Mechanisms Regulating CXCR4 Expression in Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4748-4748
Author(s):  
Seong-Woo Kim ◽  
Ha-Yeon Kim ◽  
Hyo-Jin Lee ◽  
Hwan-Jung Yun ◽  
Samyong Kim ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Cxcr4 Expression ◽  
Cell Surface Expression ◽  
Apoptotic Cells ◽  
Myeloma Cells ◽  
Hypoxic Conditions ◽  
Tnf Α

Abstract The chemokine receptor CXCR4 plays roles in the homing of myeloma cells to the bone marrow (BM) and in the progression of the disease. However, the regulation of CXCR4 expression in myeloma cells is poorly defined. This study investigated the mechanisms regulating CXCR4 expression in myeloma cells. RPMI8226 and U266 myeloma cells strongly expressed CXCR4 on the cell surface, whereas ARH77 myeloma cells expressed minimal CXCR4 on the cell surface, as determined by flow cytometry using three different monoclonal antibodies to CXCR4. However, Western blot analysis, flow cytometry after permeabilization, and immunofluorescence staining reveled that ARH77 cells have abundant CXCR4 in the cytoplasm, in amounts similar to those in RPMI8226 and U266 cells. The cell surface expression of CXCR4 in primary CD138+ cells obtained from the BM of multiple myeloma patients differed among different specimens. Similar to myeloma cell lines, the primary myeloma cells that expressed minimal CXCR4 on the cell surface had abundant CXCR4 in the cytoplasm. The transmigration of the cells induced by stromal cell-derived factor-1 (SDF-1) was correlated with the cell surface expression of CXCR4, indicating that only the CXCR4 on the cell surface is functional. These findings suggest that myeloma cells have their own intrinsic mechanisms for regulating CXCR4 expression on the cell surface. In all three myeloma cell lines and in some primary BM CD138+ cells, dexamethasone (Dex) enhanced CXCR4 expression both in the cytoplasm and on the cell surface while downregulating SDF-1; this led to enhanced cell migration in response to SDF-1. Cell surface CXCR4 expression was more prominent in annexin V-positive apoptotic cells. VEGF and proinflammatory cytokines, including TNF-α and TGF-β1, also upregulated the cell surface expression of CXCR4 in RPMI8226 and some primary BM CD138+ cells, but not in U266 and ARH77 cells. Myeloma cells, including all the three cell lines and some primary BM CD138+ cells, incubated under hypoxic conditions (1% O2) exhibited upregulation of CXCR4 both in the cytoplasm and on the cell surface. Again, surface CXCR4 expression was stronger in apoptotic cells than in non-apoptotic cells, suggesting that the upregulation of CXCR4 is a counter-regulatory phenomenon for stimuli causing cell damage. As proven previously for other cell types, hypoxia induced the accumulation of HIF-1α in myeloma cells. Topotecan, which inhibits HIF-1α, attenuated the hypoxia-induced upregulation of CXCR4, whereas the proteasome inhibitor bortezomib slightly enhanced it. Dex, VEGF, TNF-α, and TGF-β1 all induced the accumulation of HIF-1α in RPMI8226 and some primary BM CD138+ cells. In addition, the effects of topotecan and bortezomib under hypoxic conditions were observed in the change of CXCR4 expression mediated by Dex and cytokines. These results indicate that complex intrinsic and extrinsic mechanisms regulate CXCR4 expression in myeloma cells and suggest that HIF-1α is a common regulatory molecule, at least in the extrinsic mechanisms.

CD9 expression enhances the susceptibility of myeloma cell lines to cell-mediated cytolysis

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 224-233
Author(s):  
Suhair Shallal ◽  
Jacki Kornbluth
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cell Surface Expression ◽  
Pokeweed Mitogen ◽  
Myeloma Cells ◽  
Enhanced Sensitivity

Myeloma tumor cells, both freshly excised and cultured, are extremely resistant to cell-mediated cytolysis. As evidence suggests that B-cell susceptibility to lysis is dependent upon its state of differentiation and activation, we tested the ability of a variety of B-cell proliferation and differentiation agents, including pokeweed mitogen (PWM), to enhance the sensitivity of myeloma cells to cell-mediated lysis. PWM was found to significantly enhance the susceptibility of myeloma cell lines and freshly isolated myeloma cells to interleukin-2 (IL-2)–activated cell-mediated cytolysis. This effect was seen with the use of both IL-2–stimulated natural killer (NK) cells and T cells as effectors. The enhanced sensitivity of myeloma cells to cytolysis correlated with an increase in their cell surface expression of CD9, a pre-B cell marker and member of the transmembrane 4 superfamily. Incubation of PWM-stimulated myeloma cells with either monoclonal antibodies or antisense oligonucleotides directed against CD9 abrogated the effect of PWM. In order to determine whether there was a direct relationship between the expression of CD9 and enhanced sensitivity to cytolysis, myeloma cell lines that lacked CD9 expression were transfected with the CD9 gene. The level of cell surface CD9 expression correlates with enhanced susceptibility to lysis. Therefore, CD9 appears to be an important component in enhancing the sensitivity of myeloma cells to lysis mediated by IL-2–activated T cells and NK cells.

