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MDM2 function
Biochimica et Biophysica Acta 1377 Ž1998. M55–M59
Mini review
MDM2 function Guillermina Lozano b
a,)
, Roberto Montes de Oca Luna
b
a UniÕersity of Texas, M.D. Anderson Cancer Center, 1515 Holcombe BlÕd., Houston, TX 77030, USA Escuela de Medicina, Instituto Tecnologico y de Estudios Superiores de Monterrey, 64849 Monterrey NL, Mexico
Received 8 October 1997; accepted 10 December 1997
Keywords: Transcription; Translation; Amplification; Tumorigenesis; Cell cycle
1. Introduction The development of human tumors is the result of multiple events including the loss of tumor suppressor genes and the activation of oncogenes. Together these events undermine the signals involved in normal growth control. The mdm2 gene was first cloned as a gene amplified on double minute particles in a transformed murine cell line, hence its name, murine double minute 2 w1x. Upon its identification as a p53 binding protein w2x, and as a gene amplified in many sarcomas w3x, MDM2 quickly rose to fame.
sible for targeting MDM2 to the nucleus. A domain that contains 40% glutamic acid and aspartic acid residues is located between amino acids 221–272. When fused to the lex A DNA binding domain, this sequence was able to activate transcription w7x. However, larger constructs of mdm2 containing this acidic region could not activate transcription. MDM2 also contains a zinc finger of the C4 class at amino acids 305–322 and a RING finger at amino acids 438–478 w8x. Zinc fingers encode DNA binding motifs and,
2. Structure Sequencing of the murine w1x, human w3x and Xenopus w4x dm2 genes reveals several motifs. Beginning at the amino terminus, the first motif encountered is a potential nuclear localization sequence between amino acids 181–185 ŽFig. 1.. While mdm2 is located in the nucleus w5,6x, no experimental data are available which indicate that this sequence is respon-
)
Corresponding author. Tel.: q1-713-792-8945; fax: q1-713794-4295
Fig. 1. A line drawing of MDM2 depicting several motifs, binding proteins, and a binding RNA. Only those MDM2 interacting proteins in which the MDM2 binding domain Žamino acids numbered underneath. has been mapped, are shown. NLS, nuclear localization signal; ZF, zinc finger; RF, ring finger.
0304-419Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 9 X Ž 9 7 . 0 0 0 3 7 - 1
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G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59
together with the observation that the acid domain has transactivation properties, suggested that MDM2 might be a transcription factor. No other data, however, exist which support this hypothesis. RING finger domains are known protein–protein interaction motifs, but may also bind DNA or RNA w9x. An RNA with predicted secondary structure and sequence binds the MDM2 RING finger w10x. A point mutation in a conserved amino acid of the RING finger eliminates binding.
3. Relationship between mdm2 and p53 By far the most important known function of MDM2 is the negative regulation of p53 function. MDM2 binds the amino terminal transactivation domain of p53 and inhibits its ability to activate transcription by masking access to the transcriptional machinery w2,7,11x. The deletion of MDM2 amino acids 1–50 eliminate p53 binding, and the minimal region that binds p53 encompasses amino acids 19– 102 w11x. In fact, the crystal structure of the p53rMDM2 complex has been determined w12x. An unusual interaction motif that is primarily dependent on van der Waals forces with only two hydrogen bonds exists between these two proteins w12x. Another mechanism by which MDM2 may inhibit p53 function is to target p53 for degradation w13,14x. In transient transfection assays, p53 was stable only in the presence of MDM2 mutants that could not bind p53. Moreover, the addition of proteosome inhibitors MG132 or lactacystin that inhibit the ubiquitin degradation pathway allowed stabilization of p53. These data were further substantiated by studies in stable transfectants of MCF7 cells in which the stability of p53 is altered by the levels of MDM2 w15x. Perhaps then initially, MDM2 binds p53 when it is bound at a promoter providing an immediate inhibitory signal, then is responsible for its degradation. However, these data are contradicted by the finding that some tumors show high levels of expression of both HDM2 and wild type p53 w16,17x, suggesting that other cellular factors might also affect either the binding of p53 to HDM2 or the ability of HDM2 to signal ubiquitin degradation of p53. For example, it is possible that mutations, or deletions of HDM2 which have
been seen in some tumors w18x, bind p53, but cannot signal degradation. The most striking example of the importance of the interaction between MDM2 and p53 was observed in vivo by examination of an mdm2 null mouse. The loss of mdm2 resulted in early embryonic lethality, a defect that was completely rescued in the absence of p53 w19,20x. These data suggested that the mdm2 null mouse dies because of its inability to down-modulate p53 and also provided the first demonstration that p53 is functional in normal development. Importantly, these data suggest the possibility that the function of p53 as the guardian of the genome is important during development to prevent the propagation of mistakes during a period of rapid cell division.
