Sphingosine-1 Phosphate (S1P) Is a Myeloma Survival Factor and Protects Myeloma Cells from Thalidomide-Induced Growth Inhibition by Counteracting Ceramide Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3509-3509
Author(s):  
Xin Li ◽  
Angela Pennisi ◽  
Shmuel Yaccoby
Keyword(s):  
Drug Resistance ◽  
Growth Inhibition ◽  
Annexin V ◽  
Myeloma Cell ◽  
Inhibitory Effect ◽  
Tunel Staining ◽  
Serum Starvation ◽  
Myeloma Cells ◽  
Sphingosine 1 Phosphate

Abstract Sphingosine 1-phosphate (S1P) and ceramide are sphingolipid metabolites and their intracellular balance impacts fate of normal and cancer cells; whereas S1P stimulates tumor cell migration, survival, proliferation and drug resistance, ceramide acts as endogenous pro-apototic factor. The role of S1P/ceramide pathway in myeloma has not been reported. Recent study demonstrated that thalidomide’s antiangiogenic activity involves increased production of ceramide in microvessels (Yabu et al., Blood 2005). In this study we tested the effect of S1P and ceramide on myeloma growth and determined the protective role of S1P against the inhibitory effect of thalidomide on myeloma cells. S1P (0.1–10 μM) stimulated growth of several human myeloma cell lines in serum-free media in time and dose dependent manners, reaching maximum response (>25%, p<0.001) at approximately 5 μM. Annexin V/PI flow analysis and TUNEL staining for detection of apoptosis revealed reduced percent of apoptotic myeloma cells following S1P treatment. S1P also consistently and significantly protected primary CD138-selected myeloma cells (n=5) from serum starvation-induced apoptosis as shown by MTT assay (p<0.01) and annexin V/PI flow cytometry (p<0.05). In contrast, synthetic ceramide (3–10 μM) inhibited growth of myeloma cells, an effect that was completely counteracted by S1P. We have previously demonstrated that osteoclasts and megakaryocytes support primary myeloma cell survival. Autotaxin, a key exo-enzyme involved in extracellular production of S1P and lysophosphatidic acid (LPA), was consistently upregulated in primary myeloma cells by 11±3 folds over baseline levels after co-culture with osteoclasts (n=17, p<0.01) and by 4±1 folds after co-culture with megakaryocytes (n=5, p<0.04), as assessed by microarray and confirmed by qRT-PCR. Furthermore, in co-cultures the S1P receptors antagonist, FTY720, at concentrations that have no direct anti-tumor response (≤1 μM), inhibited the stimulatory effects of osteoclasts and megakaryocytes on survival of our BN-stroma dependent myeloma cell line by 30% to 50% (p<0.001) suggesting that S1P is an important microenvironmental factor in myelomatous bone. Since metabolic activation of thalidomide is required for its anti-tumor activity, thalidomide (10–100 μM) was incubated with active human microsomes while control non-metabolized thalidomide was similarly prepared using inactivated microsomes. Metabolized but not non-metabolized thalidomide effectively inhibited growth of myeloma cells by >40% (p<0.001). Addition of S1P (1 μM) completely abrogated the inhibitory effect of metabolized thalidomide in all tested doses. We conclude that S1P is a myeloma survival and growth factor and protects myeloma cells from thalidomide-induced growth inhibition, presumably through counteracting increased ceramide activity by thalidomide. Pharmacological modulation of enzymes involved in regulation of the ceramide to S1P ratio and blocking S1P activity could constitute novel therapeutic approaches for treating and overcoming drug resistance in myeloma.

Enforced CD19 Expression Leads to Growth Inhibition and Reduced Tumorigenicity

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3551-3558 ◽  
Author(s):  
Maged S. Mahmoud ◽  
Ryuichi Fujii ◽  
Hideaki Ishikawa ◽  
Michio M. Kawano
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Myeloma Cell ◽  
Inhibitory Effect ◽  
Myeloma Cell Line ◽  
Growth Inhibitory Effect ◽  
Myeloma Cells ◽  
Growth Inhibitory ◽  
Human Myeloma Cell Line