CD9 expression enhances the susceptibility of myeloma cell lines to cell-mediated cytolysis

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 224-233 ◽  
Author(s):  
Suhair Shallal ◽  
Jacki Kornbluth
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cell Surface Expression ◽  
Pokeweed Mitogen ◽  
Myeloma Cells ◽  
Enhanced Sensitivity

Abstract Myeloma tumor cells, both freshly excised and cultured, are extremely resistant to cell-mediated cytolysis. As evidence suggests that B-cell susceptibility to lysis is dependent upon its state of differentiation and activation, we tested the ability of a variety of B-cell proliferation and differentiation agents, including pokeweed mitogen (PWM), to enhance the sensitivity of myeloma cells to cell-mediated lysis. PWM was found to significantly enhance the susceptibility of myeloma cell lines and freshly isolated myeloma cells to interleukin-2 (IL-2)–activated cell-mediated cytolysis. This effect was seen with the use of both IL-2–stimulated natural killer (NK) cells and T cells as effectors. The enhanced sensitivity of myeloma cells to cytolysis correlated with an increase in their cell surface expression of CD9, a pre-B cell marker and member of the transmembrane 4 superfamily. Incubation of PWM-stimulated myeloma cells with either monoclonal antibodies or antisense oligonucleotides directed against CD9 abrogated the effect of PWM. In order to determine whether there was a direct relationship between the expression of CD9 and enhanced sensitivity to cytolysis, myeloma cell lines that lacked CD9 expression were transfected with the CD9 gene. The level of cell surface CD9 expression correlates with enhanced susceptibility to lysis. Therefore, CD9 appears to be an important component in enhancing the sensitivity of myeloma cells to lysis mediated by IL-2–activated T cells and NK cells.

Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infection

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3449-3456 ◽  
Author(s):  
Yasuhiko Munakata ◽  
Takako Saito-Ito ◽  
Keiko Kumura-Ishii ◽  
Jie Huang ◽  
Takao Kodera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Parvovirus B19 ◽  
Erythroid Cells ◽  
Interfering Rna

AbstractHuman parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.

