Cellular Transformation of Mouse Embryo Fibroblasts in the Absence of Activator E2Fs Tushar Gupta, Maria Teresa Sáenz Robles, James M. Pipas Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA ABSTRACT The E2F family of transcription factors, broadly divided into activator and repressor E2Fs, regulates cell cycle genes. Current models indicate that activator E2Fs are necessary for cell cycle progression and tumorigenesis and are also required to mediate transformation induced by DNA tumor viruses. E2Fs are negatively regulated by the retinoblastoma (RB) family of tumor sup- pressor proteins, and virus-encoded oncogenes disrupt the RB-E2F repressor complexes. This results in the release of activator E2Fs and induction of E2F-dependent genes. In agreement, expression of large tumor T antigens (TAg) encoded by polyomavi- ruses in mammalian cells results in increased transcriptional levels of E2F target genes. In addition, tumorigenesis induced by transgenic expression of simian virus 40 (SV40) TAg in choroid plexus or intestinal villi requires at least one activator E2F. In contrast, we show that SV40 TAg-induced transformation in mouse embryonic fibroblasts is independent of activator E2Fs. This work, coupled with recent studies showing that proliferation in stem and progenitor cells is independent of activator E2Fs, sug- gests the presence of parallel pathways governing cell proliferation and tumorigenesis. IMPORTANCE The RB-E2F pathway is altered in many cancers and is also targeted by DNA tumor viruses. Viral oncoprotein action on RBs re- sults in the release of activator E2Fs and upregulation of E2F target genes; thus, activator E2Fs are considered essential for nor- mal and tumorigenic cell proliferation. However, we have observed that SV40 large T antigen can induce cell proliferation and transformation in the absence of activator E2Fs. Our results also suggest that TAg action on pRBs regulates both E2F-dependent and -independent pathways that govern proliferation. Thus, specific cell proliferation pathways affected by RB alterations in cancer may be a factor in tumor behavior and response to therapy. C ell proliferation is a highly regulated process involving four well-coordinated phases of the cell cycle. Each phase is regu- lated by various pro- and antiproliferation pathways, whose dis- ruption leads to either cell death or uncontrolled cell proliferation and cancer. One of those controllers is the retinoblastoma (RB)/ E2F pathway, and mutations in members of this pathway are often found in human cancers, leading to enhanced E2F activity and upregulation of E2F target gene (1). Furthermore, various viral oncoproteins—including the large tumor (T) antigen (TAg) from polyomaviruses, E1A from adenoviruses, and E7 from papilloma- viruses— have independently evolved to target this pathway, in- dicating its central role in cell cycle regulation (2–4). E2Fs are transcription factors that regulate cell proliferation by controlling the expression of cell cycle genes. In a growth-arrested cell, the E2Fs are bound to and negatively regulated by hypophos- phorylated members of the retinoblastoma (RB) protein family (pRb, p107, p130) (1). Upon receiving mitogenic signals, pRBs become hyperphosphorylated and release E2Fs, which then in- crease the transcription of cell cycle genes. There are nine known mammalian E2Fs, broadly divided into transcriptional activators (E2F1, E2F2, and E2F3a [E2F1-2-3a]) and repressors (E2F3b-4-8) (1). E2F proteins are functionally very similar and often can com- pensate for each other, both within each subclass and even be- tween the two subclasses, thus complicating their study (1, 5, 6). Previous reports have suggested an essential role of the activa- tor E2Fs in cell survival and proliferation: genetic ablation of E2F1-2-3 in mice leads to embryonic lethality at 9.5 days, and their depletion from mouse embryonic fibroblasts (MEFs) in cell cul- ture leads to growth arrest and death (5, 7). Similarly, while MEFs derived from E2F single-knockout or double-knockout (DKO) mice are able to survive and proliferate in cell culture, removal of the third activator E2F—the E2F1-2-3 triple-knockout (TKO) MEFs—results in growth arrest and death (5, 7). However, certain murine cell types, both in vivo (the progenitor cells of retina, lens, and small intestinal crypts) and in cell culture (embryonic stem cells), proliferate in the absence of the three activator E2Fs (8–10). Furthermore, several rounds of cell proliferation take place in E2F1-2-3 null embryos until they die by day 9.5. These results suggest that, at least in some instances, mechanisms other than activator E2Fs control cell survival and proliferation. The large tumor antigen (TAg) of polyomavirus simian virus 40 (SV40) is a multidomain oncoprotein that interacts with and disrupts the function of several host proteins, thereby interfering with specific cellular pathways (11, 12). TAg expression causes transformation of several primary cell types in cell culture and also induces tumors in model organisms. Studies investigating the Received 15 December 2014 Accepted 18 February 2015 Accepted manuscript posted online 25 February 2015 Citation Gupta T, Sáenz Robles MT, Pipas JM. 2015. Cellular transformation of mouse embryo fibroblasts in the absence of activator E2Fs. J Virol 89:5124 –5133. doi:10.1128/JVI.03578-14. Editor: M. J. Imperiale Address correspondence to James M. Pipas, pipas@pitt.edu. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03578-14 5124 jvi.asm.org May 2015 Volume 89 Number 9 Journal of Virology Downloaded from https://journals.asm.org/journal/jvi on 13 January 2022 by 35.170.82.111.