Article |
Address correspondence to Eiji Hara, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK. Tel.: 44-161-446-3122. Fax: 44-161-446-3075. E-mail: Ehara{at}picr.man.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cell cycle; immortalization; Cdk; Ets; senescence
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epstein-Barr virus (EBV) is a prevalent human herpes virus. It is frequently associated with a number of human proliferative and malignant diseases, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, and gastric carcinoma (for review see Farrell, 1995; Thorley-Lawson, 2001). Nine viral oncoproteins are expressed in EBV-established lymphoblastoid cell lines, five of which appear to be absolutely required for B cell immortalization (Hammerschmidt and Sugden, 1989, Cohen et al., 1991; Kaye et al., 1993; Tomkinson et al., 1993; Kilger et al., 1998). Among these oncoproteins, latent membrane protein 1 (LMP1) has been shown to transform established rodent fibroblasts and immortalize primary rodent fibroblasts (Wang et al., 1985; Yang et al., 2000a, b; Eliopoulos and Young, 2001). Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, such as human papillomavirus E7 or adenovirus E1A (Jansen-Durr, 1996; Classon and Harlow, 2002), LMP does not bind to the pRB family proteins. Recently, LMP1 was shown to block the induction of p16INK4a and to prevent Ras-induced senescence in human fibroblasts, suggesting that the p16INK4a could be an important target of LMP1 in fibroblasts (Yang et al., 2000b). However, until now, it has been unclear how LMP1 blocks induction of p16INK4a expression in primary human fibroblasts.
To obtain mechanistic insight into how LMP1 inhibits p16INK4a expression, we examined the effect of LMP1 on Ets2, which is an important transcription factor inducing p16INK4a expression in Ras-induced senescence (Ohtani et al., 2001). Here, we report that LMP1 inactivates Ets2 by promoting the intracellular redistribution of Ets2 from the nucleus to the cytoplasm in a CRM1-dependent manner. Furthermore, we find here that LMP1 also inactivates E2F4 and E2F5 (E2F4/5), which are essential downstream mediators of the p16INK4aRB growth arrest pathway (Gaubatz et al., 2000), also through promoting the CRM1-dependent intracellular redistribution of E2F4/5. These findings reveal a novel activity of the LMP1 oncoprotein and would facilitate understanding of how LMP1 oncoprotein of EBV perturbs p16INK4aRB pathway.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
CTAR1 and CTAR2 domains are required for LMP1-induced intracellular redistribution of E2F4 from the nucleus to the cytoplasm
LMP1 is composed of six transmembrane domains and a long carboxy-terminal cytoplasmic segment. The region containing the six transmembrane domains mediates its oligomerization in the cytoplasmic membrane, resulting in the constitutive activation of the downstream signals (Eliopoulos and Young, 2001). There are at least two functional domains (CTAR1 and CTAR2) in the cytoplasmic tail of LMP1, which activate multiple signal transduction pathways (Brown et al., 2001; Schultheiss et al., 2001; Thorley-Lawson, 2001). Therefore, we examined the effect of a series of LMP1 mutants lacking CTAR1 and/or CTAR2 domain on the subcellular localization of E2F4 (Fig. 7 A). As shown in Fig. 7 B, LMP1 mutants lacking CTAR1 and/or CTAR2 domain failed to induce cytoplasmic accumulation of E2F4, suggesting that the signaling from both CTAR1 and CTAR2 domains of LMP1 are required for intracellular redistribution of E2F4. This is consistent with a previous observations that the mutant LMP1 lacking CTAR2 failed to immortalize MEFs (Xin et al., 2001), and both CTAR1 and CTAR2 domains are necessary for efficient B cell immortalization (Eliopoulos and Young, 2001).