In multiple myeloma (MM), the cell surface protein, CD19, is specifically lost while it continues to be expressed on normal plasma cells. To examine the biological significance of loss of CD19 in human myeloma, we have generated CD19 transfectants of a tumorigenic human myeloma cell line (KMS-5). The CD19 transfectants showed slower growth rate in vitro than that of control transfectants. They also showed a lower capability for colony formation as evaluated by anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was CD19-specific and probably due to CD19 signaling because this effect was not observed in cells transfected with a truncated form of CD19 that lacks the cytoplasmic signaling domain. The in vitro growth-inhibitory effect was confirmed in a nontumorigenic human myeloma cell line (U-266). However, introduction of the CD19 gene into a human erythroleukemia cell line (K-562) also induced growth inhibition, suggesting that this effect is CD19-specific, but not restricted to myeloma cells. These data suggest that the specific and generalized loss of CD19 in human myeloma cells could be an important factor contributing to the proliferation of the malignant plasma cell clones in this disease.

Emodin Enhances the Chemosensitivity of Endometrial Cancer by Inhibiting ROS-Mediated Cisplatin-resistance

2018 ◽  
Vol 18 (7) ◽  
pp. 1054-1063 ◽  
Author(s):  
Ning Ding ◽  
Hong Zhang ◽  
Shan Su ◽  
Yumei Ding ◽  
Xiaohui Yu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Endometrial Cancer ◽  
Cancer Cells ◽  
Chemotherapeutic Agent ◽  
Annexin V ◽  
Chemotherapeutic Agents ◽  
Endometrial Cancer Cell ◽  
Animal Survival ◽  
Apoptosis Level

Background: Endometrial cancer is a common cause of death in gynecological malignancies. Cisplatin is a clinically chemotherapeutic agent. However, drug-resistance is the primary cause of treatment failure. Objective: Emodin is commonly used clinically to increase the sensitivity of chemotherapeutic agents, yet whether Emodin promotes the role of Cisplatin in the treatment of endometrial cancer has not been studied. Method: CCK-8 kit was utilized to determine the growth of two endometrial cancer cell lines, Ishikawa and HEC-IB. The apoptosis level of Ishikawa and HEC-IB cells was detected by Annexin V / propidium iodide double-staining assay. ROS level was detected by DCFH-DA and NADPH oxidase expression. Expressions of drug-resistant genes were examined by real-time PCR and Western blotting. Results: Emodin combined with Cisplatin reduced cell growth and increased the apoptosis of endometrial cancer cells. Co-treatment of Emodin and Cisplatin increased chemosensitivity by inhibiting the expression of drugresistant genes through reducing the ROS levels in endometrial cancer cells. In an endometrial cancer xenograft murine model, the tumor size was reduced and animal survival time was increased by co-treatment of Emodin and Cisplatin. Conclusion: This study demonstrates that Emodin enhances the chemosensitivity of Cisplatin on endometrial cancer by inhibiting ROS-mediated expression of drug-resistance genes.

scholarly journals A critical role of autocrine sonic hedgehog signaling in human CD138+ myeloma cell survival and drug resistance

Blood ◽  
2014 ◽  
Vol 124 (13) ◽  
pp. 2061-2071 ◽  
Author(s):  
Zhiqiang Liu ◽  
Jingda Xu ◽  
Jin He ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Survival ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Critical Role ◽  
Myeloma Cell ◽  
Sonic Hedgehog Signaling ◽  
Key Points

Key Points CD138+ MM cells are a major source of SHH. Autocrine SHH enhances MM drug resistance.

scholarly journals Resveratrol Targets Sphingolipid Metabolism to Induce Growth Inhibition in FLT3 ITD Acute Myeloid Leukemia

Proceedings ◽  
2019 ◽  
Vol 40 (1) ◽  
pp. 4
Author(s):  
Ersöz ◽  
Adan
Keyword(s):  
Growth Inhibition ◽  
Cell Fate ◽  
De Novo ◽  
Therapeutic Potential ◽  
Annexin V ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Cytotoxic Effects ◽  
Sphingosine 1 Phosphate ◽  
First Time

