regulation oftranscriptional activation of mdm2 genebyp53...

Vol. 9, 119-130, February 1998 Cell Growth & Differentiation 119

Regulation of Transcriptional Activation of mdm2 Gene by p53in Response to UV Radiation1

Leslie J. Saucedo, Brian P. Carstens,2Scott E. Seavey, Lee D. Albee Il,� andMary Ellen Perry”Department of Oncology, McArdle Laboratory for Cancer Research,

University of Wisconsin Medical School, Madison, Wisconsin 53706

AbstractThe mdm2 oncogene is expressed at elevated levels ina variety of human tumors, and its product inactivatesthe p53 tumor suppressor protein. MDM2 forms anautoregulatory loop with p53, because the mdm2 genecontains a promoter that is responsive to p53.Synthesis of MDM2 protein increases in a p53-dependent manner in response to DNA-damagingagents such as UV light. Afthough this increase likelyresults from enhanced transcription, the amount ofMDM2 protein does not correspond to the amount ofp53 protein in cells exposed to UV light. Here we showthat the p53-specific internal promoter in the mdm2

gene is induced after exposure to UV light, whereasthe upstream constitutive promoter is not induced. Theamount of the mdm2 transcript does not parallel theability of p53 to bind DNA, indicating that transcriptionis regulated at a step distinct from activation of theDNA-binding function of p53.

IntroductionThe function of the p53 tumor suppressor protein is impor-tant for prevention of cancer in people and in mice (1 , 2). Inmore than 50% of human tumors, both alleles of p53 are

mutated or deleted, indicating that loss of function of p53

contributes to tumorigenesis (2). Studies in mice substantiatea role for p53 in preventing cancer; the incidence of tumorformation approaches 1 00% in mice genetically engineered

to lack p53 (1). The tumor-suppressive effects of p53 seem

to be mediated through the ability of the protein to bind DNA,

because most mutations in human tumors result in loss of

Received 1 0/1 0/97; revised 1 2/23/97; accepted 12/28/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1 734 solely to mdi-cate this fact.1 Supported by developmental funds from Cancer Center Support GrantCA-07175 to the McArdle Laboratory for Cancer Research and by NIHGrant CA-70781 (to M. E. P.). L J. S. and L D. A. received support fromUSPHS Grant CA-09135 from the National Cancer Institute.2 Present address: Physician Assistants Program, University of Iowa, IowaCity, IA 52246.3 Present address: Section of Cancer Biology, Radiation OncologyCenter, Washington University, St. Louis, MO 63108.4 To whom requests for reprints should be addressed, at 207A McArdleLaboratory for Cancer Research, 1400 University Avenue, Madison, WI53706. Phone: (608) 265-5537; Fax: (608) 262-2824; E-mail: [email protected].

this function (3). The sequence-specific DNA-binding func-tion of p53 is essential for transcriptional activation (4) of a

number of genes that contain sequences to which wild-type,but not tumor-derived, mutant p53 binds (5-8). Several ofthese genes encode proteins that seem to participate in the

arrest of cell division or the stimulation of apoptosis induced

by p53 (reviewed in Ref. 9). Expression of these genes maybe regulated through interactions between p53 and compo-nents of the basal transcription factor TFIID, because p53mutants lacking the ability to interact with the TATA box-

binding protein-associated factors TAFII32 and TAFII7O are

unable to activate transcription, although they are able tobind DNA (10, 1 1). Alternatively, transcription may be influ-enced by interactions between p53 and coactivators such as

p300 and CBP, because p53 mutants that fail to interact withthese coactivators fail to transactivate (1 2). The mechanismsthat normally regulate p53 function are not well understood.

