CD200 Is Preferentially Expressed in CD138 dim Multiple Myeloma Cells: Implications for a Myeloma Stem Cell Subpopulation with Potential Immune-Resistance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4877-4877
Author(s):  
Xiaochuan Chen ◽  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Cancer Stem Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Population ◽  
Cell Subpopulation ◽  
Myeloma Cells ◽  
Expression Levels

Abstract Abstract 4877 Introduction The CD138negCD20+ cell population has been suggested as the putative multiple myeloma (MM) cancer stem cells, both in MM cell lines and in patient specimens. CD200 is an immunosuppressive membrane glycoprotein reported to be co-expressed with other stem-cell markers in prostate, breast, brain, and colon cancers. Also, CD200 is a negative prognostic factor for MM. It remains unknown whether CD200 is expressed in the CD138negCD20+ MM cell population, the putative MM cancer stem cells. Here we addressed this question by assessing CD200 expression in different subpopulations of MM cells according to their CD138 expression levels. Methods Two MM cell lines (RPMI8226 and NCI-H929) were co-stained with FITC-labeled anti-CD200, PE-labeled anti-CD20, and APC-labeled anti-CD138 antibodies for multicolor flow cytometry analysis. The cells were gated into 4 subpopulations (h1>h2>h3>h4 corresponding to CD138 expression levels: high>dim>low>negative). In each gated cell population, CD200 expression was assessed, and the CD200+ and CD200neg cell subpopulations were further gated for analysis of their CD20 expression levels. Results In the RPMI 8226 cell line, the distribution of gated h1, h2, h3, and h4 populations was 87.51%, 5.86%, 5.26%, and 1.29%, respectively. In the h2 (CD138dim) population, 22.88% of the cells were CD200+. In contrast, CD200 was expressed at much lower levels in the other three populations, ranging from 7.28% (h1), 6.65% (h3), to 0.48% (h4). In CD200+ cells from the gated h1, h2, h3, and h4 population, CD20+ cells were 19.50%, 23.03%, 27.67%, and 18.75%, respectively, while in the CD200neg cells, CD20+ cells were 20.79%, 22.50%, 25.08%, and 28.02%, respectively. In the NCI-H929 cell line, 18.59% of the cells in the h2 population were CD200+, whereas only 1.61%, 1.69%, and 0.47% of the cells in the h1, h3, and h4 populations were CD200+. In each population, there were more CD20+ cells in CD200+ cells than in CD200neg cells, which were 24.52% vs 0.04% (h1), 41.94% vs 11.34% (h2), 11.11% vs 3.06% (h3), and 50.00% vs 2.25% (h4), respectively. Conclusions These results demonstrate that the immunosuppressive molecule CD200 is preferentially expressed in a CD138dim subpopulation of multiple myeloma cells. Depending on cell line, the putative myeloma stem cell marker CD20 is either preferentially, or not preferentially, expressed in the CD200+ cells, suggesting that an immune-resistant subpopulation of MM stem cells might exist, which may have important implications for designing immunotherapeutic approaches to treat this disease. Disclosures Goldenberg: Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

How to Overcome Myeloma Stem Cell Resistance to Therapy - Targeting the Stem Cell Niche

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-6-SCI-6
Author(s):  
Constantine S. Mitsiades
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Stem Cell ◽  
Cancer Stem Cells ◽  
Cancer Stem Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cell Population ◽  
Research Funding ◽  
Accessory Cells

