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Simon J. Gaskell - One of the best experts on this subject based on the ideXlab platform.
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Proteomic analysis of the adaptation of the host NS0 Myeloma Cell line to a protein-free medium
2020Co-Authors: Kathya R De La Luz-hernández, Luis Rojas-del Calvo, Svieta Victores-sarasola, Agustín Lage-castellanos, Sarah R. Hart, Lila Castellanos-serra, Adolfo Castillo-vitlloch, Simon J. Gaskell, Claire E. Eyers, Michael BarberAbstract:The NS0 Myeloma Cell line is often used for the production of monoclonal antibodies (Mabs) and other recombinant proteins. The growth of these mammalian Cells in a protein-free media has several advantages including cost, safety, consistency, efficiency and regulatory approval. However, the adaptation of the NS0 Myeloma Cell line so as to grow in a protein-free medium is poorly understood. In order to better understand this process, we applied proteomic techniques, specifically two-dimensional electrophoresis (2DE) and mass spectrometry (MS), to identify the key pathways involved in this adaptation. The analysis of changes in protein expression between the host Myeloma Cell line and two recombinant NS0 Cell lines expressing different humanized Mabs has primarily revealed changes in proteins associated with carbohydrate metabolism, energy production, protein synthesis and folding, membrane transport and Cell proliferation. Other factors that may be involved in the adaptation of NS0 Myeloma Cells to a protein-free medium are reported.
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Using the “OMICS” Technologies as Complementary Tools to Study the Molecular Mechanisms Involved with the Adaptation of Myeloma Cell Line to Protein-Free Medium
Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT) Dublin Ireland June 7-10 2009, 2011Co-Authors: Kathya R De La Luz-hernández, Agustín Lage-castellanos, Lila Castellanos-serra, Adolfo Castillo-vitlloch, Y. Rabasa-legón, J. Díaz-brito, Simon J. GaskellAbstract:Production of recombinant therapeutic proteins, especially monoclonal antibodies (Mab), in Myeloma Cell lines represents a significant segment of the pharmaceutical market, and therefore striving for increased productivity of these lines represents a major investment of resources. The elucidation of biologically important markers for the adaptation of NS0 Myeloma Cell line to protein-free medium and the recombinant protein production are a major emphasis of our research. These markers could potentially be used in a variety of ways to improve culture conditions, including active approaches to agonize/antagonize important pathways within a medium formulation or diagnostic approaches indicative of improved conditions during the culture. In this work, we used two-dimensional electrophoresis/mass spectrometry and the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 Myeloma Cell line. Several proteins with differential expression profile were characterized and quantified. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation by proteomic analysis. The same results were obtained using flux balance analysis in a murine metabolic network with selected medium conditions.
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metabolic and proteomic study of ns0 Myeloma Cell line following the adaptation to protein free medium
Journal of Proteomics, 2008Co-Authors: K R De La Luzhernandez, Rojasdel L Calvo, Y Rabasalegon, A Castillovitlloch, Agustin Lagecastellanos, J Diaz, Simon J. GaskellAbstract:Abstract Proteomics and metabolomics technologies are potentially useful tool for the study of the very complex process of Cell adaptation to protein-free medium. In this work, we used the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 Myeloma Cell line. Several proteins with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic.
K R De La Luzhernandez - One of the best experts on this subject based on the ideXlab platform.
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metabolic and proteomic study of ns0 Myeloma Cell line following the adaptation to protein free medium
Journal of Proteomics, 2008Co-Authors: K R De La Luzhernandez, Rojasdel L Calvo, Y Rabasalegon, A Castillovitlloch, Agustin Lagecastellanos, J Diaz, Simon J. GaskellAbstract:Abstract Proteomics and metabolomics technologies are potentially useful tool for the study of the very complex process of Cell adaptation to protein-free medium. In this work, we used the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 Myeloma Cell line. Several proteins with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic.
Chor Sang Chim - One of the best experts on this subject based on the ideXlab platform.
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Establishment of a bortezomib-resistant Chinese human multiple Myeloma Cell line: MMLAL
Cancer Cell International, 2013Co-Authors: Kwan Yeung Wong, Chi Chiu So, Chor Sang ChimAbstract:Background A new human Myeloma Cell line, MMLAL, was established from the Myelomatous pleural effusion of a 73-year-old Chinese patient suffering from symptomatic International stage III IgG/lambda Myeloma. After a brief period of complete remission, he developed aggressive systemic relapse complicated by malignant pleural effusion with exclusive plasma Cell infiltration. His disease remained chemo-refractory, and died six months after relapse.
