Di€Erential Patterns of Cell Cycle Regulatory Proteins Expression in Transgenic Models of Thyroid Tumours
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Oncogene (1998) 17, 631 ± 641 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Dierential patterns of cell cycle regulatory proteins expression in transgenic models of thyroid tumours Fre de rique Coppe e1, Fabienne Depoortere1, Jiri Bartek2, Catherine Ledent1, Marc Parmentier1 and Jacques E Dumont1 1IRIBHN, Universite Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium; 2Danish Cancer Society, Division of Cancer Biology, 49 Strand boulevarden, DK-2100 Copenhagen, Denmark Cell cycle proteins regulate the transitions from G1 to S result of the continuous release of adenosine by most and G2 to M phases. In higher eukaryotes, their function cell types (Maenhaut et al., 1990). The Tg-A2aR is controlled by intracellular cascades regulated by transgene promotes in mice, as the cyclic AMP extracellular growth factors. We have studied in (cAMP) cascade in dog thyroid epithelial cells in previously described transgenic mouse models for thyroid primary culture, both cell proliferation and function proliferative diseases the expression of the key proteins (Roger et al., 1982; Roger and Dumont, 1984; Dumont regulating the cell cycle by Western blotting and et al., 1992), resulting in the appearance of a immunohistochemistry, and have correlated the observa- dierentiated goiter and severe hyperthyroidism tions with the known actions of the transgenes on the (Ledent et al., 1992). This model, as the model of signal transduction cascades. In the adenosine A2a mice expressing in the thyroid a Gsa mutant (Michiels receptor model, the cyclic AMP pathway, upstream of et al., 1994), mimics the human autonomous hyper- the Rb family cell division block, is constitutively functional adenomas and non-autoimmune familial activated. In the model expressing HPV 16 E7 protein, hyperthyroidism due to mutations activating the the Rb-like proteins are inhibited. Cyclin-dependent cAMP pathway, such as the constitutive activation of kinases cdk4, cdk2 and cdc2, and the associated cyclins the thyrotropin receptor (Parma et al., 1996; Vassart et D, E and A have been studied. Cyclin D3 appears as the al., 1996), or of the Gsa protein (Lyons et al., 1990; major cyclin D subtype expressed in mouse thyroid Suarez et al., 1991). The major function attributed to epithelial cells in normal and transgenic mice. In the the E7 oncoprotein of the human papillomavirus type adenosine A2aR model, all cell cycle proteins tested were 16 (HPV 16) is to bind and inactivate various proteins accumulated. In the E7 model, all cell cycle proteins of the Rb family (p105-Rb, p107 and p130) (reviewed except for D-type cyclins and cdk4 were also accumu- in Zacksenhaus et al., 1993), allowing the release of lated. A similar pattern was observed in thyroids transcriptional factors of the E2F family that promote coexpressing both transgenes, suggesting a dominant entry into the S phase of the cell cycle (reviewed in eect of E7 over the consequences of the cAMP cascade Nevins 1992; Farnham et al., 1993). The Tg-E7 activation. The cyclin-dependent kinase inhibitors transgenic mice develop a large dierentiated goiter p21cip1/waf1 and p27kip1 were not downregulated in these constituted of functionally active thyrocytes (Ledent et proliferating thyroids which suggest other roles than the al., 1995). In both models, malignant tumours only inhibition of the cell cycle progression. appear in some very old animals. By interbreeding the Tg-A2aR and Tg-E7 transgenic lines, a model of highly Keywords: cyclins; cdk(s); CKI; thyroid; HPV 16 E7; proliferative and hyperfunctional goiter was obtained, cAMP a model of dierentiated thyroid carcinomas character- ized by a high incidence and early occurrence of lung metastases (Coppe e et al., 1996). Both transgenes thus clearly cooperated in promoting proliferation and the Introduction development of early malignant tumours. Although growth factors and oncogenes activate cell Thyroid tumours result from the accumulation of proliferation through various pathways, their action genetic events that activate proliferation pathways in generally converges on the activation of the cyclin the normally `quiescent' thyrocytes (Wynford-Thomas, dependent kinases (cdk). The activity of cdk(s) is 1997). In order to develop models for thyroid tumours, regulated by association with cyclins, by stimulatory or transgenic mouse lines have been generated in our inhibitory phosphorylation events, and by association laboratory, by directing speci®cally the expression of with speci®c inhibitory proteins (reviewed in GraÄ na viral oncogenes or signalling proteins to thyroid and Reddy, 1995; Palmero and Peters, 1996). The epithelial cells (thyrocytes), under the control of the activated cdk(s) catalyze the phosphorylation and the bovine thyroglobulin gene promoter (Tg-) (Christophe inactivation of the Rb family proteins which are a key and Vassart, 1990; Ledent et al., 1990). element in the control of cell proliferation, including in Ectopic expression of the adenosine A2a receptor thyrocytes (Coulonval et al., 1997). Dierent cyclin-cdk leads to permanent stimulation of adenylyl cyclase, as a complexes are sequentially formed and activated during the cell cycle, allowing progression through the dierent checkpoints of this cycle: cyclin D-cdk4 or Correspondence: F Coppe e -cdk6 controls progression through late G1, cyclin E- Received 13 August 1997; revised 11 March 1998; accepted 11 March cdk2 acts at the initiation of S phase, cyclin A-cdk2 1998 and then cdc2 act in S and G2 phases and the cyclin Cell cycle proteins in thyroid transgenic mice F CoppeÂe et al 632 B1-cdc2 at M phase (GraÄ na and Reddy, 1995; Palmero morphology in Tg-A2aR/Tg-E7 animals expressing and Peters, 1996). There are three subtypes of D both transgenes remains homogeneous with age and cyclins (D1, D2 and D3), encoded by dierent genes, is characterized by a high cellular density and narrow but sharing sequence homology (Inaba et al., 1992). colloid lumina. The variable contribution of thyroglo- They are dierentially expressed during G1, depending bulin into the thyroid total protein content of the on the cell types considered (Inaba et al., 1992; dierent models could in¯uence markedly the quantita- Palmero et al., 1993; Ando et al., 1993; Sherr, 1993; tion of cell cycle proteins. To reduce this source of Bartkova et al., 1994; Sweeney et al., 1997). Promoter error, we have separated the cellular fraction from analysis of cyclins D2 and D3 revealed marked colloid by gentle homogenization of thyroid tissue in a dierences in the control of gene expression following non denaturating buer disrupting follicles without the stimulation by growth factors (Brooks et al., 1996). substantial cell damage, followed by low speed Moreover, various observations suggest that individual centrifugation (`cellular' extracts, see Materials and D-type cyclins have distinct functions in dierent cell methods). Equal amounts of the various protein types in addition to activating cdk4/6 (Ewen et al., extracts were run on SDS-polyacrylamide gels, and 1993; Kato and Sherr, 1993; Lucibello et al., 1993; silver staining did not show signi®cant dierences in Sherr, 1993; Han et al., 1996; Kranenburg et al., 1996; the protein pattern among the various transgenic and Sofer-Levi and Resnitzki, 1996). control groups (data not shown). Immunodetection of The activity of the dierent cyclin-cdk complexes is thyroglobulin on Western blots from 1 month-old repressed by two families of Cyclin-dependent Kinase animals showed that most thyroglobulin was eciently Inhibitors (CKI): 1/p21cip1/waf1, p27kip1 and p57kip2 which removed from `cellular' extracts in the various models. negatively regulate the activity of all cyclin-cdk In older animals (5 months), large amounts of complexes in vitro, and probably in vivo, 2/ speci®c thyroglobulin were found in the supernatants, espe- inhibitors of cdk4/6 such as p15INK4b, p16INK4a, p18INK4c cially in the Tg-E7 thyroid extracts (Figure 1). and p19INK4d (reviewed in Sherr and Roberts, 1995). Paradoxically, the cAMP cascade induces the expres- Expression of cyclins and cdks sion of p27 CKI in cells in which it inhibits cell proliferation (Kato et al., 1994; Ward et al., 1996) as The proteins analysed could be expressed in other cell well as in our dog thyrocytes in vitro in which it types than the epithelial cells and contaminate the stimulates it (Roger and Dumont, 1984; Depoortere et protein extracts. However, the epithelial cells represent al., 1996). the majority cells of thyroid cells. Moreover, other cells As most of the work concerning the expression of cell cycle proteins has been carried out in unphysiolo- gical cell system cultures, we have analysed by Western blotting and immunohistochemistry the expression of cdk(s), cyclins and p27 proteins as p21 messenger in the in vivo Tg-A2aR, Tg-E7 and Tg-A2aR/Tg-E7 transgenic models of thyroid proliferation and tumor- igenesis. Results Normalization of thyroglobulin content in protein extracts The functional unit of thyroid tissue is the follicle, a single cuboidal epithelium lining a lumen ®lled with colloid, which normally is the major constituent of the total thyroid mass. Colloid is made essentially of the thyroid hormone precursor thyroglobulin. In our transgenic models, the follicular structure is pre- served, but the ratio between cells and colloid varies according to the model and the age of the animals (Coppe e et al., 1996). The Tg-A2aR mice develop thyroid hyperfunction,