Induction of CD45 Expression and Proliferation in U-266 Myeloma Cell Line by Interleukin-6

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3887-3897 ◽  
Author(s):  
Maged S. Mahmoud ◽  
Hideaki Ishikawa ◽  
Ryuichi Fujii ◽  
Michio M. Kawano
Keyword(s):  
Cell Line ◽  
Expression Pattern ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Cellular Proliferation ◽  
Tyrosine Phosphatase ◽  
Biological Significance ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Gradual Loss

Recently, there has been an increasing interest in the expression pattern and biological significance of the CD45 molecule in myeloma cells. In this study, we have further defined the phenotypic pattern of CD45 expression on myeloma cells. Using a panel of myeloma cell lines, we showed that CD45 showed a remarkably heterogeneous pattern of expression. Whereas some cell lines were CD45+ and others were CD45−, the U-266 cell line, although predominantly CD45−, still had a considerable subpopulation of CD45+ cells. Among the myeloma cell lines examined, there was a direct correlation between interleukin-6 (IL-6) dependency and CD45 positivity. Moreover, we showed that IL-6 stimulation led to the induction of expression of CD45 and cellular proliferation. Using independent experimental approaches, we could show that the IL-6–induced effects were closely linked to CD45 expression. First, sorting out CD45+ and CD45− subsets of U-266 cell line followed by IL-6 stimulation, only the CD45+ cells showed a proliferative advantage after IL-6 stimulation. Second, IL-6 stimulation of sorted CD45−cells was gradually followed by phenotypic conversion to CD45+ cells that started after 2 days as judged by the detection of CD45 mRNA by reverse transcription polymerase chain reaction (RT-PCR) and immunophenotypic analysis by flow cytometry. Withdrawal of IL-6 from the medium led to gradual loss of CD45 expression in CD45+ flow-sorted U-266 cells. Third, the use of vanadate, a potent inhibitor of protein tyrosine phosphatase (PTP), abrogated the IL-6–induced proliferation in the CD45+ myeloma cells. On the other hand, cellular proliferation induced by IL-6 was not affected by the serine-threonine phosphatase inhibitor okadaic acid. Our data show that the expression pattern of CD45 in myeloma cell lines is heterogeneous and show for the first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in myeloma by determining the proliferative population among myeloma cells.

Induction of CD45 Expression and Proliferation in U-266 Myeloma Cell Line by Interleukin-6

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3887-3897 ◽  
Author(s):  
Maged S. Mahmoud ◽  
Hideaki Ishikawa ◽  
Ryuichi Fujii ◽  
Michio M. Kawano
Keyword(s):  
Cell Line ◽  
Expression Pattern ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Cellular Proliferation ◽  
Tyrosine Phosphatase ◽  
Biological Significance ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Gradual Loss

Abstract Recently, there has been an increasing interest in the expression pattern and biological significance of the CD45 molecule in myeloma cells. In this study, we have further defined the phenotypic pattern of CD45 expression on myeloma cells. Using a panel of myeloma cell lines, we showed that CD45 showed a remarkably heterogeneous pattern of expression. Whereas some cell lines were CD45+ and others were CD45−, the U-266 cell line, although predominantly CD45−, still had a considerable subpopulation of CD45+ cells. Among the myeloma cell lines examined, there was a direct correlation between interleukin-6 (IL-6) dependency and CD45 positivity. Moreover, we showed that IL-6 stimulation led to the induction of expression of CD45 and cellular proliferation. Using independent experimental approaches, we could show that the IL-6–induced effects were closely linked to CD45 expression. First, sorting out CD45+ and CD45− subsets of U-266 cell line followed by IL-6 stimulation, only the CD45+ cells showed a proliferative advantage after IL-6 stimulation. Second, IL-6 stimulation of sorted CD45−cells was gradually followed by phenotypic conversion to CD45+ cells that started after 2 days as judged by the detection of CD45 mRNA by reverse transcription polymerase chain reaction (RT-PCR) and immunophenotypic analysis by flow cytometry. Withdrawal of IL-6 from the medium led to gradual loss of CD45 expression in CD45+ flow-sorted U-266 cells. Third, the use of vanadate, a potent inhibitor of protein tyrosine phosphatase (PTP), abrogated the IL-6–induced proliferation in the CD45+ myeloma cells. On the other hand, cellular proliferation induced by IL-6 was not affected by the serine-threonine phosphatase inhibitor okadaic acid. Our data show that the expression pattern of CD45 in myeloma cell lines is heterogeneous and show for the first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in myeloma by determining the proliferative population among myeloma cells.

