![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Shunbin Xiong,
Ana C. Elizondo-Fraire,
John M. Parant, and
Guillermina Lozano*
The University of Texas Graduate School of Biomedical Sciences and Department of Molecular Genetics, Section of Cancer Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Received 20 June 2005/ Returned for modification 31 July 2005/ Accepted 12 October 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The activity of p53 is negatively regulated by numerous proteins (22). The functional significance of two of these, Mdm2 and Mdm4, has been examined with mouse models. Mice lacking Mdm2 die early in development (18, 25). Embryonic death occurs before implantation as a result of the activation of the p53-dependent apoptotic pathway in blastocysts (3). Concomitant deletion of p53 completely rescues this lethal phenotype. Loss of Mdm4, the gene encoding a second p53 inhibitor, also results in an embryonic lethal phenotype that is rescued by loss of p53 (7, 23, 28).
While clearly Mdm2 and Mdm4 are both potent p53 inhibitors, the relationship between them is complex and entangled. Mdm2 is an E3 ubiquitin ligase that catalyzes ubiquitination of itself and p53 (13, 15, 20). Mdm4, on the other hand, does not appear to ubiquitinate p53 (17). Both Mdm2 and Mdm4 bind the same p53 domain with similar affinities (2). The relationship between Mdm2 and Mdm4 is more complex, as Mdm4 was identified with yeast two-hybrid screens by using Mdm2 as bait (30, 32). In transient-transfection experiments, Mdm2 and Mdm4 interact through their RING domains. This interaction has two major effects: (i) it pulls Mdm4, a cytoplasmic protein, into the nucleus, and (ii) it inhibits the E3 ligase activity of Mdm2, allowing stabilization of p53 and Mdm2. Since Mdm2 and Mdm4 interact, bind the same domain of p53, and yet have different effects on p53, the ratio of these two proteins to each other should determine the outcome of p53 regulation. Indeed, when cells have higher levels of Mdm2 than of Mdm4, p53 is ubiquitinated and unstable. When cells have higher levels of Mdm4 than of Mdm2, stable but inactive p53 is present (12). One must keep in mind, though, that all of these data are from overexpression experiments producing supraphysiological levels of Mdm2 and Mdm4 and that most studies use tagged versions of the proteins, which may affect their activities as well. Nevertheless, these in vitro studies raised questions as to the ratios of Mdm2 and Mdm4 in normal development and in specific tissues in the regulation of p53 activity.
A delicate balance, controlled by multiple pathways, is critical to maintaining p53 at appropriate levels. While absence of p53 results for the most part in a normal mouse embryo, too much p53 results in developmental abnormalities. The Mdm2 and Mdm4 null phenotypes in mice are classic examples of developmental defects due to constitutive p53 activity. Mdm2 null mice die by initiating p53-dependent apoptosis at embryonic day 3.5 (E3.5), while two different alleles of Mdm4 initiate p53-dependent cell cycle arrest and/or apoptosis at later developmental time points (3, 23, 28). Interestingly, these results suggest that the role of the p53 inhibitors in regulating p53 function in apoptosis and cell cycle arrest during embryonic development may vary in a temporal and tissue-specific manner. To examine this possibility, we generated Mdm2 (11) and Mdm4 conditional alleles (this study).
To probe the functional significance of Mdm2 and Mdm4 on apoptosis and proliferation, we used the 
-myosin heavy chain promoter driving Cre expression in the developing heart. During embryonic development, mononucleated contractile cardiomyocytes proliferate (29). Differentiation of cardiomyocytes occurs shortly after birth, and eventually these cells withdraw from the cell cycle. Shaping of the embryonic heart involves a balance of apoptosis and proliferation, which continue postnatally in cardiomyocytes but cease by adulthood (5, 19). We chose the -myosin heavy chain because it is cardiac specific, it is expressed beginning at E8.5, and its expression lasts throughout development (6, 21, 27). We deleted Mdm2 and Mdm4 in cardiomyocytes by crossing the -myosin heavy chain promoter-driven Cre mouse (MyHC-Cre) (1) with Mdm2 and Mdm4 conditional alleles, and we show tissue-specific differences for the roles of Mdm2 and Mdm4 in heart development.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 mice.
