mdm2-a, a common mdm2 splice variant, causes perinatal ... · introduction p53 is a tumor...

INTRODUCTIONp53 is a tumor suppressor protein that plays a central role incontrolling cell cycle progression (Kastan et al., 1991) andapoptosis (Yonish-Rouach et al., 1991). The expression andactivity of p53 are precisely regulated in order to maintainappropriate levels of cellular proliferation and death (Momandet al., 1992). MDM2 is the best-characterized negative regulatorof p53 and itself is classified as an oncogene (Fakharzadeh et al.,1991). Inhibition of p53 by MDM2 results in strict control of p53protein levels through MDM2-mediated ubiquitylation andproteasomal degradation (Li et al., 2003). Alterations in MDM2levels lead to shifts in p53 activity, which result in dramaticbiological outcomes (Donehower, 2002). At one end of thespectrum, when MDM2 is highly overexpressed, p53 cannotsuppress growth sufficiently and transformation results (Olineret al., 1992; Ladanyi et al., 1993; Reifenberger et al., 1993; Corviet al., 1995). At the other end of the spectrum, the deletion ofMdm2, as observed in Mdm2-null embryos, results in uncheckedp53 activity, and the enhanced growth inhibition consequentlyhinders organ development (Jones et al., 1995; Montes de OcaLuna et al., 1995). Additionally, several mouse models have beendeveloped that exhibit varying levels of p53 activity. As expected,reduced p53 activity leads to tumor susceptibility (Donehower etal., 1992; Jacks et al., 1994), whereas enhanced p53 activityconfers tumor protection (Garcia-Cao et al., 2002; Tyner et al.,2002; Maier et al., 2004; Mendrysa et al., 2006), and surprisingly,

in certain model systems, accelerates aging (Tyner et al., 2002;Maier et al., 2004).

Many splice variants of Mdm2 have been identified both intumors (Sigalas et al., 1996; Bartel et al., 2002a) and in normaltissues (Bartel et al., 2004); however, the functionalcharacterization of the MDM2 isoforms encoded by these variantsis limited. Mdm2-a is one of the most common variants identifiedto date (Bartel et al., 2002a) and the most common variantdetected in pediatric rhabdomyosarcoma tumors (Bartel et al.,2002b). The protein encoded by this particular variant has adeletion of a large portion of the amino terminus and, therefore,lacks a functional p53-binding domain (Sigalas et al., 1996; Bartelet al., 2002b). However, it maintains the central acidic domainand the carboxy-terminal RING finger domain, which containimportant sequences for binding to other proteins and for theubiquitylation of p53 (Haupt et al., 1997; Honda et al., 1997;Kubbutat et al., 1997; Bartel et al., 2002b). Previous data havesuggested that, unlike several similar MDM2 isoforms, MDM2-A [or Δ28-220 as referred to by Fridman et al. (Fridman et al.,2003)] does not enhance tumorigenesis (Fridman et al., 2003).These data are consistent with a model proposed previouslysuggesting that splice variants with an intact C-terminal RINGdomain bind to full-length MDM2 protein, resulting in increasedp53 activity and a growth-inhibitory, rather than growth-promoting, phenotype (Evans et al., 2001; Dang et al., 2002).However, these data are inconsistent with the frequent expressionof MDM2 splice variants in tumors.

In order to assess the physiological function of MDM2-A in vivo,we generated an Mdm2-a transgenic mouse model. These mice,and mouse embryonic fibroblasts (MEFs) derived from them,provide evidence of a p53-dependent phenotype of MDM2-A anddemonstrate the impact of this splice variant on growth inhibition,senescence and longevity.

RESEARCH ARTICLE

Disease Models & Mechanisms 47

Disease Models & Mechanisms 2, 47-55 (2009) doi:10.1242/dmm.000992

MDM2-A, a common Mdm2 splice variant, causesperinatal lethality, reduced longevity and enhancedsenescenceErin L. Volk1,*, Katja Schuster2,*, Katie M. Nemeth1, Liying Fan1 and Linda C. Harris1,‡

SUMMARY

MDM2 is the predominant negative regulator of p53 that functions to maintain the appropriate level of expression and activity of this central tumorsuppressor. Mdm2-a is a commonly identified splice variant of Mdm2; however, its physiological function is unclear. To gain insight into the activityof MDM2-A and its potential impact on p53, an Mdm2-a transgenic mouse model was generated. Mdm2-a transgenic mice displayed a homozygous-lethal phenotype that could be rescued by a reduction in p53 expression, demonstrating a dependence upon p53. Mdm2-a hemizygous mice exhibitedreduced longevity, and enhanced senescence was observed in their salivary glands. In addition, the transgenic mice lacked typical, acceleratedaging phenotypes. Growth of transgenic mouse embryonic fibroblasts (MEFs) was inhibited relative to wild-type MEFs, and MDM2-A was shown tobind to full-length MDM2 in an interaction that could increase p53 activity via reduced MDM2 inhibition. Evidence of p53 activation was shown inthe Mdm2-a transgenic MEFs, including p53-dependent growth inhibition and elevated expression of the p53 target protein p21. In addition, MDM2-A increased senescence in a p21-independent manner. In conclusion, unexpected roles for MDM2-A in longevity and senescence were identifiedin a transgenic mouse model, suggesting that Mdm2 splice variants might be determinants of these phenotypes in vivo.

1Department of Molecular Pharmacology, St Jude Children’s Research Hospital, 262Danny Thomas Place, Memphis, TN 38105, USA2Simmons C. Cancer Center, University of Texas Southwestern Medical Center,5323 Harry Hines Boulevard, Dallas, TX 75390, USA*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

http://dmm.biologists.org/lookup/doi/10.1242/dmm.000992Access the most recent version at First posted online on 22 December 2008 as 10.1242/dmm.000992

http://dmm.biologists.org/lookup/doi/10.1242/dmm.000992

RESULTSMdm2-a transgenic mice are homozygous lethal in a p53-dependent mannerTransgenic mice expressing the Mdm2-a cDNA were generated bystandard pronuclear injection methods, as detailed in the Methods.Despite multiple rounds of microinjection of the pCAGGS–Mdm2-a construct, only one founder line was obtained that integrated theMdm2-a cDNA without any mutations (Schuster and Harris,2007). In addition, despite the use of a ubiquitous promoter,expression of the transgene was restricted to only a few tissues (Fig.1). Analysis of transgene expression in Mdm2-a mice revealed highlevels of MDM2-A in muscle, the heart and salivary glands,moderate levels in the brain and lungs, and low levels of expressionin the kidney and spleen (Fig. 1). To rule out any chromosomalintegration positional effects of the transgene, Mdm2-a waslocalized to a region on chromosome 13 that was at least 150 kb,5� and 3�, from the nearest coding regions.