scholarly journals Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 517-526 ◽  
Author(s):  
B Klein ◽  
XG Zhang ◽  
M Jourdan ◽  
J Content ◽  
F Houssiau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Neutralizing Antibodies ◽  
Myeloma Cell ◽  
B Cell Differentiation ◽  
Myeloma Cells ◽  
Autocrine Regulation ◽  
Spontaneous Proliferation ◽  
Human Myeloma Cell

Abstract To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.

Biological and Molecular Characterization by Integrative Genomics Approach of CMA-03/06, a Newly Established Interleukin-6 Independent Variant of the CMA-03 Human Myeloma Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1814-1814
Author(s):  
Donata Verdelli ◽  
Lucia Nobili ◽  
Katia Todoerti ◽  
Laura Mosca ◽  
Sonia Fabris ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Molecular Characterization ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Growth And Survival ◽  
Genome Wide

Abstract Abstract 1814 Poster Board I-840 Background The growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. Aims. To perform a biological and molecular characterization of this novel cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. Methods. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip® Human Mapping 250K Nsp arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the SAM software version 3.0. Results Unlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The immunophenotypic analysis showed a significant difference in the expression of three antigens in the 2 cell lines: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 1 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. Conclusions Our data show the IL-6 independence of CMA-03/06 cell line in the absence of an autocrine IL-6 loop; the cells, however, maintain the IL-6 signaling pathway responsiveness. A consistent number of genes particularly relevant for MM biology were found deregulated in CMA-03/06 cell line compared with CMA-03. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. Disclosures No relevant conflicts of interest to declare.

MYC Responsible for down-Regulation of CD33 Expression in Human Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5116-5116
Author(s):  
Karim Shamsasenjan ◽  
Ken-ichiro Otsuyama ◽  
Mohd S. Iqbal ◽  
Maged S. Mahmoud ◽  
Michio M. Kawano
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Stat3 Phosphorylation ◽  
Serum Crp ◽  
Human Myeloma Cells

Abstract Human myeloma cells from about 10% of cases with multiple myeloma expressed CD33 and have monocytoid morphology with convoluted nuclei, and all these patients had no increase in serum CRP values. In CD33(+) myeloma cells as well as myeloma cell lines, CD33 expression levels were correlated with the increased expression levels of CEBPA (C/EBPα) gene. This correlation was confirmed by the finding that transfection with the CEBPA gene induced CD33 expression in a CD33(−) myeloma cell line. As suggested by the lack of an increase in serum CRP values in CD33(+) myelomas, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines along with the down-regulation of CEBPA gene expression. Cucurbitacin I (STAT3 inhibitor) but not U0126 (MAPK inhibitor) could abolish the effect of IL-6. Furthermore, IL-6 up-regulated the expression of MYC via STAT3 phosphorylation and MYC bound to the promoter region of CEBPA gene followed by the down-regulation of the CEBPA expression. It was confirmed that introduction of shRNA for MYC into a CD33(+) myeloma cell line blocked the IL6-induced down-regulation of CD33 and CEBPA expression. Therefore, these results indicate that IL-6 can reverse the expression level of CD33 by up-regulating MYC followed by the down-regulation of CEBPA expression.

