Failure of viral oncoproteins to target the p53-homologue p51A

Judith Roth1 and Matthias Dobbelstein2

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


   Abstract
Top
Abstract
Main text
References
 
The p51/p63/KET proteins were identified based on their strong homology to the tumour suppressor p53 and a related set of proteins termed p73. All these protein species were shown to activate transcription from at least some p53-responsive promoters. To evaluate a possible role of the transcriptionally active splicing variant p51A/p63{gamma} in tumour suppression, we determined whether viral oncoproteins that inactivate p53 might also target p51A. Neither the large T-antigen of simian vacuolating virus 40 (SV40) nor the E6 protein from human papillomavirus type 18 were found to inhibit p51A-mediated transcription, whereas they strongly suppress the activity of p53. Further, SV40 T-antigen directly interacts with p53 but not detectably with p51A. Finally, a cytoplasmic mutant (K128A) of SV40 T-antigen relocalizes p53 from the nucleus to the cytoplasm, but p51A remains in the nucleus when coexpressed with cytoplasmic T-antigen. These results strongly suggest that the inhibitory effect of these viral oncoproteins is specific for p53 and does not measurably affect p51A. Thus, unlike p53, p51A does not appear to be a necessary target in virus-induced cell transformation and may not exert a role comparable to p53 in tumour suppression.


   Main text
Top
Abstract
Main text
References
 
The p53 gene is the most frequently mutated tumour suppressor gene in human malignancies, and all known small DNA tumour viruses express proteins that inactivate the p53 protein. These and many other lines of evidence support the view that p53 is a central factor in the control of malignant growth (Levine, 1997 ). More recently, it was discovered that p53 is not the only factor of its kind in mammalian cells. Rather, gene products with strong homologies to p53 were identified. These p53 homologues were each found to be expressed in several splicing variants. The first p53 homologue to be reported was termed p73 (Kaghad et al., 1997 ). p73 was subsequently shown to induce transcription and apoptosis in a manner similar to p53 (Jost et al., 1997 ), but some splicing variants of p73 lack these activities (De Laurenzi et al., 1998 ). Another protein (and its splicing variants) with strong homologies to p53 and even more pronounced similarity to p73 was identified from human, murine and rat cells, and termed p51 (Osada et al., 1998 ), p40 (Trink et al., 1998 ), p63 (Yang et al., 1998 ), p73L (Senoo et al., 1998 ) or KET (Schmale & Bamberger, 1997 ). A particularly active splicing variant of this factor was termed p51A (Osada et al., 1998 ) or p63{gamma} (Yang et al., 1998 ), and this splicing form was examined in the present study. While this and other splicing variants of p51 have been shown to activate transcription in a manner analogous to p53 and p73 (Osada et al., 1998 ), it remains to be shown whether p51 is a growth regulator that inhibits the development of malignant tumors, like p53, or whether it performs functions other than tumour suppression. Mice lacking the p51/p63 gene have been developed. These mice are born alive but carry severe developmental defects (Mills et al., 1999 ; Yang et al., 1999 ) and may therefore not live up to the formation of tumors, even if tumour development was facilitated by the absence of p51. Therefore, the role of p51 in cancer needs to be evaluated by different approaches. One way of doing so is to determine if viral oncoproteins evolved to target p51 in addition to p53. This approach was previously used to examine p73. It was shown that the E1B 55 kDa protein from adenovirus (Higashino et al., 1998 ; Marin et al., 1998 ; Roth et al., 1998 ; Steegenga et al., 1999 ), the large T-antigen from simian vacuolating virus 40 (SV40) (Dobbelstein & Roth, 1998 ; Higashino et al., 1998 ; Marin et al., 1998 ; Reichelt et al., 1999 ) and the E6 proteins from oncogenic human papillomaviruses (HPV) (Marin et al., 1998 ; Prabhu et al., 1998 ) all fail to functionally interact with p73 but do efficiently bind and inactivate p53. These results suggest that virus-mediated malignant transformation can occur without inactivating p73 but usually requires the targeting of p53. In this study, we examined the ability of SV40 T-antigen and HPV-18 E6 protein to interact with p51A. Our results suggest that p51A, like p73, does not represent a target for these viral oncoproteins.