Interestingly, when Mdm2-a transgenic mice were bred, onlyhemizygous mice were viable (Table 1). Analysis of newborn littersrevealed that Mdm2-a homozygous pups were born at the expectedMendelian ratio and were anatomically indistinguishable fromhemizygous and wild-type littermates; however, the homozygouspups died of unknown causes several hours after birth. Earlierembryonic stages were evaluated to determine whether anyabnormalities could be observed. At embryonic day 14.5 (E14.5),all Mdm2-a homozygotes begin to display marked edema,specifically in the subcutaneous tissue of the nuchal region, whichis absent in both hemizygous and wild-type embryos (Fig. 2A-E).The fluid in this region contained foamy macrophages indicativeof active clearance of the edema (Fig. 2E). The edematousphenotype suggested a possible lymphatic defect, but detailedpathological analyses of lymph and blood vessels, as well ascomplete blood counts from both Mdm2-a homozygous neonatesand embryos, revealed neither gross anatomical malformation (Fig.2F,G) nor hematopoietic defects (data not shown). In addition, allorgan systems appeared normal in the homozygous embryos andneonates. Consequently, the actual cause of death of the Mdm2-ahomozygous neonates remains unresolved.

The only function currently assigned to Mdm2 splice variantsis the aforementioned p53 activation that occurs as a consequenceof splice variant binding to full-length MDM2 (Evans et al., 2001;Dang et al., 2002). To assess directly the role of p53 on the MDM2-A-mediated perinatal lethality, transgenic mice were crossed ontoa p53-null background. Interestingly, Mdm2-a homozygous

animals were viable and present at the expected Mendelian ratio,but only in the presence of a concomitant reduction in p53 genedosage (Table 1). These data demonstrate that the perinatallethality of the Mdm2-a homozygous mice was mediated by p53and that MDM2-A activities in vivo are, at least in part, p53dependent.

Mdm2-a transgenic mice exhibit reduced longevityIn contrast to their homozygous counterparts, Mdm2-ahemizygous mice were viable with no obvious phenotype comparedwith wild-type littermates. Therefore, to assess the in vivo role ofthis splice variant further, these animals were used in all subsequentexperiments. Mdm2-a transgenic mice and their wild-typelittermates were observed for any health problems for up to 30months.

After reaching 1 year of age, many animals developed typicalage-related health problems that required euthanasia, such asuterine hyperplasia, inguinal hernia and retinal degeneration. Toofew tumors developed in either the wild-type or Mdm2-a transgenicanimals to allow any statistical analysis; however, both the frequencyand tumor type appeared to be similar between the two groups ofmice (Table 2). Interestingly, although there were no differences inthe causes of death between transgenic and wild-type mice, Mdm2-a mice had significantly shorter life spans as shown by the Kaplan-Meier survival curve (Fig. 3). The median survival of wild-type micewas 25 months, whereas the Mdm2-a mice had a median survivalof only 20 months.

MDM2-A-mediated reduction in longevity was not the result ofaccelerated agingTo determine the role of MDM2-A in the shortened life span ofthe transgenic animals, we assessed various aging parameters incohorts of young (3 months) and aged (18-20 month) mice. Totalbody mass, various organ masses, bone density, dermal adiposethickness and shaved hair re-growth were measured and comparedbetween transgenic and wild-type mice. As expected, total bodymass, as well as that of most organs, increased as both wild-typeand Mdm2-a transgenic mice progressed from 3 to 20 months ofage (Fig. 4A). There was a trend towards reduced body and organmasses (Fig. 4A), as well as a reduced rate of hair growth (data notshown), in the Mdm2-a transgenic mice at both 3 and 20 monthsof age; however, the differences were not statistically significant.Additionally, there was no detectable difference in either bonedensity or dermal adipose thickness between transgenic and wild-

dmm.biologists.org48

MDM2-A reduces longevity and promotes senescenceRESEARCH ARTICLE

Fig. 1. The Mdm2-a transgene has limited tissue expression. Western blot analysis of various tissues from either wild-type (w) or Mdm2-a transgenic (t) micewere probed with antibodies to detect either full-length MDM2 (MDM2-FL) and its splice variant, MDM2-A, or the loading control α-tubulin.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

type animals (data not shown). Thus, despite their shortened lifespan, there appeared to be only subtle changes in the aging ofMdm2-a mice.

Tissues from Mdm2-a transgenic mice display enhancedsenescenceAlthough there were no significant differences in various physicalparameters of aging, it remained possible that there werebiochemical differences in tissues that might have contributed tothe reduced life span of Mdm2-a transgenic animals. To this end,the role of MDM2-A in senescence was evaluated in vivo. Theactivity of β-galactosidase at pH 6.0 is the current standard markerof cellular senescence (Dimri et al., 1995). Therefore, senescence-associated β-galactosidase (SA-βgal) assays were performed onsalivary gland, muscle, heart, liver and brain tissue samples fromboth transgenic and wild-type mice. Although SA-βgal-positivecells could be detected in several organs, no significant differenceswere found between wild-type and transgenic tissues, with one

exception. In the salivary gland tissue from Mdm2-a mice, SA-βgalactivity increased appropriately with age, and stained more robustlythan that of wild-type littermates, particularly in aged tissue (Fig.4B). Even though we observed enhanced senescence in only thesalivary gland, these data suggest that there might have been othertissues in which senescence was enhanced by MDM2-A expression,potentially contributing to the reduction in longevity observed inthe Mdm2-a mice.

MDM2-A causes growth inhibition in vitro and interacts with full-length MDM2To gain further insight into the function of MDM2-A, MEFs wereisolated from transgenic and wild-type mice for in vitro analysesof cellular growth and p53 activation. Western blot analyses showedthat neither p53 nor full-length MDM2 protein levels were alteredby the presence of MDM2-A (Fig. 5A). However, cellular growthassays demonstrated that MEFs expressing MDM2-A grew at aslower rate than wild-type MEFs (Fig. 5B), suggesting that MDM2-A confers a growth-inhibitory phenotype.