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.

scholarly journals Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3712-3720 ◽  
Author(s):  
H Uchiyama ◽  
BA Barut ◽  
AF Mohrbacher ◽  
D Chauhan ◽  
KC Anderson
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Rgd Peptide ◽  
Beta 1 Integrin ◽  
Beta 2

Previous studies show that human myeloma-derived cell lines specifically adhere to fibronectin (FN) through very late antigen-4 (VLA-4; alpha 4 beta 1 integrin complex) and RGD-peptide mechanisms, which may contribute to the localization of tumor cells in bone marrow (BM). In these studies, we characterized the adhesion of myeloma- derived cell lines to both normal and myeloma BM stromal cells (BMSCs) and the effect of adhesion on DNA synthesis. Because interleukin-6 (IL- 6) plays an important role in the pathogenesis of multiple myeloma, we also examined the effects of tumor cell adhesion on IL-6 secretion by BMSCs. In 51chromium binding assays, the U266, ARH-77, and IM-9 cell lines showed 52% +/- 12%, 55% +/- 6%, and 47% +/- 7% specific adherence, respectively, to normal BMSCs and 74% +/- 4%, 60% +/- 3%, and 61% +/- 6% specific adherence, respectively, to myeloma BMSCs. In contrast, only 12% to 13% specific binding of HS-Sultan cells to BMSCs was noted. The binding of myeloma cells to BMSCs was partially blocked with anti-beta 1 monoclonal antibody (MoAb), anti-beta 2 integrin MoAb, and excess RGD peptide, suggesting multiple mechanisms for the adhesion of myeloma cell lines to BMSCs. Binding of cell lines to FN or myeloma BMSCs did not affect cell line proliferation; however, adhesion of myeloma cell lines to normal BMSCs decreased DNA synthesis, ie, stimulation indices are 0.1 +/- 0.04, 0.2 +/- 0.1, 0.2 +/- 0.07, and 0.1 +/- 0.06 for the adherent non-IL-6-dependent U266, ARH-77, HS- Sultan, and IM-9 cells, respectively (n = 5, P < .01). In contrast, adherence of IL-6-dependent B9 cells increased their proliferation (stimulation index, 3.2 +/- 0.7). Significant (twofold to eightfold) increases in IL-6 secretion were evident in cell line-adherent (> or = 12 hours) normal and myeloma BMSC cultures. Paraformaldehyde fixation of BMSCs before adhesion completely abrogated IL-6 secretion, suggesting that IL-6 secretion was triggered in BMSCs rather than in cell lines. Partial blocking of cell line adhesion to BMSCs, using anti- beta 1 integrin and anti-beta 2 integrin MoAbs and RGD peptide, also partially blocked the triggering of IL-6 secretion by BMSCs. When cell lines were placed in Transwell inserts and then cultured with either normal or myeloma BMSCs, permitting juxtaposition without cell to cell contact between myeloma cell lines and BMSCs, no increase in IL-6 secretion was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Sphingolipids as a novel target for the treatment of multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19534-e19534
Author(s):  
Yubin Kang ◽  
Jagadish Kummetha Venketa
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

HLA-Cw6 Alloreactive CTL Recognizing HLA-CW6+ Myeloma Cells but Not Non-Myeloma HLA-Cw6+ Cells Are Peptide Dependent.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2474-2474
Author(s):  
Alexandrine Geffroy ◽  
Agnes Moreau-Aubry ◽  
Regis Bataille ◽  
Catherine Pellat-Deceunynck
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Unrelated Donor ◽  
Myeloma Cell Line ◽  
Specific Lysis ◽  
Myeloma Cells ◽  
Target Ratio ◽  
Effector Target

