Classical Hodgkin Lymphoma Cell Lines Research Articles

Analysis of CCL5 expression in classical Hodgkin's lymphoma L428 cell line

CCL5 is one of the chemoattractant cytokines involved in inflammatory observed in both diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin's lymphoma (CHL). However, the pathological effects of CCL5 remain unclear. To gain a better understanding of the role of CCL5 in CHL and DLBCL, we examined the expression of CCL5 in the CHL cell line L428 and the DLBCL cell lines Ly1 and Ly8, as well as its chemotactic effect on CD4+ T cells. CCL5 mRNA expression was detected by real-time quantitative RT-PCR. Intracellular CCL5 protein expression was analyzed using confocal microscopy, and CCL5 protein secretion was detected by ELISA. The chemotactic function of CCL5 was assessed using a Transwell coculture system, and the number of migrated CD4+ T cells was counted. Moreover, the p-iкBα and p65 levels of NF-кB signaling molecules in these lymphoma cell lines were detected by Western blotting. The results showed that CCL5 mRNA and protein expression in the L428 cells was significantly higher than in Ly1 and Ly8 cells (p<0.05). L428 cells secreted more CCL5 than the Ly1 or Ly8 cells, and the secreted CCL5 was capable of inducing CD4+ T cell migration. The expression levels of the NF-кB transcription factors p65 and p-iкBα were examined in these lymphoma cells. L428, Ly1 and Ly8 cells expressed similar levels of p65, while p-iкBα expression was higher in the L428 cells than in the Ly1 or Ly8 cells, indicating that a high CCL5 expression may be related to the increased activity of the NF-кB signaling pathway in L428 cells.

Glioma-associated oncogene homologue 3, a hedgehog transcription factor, is highly expressed in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma

The hedgehog signaling pathway has been shown to play a pathogenic role in diffuse large B-cell lymphoma and anaplastic large cell lymphoma, but has not been assessed in classical Hodgkin lymphoma. Glioma-associated oncogene homologues 1, 2, and 3 are transcriptional effectors of the hedgehog pathway. In this study, we first used real-time quantitative polymerase chain reaction to investigate the expressions of GLI1, GLI2, and GLI3 in 3 classical Hodgkin lymphoma cell lines. GLI1 and GLI2 were variably expressed, but GLI3 was highly expressed in all cell lines. We then used immunohistochemistry to assess glioma-associated oncogene homologues 1, 2, and 3 in 39 classical Hodgkin lymphoma patient samples. Glioma-associated oncogene homologues 1 and 2 were weakly to variably expressed in a subset of classical Hodgkin lymphoma patient samples. In contrast, glioma-associated oncogene homologue 3 showed strong, uniform nuclear expression in virtually all Hodgkin/Reed-Stenberg cells. We then performed an immunohistochemical survey of glioma-associated oncogene homologue 3 expression in 13 cases of nodular lymphocyte predominant Hodgkin lymphoma and 218 non-Hodgkin lymphomas. Most other lymphoma types showed variable or no expression of glioma-associated oncogene homologue 3, with a minor subset of cases of nodular lymphocyte predominant Hodgkin lymphoma, ALK-positive and ALK-negative anaplastic large cell lymphoma, and B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma showing a glioma-associated oncogene homologue 3 staining pattern indistinguishable from classical Hodgkin lymphoma. Our data provide a rationale to further investigate the biologic significance of glioma-associated oncogene homologue 3 in classical Hodgkin lymphoma biology.

BAY 43‐9006/Sorafenib blocks CSF1R activity and induces apoptosis in various classical Hodgkin lymphoma cell lines

BAY 43‐9006/Sorafenib blocks CSF1R activity and induces apoptosis in various classical Hodgkin lymphoma cell lines

Expression of Grb2 distinguishes classical Hodgkin lymphomas from primary mediastinal B-cell lymphomas and other diffuse large B-cell lymphomas

Growth factor receptor-bound protein 2 is an adaptor molecule that mediates B-cell receptor (BCR) signaling pathways, but the expression of growth factor receptor-bound protein 2 in lymphoma tissues has not been reported. We sought to characterize growth factor receptor-bound protein 2 protein expression in reactive tonsillar tissues and lymphoma tissues obtained from diagnostic biopsies of classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and 20 low-grade B-cell lymphomas. Growth factor receptor-bound protein 2 expression was assessed in tissues by immunohistochemistry and in lymphoma cell lines by immunoblotting. In reactive lymphoid tissues, growth factor receptor-bound protein 2 was expressed in the cytoplasm of B-cells and histiocytes but not T-cells. Strong, cytoplasmic growth factor receptor-bound protein 2 expression was seen in the neoplastic cells of follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, splenic marginal zone lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, and nodular lymphocyte predominant Hodgkin lymphoma. In contrast, only 10% of the classical Hodgkin lymphomas showed growth factor receptor-bound protein 2 expression in the neoplastic cells. Growth factor receptor-bound protein 2 protein expression was detected by Western blotting in all lymphoma cell lines tested with higher levels in primary mediastinal large B-cell lymphoma compared with classical Hodgkin lymphoma cell lines. These findings support a role for growth factor receptor-bound protein 2 in the diagnostically challenging workup of classical Hodgkin lymphoma versus primary mediastinal large B-cell lymphoma and warrant further studies to evaluate the biologic significance of growth factor receptor-bound protein 2 in the pathogenesis of classical Hodgkin lymphoma.