Abstract Abstract SCI-6 The concept of cancer stem cells has attracted again intense research interest, as the drug resistance attributed to this infrequent subpopulation of tumor cells could explain how patients can relapse even after prolonged complete clinical, biochemical, radiologic or even molecular remissions. In multiple myeloma (MM), several aspects of the cancer stem cell concept remain to be elucidated, including the potential heterogeneity of this cell subpopulation or whether CD138+ expression is incompatible or not with a MM stem cell. As these questions are being resolved, emerging data highlight that the drug resistance of MM cells with clonogenic/stem cell-like features is heavily influenced by interactions with non-malignant accessory cells of the local microenvironment, including bone marrow stromal cells (BMSCs). Indeed, transcriptional signatures of “stemness”, as identified in normal stem cells or cancer stem cells from other neoplasias, are detected in the bulk population of MM cell lines and are upregulated after MM cell interaction with BMSCs. MM cell lines and primary tumor cells contain subpopulations with clonogenic potential, such as the side population (SP) cells. SP cells, detected by low intracellular accumulation of Hoechst 33342 dye (in contrast to the tumor's “main population” (MP), are considered an enriched source of tumor-initiating cells in diverse neoplasias and were detected among CD138− CD20+ CD27+ clonogenic cells in primary MM samples. Interaction with BMSCs increases the viability of SP cells and their percentage within the MM cell population. While interaction with BMSCs or other accessory cells of the microenvironmental niche suppresses the anti-MM activity of glucocorticoids, conventional chemotherapeutics and certain investigational agents, other agents (e.g. immunomodulatory thalidomide derivatives (IMIDs), such as lenalidomide) have increased activity against MM SP cells in the context of this tumor-microenvironment interaction. These observations suggest that MM cells with stem cell-like features exhibit functional plasticity depending on which specific microenvironmental niche they interact with. The Hedgehog, Wnt and Notch pathways, as well as regulators of chromatin remodeling, e.g. histone demethylases, have emerged as putative links between drug resistance, “cancer stemness” and how these functional outcomes are modulated by the local microenvironment in MM. These pathways, as well as embryonic stem cell-associated antigens (e.g. SOX2), represent intriguing targets for investigational therapies. However, clinical translation of such treatments has notable challenges, as conventional criteria for response assessment may not accurately reflect the treatment's impact on clonogenic tumor cells. Progression-free survival is considered a more appropriate endpoint for cancer stem-cell targeting agents, its assessment, however, may be confounded without concomitant treatment that suppresses the bulk of the tumor. Consequently, candidate cancer stem cell-targeting agents may have to be evaluated in combination with regimens (including lenalidomide-bortezomib-Dex (RVD) or other combinations built around the therapeutic “backbone” of proteasome inhibition and IMIDs) which potently target the bulk MM cell population and induce high rates of complete/near complete responses. Further improvements of this dual targeting of clonogenic and bulk tumor cells may be facilitated by recently developed high-throughput platforms (e.g. compartment-specific bioluminescence imaging, CS-BLI) which screen, in the presence vs. absence of stroma or other accessory cells, large numbers of anti-tumor agents and combinations thereof against the bulk tumor cell population or its clonogenic compartments. These new platforms will inform the rational design of regimens that will hopefully improve the long-term outcome of MM patients by suppressing a clonogenic/stem cell-like tumor compartment and circumventing microenvironment-dependent drug resistance. Disclosures: Mitsiades: Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

scholarly journals Investigation of the expression of RIF1 gene on head and neck, pancreatic and brain cancer and cancer stem cells

2016 ◽  
Vol 39 (6) ◽  
pp. 43 ◽  
Author(s):  
Hacer E GursesCila ◽  
Muradiye Acar ◽  
Furkan B Barut ◽  
Mehmet Gunduz ◽  
Reidar Grenman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Cancer ◽  
Stem Cells ◽  
Stem Cell ◽  
Cancer Stem Cells ◽  
Head And Neck ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines

Purpose: Recent studies have shown that cancer stem cells are resistant to chemotherapy. The aim of this study was to compare RIF1 gene expression in head and neck, pancreatic cancer and glioma cell lines and the cancer stem cells isolated from these cell lines. Methods: UT-SCC-74 from Turku University and UT-SCC-74B primary tumor metastasis and neck cancer cell lines, YKG-1 glioma cancer cell line from RIKEN, pancreatic cancer cell lines and ASPC-1 cells from ATCC were grown in cell culture. To isolate cancer stem cells, ALDH-1 for UT-SCC-74 and UT-SCC-74B cell line, CD-133 for YKG-1 cell line and CD-24 for ASPC-1 cell line, were used as markers of cancer stem cells. RNA isolation was performed for both cancer lines and cancer stem cells. RNAs were converted to cDNA. RIF1 gene expression was performed by qRT-PCR analysis. RIF1 gene expression was compared with cancer cell lines and cancer stem cells isolated from these cell lines. The possible effect of RIF1 gene was evaluated. Results: In the pancreatic cells, RIF1 gene expression in the stem cell-positive cell line was 256 time that seen in the stem cell-negative cell line. Conclusion: Considering the importance of RIF1 in NHEJ and of NHEJ in pancreatic cancer, RIF1 may be one of the genes that plays an important role in the diagnoses and therapeutic treatment of pancreatic cancer. The results of head and neck and brain cancers are inconclusive and further studies are required to elucidate the connection between RIF1 gene and these other types of cancers.