Other MDM2 splice variants have been shown to bind to full-length MDM2 and result in growth inhibition as a consequence ofincreased p53 activity. Technical difficulties precluded analysis ofthe interaction between full-length MDM2 and MDM2-A in thetransgenic MEFs. Therefore, to evaluate their potential interaction,FLAG-tagged MDM2-A was retrovirally expressed in wild-typeMEFs and immunoprecipitated with an anti-FLAG antibody. Byprobing the western blot with an anti-MDM2 antibody, full-lengthMDM2 was found to co-immunoprecipitate weakly with MDM2-A (Fig. 5C), demonstrating that the MDM2-A splice variant iscapable of binding to full-length MDM2 protein. This interactioncould potentially disrupt the endogenous protein interactions of


MDM2-A reduces longevity and promotes senescence RESEARCH ARTICLE

p53 genotype of the Mdm2-a+/+ mice

Mdm2-a genotype Wild-type p53+/– p53 –/– Total

Mdm2-a+/+ 0 5 2 7

Table 1. Mdm2-a homozygous mice are viable only when p53 gene

dosage is reduced

Mdm2-a genotype

Cross Wild-type Mdm2-a+/– Mdm2-a+/+ Total

Mdm2-a+/– intercross 90 115 0 205

p53+/–/Mdm2-a+/–

intercross

9 13 7* 29

*The lower half of this table shows the p53 genotype of these seven mice.

Fig. 2. Mdm2-a homozygous embryosexhibit edema. Wild-type (A) and Mdm2-ahomozygous (B) embryos harvested at E14.5show Mdm2-a-specific edema. Cross-sectionsthrough the nuchal regions of wild-type (C)and Mdm2-a homozygous (D) E14.5 embryos.Bar, 600 μm. (E) Edematous region of Mdm2-ahomozygous embryo. Arrowheads designatefoamy macrophages within the region ofedema. Bar, 100 μm. Whole-body cross-sections of wild-type (F) and Mdm2-atransgenic (G) E18.5 neonates exemplify theapparent normal development of Mdm2-aneonates. Bar, 2.5 mm.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

full-length MDM2, particularly with p53, to mediate a growth-inhibitory phenotype.

Exogenous expression of MDM2-A in wild-type MEFs followingretroviral transduction could also inhibit cell growth (Fig. 5D).MEFs transduced with retroviral vectors expressing either full-length MDM2 or MDM2-M3 (the RING domain only) were alsoevaluated. As expected, full-length MDM2 enhanced growth owingto the inhibition of p53 activity. MDM2-M3 suppressed growth ina similar manner to MDM2-A when compared with vector-control-transduced cells.

MDM2-A-mediated activation of p53To assess the role of p53 in the Mdm2-a-mediated in vitro growthinhibition, p53-null MEFs were generated with, and without,Mdm2-a transgene expression, and assayed for cell growth. Incontrast to the growth inhibition observed in MEFs containingMdm2-a and expressing wild-type p53 (Fig. 5B), p53-null MEFsgrew at the same rate in the presence or absence of MDM2-A(Fig. 6A). Thus, when p53 is deleted, the growth inhibition ofMdm2-a-expressing MEFs is abolished, demonstrating that thisphenotype is p53 dependent. In addition, p53-null MEFs wereinsensitive to the growth-inhibitory effects of exogenous MDM2-A expression, again confirming the dependence on p53 (data notshown).

To assess directly p53 activation in Mdm2-a MEFs, luciferasereporter assays were performed using p53-responsive promoters.Surprisingly, when using either the p21WAF1/CIP1 promoter (Fig. 6B)or the bax promoter (data not shown), there was no significantdifference in p53 transcriptional activity in MEFs that expressMDM2-A relative to wild-type MEFs.

To explore further the potential p53 activation in the presenceof MDM2-A, several p53 target genes were assessed for alterationsin their expression in wild-type and transgenic MEFs. Levels ofp21WAF1/CIP1, a cyclin-dependent kinase inhibitor (El-Deiry et al.,



Table 2. Tumors detected in Mdm2-a hemizygous transgenic mice and wild-type littermates

Mouse line Number of tumors/total (%) Tumor types (number of animals) Age at death (months)

Wild-type 2/55 (4) Squamous cell carcinoma (1)

Soft tissue sarcoma (1)

25

24

Mdm2-a transgenic 4/64 (6) Alveolar bronchiolar adenoma (1)

Soft tissue sarcoma (1)

Osteoma (1)

Soft tissue mass* leg (1)

12

19

8

13

*Diagnosis unclear.

Fig. 3. Mdm2-a transgenic mice display reduced longevity. Kaplan-Meiersurvival curve of wild-type (bold line) and Mdm2-a transgenic (thin line) mice(p=0.029). The survival curve is based on data from 55 wild-type and 64 Mdm2-a transgenic mice.

Fig. 4. Mdm2-a transgenic mice do not show accelerated aging, butdisplay enhanced senescence in aged tissue. (A) Tissue and total body massfrom 3-month-old wild-type (light gray bars) and Mdm2-a (white bars) malemice, and from 20-month-old wild-type (black bars) and Mdm2-a (dark graybars) male mice. Female mice exhibited the same trend (data not shown).(B) SA-βgal staining of salivary gland tissue from young and aged wild-typeand Mdm2-a transgenic mice. Bar, 50 μm.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

1994), were elevated in the presence of MDM2-A, whereas theexpression of endogenous MDM2 (Fig. 5A) and the pro-apoptoticproteins Bax (Miyashita and Reed, 1995) and PUMA (Nakano andVousden 2001; Yu et al., 2001) were unaltered by the transgene (Fig.6C). The elevation of p21 was shown to be p53 dependent becauseMDM2-A transgenic MEFs that were p53-null showed no p21activation (Fig. 6C). As expected, p53-null MEFs displayed reducedexpression of the p53 target genes bax and PUMA. These datademonstrate that specific p53 targets are affected by MDM2-A,but there was no global increase in the expression of p53-responsivegenes in the presence of MDM2-A.