PCR genotyping.
ES clones were amplified using primer F1, 5'-TTAACCAGTTGGGTCCATCTCTTCTGG-3', and a primer within neo cassette neo19, 5'-GCTATCAGGACATAGCGTTGGC-3'. The following primers were used to distinguish between the Mdm4 conditional and null alleles: F4, 5'-TAGAATCTGGAATTACAGACAG-3'; In1re, 5'-TGTCTTTAGCATTTACTAAGAGCT-3'; and In2re, 5'-TATCCAGTGTCCTCTTCTGGCTT-3'. Another set of primers was used to distinguish Mdm4 wild-type and conditional alleles: F3, 5'-GGTGTCCTTGAACTTGCTGTGTAGAA-3', and E2re, 5'-CTGGGCCGAGGTGGAATGTGATGT-3'. The Cre transgene was genotyped using the following primers: cre-up, 5'-TCCAATTTACTGACCGTACACCAA-3', and cre-dn, 5'-CCTGATCCTGGCAATTTCGGCTA-3'. The p53 wild-type and mutant alleles were genotyped as previously described (16). Primers A, B, and C were previously described and amplify the following alleles: wild-type Mdm2, Mdm2exons 5-6, and Mdm2FM (11).
ß-Galactosidase (ß-gal) staining of embryos. Embryos at various stages of development were fixed for 1 h, rinsed, and stained overnight as described previously (26). Embryos were embedded in paraffin, and sections (7 µm thick) were mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Slides were counterstained with eosin.
Histology, TUNEL staining, and immunohistochemistry. Embryos at E9.5 were fixed overnight in 4% (vol/vol) paraformaldehyde and were embedded in paraffin. Sections (7 µm thick) were mounted on Superfrost Plus slides and were stained with eosin and hematoxylin. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were carried out on paraffin-embedded embryo sections as described previously (9), modified by using ABC and DAB kits from Vector Laboratories (Burlingame, CA). Slides were counterstained with methyl green.
The mouse sections were subjected to immunohistochemistry by using commercial monoclonal antiactin, smooth-muscle immunoglobulin G (1:200) (Sigma, St. Louis, MO), CM-5 (1:200) (Vector Laboratories, Burlingame, CA), and Ki-67 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA). Visualization was performed using the ABC and DAB kits named above. Counterstaining was performed with nuclear fast red.
Western blot analysis.
Cell lysates (1or 2 mg) from wild-type and p53–/– Mdm42/2 mouse embryo fibroblasts were precleared with protein A agarose (Sigma, St. Louis, MO) and immunoprecipitated with an Mdm4 polyclonal antibody (J. A. Barboza and G. Lozano, unpublished data) or a green fluorescent protein antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as the control, overnight. Western blot analysis was performed with the same Mdm4 antibody.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
exons 5-6 allele that results from Cre recombination in the germ line. When heterozygous Mdm2exons 5-6 mice were mated with each other, no homozygous Mdm2exons 5-6 mice were born, indicating that this allele also resulted in embryonic lethality. Dissection of embryos at E8.5 showed approximately 25% empty deciduas, a phenotype identical to that of the Mdm2 null embryo. Loss of p53 also rescued the Mdm2exons 5-6 homozygous phenotype. Thus, loss of Mdm2 from the conditional allele led to the same embryonic lethal phenotype as was previously described for an Mdm2 null allele (25).
|
To generate mice with a deletion of Mdm4 from the conditional allele and examine their phenotype, we crossed the Mdm4+/FX mouse to a CMV-Cre mouse. Mice heterozygous for this Mdm42 allele were normal. Mdm4+/2 mice were intercrossed to examine the phenotype of Mdm42/2 mice. None of the 42 offspring born were Mdm42/2, indicating that the deletion of exon 2 of Mdm4 results in an embryonic lethal phenotype. Dissection of embryos at E9.5 showed that all Mdm42/2 embryos were abnormal, similar to the phenotype for the original Mdm4 null mutation (28). We also tested whether the embryonic lethality of Mdm42/2 mice can be rescued by loss of p53. The Mdm42 allele was bred into a p53 null background. The Mdm42/2 p53–/– mice were viable and born at approximately the expected ratio. We also performed Western blot analysis using mouse embryo fibroblasts from Mdm42/2 p53–/– mice and detected no Mdm4 protein (Fig. 1E). Thus, extensive analyses of the Mdm2 and Mdm4 conditional alleles indicated that deletion with Cre recombinase resulted in null alleles that had phenotypes identical to those of the original null alleles.