Cancer Stem Cell Activity in Mantle Cell Lymphoma Is Inhibited by TLR9 Activation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 884-884
Author(s):  
Sarah Brennan ◽  
Brooke Meade ◽  
Qiuju Wang ◽  
George Weiner ◽  
William Matsui
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Cancer Stem Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Cell Lymphoma ◽  
Cpg Odn ◽  
Mantle Cell

Abstract Patients with Mantle Cell Lymphoma (MCL) often respond to initial cytotoxic therapy, but invariably relapse. This clinical pattern suggests that standard therapies fail to eliminate a population of chemoresistant cells responsible for tumor regrowth and disease relapse. Studies in myeloid leukemias and multiple myeloma have suggested that cancer stem cells (CSC) are relatively quiescent and that this property promotes drug resistance. Therefore, the induction of CSC proliferation may increase their susceptibility to anti-cancer treatment. Unmethylated CpG DNA sequences induce the activation and differentiation of normal B cells through toll like receptor 9 (TLR9), and synthetic CpG oligonucleotides (ODN) are currently undergoing clinical trials in MCL and other B cell non-Hodgkin’s lymphomas. We initially examined the effects of TLR9 agonists on the clonogenic growth of the human MCL cell lines Granta 519, Jeko-1, and Rec-1 and found that the unmethylated CpG ODN 2006 (5ug/mL, 48 hours) significantly inhibited the ability of each of these cell lines to form colonies in methylcellulose (p<0.01). We previously demonstrated that clonogenic CSC in multiple myeloma and Hodgkin’s lymphoma may be identified based on the relative activity of the intracellular detoxification enzyme aldehydedehydrogenase (ALDH). Similarly, each of the MCL cell lines contained small populations (0.5−2.3%) of ALDH+ cells that were enriched for in vitro clonogenic growth upon primary and secondary plating in methylcellulose. Following treatment with CpG ODN 2006, the population of ALDH+ cells was significantly reduced in each cell line (p<0.05). The self-renewal potential of normal and cancer stem cells is lost through the process of differentiation, and treatment of the MCL cell lines with CpG ODN 2006 resulted in the loss of CD19 and CD20 expression and induction of the plasma cell antigen CD138 by FACS. Similarly, RT-PCR for regulators of B cell differentiation revealed decreased PAX-5 and BCL-6 expression coupled with increased transcription of BLIMP-1 and XBP-1. We hypothesized that the plasma cell differentiation induced by TLR9 agonists may improve the efficacy of anti-tumor agents with known activity against plasma cells, specifically the proteosome inhibitor bortezomib. Treatment with CpG ODN 2006 or bortezomib (10–100nM, 48 hours) alone induced apoptosis in a small percentage of cells within each MCL line, but the combination of these agents synergistically increased the proportion of Annexin V+ cells. In summary, cellular activation by CpG ODN 2006 reduces MCL clonogenicity and stem cell function through the induction of differentiation. In turn, plasma cell maturation appears to increases the sensitivity of MCL to bortezomib and suggests a novel mechanism for the anti-tumor activity of CpG ODN.

scholarly journals P04.01 Dendritic-cell based immunotherapy targeting pancreatic and NSCLC cancer stem cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A36-A36
Author(s):  
J Calmeiro ◽  
M Carrascal ◽  
L Mendes ◽  
IF Duarte ◽  
C Gomes ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Cancer Immunotherapy ◽  
Cell Lines ◽  
Cell Population ◽  
Culture Media ◽  
Small Cells ◽  
Antigen Uptake ◽  
Part Time

