Abstract
Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.
A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression