Biology and Microenvironment
Rianne Veenstra, Joost Kluiver, Lydia Visser, Arjan Diepstra and Anke van den Berg
University of Groningen, University Medical Center Groningen, Department of Pathology & Medical Biology, Groningen, Netherlands
Introduction: TCF3 is a transcription factor, which plays an important role in the development of normal B and T cells. Activity of TCF3 can be inhibited by the ID2 and ABF-1 proteins that both are highly expressed in classical Hodgkin Lymphoma (cHL). Lower TCF3 levels or activity might contribute to the loss-of-B-cell phenotype, which is a defining feature of cHL tumor cells.
Methods: The effect of modulating TCF3, ID2 and ABF-1 on growth of cHL cell lines L428, L1236 and KMH2 was assessed in a GFP competition assay. For TCF3 overexpression an empty vector control was used as a negative control. For knockdown of TCF3, ID2 and ABF-1 two shRNA constructs were made and a non-targeting shRNA was used as negative control. All constructs contained a GFP gene, and the effect on cell growth was determined by measuring the GFP+ cell percentage in a mixed population over a period of three weeks. Gene expression profiling was performed to identify cHL specific downstream targets for TCF3.
Results: Endogenous levels of TCF3 were significantly decreased in cHL cell lines compared to germinal center (GC) B cells. Endogenous levels of the two main inhibitors of TCF3, ID2 and ABF-1, were significantly increased in cHL cell lines compared to GC B cells. TCF3 overexpression was validated at the mRNA and protein level and induced a strong negative effect on cell growth in all three cHL cell lines, with a 50% reduction in GFP% cells in six days. Knockdown of TCF3 or ID2 did not affect growth of cHL cell lines. ABF-1 knockdown had a negative effect on cell growth in 2 cHL cell lines, with a 50% reduction in GFP+ cells in 20 to 27 days. Knockdown of ABF-1 in KMH2 cells did not affect cell growth. Using a 2-fold cut-of criterion and a p-value of <0.05 we identified 143 and 18 differentially expressed genes upon ABF-1 knockdown in L428 and L1236 cells.
Conclusion: Ectopic expression of TCF3 had a strong negative effect on cell growth in all cHL cell lines, suggesting a pro-apoptotic and/or anti-proliferative role for TCF3 in cHL. Downregulation of ABF-1, resulting in a functional restoration of TCF3 also had a negative effect on cell growth. Inhibition of ID2 had no effect in cHL cell lines, consistent with its overall low expression levels. A variable number of genes responded to ABF-1 knockdown in the two cHL cell lines. We are currently analyzing the functions and involved pathways of the TCF3-regulated genes to further understand the role of TCF3 in cHL.