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c-JUN N-terminal Kinase (JNK) is activated/phosphorylated by upstream MAPK kinases (MKK), and, in turn, phosphorylates and activates its major substrate c-JUN, a member of the activator protein-1 (AP-1) transcription factors. c-JUN is overexpressed and activated in Hodgkin and Reed Sternberg cells (HRS) of classical Hodgkin lymphoma (cHL), however, the mechanism of its activation remains unknown. JNK activation was immunohistochemically assessed in 60 cases of HL and in a control group of 151 B-cell non-Hodgkin lymphomas. The biologic effects of JNK activation in cultured HRS cells were investigated using colony formation, cell growth and viability assays and cell cycle analysis by flow cytometry. Western blotting was used to assess protein levels. p-JNK was expressed in 90% of HL, 83% of Burkitt lymphomas, 28% of mantle cell lymphomas, 23% of diffuse large B-cell lymphomas, 19% of follicular lymphomas, and 18% of extranodal marginal zone lymphomas of MALT type. None of the 48 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma and 18 cases of plasma cell myeloma showed JNK phosphorylation (P < 001, Kruskall-Wallis test). Pharmacological inhibition of JNK activity in cultured HRS cells resulted in a significant decrease of cell growth, which was associated with cell cycle arrest at the G2/M phase. The cell cycle effects were linked to deactivation of c-JUN and upregulation of its known target, the cyclin-dependent kinase inhibitor p21. JNK is highly activated in HRS cells, and may contribute to uncontrolled cell cycle progression and proliferation of tumor cells in cHL., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fas-associated death domain protein is a key component of the extrinsic apoptotic pathway. In addition, in animal models, Fas-associated death domain protein phosphorylation at serine 194 has been shown to affect cell proliferation, especially in T lymphocytes. The importance of Fas-associated death domain protein phosphorylation at serine 194 for the proliferation of B lymphocytes, however, is uncertain. Here we show in reactive lymph nodes that serine 194 phosphorylated Fas-associated death domain protein is expressed predominantly in the dark (proliferative) zone of germinal centers. In B-cell non-Hodgkin lymphoma cell lines, serine 194 phosphorylated Fas-associated death domain protein levels are substantially higher in highly proliferating cells and lower in serum-starved cells. We also used immunohistochemical analysis to assess Fas-associated death domain protein phosphorylation at serine 194 expression in 122 B-cell non-Hodgkin-type lymphomas. The mean percentage of serine 194 phosphorylated Fas-associated death domain protein positive tumor cells was 81% in Burkitt lymphoma, 41% in diffuse large B-cell lymphoma, 18% in follicular lymphoma, 18% in plasma cell myeloma, 12% in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, 11% in mantle cell lymphoma, and 2% in chronic lymphocytic leukemia/small lymphocytic lymphoma (P < .0001, Kruskal-Wallis test). Furthermore, in chronic lymphocytic leukemia/small lymphocytic lymphoma, serine 194 phosphorylated Fas-associated death domain protein was detected predominantly in proliferation centers. In the entire study group, the percentage of cells positive for serine 194 phosphorylated Fas-associated death domain protein correlated significantly with the proliferation index Ki-67 (Spearman R = 0.9, P < .0001). These data provide evidence that serine 194 phosphorylated Fas-associated death domain protein is involved in the proliferation of normal and neoplastic B cells and has features of a novel proliferation marker., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mantle cell lymphoma is a distinct type of B-cell lymphoma characterized by the t(11;14)(q13;q32). Mantle cell lymphomas exhibit a spectrum of morphologic findings, of which a subset of tumors is clinically aggressive with a high proliferation rate. These neoplasms are known as aggressive variants of which there are blastoid and pleomorphic subsets. CKS-1B (CDC28 protein kinase regulatory subunit 1B) is essential for the ubiquitination and degradation of p27 and cell cycle progression. We analyzed CKS-1B expression in mantle cell lymphoma cell lines and tumors by Western blot and immunohistochemical analysis. In 4 mantle cell lymphoma cell lines, CKS-1B was expressed at variable levels and correlated inversely with p27 expression. In mantle cell lymphoma tumors, CKS-1B was positive in 10 (28.6%) of 35 typical versus 14 (87.5%) of 16 