Mdm2-a MEFs display enhanced senescenceIn vivo data suggested that Mdm2-a plays a role in enhancingsenescence in the tissues of Mdm2-a transgenic mice (Fig. 4B). Toassess this phenotype in vitro, Mdm2-a and wild-type MEFs werestained for SA-βgal from passages 4-16. As expected, both wild-type and Mdm2-a MEFs contained low levels of SA-βgal-positive

cells at both very early and late passages owing to their mitoticallyactive or immortal states, respectively (data not shown). A peak inthe proportion of senescent cells was observed at passage 6, andsignificant differences in the degree of senescence between Mdm2-a and wild-type MEFs were observed (Fig. 7A,B). Mdm2-a MEFsdisplayed a greater fraction of senescent cells compared with wild-type MEFs, which probably contributed to their phenotype ofslower growth.

Because p53 was shown to be responsible for the growthinhibition observed in the Mdm2-a MEFs, the role of p53 inMDM2-A-mediated senescence was also evaluated. When p53 wasdeleted, little or no senescence was observed in either wild-typeor Mdm2-a MEFs (Fig. 7B), confirming the p53-dependence of thisphenotype (Harvey et al., 1993). However, because senescence wassuppressed in both wild-type and transgenic MEFs, it was difficultto assess the contribution of p53 to the increased senescenceobserved in the presence of Mdm2-a.

To determine whether p21 was the p53 target gene involved inthe MDM2-A-mediated growth inhibition and senescence, both thewild-type and the Mdm2-a phenotypes were assessed in the absenceof p21. In p21-null MEFs that were generated with and without thetransgene, Mdm2-a-mediated growth inhibition persisted, suggestingthat p21 was not involved (Fig. 7C). Senescence was also quantitatedin p21-null MEFs with and without MDM2-A, and overall thisphenotype was suppressed in the absence of p21. However, therewas a slight increase in SA-βgal-positive staining in Mdm2-a–p21-null MEFs compared with Mdm2–p21-null MEFs – a trend similarto that observed in MEFs containing wild-type p21. Even thoughthis difference was not significant (Fig. 7B), these data suggested thatp21 was probably not responsible for the enhanced senescenceobserved in the presence of Mdm2-a.

DISCUSSIONMdm2 splice variants have long been considered to be either tumor-specific isoforms that actively promote tumorigenesis or the passiveby-products of deregulated splicing in transformed cells (Bartel etal., 2002a). Here, we provide evidence for a novel and apparentlycontrasting role for the Mdm2-a splice variant in normal tissues.We show that MDM2-A causes growth inhibition, resulting ineither p53-dependent perinatal lethality of Mdm2-a transgenichomozygotes or reduced longevity of hemizygous mice in vivo.These phenotypes are a consequence of p53 activation, whichprobably leads to elevation of specific downstream targets.Additionally, and in accordance with growth inhibition and reducedlife span, MDM2-A expression was shown to enhance senescencein MEFs and in certain aged tissues of the transgenic mice.

As has been described previously for other MDM2 splicevariants, the presence of MDM2-A appears to reduce the inhibitorycapacity of full-length MDM2 by disrupting its normal interactionwith p53, which in turn results in increased p53 activity (Evans etal., 2001). The impact of MDM2-A in vivo mirrors other previouslydescribed mouse models in which the MDM2-p53 balance isaltered. The MDM2-A-mediated perinatal lethality, which can berescued by p53 reduction, is similar to, albeit less robust than, theembryonic lethality observed when Mdm2 is deleted. Mdm2-nullembryos die at an early developmental stage shortly afterimplantation and can only be rescued by deletion of p53 (Jones etal., 1995; Montes de Oca Luna et al., 1995), whereas Mdm2-a



Fig. 5. MDM2-A is growth-inhibitory and binds to full-length MDM2 invitro. (A) Western blot analysis of MEFs to detect expression of full-lengthMDM2 (MDM2-FL), the MDM2-A transgene, p53 and the loading control TFIID.(B) Growth curves for wild-type (circles, solid line) and MDM2-A transgenic(squares, dotted line) MEFs isolated from embryos from the same litter.(C) Immunoprecipitation (IP) of FLAG-tagged MDM2-A that was retrovirallyexpressed in wild-type MEFs. Immunoblot (IB) analysis with anti-MDM2antibody was used to visualize the full-length MDM2 (MDM2-FL) and MDM2-Aproteins. The faint band visible in the vector control (VC) lane representsantibody heavy chain. (D) Transduction of wild-type MEFs with retroviralvectors containing cDNAs of different MDM2 isoforms. MDM2-A and MDM2-M3 inhibit the growth of wild-type MEFs, whereas full-length MDM2 results inenhanced cell growth. Symbols: MDM2-A (squares, dotted line); vector control(circles, solid line); full-length MDM2 (diamonds, solid line); truncated MDM2-M3 (triangles, solid line).D

iseas

e M

odel

s & M

echa

nism

s

DM

M

homozygotes die shortly after birth and survive only when miceare concomitantly heterozygous or null for p53.

Reduced longevity has also been observed in models of p53activation that have been described previously. Mutant p53 mice(p53+/m) that express a truncated form of p53, which is capable ofactivating the wild-type allele, display reduced longevity, enhancedaging and reduced tumor formation (Tyner et al., 2002). Similarly,p44 mice express a highly active p53 isoform and display ashortened life span accompanied by accelerated aging, activationof p53 target genes and enhanced senescence (Maier et al., 2004).The phenotype of Zmpste24-null mice, which lack a keymetalloproteinase involved in nuclear architecture, was alsoproposed to be the result of p53 activation (Varela et al., 2005).These animals display reduced longevity, enhanced aging andupregulation of several p53 target genes (Varela et al., 2005).However, not all models in which p53 is elevated have shown thesame phenotypes (Garcia-Cao et al., 2002; Mendrysa et al., 2006).The cause of phenotypic differences between the mice within eachmodel system is unclear, but probably reflects variation in thedegree of p53 activation in specific tissues. The modest changesobserved in the Mdm2-a mice are probably a consequence of thelimited tissue expression of the transgene in a hemizygous animal.

Interestingly, p53 activation in the presence of MDM2-A was notevident in a p53-responsive reporter assay in MEFs, and p21 was theonly target gene observed to display elevated expression. However,this result is consistent with previous observations of enhanced p53

activity. For instance, the mutant p53 expressed in the p53+/m miceonly showed increased p53 activity in a luciferase reporter assay whenthe mutant allele was ectopically expressed in Saos-2 cells (Tyner etal., 2002). Additionally, not all p53 target genes evaluated were foundto be elevated in p44- and Zmpste24-null cells (Maier et al., 2004;Varela et al., 2005). Remarkably, despite multiple indirect indicatorsof p53 activation, these models, including that of Mdm2-a, fail todemonstrate any changes in the p53 protein itself, or a globalactivation of p53-responsive genes. However, these models dosupport the hypothesis that p53 target genes can be specifically andselectively upregulated depending on the cellular context and degreeof p53 activation (Bouvard et al., 2000). Therefore, p21 upregulationtogether with reduced longevity, p53-dependent growth inhibitionand perinatal lethality, which are all observed in the presence ofMDM2-A, are all indicators of p53 activation.