Abstract We investigated the generation of myeloma-specific cytotoxic T lymphocytes (CTL) from unrelated normal donors non-HLA matched with the myeloma cell line SBN. The aim was to obtain alloreactive CTL specific of peptides to identify genes preferentially expressed by to myeloma cells. After coculture of SBN with PBL of unrelated donor, the T-cell line obtained was cloned and each CTL was assessed against SBN and SB-EBV (B-EBV cell line obtained by infecting B cells of SB patient with EBV) simultaneously. Only the CTL recognizing SBN but not SB-EBV were kept for further study. Among more than 200 clones screened, we isolated two different CTL CD8+ recognizing SBN only (60% of specific lysis at effector: target ratio 5). Their cytotoxicity was blocked by antibodies against HLA-I molecules and more precisely by mAb against HLA-B/Cw molecules. Both CTL recognized also other MM cell lines but only those matched with SBN for HLA-Cw0602 (XG6, BCN) suggesting an HLA-Cw6 recognition. Furthermore, antiserum directed against HLA-Cw6 abrogated the recognition of SBN, XG6 and BCN. Both CTL were not cytotoxic against BC-EBV nor against 3 other B-EBV cell lines derived from HLA-Cw0602 donors. This unreactivity was not restricted to B-EBV cell lines since PBL from 2 normal HLA-Cw0602 donors were also not recognized (cytotoxicity, TNF secretion). We directly measured cell surface HLA-Cw6 expression of both B-EBV and myeloma cells with a scFv directed against HLA-Cw6 and we showed that HLA-Cw6 was more expressed by B-EBV cells as compared to myeloma cells (2fold). So, the lack of recognition of B-EBV cells was not related to HLA-Cw6 expression level. We also looked for KIR expression by the CTL since KIR molecules could have blocked B-EBV recognition that expressed more HLA-Cw6 molecules as compared to myeloma cells. No KIR molecules (p58.1, p58.2, CD94) were found to be expressed by the CTL. To investigate whether the recognition of myeloma cells was peptide dependent, we performed two experiments i.e., acid elution and cold target competition. Acid elution (glycine 0.3M, pH=2.5) abrogated myeloma recognition and cold target competition showed that B-EBV cells were not recognized by the CTL. These data suggest that the CTL recognize a peptide or a set of peptides restricted to myeloma cells or that the CTL recognition is activated by tumor cells only. Anyway, such alloreactive CTL could be helpful for GVL reaction in allotransplanted patients with myeloma.

Bortezomib Induces Rapid Upregulation of Topoisomerase IIα and Demonstrates Schedule-Dependent Synergistic Cytotoxicity with Anthracyclines and Etoposide in Acute Myeloid Leukemia and Myeloma Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2478-2478
Author(s):  
Michael P. Carroll ◽  
Olin Feuerbacher ◽  
Andrew M. Yeager ◽  
Terry H. Landowski
Keyword(s):  
Enzymatic Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Antitumor Agents ◽  
Myeloma Cell ◽  
Targeted Agents ◽  
Topoisomerase Iiα ◽  
Myeloma Cells ◽  
Synergistic Cytotoxicity ◽  
Proteasomal Inhibition

