Hodgkin Lymphoma Cell Line L428 Research Articles

Identification of Differentially Expressed Human Endogenous Retrovirus Families in Human Leukemia and Lymphoma Cell Lines and Stem Cells.

Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB.

Proteomic analysis and verification of protein expression after upregulation of human CD99 in Hodgkin lymphoma cell line L428

目的探索稳定过表达CD99前后霍奇金淋巴瘤细胞株L428细胞中差异表达的蛋白，并分析验证差异蛋白的功能。方法采用荧光差异凝胶双向电泳和质谱分析技术检测L428细胞稳定过表达CD99前后蛋白表达的差异，采用GOfact开放软件对差异蛋白进行聚类分析，筛选在细胞转化过程中起重要作用的蛋白，并分析验证其功能。结果筛选鉴定得到38个差异蛋白，其中在稳定过表达CD99的霍奇金淋巴瘤细胞株L428（L428-CD99细胞）中与CD99表达呈正相关的蛋白21个，呈负相关的蛋白17个，所得的38个差异蛋白中有32个蛋白参与了细胞的生物过程，35个蛋白参与了细胞的组成和构建，28个参与了抗氧化、蛋白结合、催化活性、酶调节、信号转导、结构分子、翻译调节和离子转运等多个方面。结论筛选得到38个差异蛋白，其中关于细胞骨架、细胞分化、信号通路、调节基因表达等蛋白的改变与H/RS细胞和B淋巴瘤细胞转化相关。

Small and big Hodgkin-Reed-Sternberg cells of Hodgkin lymphoma cell lines L-428 and L-1236 lack consistent differences in gene expression profiles and are capable to reconstitute each other.

The hallmark of classical Hodgkin lymphoma (cHL) is the presence of giant, mostly multinucleated Hodgkin-Reed-Sternberg (HRS) cells. Whereas it has recently been shown that giant HRS cells evolve from small Hodgkin cells by incomplete cytokinesis and re-fusion of tethered sister cells, it remains unsolved why this phenomenon particularly takes place in this lymphoma and what the differences between these cell types of variable sizes are. The aim of the present study was to characterize microdissected small and giant HRS cells by gene expression profiling and to assess differences of clonal growth behavior as well as susceptibility toward cytotoxic intervention between these different cell types to provide more insight into their distinct cellular potential. Applying stringent filter criteria, only two differentially expressed genes between small and giant HRS cells, SHFM1 and LDHB, were identified. With looser filter criteria, 13 genes were identified to be differentially overexpressed in small compared to giant HRS cells. These were mainly related to energy metabolism and protein synthesis, further suggesting that small Hodgkin cells resemble the proliferative compartment of cHL. SHFM1, which is known to be involved in the generation of giant cells, was downregulated in giant RS cells at the RNA level. However, reduced mRNA levels of SHFM1, LDHB and HSPA8 did not translate into decreased protein levels in giant HRS cells. In cell culture experiments it was observed that the fraction of small and big HRS cells was adjusted to the basic level several days after enrichment of these populations via cell sorting, indicating that small and big HRS cells can reconstitute the full spectrum of cells usually observed in the culture. However, assessment of clonal growth of HRS cells indicated a significantly reduced potential of big HRS cells to form single cell colonies. Taken together, our findings pinpoint to strong similarities but also some differences between small and big HRS cells.

Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

BackgroundCannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.Design and MethodsWe examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated.ResultsA predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.ConclusionsThe present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Ets-1 Activates Overexpression of JunB and CD30 in Hodgkin's Lymphoma and Anaplastic Large-Cell Lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

NK Cells in Hodgkin Lymphoma Are Impaired but Can Be Activated

Abstract 2182 Introduction:NK cells represent the key component of the innate immune system to recognize and eliminate cancer cells. Defects in NK cell function including impaired cytotoxicity/cytokine secretion, aberrant receptor expression profile, NK cell number and NK cell anergy are reported in non Hodgkin lymphoma and correlate with a bad prognosis. So far, nothing is known about the phenotype of peripheral NK cells and serum levels of ligands for NK cell receptors in Hodgkin Lymphoma (HL) patients. Here, cytotoxicity, expression pattern of activating NK cell receptors and the serum levels of several ligands for the key cytotoxic receptors NKG2D and NKp30 are determined. Methods:The cytotoxicity of NK cells isolated from HL patients was analysed by europium release assay using the HL cell line L428 as target cells. The serum level of the NKp30-ligand BAT3 and ligands for NKG2D (MICA, MICB and ULBP1,2,3) was estimated in sera of 117 HL patients and 40 healthy donors by ELISA. The expression pattern of NKp30, NKp44, NKp46, CD16 and the activation markers CD25, CD69 and CD71 was determined by 4-colour FACS analysis of peripheral blood lymphocytes. Results:The cytotoxicity assays reveal a significantly reduced killing efficacy of NK cells from HL patients against the Hodgkin cell line L428 in comparison to NK cells from healthy donors. Correlating with the impaired NK cell function, we observed that the serum level for BAT3 and MICA was significantly elevated in HL patients, whereas other ligands (MICB and ULBP1,2,3) remained unchanged. NKG2D showed a significantly decreased expression on NK cells of HL patients. No significant difference was observed for all other receptors and activation markers tested. Conclusion:Our results suggest that soluble BAT3 and MICA, ligands for NKp30 and NKG2D, contribute to the NK cell inhibition in HL patients. Since soluble ligands for NK cell receptors are known to inhibit NK cell-cytotoxicity, the release of these ligands might represent an immune escape mechanism of HL tumors to avoid detection and killing by the innate immune system. To overcome NK cell inhibition in HL patients we design, express and purify bispecific proteins (immunoligands) that target NKG2D and a HL-specific tumorantigen. Work to activate HL-derived NK cells with immunoligands ex vivo will be discussed. Disclosures:Engert:Affimed Therapeutics AG: Honoraria, Research Funding.