Recent evidence indicates that tumors expressing wild-type p53 have evolved multiple mechanisms to inhibit theability of p53 to transactivate gene expression (9). In 10-

60% of sarcomas, leukemias, and breast cancers (1 3-1 5), aninhibitor of the transcriptional activation function of p53 is

expressed at elevated levels. The inhibitory protein, the Iarg-

est product of the human homologue of the murine double

minute 2 gene (mdm2), p90, binds to the transactivationdomain of p53 and inhibits expression of constructs contain-

ing p53-responsive promoters (1 6-1 8). In other tumors, such

as neuroblastomas and some breast cancers, the wild-typep53 protein is retained in the cytoplasm, where it cannotfunction as a transcriptional activator (1 9, 20). For other

tumor types, the mode of inhibition of p53 is less clear. For

example, in cell lines derived from teratocarcinomas, high

levels of wild-type p53 protein are present in the nucleus, but

there are low levels of expression of reporter and endoge-

nous genes containing p53 REs5 (21). The specific activity ofthe p53 protein, defined as the transcriptional response permole of p53 protein, seems to be regulated in teratocarci-

noma cell lines, because expression of p53-responsive

genes can be induced on differentiation of these cells, whilethe amount of p53 protein decreases (21). Differentiation ofnormal keratinocytes also results in decreased levels of p53protein, concurrent with increased expression of p53-re-

sponsive genes (22). Together, these studies indicate that

there are uncharacterized mechanisms that regulate the spe-cific activity of p53 as an inducer of gene expression.

Treatment of cultured cells with UV light results in a dis-

cordance between the amount of p53 protein and theamount of mRNA expressed from p53-responsive genes (23,

5 The abbreviations used are: RE, response element; EMSA, electromo-bility shift assay; PBSBT, PBS containing 0.5% BSA and 0.05% Tween20; Ab, antibody.

120 UV Light Induces a p53-Specific Transcript of MDM2

6 M. E. Perry, unpublished observations.

24). The response of cells to UV light provides an opportunity

to study the regulation of the transcriptional activation func-

tion of p53, because the levels of p53 protein rise in adose-dependent manner (24, 25). In addition, exposure touV light results in enhanced expression of reporter and en-dogenous p53-responsive genes (24, 25). Although some

p53-responsive genes such as GADD45 (26) and p21 WAF1

(Ref. 27; this paper) do not require p53 for induction in

response to UV light, others, such as mdm2, do require p53function (23). Paradoxically, the magnitude ofthe induction in

mdm2 expression, measured by Northern analysis and bypulse-labeling of MDM2 protein, does not correspond withthe amount of p53 protein in cells exposed to different dosesof UV light. These findings indicated that there may be reg-

ulation of the ability of p53 to mediate gene expression afterexposure of cells to UV light. Indeed, Hupp et a!. (28) re-ported that 16 h after exposure of mammalian cells to UV

light, there was elevated expression of a p53-responsive

reporter gene without an increase in the amount of p53

protein. A model was proposed in which the specific activity

of the p53 protein as a sequence-specific DNA-binding pro-tom is increased by exposure of cells to UV light (28). Con-sistent with this model is the observation that the DNA-

binding activity of purified p53 protein can be stimulated by

single-stranded DNA molecules similar in length to thoseoligonucleotides excised during repair of UV-induced DNA

damage (29). In this study, we determine that exposure ofcells to UV light results in regulation of the ability of p53 totransactivate gene expression at a level distinct from the

ability of p53 to bind DNA in a sequence-specific manner. We

examined the regulation of expression of the mdm2 gene in

response to UV light because this gene contains two pro-

moters, one of which (P2) is dependent on p53 for activity

(30). Thus, expression of transcripts initiated from the P2promoter reflects the ability of p53 and the effectors of its

function to stimulate transcription. We found that the P2

promoter in the mdm2 gene responds to UV light, whereasthe basal upstream promoter does not respond. Although

induction of the P2 promoter by UV light requires p53, there

is a discordance between the amount of p53 and the amountof transcript initiated at the P2 promoter. Furthermore, wemeasured the ability of p53 to bind DNA in a sequence-

specific manner and found that the amount of transcriptinitiated at the P2 promoter does not correlate with the

capacity of p53 to bind DNA in a sequence-specific manner.Thus, the ability of p53 to stimulate the activity of the P2promoter of mdm2 is mediated in part by changes distinctfrom those that regulate the DNA-binding function of p53.