Abstract The proteasome inhibitor bortezomib (BZ) has significant antineoplastic activity in multiple myeloma, lymphoma and acute leukemia. Recently, it has been shown that BZ can potentiate the activity of other antitumor agents in vitro. Topoisomerase IIα (topo IIα) is known to be regulated by proteasomal degradation, suggesting that proteasomal inhibition might increase intracellular topo IIα levels and thereby enhance sensitivity to topo IIα-targeted agents, including etoposide, daunorubicin and mitoxantrone. Incubation with sublethal concentrations of BZ led to rapid upregulation of topo IIα protein levels in the human TF-1 AML cell line, the human 8226 myeloma cell line and the 8226-derived, topo IIα-deficient, anthracycline-resistant Dox1V line (kindly provided by William S. Dalton), as measured by Western blot assay. Enhanced topo IIα expression was evident at 3 hrs, peaked at 6 hrs and fell off markedly 24 hrs after BZ addition. Maximal upregulation was 25–90 fold as measured by optical densitometry. BZ induced a shift in the apparent molecular weight of topo IIα, suggesting that upregulation resulted, at least in part, from the stabilization of the ubiquitinated form(s) of the enzyme following proteasomal inhibition. The kinetics of topo IIα upregulation inversely correlated with measured 20S proteasomal activity, further supporting a post-translational mechanism. Nuclear extracts prepared from BZ-treated cells showed a corresponding 36-fold increase in topo IIα enzymatic activity. The cytotoxicity of BZ in combination with other antineoplastic agents was evaluated by MTT assay. Synergistic cytotoxicity was observed for BZ plus mitoxantrone, etoposide or daunorubicin, but no significant augmentation was observed in this system for BZ plus cytarabine, melphalan, camptothecin, dexamethasone, vincristine or fludarabine. In TF-1 AML cells, sublethal doses of BZ reduced the IC50 for daunorubicin, mitoxantrone and etoposide 16-, 52- and 22-fold, respectively. In 8226 myeloma cells, BZ enhanced sensitivity to daunorubicin and etoposide 11- and 4-fold, respectively. Importantly, BZ treatment overcame chemoresistance in topo IIα-deficient Dox1V myeloma cells, enhancing the sensitivity to daunorubicin and etoposide 110- and 7- fold, respectively. The observed synergy was sequence-dependent and correlated with BZ-induced upregulation of topo IIα. Maximal synergy occurred within 3–6 hrs of BZ pretreatment and diminished after 24 hrs. Minimal synergy occurred when topo IIα-targeted agents preceded BZ by ≥ 6 hrs. These findings demonstrate that BZ has synergistic cytotoxic effects with topo IIα-targeted agents in AML and myeloma cell lines, which correlate with BZ-induced upregulation of topo IIα protein and nuclear enzymatic activity. Furthermore, these data provide a rationale for timed-sequential cytotoxic therapy in patients with hematologic malignancies.

scholarly journals Myeloma cells upregulate interleukin-6 secretion in osteoblastic cells through cell-to-cell contact but downregulate osteocalcin

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3151-3159 ◽  
Author(s):  
S Barille ◽  
M Collette ◽  
R Bataille ◽  
M Amiot
Keyword(s):  
Cell Lines ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Osteoblastic Cells ◽  
Osteoblastic Cell ◽  
Cell Contact ◽  
Osteosarcoma Cell ◽  
Bone Microenvironment ◽  
Myeloma Cells ◽  
Coculture System

Previous studies have shown that bone marrow, especially the bone microenvironment, may play an important role in the pathogenesis of multiple myeloma (MM). To elucidate the relationship between myeloma cells and bone cells, mainly osteoblasts, we have established a coculture system between two interleukin-6 (IL-6)-dependent myeloma cell lines, XG1 and XG6, and the osteosarcoma cell lines Saos-2 and MG63. Both osteosarcoma cell lines have retained major functions of normal osteoblasts; principally, the capacity to produce hematopoietic growth factors (including IL-6) and osteocalcin, a noncollagenic protein essential in the bone formation process. Because IL-6 is a critical growth factor in MM, we have examined the IL-6 osteoblastic cell production in our coculture system. XG1 cells strongly upregulate IL-6 production by MG63 and Saos-2 cells. Of major interest, the triggering of IL-6 is totally dependent on the physical contact between myeloma cells and osteoblastic cells, contact that is partly mediated by CD44, CD56, and fibronectin interactions. Osteocalcin production by MG63 and Saos-2 cells has previously been shown to be dependent on 1,25-(OH)2D3. We demonstrate that XG1 and XG6 cells reduced the amount of osteocalcin in MG63 coculture cell supernatants, a reduction that is partly mediated by a soluble factor and by cell-to-cell contact. Notably, whereas one of the myeloma cell lines, XG6, has lost its capacity to stimulate IL-6 production by osteoblastic cell lines, both XG1 and XG6 cell lines remain able to reduce the osteocalcin amount, indicating that IL-6 and osteocalcin levels are regulated by two different pathways. In conclusion, these data strongly support the concept that the bone microenvironment is directly modified by contact with myeloma cells and are consistent with the characteristics observed in vivo in patients with MM patients, ie, abnormally high IL-6 and low osteocalcin levels, respectively.

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