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.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Transcriptional activities of p53 and p51A in the presence of SV40 T-antigen and the HPV-18 E6 oncoprotein. Expression plasmids for p53 or p51A (30 ng each) were transfected into H1299 cells using FuGene 6 reagent (Roche), along with expression plasmids for SV40 T-antigen or HPV-18 E6 protein (1·3 µg) and a reporter plasmid (pBP100luc; Freedman et al., 1997 ) containing the p53-responsive element from the mdm2 promoter and the luciferase gene (100 ng). The expression plasmids were combined as indicated below the lanes, and in the negative control experiments, expression plasmids were replaced by the corresponding ‘empty’ vector plasmids. After 24 h, the cells were harvested and luciferase activity was determined. The value obtained with p53 alone was set 100% and the other values were normalized accordingly. The error bars represent the standard errors obtained from at least three independent experiments.

 
In a similar experiment, the E6 protein from HPV-18 was expressed with p53 and p51A using a suitable expression plasmid (gift from M. Scheffner). E6 reduced p53-driven luciferase expression (Fig. 1, compare lanes 4 and 6) but not p51A-mediated reporter expression (Fig. 1, compare lanes 7 and 9). Based on this observation, we conclude that the inhibitory action of E6 is targeted to p53 and affects p51A only to a far lesser extent or not at all.

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 1–4). 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 p53–T-antigen interactions. The p73{beta} protein was prepared (Fig. 2, lane 3) for use as a negative control, since p73{beta} 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 A–Sepharose (Sigma), and extensive washing, the associated proteins were analysed by autoradiography (Fig. 2, lanes 5–8). 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 9–12). Taken together, these results indicate that SV40 T-antigen selectively interacts with p53 but fails to interact efficiently with its homologues p73{beta} and p51A.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2. Physical interaction of p53 but not p51A with SV40 T-antigen. p53, a mutant form of p53 (L22Q/W23S), and the p53 homologues p73{beta} and p51A were synthesized by in vitro transcription and translation using T7 RNA polymerase and rabbit reticulocyte lysate (Promega). The proteins were radioactively labelled by incorporation of [35S]methionine. The products of this reaction were separated by SDS–PAGE and visualized by autoradiography (lanes 1–4). These samples were incubated with lysate of COS-7 cells that constitutively express SV40 T-antigen (lanes 5–8) or with equal amounts of 293 cell lysate that does not contain T-antigen. The incubation was followed by immunoprecipitation with a monoclonal antibody (Pab 419) against T-antigen as described previously (Dobbelstein & Roth, 1998 ). The precipitated material was analysed by SDS–PAGE and autoradiography (lanes 5–12).

 
To confirm this result, a mutant form of T-antigen (K128A) was employed that localizes almost exclusively to the cytoplasm due to a defective nuclear localization signal (Kalderon et al., 1984ab ). Both p53 and p51A were transiently expressed in H1299 cells with a HA tag appended to the carboxy terminus of both proteins to allow detection with the same antibody. As expected, both proteins were found to accumulate in the nucleus (Fig. 3a, c). However, upon co-expression of p53 with cytoplasmic T-antigen, p53 was found predominantly in the cytoplasm (Fig. 3e), as reported previously (Dobbelstein & Roth, 1998 ). In contrast, p51A remained in the nucleus of the cell (Fig. 3, compare c and g), regardless of the expression of cytoplasmic T-antigen. Thus, cytoplasmic T-antigen relocalizes p53 from the nucleus to the cytoplasm but does not affect the location of p51A. This result further supports the view that p51A does not represent a binding partner for SV40 T-antigen.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3. Intracellular relocalization of p53 but not p51A by a cytoplasmic mutant of SV40 T-antigen. H1299 cells were transiently transfected (FuGene 6, Roche) to express carboxy-terminally HA-tagged p53 (a, b, e, f) or p51A (c, d, g, h) using 100 ng of the expression plasmid in each case. In addition, SV40 T-antigen carrying a mutation (K128A) that abolishes nuclear localization (e–h) or the corresponding ‘empty’ vector plasmid (a–d) was transfected (400 ng of expression plasmid). The cells were stained simultaneously with antibodies directed against the HA tag (Santa Cruz Y-11) (a, c, e, g) and against T-antigen (Pab 419) (b, d, f, h).