Because p21 was elevated upon MDM2-A expression and isknown to play a role in growth inhibition and senescence (Nodaet al., 1994), we evaluated p21 as a candidate p53 target gene thatcould potentially mediate these phenotypes as a consequence ofMDM2-A expression. The data demonstrate that MDM2-Aremains growth-inhibitory in p21-null MEFs, indicating that p21is not involved. In addition, although p21 is important forsenescence of both wild-type and MDM2-A transgenic MEFs, itappears that p21 does not play a role in the MDM2-A-phenotype-enhanced senescence. Therefore, growth inhibition and potentiallythe senescence observed in the presence of MDM2-A are p53mediated; however, the downstream effectors responsible for thesephenotypes remain unknown.

The data described here indicate that MDM2-A acts throughp53 to slow growth and increase senescence, resulting inphenotypes that are potentially tumor protective (Campisi, 2001).However, Mdm2-a mice were generally healthy and developed fewtumors. The extremely low level of tumorigenesis in these miceprobably obscured any tumor protection conferred by MDM2-A.It will be important in the future to investigate the impact ofMDM2-A on both tumorigenesis and carcinogenesis to evaluatewhether MDM2-A expression is tumor-protective when theincidence of tumor formation is increased.

In conclusion, to the best of our knowledge, we have identifieda novel physiological role for the Mdm2 splice variant MDM2-A.This splice variant, and potentially other Mdm2 splice variants,might be important determinants of aging, senescence andlongevity. However, the data presented here are in contrast to thoseof others describing an oncogenic phenotype for other, similar,MDM2 variants (Fridman et al., 2003; Steinman et al., 2004). Infuture work, the challenge will be to evaluate the expression levelsof different variants in specific tissue types and to determine theconsequences of their coordinated expression on aging, senescenceand transformation when expressed at physiological levels.

METHODSGeneration of the Mdm2-a cDNA expression plasmidThe Mdm2-a cDNA was generated by the PCR-based method ofsplicing by overlapping extension. The murine full-length Mdm2cDNA (provided by G. Lozano, MD Anderson, TX) was used as atemplate, and exons 4-9 (Δ28-220 amino acids) were deleted togenerate a murine sequence equivalent to the human MDM2-Asequence (GenBank U33199). Primers flanking the N-terminal and



Fig. 6. p53 activation in the presence of MDM2-A. (A) Growth-curve analysisof p53-null (circle, solid line) and Mdm2-a–p53-null (squares, dotted line) MEFs.(B) Relative luciferase activity of the p53-responsive p21WAF1/CIP1 promoterlinked to the luciferase reporter gene in both wild-type and MDM2-Atransgenic MEFs. (C) Western blot analysis of expression of the p53 targetgenes p21, bax and PUMA in both wild-type and p53-null MEFs with, andwithout, MDM2-A. Tubulin is shown as a loading control.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

C-terminal regions of Mdm2-a included EcoRI and XhoI restrictionsites, respectively (underlined), and were as follows: N-terminalprimer, 5�-CCGAATTCCGCCAATGTGCAATACCAACAT-3�and C-terminal primer, 5�-CCGCTCGAGGCTAGTTGAAG-TAACTTAGCACA-3�. Mutually complementary super-primersthat overlap the junction between exon 3 and exon 10 weregenerated: antisense, 5�-CTTACGCCATCGTCAAGATCCA-GAGTCTCTCTTGTTCCGAAG-3� and sense, 5�-CTTCGGAA-CAAGAGACTCTGGATCTTGACGATGGCGTAAG-3�. Therecombinant Mdm2-a cDNA was inserted into the pCAGGS/MCSvector (Niwa et al., 1991) (provided by J. Cunningham, St JudeChildren’s Research Hospital, TN) to potentially allow constitutiveubiquitous expression of MDM2-A protein in transgenic mice. Theplasmid was digested with HincII and PstI to remove unnecessaryplasmid DNA. A 3.1 kb DNA fragment containing the chicken actinpromoter, CMV enhancer, Mdm2-a cDNA and rabbit β-globinpolyadenylation signal was generated for microinjection.

Generation of Mdm2-a transgenic miceTransgenic mice were generated in the Transgenic Core Unit at StJude by standard microinjection of DNA into the pronuclei offertilized single-cell mouse embryos (FVB/NJ) (Hogan et al., 1994).The Institutional Animal Care and Use Committee (IACUC)approved all animal work, and all experiments conformed to theapplicable regulatory standards. Transgenic mice and their wild-type littermates were monitored for tumor development anddisease. Animals showing signs of pain or distress were euthanizedaccording to IACUC guidelines. Euthanized mice were examinedfor lesions and tissue abnormalities.

Transgene genomic localization and genotypingThe Mdm2-a insertion site was mapped within the mouse genomeusing the BD GenomeWalker Universal Kit (BD Biosciences, PaloAlto, CA) according to the manufacturer’s protocol. Briefly,genomic DNA was prepared from tail tips using the DNEasy kit(Qiagen, Valencia, CA) and digested individually with DraI,EcoRV, PvuII and StuI blunt-cutting restriction enzymes togenerate four DNA libraries. BD GenomeWalker adaptors weresubsequently ligated to the purified DNA, and primary andsecondary PCRs were performed. PCR reactions were preparedusing the HotStar Taq Master Mix kit (Qiagen) and used thefollowing gene-specific primers in combination with adaptor-specific primers: TG2, 5�-AGGTGGCTATAAAGAGGTC -

ATCAGTA-3�; TG2N, 5�-AGA TT TTTCCTCCTCTCCTGA -CTACT-3�. The resultant amplicons were isolated from gels usingthe QiaexII kit (Qiagen) and sequenced and mapped using BLASTsearches of the mouse genome. Localization of the transgene toa region of chromosome 13 allowed for the construction of thefollowing flanking and internal primers for determination ofzygosity: Mdm2-aTg, 5�-TTCATTGCAATAGTGTGTTGGA-3�;Chr13A, 5�-TGCAT CATTTTGAATCACAGC-3�; and Chr13B,5�-TTAA GC AATCACCTGCCAAT-3�. A 311 bp productidentifies wild-type animals, whereas a 587 bp product is presentin Mdm2-a homozygous animals; hemizygous mice contain bothproducts.

