Institut für Virologie, Philipps-Universität Marburg, Robert Koch Str. 17, 35037 Marburg, Germany1
Author for correspondence: Matthias Dobbelstein. Fax +49 6421 28 68962. e-mail dobbelst{at}mailer.uni-marburg.de
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Abstract |
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Introduction |
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Adenovirus and simian virus 40 (SV40) mediate the malignant transformation of cells and induce tumours in animals. The mechanisms underlying this phenomenon have been studied for decades, establishing many of the principles relevant to molecular tumourigenesis (Shenk, 1996 ). Understanding how tumour viruses induce and maintain malignancy has frequently elucidated virus and non-virus carcinogenesis in general. Recently, the investigation of virus-mediated transformation was intensified by the observation that human primary cells can be transformed by a limited set of overexpressed genes, i.e. the telomerase catalytic subunit, oncogenic Ras and SV40 large tumour (T) antigen. These oncogenes were found to be sufficient to transform human fibroblasts (Hahn et al., 1999
) and human mammary epithelial cells (Elenbaas et al., 2001
). While these studies may reveal a limited set of oncogenic changes needed for human carcinogenesis, the usefulness of the system depends on a full understanding of the effects caused by the continuous expression of the SV40 T antigen.
The Ad E1 region encodes the E1A proteins, which share common domains, and two entirely distinct E1B proteins, namely the E1B 55 kDa protein (E1B 55K) and the E1B 19 kDa protein (E1B 19K). These three protein entities co-operate to transform primary cells. Ad type 5 (Ad5) E1B 55K forms a specific complex with p53 (Sarnow et al., 1982a ) and, at least transiently, blocks p53-mediated transcription, an activity that co-segregates with the transforming potential of E1B 55K in mutational analysis (Yew & Berk, 1992
). Ad5 E1B 55K binds to the amino-terminal portion of p53 that is responsible for transcriptional activation (Lin et al., 1994
), actively represses p53-mediated transcription (Yew et al., 1994
) and sequesters p53 to characteristic perinuclear clusters (Blair Zajdel & Blair, 1988
; Zantema et al., 1985a
). Given the close correlation between the ability of E1B 55K to bind p53 and its transforming potential (Yew & Berk, 1992
), it is widely assumed that E1B 55K constantly inactivates p53 in Ad E1-transformed cells (Grand et al., 1995
). This view was further supported by the observation that peptides blocking the interaction between E1B 55K and p53 at least temporarily inhibit the growth of Ad E1-transformed cells (Hutton et al., 2000
).
A homologue of p53, termed p73, is expressed in mammalian cells (Kaghad et al., 1997 ). Many activities are shared between p53 and p73, such as transcriptional activation and the induction of apoptosis (Jost et al., 1997
). However, unlike p53, p73 does not detectably interact with Ad E1B 55K (Higashino et al., 1998
; Marin et al., 1998
; Roth et al., 1998
; Steegenga et al., 1999; Wienzek et al., 2000
). Based on this finding, a chimera of p53 and p73 was constructed, replacing the five residues at positions 2428 of p53 with the corresponding amino acids from p73, to create the mutant p53mt2428 (Roth et al., 1998
). This p53 mutant is fully active, inducing the transcriptional activation of p53-responsive promoters to the same extent as wild-type p53. However, it cannot be bound and inhibited by the E1B 55K proteins of Ad5 or Ad12 (Koch et al., 2001
; Roth et al., 1998
; Wienzek et al., 2000
). It also remains stable and active throughout an Ad infection (Koch et al., 2001
). Given these properties, p53mt2428 represents a tool to analyse the behaviour of cells in the simultaneous presence of E1B 55K and transcriptionally active p53.
SV40 employs a similar strategy as Ad to inactivate p53. The large T antigen of SV40 binds p53 and prevents it from activating transcription (Jiang et al., 1993 ; McCormick et al., 1981
; Sarnow et al., 1982a
) but does not decrease the stability of p53 (Zantema et al., 1985b
). In contrast, when cells were transformed by oncogenic human papillomaviruses (HPVs), p53 is destabilized and virtually eliminated. This effect is due to enhanced p53 ubiquitination in the presence of oncogenic HPV E6 proteins (Huibregtse et al., 1991
; Scheffner et al., 1990
, 1993
).
p53 induces the expression (Barak et al., 1994 ; Juven et al., 1993
) of its cellular antagonist, Mdm2 (Oliner et al., 1993
), which mediates the degradation of p53 (Haupt et al., 1997
; Kubbutat et al., 1997
), thereby establishing a regulatory feedback loop (Wu et al., 1993
). In cells that express Ad E1 or SV40 T antigens, however, p53 is at least temporarily inactivated by either E1B 55K or SV40 T antigen and this can be expected to interrupt the activation of mdm2 expression. Accordingly, p53 is stabilized in the presence of E1B 55K (Zantema et al., 1985b
) or SV40 T antigen (Reich et al., 1983
).
