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Regulation of the Cdc25a Gene by the Human Papillomavirus Type 16 E7 Oncogene

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Regulation of the Cdc25a Gene by the Human Papillomavirus Type 16 E7 Oncogene Oncogene (2001) 20, 543 ± 550 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc ORIGINAL PAPERS Regulation of the Cdc25A gene by the human papillomavirus Type 16 E7 oncogene Stephanie C Katich1, Karin Zerfass-Thome1 and Ingrid Homann*,1 1Angewandte Tumorvirologie (F0400), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany Cdc25A is a tyrosine phosphatase that is involved in the (Homann et al., 1993). Recent work indicates that regulation of the G1/S phase transition by activating Cdc25B seems to play the role of a `starter phosphatase' cyclin E/Cdk2 and cyclin A/Cdk2 complexes through by activating cyclin B/Cdk1 in order to initiate the removal of inhibitory phosphorylations. The E6 and E7 positive feedback mechanism (Lammer et al., 1998; oncoproteins of the high-risk human papillomaviruses Karlsson et al., 1999). Cdc25 phosphatases are main (HPV) interact with and functionally abrogate the p53 players of the G2 arrest caused by DNA damage or in the and pRB proteins, respectively. In the present study we presence of unreplicated DNA (Sanchez et al., 1997; have investigated the regulation of the Cdc25A promoter Peng et al., 1997). Cdc25A and B are putative human during G1 and S-phases of the cell cycle and by the oncogenes (Galaktionov et al., 1995, 1997). The Cdc25A HPV-16 E7 oncoprotein. Serum induction leads to a phosphatase plays a crucial role at the G1/S phase derepression of the Cdc25A promoter and can be transition as shown by microinjection experiments using mediated through two E2F binding sites, E2F-A and Cdc25A speci®c antibodies (Homann et al., 1994) and E2F-C. While E2F-A is by both E2F1 and E2F4, E2F-C by conditional overexpression of Cdc25A (Blomberg and is regulated only by E2F1. The Cdc25A promoter is Homann, 1999). Cdc25A functions by activating cyclin transactivated by the E7 oncogene of HPV-16. Further- E- and cyclin A-dependent kinases (Blomberg and more, Cdc25A levels are highly increased in E7- Homann, 1999). expressing cell lines. Inducible expression of E7 leads Studies on human papillomavirus type 16 have to an immediate increase in Cdc25A protein levels. These demonstrated that the product of the early gene E7, data suggest that Cdc25A may be a critical target of plays a key role in the immortalization and malignant HPV-16 E7 in the disruption of the G1/S phase transformation of the host cell. The E7 protein is the transition. Oncogene (2001) 20, 543±550. major oncogenic protein produced by cervical cancer- associated HPV-16 (reviewed in zur Hausen, 1999). The Keywords: Cdc25; cell cycle; human papillomavirus majority of the transforming potential of E7 protein has type 16; E7 oncoprotein been ascribed to its ability to bind to, and down-regulate the retinoblastoma (RB) gene product (Boyer et al., 1996; Lee et al., 1998). Loss of RB function causes the Introduction release and activation of multiple E2F transcription factor family members, leading to cell cycle progression Transitions in the mammalian cell cycle are governed by through the G1/S phase transition. (Funk et al., 1997). cyclin-dependent kinases (Cdks) whose activities are Ectopic expression of E2F1, E2F2, E2F3 and to a lesser determined by cyclin binding, small Cdk inhibitor extent E2F4 is sucient to induce S-phase in immorta- proteins and both positive and negative regulatory lized quiescent rat ®broblasts (Lukas et al., 1996; phosphorylations (Nilsson, 2000). The Cdc25 phospha- DeGregori et al., 1997). Genes that are responsive to tases are members of the tyrosine phosphatase family E2F include well-studied cell cycle regulators including and catalyze dephosphorylation and activation of cyclin/ cyclin E, cyclin A, Cdk1 and Cdk2 (Dalton, 1992; Ohtani Cdk complexes through removal of inhibitory phosphor- et al., 1995; Schulze et al., 1995; Botz et al., 1996; Arata et ylations. In human cells, three Cdc25 homologs called al., 2000). Recently it has been shown that TGFb- Cdc25A, Cdc25B and Cdc25C have been identi®ed mediated down-regulation of Cdc25A in keratinocytes is (Sadhu et al., 1990; Galaktionov and Beach, 1991). mediated by an E2F site within the Cdc25A promoter Cdc25C dephosphorylates cyclin B/Cdk1, thereby acti- (Iavarone and Massague, 1999). Cdc25A is an essential vating its kinase activity. Cdc25C in turn becomes target for E2F1, since its activity is required for ecient phosphorylated by cyclin B/Cdk1 thus creating a induction of S phase by E2F1 and RB family members positive feedback loop leading to entry into mitosis (Vigo et al., 1999; Chen and Prywes, 1999). In this study we show that the human Cdc25A promoter can be transactivated by the E7 oncogene of human papillomavirus type 16. This