Gastroenterologie und Stoffwechsel, Zentrum Innere Medizin, Klinikum der Universität Marburg, Baldingerstraße, 35043 Marburg, Germany1
Institut für Virologie, Universität Marburg, Robert Koch Str. 17, 35037 Marburg, Germany2
Author for correspondence: Matthias Dobbelstein. Fax +49 6421 286 8962. e-mail dobbelst{at}mailer.uni-marburg.de
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Abstract |
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We first determined if the expression of viral oncoproteins inhibits the transcriptional activity of p51A. For these experiments, the p51A splicing form was chosen since it shows considerably stronger transcriptional activity in reporter assays when compared to the p51B splicing form (Osada et al., 1998 ; our unpublished observations). The p51A cDNA (generous gift from S. Ikawa) was cloned into the expression vector pcDNA3 (Invitrogen) along with the 5' untranslated region of lamin mRNA to enhance expression. Additionally, a C-terminal HA epitope was appended to the protein to facilitate detection. This construct was transfected into H1299 cells (a p53 -/- cell line derived from a lung adenocarcinoma) along with the reporter construct pBP100luc (Freedman et al., 1997
) that contains a fragment of the p53-responsive mdm2-promoter, controlling the expression of luciferase. Quantification of the luciferase enzyme revealed p51A-mediated activation of this promoter (Fig. 1
, lane 7). Similarly, transient expression of p53 yielded enhanced luciferase expression (Fig. 1
, lane 4). This activity of p53 was profoundly suppressed by SV40 T-antigen that was transiently expressed from a co-transfected plasmid (obtained from I. Pastan) (Fig. 1
, lane 5). However, T-antigen failed to suppress p51A-mediated luciferase expression (Fig. 1
, lane 8), strongly suggesting that T-antigen does not inactivate p51A to an extent comparable to the suppression of p53 activity by this viral protein.
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The inhibitory effect of SV40 T-antigen on p53 is apparently a result of direct interaction between the two proteins. Therefore, we determined whether the failure of T-antigen to block the activity of p51A can be explained by specific binding of T-antigen to p53 but not to p51A. p53 and its homologues were prepared and radioactively labelled by in vitro transcription and translation using rabbit reticulocyte lysate (Promega) (Fig. 2, lanes 14). A mutant version of p53 (L22Q/W23S) was prepared (Fig. 2
, lane 2) that lacks the ability to bind mdm2 (Lin et al., 1994
). This mutant version of p53 was included in the experiment since mdm2 was recently shown to form a trimeric complex with p53 and T-antigen (Henning et al., 1997
) and may thus contribute to the stability of p53T-antigen interactions. The p73
protein was prepared (Fig. 2
, lane 3) for use as a negative control, since p73
was previously shown to lack T-antigen binding activity (Dobbelstein & Roth, 1998
; Marin et al., 1998
; Reichelt et al., 1999
). The labelled proteins were then incubated as described previously (Dobbelstein & Roth, 1998
) with nuclear extracts of COS-7 cells, a cell line that constitutively expresses SV40 T-antigen. After further incubation with a monoclonal antibody to T-antigen (Pab 419, Calbiochem) and protein ASepharose (Sigma), and extensive washing, the associated proteins were analysed by autoradiography (Fig. 2
, lanes 58). As shown in Fig. 2
(lane 5) p53 bound to T-antigen. A p53 mutant with amino acid exchanges at positions 22 and 23 bound equally well (Fig. 2
, lane 6), arguing that the binding efficiency of p53 to T-antigen is not enhanced by any mdm2 that may be present in the COS-7 or reticulocyte lysates. p73 (Fig. 2
, lane 7) and p51A (Fig. 2
, lane 8) failed to co-precipitate with T-antigen. None of the labelled proteins was precipitated when a lysate of 293 cells (lacking SV40 T-antigen) was used (Fig. 2
, lanes 912). Taken together, these results indicate that SV40 T-antigen selectively interacts with p53 but fails to interact efficiently with its homologues p73
and p51A.
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While our results show that the HPV-18 E6 protein fails to downregulate p51A-mediated transcription, the reason for this is currently unknown. It is well established that p53 is inactivated by HPV-18 E6 through a complex mechanism that not only includes a direct interaction between p53 and HPV-18 E6, but also the association with another cellular protein termed E6AP. p53 is then ubiquitinylated and degraded (Huibregtse et al., 1991 ; Scheffner et al., 1990
, 1993
; Werness et al., 1990
). It is subject to further investigations whether all of these steps occur specifically on p53 but not p51A, or whether HPV-18 E6 might still form a complex with p51A and E6AP5 without mediating the intracellular degradation of p51A.
