Rituximab Resistance Is Associated with Global Downregulation of CD20 and CD54 In B-Cell Lymphoma Cells: Implication of CD20 Signaling on CD54

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5077-5077
Author(s):  
Ping-Chiao Tsai ◽  
Peter Dvorak ◽  
Naveen Bangia ◽  
Scott Olejniczak ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Adhesion ◽  
B Cells ◽  
B Cell ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Cell Aggregation ◽  
Cell Clustering ◽  
In Vitro Exposure

Abstract Abstract 5077 Cell adhesion plays an important role in the cell-cell communication and provides important signals for cell survival, migration and aggregation. Pre-clinical studies have been conducted to investigate differences in the expression of adhesion molecules on the surface of malignant B-cells in an attempt to explain differences in the clinical behavior and patterns of disease dissemination between non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Of interest CLL cells have lower levels of both adhesion molecules and CD20 when compared to follicular lymphomas (FL). It is unclear if the expression of adhesion molecules affects rituximab activity or if rituximab disrupts cell-to-cell interactions. To this end, we studied cell aggregation and expression of adhesion molecules in a panel of rituximab-senstive (RSCL) and rituximab-chemotherapy lymphoma cell lines (RRCL) that have been extensively characterized by our group (Czuczman MS. et al. Clin Cancer Res. 2008; 14:1561-70). Homotypic adhesion of B-cells is known to due to the interaction of ICAM-1(CD54) and LFA-1(CD11a). Expression of CD54 and its ligand CD11a was studied by Amnis ImageStream Technology analysis not only in RSCL and RRCL, but also in several intermediate passages obtained during the generation of RRCL process. To further define the role of CD54 in B-cell aggregation and rituximab activity, RSCL were exposed to RPMI, rituximab (10mg/ml), isotype (10mg/ml) with or without a blocking anti-CD54 monoclonal antibody (mAb) (0.25mg/ml) and patterns of cell aggregation were evaluated by inverted light microscopy and photographs were captured at different time intervals. Cell death and down-stream signaling was evaluated at various times after rituximab treatment. Differences in the expression levels of CD54 correlated with CD20 levels in NHL samples. RRCL were found to have lower mean CD54 density. Gradual loss of CD54 was observed during repeated exposure to escalating doses of rituximab, indicating potential CD54 regulation through rituximab-CD20 signaling. No difference in the CD11a was observed between RSCL and RRCL. RSCL aggregate and form clusters under typical culture conditions whereas RRCL do not aggregate in vitro. Exposure of RSCL to rituximab induced a rapid cell clustering in RSCL. Blocking CD54 using mAbs prevented spontaneous and rituximab-induced cell clustering, resulting in a phenotype similar to the RRCL. Of interest in vitro exposure to anti-CD54 mAb resulted in apoptosis of RSCL, suggesting cell adhesion is important for survival in B-cell lymphomas. The decrease in cell aggregation following CD54 blocking was not reduced by inhibition of caspase activation suggesting that cell death was not the dominant factor in preventing cell clustering in CD54-neutralized RSCL. In summary, we observed a loss of CD54, CD20 and cell aggregation during the process of acquiring resistance to rituximab. Furthermore, blocking of CD54 appears to abolish the clustering effects of rituximab in vitro. Loss of CD54 is observed in rituximab-chemotherapy cross-resistant cell lines and may disrupt signaling events thereby contributing to resistance to rituximab and chemotherapy drugs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential attachment of human neoplastic B cells to purified extracellular matrix molecules

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1586-1594 ◽  
Author(s):  
D Segat ◽  
C Pucillo ◽  
G Marotta ◽  
R Perris ◽  
A Colombatti
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Philadelphia Chromosome ◽  
Maturation State ◽  
Neoplastic Lymphocytes