BackgroundThe field of cancer immunotherapy is growing at a fast pace, with new developments in this field leading to a change in cancer therapy. Dendritic cells (DCs) are one of the central tools in cellular anti-tumour immunotherapy and the production of clinical grade monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. However, there is a large space for improvement of protocols and a clear need for the establishment of clinical standard operating procedures (CSOP). Cancer stem cells (CSCs) are a recently identified small cell population present in the tumour, resistant to radio/chemotherapy and known to be responsible for disease recurrence. Here, we aim to contribute to the standardization of CSOPs and to target and eradicate CSCs by developing a DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing DC loading with CSCs vs. classical tumour lysates.Materials and MethodsCSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were isolated and characterized by RT-PCR and flow citometry. CSCs resistance to chemotherapy was also assessed. In vitro anti-tumour cytotoxicity assays were performed. We also defined and compared the effect of 4 culture media during human Mo-DCs production. Three Good Manufacturing Practice (GMP) serum-free culture media for clinical use were tested - DendriMACS, AIM-V and X-VIVO 15. RPMI was used as a comparative term given that it is largely used in pre-clinical research. We characterized DC viability, differentiation, maturation, internalization of tumour lysates, cytokines production and autologous T cell stimulatory capacity, as well as metabolomic profiles by Nuclear Magnetic Resonance (NMR) spectroscopy.ResultsCSCs from PANC-1 and A549 cell lines were successfully isolated and overexpressed the stem-like markers NANOG, OCT4, SOX2 and CD133, with resistance to gemcitabine. In terms of differentiation, maturation, antigen uptake capacity and metabolic profiles, AIM-V and X-VIVO 15 present similar results. However, the use of X-VIVO 15 shows an enhanced DC production of IL-12. DCs cultured in X-VIVO 15 and AIM-V media are able to induce a superior stimulation of T cells (CTLs and Th1 responses) while DCs cultured in DendriMACS are more prone to induce Treg polarization. Our data show that X-VIVO 15 and AIM-V culture media are preferable to support the differentiation of DCs to be used in immunostimulatory approaches such as in cancer immunotherapy.ConclusionsOverall, our results demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. This highlights the need of critically defining the culture medium to be used in DC cancer immunotherapy, attaining the desired cell characteristics and consequent robust clinical responses. We are now assessing in vitro anti-tumour cytotoxicity to evaluate if DC loading with CSC antigens can be an efficient immunotherapy strategy to target and eliminate this specific and resistant cancer cell population.FundingImmunoDCs@CancerStemCells: Cellular Immunotherapy towards the elimination of cancer stem cells (Ref.: POCI-01-0247-FEDER-033532), co-funded by FEDER, COMPETE2020 and University of Coimbra.Disclosure InformationJ. Calmeiro: None. M. Carrascal: A. Employment (full or part-time); Significant; Tecnimede Group. L. Mendes: None. I.F. Duarte: None. C. Gomes: None. J. Serra: A. Employment (full or part-time); Significant; Tecnimede Group. A. Falcão: None. M.T. Cruz: None. B.M. Neves: None.

PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3417-3417
Author(s):  
Yutaka Okuno ◽  
Hiro Tatetsu ◽  
Shikiko Ueno ◽  
Hiroyuki Hata ◽  
Yasuhiro Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Promoter Region ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Upstream Region ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Expression Levels

Abstract It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.

Elimination of CD20-Expressing Cells in Multiple Myeloma by Iodine I-131 Tositumomab (Bexxar®) Correlates with Response to Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5176-5176 ◽  
Author(s):  
Andrzej J. Jakubowiak ◽  
Malathi Hari ◽  
Tara Kendall ◽  
Yasser Khaled ◽  
Shin Mineishi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Partial Response ◽  
Clinical Outcomes ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Myeloma Cells ◽  
Consolidation Treatment ◽  
Anti Cd20

