High Incidence of Thymic Epithelial Tumors in E2F2 Transgenic Mice
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m313682200
ISSN1083-351X
AutoresBlanca Scheijen, Marieke Bronk, Tiffany van der Meer, Daphne de Jong, René Bernards,
Tópico(s)Glioma Diagnosis and Treatment
ResumoIn virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the Eμ and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. Eμ-pp-E2F2 mice start to develop cytokeratin- and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4+CD8+ double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, Eμ-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells. In virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the Eμ and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. Eμ-pp-E2F2 mice start to develop cytokeratin- and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4+CD8+ double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, Eμ-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells. E2F transcription factors are key regulators of cell division and function by controlling the expression of genes that are critical for DNA synthesis, DNA repair, and mitosis (1Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (495) Google Scholar, 2Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Crossref PubMed Scopus (903) Google Scholar, 3Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (629) Google Scholar). Six distinct genes encode seven different E2F proteins (E2F1, E2F2, E2F3a, E2F3b, and E2F4 to E2F6), which form heterodimers with DP1, or one of the four different splice variants (α, β, γ, and δ) of human DP2 (4Rogers K.T. Higgins P.D. Milla M.M. Phillips R.S. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7594-7599Crossref PubMed Scopus (37) Google Scholar) or mouse DP3 (5de la Luna S. Burden M.J. Lee C.W. La Thangue N.B. J. Cell Sci. 1996; 109: 2443-2452Crossref PubMed Google Scholar, 6Ormondroyd E. de la Luna S. La Thangue N.B. Oncogene. 1995; 11: 1437-1446PubMed Google Scholar). However, E2F7, which binds to consensus E2F DNA recognition sites and acts as a transcriptional repressor, lacks the residues necessary for DP dimerization (7de Bruin A. Maiti B. Jakoi L. Timmers C. Buerki R. Leone G. J. Biol. Chem. 2003; 278: 42041-42049Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Di Stefano L. Jensen M.R. Helin K. EMBO J. 2003; 22: 6289-6298Crossref PubMed Scopus (207) Google Scholar). E2F1, E2F2, and E2F3a are potent activators of E2F-responsive genes, but their transcriptional activity is inhibited by binding to the retinoblastoma protein (pRb). 1The abbreviations used are: pRb, retinoblastoma protein; Cdk, cyclin-dependent kinase; DP, double positive; Eμ, immunoglobulin heavy chain enhancer; HA, hemagglutinin; K5, keratin 5; LTR, long terminal repeat; pp, pim1 promoter; TEC, thymic epithelial cell; DAB, 3,3′-diaminobenzidine tetrahydrochloride.1The abbreviations used are: pRb, retinoblastoma protein; Cdk, cyclin-dependent kinase; DP, double positive; Eμ, immunoglobulin heavy chain enhancer; HA, hemagglutinin; K5, keratin 5; LTR, long terminal repeat; pp, pim1 promoter; TEC, thymic epithelial cell; DAB, 3,3′-diaminobenzidine tetrahydrochloride. pRb is functionally inactivated at the G1-S transition by cyclin D-cyclin dependent kinase 4 (Cdk4)/Cdk6 and cyclin E-Cdk2-mediated phosphorylation (9Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (852) Google Scholar, 10Harbour J.W. Luo R.X. Dei Santi A. Postigo A.A. Dean D.C. Cell. 1999; 98: 859-869Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar), thus enabling E2Fs to activate their target genes. Besides promoting cell division, E2F1, and to a lesser extent E2F2 and E2F3a, also induce programmed cell death through both p53-dependent and p53-independent mechanisms. Studies performed on E2F gene-targeted mice have demonstrated both unique and redundant functions of the different E2Fs (11DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar). E2f1-/- and E2f2-/- mice are fully viable, and each mutant shows distinct cell lineage-specific phenotypes: E2f1-/- thymocytes are defective in T cell receptor-mediated apoptosis (12Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. 