Expression of the c-Met Oncogene Correlates with Favorable Progression Free Survival In Classical Hodgkin Lymphoma

AbstractAbstract 3880 BackgroundThe HGF/c-Met signaling pathway regulates a variety of biological processes, including proliferation, survival and migration. Aberrant c-Met activation has been implicated in the pathogenesis of many human cancers. Furthermore, c-Met is of prognostic significance in several types of cancer such as diffuse large B cell lymphoma, bladder cancer, breast cancer, colorectal cancer and ovarian cancer. Expression of c-Met in Hodgkin lymphoma (HL) patients, as well as increased levels of HGF in the urine of HL patients has been reported, but no prognostic studies or functional data have been reported. MethodsWe studied the prognostic significance of c-Met expression by immunohistochemistry on paraffin sections of classical HL (cHL) patients from two independent patient cohorts. Functional studies were performed on HL cell line L428 that showed high c-Met levels, constitutive phosphorylated c-Met and no HGF expression. The effect of stimulation by HGF and inhibition by c-Met kinase inhibitor SU11274 was investigated by Western blot, cell proliferation and cell cycle progression analysis. ResultsExpression of c-Met was detected in Hodgkin Reed-Sternberg (HRS) cells in 52% (79/152) of primary cHL tissue samples and expression of its ligand, hepatocyte growth factor (HGF), was detected in HRS cells in 8% (10/120) of the cHL patients. Co-expression of HGF and c-Met in HRS cells was observed in only 3% (4/120) of cHL patients. A variable percentage of infiltrating cells stained positive for HGF, supporting a predominant paracrine activation route. In contrast to its adverse prognostic impact in other cancers, high c-Met expression significantly correlated with favorable 5 year progression free survival in both patient cohorts. To explain these unexpected findings we studied the c-Met/HGF signaling pathway in the L428 cHL cell line. The levels of phosphorylated c-Met, Akt, and Erk1/2 were upregulated upon HGF stimulation and this induction could be blocked by inhibiting c-Met activation with SU11274. Activation with HGF did not effect cell growth, while SU11274 alone suppressed cell growth. SU11274, as well as inhibitors of PI3K, MEK1/2 and Erk1/2 (downstream targets of the HGF/c-Met signaling pathway) induced G2/M cycle arrest. ConclusionExpression of c-Met in tumor cells was observed in 52% of cHL patients. In contrast to its prognostic value in other cancers, expression of c-Met in HRS cells of cHL patients correlated with favorable prognosis in two independent cohorts. Although triggering of c-Met in L428 cells with HGF induced activation of its downstream factors, no effect was observed on proliferation. Remarkably, the c-Met inhibitor did suppress cell growth of L428 cells. Disclosures:No relevant conflicts of interest to declare.

The Epstein-Barr Virus (EBV) Encoded Oncoprotein LMP1 Mediates Down Regulation of Shelterin Proteins, Formation of Telomere Aggregates and Multinuclearity.