|
To evaluate the impact of the LMP1-induced intracellular redistribution of E2F4 on cell growth, we next tested whether or not ectopic expression of NLSE2F4 can counteract LMP1-induced cell proliferation. Because E2F4 acts as a repressor complex through interacting with pRB family proteins, we coexpressed unphosphorylated form of pRB with NLSE2F4 in early passage TIG-3 cells. LMP1 expression significantly increased the cell number even in the presence of unphosphorylated form of pRB (Fig. 7 D, lane 3). This effect was completely blocked by coexpression of NLSE2F4, whereas coexpression of wild-type E2F4 did not have a significant effect on cell growth (Fig. 7 D, lanes 4 and 5). These results demonstrate the relevance of LMP1-induced intracellular redistribution of E2F4 to LMP1-dependent cell proliferation.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here, we show that LMP1 blocks Ets2 transcriptional activity through promoting a CRM1-dependent intracellular redistribution of Ets2 from the nucleus to the cytoplasm, thereby reducing the level of p16INK4a expression (Figs. 1, 2, 5, and 6). Because p16INK4a expression is also regulated by other factors such as bmi-1, JunB, 143-3, and SNF5 (Jacobs et al., 1999; Dellambra et al., 2000; Passegue and Wagner, 2000; Betz et al., 2002), LMP1 may affect these transcription factors as well. However, we found here that LMP1 also targets downstream mediators of p16INK4aRB pathway. It has been suggested that the p16INK4a-induced growth arrest requires a function provided by a complex that contains p107 or p130, and E2F4 or E2F5 (Bruce et al., 2000; Gaubatz et al., 2000). Although inactivation of all three activator E2Fs, E2F13, causes a G1 arrest in MEFs (Wu et al., 2001), MEFs lacking both repressor E2Fs, E2F4/5, grow normally but are insensitive to a p16INK4a-induced G1 arrest (Gaubatz et al., 2000). This suggests that E2F4/5 are essential downstream mediators of p16INK4a-induced growth arrest pathway.
Our results shown here clearly demonstrate that LMP1 blocks the function of E2F4/5 by promoting a CRM1-dependent intracellular redistribution of E2F4/5 from the nucleus to the cytoplasm. Because E2F4/5 lacks an NLS, E2F4/5 requires binding to NLS-containing proteins for nuclear localization (Trimarchi and Lees, 2002). Interaction between E2F4 and pRB family proteins seems to be a key for its nuclear localization, because E2F4 only localizes in the cytoplasm in MEFs lacking both p107 and p130 (Rayman et al., 2002). Indeed, we observed that LMP1 induces dissociation of E2F4 from pRB family proteins (Fig. 6 A). Moreover, LMP1 failed to promote cytoplasmic accumulation of E2F4 if E2F4 is fused to NLS (Fig. 6 C). This evidence strongly suggests that dissociation of E2F4 from pRB family proteins is essential for LMP1-induced intracellular redistribution of E2F4 from the nucleus to the cytoplasm. However, subcellular localization of cellular proteins is generally dependent on the ratio of nuclear import and export. Thus, nuclear import/export machinery can be affected by LMP1. Indeed, overexpression of CRM1 alone was sufficient to promote cytoplasmic accumulation of E2F4 and mutation of NES sequences in E2F4 or treatment with LMB rendered E2F4 insensitive to LMP1-induced intracellular redistribution (Figs. 4 A and 6 C). Moreover, expression of LMP1 significantly increased the binding between endogenous CRM1 and endogenous E2F4 (Fig. 6 D). Therefore, it is possible that the increased binding between E2F4 and CRM1 is a key for LMP1-induced cytoplasmic accumulation of E2F4, although LMP1 might dissociate E2F4 from pRB family protein in a parallel pathway. Both Ets2 and E2F5 do not contain typical NES sequences (Boulukos et al., 1989; Graves and Petersen, 1998; Ducret et al., 1999; Gaubatz et al., 2001; Sharrocks, 2001). However, it is quite possible that both proteins contain unidentified NES sequences, because NES is not a well-defined sequence (la Cour et al., 2003). Indeed, we were able to see significant interaction between endogenous Ets2 and endogenous CRM1 in LMP1-expressing cells (Fig. 6 E, lanes 1 and 2). This evidence strongly suggests that LMP1 induces intracellular redistribution of Ets2 through, at least partly, increasing the binding between Ets2 and CRM1. It is also important to note that we were unable to see cytoplasmic accumulation of Ets2 in serum-stimulated cells (unpublished data). Moreover, LMP1-induced intracellular redistribution of Ets2 and E2F4 was also seen in the cells arrested in G1 phase (unpublished data), precluding the possibility that these effects may be secondary consequences of cell cycle progression induced by LMP1.