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In contrast, E2F2-deficient thymocytes show normal apoptosis characteristics, whereas E2f2-/- splenic T cells display accelerated G1-S phase progression (15Murga M. Fernandez-Capetillo O. Field S.J. Moreno B. Borlado L.R. Fujiwara Y. Balomenos D. Vicario A. Carrera A.C. Orkin S.H. Greenberg M.E. Zubiaga A.M. Immunity. 2001; 15: 959-970Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Zhu J.W. Field S.J. Gore L. Thompson M. Yang H. Fujiwara Y. Cardiff R.D. Greenberg M. Orkin S.H. DeGregori J. Mol. Cell. Biol. 2001; 21: 8547-8564Crossref PubMed Scopus (82) Google Scholar). On the other hand, erythropoiesis and pre-B cell differentiation are defective in E2f2-/- mice, whereas there is increased B cell maturation in E2f1-/- animals (17Li F.X. Zhu J.W. Hogan C.J. DeGregori J. Mol. Cell. Biol. 2003; 23: 3607-3622Crossref PubMed Scopus (73) Google Scholar). E2F3 deficiency dramatically impairs cell proliferation of mouse embryonic fibroblasts and results in reduced neonatal viability (18Humbert P.O. Verona R. Trimarchi J.M. Rogers C. Dandapani S. Lees J.A. Genes Dev. 2000; 14: 690-703PubMed Google Scholar). E2F1 and E2F2 share redundant roles with E2F3 during embryonic development because deficiency for either E2F1 or E2F2 exacerbates the lethal phenotype observed in E2f3-/- embryos (19Cloud J.E. Rogers C. Reza T.L. Ziebold U. Stone J.R. Picard M.H. Caron A.M. Bronson R.T. Lees J.A. Mol. Cell. Biol. 2002; 22: 2663-2672Crossref PubMed Scopus (73) Google Scholar, 20Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (492) Google Scholar). Although E2f1-/-; E2f2-/- mouse embryonic fibroblasts proliferate efficiently, the combined loss of E2F1, E2F2, and E2F3 completely abolishes entry into S phase and further cell division (20Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (492) Google Scholar). E2f1+/- as well as E2f1-/- mice develop a similar spectrum of tumor types after a latency of 16 months, including lymphomas, hemangiosarcomas, and histiocytic sarcomas, arguing that E2f1 acts as a tumor-suppressor gene (21Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). The tumor-inhibitory function of E2F1 is illustrated further by the finding that overexpression of E2F1 inhibits glioma tumor growth (22Fueyo J. Gomez-Manzano C. Yung W.K. Liu T.J. Alemany R. 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Importantly, loss of E2F3 expression harbors no increased susceptibility for the development of tumors, both in wild type and E2f1 mutant mice. On the other hand, enforced expression of E2F1 in the liver results in hepatocellular adenomas as well as large cell dysplasia (28Conner E.A. Lemmer E.R. Omori M. Wirth P.J. Factor V.M. Thorgeirsson S.S. Oncogene. 2000; 19: 5054-5062Crossref PubMed Scopus (127) Google Scholar). Transgenic K5-E2F1 expression induces spontaneous tumors that originate from epithelial basal cells (29Pierce A.M. Schneider-Broussard R. Gimenez-Conti I.B. Russell J.L. Conti C.J. Johnson D.G. Mol. Cell. Biol. 1999; 19: 6408-6414Crossref PubMed Scopus (139) Google Scholar), accelerates formation of benign skin papillomas in collaboration with a v-Ha-ras transgene (30Pierce A.M. Fisher S.M. Conti C.J. Johnson D.G. Oncogene. 1998; 16: 1267-1276Crossref PubMed Scopus (129) Google Scholar), and enhances skin tumorigenesis in p53-/- mice (31Pierce A.M. Gimenez-Conti I.B. 