4. mdm2 and tumorigenesis Initially cloned as a gene amplified on double minute chromosomes, mdm2 overexpression in nontransformed cells such as NIH3T3 or RAT2 cells resulted in a transformed phenotype w1x. The overexpression of mdm2 alone in primary rat embryo fibroblasts resulted in immortalization, or in transformation when cotransfected with the activated ras gene w21x. Neonatal rat astrocytes also became tumorigenic upon overexpression of mdm2 w22x. Moreover, analysis of the transformed cells showed enhanced expression of the angiogenic mitogens basic fibroblast growth factor and platelet-derived growth factor. In vivo, the overexpression of mdm2 in breast epithelium of transgenic mice leads to the development of tumors w23x. These data support the hypothesis that mdm2 functions as an oncogene in the process of cell transformation. The mapping of the hdm2 gene to chromosome 12, a region often amplified in sarcomas, led to the identification of hdm2 amplification in greater than 30% of sarcomas w3x. The amplification of the hdm2 gene has been seen in many other types of tumors and is summarized in a recent review w24x. Other mechanisms, however, appear to also result in increased HDM2 protein. Immunohistochemical and western analyses of human tumors and cell lines showed that some tumors overexpressed HDM2, but did not show amplification of the gene w17,25x. In one
G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59
set of experiments, Landers et al. w17x showed that enhanced translation of hdm2 was responsible for increased HDM2 protein levels. Thus, it appears that mechanisms other than amplification may also result in the overproduction of HDM2. The finding that HDM2 can bind and inhibit p53 function suggests that it might be an impediment to gene therapy using p53 constructs. Several trials are ongoing that introduce retroviral vectors expressing wild type p53 into tumors. If a tumor contains high levels of HDM2, the introduction of p53 may not have an affect on tumor growth. For those tumors overproducing HDM2 and wild type p53, it may be more effective to inhibit the ability of HDM2 to bind p53, yielding a functional p53. Several small peptides have been shown to inhibit the interaction of p53 and HDM2 w26x and may, in combination with p53, provide a better therapy.
5. p53 independent functions of mdm2 Since the identification of the MDM2rp53 interaction and the amplification of hdm2 in sarcomas without concomitant mutation of p53, it has been postulated that the overexpression of hdm2 is another mechanism of inactivating p53 w3x. However, since the initial observations, some tumors have both amplification of hdm2 and mutations in p53 w27–29x. Patients with these tumors had significantly reduced survival as compared to those with only one of these alterations. These data are indicative of independent functions for the two genes. Experiments in culture and in mice suggest that mdm2 can itself confer a growth advantage in the absence of p53 or has functions independent of p53. The overexpression of mdm2 in cells lacking p53 yielded increased anchorage-independent growth w30x. In addition, Sigalas et al. w18x cloned several hdm2 cDNAs with transforming activities that lacked portions of the p53 binding domain. The clearest demonstration that mdm2 has functions independent of p53 is the generation of transgenic mice that overexpress mdm2 in breast epithelium w23x. The overexpression of mdm2 results in increased BrdU incorporation and PCNA staining, indicative of DNA replication, at a time during lactation when the cells of the mammary gland should be
M57
fully differentiated and nondividing. The cells were normally large and multinucleated, and ploidy analysis suggested that they were undergoing multiple rounds of DNA replication without cytokinesis. Importantly, this phenotype was not altered in mdm2 transgenic mice in the absence of p53, indicating independence of p53. No one doubts the ability of MDM2 to inhibit p53 function, but given the data, it seems reasonable that both p53 and MDM2 can affect growth control independent of each other.