Abstract It has been proposed that treatment failures in multiple myeloma (MM) are related to the chemoresistance of a subset of malignant myeloma cells with clonogenic potential to the anti-myeloma drugs (Matsui et al., Cancer Research2008, 68:190). Studies suggest that these putative myeloma stem cells (MSC) are CD138neg and express CD20, which is usually absent in more differentiated malignant plasma cells (Matsui et al., Blood2004, 15:2332). These findings provided a rationale for a treatment strategy to eliminate chemoresistant clonogenic MSC using anti-CD20 antibodies. To evaluate the clinical effects of anti-CD20 treatment, we developed a phase II trial with Bexxar as a consolidation treatment for MM. We hypothesized that Bexxar would be more efficacious than unlabeled antibody in the eradication of highly radiosensitive myeloma cells by both direct and cross-fire effects. Preclinical studies showed that tositumomab, the antibody used in Bexxar, inhibited colony formation of clonogenic myeloma cells. To be eligible for Bexxar treatment, patients must have completed at least 4 cycles of therapy (1st to 3rd line) and have measurable disease in a plateau of at least partial response (PR). To date, 10 of 30 patients have been enrolled, of which 5 were treated with Bexxar after completion of initial therapy prior to proceeding to autologous stem cell transplant (ASCT). Patients proceeding to ASCT required hematopoietic stem cells collection prior to Bexxar and 3 months after Bexxar. Eight patients are evaluable for response and toxicities. At 3 months post Bexxar, 1 patient achieved a partial response (PR), 4 patients had stable disease (SD), and 1 patient progressed (PD). At 1 year post Bexxar, 1 patient with initial PR achieved CR and remains in CR, 1 patient is in unconfirmed CR, 2 are in partial response (PR), and 3 remain in SD. After a median 20 months of followup (range 4–24), all patient are alive, 4 in continued response, 3 with SD. Hematological toxicities were mild to moderate (1 patient grade 3 and 4 patients grade 2 thrombocytopenia, 4 patients grade 2 neutropenia). Non-hematological toxicity was limited to HAMA (6 patients). Out of patients who received Bexxar prior to transplant, 3 collected stem cells post-Bexxar without problems, one requires re-collection. Three patients who proceeded to ASCT to date using the post-Bexxar stem cell collection, engrafted at 11–12 days, and had no unexpected toxicities with ASCT. We also analyzed CD20+ cells in bone marrow aspirates (BM) and stem cell collections (SCC) using samples collected from 4 patients before and after Bexxar. Bexxar treatment eliminated a median 80% of CD20+ cells (range 23–97). For a given patient, elimination of CD20+ cells from SCC correlated with elimination of CD20+ cells from BM. The most complete elimination of CD20+ cells from BM was observed in 2 patients who at 1 year achieved CR (94% and 97%), compared to patients who achieved PR (23% and 68%). We conclude that anti-CD20 consolidation treatment of myeloma patients with Bexxar used as targeted therapy against clonogenic myeloma cells is feasible and well tolerated. Clinical outcomes to date are encouraging considering that clonogenic MSC represent &lt;5% of malignant plasma cells and delayed responses observed in this study could be expected. While early, clinical outcomes appear to correlate with the efficacy of the CD20+ cell elimination by Bexxar treatment in myeloma.

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.

YM155 Exerts Potent Cytotoxic Activity Against Quiescent (G0/G1) Multiple Myeloma and Bortezomib Resistant Cells Via Inhibition of Mcl-1 and Survivin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3023-3023
Author(s):  
Miyuki Ookura ◽  
Tatsuya Fujii ◽  
Shinji Kishi ◽  
Hiroko Shigemi ◽  
Naoko Hosono ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cytotoxic Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Myeloma Cells ◽  
Ic50 Value ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin. Disclosures No relevant conflicts of interest to declare.

Colorectal cancer stem cells: Characterization and functional analysis

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 4124-4124
Author(s):  
T. Yeung ◽  
J. Wilding ◽  
W. Bodmer
Keyword(s):  
Colorectal Cancer ◽  
Stem Cells ◽  
Stem Cell ◽  
Cancer Stem Cells ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Tumour Growth ◽  
Colorectal Cancer Cell Lines ◽  
Colorectal Cancer Stem Cells