Sphingolipids are important signaling lipids which play crucial roles to determine the cell fate. Ceramide, apoptotic central molecule of sphingolipid metabolism, which is produced through de novo pathway by serine palmitoyl transferase (SPT) and can be converted to antiapoptotic sphingosine-1-phosphate (S1P) and glucosyl ceramide (GC) by sphingosine kinase (SK) and glucosyl ceramide synthase (GCS), respectively. It is aimed to investigate therapeutic potential of resveratrol on FLT3-ITD (Internal Tandem Duplication) AML cells and to identify potential mechanism behind resveratrol-mediated growth inhibition by targeting of ceramide metabolism. The cytotoxic effects of resveratrol, SPT inhibitor (myricoin), SK-1 inhibitor (SKI II), GCS inhibitor (PDMP), resveratrol: SPT inhibitor, resveratrol: SK-1 inhibitor and resveratrol: GCS inhibitor combinations on MOLM-13 and MV4-11 FLT3 ITD AML cells were investigated by cell proliferation assay. Apoptosis was evaluated by annexin V/PI double staining. There were synergistic cytotoxic effects of resveratrol with co-administration of SPT inhibitor, SK-1 inhibitor and GCS inhibitor and apoptosis was synergistically induced for resveratrol and its combinations. This preliminary data showed for the first time that resveratrol might inhibit the growth of FLT3 ITD AML cells through targeting ceramide metabolism.

Role of erythrocyte phosphatidylserine in sickle red cell–endothelial adhesion

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1564-1571 ◽  
Author(s):  
B. N. Yamaja Setty ◽  
Surekha Kulkarni ◽  
Marie J. Stuart
Keyword(s):  
Annexin V ◽  
Inhibitory Effect ◽  
Vascular Pathology ◽  
Membrane Asymmetry ◽  
Adhesion Receptor ◽  
Sickle Erythrocyte ◽  
Erythrocyte Surface ◽  
Erythrocyte Adhesion ◽  
The Individual

Phosphatidlyserine (PS) exposure on the erythrocyte surface endows the cell with the propensity of adhering to vascular endothelium. Because individuals with sickle cell disease (SCD) manifest loss of erythrocyte membrane asymmetry with PS exposure, we have assessed the contribution of this marker to the process of sickle erythrocyte–microendothelial adhesion. Assays for plasma-induced adhesion were conducted on unactivated endothelium, in the absence of immobilized ligands, such that PS was compared to the erythrocyte adhesion receptor CD36. Blocking studies with erythrocytes pretreated with annexin V (to cloak PS) or anti-CD36 or both revealed an inhibitory effect on adhesion of 36% ± 10% and 23% ± 8% with blocking of both sites suggestive of an additive effect. We next evaluated 87 blood samples from patients with SCD and grouped them into 4 categories based on adhesion marker (CD36 and PS) levels. Results revealed a striking correlation between erythrocyte PS positivity and adhesion. Analyses of the individual patient data demonstrated a positive correlation between PS and adhesion (R = 0.52,P &lt; .000 001), whereas none was noted between adhesion and CD36 (R = 0.2, P &gt; .07). The effect of PS on adhesion appears to be related to the quantitative differences in erythrocyte markers in SCD, with PS the predominant marker when compared to CD36 both in the total erythrocyte population, and when the adherence-prone erythrocyte, the CD71+ stress reticulocyte, was evaluated. Our study signals the entrance of an important new contributor to the field of sickle erythrocyte–endothelial adhesion. The implications of erythrocyte PS exposure in relation to the vascular pathology of SCD need to be assessed.

Multiple Myeloma: Interleukin-15 Antagonizes Bone Morphogenetic Protein-Induced Apoptosis and Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3444-3444
Author(s):  
Magne Rekvig ◽  
Anne-Tove Brenne ◽  
Torstein Baade Ro ◽  
Anders Waage ◽  
Magne Borset ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Inhibition ◽  
Cell Line ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Stimulus Figure ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract Multiple myeloma has two distinct features: Expansion of malignant plasma cells within the bone marrow accompanied by skeletal destruction. Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and inhibit growth in myeloma cells. BMPs are members of the TGF-β superfamily of proteins capable of inducing bone formation, and regulate proliferation, differentiation and apoptosis. We have investigated myeloma cell apoptosis and proliferation with BMP-4 and −6 in concert with the myeloma cell growth factors interleukin (IL)-2, IL-6, IL-10, IL-15, IL-21, tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-1. Eight samples of highly purified myeloma cells from patients and a human myeloma cell line, IH-1 (Brenne AT et al. Blood. 2002 May 15;99(10):3756–62.), were used in this study. Cytokine concentrations used in the referred experiments were for BMP-4 20ng/ml, BMP-6 250ng/ml, IL-15 20ng/ml and IL-6 0,1ng/ml, respectively. Growth inhibition was measured in a proliferation assay by methyl-[3H]-thymidine incorporation and apoptosis by annexin V- FITC-binding/PI-uptake on flow cytometry. IL-15 antagonized growth inhibition (Figure A) and prevented apoptosis induced by BMP-4 (Figure B) and BMP-6 in the myeloma cell line IH-1. IL-15 also antagonized the growth inhibition induced by BMP-4 and/or BMP-6 in three out of eight patient samples. Neither IL-6, nor any of the other investigated cytokines were able to rescue the myeloma cells from growth inhibition and apoptosis induced by BMP-4 and -6. Among the investigated cytokines, we found that IL-15 has a unique capability to antagonize BMP- induced apoptosis and growth inhibition in myeloma cells. We examined cleavage of the proapoptotic protein caspase-3 and found that BMP-4 activated caspase-3 in the IH-1 cell line. This activation of caspase-3 was blocked by IL-15 but not by IL-6. We have demonstrated a possible mechanism for myeloma cells to escape apoptosis and growth-inhibition within the bone marrow. Intramedullar levels of IL-15 and BMPs may play a role in the pathogenesis of multiple myeloma. Figure A. Proliferation in response to BMP-4 stimulus Figure A. Proliferation in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus

Expression of Wnt-3a in Myeloma Cells Inhibits the Osteolytic Phenotype In-Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3420-3420
Author(s):  
Ya-Wei Qiang ◽  
Shmuel Yaccoby ◽  
John D. Shaughnessy
Keyword(s):  
Bone Resorption ◽  
Wnt Signaling ◽  
Myeloma Cell ◽  
Beta Catenin ◽  
Canonical Wnt Signaling ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Canonical Wnt

Wnt signaling is a highly conserved signal transduction pathway involved in embryonic development. Inappropriate canonical Wnt signaling resulting in beta-catenin stabilization, is associated with several types of human cancers. Multiple myeloma plasma cells express Wnt receptors, Wnt ligands and soluble Wnt inhibitors. Wnt signaling is central to osteoblast and osteoclasts development and secretion of Wnt signaling inhibitors by myeloma cells is thought to contribute to the osteolytic phenotype seen in this disease and prostate cancer. While it is now clear that MM cells can signal through both canonical and non-canonical mechanisms, there are conflicting data as to the direct role of Wnt signaling in myeloma cell biology. Others have shown that Wnts cause proliferation of myeloma cells; while we have shown that canonical Wnts cause morphological changes and migration, but not cell proliferation. To further elucidate the role of canonical Wnt signaling in myeloma and myeloma bone disease we used limiting dilutions in the presence of G418 to create two independent stable clones of the myeloma cell line NCI-H929 expressing Wnt-3A (H929/W3A), which is not expressed in myeloma, and an empty vector (H929/EV). Because Wnt antibodies are not available we cloned Wnt-3A as a fusion protein with hemagglutinin (HA). Western blots against HA revealed a positive band of the expected size only in the H929/W3A clones. GST-E-cadherin binding assay and Western blot analysis revealed elevated levels of total and free beta-catenin in H929/W3A relative to H929/EV, however, there this was not associated with increased growth or proliferation by MTT assay. To determine the in-vivo growth characteristics and effects on bone resorption of Wnt-3A producing cells, we transplanted the lines into a human bone implanted the flank of SCID mice. Tumor growth rate as determined by increased production of human immunoglobulin in mice serum was significantly slower in the Wnt-3A transfected cells relative to controls (P < .05). Loss of bone mineral density (BMD) of the implanted bones engrafted with H929/W3A cells was lower than in bones engrafted with H929/EV cells (P < .05). Reduced tumor burden and BMD loss was also visualized on x-ray radiographs. Taken together these data indicate that all factors promoting bone resorption produced by or elicited by the myeloma cell line H929 are subordinate to canonical Wnt signaling and that prevention of bone destruction may help control myeloma progression.