6. mdm2 binds other proteins As mentioned above, evidence has accumulated indicating that MDM2 has separate functions in addition to its interaction and inhibition of p53 function. Recent developments suggest that MDM2 binds other factors implicated in growth control. MDM2 binds another tumor suppressor, the retinoblastoma Ž Rb. gene product w31x This interaction also disrupts Rb function as a growth suppressor and inhibitor of transcription. In addition, a complex was detected between MDM2 and E2F1, a transcription factor important for the G1rS transition w32x. In this case, however, the interaction further stimulated the activity of E2F1. Thus, MDM2 appears to inhibit Rb and p53 function, but its interaction with E2F1 suggests that in addition to inactivation of tumor suppressors, MDM2 can augment cell proliferation by activating genes involved in S-phase progression. These interactions have yet to be confirmed by other investigators. Another mechanism of transcriptional inhibition by MDM2 appears to be the presence of an inhibitory domain that can directly repress basal transcription by binding monomeric TATA-binding protein ŽTBP. and the 34 KDa subunit of TFIIE w33x. The functional significance of the MDM2rTBP interaction is unknown since TBP, in general, is a sticky protein and since the TFIID complex, which includes TBP, does not bind MDM2 w33x. Thus, the TBP domain that interacts with MDM2 may be inaccessible. The interaction of MDM2 with TFIIE may physically impede binding of TFIIE to general transcription factors, thereby inhibiting transcription independent of its interaction with p53. These data, coupled with the observation that MDM2 also contains a zinc finger DNA binding domain, suggest that MDM2 might
M58
G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59
repress transcription of other genes by binding directly to their promoters. The possibility also exists that MDM2 functions in translation since it interacts with the ribosomal proteins L5 w34x and L11 Ž Colman and Finlay, unpublished observations.. We must keep in mind however, that whatever other functions MDM2 might have, they are not important for viability; the absence of both mdm2 and p53 yields viable mice whose major problem is the development of tumors due to lack of p53 w35x.
7. Alternative transcripts and a homolog In some systems, the overexpression of mdm2 results in multiple proteins and an important question is whether these alternate products are necessary andror required for tumorigenesis. The full length MDM2 encodes a predicted 52 kd protein that migrates at 90 kd in SDS-PAGE. MDM2 proteins ranging in size from 57 kd to 85 kd have been observed in cells overexpressing mdm2 w6,18,36x. Different cDNAs have been cloned which contain different regions of mdm2, not all of which correspond to exonrintron boundaries w36,37x. The abnormally spliced transcripts of mdm2 and resulting proteins have not been seen in nontransformed cells, nor in multiple tissues or during embryogenesis in normal mice w18,19,37x. Regardless, the expression of the full length p90 MDM2 protein is sufficient to induce tumors in transgenic mice that contain an mdm2 minigene w23x. Recently, an mdm2 homolog called mdmx was identified by screening an expression library using radioactively labeled p53 w38,39x. The domains most highly conserved are the p53-binding domain at the amino terminus, and the zinc and RING fingers. Like expression of mdm2, mdmx expression inhibits p53 transcriptional activation. However, mdmx does not appear to be able to substitute for mdm2 in early development, perhaps because it is not expressed at that point. A deletion of the mdmx gene would yield some insight into the function of this mdm2 homolog. In summary, the discovery of mdm2 caught everyone’s attention because it inhibited the function of the
tumor suppressor p53. And while this is true, many questions remain as to other functions of MDM2 in tumorigenesis.