 
Our results show that viral oncoproteins from HPV and SV40 do not detectably antagonize the activity of p51A but specifically inactivate p53. The E1B 55 kDa protein of adenovirus type 5 seems to display a similar specificity (our unpublished observations). This does not preclude that other viral or cellular proteins might target p51A. However, at least the SV40 T-antigen alone is already sufficient to mediate the transformation of various cell types, yet does not bind p51A. This argues that p51A inactivation is not a prerequisite for virus-mediated malignant transformation, whereas all known small DNA tumour viruses have devised a mechanism to control p53. Therefore, it seems likely that p53 and p51A do not perform equivalent roles in tumour suppression.

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.


   Acknowledgments
 
We thank H.-D. Klenk and R. Arnold for their generous support, and S. Ikawa, M. Scheffner and I. Pastan for plasmids. This work was supported by the German Research Foundation and the P. E. Kempkes foundation. M.D. was a recipient of the Stipendium für Infektionsbiologie of the German Cancer Research Center.


   References
Top
Abstract
Main text
References
 
Bian, J. & Sun, Y. (1997). p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: a third member of the p53 family? Proceedings of the National Academy of Sciences, USA 94, 14753-14758.[Abstract/Free Full Text]

De Laurenzi, V., Costanzo, A., Barcaroli, D., Terrinoni, A., Falco, M., Annicchiarico-Petruzzelli, M., Levrero, M. & Melino, G. (1998). Two new p73 splice variants, gamma and delta, with different transcriptional activity. Journal of Experimental Medicine 188, 1763-1768.[Abstract/Free Full Text]

Dobbelstein, M. & Roth, J. (1998). The large T antigen of simian virus 40 binds and inactivates p53 but not p73. Journal of General Virology 79, 3079-3083.[Abstract]

Dobbelstein, M., Wienzek, S., König, C. & Roth, J. (1999). Inactivation of the p53-homologue p73 by the mdm2-oncoprotein. Oncogene 18, 2101-2106.[Medline]

Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A.Jr, Butel, J. S. & Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215-221.[Medline]

Freedman, D. A., Epstein, C. B., Roth, J. C. & Levine, A. J. (1997). A genetic approach to mapping the p53 binding site in the MDM2 protein. Molecular Medicine 3, 248-259.[Medline]

Henning, W., Rohaly, G., Kolzau, T., Knippschild, U., Maacke, H. & Deppert, W. (1997). MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection. Journal of Virology 71, 7609-7618.[Abstract]

Higashino, F., Pipas, J. M. & Shenk, T. (1998). Adenovirus e4orf6 oncoprotein modulates the function of the p53-related protein, p73. Proceedings of the National Academy of Sciences, USA 95, 15683-15687.[Abstract/Free Full Text]

Huibregtse, J. M., Scheffner, M. & Howley, P. M. (1991). A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO Journal 10, 4129-4135.[Abstract]

Jost, C. A., Marin, M. C. & Kaelin, W. G.Jr (1997). p73 is a human p53-related protein that can induce apoptosis. Nature 389, 191-194.[Medline]

Kaelin, W. G.Jr (1998). Another p53 Doppelganger? Science 281, 57-58.[Free Full Text]

Kaghad, M., Bonnet, H., Yang, A., Creancier, L., Biscan, J. C., Valent, A., Minty, A., Chalon, P., Lelias, J. M., Dumont, X., Ferrara, P., McKeon, F. & Caput, D. (1997). Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 90, 809-819.[Medline]

Kalderon, D., Richardson, W. D., Markham, A. F. & Smith, A. E. (1984a). Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311, 33-38.[Medline]

Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984b). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509.[Medline]

Levine, A. J. (1997). p53, the cellular gatekeeper for growth and division. Cell 88, 323-331.[Medline]

Lin, J., Chen, J., Elenbaas, B. & Levine, A. J. (1994). Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes & Development 8, 1235-1246.[Abstract]

Mai, M., Yokomizo, A., Qian, C., Yang, P., Tindall, D. J., Smith, D. I. & Liu, W. (1998). Activation of p73 silent allele in lung cancer. Cancer Research 58, 2347-2349.[Abstract]

Marin, M. C., Jost, C. A., Irwin, M. S., DeCaprio, J. A., Caput, D. & Kaelin, W. G.Jr (1998). Viral oncoproteins discriminate between p53 and the p53 homolog p73. Molecular and Cellular Biology 18, 6316-6324.[Abstract/Free Full Text]

Mills, A. A., Zheng, B., Wang, X. J., Vogel, H., Roop, D. R. & Bradley, A. (1999). p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708-713.[Medline]