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establishment of a bortezomib resistant chinese human multiple Myeloma Cell line mmlal
Cancer Cell International, 2013Co-Authors: Kwan Yeung Wong, Chi Chiu So, Chor Sang ChimAbstract:Background: A new human Myeloma Cell line, MMLAL, was established from the Myelomatous pleural effusion of a 73-year-old Chinese patient suffering from symptomatic International stage III IgG/lambda Myeloma. After a brief period of complete remission, he developed aggressive systemic relapse complicated by malignant pleural effusion with exclusive plasma Cell infiltration. His disease remained chemo-refractory, and died six months after relapse. Methods: Purified mononuclear Cells from the pleural effusion of the patient were cultured in the presence of IL-6. Continually growing Cells were characterized by morphological, immunophenotypic, cytogenetic, fluorescence in situ hybridization (FISH) and TP53 mutation analyses. Cell proliferation was measured and compared with other Myeloma Cell lines by Cell counting at day 3, 6, 9, and 12. Drug resistance against bortezomib, a proteasome inhibitor approved as a frontline chemotherapy for eligible Myeloma patients, was evaluated and compared with other Myeloma Cell lines by MTT assay. Results: Immunophenotypic analysis of the Myeloma Cells confirmed strong expression of plasma Cell markers CD38 and CD138 but not T-Cell or natural killer-Cell marker CD56. Cytogenetic analysis of the Myeloma Cells showed a hypodiploid composite karyotype including loss of chromosome 13 and 17 or deletion of the short arm of chromosome 17, i.e. del(17p), in the form of isochromosome 17q10. FISH confirmed a hypodiploid karyotype with TP53 deletion but absence of t(4;14). Sequencing analysis of the TP53 gene indicated absence of mutation. Cell counting revealed that the maximum viable Cell density was about 2.5 X 10 6 Cells/ml. Upon bortezomib treatment, MTT assay reported an IC50 of 72.17nM, suggesting a strong bortezomib resistance. Conclusion: A hypodiploid with loss of chromosome 13 and loss or del(17p) human Myeloma Cell line, MMLAL, was established from the pleural effusion of a Chinese Myeloma patient.
Lawrence H. Boise - One of the best experts on this subject based on the ideXlab platform.
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A Role for Syntenin-1 in Multiple Myeloma Cell Survival
Blood, 2018Co-Authors: Tyler Moser-katz, Catherine M. Gavile, Benjamin G. Barwick, Sagar Lonial, Lawrence H. BoiseAbstract:Multiple Myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma Cells that, despite recent therapeutic advances, remains incurable. Myeloma Cells retain numerous characteristics of normal plasma Cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma Cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of Myeloma Cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in Myeloma Cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the Cell. Syntenin-1 regulates the surface expression of CD138, a plasma/Myeloma Cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer Cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple Myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on Myeloma Cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of Myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in Myeloma Cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s Myeloma Cell lines. We then determined the amount of Cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased Cell death in syntenin-1-silenced Cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s Cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for Myeloma Cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of Myeloma Cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P Our data supports a novel role for syntenin-1 in Myeloma Cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple Myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial:Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.
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CD28 and CD86 Are Necessary for Myeloma Cell Survival.
Blood, 2012Co-Authors: Catherine M. Gavile, Sagar Lonial, Jayakumar R. Nair, Lawrence H. BoiseAbstract: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.