Embryo/neonate sectioning and stainingBoth embryos and neonates were fixed in 10% formalin overnight,embedded in paraffin and sectioned in serial 4-micron intervals.Sections were stained with hematoxylin and eosin using standardmethods and evaluated by the St Jude diagnostic laboratory.

Aging analysisSeveral aging parameters were assessed in both young (3-month)and aged (18–20-month) wild-type and Mdm2-a transgenic maleand female mice. For hair re-growth analysis, approximately 1square inch of dorsal fur was shaved and, after 3 weeks growth,scored as either positive or negative. Mice were then euthanizedand X-rayed (Faxitron X-ray, Wheeling, IL) at 35 kV for 10 secondsfor bone density analysis. Whole-body and individual organ masseswere determined and organs were either cryopreserved or fixed.Brain, heart, kidney and liver were cryopreserved for sectioningand subsequent SA-βgal staining, as described below. Skin sampleswere fixed in formalin, sectioned and stained with hematoxylin andeosin. Muscle, adipose and epidermal layers of the skin weremeasured and the average of at least 30 measurements of each wasused for comparison.

MEF preparation and growth curvesMEFs were generated by standard methods (Hogan et al., 1994)from E14.5 embryos and cultured in DMEM containing 10% FBS,2 mM glutamine, 0.1 mM β-mercaptoethanol, 100 U/mlpenicillin/streptomycin and non-essential amino acids. Growthcurves were generated using P3-P5 MEFs. Cells were plated intriplicate at 5�104 cells/well in 6-well dishes and counted daily for5 days.



Fig. 7. MDM2-A enhances senescencein a p21-independent manner. (A) SA-βgal staining in wild-type and Mdm2-atransgenic MEFs. (B) Histogram ofpassage 6, SA-βgal-positive cells in wild-type, p53-null and p21-null MEFs with,and without, MDM2-A. (C) Growth curveof p21-null (circles, solid line) and Mdm2-a–p21-null (squares, dotted line) MEFs.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Protein extraction, immunoprecipitation and western blottingTo generate cell extracts, MEFs were pelleted, suspended insonication buffer (50 mM Tris pH 8.0, 300 mM NaCl, 1 mMEDTA, 0.5 mM DTT, 0.1% NP40, 1 mM sodium orthovanadate,10 mM NaF, 200 μM PMSF and 10 μg/ml of each of the proteaseinhibitors leupeptin, aprotinin and antipain), freeze-thawed threetimes and centrifuged at 20,000 g at 4°C to remove the insolublefraction. To obtain protein extracts from mouse tissues, sampleswere homogenized in pre-chilled sonication buffer and sonicatedtwice at 4°C for 10 seconds before centrifugation at 20,000 g at4°C. Protein concentrations were determined by measuring thecolor intensity of protein assay dye reagent (Bio-Rad; Hercules,CA) at 595 nm. Protein samples were separated by electrophoresison a Novex 4-20% gradient Tris-glycine polyacrylamide gel(Invitrogen, Carlsbad, CA) and transferred to apolyvinylidenedifluoride (PVDF) membrane (Immobilon,Millipore; Billerica, MA) by electroblotting. Forimmunoprecipitation (IP), pelleted MEFs were resuspended incooled IP-lysis buffer [50 mM Tris (pH 8.0), 5 mM EDTA, 150mM NaCl, 1% NP40 and protease inhibitors, as described forprotein extract preparation], immunoprecipitated with an anti-FLAG-M2 agarose affinity gel (Sigma, St Louis, MO) and washedthree times in cooled IP wash buffer [5% sucrose, 5 mM Tris (pH7.4), 5 mM EDTA, 0.5 M NaCl, 1% NP40 and protease inhibitors]as described previously (McKenzie et al., 2002). Proteins werevisualized by immunoblotting with the following antibodies: anti-hMDM2 (R&D Systems, Minneapolis, MN), anti-MDM2 (C-18,Santa Cruz Biotechnology, Santa Cruz, CA), anti-p53 (JA1308,Calbiochem, La Jolla, CA), anti-p21 (F-5, Santa CruzBiotechnology), anti-bax (P-19, Santa Cruz Biotechnology), anti-PUMA (4976, Cell Signaling, Danvers, MA), anti-α-tubulin (B-7, Santa Cruz Biotechnology), anti-β-tubulin (clone 2.1, Sigma)or anti-TFIID (N-12, Santa Cruz Biotechnology). Secondaryantibodies conjugated to horseradish peroxidase were obtainedfrom Amersham (Piscataway, NY) and visualized by incubationof the membrane with either ECL reagents (Amersham) orSuperSignal (Pierce, Rockford, IL), and subsequent exposure toBioMax MR film (Eastman Kodak, Rochester, NY).

Senescence-associated β-galactosidase stainingTissue cryosections and plated MEFs were stained using the β-galactosidase staining kit (US Biological, Swampscott, MA)according to the manufacturer’s protocol. Briefly, cells were washedin PBS, fixed in 2% formaldehyde and 0.2% glutaraldehyde in PBSfor 5 minutes, and then washed again in PBS. Cells were incubatedin staining solution [1 mg/ml X-gal, 40 mM citric acid/sodiumphosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mMpotassium ferricyanide, 150 mM NaCl, 2 mM magnesium chloride]and staining was quantitated after 24 hours.

Retroviral expressionSubconfluent 293T cells were plated and co-transfected with thehelper pEQECO packaging vector (provided by Dr E. Vanin) andMSCV-IRES-GFP retroviral vectors containing the Mdm2-a-FLAGcDNA. The medium was changed after 24 hours and virus-containing supernatant was harvested at 48-72 hours post-transfection. Early-passage (≤4) wild-type MEFs were transducedwith filtered retroviral supernatant containing 1 μg/ml polybrene

(Sigma). Two days after transduction, cells were sorted for GFPexpression by flow cytometry under sterile conditions, and GFP-positive cells were used to prepare cell extracts.