At present, it remains possible that the p51 gene does not represent a tumour suppressor gene at all. If so, what other function(s) might be associated with this protein? Studies on mice carrying a targeted disruption of the p51/p63 gene suggest that p51 proteins are required for proper development of the limb and ectodermal structures (Mills et al., 1999 ; Yang et al., 1999
). Such a role in development has not been reported for p53 (Donehower et al., 1992
). Thus, the p51 gene and its products may carry out important functions during development, but the limited life span of these mice makes it difficult to draw any conclusions on its role in tumorigenesis. The results presented here suggest that its function in tumour suppression may not be comparable to the role of p53. The ultimate answer on the importance of p51 in tumour development can be expected from detailed studies on p51 mutations and expression patterns in tumour tissue. In the case of the first p53 homologue to be identified, p73, such studies revealed that the corresponding gene does not display the features of a bona fide tumour suppressor gene in the tumour species examined (Mai et al., 1998
; Nomoto et al., 1998
; Sunahara et al., 1998
; Takahashi et al., 1998
). The failure of viral oncoproteins to interact with p51A suggests that the same might turn out to be true for the p51 gene. However, until the mutational status of the p51 gene and other p53 homologues that might exist (Bian & Sun, 1997
; Zeng et al., 1998
) are fully characterized, it is still not certain whether p53 may have one or several Doppelgänger (Kaelin, 1998
) that can actually replace some of its tumour suppressing functions.
Despite the functional differences between p53 and its homologues encoded by the p73 and p51 genes, we and others have shown that the mdm2 protein antagonizes p73-mediated transcription in addition to regulating p53 (Dobbelstein et al., 1999 ; Zeng et al., 1999
). We have tested the ability of mdm2 to bind p51A (our unpublished observations). While p51A can be shown to bind mdm2 in vitro, we did not observe inhibitory effects of mdm2 on p51A-mediated transcription in our assays (data not shown). Differences in turnover and/or DNA binding affinities between p53 and p51A may explain this phenomenon. At present, it is unclear whether mdm2 can actually inhibit p51A in a physiological setting. However, it is tempting to speculate that antagonizing effects of mdm2 on p73 and possibly p51A may affect embryonic development.
How can the different functional roles of p53 and its homologues be explained on a mechanistic level? First, despite the abilities of all three protein species to activate transcription from different promoters, quantitative differences in promoter specificity between p53 and p73 have been demonstrated (Zhu et al., 1998 ). Such differences may also exist between p53 and p51A. Nonetheless, all three protein species not only activate p53-responsive promoters but also induce apoptosis (Jost et al., 1997
; Osada et al., 1998
; Yang et al., 1998
), which is generally thought to be an essential activity for p53-mediated tumour suppression. However, another important feature that determines the biological role of these proteins is likely to be in the regulation of their expression. Indeed, it has been shown that p53 accumulation can be induced by genotoxic stress, while the levels of p73 do not seem to be upregulated under such circumstances (Kaghad et al., 1997
). Further, p53 is found at low but ubiquitous levels, whereas tissue-specific expression was observed for the p73 and p51 genes (Kaghad et al., 1997
; Osada et al., 1998
). In addition to overall expression, p73 and p51 (but not human p53) can be regulated by alternative splicing, resulting in transcriptionally inactive forms (De Laurenzi et al., 1998
; Yang et al., 1998
) that may even exert dominant negative effects on the transcription from p53-responsive promoters. Taken together, these observations suggest that p53 homologues may, in principle, be able to carry out the biochemical activities needed for tumour suppression, but that their expression may not generally be regulated in a way appropriate to achieve this. In such a scenario, the growth of tumour cells carrying mutant p53 might be controlled by a strategy aimed at the expression of transcriptionally active p73 and p51 proteins in these cells. If the expression of such p53 homologues could be achieved, this would represent a novel therapeutic approach to suppress tumour growth. The failure of viral oncoproteins, like HPV E6, to inhibit p51A and p73 may then represent an advantage that facilitates the reconstitution of growth control.
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References |
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Received 22 June 1999;
accepted 12 August 1999.