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Interestingly, the absence of E2F1 reverts the UVB-induced apoptosis defect in primary fibroblasts and early onset thymic lymphomas seen in p53-/- mice, arguing that E2F1 acts functionally downstream of p53 (34Wikonkal N.M. Remenyik E. Knezevic D. Zhang W. Liu M. Zhao H. Berton T.R. Johnson D.G. Brash D.E. Nat. Cell Biol. 2003; 5: 655-660Crossref PubMed Scopus (73) Google Scholar). These data demonstrate that in vivo E2F1 has both tumor-suppressive as well as tumor-promoting activity depending on cell lineage, stage of differentiation, status of the p53/ARF/MDM2 pathway, and expression level of pRb. However, there is much less information about the potential diverse functions of E2F2 in different cell lineages. E2F2 has an unexpected negative role in controlling antigen-stimulated T cell proliferation (15Murga M. Fernandez-Capetillo O. Field S.J. Moreno B. Borlado L.R. Fujiwara Y. Balomenos D. Vicario A. Carrera A.C. Orkin S.H. Greenberg M.E. Zubiaga A.M. Immunity. 2001; 15: 959-970Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Zhu J.W. Field S.J. Gore L. Thompson M. Yang H. Fujiwara Y. Cardiff R.D. Greenberg M. Orkin S.H. DeGregori J. Mol. Cell. Biol. 2001; 21: 8547-8564Crossref PubMed Scopus (82) Google Scholar), whereas in mouse embryonic fibroblasts endogenous E2F2 contributes to promote cell division (20Wu L. Timmers C. Maiti B. Saavedra H.I. Sang L. Chong G.T. Nuckolls F. Giangrande P. Wright F.A. Field S.J. Greenberg M.E. Orkin S. Nevins J.R. Robinson M.L. Leone G. Nature. 2001; 414: 457-462Crossref PubMed Scopus (492) Google Scholar), and in hematopoietic progenitor cells E2F2 is required for efficient S phase progression (17Li F.X. Zhu J.W. Hogan C.J. DeGregori J. Mol. Cell. Biol. 2003; 23: 3607-3622Crossref PubMed Scopus (73) Google Scholar). In postmitotic lens fibers overexpression of E2F2 induces cell cycle entry and subsequent apoptosis (35Chen Q. Hung F.C. Fromm L. Overbeek P.A. Investig. Ophthalmol. Vis. Sci. 2000; 41: 4223-4231PubMed Google Scholar). In this study, we provide evidence that E2F2 has a distinct function in promoting oncogenic transformation of nonlymphoid thymic epithelial cells (TECs). E2F2 Transgenic Mice—For the generation of Eμ-pp-E2F2 mice, a 1.3-kb HA-tagged human E2F2 cDNA was inserted into the SacI-KpnI site of pJ3Ω. The NotI-HpaI fragment containing HA-E2F2, followed by splice donor and acceptor elements derived from SV40 small t antigen intervening sequences present in pJ3Ω, was cloned in EagI-HpaI site of Eμ-pim1 promoter-Moloney murine leukemia virus LTR transgenic vector. The assembled transgene was liberated with HindIII from the vector backbone, microinjected into pronuclei of FVB zygotes, and transferred to (B6 × DBA)F1 foster mice. Eμ-pp-E2F founder mice and subsequent transgenic progeny were identified with PCR analysis on genomic DNA isolated from tail biopsies with transgene specific primers TDK5′ 5′-CGGCCTTTGATGGCTTTG-3′ and EMU3′ 5′-AGGGTATGAGAGAGCCTC-3′. Northern and Southern Blot Analyses—Total RNA was isolated from frozen sections of normal and hyperplastic thymi or isolated TECs and thymocytes using TRIzol (Invitrogen). TECs were isolated as described previously (36Izon D.J. Nieland J.D. Godfrey D.I. Boyd R.L. Kruisbeek A.M. Int. Immunol. 1994; 6: 31-39Crossref PubMed Scopus (19) Google Scholar). Samples of 20 μg of total RNA were separated on a 1% paraformaldehyde-containing agarose gel, transferred to Protran nitrocellulose filter (Schleicher & Schuell), and hybridized to random priming labeled [α-32P]dATP U3LTR or 0.8-kb β-actin cDNA probes. Genomic DNA (15 μg), isolated from frozen tissue samples, was digested with PvuII, separated on 0.7% 1 × TAE-agarose gel, blotted on Protran nitrocellulose filter, and hybridized to probe J15, a 900-bp ClaI-EcoRI fragment of Jβ2 locus, to check for T cell receptor rearrangements. Western Blot Analysis—Total cell extracts were generated by lysis of frozen thymic tissues in ice-cold ELB buffer (250 mm NaCl, 0.1% Nonidet P-40, 50 mm HEPES pH 7.0, and 5 mm EDTA) supplemented with protease inhibitors (Complete, Roche Applied Science). Samples were cleared by centrifugation for 10 min at 14,000 rpm, lysates separated with SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Polyclonal antibodies against E2F2 (C20), cyclin A (C19), cyclin E (M20), and actin (C11) (Santa Cruz) were used for immunoblotting. Results were visualized by Enhanced Chemiluminescence (Amersham Biosciences). Histology and Immunohistochemistry—For histopathological examination, thymi were removed, fixed with 10% buffered formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin. For immunohistology of frozen sections, tissues were immersed in optimal cutting temperature compound and snap frozen in liquid nitrogen. 