Together, it is evident that LMP1-induced intracellular redistribution has at least two effects on the p16INK4aRB pathway: (1) inhibition of p16INK4a expression and (2) blocking the function of downstream mediators of the p16INK4aRB pathway (Fig. 7 E, model). It is interesting to note that other NES-containing proteins, such as p27Kip1 (Fig. 2 B, 3 and 4), are resistant to LMP1-induced intracellular redistribution. Moreover, we were unable to see any increase of binding between CRM1 and p27Kip1 (Fig. 6 D). Similar results were seen in interaction between CRM1 and cyclinB1, which is also known as another NES-containing protein (Fig. 6 D), suggesting that there must be some target specificity of LMP1-induced intracellular redistribution. Because both CTAR1 and CTAR2 (CTAR1/2) domains are required for LMP1-induced intracellular redistribution, multiple signal transduction pathways are likely to be involved in LMP1-induced intracellular redistribution of Ets2 and E2F4/5 (Fig. 7, AC). U0126 and U0125, both are specific inhibitors of MEK1/2 pathway, efficiently attenuated the activity of LMP1 on intracellular redistribution of E2F4, whereas other pharmacological inhibitors did not have significant impact on LMP1 activity (Fig. 7 C). This suggests that LMP1 may induce intracellular redistribution of transcription factors, at least partly, through MEK1/2 pathways. Although further work is required to understand how signaling activated by CTAR1/2 induces Ets2 and E2F4 binding to CRM1 in future studies, our work reveals the novel activity of LMP1 oncoprotein. In conclusion, this paper provides the first evidence that the viral oncoprotein blocks p16INK4aRB pathway through targeting certain transcription factors for CRM1-dependent intracellular redistribution. These findings would provide a new insight into how viral oncoprotein can deregulate cell proliferation leading to cancer.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Luciferase reporter assays
Luciferase reporter activities driven by the human p16INK4A gene promoter (Ohtani et al., 2001), the human Rb gene promoter, and 36 tandem repeats of Ets binding sites were assayed using SVts8 cells as described previously (Ohtani et al., 2001). Effector plasmids were cotransfected as indicated in the figures, along with a standard amount of the MMLV-lacZ control plasmid. Cells were harvested 48 h after transfection and assayed for luciferase and ß-galactosidase. Luciferase activities were normalized to the corresponding ß-galactosidase activity.
ChIPs assay
ChIP assays were performed as reported previously (Ohtani et al., 2001). After immunoprecipitation with a polyclonal antiserum (#57) against Ets2 (Ohtani et al., 2001), the recovered DNA was analyzed by PCR with primers flanking the putative Ets binding site in the p16 promoter: 5'-TGCTCGGAGTTAATAGCACC-3' and 5'-CTCCATGCTGCTCCCCGCCG-3'.
Antibodies and protein analysis
Immunoblotting and immunoprecipitation were performed as described previously (Sugimoto et al., 1999) with primary antibodies against p16INK4A (Oncogene Research Products), Ras (Calbiochem), MEK1/2 (New England Biolabs, Inc.), phospho-MEK1/2 (New England Biolabs, Inc.), Ets2 (polyclonal antibody; Santa Cruz Biotechnology, Inc.; mAb: 10B3; Sanij et al., 2003), LMP1 (LMP025) Lamin A/C (Santa Cruz Biotechnology, Inc.), E2F4 (Santa Cruz Biotechnology, Inc.), -tubulin (Sigma-Aldrich), Flag M2 (Sigma-Aldrich), RB (BD Biosciences), Phospho-RB (Ser780; Cell Signaling), Phospho-RB (Ser795; Cell Signaling), Phospho-RB (Ser807/811; Cell Signaling), p107 (Santa Cruz Biotechnology, Inc.), p130 (Santa Cruz Biotechnology, Inc.), Sp1 (Santa Cruz Biotechnology, Inc.), and CRM1 (Santa Cruz Biotechnology, Inc.). The nuclear and cytoplasmic fractions were prepared using NE-PER nuclear cytoplasmic extraction reagents (Pierce Chemical Co.) as described previously (Chen et al., 2002).
Immunofluorescence and BrdU incorporation
Immunofluorescence analyses were performed as described previously (Llanos et al., 2001) using primary antibodies against Ets2 (Santa Cruz Biotechnology, Inc.), E2F4 (Santa Cruz Biotechnology, Inc.), Id1 (Santa Cruz Biotechnology, Inc.), LMP1(LMP025), Flag M2 (Sigma-Aldrich), and HA (Roche). Alexa Fluor546 and 488 (Molecular Probes) and tetramethylrhodamine (DakoCytomation) were used as second antibodies. BrdU incorporation assays were performed as reported previously (Gaubatz et al., 2001).