Luciferase reporter assayEarly passage (≤4) MEFs were co-transfected with the p53-responsive p21WAF1/CIP1 promoter firefly luciferase reporter plasmid(a gift from Wafik El-Diery, University of Pennsylvania, PhiladelphiaPA) and the pRL-SV40 transfection control renilla plasmid(Promega, Madison, WI) using Fugene 6 (Roche, Indianapolis, IN)and standard methods. At 48-72 hours post-transfection, cellextracts were harvested and assayed according to themanufacturer’s protocol using the dual luciferase reporter assaysystem (Promega) and the Optocomp I luminometer (GEMBiomedical, Hamden, CT). After normalizing data for transfectionefficiency based upon expression from the pRL-SV40 plasmid, datawere presented relative to the activity measured in wild-typeMEFs.ACKNOWLEDGEMENTSWe thank John Raucci, George Heath and the Transgenic Core Facility at St Judefor the generation of the transgenic mice; Misty Cheney, June Bursi and theAnimal Resource Center for their technical assistance; and Dr Kelli Boyd and theDiagnostic Lab for evaluation of mouse tissues. We also acknowledge the St JudeHartwell Center for primer production and sequence analysis and the St Jude FlowCytometry Lab for sorting of GFP-positive cells. We would also like to thank Julie



TRANSLATIONAL IMPACT

Clinical issueStrict control of cell growth is essential for embryonic development as well asfor cancer prevention. The protein MDM2 plays a key role in cell growth byinhibiting the activity of the tumor suppressor protein p53. Many shortenedversions of MDM2 have been identified in both normal and tumor tissues;however, their function in either context is unknown. One short form of MDM2known as MDM2-A has been frequently detected in tumors, particularly inchildhood rhabdomyosarcomas, which are tumors made up of cells that wouldnormally develop into skeletal muscle. Thus, determining MDM2-A functionmay provide an insight into how these tumors develop and how the diseasemight respond to treatment.

ResultsHere, the authors generate a transgenic mouse expressing MDM2-A protein innormal tissues. Although this protein was detected in tumors, the MDM2-Amouse was not tumor prone. Rather, the most striking phenotype was thatmice expressing high levels of MDM2-A did not survive beyond birth and thatmice expressing lower levels of MDM2-A had a shortened life span. Furtheranalysis of cells and tissues from MDM2-A mice demonstrated that MDM2-Aexpression activates the tumor suppressor protein p53 and results in increasedcell senescence and earlier cell death.

Implications and future directionsThese results demonstrate that shorter forms of MDM2, such as MDM2-A, canbe detrimental with respect to enhancing cellular senescence and reducing lifeexpectancy. In contrast, because MDM2-A is implicated in the activation of thep53 tumor suppressor protein, it is possible that MDM2-A expression canprotect against tumor formation. Future studies using tissue-specific MDM2-Aexpression will further clarify the physiological effects of MDM2-A.Additionally, since activation of p53 enhances the sensitivity of cells to certaincancer drugs, MDM2-A may serve as a marker of tumor response tochemotherapeutic agents.

doi:10.1242/dmm.002089

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Groff of the St Jude Biomedical Communication Department for preparing thefigures and Dr Martine Roussel for the full-length MDM2 and MDM2-M3 retroviralconstructs. This work was supported by NIH grants CA92401, CA21765 and theAmerican Lebanese Syrian Associated Charities (ALSAC).

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSE.L.V., K.S. and L.C.H. conceived and designed the experiments, analyzed the dataand wrote the paper; E.L.V., K.S., L.F. and K.M.N. performed the experiments. Allauthors discussed the data and edited the manuscript.

Received 19 June 2008; Accepted 10 October 2008.

REFERENCESBartel, F., Taubert, H. and Harris, L. C. (2002a). Alternative and aberrant splicing of

MDM2 mRNA in human cancer. Cancer Cell 2, 9-15.Bartel, F., Taylor, A. C., Taubert, H. and Harris, L. C. (2002b). Novel mdm2 splice

variants identified in pediatric rhabdomyosarcoma tumors and cell lines. Oncol. Res.12, 451-457.

Bartel, F., Pinkert, C. A., Fiedler, W., Wurl, P., Schmidt, H. and Taubert, H. (2004).Expression of alternatively and aberrantly spliced transcripts of the MDM2 mRNA isnot tumor-specific. Int. J. Cancer 24, 143-151.

Bouvard, V., Zaitchouk, T., Vacher, M., Duthu, A., Canivet, M., Choisy-Rossi, C.,Nieruchalski, M. and May, E. (2000). Tissue and cell-specific expression of the p53-target genes: bax, fas, mdm2 and waf1/p21, before and following ionising irradiationin mice. Oncogene 19, 649-660.

Campisi, J. (2001). Cellular senescence as a tumor-suppressor mechanism. Trends CellBiol. 11, S27-S31.

Corvi, R., Savelyeva, L., Breit, S., Wenzel, A., Handgretinger, R., Barak, J., Oren, M.,Amler, L. and Schwab, M. (1995). Non-syntenic amplification of MDM2 and MYCN inhuman neuroblastoma. Oncogene 10, 1081-1086.

Dang, J., Kuo, M. L., Eischen, C., Stepanova, L., Sherr, C. and Roussel, M. (2002). TheRING domain of Mdm2 can inhibit cell proliferation. Cancer Res. 62, 1222-1230.

Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E.,Linskens, M., Rubelj, I., Pereira-Smith, O. et al. (1995). A biomarker that identifiessenescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA92, 9363-9367.

Donehower, L. A. (2002). Does p53 affect organismal aging? J. Cell Physiol. 192, 23-33.Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr,

Butel, J. S. and Bradley, A. (1992). Mice deficient for p53 are developmentallynormal but susceptible to spontaneous tumours. Nature 356, 215-221.

El-Deiry, W. S., Harper, J. W., O’Conner, P. M., Velculescu, V. E., Canman, C. E.,Jackman, J., Pietenpol, J. A., Burrell, M., Hill, D. E., Wang, Y. et al. (1994).WAF1/CIP1 is induced upon p53-mediated growth arrest and apoptosis. Cancer Res.54, 1169-1174.

Evans, S. C., Viswanathan, M., Grier, J. D., Narayana, M., El-Naggar, A. K. andLozano, G. (2001). An alternatively spliced HDM2 product increases p53 activity byinhibiting HDM2. Oncogene 20, 4041-4049.