Recirculation of normal and neoplastic lymphocytes occurs via binding to the endothelial luminar surface, followed by extravasation and the subsequent interaction of the cells with components of the underlying basement membrane and stromal extracellular matrix (ECM). To identify matrix constituents that could be involved in the tissue dissemination of neoplastic B cells, we have examined the ability of three lymphoma B- cell lines and one Philadelphia chromosome (Ph1)-positive cell line established from the lymphoid transformation of a chronic myeloid leukemia (CML) to adhere to a range of purified ECM molecules. Immunophenotyping with a panel of markers suggested that the lines derived from cells that had undergone transformation at distinct stages of B-cell maturation. The four cell lines displayed a differential ability to adhere to the ECM molecules tested. BV-173, Ci-1, and Sc-1 cells attached to various degrees to fibronectin (FN). Ri-1, Ci-1, and Sc-1 cells attached to human laminin (LN) variants, whereas only Ci-1 and Sc-1 cells showed some affinity for collagen (Col) type VI. All four cell lines interacted with fibrillar Col I, but only BV-173 and Ri- 1 cells attached to fibrillar Col III. The subendothelial Col VIII only was active as a substratum for BV-173 cells. In all cases, cells bound to fibrillar collagens when they were assembled into polymeric fibrils, and were incapable of adhering to monomeric and denatured collagen. In contrast to cell adhesion to FN and LN, which showed a plateau at high substrate concentrations, adhesion to fibrillar Col I reached a peak at intermediary concentrations and decreased thereafter, suggesting that cells respond to a definite macromolecular arrangement of collagenous fibrils. Adhesion to individual ECM molecules was not directly correlated with the apparent maturation state of the cells, nor with the relative density of known ECM receptors. Taken together, these results suggest that interaction of neoplastic B cells with selected matrix components may influence their dispersion throughout tissues. We further suggest that the use of quantitative cell adhesion assays in vitro may provide means of defining the behavioral traits of neoplastic B cells in vivo.

The Acquirement of Rituximab Resistance Is Associated with a Global Downregulation of CD54 in B-Cell Lymphoma Cells: The Potential Role of Adhesion Molecules in Rituximab Anti-Tumor Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2607-2607
Author(s):  
Ping-Chiao Tsai ◽  
Naveen Bangia ◽  
Scott Olejniczak ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Myron Czuczman
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Adhesion Molecules ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Aggregation ◽  
B Cell Lymphomas ◽  
Cell Clustering

Abstract Cell adhesion plays an important role in the cell-cell communication and provides important signals for cell survival, migration, aggregation, or other cell functions. Preclinical studies have been conducted to investigate the expression profiles of different adhesion molecules on the surface of malignant B-cells in an attempt to explain differences in the clinical behavior and patterns of spread between non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Of interest, CLL cells have lower levels of both adhesion molecules and CD20 when compared to follicular lymphomas (FL). Recently, knockout studies had demonstrated that CD26, an adhesion molecule, modified responses to chemotherapy in B-cell lymphomas. It is unclear if the expression of adhesion molecules affects rituximab activity. To this end, we studied the patterns of cell aggregation and expression of adhesion molecules in a panel of rituximab-sensitive (RSCL) and rituximab-chemotherapy lymphoma cell lines (RRCL) that had been extensively characterized by our group (Czuczman S. et al. Clin Cancer Res.2008; 14:1561–70). Homotypic adhesion of B-cells is known to, due to the interaction of ICAM-1(CD54) and LFA-1(CD11a). Expression of CD54 and its ligand CD11a was studied by flow cytometry analysis and polymerase chain reaction (PCR, CD54 only). Patterns of cell aggregation in RSCL and RRCL in resting conditions were studied by inverted light microscopy. To define further the role of CD54 in B-cell aggregation and rituximab activity, RSCL (Raji and RL cells) were exposed to RPMI, rituximab (10mg/ml), isotype (10mg/ml) with or without a blocking anti-CD54 monoclonal antibody (0.25mg/ml) and patterns of cell aggregation were evaluated by inverted light microscopy, and photographs were captured at different time intervals. Experiments were conducted with or without the potent pan-caspase inhibitor Q-VD-OPh and performed in triplicates. Cell death was detected by propidium iodine staining and quantified by flow cytometry. Differences in the expression levels of CD54 were observed in the NHL cells tested. RRCL were found to have lower levels of CD54 at the surface protein and gene level. No differences in the CD11a were observed. RSCL aggregate and form clusters under culture conditions whereas RRCL do not aggregate in vitro. In vitro exposures to rituximab lead to a rapid cell clustering in RSCL. Blocking CD54 using mAbs prevented spontaneous and rituximab induced cell clustering, resulting in a phenotype similar to the RRCL. Of interest, in vitro exposure to anti-CD54 mAb and to a lesser degree rituximab resulted in apoptosis of RSCL, suggesting that cell adhesion is important for survival in B-cell lymphomas. The decrease in cell aggregation following CD54 blocking was not reduced by inhibition of caspase activation suggesting that cell death was not the dominant factor in preventing cell clustering in RSCL. In summary, our data suggests that CD54 is important for B-cell lymphoma cell aggregation and survival. Furthermore, blocking of CD54 appears to abolish the clustering effects of rituximab in vitro. Loss of CD54 is observed in rituximab-chemotherapy cell lines and may disrupt signaling events that control cell proliferation (i.e. pro- or anti-apoptotic proteins) rendering these cells resistant to rituximab and chemotherapy drugs. Ongoing studies in lymphoma severe combined immunodeficiency mice (SCID) are underway to further define the role of CD54 in the progression of B-cell lymphomas and responses to rituximab activity in vivo.