ResultsThe Increase in the Rate of MDM2 Protein Synthesis afterExposure of Cells to UV Light Does Not Correlate with theAmount of p53 Protein in the Cell. The induction of ex-pression of the MDM2 protein in response to UV light re-quires p53 function; however, the magnitude of the increasein the level of MDM2 protein does not parallel the magnitude

of the increase in the level of p53 protein as determined byWestern analysis (23). To determine whether the rate of

synthesis of MDM2 protein correlates with the amount of p53

protein in cells exposed to UV light, we measured the in-

crease in the amount of newly synthesized MDM2 and corn-pared it to the increase in the total amount of p53 proteinafter exposure of cells to UV light. The p53 protein normally

has a short half-life of about 20 mm that becomes lengthenedto several hours on exposure of cells to UV light (25). Thus,the amount of p53 can be tracked by radiolabeling cells for

several half-lives before exposure to UV light, by which timep53 is labeled to steady state, and continuing to label for thelength of time of interest. The continuous labeling procedure

was used to track the amount of p53 protein, because it ismore sensitive than Western analysis and allowed us to

analyze several parameters in each cell lysate (see below). To

ensure that the assay was capable of measuring the steady-state level of p53 over a 9-h labeling period, we radiolabeled

cells in the presence of unlabeled methionine (2 x 1 O� M inDMEM) for 3 or 9 h, in the absence of UV exposure, andfound comparable amounts of radiolabeled p53 in the result-ing lysates. Thus, this method is capable of radiolabeling p53to steady state in the absence of exposure to UV light.

Furthermore, we compared the magnitude of the increases in

the level of p53 measured by the continuous labeling method

to those by Western analysis and found them to be similar.6

We treated Cl 27 cells with a dose of UV light that resultsin greater than 97% survival (4 J/m�) or a dose that results inonly 10% survival (20 J/m�).6 For simplicity, we will refer to

these doses as the low and high doses of UV light. The

amount of p53 protein present after continuous labeling was

compared to the amount of MDM2 protein synthesized dur-ing a 30-mm pulse labeling (Fig. 1). Two h aftertreatment withthe low dose of UV light, the level of p53 protein had risen

2-fold (Fig. lA), and the rate of MDM2 protein synthesis hadrisen 6-fold (Fig. 1B). By 8 h, the amount of p53 protein haddecreased, as had the rate of synthesis of MDM2 protein. Incontrast, after exposure of cells to the high dose of UV light,

p53 levels rose about 3-fold by 2 h, but MDM2 levels had notchanged. Eight h after exposure, the amount of p53 proteinhad increased to 8 times the normal level (Fig. 1A), and the

rate of synthesis of MDM2 was increased 13-fold (Fig. 1B).Therefore, although both p53 and MDM2 increase in re-sponse to low and high doses of UV light, there is not acorrelation between the level of p53 protein and the rate of

synthesis of MDM2 protein 2 h after exposure to UV light.

Instead, there is a delay in the induction of MDM2 expressionrelative to the rise in p53 after exposure to the high dose ofUV light (20 J/m�).

This discrepancy is not due to the decrease in total cellular

protein synthesis after exposure to UV light, because equiv-

alent amounts of radioactive protein were incubated with

Abs to MDM2, yet less radiolabeled MDM2 protein wasrecovered 2 h after the high dose than 2 h after the low dose.

Thus, relative to the rate of synthesis of other proteins, the

synthesis of MDM2 protein was slower 2 h after exposure to

a high dose of UV light than 2 h after exposure to a low doseof UV light. The lack of an induction of MDM2 2 h afterexposure to the high dose could not be ascribed to death of

CM5M(-)

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Uv dose (J/m2):h labeled:

A

B

0 4 4 20 20

2 2 8 2 8

M (-) 628

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E� �1�exon � I

Pl__#{248}’#{149}#{149} P2�’ �primer

BC127 (-) (+)

;� .:� ‘ - 325I

P2-�- ::; � � -165

Uv dose (J/m2): 0 4 4 20 20

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Cell Growth & Differentiation 121