STAT6 and STAT1 are essential antagonistic regulators of cell survival in classical Hodgkin lymphoma cell line

Classical Hodgkin lymphoma (cHL) is a malignant lymphoid disorder characterized by aberrant activation of signaling pathways. Constitutive activation of several components of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been observed in Hodgkin and Reed/Sternberg cells, the tumor cells of cHL. In this study, we investigate the function of STAT6 in cHL cell lines and show that STAT6 promotes survival of these cells. Microarray expression analysis of STAT6-shRNA (short hairpin RNA)-expressing cHL cell lines was carried out to analyze the STAT6-mediated survival mechanism. Some of the identified genes with potentially important regulatory functions were also interleukin (IL)-4 dependently regulated in Ramos B cells and binding of STAT6 to the regulatory regions of several genes could be confirmed, indicating that these are direct STAT6 target genes. Importantly, STAT6 knockdown increased the expression and activation of STAT1 as well as the expression of known STAT1 target genes, indicating a cross-regulation between these signaling molecules. Forced expression of STAT1 was able to induce apoptosis in cHL cell line L1236. These findings indicate that both STAT6 and STAT1 can act as important antagonistic regulators in the pathogenesis of cHL.

Identification of candidate tumour suppressor gene loci for Hodgkin and Reed‐Sternberg cells by characterisation of homozygous deletions in classical Hodgkin lymphoma cell lines

Several tumour suppressor genes (TSG) have been identified as a result of mapping homozygous deletions in cancer cells. To identify putative TSG involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we investigated four cHL cell lines (L428, HDLM2, KMH2, L1236) using four different array-Comparative Genomic Hybridisation (array-CGH) platforms and focused on high resolution identification of homozygous deletions. Out of 79 candidate regions of bi-allelic loss identified by array-CGH, besides previously described regions, 28 novel regions of homozygous deletions could be verified by polymerase chain reaction. These regions ranged from 13 kb to 619 kb in size. Eleven of the 28 novel bi-allelic losses were putative copy number polymorphisms. This left 17 regions that might harbour novel tumour suppressors involved in Hodgkin lymphoma. Expression profiling with two different platforms confirmed lack of expression of the majority of the genes located in the homozygous deletions. Furthermore, analysis of ontology annotations of genes located in the homozygously deleted regions indicated an enrichment of genes involved in apoptosis and cell death. In summary, through the mapping of homozygous deletions in cell lines this study identified a series of genes, such as SEPT9, GNG7 and CYBB, which might encode candidate tumour suppressors involved in the pathogenesis of cHL.

Expression of the Human Germinal Center Associated Lymphoma (HGAL) Protein Identifies a Subset of Classical Hodgkin Lymphoma of Germinal Center Derivation and Improved Outcome.

Classical Hodgkin lymphoma (CHL) exhibit crippled somatic mutations in rearranged Ig genes and an immunophenotype unlike any other hematolymphoid malignancy. Expression profiles of CHL cell lines show that CHL is most closely related to EBV-transformed peripheral blood B-cells with marked loss of B lineage-specific gene expression. The Human Germinal center-Associated Lymphoma (HGAL) gene was identified as a candidate molecule that predicted improved outcome in diffuse large B-cell lymphoma. Previously, we showed that HGAL mRNA and protein are expressed specifically in germinal centers (GC), GC-derived lymphomas and a substantial proportion of CHL. Here, we sought to investigate whether HGAL protein expression could serve to distinguish biologically distinct subgroups of CHL. Ninety informative cases of CHL on a tissue microarray of 1.0 mm representative cores of paraffin embedded tissue were used. Staining was performed and the percent of stained Hodgkin cells was scored on a 3-tiered scale as negative (0%), weak (<30%), and strong (>30%), as previously described (Natkunam et al. Blood 2005, 105:3979–86). These cutoffs were chosen before results were correlated with clinical endpoints. Twenty-five patients (28%) showed no staining for HGAL and 25 patients (28%) had weak staining; HGAL was strongly expressed in 40 patients (44%). Thirteen of the 90 patients had died and 19 had failed (i.e. relapsed or died). The overall 5-year survival (OS) and failure-free survival (FFS) rates were 87% + 4% and 79% + 4%, respectively. HGAL expression was associated with improved OS in univariate analysis (p=0.04, no vs weak/strong staining), as were age < 45 years (p<0.001), stage I or II (p=0.02), and possibly histology (nodular sclerosing vs other, p=0.09). However, in multivariate analysis with age, stage and histology, HGAL expression no longer had an impact (p=0.93). HGAL did not impact FFS in either univariate (p=0.13) or multivariate analysis (p=0.87). The expression of the GC-specific marker HGAL in a subset of CHL suggests that a substantial proportion of CHL retain characteristics of lymphomas of GC derivation. The association with improved OS in univariate but not multivariate analysis suggests HGAL may be a biologic marker related to known clinical parameters of improved OS and FFS. Comparative studies with well-characterized GC markers, BCL6 and CD10, and with BCL2, MUM1 and Blimp-1 are underway to further define biologically distinct subsets of CHL. Analysis of additional cases of CHL is also on-going to substantiate these findings in multivariate analysis.