![]() |
Acknowledgments |
---|
This work was supported by grants from the Cancer Research UK and the Association for International Cancer Research to E. Hara (grant 02028). N. Ohtani is partly supported by the Uehara Memorial Foundation.
Submitted: 14 February 2003
Revised: 8 May 2003
Accepted: 3 June 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alcorta, D.A., Y. Xiong, D. Phelps, G. Hannon, D. Beach, and J.C. Barrett. 1996. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence. Proc. Natl. Acad. Sci. USA. 93:1374213747.
Betz, B.L., M.W. Strobeck, D.N. Reisman, E.S. Knudsen, and B.E. Weissman. 2002. Re-expression of hSNF5/INI1/BAF47 in pediatric tumor cells leads to G1 arrest associated with induction of p16ink4a and activation of RB. Oncogene. 21:51935203.[CrossRef][Medline]
Boulukos, K.E., P. Pognonec, B. Rabault, A. Begue, and J. Ghysdael. 1989. Definition of an Ets1 protein domain required for nuclear localization in cells and DNA-binding activity in vitro. Mol. Cell. Biol. 9:57185721.[Medline]
Brookes, S., J. Rowe, M. Ruas, S. Llanos, P. A. Clark, M. Lomax, M.C. James, R. Vatcheva, S. Bates, K.H. Vousden, D. Parry, N. Gruis, N. Smit, W. Bergman, and G. Peters. 2002. INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. EMBO J. 21:29362945.
Brown, K.D., B.S. Hostager, and G.A. Bishop. 2001. Differential signaling and tumor necrosis factor receptor-associated factor (TRAF) degradation mediated by CD40 and Epstein-Barr Virus oncoprotein latent membrane protein 1 (LMP1). J. Exp. Med. 193:943954.
Bruce, J.L., R.K. Hurford, Jr., M. Classon, J. Koh, and N. Dyson. 2000. Requirements of cell cycle arrest by p16INK4a. Mol. Cell. 6:737742.[Medline]
Campisi, J. 2001. Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol. 11:S27S31.[CrossRef][Medline]
Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:27452752.[Medline]
Chen, C.R., Y. Kang, P.M. Siegel, and J. Massague. 2002. E2F4/5 and p107 Smad cofactors linking the TGFbeta receptor to c-myc repression. Cell. 110:1932.[Medline]
Classon, M., and E. Harlow. 2002. The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer. 2:910917.[CrossRef][Medline]
Cohen, J.L., F. Wang, and E. Kieff. 1991. Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation. J. Virol. 65:25452554.[Medline]
Dajee, M., M. Lazarov, J.Y. Zhang, T. Cal, C.L. Green, A.J. Russell, M.P. Marinkovich, S. Tao, Q. Lin, Y. Kubo, and P.A. Khavari. 2003. NF-kappaB blockade and oncogenic Ras trigger invasive human epidermal neoplasia. Nature. 421:639643.[CrossRef][Medline]
Dellambra, E., O. Golisano, S. Bondanza, E. Siviero, P. Lacal, M. Molinari, S. D'Atri, and M. de Luca. 2000. Down regulation of 14-3-3 prevents clonal evolution and leads to immortalization of primary human keratinocytes. J. Cell Biol. 149:11171129.
DePinho, R.A. 2000. The age of cancer. Nature. 408:248254.[CrossRef][Medline]
Dowson, C.W., G. Tramountanis, A.G. Eliopoulos, and L.S. Young. 2003. Epstein-Barr virus latent memebrane protein 1 (LMP1) activates the phosphatidylinositol 3-kinase/Akt pathway to promote cell survival and induce actin filament remodeling. J. Biol. Chem. 278:36943704.
Drayton, S., and G. Peters. 2002. Immortalisation and transformation revisited. Curr. Opin. Genet. Dev. 12:98104.[CrossRef][Medline]
Ducret, C., S.M. Maira, A. Dierich, and B. Wasylyk. 1999. The Net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock. Mol. Cell. Biol. 19:70767087.
Eliopoulos, A.G., and L.S. Young. 2001. LMP1 structure and signal transduction. Semin. Cancer Biol. 11:435444.[CrossRef][Medline]
Farrell, P.J. 1995. Epstein-Barr virus immortalizing genes. Trends Microbiol. 3:105109.[CrossRef][Medline]
Foos, G., J.J. Garcia-Ramirez, C.K. Galang, and C.A. Hauser. 1998. Elevated expression of Ets2 or distinct proteins of Ets2 can reverse ras-mediated cellular transformation. J. Biol. Chem. 273:1887118880.