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UV dose (J/m2): 0 4 4

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Fig. 1. Comparison of the level of p53 (A) to the rate of synthesis ofMDM2 (B) in UV-treated Cl 27 cells. To determine the levels of p53 (A),cells were labeled with [�5S]methionine for 1 h before exposure to a doseof UV light of 0, 4, or 20 J/m2 and subsequently labeled for 2 or 8 h (hlabeled). Equivalent amounts of total cell protein were incubated with thepolyclonal Ab CM5 to immunoprecipitate p53. 14C-labeled molecular sizemarkers are indicated in kilodaltons (M). An Ab that recognizes SV4O largeT antigen was used as a negative control (-). To measure the rate ofsynthesis of MDM2 protein (B), cells were incubated with r5S]methioninefor 30 mm either 2 or 8 h after exposure to a dose of UV light of 0, 4, or20 J/m2. Equivalent amounts of acid-precipitable counts were incubatedwith the polyclonal Ab 628 to immunoprecipitate MDM2. The markers (M)and negative control N) are as described in A.

the cells, because MDM2 was induced 6 h later (Fig. 1B).

These observations indicate that p53 may be less effective atstimulating MDM2 expression 2 h after exposure to a highdose of UV light than 2 h after exposure to a low dose of UV

light.

A Specific p53-responsive Promoter Is Induced by UVLight. Expression of mdm2 RNA is induced by DNA damage

(31 , 32). If the delay in the increase in the rate of MDM2

protein synthesis after exposure to a high dose of UV lightwas due to a lag between the time p53 was stabilized by UV

light and the time mdm2 RNA was induced, then the delaymight reflect an inhibition of the ability of p53 to stimulate

transcription. The first intron of the mdm2 gene contains two

binding sites for p53 that confer responsiveness to p53 on

reporter genes (7). When cells expressing a temperature-

sensitive mutant of p53 (alal 35va1) are shifted from the non-permissive to the permissive temperature, transcription ofmdm2 from an internal promoter (P2) is stimulated (33), buttranscription from an upstream constitutive promoter (P1) is

Fig. 2. Primer extension analysis of mdm2 transcripts induced by UVlight. Schematic of mdm2 gene (A) with the two binding sites or REs forp53 in the first intron and both promoters (P1 and P2) indicated. Inductionof the P2 promoter of mdm2 by UV light (B). Cells were exposed to a doseof UV light of 0, 4, or 20 J/m2, and total RNA was isolated 2 or 8 h later (hafter U(A. A radiolabeled primer complementary to exon 3 of mdm2 washybridized to RNA and extended by reverse transcriptase. A product ofthe size predicted from the length of the transcript initiated from the P2promoter of mdm2 (165 bases) is indicated (P2). No product of the sizeexpected for the product of the P1 promoter (283) was detected. Twospecies longer than that (300 and 325) did not contain sequences fromexon 1 of mdm2. Cells harboring a temperature-sensitive allele of p53served as a negative control (-) at the nonpermissive temperature and apositive control (+) at the permissive temperature.

not increased. Thus, the P2 promoter responds to p53 func-

tion, whereas the P1 promoter does not. To test whether the

P2 promoter, but not the P1 promoter, was induced by UV

light, we attempted to measure the amount of mdm2 mRNA

initiating at each promoter using primer extension. The two

transcripts differ by the presence of exon 1 (30), such that theamount of mdm2 mRNA initiated at each promoter should be

revealed by primer extension analysis using an oligonucleo-tide that hybridizes to exon 3 (Fig. 2.4). After exposure of

C127 cells to either a low or a high dose of UV light, a

mdm2-specific transcript initiating at bp 3-5 of exon 2 was

induced (Fig. 2B), indicating that the P2 promoter was stim-

ulated by exposure of cells to UV light. Transcripts initiating

at P2 were not detected in untreated cells, confirming the

correlation of P2 activity with p53 function, which is in-

creased in cells exposed to UV light. No product correspond-

ing to the size predicted for the transcript from the P1 pro-

moter (283 bp) was detected by this assay before or afterexposure of cells to UV light. Two products larger than thatexpected from a RNA initiated at the P1 promoter were

C127 (10)3 (-) (+)

P1 � � � � � F

P2�” � . X2

Uv dose (J/m): 0

hafterUV: 0

a Mean fold increase over no UV treatment (n = 3).b Rate of MDM2 protein synthesis measured by pulse labeling with

r5S]methionine.CAmount of mdm2 transcript from P1 promoter, measured by 51 nude-

ase digestion.d Amount of mdm2 transcript from P2 promoter, measured by Si nude-

ase digestion.e Amount of p53 protein as measured by continuous labeling.