AbstractAbstract 3625 Introduction:Tumour cells in classical Hodgkin's lymphoma (HL) and Burkitt's lymphoma (BL) are derived from germinal center B-cells and may or may not harbour the EBV genome. In both, EBV-negative and EBV-associated HL, the 3D nuclear organization of telomeres marks the transition from mononuclear Hodgkin (H-cell) to diagnostic multinuclear Reed-Sternberg (RS-cell) cells. Moreover, transient expression of the EBV-encoded oncoprotein LMP1 in the HL cell lines L-428 and HD-MyZ significantly increases the percentage of RS cells. However, the molecular mechanisms through which this selective LMP1 oncoprotein expression promotes the transition from H- to RS-cells is still unknown. In order to answer this question we analyzed the 3D telomere dynamics and the expression of key shelterin proteins at the transcriptional, translational and topographical localization level in the EBV-negative Burkitt cell line BJAB, stably transfected with an inducible LMP1 system (tetracycline on/off system). To mimic the permanent LMP1 expression as observed in tumour cells of EBV-associated Hodgkin's lymphoma, we extended the period of LMP1 expression up to 21 days and examined key proteins of the shelterin complex, 3D telomere dynamics and formation of multinuclear cells in interphase nuclei of BJAB Burkitt's lymphoma cells at day 1, 3, 7, 10, 14 and 21. Statistical analysis was performed using nested or two-way analysis of variance. Results:Already at day 3 LMP1 induced substantial down regulation of the shelterin key-components TRF1, TRF2 and POT1 at the transcriptional and protein level. This down regulation was reversible after LMP1 suppression; in particular, suppression of LMP1 induction at day 7 returned shelterin-key components to the initial level of expression at day 14. Massive LMP1 mediated down regulation of shelterin proteins was also confirmed by immunofluorescence. At day 7, stable LMP1 expression led to a significant increase of 3D telomere aggregates, nuclear volume and polyploid cells, most of them multinucleated, when compared to non-LMP1 expressing BJAB controls (p < 0.02). These changes progressed over time and at day 21 LMP1 expressing BJAB cells were characterized by highly significant increase of 3D telomere aggregates (p = 0.0005) and increased nuclear volume (p < 0.0001), compared to the LMP1-suppressed control. Discussion: In an experimental setting mimicking the in vivo conditions of EBV-associated HL, i.e. a permanent LMP1 expression in the H-cells, the LMP1 oncoprotein deregulates shelterin-expression, leads to telomere aggregates and mediates multinuclearity. Our findings point to a shelterin/telomere related key function of LMP1 in Reed-Sternberg cell formation in EBV-associated Hodgkin's lymphoma and may explain the four fold increased risk to develop HL within a median incubation time of four years after symptomatic EBV infection. The LMP1-dependent mechanisms involved are probably unprotected telomeres (shelterin down regulation) favouring chromosomal rearrangements as a result of telomeric aggregate formation and ongoing bridge-fusion- bridge cycles which lead to disturbed cytokinesis and finally multinuclearity. Disclosures:No relevant conflicts of interest to declare.

Nutlin-3A, an MDM2 Antagonist, Induces p53-Dependent Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma (HL).

p53 is the most frequently mutated tumor suppressor gene in human cancer. However, in Hodgkin lymphoma (HL) p53 is mutated only in a small subset of cases suggesting that modulation of wild-type-p53 (wt-p53) levels in Hodgkin and Reed-Sternberg (HRS) cells may have therapeutic implications in these patients. MDM2 (HDM2 in humans) is a physiologic negative regulator of p53 levels through a well-established auto-regulatory feedback loop. Nutlin-3A is a recently developed small molecule, which antagonizes mdm2 through disruption of p53-MDM2 interaction resulting in p53 stabilization. We hypothesized that nutlin 3A may stabilize p53 in HRS cells carrying wt-p53 gene, thus leading to p53-dependent apoptosis and G1-S cell cycle arrest. We used two novel classical HL cell lines recently established in our Institution, MDA-V and MDA-E, which have been shown to carry wt-p53 gene. As a control, we used a HL cell line L-428 harboring a mutant p53 (mt-p53) gene product (deletion at exon 4). We investigated effects on apoptosis and cell cycle arrest after treatment of cultured HRS cells with nutlin-3A or a 150-fold less active enantiomere, nutlin-3B. Treatment with nutlin-3A resulted in substantial cell death (up to 65%) in a concentration-dependent manner associated with increased apoptosis as shown by apoptotic morphology (DAPI immunofluorescence), annexin V binding (flow cytometry) and caspase activation (Western blot analysis) in MDA-V and MDA-E cells, but not in L-428 cells. Nutlin-3A-induced apoptotic cell death was accompanied by stabilization of p53 protein as detected by western blot analysis and immunofluorescence and up-regulation of pro-apoptotic Bax, a known target of p53. Inhibition of nuclear export by leptomycin B stabilized p53 at a similar level as compared to nutlin-3A treatment in these cells, suggesting that nutlin-3A stabilized p53 through inhibition of MDM2-mediated degradation of the protein. By contrast, no changes in cell viability, growth or apoptosis were seen after treatment with the inactive nutlin-3B small molecule. Treatment with nutlin-3A also resulted in a significant decrease (up to 85%) of cells in S-phase and a dose-dependent increase of cells in G1 phase of cell cycle as detected by flow cytometry, in MDA-V and MDA-E cells, but not in L-428 cells. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21, a transcriptional target of p53. In contrast, treatment of HRS cells with nutlin-3B had no effects on the cell cycle irrespective of p53 mutation status. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed synergistic effects and enhanced cytotoxicity in HRS cells with wt-p53 gene. Targeting MDM2 with the specific antagonist nutlin-3A that leads to non-genotoxic p53 activation, apoptosis induction and cell cycle inhibition may provide a new therapeutic approach for patients with HL.