Here we report the isolation of a cDNA encoding a new p53‐associating protein. This new protein has been called MDMX on the basis of its structural similarity to MDM2, which is especially notable in the p53‐binding domain. In addition, the putative metal binding domains in the C‐terminal part of MDM2 are completely conserved in MDMX. The middle part of the MDMX and MDM2 proteins shows a low degree of conservation. We can show by co‐immunoprecipitation that the MDMX protein interacts specifically with p53 in vivo. This interaction probably occurs with the N‐terminal part of p53, because the activity of the transcription activation domain of p53 was inhibited by co‐transfection of MDMX. Northern blotting showed that MDMX, like MDM2, is expressed in all tissues tested, and that several mRNAs for MDMX can be detected. Interestingly, the level of MDMX mRNA is unchanged after UV irradiation, in contrast to MDM2 transcription. This observation suggests that MDMX may be a differently regulated modifier of p53 activity in comparison with MDM2. Our study indicates that at least one additional member of the MDM protein family exists which can modulate p53 function.

It has recently been shown that an adenovirus mutant lacking expression of the large E1B protein (DE1B) selectively replicates in p53 de®cient cells. However, apart from the large E1B protein the adenovirus early region encodes the E1A and E4orf6 proteins which also have been reported to aect p53 expression as well as its functioning. After infection with wild-type adenovirus we observed a dramatic decrease in wild-type p53 expression while no down-regulation of p53 could be detected after infection with the DE1B virus. The dierent eects of the wild-type and DE1B adenovirus on p53 expression were not only found in cells expressing wild-type p53 but were also observed when tumor cells expressing highly stabilized mutant p53 were infected with these two viruses. Infection with dierent adenovirus mutants indicated the importance of a direct interaction between p53 and the large E1B protein for reduced p53 expression after infection. Moreover, coexpression of the E4orf6 protein was found to be required for this phenomenon, while expression of E1A is dispensable. In addition, we provide evidence that p53 is actively degraded in wild-type adenovirus-infected cells but not in DE1B-infected cells.

Multiple adenovirus (Ad) early proteins have been shown to inhibit transcription activation by p53 and thereby to alter its normal biological functioning. Since these Ad proteins affect the activity of p53 via different mechanisms, we examined whether this inhibition is target gene specific. In addition, we analyzed whether the same Ad early proteins have a comparable effect on transcription activation by the recently identified p53 homologue p73. Our results show that the large E1B proteins very efficiently inhibited the activity of p53 on the Bax, p21Waf1 , cyclin G, and MDM2 reporter constructs but had no effect on the activation of the same reporter constructs by p73, with the exception of some inhibition of the Bax promoter by Ad12 E1B. The repressive effect of the E1A proteins on p53 activity is less than that seen with the large E1B proteins, but the E1A proteins inhibit the activity of both p53 and p73. We could not detect significant inhibition of p53 functions by E4orf6, but a clear repression of the transcription activation by p73 by this Ad early protein was observed. In addition, we found that stable expression of the Ad5 E1A and that of the E1B protein both caused increased p73 protein expression. The large E1B and the E4orf6 proteins together do not target the p73 protein for rapid degradation after adenoviral infection, as has previously been found for the p53 protein, probably because the large E1B protein does not interact with p73. Our results suggest that the p53 and p73 proteins are both inactivated after Ad infection and transformation but via distinct mechanisms.By regulating the expression of different target genes, p53 can affect important cellular processes like cell cycle progression and apoptosis. Multiple adenovirus early (Ad E) proteins have been shown to inhibit the transcription activation potential of p53 via different mechanisms and thereby to impair its normal biological functioning. The first Ad E protein identified as inhibiting p53 activity was the large E1B protein (38). Yew and colleagues have shown that in the presence of the large E1B protein p53 can still bind to its consensus sequence but that the transcription-repressive regions present in the large E1B protein inhibit the transcription activation potential of p53 (39). In addition, we and others have shown that the E1A proteins also can inhibit transcription activation by p53 (27,30). The E1A proteins can directly interact with the p300 protein, which not only serves as a cofactor for p53 transactivation but also activates its sequence-specific DNA binding by acetylation of the C terminus of p53 (6, 7, 12). The third Ad E protein which has been reported to inhibit transcription activation by p53 is the E4orf6 protein. Dobner and coworkers reported that E4orf6 inhibits the activity of p53 by a direct interaction with the C terminus of p53 which inhibits binding of TAF32 to the transcription activation domain of p53 (2). It is currently unclear why Ads produce such a number of different proteins all causing repression ...

p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo-or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G 1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.Cell proliferation is a tightly regulated process in which the p53 tumor suppressor protein plays an important role. A possible mechanism by which p53 can, at least partially, fulfill its tumor suppressor function is by regulating the expression of a set of target genes. p53 is known to be able to down-regulate the expression of a number of genes (15,33,46,53), probably by interacting with the basal transcription machinery (1,41,48). On the other hand, p53 has also been found to activate the transcription of genes containing a p53-responsive element (12,43,57). The list of p53-inducible genes at the moment features genes like Waf1 (11), MDM2 (2, 64), GADD45 (19), the cyclin G gene (38, 67), EGFR (7), and Bax (36, 47), but probably many more p53-responsive genes will be identified in the near future. Most naturally occurring p53 mutants have lost their normal transcription-regulatory functions (20). In addition, a gain of function has been shown for certain p53 mutants (9, 24).We have used the adenovirus transformation system to obtain more insight into the mechanism of regulation of transcription by p53 and to answer the question whether structural changes of p53 play a role in these modulations. It has been shown that the two major adenovirus E1B proteins have different effects on the transcription-regulatory functions of p53. The small E1B protein can inhibit only the transcription-inhibitory function of p53 (44,49,51), whereas the large E1B protein can inhibit the stimulation (66) as well as the repression of transcription by p53 (51). We have previously shown that in adenovirus type 12 (Ad12)-transformed cells, p53 is present in complexes of increased molecular weight compared with those...

The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc-®nger family and is homozygously mutated or deleted in a subset of Wilms' tumors. Through alternative mRNA splicing, the gene is expressed as four main polypeptides that dier by a stretch of 17 amino acids just N-terminal of the four zinc-®ngers and three amino acids between zinc ®ngers 3 and 4. We have previously shown that expression of the WT1(7/7) isoform, lacking both inserts, increases the tumor growth rate of the adenovirus-transformed baby rat kidney (AdBRK) cell line 7C3H2, whereas expression of the WT1(7/+) isoform, lacking the 17aa insert, strongly suppresses the tumorigenic phenotype. In the present study we show that expression of these splice variants does not aect the tumorigenic potential of the similar AdBRK cell line, 7C1T1. In contrast to the 7C3H2 cell line, this AdBRK cell line expresses high endogenous levels of EGR-1 (early growth response-1) protein, a transcription factor structurally related to WT1. Ectopic expression of EGR-1 in the 7C3H2 AdBRK cells signi®cantly increases their in vivo growth rate and nulli®es the tumor suppressor activity of the WT1(7/+) protein. Furthermore, we ®nd that EGR-1 levels are elevated in some Wilms' tumors. These data are the ®rst to show that EGR-1 overexpression causes enhanced tumor growth and that WT1 and EGR-1 exert antagonizing eects on growth regulation in baby rat kidney cells, which might re¯ect the situation in some Wilms' tumors. Oncogene (2000) 19, 791 ± 800.