transactivation is *Correspondence: I Homann Received 18 May 2000; revised 14 November 2000; accepted 23 mediated through two functional E2F binding sites November 2000 within the promoter. Furthermore Cdc25A levels are Regulation of Cdc25A by HPV-16 E7 SC Katich et al 544 highly increased in E7-expressing cell lines. Inducible prototype E2F site in nucleotide sequence and protein expression of E7 leads to an immediate increase in binding. An E2F-binding site with a similar sequence Cdc25A protein levels. These results suggest that the motif was previously found in the cyclin A promoter transcriptional regulation of the Cdc25A gene con- (Schulze et al., 1995; Figure 1, middle panel). To tributes to the control of cell cycle progression in determine which of the E2F sites is functional we virally transformed ®broblasts. transfected wild-type or mutant Cdc25A promoter constructs (nt 7457/+121) driving a luciferase reporter gene into NIH3T3 cells that were serum-starved and then released from the block. Samples were taken at the Results indicated time points. In Figure 2a the percentage of cells in G1, S and G2 or M phase are shown. While the Characterization of the functional E2F sites in the human Cdc25A promoter is repressed during G0 (Iavarone and Cdc25A promoter Massague, 1999; Figure 2b), the Cdc25A wild-type The region from 7457 to +121 of the human Cdc25A promoter construct was induced threefold at 16 h after promoter contains potential binding sites for known serum-induction when the cells were in mid S-phase. transcription factors, among them an E2F binding site These results are in agreement with previously published denoted E2F-A, which is located 60 bp upstream of the results (Chen and Prywes, 1999). Mutation of the E2F- transcriptional start site (Iavarone and Massague, A binding site (MUT 1) (Figure 1, lower panel) leads to 1999). The same authors also identi®ed a second E2F a similar fold of induction as found for the wild-type site (E2F-B) spanning the transcriptional start site. We promoter activity in such an experiment. This would have isolated the human Cdc25A promoter and suggest that another site of induction is present in the identi®ed an additional putative E2F binding site in the promoter, E2F-C, which is located at 160 bp upstream of the transcriptional start site in the promoter (Figure 1, upper panel). This site diers from the a b Figure 2 Cell cycle regulation of the Cdc25A promoter. (a) NIH3T3 cells were transfected with either the wild-type Cdc25A promoter or promoter constructs with a mutation in the E2F-A site (MUT1), E2F-C (MUT2) or mutations in both sites (MUT3). Figure 1 Sequence of the human Cdc25A promoter. Three E2F Cells were subsequently serum-starved for 3 days. After re- sites (denoted E2F-A, E2F-B and E2F-C) are boxed (upper addition of serum synchronized cells were harvested at dierent panel). Comparison of the dierent E2F binding sites found in the time points. Relative luciferase activity was measured. The results human Cdc25A promoter (middle panel) with the E2F consensus are the mean of three independent experiments. (b) Cell cycle sequence and a functional E2F binding site of the human cyclin A distribution. NIH3T3 cells were synchronized by serum-starvation promoter. Lower panel; Schematic representation of the E2F-site and released by addition of 10% calf serum. Samples were taken mutations within the Cdc25A promoter for ¯ow cytometry at the indicated time points Oncogene Regulation of Cdc25A by HPV-16 E7 SC Katich et al 545 a Cdc25A promoter. To test this we mutated the E2F-C site, MUT 2 (Figure 1, lower panel), and determined the promoter activity. Mutation of both E2F binding sites (MUT3) (Figure 1, lower panel) only showed a slight increase in promoter activity (Figure 2b) which could be due to the third E2F site that is present in the promoter, E2F-B (Vigo et al., 1999). Taken together these results show that the E2F-A and E2F-C sites are both functional and required for E2F-induced S-phase entry. Transactivation of the Cdc25A gene by the E7 oncoprotein of human papillomavirus type 16 Since we identi®ed functional E2F binding sites in the Cdc25A promoter we asked if expression of HPV-16 E7 would lead to an activation of Cdc25A transcrip- tion in NIH3T3 cells. An expression vector for HPV-16 E7 was co-transfected with the Cdc25A promoter construct into NIH3T3 cells and the relative luciferase activity was determined. As shown in Figure 3a a threefold increase of Cdc25A promoter activity was observed when HPV-16 E7 was co-transfected in b comparison to the co-transfection of an empty vector. In order to demonstrate that only a high-risk papillomavirus oncogene as HPV-16 E7 is able to transactivate the Cdc25A promoter we also co- transfected a protein from the low-risk papilloma virus, HPV-11 E7. As expected, HPV-11 E7 is not able to transactivate the Cdc25A promoter.
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