Loss of Mdm2 in embryonic heart is lethal.
Since shaping of the embryonic heart involves a balance of apoptosis and proliferation, we decided to examine the roles of the p53 inhibitors Mdm2 and Mdm4 in heart development. We deleted Mdm2 and Mdm4 in the mouse embryonic heart by using the MyHC-Cre mouse (1). MyHC-Cre mice contain the -myosin heavy-chain promoter driving expression of Cre recombinase in mature cardiomyocytes. To formally test whether the MyHC-Cre transgene expressed Cre in the heart during embryogenesis, we crossed the MyHC-Cre mouse to the ROSA26 reporter line that contains a lacZ gene that is not expressed unless Cre recombination occurs (31). MyHC-Cre ROSA26 embryos were collected at various stages and stained for ß-gal activity. LacZ staining was first observed at E9.5 and was specific for the heart (Fig. 2A and B). Staining became more prominent at later embryonic stages but remained heart specific (Fig. 2C and D). Thus, the MyHC-Cre transgene expressed Cre beginning at E9.5 and expression was cardiac specific.
|
MyHC-Cre gene. From this cross, 38 total progeny were genotyped and none was Mdm2–/FM MyHC-Cre (Table 1). If viable, 25% of the mice (approximately 10 mice) from this cross should have been Mdm2–/FM MyHC-Cre. Thus, loss of Mdm2 in the heart resulted in an embryonic lethal phenotype.
|
MyHC-Cre male to determine the timing of embryonic lethality. All Mdm2–/FM MyHC-Cre embryos died by E13.5, and most lacked any visible signs of a heart (data not shown). All embryos at E9 were histologically normal (see Fig. S1 in the supplemental material). Abnormalities appeared only in Mdm2–/FM MyHC-Cre embryos as early as E9.5, the same time that heart-specific Cre recombination was first detected. Embryos at E9.5 showed a significant thinning of the myocardial layer in the ventricles, which eventually led to heart failure and embryonic death (Fig. 3A and B). Immunohistochemistry with an smooth-muscle actin antibody, a marker that stains cardiomyocytes at this embryonic stage, showed the presence of cardiomyocytes in the mutant embryo at E9.5, but the heart mass was smaller in size (Fig. 3C and D). The embryonic cardiomyocytes in the ventricles were affected more than those in the atrium of the heart, as shown by LacZ staining with the ROSA26 reporter (data not shown). Careful review of these data showed Cre recombination was strongest in the ventricles. Many of the mutant embryos were also smaller than wild-type embryos, probably due to lack of blood flow. However, mutant embryos developed to the correct developmental stage based on somite count.
|
MyHC-Cre embryos at E13.5 that still had a heart showed abnormal trabeculation in the ventricle and abnormal ventricular wall structure (Fig. 3E to H). The mutant heart failed to function properly, as indicated by blood leaking outside the heart and the backup of blood in the atrium (Fig. 3H and I). In addition, the decreased blood flow resulted in congestion and backup of blood in other organs (Fig. 3J).