References w1x S.S. Fakharzadeh, S.P. Trusko, D.L. George, EMBO J. 10 Ž1991. 1565–1569. w2x J. Momand, G.P. Zambetti, D.C. Olson, D. George, A.J. Levine, Cell 69 Ž1992. 1237–1245. w3x J.D. Oliner, K.W. Kinzler, P.S. Meltzer, D.L. George, B. Vogelstein, Nature 358 Ž1992. 80–83. w4x V. Marechal, B. Elenbaas, L. Taneyhill, J. Piette, M. Mechali, J.-C. Nicolas, A.J. Levine, J. Morreau, Oncogene 14 Ž1997. 1427–1433. w5x F.S. Leach, T. Tokino, P. Meltzer, M. Burrell, J.D. Oliner, S. Smith, D.E. Hill, D. Sidransky, K.W. Kinzler, B. Vogelstein, Cancer Res. 53 Ž1993. 2231–2234. w6x D.C. Olson, V. Marechal, J. Momand, J. Chen, C. Romocki, A.J. Levine, Oncogene 8 Ž1993. 2353–2360. w7x J.D. Oliner, J.A. Pietenpol, S. Thiagalingam, J. Gyuris, K.W. Kinzler, B. Vogelstein, Nature 362 Ž1993. 857–860. w8x M.N. Boddy, P.S. Freemont, K.L.B. Borden, TIBS 19 Ž1994. 198–199. w9x A.J. Saurin, K.L.B. Borden, M.N. Boddy, P.S. Freemont, TIBS 21 Ž1996. 208–214. w10x B. Elenbaas, M. Dobbelstein, J. Roth, T. Shenk, A.J. Levine, Mol. Med. 2 Ž1996. 439–451. w11x J. Chen, V. Marechal, A.J. Levine, Mol. Cell. Biol. 13 Ž1993. 4107–4114. w12x P.H. Kussie, S. Gorina, V. Marechal, B. Elenbaas, J. Moreau, A.J. Levine, N.P. Pavletich, Science 274 Ž1996. 948–953. w13x Y. Haupt, R. Maya, A. Kazaz, M. Oren, Nature 387 Ž1997. 296–299. w14x M.H.G. Kubbutat, S.N. Jones, K.H. Vousden, Nature 387 Ž1997. 299–303. w15x C.A. Midgley, D.P. Lane, Oncogene 15 Ž1997. 1179–1189. w16x J. Keleti, M.M. Quezado, M.M. Abaza, M. Raffeld, M. Tsokos, Am. J. Pathol. 149 Ž1996. 143–151. w17x J.E. Landers, S.L. Cassel, D.L. George, Cancer Res. 57 Ž1997. 3562–3568. w18x I. Sigalas, A.H. Calvert, J.J. Anderson, D.E. Neal, J. Lunec, Nat. Med. 2 Ž1996. 912–917. w19x R. Montes de Oca Luna, D.S. Wagner, G. Lozano, Nature 378 Ž1995. 203–206. w20x S.N. Jones, A.E. Roe, L.A. Donehower, A. Bradley, Nature 378 Ž1995. 206–208. w21x C.A. Finlay, Mol. Cell. Biol. 13 Ž1993. 301–306. w22x S. Kondo, T. Morimura, G.H. Barnett, Y. Kondo, R.W. Peterson, R. Kaakaji, J. Takeuchi, S.A. Toms, J. Liu, B. Werbel, B.P. Barna, Oncogene 13 Ž1996. 1773–1779. w23x K. Lundgren, R. Montes de Oca Luna, Y.B. McNeill, E.P. Emerick, B. Spencer, C.R. Barfield, G. Lozano, M.P. Rosenberg, C.A. Finlay, Genes Dev. 11 Ž1997. 714–725.
G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59 w24x J. Momand, G.P. Zambetti, J. Cell. Biochem. 64 Ž1997. 343–352. w25x M.S. Sheikh, Z.-M. Shao, A. Hussain, J.A. Fontana, Cancer Res. 53 Ž1993. 3226–3228. w26x A. Bottger, V. Bottger, C. Garcia-Echeverria, P. Chene, ¨ ¨ ` H.-K. Hochkeppel, W. Sampson, K. Ang, S.F. Howard, S.M. Picksley, D.P. Lane, J. Mol. Biol. 269 Ž1997. 744–756. w27x C. Cordon-Cardo, E. Latres, M. Drobnjak, M.R. Oliva, D. Pollack, J.M. Woodruff, V. Marechal, J. Chen, M.F. Brennan, A.J. Levine, Cancer Res. 54 Ž1994. 794–799. w28x M. Zou, Y. Shi, S. Al-Sedairy, S.S. Hussain, N.R. Farid, Cancer 76 Ž1995. 314–318. w29x T. Gunther, R. Schneider-Stock, J. Rys, A. Niezabitowshi, A. Roessner, J. Cancer Res. Clin. Oncol. 123 Ž1997. 388– 394. w30x M.C. Dubs-Poterszman, B. Tocque, B. Wasyiyk, Oncogene 11 Ž1995. 2445–2449. w31x Z.-X. Xiao, J. Chen, A.J. Levine, N. Modjtahedi, J. Xing, W.R. Sellers, D.M. Livingston, Nature 375 Ž1995. 694–698. w32x K. Martin, D. Trouche, C. Hagemeier, T.S. Sorensen, N.B. La Thangue, T. Kouzarides, Nature 375 Ž1995. 691–694.