Nomoto, S., Haruki, N., Kondo, M., Konishi, H. & Takahashi, T. (1998). Search for mutations and examination of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers. Cancer Research 58, 1380-1383.[Abstract]

Osada, M., Ohba, M., Kawahara, C., Ishioka, C., Kanamaru, R., Katoh, I., Ikawa, Y., Nimura, Y., Nakagawara, A., Obinata, M. & Ikawa, S. (1998). Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nature Medicine 4, 839-843.[Medline]

Prabhu, N. S., Somasundaram, K., Satyamoorthy, K., Herlyn, M. & El-Deiry, W. S. (1998). p73, unlike p53, suppresses growth and induces apoptosis of human papillomavirus E6-expressing cancer cells. International Journal of Oncology 13, 5-9.[Medline]

Reichelt, M., Zang, K. D., Seifert, M., Welter, C. & Ruffing, T. (1999). The yeast two-hybrid system reveals no interaction between p73 alpha and SV40 large T-antigen. Archives of Virology 144, 621-626.[Medline]

Roth, J., König, C., Wienzek, S., Weigel, S., Ristea, S. & Dobbelstein, M. (1998). Inactivation of p53 but not p73 by adenovirus type 5 E1B 55-kilodalton and E4 34-kilodalton oncoproteins. Journal of Virology 72, 8510-8516.[Abstract/Free Full Text]

Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M. (1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129-1136.[Medline]

Scheffner, M., Huibregtse, J. M., Vierstra, R. D. & Howley, P. M. (1993). The HPV-16 E6 and E6–AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75, 495-505.[Medline]

Schmale, H. & Bamberger, C. (1997). A novel protein with strong homology to the tumor suppressor p53. Oncogene 15, 1363-1367.[Medline]

Senoo, M., Seki, N., Ohira, M., Sugano, S., Watanabe, M., Tachibana, M., Tanaka, T., Shinkai, Y. & Kato, H. (1998). A second p53-related protein, p73L, with high homology to p73. Biochemical and Biophysical Research Communications 248, 603-607.[Medline]

Steegenga, W. T., Shvarts, A., Riteco, N., Bos, J. L. & Jochemsen, A. G. (1999). Distinct regulation of p53 and p73 activity by adenovirus E1A, E1B, and e4orf6 proteins. Molecular and Cellular Biology 19, 3885-3894.[Abstract/Free Full Text]

Sunahara, M., Ichimiya, S., Nimura, Y., Takada, N., Sakiyama, S., Sato, Y., Todo, S., Adachi, W., Amano, J. & Nakagawara, A. (1998). Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas. International Journal of Oncology 13, 319-323.[Medline]

Takahashi, H., Ichimiya, S., Nimura, Y., Watanabe, M., Furusato, M., Wakui, S., Yatani, R., Aizawa, S. & Nakagawara, A. (1998). Mutation, allelotyping, and transcription analyses of the p73 gene in prostatic carcinoma. Cancer Research 58, 2076-2077.[Abstract]

Trink, B., Okami, K., Wu, L., Sriuranpong, V., Jen, J. & Sidransky, D. (1998). A new human p53 homologue. Nature Medicine 4, 747-748.[Medline]

Werness, B. A., Levine, A. J. & Howley, P. M. (1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76-79.[Medline]

Yang, A., Kaghad, M., Wang, Y., Gillett, E., Fleming, M. D., Dotsch, V., Andrews, N. C., Caput, D. & McKeon, F. (1998). p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Molecular Cell 2, 305-316.[Medline]

Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R. T., Tabin, C., Sharpe, A., Caput, D., Crum, C. & McKeon, F. (1999). p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714-718.[Medline]

Zeng, X., Levine, A. J. & Lu, H. (1998). Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53. Proceedings of the National Academy of Sciences, USA 95, 6681-6686.[Abstract/Free Full Text]

Zeng, X., Chen, L., Jost, C. A., Maya, R., Keller, D., Wang, X., Kaelin, W. G.Jr, Oren, M., Chen, J. & Lu, H. (1999). MDM2 suppresses p73 function without promoting p73 degradation. Molecular and Cellular Biology 19, 3257-3266.[Abstract/Free Full Text]

Zhu, J., Jiang, J., Zhou, W. & Chen, X. (1998). The potential tumor suppressor p73 differentially regulates cellular p53 target genes. Cancer Research 58, 5061-5065.[Abstract]

Received 22 June 1999; accepted 12 August 1999.