blastoid/pleomorphic cases (Fisher exact test, P = .0002). Analyzed as a continuous variable, the percentage of CKS-1B-positive cells significantly correlated with blastoid/pleomorphic morphology (Mann-Whitney U test, P = .001). Twelve (23.5%) of 51 mantle cell lymphoma tumors expressed p27. Proliferation rate (Ki-67) was higher in blastoid/pleomorphic variants than in typical mantle cell lymphoma tumors and was inversely associated with p27 levels in typical mantle cell lymphoma. However, CKS-1B expression did not correlate with p27 expression, proliferation rate, or prognosis in the entire study group. Fluorescence in situ hybridization analysis of 10 CKS-1B-positive mantle cell lymphoma tumors showed no evidence of CKS-1B gene amplification. We conclude that CKS-1B is commonly expressed in mantle cell lymphoma, particularly in aggressive histologic variants, and may be involved in pathogenesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The Mix1 homeobox-like (MIXL1) gene encodes a paired class homeobox transcription factor that is involved in embryogenesis. Previous studies have shown that the MIXL1 gene product is expressed in B- and T-cell progenitors of normal bone marrow and, in some cell lines derived from hematopoietic neoplasms. The status of MIXL1 expression and subcellular localization in human lymphomas is unknown. Using a highly specific antibody, we assessed for MIXL1 expression in lymphoma cell lines of B- and T-cell lineage by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. We also assessed for MIXL1 expression using immunohistochemical methods in 193 lymphoid tumors, including 140 B-cell non-Hodgkin lymphomas (NHL), 36 T-cell NHL, and 17 Hodgkin lymphomas (HL). MIXL1 was detected predominantly in the nuclear fraction of all cell lines tested and was predominantly nuclear in primary tumor specimens. Based on the distribution of the staining results (histogram), a 50% cutoff was selected for high versus low MIXL1 expression. High MIXL1 expression was detected more frequently in Burkitt lymphoma and diffuse large B-cell lymphoma compared with other types of B-cell NHL (P < .0001, chi(2) test). Most cases of T-cell NHL and all cases of HL also highly expressed MIXL1. Most plasma cell myelomas were negative for MIXL1, but rare cases had low MIXL1 expression. MIXL1 expression significantly correlated with proliferation index (Ki-67) in B-cell NHL (P < .0001). The frequent and high expression of MIXL1 in aggressive B-cell NHL, T-cell NHL, and HL suggests that MIXL1 may be involved in lymphomagenesis.

Anaplastic large cell lymphoma (ALCL) includes a subset of tumors that has abnormalities of chromosome 2p23, resulting in overexpression of anaplastic lymphoma kinase (ALK). Previous studies have reported differences in apoptotic rate and expression levels of apoptosis regulatory proteins between ALK+ and ALK- ALCL. In this study, we assessed for expression of the intrinsic apoptotic pathway proteins cytochrome c, apoptosis protease-activating factor 1, and procaspase 9 in 2 ALK+ ALCL cell lines and 42 ALCL tumors (17 ALK+, 25 ALK-). We used the Karpas 299 and SU-DHL-1 cell lines, and the inhibitors Z-LEHD-FMK (specific for caspase 9) and Boc-D-FMK (general caspase inhibitor) to investigate the role of caspase 9 activation in chemotherapy-induced apoptotic cell death. Caspase 9 activity was significantly increased in Karpas-299 and SU-DHL-1 cells after chemotherapy treatment, but remained as low as control levels with addition of either caspase inhibitor. Both caspase inhibitors rescued a substantial fraction of Karpas 299 and SU-DHL-1 cells from drug-induced cell death. In ALCL tumors, expression of cytochrome c, apoptosis protease-activating factor 1, and procaspase 9 was also assessed and correlated with apoptotic rate and activated caspase 3 levels. Cytochrome c was expressed in all 13 (100%) ALK+ and 18 (95%) of 19 ALK- ALCL tumors. Apoptosis protease-activating factor 1 was detected in 14 (88%) of 16 ALK+ and 19 (79%) of 24 ALK- ALCL tumors. Procaspase 9 was expressed in 5 (30%) of 17 ALK+ and 2 (8%) of 25 ALK- ALCL tumors (P = .09). In the entire study group (ALK+ and ALK- ALCL), procaspase 9 expression levels significantly correlated with apoptotic rate (P = .02) and activated caspase 3 levels (P = .05). This correlation could not be shown in the ALK+ or ALK- ALCL subgroups, presumably because of the small sample size. In conclusion, chemotherapy-induced cell death in ALK+ ALCL cells involves the intrinsic apoptotic pathway, and apoptosome function may be an important determinant of apoptosis in ALCL tumors.