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w33x C.J. Thut, J.A. Goodrich, R. Tjian, Genes Dev. 11 Ž1997. 1974–1986. w34x V. Marechal, B. Elenbaas, J. Piette, J.-C. Nicolas, A.J. Levine, Mol. Cell. Biol. 14 Ž1994. 7414–7420. w35x S.N. Jones, A.T. Sands, A.R. Hancock, H. Vogel, L.A. Donehower, S.P. Linke, G.M. Wahl, A. Bradley, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 14106–14111. w36x D.S. Haines, J.E. Landers, L.J. Engle, D.L. George, Mol. Cell. Biol. 14 Ž1994. 1171–1178. w37x R. Montes de Oca Luna, A.D. Tabor, H. Eberspaecher, D.L. Hulboy, L.L. Worth, M.S. Colman, C.A. Finlay, G. Lozano, Genomics 33 Ž1996. 352–357. w38x A. Shvarts, W.T. Steegenga, N. Riteco, T. van Laar, P. Dekker, M. Bazuine, R.C.A. van Ham, W. van der Houven van Oordt, G. Hateboer, A.J. van der Eb, A.G. Jochemsen, EMBO J. 15 Ž1996. 5349–5357. w39x A. Shvarts, M. Bazuine, P. Dekker, Y.F.M. Ramos, W.T. Steegenga, G. Merckx, R.C.A. van Ham, W. van der Houven van Oordt, A.J. van der Eb, A.G. Jochemsen, Genomics 43 Ž1997. 34–42.
Mini review
MDM2 function Guillermina Lozano b
a,)
, Roberto Montes de Oca Luna
b
a UniÕersity of Texas, M.D. Anderson Cancer Center, 1515 Holcombe BlÕd., Houston, TX 77030, USA Escuela de Medicina, Instituto Tecnologico y de Estudios Superiores de Monterrey, 64849 Monterrey NL, Mexico
Received 8 October 1997; accepted 10 December 1997
Keywords: Transcription; Translation; Amplification; Tumorigenesis; Cell cycle
1. Introduction The development of human tumors is the result of multiple events including the loss of tumor suppressor genes and the activation of oncogenes. Together these events undermine the signals involved in normal growth control. The mdm2 gene was first cloned as a gene amplified on double minute particles in a transformed murine cell line, hence its name, murine double minute 2 w1x. Upon its identification as a p53 binding protein w2x, and as a gene amplified in many sarcomas w3x, MDM2 quickly rose to fame.
sible for targeting MDM2 to the nucleus. A domain that contains 40% glutamic acid and aspartic acid residues is located between amino acids 221–272. When fused to the lex A DNA binding domain, this sequence was able to activate transcription w7x. However, larger constructs of mdm2 containing this acidic region could not activate transcription. MDM2 also contains a zinc finger of the C4 class at amino acids 305–322 and a RING finger at amino acids 438–478 w8x. Zinc fingers encode DNA binding motifs and,
2. Structure Sequencing of the murine w1x, human w3x and Xenopus w4x dm2 genes reveals several motifs. Beginning at the amino terminus, the first motif encountered is a potential nuclear localization sequence between amino acids 181–185 ŽFig. 1.. While mdm2 is located in the nucleus w5,6x, no experimental data are available which indicate that this sequence is respon-
)
Corresponding author. Tel.: q1-713-792-8945; fax: q1-713794-4295
Fig. 1. A line drawing of MDM2 depicting several motifs, binding proteins, and a binding RNA. Only those MDM2 interacting proteins in which the MDM2 binding domain Žamino acids numbered underneath. has been mapped, are shown. NLS, nuclear localization signal; ZF, zinc finger; RF, ring finger.
0304-419Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 9 X Ž 9 7 . 0 0 0 3 7 - 1
M56
G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59
together with the observation that the acid domain has transactivation properties, suggested that MDM2 might be a transcription factor. No other data, however, exist which support this hypothesis. RING finger domains are known protein–protein interaction motifs, but may also bind DNA or RNA w9x. An RNA with predicted secondary structure and sequence binds the MDM2 RING finger w10x. A point mutation in a conserved amino acid of the RING finger eliminates binding.