Fakharzadeh, S. S., Trusko, S. P. and George, D. L. (1991). Tumorigenic potentialassociated with enhanced expression of a gene that is amplified in a mouse tumorcell line. EMBO J. 10, 1565-1569.

Fridman, J. S., Hernando, E., Hemann, M. T., de Stanchina, E., Cordon-Cardo, C.and Lowe, S. W. (2003). Tumor promotion by Mdm2 splice variants unable to bindp53. Cancer Res. 63, 5703-5706.

Garcia-Cao, I., Garcia-Cao, M., Martin-Caballero, J., Criado, L. M., Klatt, P., Flores, J.M., Weill, J. C., Blasco, M. A. and Serrano, M. (2002). “Super p53” mice exhibitenhanced DNA damage response, are tumor resistant and age normally. EMBO J. 21,6225-6235.

Harvey, M., Sands, A. T., Weiss, R. S., Hegi, M. E., Wiseman, R. W., Pantazis, P.,Giovanella, B. C., Tainsky, M. A., Bradley, A. and Donehower, L. A. (1993). In vitrogrowth characteristics of embryo fibroblasts isolated from p53-deficient mice.Oncogene 8, 2457-2467.

Haupt, Y., Maya, R., Kazaz, A. and Oren, M. (1997). Mdm2 promotes the rapiddegradation of p53. Nature 387, 296-299.

Hogan, B., Beddington, R., Costnatini, F. and Lacy, E. (1994). Manipulating the MouseEmbryo: A Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory Press.

Honda, R., Tanaka, H. and Yasuda, H. (1997). Oncoprotein MDM2 is a ubiquitin ligaseE3 for tumor suppressor p53. FEBS Lett. 420, 25-27.

Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T.and Weinberg, R. A. (1994). Tumor spectrum analysis in p53-mutant mice. Curr. Biol.4, 1-7.

Jones, S. N., Roe, A. E., Donehower, L. A. and Bradley, A. (1995). Rescue ofembryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208.

Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B. and Craig, R. W. (1991).Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 51,6304-6311.

Kubbutat, M. H., Jones, S. N. and Vousden, K. H. (1997). Regulation of p53 stabilityby Mdm2. Nature 387, 299-303.

Ladanyi, M., Cha, C., Lewis, R., Jhanwar, S. C., Huvos, A. G. and Healey, J. H. (1993).MDM2 gene amplification in metastatic osteosarcoma. Cancer Res. 53, 16-18.

Li, M., Brooks, C. L., Wu-Baer, F., Chen, D., Baer, R. and Gu, W. (2003). Mono- versuspolyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972-1975.

Maier, B., Gluba, W., Bernier, B., Turner, T., Mohammad, K., Guise, T., Sutherland,A., Thorner, M. and Scrable, H. (2004). Modulation of mammalian life span by theshort isoform of p53. Genes Dev. 18, 306-319.

McKenzie, P. P., McPake, C. R., Ashford, A. A., Vanin, E. F. and Harris, L. C. (2002).MDM2 does not influence p53-mediated sensitivity to DNA-damaging drugs. Mol.Cancer Ther. 1, 1097-1104.

Mendrysa, S. M., O’Leary, K. A., McElwee, M. K., Michalowski, J., Eisenman, R. N.,Powell, D. A. and Perry, M. E. (2006). Tumor suppression and normal aging in micewith constitutively high p53 activity. Genes Dev. 20, 16-21.

Miyashita, T. and Reed, J. C. (1995). Tumor suppressor p53 is a direct transcriptionalactivator of the human bax gene. Cell 80, 293-299.

Momand, J., Zambetti, G. P., Olson, D. C., George, D. and Levine, A. J. (1992). TheMDM2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69, 1237-1245.

Montes de Oca Luna, R., Wagner, D. S. and Lozano, G. (1995). Rescue of earlyembryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206.

Nakano, K. and Vousden, K. H. (2001). PUMA, a novel proapoptotic gene, is inducedby p53. Mol. Cell 7, 683-694.

Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-199.

Noda, A., Ning, Y., Venable, S. F., Pereira-Smith, O. M. and Smith, J. R. (1994).Cloning of senescent cell-derived inhibitors of DNA synthesis using an expressionscreen. Exp. Cell Res. 211, 90-98.

Oliner, J. D., Kinzler, K. W., Meltzer, P. S., George, D. L. and Vogelstein, B. (1992).Amplification of a gene encoding a p53-associated protein in human sarcomas.Nature 358, 80-83.

Reifenberger, G., Liu, L., Ichimura, K., Schmidt, E. E. and Collins, V. P. (1993).Amplification and overexpression of the MDM2 gene in a subset of humanmalignant gliomas without p53 mutations. Cancer Res. 53, 2736-2739.

Schuster, K. and Harris, L. C. (2007). Selection for mutations in the cDNAs oftransgenic mice upon expression of an embryonic lethal protein. Transgenic Res. 16,527-530.

Sigalas, I., Calvert, A. H., Anderson, J. J., Neal, D. E. and Lunec, J. (1996).Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences:transforming ability and frequent detection in human cancer. Nat. Med. 2, 912-917.

Steinman, H. A., Burstein, E., Lengner, C., Gosselin, J., Pihan, G., Duckett, C. S. andJones, S. N. (2004). An alternative splice form of Mdm2 induces p53-independentcell growth and tumorigenesis. J. Biol. Chem. 279, 4877-4886.

Tyner, S. D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H.,Lu, X., Soron, G., Cooper, B., Brayton, C. et al. (2002). p53 mutant mice that displayearly ageing-associated phenotypes. Nature 415, 45-53.

Varela, I., Cadinanos, J., Pendas, A. M., Gutierrez-Fernandez, A., Folgueras, A. R.,Sanchez, L. M., Zhou, Z., Rodriguez, F. J., Stewart, C. L., Vega, J. A. et al. (2005).Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53signalling activation. Nature 437, 564-568.

Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A. and Oren, M.(1991). Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibitedby interleukin-6. Nature 352, 345-347.

Yu, J., Zhang, L., Hwang, P. M., Kinzler, K. W. and Vogelstein, B. (2001). PUMAinduces the rapid apoptosis of colorectal cancer cells. Mol. Cell 7, 673-682.


MDM2-A reduces longevity and promotes senescence RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

mdm2-a, a common mdm2 splice variant, causes perinatal ... · introduction p53 is a tumor...

Documents