E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

Abstract

EBV latent antigen EBNA3C is indispensible for in vitro B-cell immortalization resulting in continuously proliferating lymphoblastoid cell lines (LCLs). EBNA3C was previously shown to target pRb for ubiquitin-proteasome mediated degradation, which facilitates G1 to S transition controlled by the major transcriptional activator E2F1. E2F1 also plays a pivotal role in regulating DNA damage induced apoptosis through both p53-dependent and -independent pathways. In this study, we demonstrate that in response to DNA damage LCLs knocked down for EBNA3C undergo a drastic induction of apoptosis, as a possible consequence of both p53- and E2F1-mediated activities. Importantly, EBNA3C was previously shown to suppress p53-induced apoptosis. Now, we also show that EBNA3C efficiently blocks E2F1-mediated apoptosis, as well as its anti-proliferative effects in a p53-independent manner, in response to DNA damage. The N- and C-terminal domains of EBNA3C form a stable pRb independent complex with the N-terminal DNA-binding region of E2F1 responsible for inducing apoptosis. Mechanistically, we show that EBNA3C represses E2F1 transcriptional activity via blocking its DNA-binding activity at the responsive promoters of p73 and Apaf-1 apoptosis induced genes, and also facilitates E2F1 degradation in an ubiquitin-proteasome dependent fashion. Moreover, in response to DNA damage, E2F1 knockdown LCLs exhibited a significant reduction in apoptosis with higher cell-viability. In the presence of normal mitogenic stimuli the growth rate of LCLs knockdown for E2F1 was markedly impaired; indicating that E2F1 plays a dual role in EBV positive cells and that active engagement of the EBNA3C-E2F1 complex is crucial for inhibition of DNA damage induced E2F1-mediated apoptosis. This study offers novel insights into our current understanding of EBV biology and enhances the potential for development of effective therapies against EBV associated B-cell lymphomas.

Author Summary

Aberrant cellular proliferation due to deregulation of E2F1 transcriptional activity as a result of either genetic or functional alterations of its upstream components is a hallmark of human cancer. Interestingly, E2F1 can also promote cellular apoptosis regardless of p53 status by activating a number of pro-apoptotic genes in response to DNA damage stimuli. Epstein-Barr virus (EBV) encoded essential latent antigen EBNA3C can suppress p53-mediated apoptotic activities. This study now demonstrates that EBNA3C can further impede E2F1 mediated apoptosis by inhibiting its transcriptional ability, as well as by facilitating its degradation in an ubiquitin-proteasome dependent manner. This is the first evidence, which shows through targeting EBNA3C function linked to the E2F1-mediated apoptotic pathway, an additional therapeutic platform could be implemented against EBV-associated human B-cell lymphomas.