Since the stability of p53 is increased in Ad E1- and SV40-transformed cells, we hypothesized that p53 accumulates to an extent that will saturate E1B 55K molecules or T antigens present in these cells and, that beyond this point, any excess of p53 is transcriptionally active. This hypothesis was tested and, indeed, active p53 was constantly present in Ad E1-transformed cells and also in SV40-transformed cells. Furthermore, the stable overexpression of E1B-resistant, active p53mt2428 was tolerated by Ad E1-transformed cells, strongly suggesting that all the relevant growth-preventing products of p53 target genes are effectively inactivated upon transformation of cells by Ad E1 proteins.
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Methods |
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Plasmids.
Expression plasmids for p53 (Lin et al., 1994 ), p53mt2428 (Roth et al., 1998
) and Ad5 E1B 55K (Dobbelstein et al., 1997
) were described previously, as well as the reporter plasmids pGL2BP100 (Freedman et al., 1997
) and pGL2RSV (Ristea et al., 2000
). For stable expression of p53 and its mutants, the p53-encoding region was excised from pRcCMVp53 (Lin et al., 1994
) or pRcCMVp53R273H (Lin et al., 1994
) with HindIII/XbaI. The ends were filled with Pfu turbo DNA polymerase (Stratagene). The plasmid pCIN4, expressing a bicistronic mRNA encoding the gene of interest along with an enzyme conferring neomycin resistance (Rees et al., 1996
), was linearized with EcoRV and ligated to the p53-encoding region in the sense orientation. The plasmid pCINp53mt2428 was made by site-directed mutagenesis using the plasmid pCINp53 and the same oligonucleotides as described previously (Roth et al., 1998
). All constructs were confirmed by sequencing.
Luciferase assays.
For transfections, 2x105 cells per assay were used. Luciferase activities were determined 24 h later using a pre-manufactured assay system (Promega) and an automated luminometer (Berthold).
Immunoblots.
Proteins were separated by 10% SDSPAGE, transferred to nitrocellulose and then incubated with antibodies diluted in PBS containing 5% milk powder and 0·1% Tween 20. Detection was carried out by chemiluminescence (Pierce), using a peroxidase-coupled secondary antibody (Jackson). PAb1801, an antibody to p53 (Calbiochem), and another antibody against Lamin B (Zymed) were diluted 1:5000. Antibody 2A10 to Mdm2 was obtained from a hybridoma supernatant (gift from A. J. Levine) and diluted 1:50.
PCR.
To determine the ratio of wild-type and mutant p53 transcripts in a cell population, total RNA was prepared from these cells using Trizol (Life Technologies) and then treated with DNase. The RNA was reverse transcribed using the p53-specific primer 5' GGGAGCAGCCTCTGGCATTCTG and the RNA-dependent DNA polymerase Superscript II (Life Technologies). This cDNA was then used as a template for PCR amplification, using Pfu turbo DNA polymerase (Stratagene) and the primers 5' ATGGAGGAGCCGCAGTCAGATC and 5' GGGAGCAGCCTCTGGCATTCTG. The DNA obtained was then treated with the restriction enzyme SacI, which cut the PCR product originating from the mutant p53 transcript but not that from the wild-type p53 transcript.
Immunofluorescence.
Cells were seeded on plastic slides suitable for microscopy (Nunc) for 24 h, washed with PBS, fixed with paraformaldehyde (4% in PBS, 15 min), permeabilized with Triton X-100 (0·2% in PBS, 25 min), blocked (10% FBS in PBS, 15 min) and incubated with antibody, as described previously (Dobbelstein et al., 1992 ). To detect E1B 55K, the murine monoclonal antibody 2A6 (Sarnow et al., 1982b
) was used. The p53 protein was stained with a polyclonal rabbit antibody (FL-393, Santa Cruz). Primary mouse antibodies were visualized by secondary antibodies coupled to Alexa 488 (Molecular Probes). Primary rabbit antibodies were detected by an Alexa 594-labelled secondary antibody (Molecular Probes). Prior to mounting (Fluroprep, bioMérieux), the cell nuclei were briefly stained using 4',6-diamidino-2-phenylindole (DAPI).