3. Relationship between mdm2 and p53 By far the most important known function of MDM2 is the negative regulation of p53 function. MDM2 binds the amino terminal transactivation domain of p53 and inhibits its ability to activate transcription by masking access to the transcriptional machinery w2,7,11x. The deletion of MDM2 amino acids 1–50 eliminate p53 binding, and the minimal region that binds p53 encompasses amino acids 19– 102 w11x. In fact, the crystal structure of the p53rMDM2 complex has been determined w12x. An unusual interaction motif that is primarily dependent on van der Waals forces with only two hydrogen bonds exists between these two proteins w12x. Another mechanism by which MDM2 may inhibit p53 function is to target p53 for degradation w13,14x. In transient transfection assays, p53 was stable only in the presence of MDM2 mutants that could not bind p53. Moreover, the addition of proteosome inhibitors MG132 or lactacystin that inhibit the ubiquitin degradation pathway allowed stabilization of p53. These data were further substantiated by studies in stable transfectants of MCF7 cells in which the stability of p53 is altered by the levels of MDM2 w15x. Perhaps then initially, MDM2 binds p53 when it is bound at a promoter providing an immediate inhibitory signal, then is responsible for its degradation. However, these data are contradicted by the finding that some tumors show high levels of expression of both HDM2 and wild type p53 w16,17x, suggesting that other cellular factors might also affect either the binding of p53 to HDM2 or the ability of HDM2 to signal ubiquitin degradation of p53. For example, it is possible that mutations, or deletions of HDM2 which have
been seen in some tumors w18x, bind p53, but cannot signal degradation. The most striking example of the importance of the interaction between MDM2 and p53 was observed in vivo by examination of an mdm2 null mouse. The loss of mdm2 resulted in early embryonic lethality, a defect that was completely rescued in the absence of p53 w19,20x. These data suggested that the mdm2 null mouse dies because of its inability to down-modulate p53 and also provided the first demonstration that p53 is functional in normal development. Importantly, these data suggest the possibility that the function of p53 as the guardian of the genome is important during development to prevent the propagation of mistakes during a period of rapid cell division.
4. mdm2 and tumorigenesis Initially cloned as a gene amplified on double minute chromosomes, mdm2 overexpression in nontransformed cells such as NIH3T3 or RAT2 cells resulted in a transformed phenotype w1x. The overexpression of mdm2 alone in primary rat embryo fibroblasts resulted in immortalization, or in transformation when cotransfected with the activated ras gene w21x. Neonatal rat astrocytes also became tumorigenic upon overexpression of mdm2 w22x. Moreover, analysis of the transformed cells showed enhanced expression of the angiogenic mitogens basic fibroblast growth factor and platelet-derived growth factor. In vivo, the overexpression of mdm2 in breast epithelium of transgenic mice leads to the development of tumors w23x. These data support the hypothesis that mdm2 functions as an oncogene in the process of cell transformation. The mapping of the hdm2 gene to chromosome 12, a region often amplified in sarcomas, led to the identification of hdm2 amplification in greater than 30% of sarcomas w3x. The amplification of the hdm2 gene has been seen in many other types of tumors and is summarized in a recent review w24x. Other mechanisms, however, appear to also result in increased HDM2 protein. Immunohistochemical and western analyses of human tumors and cell lines showed that some tumors overexpressed HDM2, but did not show amplification of the gene w17,25x. In one
G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59
set of experiments, Landers et al. w17x showed that enhanced translation of hdm2 was responsible for increased HDM2 protein levels. Thus, it appears that mechanisms other than amplification may also result in the overproduction of HDM2. The finding that HDM2 can bind and inhibit p53 function suggests that it might be an impediment to gene therapy using p53 constructs. Several trials are ongoing that introduce retroviral vectors expressing wild type p53 into tumors. If a tumor contains high levels of HDM2, the introduction of p53 may not have an affect on tumor growth. For those tumors overproducing HDM2 and wild type p53, it may be more effective to inhibit the ability of HDM2 to bind p53, yielding a functional p53. Several small peptides have been shown to inhibit the interaction of p53 and HDM2 w26x and may, in combination with p53, provide a better therapy.