scholarly journals Differential attachment of human neoplastic B cells to purified extracellular matrix molecules

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1586-1594 ◽  
Author(s):  
D Segat ◽  
C Pucillo ◽  
G Marotta ◽  
R Perris ◽  
A Colombatti
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Philadelphia Chromosome ◽  
Maturation State ◽  
Neoplastic Lymphocytes

Abstract Recirculation of normal and neoplastic lymphocytes occurs via binding to the endothelial luminar surface, followed by extravasation and the subsequent interaction of the cells with components of the underlying basement membrane and stromal extracellular matrix (ECM). To identify matrix constituents that could be involved in the tissue dissemination of neoplastic B cells, we have examined the ability of three lymphoma B- cell lines and one Philadelphia chromosome (Ph1)-positive cell line established from the lymphoid transformation of a chronic myeloid leukemia (CML) to adhere to a range of purified ECM molecules. Immunophenotyping with a panel of markers suggested that the lines derived from cells that had undergone transformation at distinct stages of B-cell maturation. The four cell lines displayed a differential ability to adhere to the ECM molecules tested. BV-173, Ci-1, and Sc-1 cells attached to various degrees to fibronectin (FN). Ri-1, Ci-1, and Sc-1 cells attached to human laminin (LN) variants, whereas only Ci-1 and Sc-1 cells showed some affinity for collagen (Col) type VI. All four cell lines interacted with fibrillar Col I, but only BV-173 and Ri- 1 cells attached to fibrillar Col III. The subendothelial Col VIII only was active as a substratum for BV-173 cells. In all cases, cells bound to fibrillar collagens when they were assembled into polymeric fibrils, and were incapable of adhering to monomeric and denatured collagen. In contrast to cell adhesion to FN and LN, which showed a plateau at high substrate concentrations, adhesion to fibrillar Col I reached a peak at intermediary concentrations and decreased thereafter, suggesting that cells respond to a definite macromolecular arrangement of collagenous fibrils. Adhesion to individual ECM molecules was not directly correlated with the apparent maturation state of the cells, nor with the relative density of known ECM receptors. Taken together, these results suggest that interaction of neoplastic B cells with selected matrix components may influence their dispersion throughout tissues. We further suggest that the use of quantitative cell adhesion assays in vitro may provide means of defining the behavioral traits of neoplastic B cells in vivo.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