Fornerod, M., M. Ohno, M. Yoshida, and I.W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 90:10511060.[Medline]
Fukuda, M., W. Kurosaki, K. Yanagihara, H. Kuratsune, and T. Sairenji. 2002. A mechanism in Epstein-Barr virus oncogenesis: inhibition of transforming growth factor-beta 1-mediated induction of MAPK/p21 by LMP1. Virology. 302:310320.[CrossRef][Medline]
Gaubatz, S., G.J. Lindeman, S. Ishida, L. Jakoi, J.R. Nevins, D.M. Livingston, and R.E. Rempel. 2000. E2F4 and E2F5 play an essential role in pocket protein mediated G1 control. Mol. Cell. 6:729735.[Medline]
Gaubatz, S., J.A. Lees, G.J. Lindeman, and D.M. Livingston. 2001. E2F4 is exported from the nucleus in a CRM1-dependent manner. Mol. Cell. Biol. 21:13841392.
Graves, B.J., and J.M. Petersen. 1998. Specificity within ets family of transcription factors. Adv. Cancer Res. 75:155.[Medline]
Gulley, M.L., J.M. Nicholls, B.G. Schneider, M.B. Amin, J.Y. Ro, and J. Geradts. 1998. Nasopharyngeal carcinomas frequently lack the p16/MTS1 tumor suppressor protein but consistently express the retinoblastoma gene product. Am. J. Pathol. 152:865869.[Abstract]
Hammerschmidt, W., and B. Sugden. 1989. Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes. Nature. 340:393397.[CrossRef][Medline]
Hara, E., R. Smith, D. Parry, H. Tahara, S. Stone, and G. Peters. 1996. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol. Cell. Biol. 16:859867.[Abstract]
Hayflick, L., and P.S. Moorhead. 1961. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 25:585621.
Hunter, T. 1997. Oncoprotein network. Cell. 88:333346.[Medline]
Huot, T.J., J. Rowe, M. Harland, S. Drayton, S. Brookes, C. Gooptu, P. Purkis, M. Fried, V. Bataille, E. Hara, et al. 2002. Biallelic mutations in p16INK4a confer resistance to Ras and Ets induced senescence in human diploid fibroblasts. Mol. Cell. Biol. 22:81358143.
Ishida, N., T. Hara, T. Kamura, M. Yoshida, K. Nakayama, and K.I. Nakayama. 2002. Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export. J. Biol. Chem. 277:1435514358.
Jacobs, J.J., K. Kieboom, S. Marino, R.A. DePinho, and M. van Lohuizen. 1999. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 397:164168.[CrossRef][Medline]
Jansen-Durr, P. 1996. How viral oncogenes make the cell cycle. Trends Genet. 12:270275.[CrossRef][Medline]
Kaye, K.M., K.M. Izumi, and E. Kieff. 1993. Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc. Natl. Acad. Sci. USA. 90:91509154.[Abstract]
Kilger, E., A. Kieser, M. Baumann, and W. Hammerschmidt. 1998. Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. EMBO J. 17:17001709.
Kiyono, T., S.A. Foster, J.I. Koop, J.K. McDougall, D.A. Galloway, and A.J. Klingelhutz. 1998. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature. 396:8488.[CrossRef][Medline]
Krimpenfort, P., K.C. Quon, W.J. Mool, A. Loonstra, and A. Berns. 2001. Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature. 413:8386.[CrossRef][Medline]
Kudo, N., B. Wolff, T. Sekimoto, E.P. Schreiner, Y. Yoneda, M. Yanagida, S. Horinouchi, and M. Yoshida. 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242:540547.[CrossRef][Medline]
la Cour, T., R. Gupta, K. Rapacki, K. Skriver, F.M. Poulsen, and S. Brunak. 2003. NESbase version 1.0: a database of nuclear export signals. Nucleic Acids Res. 31:393396.
Lin, A.W., M. Barradas, J.C. Stone, L. van Aelst, M. Serrano, and S.W. Lowe. 1998. Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes Dev. 12:30083019.