5 μm frozen sections were acetone fixed and stained with mouse monoclonal pan-cytokeratin (Sigma), rabbit polyclonal E2F2 (Santa Cruz), or rat monoclonal (ER-TR4, ER-TR5, and ER-TR7) (37Van Vliet E. Melis M. Van Ewijk W. Eur. J. Immunol. 1984; 14: 524-529Crossref PubMed Scopus (194) Google Scholar) antibodies. Primary antibodies were visualized with fluorescein isothiocyanate-conjugated secondary antibodies. For paraffin-embedded sections tissues were processed using a standard protocol for DAB immunostaining, incorporating an antigen retrieval step, and using a 1:250 dilution of the mouse monoclonal HA.11 antibody (Covance). DAB-stained sections were counterstained with hematoxylin. Flow Cytometry—Single cell suspensions were freshly prepared from wild-type thymus or hyperplastic thymi, and 106 cells were resuspended in 2% fetal calf serum, 5 mm HEPES in phosphate-buffered saline. Cells were stained with fluorescein isothiocyanate-conjugated CD4 (RM4-5) and phycoerythrin-conjugated CD8α (53-6.7) (BD Pharmingen), washed and analyzed on FACScan (BD Biosciences) with CellQuest software package. Generation of Eμ-pp-E2F Transgenic Mice—To compare their ability to promote cell proliferation and regulate apoptosis when overexpressed in vivo, E2F1 and E2F2 transgenic mice were generated. For this purpose, human E2F1 and E2F2 cDNAs were subcloned in a vector containing a duplicated version of the immunoglobulin heavy chain enhancer (μ) inserted into the murine pim1 promoter (pp) region, and the Moloney murine leukemia virus LTR, which encodes a polyadenylation signal (Fig. 1A). The Eμ-pp transgenic promoter has been shown to direct ubiquitous expression during embryonic development (38Alkema M.J. van der Lugt N.M. Bobeldijk R.C. Berns A. van Lohuizen M. Nature. 1995; 374: 724-727Crossref PubMed Scopus (151) Google Scholar) and confers high expression to different hematopoietic cell lineages in adult mice (39Alkema M.J. Jacobs H. van Lohuizen M. Berns A. Oncogene. 1997; 15: 899-910Crossref PubMed Scopus (111) Google Scholar, 40Allen J.D. Verhoeven E. Domen J. van der Valk M. Berns A. Oncogene. 1997; 15: 1133-1141Crossref PubMed Scopus (139) Google Scholar). Four founders containing the Eμ-pp-E2F1 (TEF1-5, -21, -43, and -62) or Eμ-pp-E2F2 (TEF2-6, -33, -35, and -37) transgene were identified by PCR analysis on genomic DNA, and each of the founders was transmitted the E2F transgene to their progeny, except founder TEF2-6. To analyze expression of the different Eμ-pp-E2F transgenes in the thymus and compare transgene-driven E2F mRNA levels, Northern blot hybridization was performed using the Moloney murine leukemia virus U3LTR probe. Both Eμ-pp-E2F1 (TEF1-5) and Eμ-pp-E2F2 (TEF2-35 and TEF2-37) mice showed significant E2F1 and E2F2 transgenic mRNA levels in thymus (Fig. 1B). Immunoblotting confirmed protein expression of transgene-derived HA-tagged E2F1 and E2F2 (data not shown). Western blotting for cyclin E and cyclin A protein levels on thymic cell extracts indicated that transgene-driven E2F2 expression, but not E2F1, resulted in enhanced levels of cyclin E (Fig. 1C). Both cyclin E1 (Ccn1) and cyclin E2 (Ccn2) gene transcriptions are controlled by the pRb/E2F pathway (3Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (629) Google Scholar), suggesting that overexpression of E2F2 in murine thymus activates cyclin E expression. Eμ-pp-E2F1 and Eμ-pp-E2F2 founder lines were mated with FVB mice to generate larger cohorts of E2F transgenic mice. Interestingly, we observed a remarkable difference in phenotypes between E2F1 and E2F2 transgenic mice. Eμ-pp-E2F1 mice displayed a strong delay in endochondral ossification with concomitant dwarfism and a relatively high perinatal lethality among Eμ-pp-E2F animals (60% of transgenic offspring) (39Alkema M.J. Jacobs H. van Lohuizen M. Berns A. Oncogene. 