Truncations in CCND1 mRNA 3′ UTR Alters microRNA Regulation in Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3184-3184
Author(s):  
Robert W. Chen ◽  
Lynne Bemis ◽  
Carol Amato ◽  
Birks Diane ◽  
Myint Han ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lymphoma ◽  
Regulation Of Gene Expression ◽  
Proliferative Potential ◽  
Transcriptional Level ◽  
Cell Cycle Regulator ◽  
Mantle Cell

Abstract Mantle Cell Lymphoma (MCL) represents only 5–10% of all non-Hodgkins lymphomas, making it an uncommon but difficult form of lymphoma to treat. It has a poor prognosis among the B cell lymphomas with median survival of three years. The genetic hallmark of MCL is the t(11,14) translocation causing amplification of cyclin D1 (CCND1), a known cell cycle regulator which is overexpressed in many other cancers. MicroRNAs (miRNA) are a new class of abundant small RNAs that play important regulatory roles at the post transcriptional level. They act by binding to the 3′ untranslated region (UTR) of mRNAs and block either their translation or initiate their degradation. Recent reports have shown truncations in the CCND1 3′ UTR occur in MCL and indicate a worse prognosis. We hypothesized that truncations in 3′ UTR of CCND1 alter it’s regulation by microRNAs. Based on bioinformatics, we identified microRNA 16 with putative docking sites in the 3′UTR of CCND1. Mir-16 has been implicated as a cell cycle regulator. We identified 2 cell lines (Jeko-1 and Z138) with truncations in CCND1 3′ UTR and demonstrated increased CCND1 mRNA expression by qRT-PCR, increased protein expression by western blot, and higher proliferative potential by cell cycle. We prepared a reporter construct by ligating the full length 3′ UTR of CCND1 to GFP. We then co-transfected this construct with mimics of mir-16 into a cancer cell line and demonstrated downregulation of CCND1 protein expression by flow cytometry. In the MCL cell line Granta-519 with non-truncated CCND1, transfection with mimics of mir-16 deminstrated decreased expression of CCND1 mRNA. These studies suggest that the overexpression of CCDN1 In MCL may result from altered regulation of gene expression from loss of a miRNA regulatory site and may give new clues into the patho-biology of this disease and insights into possible new therapies.

FTY720 Demonstrates Promising in-Vitro and in-Vivo Pre-Clinical Activity by Down-Modulation of Cyclin D1 and Pakt in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3728-3728
Author(s):  
Lapo Alinari ◽  
Qing Liu ◽  
Ching-Shih Chen ◽  
Fengting Yan ◽  
James T Dalton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lymphoma ◽  
Tumor Burden ◽  
Time Dependent ◽  
Control Group ◽  
Mantle Cell

Abstract Abstract 3728 Poster Board III-664 Over-expression of Cyclin D1 and constitutive phosphorylation of Akt has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Here we describe FTY720 (fingolimod), an immunosuppressive agent currently being explored in phase III studies in renal transplantation and multiple sclerosis patients, to mediate time- and dose-dependent cell death in primary MCL cells (6 patients) and MCL cell lines, Jeko and Mino. FTY720-induced apoptosis was associated with reactive oxygen species (ROS) generation, Bax up-regulation but not associated with caspase 3 activation in MCL. FTY720 treatment resulted in time-dependent down-modulation of Cyclin D1 and phospho Akt (p-Akt) protein level, two critical disease-relevant molecules in the pathogenesis of MCL. Consistent with the modulation of Cyclin D1, FTY720-induced cell cycle arrest with accumulation of cells in G0/G1 and G2/M phases of the cell cycle with concomitant decrease in S phase entry. Importantly, FTY720 treatment was also associated with a time-dependent phospho Erk (p-Erk) induction in Mino and Jeko cells. To determine the in vivo efficacy of FTY720, we developed a preclinical, in vivo xenograft model of human MCL where MCL cell lines (Jeko, Mino and SP53) were engrafted into severe combined immune deficient (SCID) mice. Cell dose titration trials identified 4 × 107 Mino or Jeko cells injected intravenously via tail vein to result in consistent engraftment and fatal tumor burden in all mice. All mice engrafted with 4 × 107 Jeko cells developed a disseminated disease within 3 weeks and had a median survival of 28 days (compared to 43 days for Mino and 51 days for SP53). Because the Jeko cell line was established from the peripheral blood of a patient with blastic variant MCL and demonstrated a more resistant phenotype to several immuno-chemoterapeutic compounds, this cell line was chosen to create a more stringent in vivo preclinical model. SCID mice were treated with the monoclonal antibody TMβ1 to deplete murine NK cells, engrafted with 4 × 107 Jeko cells and observed daily for signs of tumor burden. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal injection of 100 μl of saline or FTY720 (5 mg/kg resuspended in 100 μl of saline), every day, for two weeks. The median survival for FTY720-treated mice (N=10) was 38 days (95% CI:30-39) compared to 26.5 days (95% CI: 26-27 days) for the control group mice (N=10). The results from the log-rank test indicated an overall statistical significant difference in survival functions between the FTY720 treatment and the control group (p=0.001). These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant the further investigation of FTY720 in combination with other agents in clinical trials treating patients with MCL. Disclosures: No relevant conflicts of interest to declare.