To examine mechanistically the loss of cardiomyocytes, we performed apoptosis and proliferation assays with embryos at E9.5, the earliest developmental stage that showed defects. To determine if the abnormal heart phenotype was due to apoptosis, TUNEL assays were performed. A rare apoptotic cell appeared in the normal heart as expected, but apoptosis was much higher in the Mdm2 mutant embryo (Fig. 4A and B). To examine proliferation, sections of normal and abnormal hearts were stained with Ki-67 (Fig. 4C and D). Even though fewer cells were present in the Mdm2–/FM MyHC-Cre embryo hearts, they retained the ability to proliferate. Immunohistochemistry was also used to examine the presence of p53. Increased p53 levels were detected in some cells of the mutant heart but not in the normal heart (Fig. 4E and F). The presence of only a small number of p53-positive cells was probably due to the fact that cells were dying. In conclusion, the absence of Mdm2 in the heart results in increased p53 levels, leading to the loss of myocardial cells, particularly in the ventricles. The loss of these myocardial cells results in an abnormal heart structure that eventually leads to embryonic lethality.
|
MyHC-Cre mouse and analyzed by PCR amplification to detect recombination. Recombination was observed only in the heart (Fig. 5A). Recombination appeared to be incomplete, but that is because cardiomyocytes represent only 15% of the cells at this stage. To examine recombination during embryogenesis, we collected DNA from E9.5 embryos. The recombined Mdm2exons 5-6 allele was present in the Mdm2+/FM MyHC-Cre embryo (Fig. 5B). Taken together, the data indicate that loss of Mdm2 specifically in the heart of a developing embryo results in a lethal phenotype.
|
MyHC-Cre mice, 6 were Mdm2–/FM p53–/– MyHC-Cre (Table 2). These mice had the same life span as p53–/– and p53–/– Mdm2–/– mice (data not shown). Importantly, haploinsufficiency at the p53 locus did not rescue the phenotype (Table 2). Thus, the absence of p53 rescued the heart lethal phenotype.
|
2 MyHC-Cre mice. Progeny from these crosses showed the correct ratio of Mdm42/FX MyHC-Cre mice at weaning (Table 3). To confirm recombination of the Mdm4FX conditional allele by the Cre transgene, DNA was purified from the organs of Mdm4FX/FX MyHC-Cre mice. As shown in Fig. 6, the null specific Mdm4 product was present only in the heart but not in the lung, liver, tail, or skeletal muscle. These data indicate that recombination occurred and was specific to the hearts of double mutant mice. Again, recombination did not appear to be very robust, but only 15% of the cells at this developmental stage are cardiomyocytes. Additionally, since we generated mice with a floxed Mdm4 allele in the presence of a null Mdm4 allele, any recombination event would result in an Mdm4 null cell. No discernible differences in the embryonic hearts of normal and Mdm4 mutant embryos were observed (Fig. 7). Thus, loss of Mdm4 in the heart was not embryonic lethal. Some Mdm42/FX MyHC-Cre mice died within 1 year. The reason for this shortened life span is unknown at the moment.
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
At least four p53 inhibitors have been identified thus far, and we do not yet understand the need for so many p53 inhibitors. We do, however, know that the levels of active p53 must be tightly regulated to avoid cell lethality. Several in vivo experiments support this hypothesis. Ectopic expression of wild-type p53 in the kidneys, mostly during embryogenesis, hinders the differentiation of the ureteric bud, leading to smaller kidneys and subsequent renal failure (10). Mice heterozygous for the p53 gene that produced a truncated active form of p53 displayed increased resistance to spontaneous tumors and early onset of aging-associated phenotypes (33). Additionally, loss of either Mdm2 or Mdm4 results in embryonic lethal phenotypes that are rescued by loss of p53 (7, 18, 23, 25, 28). The p53 dependency suggests that the inability to down-modulate p53 activity in the absence of Mdm2 or Mdm4 is lethal. Thus, in several examples, too much p53 results in cell death.
Biochemical experiments have indicated that Mdm2 and Mdm4 form a heterocomplex (30, 32) and that this complex is critical for inactivation of p53 (12). In fact, in vitro studies by Gu et al. (12) suggest that Mdm2 and Mdm4 are mutually dependent. Mdm2 is functionally inefficient in the absence of Mdm4 due to the targeting of itself for degradation. Heterocomplex formation between the two proteins results in an increase in Mdm2 stability by hindering autoubiquination, thus enhancing Mdm2's ability to target p53 for degradation. In some studies, Mdm4, which lacks a nuclear localization signal, is dependent on Mdm2 to take it to the nucleus (24). The studies described here indicate that Mdm2 can, in the absence of Mdm4, function as a perfectly good p53 inhibitor. The reverse was not true; Mdm4 could not compensate for loss of Mdm2.