Clarithromicin Induces Autophagy in Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3509-3509 ◽  
Author(s):  
Miki Nakamura ◽  
Takahiro Kamimoto ◽  
Tamotsu Yoshimori ◽  
Hiroaki Mitsuya ◽  
Hiroyuki Hata
Keyword(s):  
Electron Microscopy ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Fluorescent Microscopy ◽  
Myeloma Cell ◽  
Pi3 Kinase ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Akt Phosphorylation ◽  
Dose Dependent

Abstract Introduction Some macrolide antibiotics exert effects other than anti-bacterial activity on the growth and viability of certain cancer cells. The presence of cytoplasmic vacuoles is one the salient features of autophagy, a cellular event believed to recycle cellular ingredients under nutrient-starved conditions. Such vacuoles (autophagosomes) fuse with lysozomes, generating autolysozomes toward later stages of autophagy, digesting organelles and degenerated proteins. Our own and others’ findings that a macrolide antibiotic clarithromicin (CAM) occasionally shows anti-myeloma effects when combined with thalidomide and/or dexamethasone prompted us to examine CAM for its effects on myeloma cells in vitro. Methods Four myeloma cell lines (12PE, KHM-11, KMM-1 and U266) and primary myeloma cells purified by CD138-conjugated immune-magnetic beads (Miltenvi Biotec, Auburn, CA) were utilized. Clarithromicin was obtained from Taisho-Toyama pharmaceuticals (Tokyo, JAPAN). Morphology was analyzed either by May-Giemza staining or electron microscopy. Autolysozome was stained with Lysotracker (Invitrogen, Carlsbad, CA) and analyzed using fluorescent microscopy. Antibody to LC3 was obtained from Dr. T. Yoshimori (Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University). Results and discussion CAM induced vacuoles in the cytoplasm of both myeloma cell lines and primary myeloma cells at concentrations ranging from 10 to 50 mg/ml at a dose-dependent manner after ~18 hours treatment. Electron microscopy revealed that those vacuoles morphologically resemble autolysozomes. To further confirm the identity of autolysozomes, cells were stained with Lysotracker, which specifically stains acid lysozome. After the treatment with CAM, the accumulation of vacuoles in the cytoplasm, stained with Lysotacker, was observed. Since initiation of autophagy depends on PI3-kinase, we investigated whether CAM induced AKT phosphorylation. AKT phosphorylation was readily observed, and moreover, the emergence of vacuoles stainable with Lysotracker was inhibited when the cells were pretreated with PI3-kinase inhibitors, 3MA or LY294002, strongly suggesting that vacuolation is indeed mediated with PI3-kinase. To further confirm that autopahgy is induced by CAM, the process of LC3-I to LC3-II, a hallmark of autophagy, was examined. We found that the induction of LC3-II by CAM occurred at a dose-dependent manner. Taken together, these findings strongly suggest that CAM induces autolysozome accumulation through activating PI3-kinase. Finally, we examined whether CAM induced apoptosis when combined with thalidomide. Three myeloma cells lines, which abundantly expressed Bcl-2, showed no growth inhibition, while KHM-11, which was defective in Bcl-2, showed marked apoptosis and growth inhibition with the combination of CAM and thalidomide, suggesting that CAM might potentially augment anti-myeloma activity of thalidomide although the mechanisms are to be determined. Taken these observations together, the manipulation of certain autophagy processes with reagents such as macrolides (i.e., CAM) might represent a new therapeutic approach in the treatment of myeloma. We hypothesize that CAM dually functions in the event of autophagy, i.e., it initiates autophagy while it suppresses autophagy at later stages. Further study under the hypothesis is currently underway.

Arsenic Trioxide-Mediated Growth Inhibition of Myeloma Cells Is Associated with An Extrinsic Signaling Pathway Involved in Caspase 3 and Caspase 8.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4899-4899
Author(s):  
Jumei Shi ◽  
Yi Wu ◽  
Siqing Wang ◽  
Xiuqin Meng ◽  
Rong Wei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Arsenic Trioxide ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Myeloma Cells

Abstract Abstract 4899 Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying molecular mechanism remains elusive. This study was performed to evaluate the molecular mechanism underlying its anti-myeloma activities. Cells from OPM2, U266, RPMI8226 myeloma cell lines and patients diagnosed with myeloma (n=6) were cultured with various concentrations of ATO for 4 days. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. ATO induced potent inhibition of myeloma cell growth compared with untreated control cells. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb, while it up-regulated p53, p21Clip1, and p27Kip1 proteins, resulting in G2/M cell cycle arrest and cell growth inhibition. ATO treatment increased mRNA levels of interferon regulatory factor-1 (IRF-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway. No significant change was detected in the expression levels of Bax, Bcl-xL caspase 9 and Bcl-2, indicating that the intrinsic signaling pathway was not involved. A pan-caspase inhibitor abrogated ATO-induced apoptosis of myeloma cells. Our data suggest that ATO induces apoptosis in MM cells most likely through an extracellular signaling pathway. Disclosures No relevant conflicts of interest to declare.