Fig. 3. Dependence of the induction of the P2 promoter by UV light onp53. The induction of the p53-dependent P2 promoter and the p53-independent P1 promoter were compared in cells expressing (C127) orlacking [(1 0)3) p53. Cells were treated as described in the Fig. 2 legend.Total RNA was protected from digestion with Si nuclease after hybrid-ization to a denatured 254 bp, radiolabeled DNA complementary to se-quences from exons 1-3 of mdm2. Transcripts from the constitutive (P1)and p53-responsive (P2) promoters are indicated. The negative control (-)

was a sample in which the probe was incubated with no RNA beforedigestion. The positive control (+) was a reaction in which the probe washybridized to two RNAs transcribed in vitro from mdm2 cDNAs of differentlength. RNA from pGEM1F contains exon 1 and reflects a transcriptinitiated at the constitutive promoter (F), whereas RNA from pGEM1X2reflects transcripts initiating from the P2 promoter (X2).


7 L. J. Saucedo, unpublished observations.

4 4 20 20 20 20

2 8 2 8 08

detected. The larger of these products was induced by UVlight, whereas the smaller was not. We had estimated thesize of the expected product from the P1 promoter to be 283

bases, based on the sequence of the longest cDNA for

mdm2 (30, 34). However, there may be multiple start sites

upstream of the 5’ end of the cDNA for transcription from the

P1 promoter (35). To determine whether the long primer

extension products contained exon 1 , cDNAs from this re-

gion of the gel were isolated, amplified by 5’ rapid amplifi-

cation of cDNA ends, and cloned.7 Sequence analysis re-

vealed that none of the cDNAs contained exon 1 of mdm2;

they were products of other genes or products of unspliced

RNA from the P2 promoter of mdm2 (containing exon 2,

intron 2, and exon 3 of mdm2). Use of oligodeoxythymidylic

acid-selected RNA resulted in loss of the largest primer

extension product, consistent with unprocessed mdm2 RNA

being the source of this species. The inability of primerextension to detect RNAs from the P1 promoter may be dueto inhibition of reverse transcription through exon 1 by the

high (73%) G-C content (34) or the use of multiple start sites,

as indicated by the work of Jones eta!. (35), in which case thesignal from the P1 promoter would be diluted. Both of these

possibilities are consistent with detection of products from

P2, but not P1 , using primers in exon 3 (Fig. 2B) or in exon 4#{149}7

To determine whether activation ofthe P2 promoter by p53

was responsible for all of the increase in MDM2 expressionin UV-treated cells, it was necessary to measure the amount

of mdm2 mRNA initiated at both the p53-independent (P1)

and p53-dependent (P2) promoters in p53-positive and p53-negative cells before and after exposure to UV light. Si

nuclease digestion of RNA hybridized to a cDNA including

Table 1 Increase in levels of mdm2 expression and amount of p53 2 hafter exposure of C127 cells to the low or high dose of UV light

2-h low dose 2-h high dose(mean ± SE)8 (mean ± SE)

Synthesis of MDM2b 6.4 ± 0.06 1 .0 ± 0.10

Amount of P1 RNAC 1.1 � 0.04 0.78 ± 0.01Amount of P2 RNAd 8.9 ± 2.5 3.5 ± 1.3Amount of p53e 2.1 ± 0.51 3.3 ± 1.1

sequences from exons 1-3 of mdm2 might be expected to

allow simultaneous detection of transcripts from the two

promoters, because the probe contains sequences from ox-

ons 1-3 of mdm2. This method has two advantages overprimer extension for detection of transcripts from the P1promoter: (a) one digestion product would be obtained from

transcripts initiated at multiple sites in the P1 promoter,because the 5’ end of the mdm2 cDNA sequences in the

probe is 3’ of all mapped start sites; and (b) there is norequirement for reverse transcription through G-C-rich se-

