title: Induction of p16(INK4a) is the major barrier to proliferation when Epstein-Barr virus (EBV) transforms primary B cells into lymphoblastoid cell lines.
abstract: To explore the role of p16(INK4a) as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16(INK4a) and p14(ARF). Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16(INK4a) protein but expressing a functional 14(ARF)-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C--in cooperation with EBNA3A--despite the absence of functional p16(INK4a). Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16(INK4a) and growth arrest, EBNA3C inactivation in the p16(INK4a)-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16(INK4a)-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16(INK4a) expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are p16(INK4a)-null, functional EBNA3C is dispensable for the outgrowth of LCLs.
authors: Section of Virology, Department of Medicine, Imperial College London, St Mary's Campus, London, United Kingdom.
authors: Lenka
authors: L
authors: Skalska
authors: Robert E
authors: RE
authors: White
authors: Gillian A
authors: GA
authors: Parker
authors: Department of Oncology, University of Cambridge, Hills  Road, Cambridge CB2 0XZ, United Kingdom and Cancer Research UK, Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE, United Kingdom
authors: Ernest
authors: E
authors: Turro
authors: Alison J
authors: AJ
authors: Sinclair
authors: Kostas
authors: K
authors: Paschos
authors: Martin J
authors: MJ
authors: Allday
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D001402:B-Lymphocytes / Q000821:virology*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D063185:Epigenetic Repression / Q000235:genetics*; D020031:Epstein-Barr Virus Infections / Q000276:immunology / Q000821:virology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism; D056426:Genetic Loci; D004854:Herpesvirus 4, Human / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008213:Lymphocyte Activation; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D061251:Primary Cell Culture; D017735:Virus Latency
chemical_list: D000956:Antigens, Viral; D019941:Cyclin-Dependent Kinase Inhibitor p16; C508151:EBNA-3A antigen; C498774:EBNA-3C, epstein-barr virus; D019309:Epstein-Barr Virus Nuclear Antigens