Lenalidomide (Revlimid®) Enhances Monoclonal Antibody-Associated Anti-Tumor Activity Against Rituximab-Sensitive and Rituximab-Resistant B-Cell Lymphoma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2522-2522 ◽  
Author(s):  
Nishitha Reddy ◽  
Raymond Cruz ◽  
Francisco Hernandez-Ilizaliturri ◽  
Joy Knight ◽  
Myron S. Czuczman
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Scid Mouse ◽  
In Vitro Exposure ◽  
Human Lymphoma ◽  
Scid Mouse Model ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Background: Lenalidomide is a potent thalidomide analogue shown to activate both the innate and adoptive immune system, inhibit angiogenesis, and modify the tumor microenvironment. While lenalidomide has received approval by the U.S. Federal Drug Administration (FDA) for the treatment of various hematological conditions, ongoing clinical trials are addressing its role in the treatment of B-cell lymphomas. There is a dire need to develop novel well-tolerated, therapies which combine various target-specific agents such as lenalidomide and monoclonal antibodies (mAbs). We previously demonstrated that lenalidomide is capable of expanding natural killer (NK) cells in a human-lymphoma-bearing SCID mouse model and improve rituximab anti-tumor activity in vivo. Methods: In our current work we studied the effects of lenalidomide on the biological activity of a panel of mAbs against various B-cell lymphomas, utilizing various rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) generated in our laboratory from Raji and RL cell lines. Functional assays including antibody-dependant cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) were performed to demonstrate changes in sensitivity to rituximab. RSCL and RRCL (1′105 cells/well) were exposed to either lenalidomide (5 μg/ml) or vehicle with or without mAb at a final concentration of 10μg/ml. The mAb panel consisted of two anti-CD20 mAbs: rituximab (Biogen IDEC, Inc.) and hA20, a humanized anti-CD20 mAb (Immunomedics, Inc.); an anti-CD80 mAb (galixumab, Biogen IDEC Inc.), and an anti-CD52 antibody (Alemtuzumab, Berlex Inc.). Changes in DNA synthesis and cell proliferation were determined at 24 and 48 hrs by [3H]-thymidine uptake. For ADCC/CMC studies, NHL cells were exposed to lenalidomide or vehicle for 24 hrs and then labeled with 51Cr prior to treatment with one of various mAbs (10 mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio, 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Changes in antigen (CD20, CD80, and CD52) expression following in vitro exposure to lenalidomide were studied by multicolor flow cytometric analysis. Results: Concomitant in vitro exposure of various RSCL and RRCL cells to lenalidomide and either galixumab, hA20 or alemtuzumab for 24 hrs resulted in improved anti-tumor activity when compared to controls. In addition, pre-incubation of both RSCL and RRCL with lenalidomide rendered cells more susceptible to alemtuzumab-, hA20- and galixumab-mediated ADCC and CMC. No antigen modulation (i.e., upregulation) was observed following in vitro exposure of lenalidomide to NHL cell lines, suggesting an alternative mechanism involved in the improvement antitumor activity observed. Conclusions: Our data suggest that the augmented antitumor effect of lenalidomide is not limited to its combination with rituximab, but also that it augments the antiproliferative and biological activity of alemtuzumab, hA20 and galixumab. Furthermore, these interactions are observed even in our RRCL. Future studies will be directed towards evaluating whether similar activity will be seen in vivo using a human lymphoma-bearing SCID mouse model. (Supported by USPHS grant PO1-CA103985 from the National Cancer Institute.)

Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4525-4525
Author(s):  
Bernardo Martinez-Miguel ◽  
Melisa A. Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Rogelio Hernandez-Pando ◽  
Cesar R. Gonzalez-Bonilla ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
B Cell Malignancies ◽  
Peptide Mimetic

Abstract The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.

A New Human CD20 Spliced mRNA as a Potential Molecular Marker for the Follow-up of B-Cell Malignancies.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1479-1479
Author(s):  
Marina Deschamps ◽  
Carole Henry ◽  
Eric Robinet ◽  
Francine Garnache-Ottou ◽  
Jean-Paul Remy-Martin ◽  
...  
Keyword(s):  
B Cells ◽  
Molecular Marker ◽  
B Cell ◽  
Cell Lines ◽  
Specific Marker ◽  
Molecular Response ◽  
Sequencing Analysis ◽  
Pcr Product ◽  
Donor And Acceptor

Abstract The human CD20 molecule (hCD20) is a B-cell lineage specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and a target for Rituximab (Rx) immunotherapy. During the course of a CD20 RTPCR on B-cell line cDNA, we unexpectedly obtained a shorter PCR product (ΔCD20) in addition to the expected 894bp PCR product (wtCD20). Sequencing analysis revealed that this additional fragment was identical to Genbank published CD20 sequence, but lacked 501bp. In silico analysis of the wtCD20 sequence, using Genesplicer and NetGene 2 softwares, showed donor and acceptor sites (nt112 & nt611 respectively, from ATG codon) matching the newly identified spliced DCD20 form and a branched site located in nt595 exon 3 of the gene respectively. The CD20 spliced mRNA form links part of the end of exon 3 to that of exon 7. The truncated sequence is on the reading frame and can code a putative protein of 130 amino-acids (~15KD) including the intracellular C- terminal domain with part of the transmembrane 1 (TM1) domain and the end of the N-terminal intracellular domain. The extracellular domain and large parts of the 4 TM segments are deleted suggesting that the putative ΔCD20 may be a non-anchored membrane protein. Using CD20 PCR assays amplifying either both wtCD20 and ΔCD20 forms or ΔCD20 alone, we detected the truncated mRNA DCD20 form in different B cell lines (n=12) but not in different T-cell lines (n=4). With a QPCR assay allowing for the specific quantification of either wtCD20 or truncated DCD20 mRNA we also detected the ΔCD20 spliced form [expressed as relative % of ΔCD20: R = (ΔCD20/wtCD20+ΔCD20) × 100] in in-vitro EBV-transformed B-cell lines (2.9 ± 4.51%, n=6); as well as in CD19+ cell sorted cells from tonsillectomy samples (9 ± 2.2%, n=7) and in-vitro B blast cells (14 ± 7.8%, n=5). Interestingly, screening of a panel of B-cell hematologic malignancies showed that the spliced form is detectable at various levels. We found a mean of 3.6 ± 5.1% in B-ALL (n=27); 3.9 ± 5.3% in follicular lymphomas (n=5); 2.9 ± 4.5% in mantle lymphomas (n=6); 3.2 ± 2.2% in high grade lymphomas (n=5); and 0.1 ± 0.2% in B-CLL (n=8). However, this spliced form was not detected in peripheral blood mononuclear cells or in immunomagnetically-sorted CD19+ or CD20+ blood cells from healthy donors. To explore clinical relevance, molecular monitoring of ΔCD20 in the marrow was performed in 2 patients. A 1rst patient with mantle lymphoma, showed an R of 4.2% at diagnosis (diag). After chemotherapy (VAD + chloraminophen) + Rx, R was 2.2% at +12 mo (×1.9 decrease from diag) and 1.8% at +18mo (×1.3 decrease/+12mo). This was in accordance with the cytological (absence of lymphoma cells) and the molecular response (absence of cyclinD1 overexpression). A second patient, with a Phi+ B-ALL, showed an ×13.7 R-decrease compared to diag (from 4.1% to 0.3%) following treatment according to the molecular response. Interestingly, R increased up to 2.6% at +8 months (×8.6/6mo) and 3% (×1.1/8mo) at +12 months after treatment, while molecular and cytological relapse was evidenced only 12 mo after treatment. In conclusion, we report evidence of a novel alternatively spliced transcript of the hCD20 gene, specifically expressed at detectable levels in leukemic, lymphoma, activated or EBV-transformed B cells, but not in normal resting B cells. Further experiments will determine whether the ΔCD20 mRNA spliced form modulates wtCD20 expression and thus influences the response to Rx treatment in hematologic B diseases. However, our initial results suggest that DCD20 quantification may be an indicator of minimal residual disease, as a potential predictive marker of relapse, especially in patients with no other molecular marker.

Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5138-5138
Author(s):  
Juan J Gu ◽  
Lianjuan Yang ◽  
Cory Mavis ◽  
Matthew J. Barth ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
B Cell ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
In Vitro Exposure ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models. Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs. Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole. Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epstein-Barr virus transformation of human pre-B cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 616-622 ◽  
Author(s):  
M Hansson ◽  
K Falk ◽  
I Ernberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Bone Marrow Cells ◽  
Fetal Bone ◽  
Barr Virus ◽  
Epstein Barr

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

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