Llanos, S., P.A. Clark, J. Rowe, and G. Peters. 2001. Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat. Cell Biol. 3:445452.[CrossRef][Medline]
Lloyd, A.C. 2002. Limits to lifespan. Nat. Cell Biol. 4:E25E27.[CrossRef][Medline]
Lundberg, A.S., W.C. Hahn, P. Gupta, and R.A. Weinberg. 2000. Genes involved in senescence and immortalization. Curr. Opin. Cell Biol. 12:705709.[CrossRef][Medline]
Lyden, D., A.Z. Young, D. Zagzag, W. Yan, W. Gerald, R. O'Reilly, B.L. Bader, R.O. Hynes, Y. Zhuang, K. Monova, and R. Benezra. 1999. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature. 401:670677.[CrossRef][Medline]
Malumbres, M., I.P. de Castro, M.I. Hernandez, M. Jimenez, T. Corral, and A. Pellicer. 2000. Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15INK4b. Mol. Cell. Biol. 20:29152925.
McConnell, B.B., M. Starborg, S. Brookes, and G. Peters. 1998. Inhibition of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr. Biol. 8:351354.[Medline]
McConnell, B.B., F.J. Gregory, F.J. Stott, E. Hara, and G. Peters. 1999. Induced expression of p16INK4a inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes. Mol. Cell. Biol. 19:19811989.
Muller, H., M.C. Moroni, E. Vigo, B.O. Petersen, J. Bartek, and K. Helin. 1997. Induction of S-phase entry by E2F transcription factors depends on their nuclear localization. Mol. Cell. Biol. 17:55085520.[Abstract]
Nevins, J.R. 2001. The Rb/E2F pathway and cancer. Hum. Mol. Genet. 10:699703.
Ohtani, N., Z. Zebedee, T.J.G. Huot, J.A. Stinson, M. Sugimoto, Y. Ohashi, A.D. Sharrocks, G. Peters, and E. Hara. 2001. Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature. 409:10671070.[CrossRef][Medline]
Ortega, S., M. Malumbres, and M. Barbacid. 2002. Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim. Biophys. Acta. 1602:7387.[CrossRef][Medline]
Passegue, E., and E.E. Wagner. 2000. JunB suppresses cell proliferation by transcriptional activation of p16INK4a expression. EMBO J. 19:29692979.
Rayman, J.B., Y. Takahashi, V.B. Indjeian, J.-H. Dannenberg, S. Catchpole, R.J. Watson, H. te Riele, and B.D. Dynlacht. 2002. E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex. Genes Dev. 16:933947.
Ren, B., H. Cam, Y. Takahashi, T. Volkert, J. Terragni, R.A. Young, and B.D. Dynlacht. 2002. E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints. Genes Dev. 16:245256.
Roberts, M.L., and N.R. Cooper. 1998. Activation of Ras-MAPK-dependent pathway by Epstein-Barr virus latent membrane protein 1 is essential for cellular transformation. Virology. 240:9399.[CrossRef][Medline]
Rodier, G., A. Montagnoli, L. Di Marcotullio, P. Coulombe, G.F. Draetta, M. Pagano, and S. Meloche. 2001. p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis. EMBO J. 20:66726682.
Roussel, M.F., R.A. Ashmun, C.J. Sherr, R.N. Eisenman, and D.E. Ayer. 1996. Inhibition of cell proliferation by the Mad1 transcriptional repressor. Mol. Cell. Biol. 16:27962801.[Abstract]
Sanij, E., B. Scott, T. Wilson, D. Xu, P. Hertzog, and E. Wolvetang. 2003. Characterization of monoclonal antibodies specific to the transcription factor Ets2 protein. Immunol. Lett. 86:6370.[CrossRef][Medline]
Schultheiss, U., S. Puschner, E. Kremmer, T.M. Mak, H. Engelman, W. Hammerschmidt, and A. Kieser. 2001. TRAF6 is a critical mediator of signal transduction by the viral oncogene latent membrane protein 1. EMBO J. 20:56785691.
Seoane, J., C. Pouponnot, P. Staller, M. Schader, M. Eilers, and J. Massague. 2001. TGFß influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b. Nat. Cell Biol. 3:400408.[CrossRef][Medline]
Serrano, M., and M.A. Blasco. 2001. Putting the stress on senescence. Curr. Opin. Cell Biol. 13:748753.[CrossRef][Medline]
Serrano, M., A.W. Lin, M.E. McCurrach, D. Beach, and S.W. Lowe. 1997. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 88:593602.[CrossRef][Medline]
Sharpless, N.E., N. Bardeesy, K.-H. Lee, D. Carrasco, D.H. Castrillon, A.J. Aguirre, E.A. Wu, J.W. Horner, and R.A. DePinho. 2001. Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature. 413:8691.[CrossRef][Medline]
Sharrocks, A.D. 2001. The Ets-domain transcription factor family. Nat. Rev. Mol. Cell Biol. 2:827837.[CrossRef][Medline]
Sherr, C.J. 1996. Cancer cell cycles. Science. 274:16721677.