4124 Background: Cancer stem cells are defined as cells within a tumour that are able to self-renew and differentiate into all cell lineages within that tumour. With our extensive panel of colorectal cell lines, our aims are: 1) To characterise and isolate cancer stem cells based on stem cell markers, morphological appearances and the ability to form multiple lineages; 2) To understand how cancer stem cells drive tumour growth and progression. Methods: 1) Fluorescent Activated Cell Sorting (FACS); 2) In vitro soft agar clonogenic and Matrigel differentiation assays; 3) In vivo tumourigenic NOD/SCID mice assay; 4) Confocal immunofluorescence imaging. Results: 1) A subpopulation of cells can differentiate into crypt-like megacolonies, retaining the ability to self-renew and differentiate. SW1222 cell line forms heterogeneous colonies when single cells are plated in Matrigel. Megacolonies can both self-renew and form terminally differentiated small colonies, whereas small colonies cannot form megacolonies. Megacolonies develop crypt-like structures and increase their expression of differentiation markers (CDX-1, CK-20) over time. Experiments are currently under way to confirm that cells from megacolonies are able to initiate tumours in NOD/SCID mice. Some cell lines retain the ability to differentiate into both neuroendocrine and epithelial lineages. 2) CD44+CD24+ enriches for the cancer stem cell population. Colorectal cancer cell lines HCT116, HT29, LS180, LS174T and SW1222 express both CD44 and CD24. The CD44+CD24+ subpopulation is the most clonogenic. In SW1222, CD44+CD24+ cells enrich for megacolonies and can reform all four CD44/CD24 subpopulations. 3) Hypoxia reduces differentiation, increases stem-like phenotype and enhances clonogenicity. Hypoxia increases the proportion of undifferentiated colorectal cancer cells when plated on Matrigel and increases clonogenicity. Conclusions: 1) Colorectal cancer cell lines contain subpopulations of cells that have the ability to self-renew, differentiate and drive tumour growth, and may be characterised by their cell surface markers and colony morphology. 2) CD44+CD24+ can be used as markers for colorectal cancer stem cells. 3) Hypoxia increases the stem-like phenotype of cancer cells, reduces differentiation and increases clonogenicity. No significant financial relationships to disclose.

The effect of BMI-1 inhibition on brain cancer stem cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13543-e13543
Author(s):  
Monal Mehta ◽  
Atif J. Khan ◽  
Hatem E. Sabaawy ◽  
Bruce George Haffty
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Stem Cell ◽  
Cancer Stem Cells ◽  
Cell Lines ◽  
Brain Cancer ◽  
Insertion Site ◽  
Stem Cell Markers ◽  
Primary Cells ◽  
Self Renewal

e13543 Background: Glioblastoma (GBM) is the most frequent and deadly brain cancer. Despite tolerance doses of radiation, control of tumor growth within the brain remains a formidable failure. Since the identification of brain cancer stem cells (BCSCs), efforts are underway to target the pathways regulating these cells. The role of Bmi-1 (B-cell specific MMLV insertion site-1), a polycomb member of chromatin-remodeling complex, in BCSCs self-renewal was elucidated. Here we utilize shRNA targeting or pharmacological inhibition of Bmi-1 in GBM cell lines and primary cells as a radiosensitizer to examine the effects of combination therapy on cell death and BCSCs differentiation. Methods: Cells were pre-treated with a Bmi-1 inhibitor before being irradiated. Serial neurosphere assay, a measure of self-renewal potential, was employed to study the effects of radiation, Bmi-1 inhibition, or the combination on BCSCs. The efficacy of this combination on cell death was assessed with MTT and clonogenic assays. Next, the abilities of the inhibitor and radiation to induce differentiation in GBM cell lines and primary cells were quantified. Further, by utilizing a novel zebrafish orthotropic xenograft model, small molecules targeting Bmi-1 and other BCSC pathways can be identified, and used to predict response to combination therapies. Results: Targeting of Bmi-1 in combination with radiation, specifically as a radiosensitizer, induced significant cell death in GBM cells, and was five-fold more effective than radiation only. Importantly, the neurosphere forming ability of BCSCs was severely compromised when the cells were treated with the combination, indicating a potent effect on the stem cell constituency. These effects may be due to loss of BCSC self-renewal potential, increased differentiation, and/or apoptosis as cells treated with the combination exhibited decreased expression of neural stem cell markers and abnormal phenotypes compared to single treatment. Conclusions: Targeting of Bmi-1 may eliminate the subpopulation of radioresistant BCSCs. Bmi-1 inhibition when combined with radiotherapy might provide an effective therapy for GBM patients specifically through its effect on BCSCs by affecting their survival, proliferation, and stem cell features.

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