1997; 15: 899-910Crossref PubMed Scopus (111) Google Scholar). Thus with great difficulty, only one Eμ-pp-E2F1 line (TEF1-5) could be maintained. In contrast, progeny of all three Eμ-pp-E2F2 founder lines (TEF2-33, -35, and -37) were born alive with normal Mendelian ratios and showed no evidence of skeletal abnormalities. Therefore, subsequent functional studies were largely restricted to E2F2 transgenic mice. Eμ-pp-E2F2 Transgene Is Highly Expressed in TECs—We decided to examine in more detail the expression pattern of the Eμ-pp-E2F2 transgene in the two major cell types present in thymus, thymocytes and TECs. Thymic epithelium forms a structurally and functionally important component of the thymus microenvironment and is required for selection of the appropriate T cell repertoire on the cell surface of thymocytes (40Allen J.D. Verhoeven E. Domen J. van der Valk M. Berns A. Oncogene. 1997; 15: 1133-1141Crossref PubMed Scopus (139) Google Scholar, 41Boyd R.L. Tucek C.L. Godfrey D.I. Izon D.J. Wilson T.J. Davidson N.J. Bean A.G. Ladyman H.M. Ritter M.A. Hugo P. Immunol. Today. 1993; 14: 445-459Abstract Full Text PDF PubMed Scopus (437) Google Scholar). Total mRNA was extracted from freshly isolated thymocytes or TECs that were cultured short term in vitro to obtain pure epithelial cells in sufficient quantity. Surprisingly, Northern blot analysis indicated that Eμ-pp-E2F2 was highly expressed in purified TECs and very low or nondetectable in primary thymocytes (Fig. 2A). Similarly, transgenic E2F2 expression was absent in mature peripheral T cells (data not shown). TECs isolated from Eμ-pp-E2F1 mice displayed reduced viability and were not able to grow in vitro, suggesting that E2F1 overexpression promotes apoptosis in these cells. Immunohistochemical analysis performed on thymic sections of Eμ-E2F2 mice confirmed HA-E2F2 nuclear staining in TECs from E2F2 transgenic thymi but not wild-type controls (Fig. 2, B and C), and high E2F2 protein levels coincided with the network of cytokeratin-positive TECs interspersed between islets of thymocytes (Fig. 2, D and E). Thus, pim1 promoter in conjunction with Eμ sequences direct high transgene expression of E2F2 in TECs. High Incidence of Spontaneous Thymic Tumors in Eμ-pp-E2F2 Transgenic Mice—The predominant phenotype observed in aging Eμ-pp-E2F2 animals of each transgenic line, which became apparent between the age of 5 and 11 months, was respiratory distress and dyspnea. Shortly thereafter, these E2F2 transgenic mice became cachectic and had to be sacrificed (Fig. 3). At autopsy, diseased Eμ-pp-E2F2 animals displayed severe hyperplastic thymi, often occupying the entire thoracic cavity (Fig. 4A). There was no evidence of enlarged secondary lymphoid tissues (spleen and lymph nodes) or macroscopic invasion of other organs. In most cases, the normal thymic bilobar structure was preserved in the solid hyperplastic thymi.Fig. 4Characterization of thymomas in Eμ-pp-E2F2 transgenic animals. A, macroscopic representation of severe hyperplastic thymus filling thoracic cavity of TEF2-35 animal at age of 8 months. The lungs and heart are almost completely obscured by the thymic tumor. The scale bar represents 8 mm. B and C, hematoxylin and eosin-stained paraffin sections of wild-type (WT) thymus at age of 4 weeks and TEF2-33 thymoma at age of 7 months. Medulla (M) and cortical (C) areas are indicated. Scale bars represent 5 mm. D, Southern blot analysis on genomic DNA isolated from thymi