Foxo 3a Signaling Mediates Apoptosis in Lymphoma Cells By the Diterpenoid Lactone Andrographolide (Andro), the Active Component of Andrographis Paniculata

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2760-2760
Author(s):  
Shuo Yang ◽  
Bo Ding ◽  
Fei Ying ◽  
Jana Svetlichnaya ◽  
Austin Tom ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Mantle Cell ◽  
Golgi Fragmentation ◽  
Bcl2 Expression

Abstract Introduction: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in lymphoma, leukemia and other solid tumor cell lines. We have shown that Andro caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples that was mediated through mitochondrial pathways and enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK (Yang et al Clin Cancer Res 2010; 16(19):4755). We hypothesized that the tumor suppressor, FOXO3a may be involved in signaling pathways that lead to apoptosis and to test that hypothesis we investigated the role of FOXO3A in Andro induced signaling in lymphoma. Methods: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the transformed follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL and MCL. We transfected shRNA FOXO3a by electroporation to build stable cells with constant knockdown of FOXO3a in Ramos and SUDHL4 cell lines. We then compared the cell viability (MTT and Golgi fragmentation), apoptosis (Annexin V by flow), c-MYC and Bcl2 expression, death receptors 4 (DR4) expression and cell cycle related proteins in wild type and FOXO3a knockdowns. Results: We found that Andro resulted in nuclear translocation of FOXO3a in Ramos at early time points. We found that shRNA stable knockdown of FOXO3a in Ramos and SUDHL4 cell lines protected cells (Ramos and SUDHL4) from Andro-induced apoptosis (Figure 1). Moreover, in multiple cell lines, we found that Andro decreased c-MYC expression, which was abrogated in part by FOXO3A knockdown compared with wild type cells. Similarly, reduction in mitochondrial membrane potential by Andro is abrogated in the FOXO 3a knockdown cells. These data suggest that FOXO3a regulates c-MYC stabilization by mitochondrial proteins (for example TFAM and MAD-1). In the Granta cell line, derived from Mantle Cell Lymphoma (MCL) and in an MCL patient sample, Andro reduced c-MYC expression. We also found that Andro induced Death Receptor 4 (DR4) at the mRNA and protein level in Granta cells in a dose-dependent manner. The cell cycle control proteins Aurora, p21, p27 (the latter 2 regulated by FOXO3a), are also increased by Andro. When cell death was measured by Golgi fragmentation and subsequent collapse, we found that Andro induced Golgi fragmentation in Granta and SUDHL4 cells Conclusion: Andro-induced lymphoma cell apoptosis is mediated through multiple signaling pathways, including FOXO3a, which appears to play a significant role, perhaps by regulating c-MYC stabilization and BCL2 expression and cell cycle proteins. These data suggest that this novel diterpenoid lactone compound deserves further pre-clinical and clinical testing in malignant lymphoma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Cell Cycle Dysregulation Represents an Early Effect of the Proteasome Inhibitor Bortezomib in Mantle Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2287-2287 ◽  
Author(s):  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Malte Rieken ◽  
Marc Weinkauf ◽  
Oliver Weigert ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
Genetic Alterations ◽  
Cell Cycle Regulators ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a distinct subtype of malignant lymphoma with an especially poor clinical outcome, a median survival time of 3 years and virtually no long-term survivors. On the molecular level, MCL is characterized by the chromosomal translocation t(11;14)(q13;q32) resulting in the constitutive overexpression of cyclin D1. However, additional genetic alterations of cell cycle regulators, e.g. deletions of the INK4A gene cluster, are detectable in the majority of cases. In various phase II studies the proteasome inhibitor bortezomib (Velcade) has demonstrated a high clinical efficacy with up to 60% remission rates in relapsed MCL. Additionally, in a previous in vitro study, the inhibitor induced a downregulation of cyclin D1 expression and a concomitant G(1) cell cycle arrest. However, little is known which molecules represent the critical targets of proteosome inhibition and how different regulators of cell cycle and apoptosis (inhibitors of CDK/INK4: p15INK4A, p16INK4B -and p14ARF and other kinase inhibitor proteins/KIP: 21CIP1, p27KIP1 and p57KIP2) are affected. 4 MCL cell lines (HBL2, GRANTA 519, Jeko-1, NCEB-1) and 2 hematological control cell lines (Jurkat, Karpas 422) were exposed to bortezomib at the minimal cytotoxic concentration (25 nmol) which corresponds to clinically achieved drug levels and results in a significant cytolysis after 48 – 72 hours. Real-time RT-PCR and protein expression levels of various CDK inhibitors (INK4s, KIPs) and cyclin D1 were determined a 0, 4, 8 and 12 hours after treatment with bortezomib. In addition, RNA- and protein expression data were compared to functional cell cycle phase (FACS) and cell apoptosis. Prelimenary data indicate that downregulation of cyclin D1 RNA expression after 12 and 24h of treatment represents a rather late event whereas alterations of other cell cycle regulators (like p21CIP1) were detected siginificantly earlier in all four MCL cell lines. Thus, expression of cell cycle regulators may indicate early events of proteasome inhibition. A comparative analysis of the cell cycle regulation network is currently being performed and will be presented at the conference.

