Altered Expressions of CD30, c-Jun N-Terminal Kinase, NF-κB and JunB Are Involved in Disease Progression in CD30-Positive Anaplastic Large Cell Lymphoma.

Abstract

Anaplastic large cell lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is an aggressive T cell lymphoma. CD30 is a characteristic cell surface protein overexpressed in ALCL cells as well as in malignant cells of Hodgkin lymphoma. CD30-mediated signals are molecular target(s) in the therapy for CD30-positive lymphoma. We studied molecular pathway(s) involved in the disease progression in two ALK- negative CD30-positive ALCL cell lines (N1, N2), which are recently established. In vitro culture, N1 and N2 represent early and advanced stage lymphoma phenotype, respectively. N2, compared to N1, was characterized by the increase of colony formation in soft agar more than 100 fold, the increase of cell surface expression of CD30 protein (p<0.0001) and the increase of activated (nuclear) NF- κB activities (p=0.0039). N2 showed significant decrease of cell surface expressions of co-stimulatory molecules such as CD80 and CD86 and decrease of adhesion molecules such as CD2, CD29 and CD49d, which suggest loss of these molecules are also involved in disease progression by allowing ALCL cells to escape immune systems of host against tumors and metastasize. Western blot analysis showed that N2 expressed CD30 protein with molecular weight of 100 Kd, whereas N1 expressed membrane molecule CD30 protein with 120Kd. These two different form of CD30 proteins were coimmunoprecipitated with TNF receptor-associated factor (TRAF)2 but not TRAF5, suggesting ligand-independent association of CD30 and TRAF2 has a function in CD30-mediated signal transduction in these cell lines. In both of N1 and N2, constitutive activation of AP-1 was demonstrated by gel shift analysis using AP-1 DNA consensus sequence. c-Jun N-terminal kinase (JNK) was highly phosphorylated in N2 compared to in N1 (p-JNK/total JNK, N1:0.14, N2:0.31). No significant difference was noted in status of extracellular-regulated kinase (ERK) and p38 kinase. Among Jun family proteins, levels of expression of JunB protein, but not c-Jun and JunD, decreased to 26% of those in N1 as judged by densitometric analysis. Three days incubation with JNK inhibitor, SP600125 (100μM) and NF- κB inhibitor, helenalin (1.0μM) inhibited cell growth of N2 to 25.4% and to51.2% of control culture, respectively. Furthermore, both of those inhibitors restored the levels of expression of JunB protein in N2 to 66% with 50μM SP600125and to 61% with 1.0μM helenalin, compared with those of N1. Increased expression of CD30 protein in N2 was not affected by the incubation with either of those inhibitors. Semi-quantitative RT-PCR analyses showed that growth inhibition by helenalin was accompanied with down-regulation of BCL2 mRNA(39.3±1.5% of control) in N2. Effect of SP600125 on BCL2 mRNA was negligible. In contrast, growth inhibition by SP600125 but not by helenalin resulted in up-regulation of CD86 mRNA(318±3% of control) in N2. Taken together, the present study showed that aberrantly activated JNK/AP-1 and NF- κB, at least in part, are responsible for disease progression in some types of CD30-positive ALCL, although details of signaling pathways triggered by altered CD30 in ALCL cells still remained to be elucidated. In addition, the data suggested that JunB, as a downstream molecule regulated by JNK and/or NF- κB, had a putative tumor suppressor function in CD30-positive lymphoma cells.