Marine and Jochemsen (22) described various models for the roles of Mdm2 and Mdm4 in negatively regulating p53 in vivo. Among them was the possibility that Mdm2 was the main regulator of p53 and Mdm4 acted as a cofactor at particular tissues or stages of differentiation, such as during the massive expansion of progenitor cells. Mdm4 would assist Mdm2 in the rapidly dividing cells to reduce p53 activity. Another proposed model is that Mdm2 and Mdm4 function independently in different cell types, although this was believed to be less likely. The analyses presented here, which indicate that loss of Mdm4 has no effect on heart development while that of Mdm2 results in abnormal heart structure and embryonic lethality, support the model that Mdm2 and Mdm4 are independent regulators of p53 in heart development. Thus, we provide the first direct evidence that Mdm2 and Mdm4 may have independent roles in a temporal and tissue-specific manner.
|
ACKNOWLEDGMENTS |
|---|
MyHC-Cre mice. This study was supported by a grant from the National Institutes of Health (CA47296) to G.L. and a training grant (CA009299) supporting J.D.G.
|
FOOTNOTES |
|---|
Supplemental material for this article may be found at http://mcb.asm.org/.
These authors contributed equally to this work.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bottger, V., A. Bottger, C. Garcia-Echeverria, Y. F. Ramos, A. J. van der Eb, A. G. Jochemsen, and D. P. Lane. 1999. Comparative study of the p53-mdm2 and p53-MDMX interfaces. Oncogene 18:189-199.[CrossRef][Medline]
3. Chavez-Reyes, A., J. M. Parant, L. L. Amelse, R. M. de Oca Luna, S. J. Korsmeyer, and G. Lozano. 2003. Switching mechanisms of cell death in mdm2- and mdm4-null mice by deletion of p53 downstream targets. Cancer Res. 63:8664-8669.
4. Farley, F. W., P. Soriano, L. S. Steffen, and S. M. Dymecki. 2000. Widespread recombinase expression using FLPeR (Flipper) mice. Genesis 28:106-110.[CrossRef][Medline]
5. Fernandez, E., Z. Siddiquee, and R. V. Shohet. 2001. Apoptosis and proliferation in the neonatal murine heart. Dev. Dyn. 221:302-310.[CrossRef][Medline]
6. Fijnvandraat, A. C., A. C. van Ginneken, P. A. de Boer, J. M. Ruijter, V. M. Christoffels, A. F. Moorman, and R. H. Lekanne Deprez. 2003. Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube. Cardiovasc. Res. 58:399-409.
7. Finch, R. A., D. B. Donoviel, D. Potter, M. Shi, A. Fan, D. D. Freed, C. Wang, B. P. Zambrowicz, R. Ramirez-Solis, A. T. Sands, and N. Zhang. 2002. Mdmx is a negative regulator of p53 activity in vivo. Cancer Res. 62:3221-3225.
8. Gan, L., M. Xiang, L. Zhou, D. S. Wagner, W. H. Klein, and J. Nathans. 1996. POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells. Proc. Natl. Acad. Sci. USA 93:3920-3925.
9. Gavrieli, Y., Y. Sherman, and S. A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.
10. Godley, L. A., J. B. Kopp, M. Eckhaus, J. J. Paglino, J. Owens, and H. E. Varmus. 1996. Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric bud and possess small kidneys. Genes Dev. 10:836-850.
11. Grier, J. D., W. Yan, and G. Lozano. 2002. Conditonal allele of mdm2 which encodes a p53 inhibitor. Genesis 32:145-147.[CrossRef][Medline]
12. Gu, J., H. Kawai, L. Nie, H. Kitao, D. Wiederschain, A. G. Jochemsen, J. Parant, G. Lozano, and Z. M. Yuan. 2002. Mutual dependence of MDM2 and MDMX in their functional inactivation of p53. J. Biol. Chem. 277:19251-19254.