Cancer-Testis Antigen MAGE-C2/CT10 Promotes Proliferation and Enhances Resistance to p53 Mediated Apoptosis in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1808-1808
Author(s):  
Nesrine Lajmi ◽  
Julia Templin ◽  
Sara Yousef ◽  
Tim Luetkens ◽  
Stefanie Spock ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Fold Increase ◽  
Myeloma Cell ◽  
Class I ◽  
Cycle Progression ◽  
Myeloma Cells ◽  
Healthy Donors ◽  
Cancer Testis

Abstract Abstract 1808 Background: Cancer-testis antigens belonging to the MAGE class I family of genes are commonly expressed in Multiple Myeloma (MM). Expression of MAGE class I genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that MAGE genes may confer a survival advantage on myeloma cells. MAGE-C2/CT10 is member of the MAGE class I family of genes thought to be a good candidate for cancer immunotherapy given its very frequent expression in primary myeloma. In normal cells, MAGE-C2/CT10 seems to suppress p53 expression by promoting its polyubiquitination and degradation. However, the function of MAGE-C2/CT10 in malignancies is completely unknown. We, therefore, investigated for the first time the role of MAGE-C2/CT10 in tumor cells derived from patients with MM. Material and Methods: MAGE-C2/CT10 expression was analysed by real-time PCR and western blot in myeloma cell lines (N=8) and in PBMC from healthy donors (N=8). For the functional evaluation of MAGE-C2/CT10 we decided to use myeloma cell line U-266 which constitutively expresses MAGE-C2/CT10 and a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The biological role of MAGE-C2/CT10 was investigated by stably silencing its expression using lentiviral short hairpin RNA (shRNA). The effects of silencing MAGE-C2/CT10 expression on myeloma cell biology were examined by determining the number of viable or apoptotic cells using a colorimetric MTT assay and annexin-V/7AAD staining followed by flow cytometry, In addition, we measured myeloma cell proliferation and the anchorage-independent growth using a BrdU incorporation assay and a colony formation assay, respectively. Finally, we investigated cell cycle phase distribution by flow cytometry and we analyzed the expression of key molecules involved in cell cycle progression and apoptosis using a real-time PCR array as well as western blot. Results: We found MAGE-C2/CT10 to be constitutively expressed in all myeloma cells lines but not in PBMC from healthy donors. Lentivirus-mediated silencing of MAGE-C2/CT10 inhibited significantly the proliferation and the anchorage-independent growth of myeloma cells. Cell cycle analysis demonstrated that the anti-proliferative effect of MAGE-C2/CT10 silencing in U-266 was due to a 70% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions and an increase in the subG0/G1 population due to an activation of apoptotic cell death. The serine-threonine checkpoint effector kinase 2 (CHK2) and its substrate, the tumor suppressor protein p53, are essential for cell cycle control, DNA repair and apoptosis. We found that the loss of MAGE-C2/CT10 expression was associated with the activation of CHK2 through phosphorylation at Thr68 as well as the activation of p53 by phosposphorylation at Ser20. Furthermore, we observed a three-fold increase in the endogenous level of p53 protein which correlated with an up-regulation of two transcriptional targets of p53, the cyclin-dependant kinase inhibitor p21WAF1 and the growth arrest and DNA-damage-inducible alpha protein (GADD45A), known to be essential for p53-induced G1 and G2 arrest, respectively. Finally, using the Human Apoptosis Profiler PCR array that contains a number of p53 target genes, we found that apoptosis induced by MAGE-C2/CT10 knockdown was due to a more than two-fold increase in the transcription of pro-apoptotic genes like BAX (Bcl2-associated × protein), BAD (BCL2-associated agonist of cell death), Cytochrome c, APAF1 (Apoptosis activating factors) as well as several caspases, which are the down-stream mediators of p53-dependant apoptosis in response to DNA damage. Conclusions: Our collected findings support an anti-apoptotic function of MAGE-C2/CT10 in MM, likely through the regulation of key molecules involved in cell cycle progression and p53-mediated apoptosis. The central role of MAGE-C2/CT10 in the biology of myeloma strongly suggest that this cancer-testis antigen represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM. Disclosures: Kröger: Fresenius Biotech: Honoraria, Research Funding.

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