Induction of Apoptosis in Jeko-1 Mantle Cell Lymphoma Cell Line by Resveratrol: A Proteomic Analysis

2008 ◽  
Vol 7 (7) ◽  
pp. 2670-2680 ◽  
Author(s):  
Daniela Cecconi ◽  
Alberto Zamò ◽  
Alice Parisi ◽  
Elena Bianchi ◽  
Claudia Parolini ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Proteomic Analysis ◽  
Cell Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cell Line ◽  
Mantle Cell Lymphoma Cell ◽  
Mantle Cell ◽  
Induction Of Apoptosis

scholarly journals Evaluation of novel cell cycle inhibitors in mantle cell lymphoma

Oncogene ◽  
2007 ◽  
Vol 26 (38) ◽  
pp. 5635-5642 ◽  
Author(s):  
I-W Park ◽  
M V R Reddy ◽  
E P Reddy ◽  
J E Groopman
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Cell Cycle Inhibitors

Cell Cycle Regulation by Iron: Novel Approaches to Mantle Cell Lymphoma Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2515-2515 ◽  
Author(s):  
Heather Gilbert ◽  
John Cumming ◽  
Josef T. Prchal
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
Lymphoma Cell ◽  
Protein Levels ◽  
Mantle Cell

Abstract Abstract 2515 Poster Board II-492 Mantle cell lymphoma is a well defined subtype of B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13 (which encodes cyclin D1) next to the immunoglobulin heavy chain gene promoter on chromosome 14q32. The result is constitutive overexpression of cyclin D1 (CD1) resulting in deregulation of the cell cycle and activation of cell survival mechanisms. There are no “standard” treatments for MCL. Despite response rates to many chemotherapy regimens of 50% to 70%, the disease typically progresses after treatment, with a median survival time of approximately 3-4 years. Mantle cell lymphoma represents a small portion of malignant lymphomas, but it accounts for a disproportionately large percentage of lymphoma-related mortality. Novel therapeutic approaches are needed. In 2007, Nurtjaha-Tjendraputra described how iron chelation causes post-translational degradation of cyclin D1 via von Hippel Lindau protein-independent ubiquitinization and subsequent proteasomal degradation (1). Nurtjaha-Tjendraputra demonstrated that iron chelation inhibits cell cycle progression and induces apoptosis via proteosomal degradation of cyclin D1 in various cell lines, including breast cancer, renal carcinoma, neuroepithelioma and melanoma. Our preliminary data show similar findings in mantle cell lymphoma. To establish whether iron chelation can selectively inhibit and promote apoptosis in mantle cell derived cell lines, the human MCL cell lines Jeko-1, Mino, Granta and Hb-12; the Diffuse Large B cell lymphoma line SUDHL-6; and the Burkitt's Lymphoma lines BL-41 and DG75 were grown with media only, with two different iron chelators (deferoxamine (DFO) and deferasirox) at various concentrations (10, 20, 40, 100 and 250 μM), and with DMSO as an appropriate vehicle control. Cells were harvested at 24, 48 and 72 hours. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V antibody and PI (BD Pharmingen, San Diego, CA). Cell growth was analyzed using the Promega MTS cytotoxicity assay. CD1 protein levels were assessed using standard Western blot techniques. At 24, 48 and 72 hours of incubation with iron chelators, the mantle cell lymphoma cell lines showed significantly increased rates of apoptosis compared to the non-mantle cell lymphoma cell lines (p<0.0001 for all time points). DFO and deferasirox inhibted cell growth with an IC50 of 18 and 12 μM respectively. All of the mantle cell lines had measurable cyclin D1 levels at baseline. None of the non-mantle cell lines expressed baseline measurable cyclin D1. In the mantle cell lines, cyclin D1 protein levels were no longer apparent on western blot after 24 hours of incubation with chelation. We then added ferrous ammonium sulfate (FAS) to DFO in a 1:1 molarity ratio and to deferasirox in a 2:1 ratio, and then treated the same lymphoma cell lines with the FAS/chelator mixture and with FAS alone for 72 hours. Adding iron to the chelators completely negated all the pro-apoptotic effects that were seen with iron chelation treatment. Treating with FAS alone had no effect on cell growth or apoptosis. Iron chelation therapy with both DFO and deferasirox results in decreased cell growth, increased cellular apoptosis, and decreased cyclin D1 protein levels in vitro in mantle cell lymphoma. The cytotoxic effects are prevented by coincubation with ferrous ammonium citrate, confirming that the effects are due to iron depletion. Proposed future research includes further defining the molecular basis of iron chelation effects; studying these therapies in combination with other cancer treatments both in vitro and in vivo; and studying iron chelation therapy in mantle cell lymphoma patients. 1. Nurtjahja-Tjendraputra, E., D. Fu, et al. (2007). “Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression.” Blood109(9): 4045–54. Disclosures: No relevant conflicts of interest to declare.

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