The c-Jun and JunB transcription factors facilitate the transit of classical Hodgkin lymphoma tumour cells through G1

Jingxi Zhang,Eugeniu Jantuan,Julinor T C Bacani,Ying-Han R Hsu,Mihye Yang,Anton Savin,Robert J Ingham,Zuoqiao Wu

doi:10.1038/s41598-018-34199-9

Abstract

Classical Hodgkin Lymphoma (cHL) is primarily a B cell lymphoid neoplasm and a member of the CD30–positive lymphomas. cHL and the other CD30–positive lymphomas are characterized by the elevated expression and/or constitutive activation of the activator protein-1 (AP-1) family transcription factors, c-Jun and JunB; however, the specific roles they play in the pathobiology of cHL are unclear. In this report we show that reducing either c-Jun or JunB expression with short-hairpin RNAs (shRNAs) reduced the growth of cHL cell lines in vitro and in vivo, primarily through impairing cell cycle transition through G1. We further investigated the effect of c-Jun and JunB knock-down on proliferation in another CD30–positive lymphoma, anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL). We found that JunB knock-down in most ALK+ ALCL cell lines examined also resulted in reduced proliferation that was associated with a G0/G1 cell cycle defect. In contrast, c-Jun knock-down in multiple ALK+ ALCL cell lines had no effect on proliferation. In summary, this study directly establishes that both c-Jun and JunB play roles in promoting HRS cell proliferation. Furthermore, we demonstrate there are similarities and differences in c-Jun and JunB function between cHL and ALK+ ALCL.

Highlights

Introduction of a JunB cDNA intoKarpas 299 cells. 5 × 106 Karpas 299 cells were transfected with 5 μg of plasmid DNA using an Amaxa nucleofector (Lonza AG; Basel Switzerland; Program A30, Kit V)
The dysregulation of multiple transcription factors are critical for the pathobiology of Classical Hodgkin Lymphoma (cHL), and in this study we show that the related activator protein-1 (AP-1) family transcription factors, c-Jun and JunB, each have important roles in promoting proliferation in this lymphoma
Knocking down either c-Jun or JunB expression was sufficient to reduce the growth rate of cHL cell lines in vitro and in vivo, which we demonstrate was a consequence of an impaired ability to transit through G1

Summary

Introduction

Introduction of a JunB cDNA intoKarpas 299 cells. 5 × 106 Karpas 299 cells were transfected with 5 μg of plasmid DNA (pcDNA3-EGFP-P2A or pcDNA3-EGFP-P2A-FLAG-JunB) using an Amaxa nucleofector (Lonza AG; Basel Switzerland; Program A30, Kit V). 5 × 106 Karpas 299 cells were transfected with 5 μg of plasmid DNA (pcDNA3-EGFP-P2A or pcDNA3-EGFP-P2A-FLAG-JunB) using an Amaxa nucleofector (Lonza AG; Basel Switzerland; Program A30, Kit V). The latter plasmid expresses a fusion protein between EGFP and JunB linked by the self-cleaving P2A peptide[53]. Seventy-two hours post transfection, cells were labelled with BrdU for 1 h, followed by LIVE/DEADTM Fixable Dead Cell Stain (Invitrogen; Carlsbad, CA) for 30 min on ice in the dark. Cells were stained with an anti-GFP antibody followed by a FITC-conjugated anti-rabbit F(ab)’ fragment to enable us to examine BrdU incorporation in transfected cells.

Methods

Results

Conclusion