Identification of a Novel Auto-Antibody Highly Prevalent in Patients with Hepatitis-Associated and Idiopathic Aplastic Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3200-3200
Author(s):  
Hiroyuki Takamatsu ◽  
Zhirong Qi ◽  
Tomoyuki Sakurai ◽  
Luis Espinoza ◽  
Naomi Sugimori ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Peptide Mass Fingerprinting ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Healthy Individuals ◽  
Peptide Mass ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract Abstract 3200 Poster Board III-137 Hepatitis-associated aplastic anemia (HAA) is a subset of acquired AA that is highly responsive to immunosuppressive therapy. The target antigens of the immune system attack in HAA are thought to be a protein shared by both liver and hematopoietic stem cells, since it is usually associated with severe hepatitis of unknown etiology. Screening sera from patients with HAA for the presence of antibodies (Abs) recognizing liver cell-derived proteins may be useful in identifying novel auto-antigens in AA. To test this hypothesis, sera from HAA patients were examined using immunoblotting with a lysate of a hepatocellular carcinoma cell line Huh7 and subsequent peptide mass fingerprinting. Methods and Results The serum of a patient with typical HAA (a 23 year-old male) possessing a small population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells was used for Western blotting (WB) with the lysates of Huh7. A distinct band of 70 kDa protein was revealed. The same band was revealed when the culture supernatant of Huh7 cells was subjected to WB. The peptide mass fingerprinting of the 70 kDa band identified this protein to be heat shock protein (HSP) 72. HSP72 is a stress-inducible protein and extracellular HSP72 enhances the cytotoxicity of CD4+ T cells and NK cells. An examination of the sera from HAA patients, idiopathic acquired AA (IAA) patients and healthy individuals with WB revealed the anti-HSP72 Abs to be detected in 10 of 12 (83%) HAA patients and in 57 of 80 (71%) IAA patients while it was detected only in 8 of 59 (14%) healthy individuals. The prevalence of anti-HSP72 Abs in AA was markedly higher than that of anti-kinectin Abs (39%), anti-PMS1 Abs (10%), anti-DRS-1 Abs (38%) or anti-moesin Abs (37%) reported previously. Anti-HSP72 Abs were frequently detectable both in patients with IAA possessing PNH-type cells (63%) and in patients without PNH-type cells (86%), a finding contrasting to the higher prevalence of anti-DRS-1 Abs and anti-moesin Abs in patients with PNH-type cells than in those without PNH-type cells reported previously. Although anti-HSP72 Abs were detectable in the sera of patients with rheumatoid arthritis and systemic lupus erythematosus, the prevalence was 15% (4 of 27) and 20% (1 of 5), respectively. In contrast to a previous report that detected anti-HSP72 Abs in 24% of patients with chronic hepatitis C, WB failed to detect the Abs in the sera of 4 patients with autoimmune hepatitis and 5 with hepatitis B or C. Ten patients with HAA were treated with immunosuppressive therapy, and 7 of the 8 responders expressed anti-HSP72 Abs. The quantification of the gene expression level of HSP72 by blood cells using real-time PCR demonstrated that the HSP72 mRNA levels were markedly higher in myeloid leukemia cell lines as well as CD34+ cells isolated from 3 healthy individuals in comparison to that in lymphoid or monocytoid leukemia cell lines. HSP72/GAPDH ratios of PBMCs and CD34+ cells from 3 healthy individuals, K562, KH88, OUN-1 were 0.51, 1.31, 1.02, 0.07 and 0.09 respectively. Other leukemia cell lines such as Daudi, Molt-4 and THP-1 did not display detectable levels of HSP72 mRNA. The cell surface expression of HSP72 was examined in various kinds of leukemia cell lines and CD34+ bone marrow (BM) cells derived from 3 healthy individuals using Ab to HSP72 (Clone C92F3A-5) because previous studies demonstrated heat-inducible expression of HSP72 by K562. Flow cytometry detected cell surface HSP72 on immature CML cell lines such as K562 but not on CD34+ BM cells, acute promyelocytic leukemia cell lines such as NB-4 and HL-60, and lymphoid leukemia cell lines such as Molt-4 and Daudi. Exposure to 42°C for 2 h increased the HSP72 expression on K562 cells and Molt-4 cells but not on CD34+ cells. Conclusion Anti-HSP72 Ab is the most prevalent auto-Ab in AA among the auto-Abs previously detected. Given the increased expression of HSP72 by immature myeloid cells as well as stress-inducible cell surface expression of the molecule, immune responses to HSP72 may thus play an essential role in the pathogenesis of HAA and IAA. Disclosures No relevant conflicts of interest to declare.

CD28 and CD86 Are Necessary for Myeloma Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2946-2946
Author(s):  
Catherine M Gavile ◽  
Jayakumar R Nair ◽  
Kelvin P Lee ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival

Abstract Abstract 2946 Multiple myeloma (MM) is a hematologic malignancy characterized by the aberrant proliferation of plasma cells. Myeloma cells retain most of the physiological characteristics of their normal counterpart – the long-lived plasma cell. Myeloma cells secrete immunoglobulin and reside in the bone marrow, where they rely heavily on interactions with the stroma for survival signals. While recent advances in therapeutics have led to an increase in median survival post-diagnosis, the disease remains incurable. Understanding the pathways which mediate growth and survival of these cells will help in identifying new targets that can potentially further improve patient outcomes. CD28 is a receptor better known for its role in T-cell signaling through interaction with its ligands, CD80 or CD86. Interaction between CD28 on T-cells and CD80/86 on antigen-presenting cells leads to survival and proliferation of T-cells. Recent work has shown that the CD80/86-CD28 pathway also plays an important role in normal plasma cell generation and survival. Interestingly, high expression of CD28 and CD86 are poor prognostic markers for myeloma patients. Previous work has shown that CD28 activation provides survival signals for myeloma cells in growth-factor deficient conditions. It has also been shown that CD28 on the myeloma cell interacts with CD80/86 on the dendritic cell, which induces secretion of IL-6 (by the DC), an important myeloma growth factor. However, it is not known if CD28 or CD86 play a role in steady state growth and survival of myeloma cells. In order to determine the role of each of these 2 molecules in myeloma physiology, we knocked-down either CD28 or CD86 on the myeloma cell via lentivirus-mediated shRNAs. We found that knockdown of CD86 leads to apoptosis in 3 myeloma cell lines (RPMI8226, MM1.s, and KMS18). Four days after infection with the lentivirus containing shCD86, 45.7±4.9 and 60.3±4.6 percent control apoptosis was observed in RPMI8226 and MM1.s respectively, while less death was observed in KMS18 (17.6±1.6). CD28-knockdown resulted in apoptosis as well (24.9±4.3 for RPMI8226, 26.8±4.1 for MM1s, 21.8±3.8 for KMS18, percent control apoptosis). Consistent with these findings, we were unable to establish a myeloma cell line with stable knockdown of either CD28 or CD86. Additionally, RPMI8226 cells stably transfected to over-express either Bcl-2, Bcl-xL, or Mcl-1 are protected from cell death induced by CD86 or CD28 silencing. These data suggest that CD28 and CD86 are essential to prevent apoptosis of myeloma cells in vitro. To confirm these findings we determined the effects of CTLA4-Ig on myeloma survival. CTLA4-Ig inhibits CD86-CD28 signaling by binding to CD86, blocking its interaction with CD28. We found that treatment of RPMI8226 and MM1.s cells with CTLA4-Ig caused apoptosis in the myeloma cells after 2 days (23.9±3.9 for RPMI8226 and 20.4±6.2 for MM1.s, percent control apoptosis). Thus like normal plasma cells, CD28 and CD86 are required for the survival of myeloma cells. To determine why silencing of CD86 has a more potent effect than CD28 silencing on myeloma cell survival in 2 out of 3 cell lines, we investigated the effects of silencing on cell surface expression of each of these proteins. CD28 and CD86 mRNA and protein levels were silenced to similar levels by their cognate hairpins. However, in MM.1s and RPMI8226 we found that silencing of CD28 resulted in an increase in CD86 surface expression. This increase was also observed at the mRNA level and in the cells over-expressing Bcl-2 family members, indicating that this is not simply due to the selection of the highest expressing cells. These data suggest a feedback loop exists to regulate CD28-CD86 signaling in myeloma cells. Surprisingly, in the KMS18 cell line, we observe the converse effect, where silencing of CD86 resulted in upregulation of CD28. This provides a likely explanation for why these cells are less susceptible to CD86 silencing than the other two lines. Interestingly, blocking CD86 with CTLA4-Ig treatment also resulted in a modest upregulation in CD28 surface expression of MM.1s and RPMI8226, which suggests that silencing CD86 and binding of CD86 with a soluble receptor are not equivalent, and that multiple signaling feedback pathways exist to regulate the expression of this receptor-ligand pair that is necessary for myeloma cell survival. Disclosures: No relevant conflicts of interest to declare.

Syndecan-1 is targeted to the uropods of polarized myeloma cells where it promotes adhesion and sequesters heparin-binding proteins

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2528-2536 ◽  
Author(s):  
Magne Børset ◽  
Øyvind Hjertner ◽  
Shmuel Yaccoby ◽  
Joshua Epstein ◽  
Ralph D. Sanderson
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Binding Proteins ◽  
Myeloma Cell ◽  
Heparin Binding ◽  
Myeloma Cells ◽  
Polarized Cells ◽  
Entire Cell ◽  
Hepatocyte Growth ◽  
Cell Cell

Syndecan-1 (CD138) is a heparan sulfate-bearing proteoglycan present on the surface of myeloma cells where it mediates myeloma cell-cell and cell-extracellular matrix adhesion. In this study, we examined myeloma cell lines for cell membrane localization of syndecan-1. On some cells we note a striking localization of syndecan-1 to a single small membrane protrusion, with the remainder of the cell surface being mostly negative for syndecan-1. Examination of cell morphology reveals that a proportion of cells from myeloma cell lines, as well as primary myeloma cells, are polarized, with a uropod on one end and lamellipodia on the other end. On these polarized cells, syndecan-1 is specifically targeted to the uropod, but in contrast, on nonpolarized cells syndecan-1 is evenly distributed over the entire cell surface. In addition to syndecan-1, several other cell surface molecules localize specifically to the uropod, including CD44 and CD54. Functional assays reveal that myeloma cell lines with a high proportion of polarized cells have a much higher migratory potential than cell lines with few polarized cells. Moreover, the uropod is the cell pole preferentially involved in aggregation of myeloma cells and in adhesion of myeloma cells to osteoblast-like cells. When polarized myeloma cells are incubated with heparin-binding proteins, like hepatocyte growth factor or osteoprotegerin, they concentrate in the uropod. These data indicate that syndecan-1 is targeted to the uropod of polarized myeloma cells and that this targeting plays a role in promoting cell-cell adhesion and may also regulate the biological activity of heparin-binding cytokines.

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