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darinaparsin induces a unique Cellular response and is active in an arsenic trioxide resistant Myeloma Cell line
Molecular Cancer Therapeutics, 2009Co-Authors: Shannon M Matulis, Alejo A Morales, Lucy Yehiayan, Claire R Croutch, Delia Gutman, Lawrence H. BoiseAbstract:Here, we report on the organic arsenical darinaparsin (ZIO-101, S -dimethylarsino-glutathione) and its anti-Myeloma activity compared with inorganic arsenic trioxide. Darinaparsin induced apoptosis in multiple Myeloma Cell lines in a dose-dependent manner, and the addition of N -acetylcysteine, which increases intraCellular glutathione (GSH), blocked cytotoxicity of both darinaparsin and arsenic trioxide. In contrast to arsenic trioxide, intraCellular GSH does not appear to be important for darinaparsin metabolism, as an inhibitor of GSH synthesis, buthionine sulfoximine, had little effect on drug activity. This discrepancy was resolved when we determined the effects of thiols on drug uptake. The addition of exogenous GSH, l-cysteine, or d-cysteine prevented darinaparsin Cellular uptake and Cell death but had no effect on the uptake or activity of arsenic trioxide, suggesting a difference in the transport mechanism of these two drugs. In addition, gene expression profiling revealed differences in the signaling of protective responses between darinaparsin and arsenic trioxide. Although both arsenicals induced a transient heat shock response, only arsenic trioxide treatment induced transcription of metal response genes and anti-oxidant genes related to the Nrf2-Keap1 pathway. In contrast to the protective responses, both arsenicals induced up-regulation of BH3-only proteins. Moreover, silencing of BH3-only proteins Noxa, Bim, and Bmf protected Myeloma Cells from darinaparsin-induced Cell death. Finally, treatment of an arsenic trioxide-resistant Myeloma Cell line with darinaparsin resulted in dose-dependent apoptosis, indicating that cross-resistance does not necessarily develop between these two forms of arsenic in multiple Myeloma Cell lines. These results suggest darinaparsin may be useful as an alternative treatment in arsenic trioxide-resistant hematologic cancers.[Mol Cancer Ther 2009;8(5):OF1–10]
Kwan Yeung Wong - One of the best experts on this subject based on the ideXlab platform.
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Establishment of a bortezomib-resistant Chinese human multiple Myeloma Cell line: MMLAL
Cancer Cell International, 2013Co-Authors: Kwan Yeung Wong, Chi Chiu So, Chor Sang ChimAbstract:Background A new human Myeloma Cell line, MMLAL, was established from the Myelomatous pleural effusion of a 73-year-old Chinese patient suffering from symptomatic International stage III IgG/lambda Myeloma. After a brief period of complete remission, he developed aggressive systemic relapse complicated by malignant pleural effusion with exclusive plasma Cell infiltration. His disease remained chemo-refractory, and died six months after relapse.
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establishment of a bortezomib resistant chinese human multiple Myeloma Cell line mmlal
Cancer Cell International, 2013Co-Authors: Kwan Yeung Wong, Chi Chiu So, Chor Sang ChimAbstract:Background: A new human Myeloma Cell line, MMLAL, was established from the Myelomatous pleural effusion of a 73-year-old Chinese patient suffering from symptomatic International stage III IgG/lambda Myeloma. After a brief period of complete remission, he developed aggressive systemic relapse complicated by malignant pleural effusion with exclusive plasma Cell infiltration. His disease remained chemo-refractory, and died six months after relapse. Methods: Purified mononuclear Cells from the pleural effusion of the patient were cultured in the presence of IL-6. Continually growing Cells were characterized by morphological, immunophenotypic, cytogenetic, fluorescence in situ hybridization (FISH) and TP53 mutation analyses. Cell proliferation was measured and compared with other Myeloma Cell lines by Cell counting at day 3, 6, 9, and 12. Drug resistance against bortezomib, a proteasome inhibitor approved as a frontline chemotherapy for eligible Myeloma patients, was evaluated and compared with other Myeloma Cell lines by MTT assay. Results: Immunophenotypic analysis of the Myeloma Cells confirmed strong expression of plasma Cell markers CD38 and CD138 but not T-Cell or natural killer-Cell marker CD56. Cytogenetic analysis of the Myeloma Cells showed a hypodiploid composite karyotype including loss of chromosome 13 and 17 or deletion of the short arm of chromosome 17, i.e. del(17p), in the form of isochromosome 17q10. FISH confirmed a hypodiploid karyotype with TP53 deletion but absence of t(4;14). Sequencing analysis of the TP53 gene indicated absence of mutation. Cell counting revealed that the maximum viable Cell density was about 2.5 X 10 6 Cells/ml. Upon bortezomib treatment, MTT assay reported an IC50 of 72.17nM, suggesting a strong bortezomib resistance. Conclusion: A hypodiploid with loss of chromosome 13 and loss or del(17p) human Myeloma Cell line, MMLAL, was established from the pleural effusion of a Chinese Myeloma patient.
