cytochem. 1978;26:921-933[Abstract]. 20. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991;139:271-279[CrossRef][Medline] [Order article via Infotrieve].
21.
Koopman G, Reutelingsperger CP, Kuijten GA, et al.
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
Blood.
1994;84:1415-1420 22. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995;80:179-185[CrossRef][Medline] [Order article via Infotrieve]. 23. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med. 2000;6:443-446[CrossRef][Medline] [Order article via Infotrieve]. 24. Almasri NM, Duque RE, Iturraspe J, et al. Reduced expression of CD20 antigen as a characteristic marker for chronic lymphocytic leukemia. Am J Hematol. 1992;40:259-263[Medline] [Order article via Infotrieve]. 25. Hultin LE, Hausner MA, Hultin PM, Giorgi JV. CD20 (pan-B cell) antigen is expressed at low levels on a subpopulation of human T-lymphocytes. Cytometry. 1993;14:196-204[CrossRef][Medline] [Order article via Infotrieve]. 26. Warzynski MJ, Graham DM, Axtell RA, Zakem MH, Rotman RK. Low level CD20 expression on T-cell malignancies. Cytometry. 1994;18:88-92[Medline] [Order article via Infotrieve].
27.
Xia Z, Dickens M, Raingeaud J, et al.
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science.
1995;270:1326-1331
28.
Lali FV, Hunt AE, Turner SJ, et al.
The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositol-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independent of p38 mitogen-activated protein kinase.
J Biol Chem.
2000;275:7395-7402 29. Shaw M, Cohen P, Alessi DR. The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Biochem J. 1998;336:241-246.
30.
Sweeney G, Somwar R, Ramlal, et al.
An inhibitor of p38 mitogen activated protein kinase prevents insulin stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myocytes.
J Biol Chem.
1999;274:10071-10078
31.
Graves JD, Draves KE, Craxton A, et al.
A comparison of signaling requirements for apoptosis of human B lymphocytes induced by the B cell receptor and CD95/Fas.
J Immunol.
1998;161:168-174
32.
Mathas S, Rickers A, Bommert K, Dörken B, Mapara MY.
Anti-CD20- and B-cell receptor-mediated apoptosis: evidence for shared intracellular signaling pathways.
Cancer Res.
2000;60:7170-7176
© 2002 by The American Society of Hematology.
| ||||||||||
 |
|
 |
|
  V. Minard-Colin, Y. Xiu, J. C. Poe, M. Horikawa, C. M. Magro, Y. Hamaguchi, K. M. Haas, and T. F. Tedder Lymphoma depletion during CD20 immunotherapy in mice is mediated by macrophage Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV Blood, August 15, 2008; 112(4): 1205 - 1213. [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]
|
|
|
|
 |
|
 M. Alfonso-Perez, S. Lopez-Giral, N. E. Quintana, J. Loscertales, P. Martin-Jimenez, and C. Munoz Anti-CCR7 monoclonal antibodies as a novel tool for the treatment of chronic lymphocyte leukemia J. Leukoc. Biol., June 1, 2006; 79(6): 1157 - 1165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. F. Israel, M. Gulley, S. Elmore, S. Ferrini, W.-h. Feng, and S. C. Kenney Anti-CD70 antibodies: a potential treatment for EBV+ CD70-expressing lymphomas Mol. Cancer Ther., December 1, 2005; 4(12): 2037 - 2044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Smith, F. Jin, and I. Joshi Enhanced efficacy of therapy with antisense BCL-2 oligonucleotides plus anti-CD20 monoclonal antibody in scid mouse/human lymphoma xenografts Mol. Cancer Ther., December 1, 2004; 3(12): 1693 - 1699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Jazirehi, M. I. Vega, D. Chatterjee, L. Goodglick, and B. Bonavida Inhibition of the Raf-MEK1/2-ERK1/2 Signaling Pathway, Bcl-xL Down-Regulation, and Chemosensitization of Non-Hodgkin's Lymphoma B Cells by Rituximab Cancer Res., October 1, 2004; 64(19): 7117 - 7126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. Eisenbeis, A. Grainger, B. Fischer, R. A. Baiocchi, L. Carrodeguas, S. Roychowdhury, L. Chen, A. L. Banks, T. Davis, D. Young, et al. Combination Immunotherapy of B-Cell Non-Hodgkin's Lymphoma with Rituximab and Interleukin-2: A Preclinical and Phase I Study Clin. Cancer Res., September 15, 2004; 10(18): 6101 - 6110. [Abstract] |
Top
Abstract
Introduction