of wild-type (WT), Moloney murine leukemia virus-induced T cell lymphoma, Eμ-pp-E2F2 thymomas of founder TEF2-6 (F2-6), and TEF2-33 (F2-33), and E2F2 transgenic animals 44, 771 (both TEF2-33), 691, and 410 (both TEF2-35). Each genomic DNA sample is digested with PvuII, blotted, and hybridized with a T cell receptor β2 probe to detect T cell receptor rearrangements. The arrow indicates the position of the germ line band. The asterisk indicates the monoclonal rearranged T cell receptor β allele. E, flow cytometry was performed on a single cell suspension of thymocytes isolated from wild-type thymus and Eμ-pp-E2F2 thymoma of founder TEF2-6, stained with anti-CD4 and anti-CD8 antibodies. The percentage of cells in each quadrant is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Hematoxylin and eosin-stained sections indicated that the functional organization of cortex and medulla was still present in all E2F2 thymic tumors examined, but darker stained lymphocyte-rich cortical areas were largely increased in size compared with their normal counterparts (Fig. 4, B and C). Pale stained medulla-like areas with Hassall corpuscle-like structures were present as isolated islets in enlarged thymi instead of one continuous area within a normal thymic lobe. These macroscopic and histological features recapitulated the characteristics of epithelial thymomas. A few Eμ-pp-E2F2 tumors showed small necrotic areas, but overall there was no evidence for high grade malignancy. None of the 18 Eμ-pp-E2F1 mice that survived into late adulthood developed thymomas or showed an enlarged thymus upon necroscopy. These findings indicate that the induction of thymomas in Eμ-pp-E2F transgenic animals results as a consequence of deregulated E2F2 expression, but not E2F1. This different ability to induce tumors in thymic epithelium may well be related to the differential ability of E2F1 and E2F2 to induce apoptosis. Phenotypic Analyses of Thymocytes in Eμ-pp-E2F2 Thymomas—To investigate whether thymocytes present in hyperplastic thymi of Eμ-pp-E2F2 transgenic mice showed evidence of transformation, T cell receptor rearrangements were assessed by Southern blotting. T cell lymphomas are normally mono- or oligoclonal in origin, which can be demonstrated by a specific and unique rearrangement pattern at the joining regions of the T cell receptor β locus (Fig. 4D, lane 2). However, Eμ-pp-E2F2 tumors contained either a regular germ line configuration, as seen in wild-type thymus (Fig. 4D, lanes 1, 3, 5, 6, and 8), or polyclonal rearrangements (Fig. 4D, lanes 4 and 7), arguing against the presence of T lymphoid tumor cells. These findings were corroborated by flow cytometric analyses, which also showed no indications for clonal outgrowth of specific thymocyte subsets in Eμ-pp-E2F2 thymic tumors as defined by CD4 and CD8 coreceptor expression (Fig. 4E). Instead, we found altered ratios of thymocyte subsets in Eμ-pp-E2F2 thymomas, with relatively more immature CD4-CD8- double negative pre-T cells as well as a higher proportion of postselected CD4+ or CD8+ single-positive thymocytes (Fig. 4E and Table I). Young Eμ-pp-E2F2 mice without thymomas showed normal CD4/CD8 thymocyte distributions (data not shown). These analyses indicate that enforced expression of E2F2 in TECs results in cortical thymomas with concomitant perturbation of T cell development.Table ILatency and distribution thymocyte subsets of Eμ-pp-E2F2 thymomas The age at which moribund Eμ-pp-E2F2 animals were sacrificed is indicated together with individual mouse identification numbers and transgenic founder lines of origin. Fraction of CD4-CD8- double negative, CD4+CD8+ double positive, CD4+CD8- single positive, and CD4-CD8+ single positive thymocyte subsets are shown as determined by flow cytometric analysis on freshly isolated thymic tumor single cell suspensions. For reference, the normal distribution in
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