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Results |
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Discussion |
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If p53 binding by E1B 55K is dispensable for the growth of Ad E1-transformed cells, what is the role of E1B 55K in transformation? One possible explanation is that p53 binding may be required only during the initial steps of cell transformation, as described by a hit-and-run scenario. However, this would not explain the maintenance of E1B 55K expression in the majority of E1-transformed cells. We therefore favour the hypothesis that E1B 55K contributes to malignant transformation by mechanisms in addition to p53 binding. Even though mutational analysis revealed a close association between p53 binding and transformation by E1B 55K (Yew & Berk, 1992 ), it should be considered that most mutants of E1B 55K have lost several functions at a time (Kao et al., 1990
; Rubenwolf et al., 1997
; Yew & Berk, 1992
; Yew et al., 1990
), presumably due to a labile conformation of the protein, which is easily disrupted even by small mutations. Therefore, mutational analysis cannot exclude the presence of at least one more, as yet unknown, biochemical activity of E1B 55K contributing to malignant transformation. A specific E1B 55K mutant that selectively abolishes p53 binding recently became available (Shen et al., 2001
). Testing this mutant in transformation assays may clarify whether E1B 55K contributes to transformation by mechanisms independent of p53 binding.
Although Ad E1-transformed cells can apparently tolerate p53, this tolerance is not without limitations, as is evident from the considerable colony suppression by p53mt2428 (Fig. 5A). Also it is clear that some, but not all, p53 molecules in these cells are inactivated by association with E1B 55K. This can explain the fact that the addition of a p53-derived peptide to Ad E1-transformed cells, and the consecutive massive activation of p53, led to a cell-cycle arrest (Hutton et al., 2000
). Nonetheless, it remains to be stated that Ad E1-transformed cells grew rapidly despite a considerable level of exogenous nuclear p53, whereas p53-/- cells did not.
To our knowledge, this is the first example of cell lines that grow despite the stable overexpression of active p53 from the cytomegalovirus major immediate early promoter one of the strongest known promoters (Boshart et al., 1985 ). This tolerance for active p53 suggests that p53 target gene products, in addition to p53 itself, are inactivated, directly or indirectly, in Ad E1-transformed cells. For some p53 targets, this was indeed found to be the case (Fig. 8B
). p53 induces the p21CIP1/WAF1 gene product (el-Deiry et al., 1993
), which inhibits the phosphorylation of the retinoblastoma protein pRb and thereby the transition from the G1 to the S phase of the cell cycle (Bartek & Lukas, 2001
). However, the Ad E1A gene products bind and inactivate pRb (Whyte et al., 1988
), thereby antagonizing p21. Likewise, p53 induces the expression of Bax, which induces apoptosis (Miyashita & Reed, 1995
). On the other hand, the E1B 19K protein interacts with Bax and inhibits cell death (Chen et al., 1996
; Han et al., 1996
). The induction of apoptosis by p53-induced Fas CD95 (Muller et al., 1998
) may be prevented by E1B 19K as well (Hashimoto et al., 1991
; Perez & White, 1998
). However, many more p53 target gene products were shown to block cell growth. These gene products include inhibitors of G2M transition, such as 14-3-3
(Hermeking et al., 1997
), MCG10 (Zhu & Chen, 2000
) and Reprimo (Ohki et al., 2000
), as well as inducers of apoptosis, such as Apaf-1 (Kannan et al., 2001
; Moroni et al., 2001
), Killer/DR5 (Wu et al., 1997
), MCG10 (Zhu & Chen, 2000
), Noxa (Oda et al., 2000a
), p53AIP1 (Oda et al., 2000b
), PUMA (Nakano & Vousden, 2001
) and many others (Vogelstein et al., 2000
; Vousden, 2000
). If Ad E1-transformed cells grow despite ongoing p53 activity, this could be explained in three ways. Firstly, the expression of most p53 target genes does not inhibit cell growth, which would be in contradiction with their previous functional analysis in many cases. Secondly, the p53 target genes might be mutated in Ad E1-transformed cells. However, it appears unlikely that such a large number of mutations should have occurred during the process of in vitro transformation. Therefore, we favour the third possibility: at least a growth-limiting subset of the p53 target gene products may be regulated, by as yet unknown mechanisms, through the action of Ad E1 proteins at levels downstream from p53 itself. This hypothesis implies that Ad E1 proteins may promote cell growth by a considerably wider variety of mechanisms than previously anticipated.
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Acknowledgments |
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References |
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Received 19 February 2002;
accepted 3 April 2002.