13. Haupt, Y., R. Maya, A. Kazaz, and M. Oren. 1997. Mdm2 promotes the rapid degradation of p53. Nature 387:296-299.[CrossRef][Medline]
14. Hollstein, M., B. Shomer, M. Greenblatt, T. Soussi, E. Hovig, R. Montesano, and C. C. Harris. 1996. Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acid Res. 24:141-146.
15. Honda, R., H. Tanaka, and H. Yasuda. 1997. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420:25-27.[CrossRef][Medline]
16. Jacks, T., L. Remington, B. O. Williams, E. M. Schmitt, S. Halachmi, R. T. Bronson, and R. A. Weinberg. 1994. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4:1-7.[CrossRef][Medline]
17. Jackson, M. W., and S. J. Berberich. 2000. MdmX protects p53 from Mdm2-mediated degradation. Mol. Cell. Biol. 20:1001-1007.
18. Jones, S. N., A. E. Roe, L. A. Donehower, and A. Bradley. 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378:206-208.[CrossRef][Medline]
19. Kim, K. K., M. H. Soonpaa, A. I. Daud, G. Y. Koh, J. S. Kim, and L. J. Field. 1994. Tumor suppressor gene expression during normal and pathologic myocardial growth. J. Biol. Chem. 269:22607-22613.
20. Kubbutat, M. H., S. N. Jones, and K. H. Vousden. 1997. Regulation of p53 stability by Mdm2. Nature 387:299-303.[CrossRef][Medline]
21. Lyons, G. E., S. Schiaffino, D. Sassoon, P. Barton, and M. Buckingham. 1990. Developmental regulation of myosin gene expression in mouse cardiac muscle. J. Cell Biol. 111:2427-2436.
22. Marine, J. C., and A. G. Jochemsen. 2004. Mdmx and Mdm2: brothers in arms? Cell Cycle 3:900-904.[Medline]
23. Migliorini, D., E. L. Denchi, D. Danovi, A. Jochemsen, M. Capillo, A. Gobbi, K. Helin, P. G. Pelicci, and J.-C. Marine. 2002. Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development. Mol. Cell. Biol. 22:5527-5538.
24. Migliorini, D., D. Danovi, E. Colombo, R. Carbone, P. G. Pelicci, and J. C. Marine. 2002. Hdmx recruitment into the nucleus by Hdm2 is essential for its ability to regulate p53 stability and transactivation. J. Biol. Chem. 277:7318-7323.
25. Montes de Oca Luna, R., D. S. Wagner, and G. Lozano. 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378:203-206.[CrossRef][Medline]
26. Nagy, A., M. Gertsenstein, K. Vintersten, and R. Behringer (ed.). 2003. Manipulating the mouse embryo: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
27. Ng, W. A., I. L. Grupp, A. Subramaniam, and J. Robbins. 1991. Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart. Circ. Res. 68:1742-1750.
28. Parant, J., A. Chavez-Reyes, N. A. Little, W. Yan, V. Reinke, A. G. Jochemsen, and G. Lozano. 2001. Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat. Genet. 29:92-95.[CrossRef][Medline]
29. Rumyantsev, P. P. 1991. Growth and hyperplasia of cardiac muscle cells. Harwood Academic Press, New York, N.Y.
30. Sharp, D. A., S. A. Kratowicz, M. J. Sank, and D. L. George. 1999. Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein. J. Biol. Chem. 274:38189-38196.
31. Soriano, P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21:70-71.[CrossRef][Medline]
32. Tanimura, S., S. Ohtsuka, K. Mitsui, K. Shirouzu, A. Yoshimura, and M. Ohtsubo. 1999. Mdm2 interacts with Mdmx through their RING finger domains. FEBS Lett. 447:5-9.[CrossRef][Medline]
33. Tyner, S. D., S. Venkatachalam, J. Choi, S. Jones, N. Ghebranious, H. Igelmann, X. Lu, G. Soron, B. Cooper, C. Brayton, S. Hee Park, T. Thompson, G. Karsenty, A. Bradley, and L. A. Donehower. 2002. p53 mutant mice that display early ageing-associated phenotypes. Nature 415:45-53.[CrossRef][Medline]
34. Vousden, K. H. 2000. p53: death star. Cell 103:691-694.[CrossRef][Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