Sotillo, R., P. Dubus, J. Martin, E. de la Cueva, S. Ortega, M. Malumbres, and M. Barbacid. 2001. Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors. EMBO J. 20:66376647.
Stade, K., C.S. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell. 90:10411050.[Medline]
Stallet, P., K. Peukert, A. Kiermaier, J. Seoane, J. Lukas, H. Karsunky, T. Moroy, J. Bartek, J. Massague, F. Hane, and M. Eilers. 2001. Repression of p15INK4b expression by Myc through association with Miz-1. Nat. Cell Biol. 3:392399.[CrossRef][Medline]
Stein, G.H., M. Beeson, and L. Gordon. 1990. Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. Science. 249:666669.[Medline]
Stein, G.H., L.F. Drullinger, A. Soulard, and V. Dulic. 1999. Differential roles of cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol. Cell. Biol. 19:21092117.
Sugimoto, M., T. Nakamura, N. Ohtani, L. Hampson, I.N. Hampson, A. Shimamoto, Y. Furuichi, K. Okumura, S. Niwa, Y. Taya, and E. Hara. 1999. Regulation of CDK4 activity by a novel CDK4 binding protein, p34SEI-1. Genes Dev. 13:30273033.
Sun, Y., A. Hildesheim, A.E. Lanier, Y. Cao, K.T. Yao, N. Raab-Traub, and C.S. Yand. 1995. No point mutation but decreased expression of the p16/MTS1 tumor suppressor gene in nasopharyngeal carcinomas. Oncogene. 10:785788.[Medline]
Thorley-Lawson, D.A. 2001. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol. 1:7582.[CrossRef][Medline]
Tomkinson, B., E. Robertson, and E. Kieff. 1993. Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. J. Virol. 67:20142025.[Abstract]
Tomoda, K., Y. Kubota, and J. Kato. 1999. Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature. 398:160165.[CrossRef][Medline]
Trimarchi, J.M., and J.A. Lees. 2002. Sibling rivalry in the E2F family. Nat. Rev. Mol. Cell Biol. 3:1120.[CrossRef][Medline]
Verona, R., K. Moberg, S. Estes, M. Starz, J.P. Vernon, and J.A. Lees. 1997. E2F activity is regulated by cell cycle-dependent changes in subcellular localization. Mol. Cell. Biol. 17:72687282.[Abstract]
Wang, D., D. Liebowitz, and E. Kieff. 1985. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell. 43:831840.[Medline]
Weinberg, R.A. 1997. The cat and mouse games that genes, viruses, and cells play. Cell. 88:573575.[CrossRef][Medline]
Wu, L., G. Timmers, B. Maiti, H.I. Saavedra, L. Sang, G.T. Chong, F. Nuckolls, P. Giangrande, F.A. Wright, S.J. Field, et al. 2001. The E2F1-3 transcription factors are essential for cellular proliferation. Nature. 414:457462.[CrossRef][Medline]
Xin, B., Z. He, X. Yang, C.P. Chan, M.H. Ng, and L. Cao. 2001. TRADD domain of Epstein-Barr virus transforming protein LMP1 is essential for inducing immortalization and suppressing senescence of primary rodent fibroblasts. J. Virol. 75:30103015.
Yang, X., J.S.T. Sham, S.W. Tsao, D. Zhang, S.W. Lowe, and L. Cao. 2000a. LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatrively transforms the cells with a p16-insensitive CDK4 oncogene. J. Virol. 74:883891.
Yang, X., Z. He, B. Xin, and L. Cao. 2000b. LMP1 of Epstein-Barr virus suppresses cellular senescence associated with the inhibition of p16INK4a expression. Oncogene. 19:20022013.[CrossRef][Medline]
Zhu, J., D. Woods, M. McMahon, and J.M. Bishop. 1998. Senescence of human fibroblasts induced by oncogenic Raf. Genes Dev. 12:29973007.
Related Article