quences in exon 1 .This method was successful, as revealed

by the pattern obtained from P1 - and P2-specific transcripts

synthesized in vitro (F and X2, respectively; Fig. 3). Tran-

scripts initiated from the P2 promoter were increased byboth doses of UV light, but transcripts from the P1 promoterwere not increased, indicating that all of the increase inmdm2 RNA was due to enhanced transcription from the P2

promoter. Transcripts from the P2 promoter were not do-tected in cells lacking p53, even after exposure to UV light.These data indicate that the induction of mdm2 by UV light

is regulated through the p53-specific P2 promoter. The

amount of P2-specific transcript thus reflects the ability of

p53 and its effectors to enhance transcription from this pro-

moter.

Delayed Activation of the P2 Promoter by p53 in Re-sponse to a High Dose of UV Light. Both the primer ex-tension (Fig. 2B) and Si nuclease digestion (Fig. 3) analyses

indicate that the amount of mdm2 transcript initiated at thep53-specific internal promoter (P2) was about 2.5 times

higher 2 h after exposure to the low dose of UV light than it

was 2 h after exposure to the high dose (Table 1). This

pattern is paralleled by the greater increase in the rate of

MDM2 protein synthesis 2 h after exposure to the low dose

than 2 h after the high dose (Fig. 1B; Table 1). Thus, thechanges in the rate of synthesis of MDM2 protein in response

to UV light correspond to the amount of P2-specific tran-

script in the cells.The magnitude of the increase in the P2-specific transcript

was greater, relative to the level of p53 protein, 2 h after

exposure to the low dose of UV light than it was 2 h afterexposure to the high dose, indicating that p53 may be more

effective as an activator of transcription after exposure of

cells to the low dose of UV light than after exposure to the


high dose (Table 1). The inefficiency of p53 in stimulating theP2 promoter 2 h after exposure to the high dose of UV lightcould be due to the general inhibition of RNA synthesis thatoccurs in cells exposed to UV light (36). To investigate

whether the timing of mdm2 expression after exposure to thehigh dose of UV light corresponded to the timing of therecovery of general RNA synthesis, we measured the incor-

poration of [�H]uridine into Cl 27 cells for 60 mm at severaltimes after exposure to UV light (36). We found that inhibitionof general RNA synthesis occurred in cells exposed to bothlow and high doses of UV light, and that the inhibition wasgreater at the higher dose. Two h after exposure to the lowdose of UV light, the rate of RNA synthesis was inhibited14%, whereas afterthe high dose, it was inhibited 55%. Theoverall rate of RNA synthesis recovered by 5 h after exposureto the low dose of UV light, but it continued to decrease for

up to 10 h after exposure to the high dose of UV light.6 Eighth after exposure to the high dose of UV light, the rate of RNAsynthesis was further decreased to 30% of normal levels. Atthis time, the amount of mdm2 RNA transcribed from the P2promoter was increased 8.9-fold. Thus, the induction of theP2 promoter after exposure to the high dose does not cor-respond to a recovery of the general rate of RNA synthesis.Instead, the P2 promoter is induced in spite of the generalinhibition of RNA synthesis and seems to be regulated inde-pendently of the recovery of total RNA synthesis. Inhibition ofgeneral RNA synthesis might be expected to affect tran-scripts from both the P1 and P2 promoters equally; however,the ratio of the level of the P2 transcript to the P1 transcriptis lower 2 h after exposure to the high dose of UV light than

2 h after exposure to the low dose (P = 0.0063). Thus, thereis specific regulation of the amount of mdm2 transcriptionthat initiates from the P2 promoter. These data indicate thatin the presence of a general inhibition of RNA synthesis, theamount of transcript from the P2 promoter rises compared tothe amount from the P1 promoter. In fact, the percentage oftotal RNA that is represented by the mdm2 transcript initi-ated at the P1 promoter is not significantly altered 2 or 8 hafter exposure to the high dose.