5. p53 independent functions of mdm2 Since the identification of the MDM2rp53 interaction and the amplification of hdm2 in sarcomas without concomitant mutation of p53, it has been postulated that the overexpression of hdm2 is another mechanism of inactivating p53 w3x. However, since the initial observations, some tumors have both amplification of hdm2 and mutations in p53 w27–29x. Patients with these tumors had significantly reduced survival as compared to those with only one of these alterations. These data are indicative of independent functions for the two genes. Experiments in culture and in mice suggest that mdm2 can itself confer a growth advantage in the absence of p53 or has functions independent of p53. The overexpression of mdm2 in cells lacking p53 yielded increased anchorage-independent growth w30x. In addition, Sigalas et al. w18x cloned several hdm2 cDNAs with transforming activities that lacked portions of the p53 binding domain. The clearest demonstration that mdm2 has functions independent of p53 is the generation of transgenic mice that overexpress mdm2 in breast epithelium w23x. The overexpression of mdm2 results in increased BrdU incorporation and PCNA staining, indicative of DNA replication, at a time during lactation when the cells of the mammary gland should be
M57
fully differentiated and nondividing. The cells were normally large and multinucleated, and ploidy analysis suggested that they were undergoing multiple rounds of DNA replication without cytokinesis. Importantly, this phenotype was not altered in mdm2 transgenic mice in the absence of p53, indicating independence of p53. No one doubts the ability of MDM2 to inhibit p53 function, but given the data, it seems reasonable that both p53 and MDM2 can affect growth control independent of each other.
6. mdm2 binds other proteins As mentioned above, evidence has accumulated indicating that MDM2 has separate functions in addition to its interaction and inhibition of p53 function. Recent developments suggest that MDM2 binds other factors implicated in growth control. MDM2 binds another tumor suppressor, the retinoblastoma Ž Rb. gene product w31x This interaction also disrupts Rb function as a growth suppressor and inhibitor of transcription. In addition, a complex was detected between MDM2 and E2F1, a transcription factor important for the G1rS transition w32x. In this case, however, the interaction further stimulated the activity of E2F1. Thus, MDM2 appears to inhibit Rb and p53 function, but its interaction with E2F1 suggests that in addition to inactivation of tumor suppressors, MDM2 can augment cell proliferation by activating genes involved in S-phase progression. These interactions have yet to be confirmed by other investigators. Another mechanism of transcriptional inhibition by MDM2 appears to be the presence of an inhibitory domain that can directly repress basal transcription by binding monomeric TATA-binding protein ŽTBP. and the 34 KDa subunit of TFIIE w33x. The functional significance of the MDM2rTBP interaction is unknown since TBP, in general, is a sticky protein and since the TFIID complex, which includes TBP, does not bind MDM2 w33x. Thus, the TBP domain that interacts with MDM2 may be inaccessible. The interaction of MDM2 with TFIIE may physically impede binding of TFIIE to general transcription factors, thereby inhibiting transcription independent of its interaction with p53. These data, coupled with the observation that MDM2 also contains a zinc finger DNA binding domain, suggest that MDM2 might
M58
G. Lozano, R. Montes de Oca Luna r Biochimica et Biophysica Acta 1377 (1998) M55–M59
repress transcription of other genes by binding directly to their promoters. The possibility also exists that MDM2 functions in translation since it interacts with the ribosomal proteins L5 w34x and L11 Ž Colman and Finlay, unpublished observations.. We must keep in mind however, that whatever other functions MDM2 might have, they are not important for viability; the absence of both mdm2 and p53 yields viable mice whose major problem is the development of tumors due to lack of p53 w35x.
7. Alternative transcripts and a homolog In some systems, the overexpression of mdm2 results in multiple proteins and an important question is whether these alternate products are necessary andror required for tumorigenesis. The full length MDM2 encodes a predicted 52 kd protein that migrates at 90 kd in SDS-PAGE. MDM2 proteins ranging in size from 57 kd to 85 kd have been observed in cells overexpressing mdm2 w6,18,36x. Different cDNAs have been cloned which contain different regions of mdm2, not all of which correspond to exonrintron boundaries w36,37x. The abnormally spliced transcripts of mdm2 and resulting proteins have not been seen in nontransformed cells, nor in multiple tissues or during embryogenesis in normal mice w18,19,37x. Regardless, the expression of the full length p90 MDM2 protein is sufficient to induce tumors in transgenic mice that contain an mdm2 minigene w23x. Recently, an mdm2 homolog called mdmx was identified by screening an expression library using radioactively labeled p53 w38,39x. The domains most highly conserved are the p53-binding domain at the amino terminus, and the zinc and RING fingers. Like expression of mdm2, mdmx expression inhibits p53 transcriptional activation. However, mdmx does not appear to be able to substitute for mdm2 in early development, perhaps because it is not expressed at that point. A deletion of the mdmx gene would yield some insight into the function of this mdm2 homolog. In summary, the discovery of mdm2 caught everyone’s attention because it inhibited the function of the
tumor suppressor p53. And while this is true, many questions remain as to other functions of MDM2 in tumorigenesis.
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