The factors that contribute to the activity of the P2 pro-moter may be involved in the timing of induction of otherpromoters after exposure to high doses of UV light (37). Forexample, some cells are capable of inducing expression ofimmediate early genes such as c-fos and c-jun within 45 mmof exposure to doses of UV light as high as 40 J/m2 (38). Infact, the c-jun gene is induced to a greater extent afterexposure to 40 J/m2 than to 10 J/m2 in HeLa cells (38). Totest whether the factors required for induction of c-jun ex-pression were rate-limiting after exposure of Cl 27 cells tohigh doses of UV light, the levels of c-jun RNA were meas-ured before and after exposure to 4 and 20 J/m2. Expressionof c-jun was induced to a greater extent 2 h after exposureto a high dose than 2 h after exposure to a low dose of UVlight,7 indicating that C127 cells can induce expression ofspecific genes to a greater degree 2 h after treatment withthe high dose than 2 h after treatment with the low dose.These results are consistent with those of others, indicatingthat specific factors regulate the activity of different UV-responsive promoters (37).

Stabilized p53 is Localized to the Nucleus of UV-treated Cells. The delay in expression of the P2-specificRNA after exposure to the high dose of UV light indicates thatthe ability of p53 to stimulate promoter activity may be in-hibited during this period. The inhibition could result from adelay in transport of the p53 protein to the nucleus afterstabilization in response to UV radiation. To determinewhether there was a delay in transport of p53 from the

cytoplasm to the nucleus after exposure of cells to a highdose of UV light, indirect immunofluorescence was used tolocate p53. By 2 h after treatment with either the low or highdose of UV light, p53 had accumulated in the nucleus (Fig. 4).The amount of p53 in the nucleus appears higher 2 h afterexposure to the high dose than 2 h after exposure to the low

dose, as predicted from the continuous labeling experiment(Fig. 1). Therefore, the delayed induction of mdm2 transcrip-tion after exposure of Cl 27 cells to the high dose of UV lightis not explained by retention of p53 in the cytoplasm.

Induction of Other p53-responsive Genes Seems De-

layed after Exposure to the High Dose of UV Light Thediscrepancy between the level of p53 protein in the nucleusand the activity of the p53-dependent P2 promoter of mdm2indicates that the ability of p53 to stimulate transcription isregulated. To determine whether other p53-responsive

genes were induced to a greater extent 2 h after exposure tothe low dose of UV light than 2 h after exposure to the highdose, we measured the levels of cydlln G1 (39) and p2l”�1

(5) RNAs before and after UV exposure. Expression of cydin

G1 was slightly greater 2 h after exposure to the low dosethan 2 h after the high dose; however, the magnitude of the

induction of cydin Gi was only about 2-fold.7 Expression of

p2l’�”� RNA was induced approximately 9-fold 2 h afterexposure to the low dose of UV light and 4-fold 2 h after thehigh dose of UV light (Fig. 5), indicating that this p53-respon-sive gene is induced in a manner similar to that of mdm2.These data indicate that the activity of p53 may be modifiedby UV light in such a way as to affect all p53-responsivegenes similarly.

To determine whether p53 function was required for theinduction of p21WAF1 by UV light, we measured the levels ofp21W��1 mRNA after UV treatment of (10)3 cells that lackp53. Expression 0fp2lW�� was induced in cells lacking p53(Fig. 5), although to a lesser extent than in cells expressing

p53. The length of the RNA induced by UV light is identical incells expressing and lacking p53 (Fig. 5), indicating that thereis not a p53-specific promoter in thep21W� gene. Thus, thelevel of p2l�’� RNA after exposure of cells to UV lightcannot be used as a measure of p53 function. Indeed, ourresults are consistent with a recent report describing p53-independent induction 0fp21WAF1 expression after exposureof human cells to UV light (27).

The Magnitude of the Increase in P2 Promoter ActivityIs Not Reflected In Changes in the Ability of p53 to BindDNA. The activity of the P2 promoter of mdm2 reflects theability of p53 to stimulate transcription, through its effectors.The amount of transcription might be determined by theamount of p53 that can bind DNA or by events independentof DNA binding. Many mechanisms have been found toregulate the ability of purified or exogenously expressed p53

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Fig. 4. Nuclear localization of p53 after exposure ofC127 cells to UV light. Cells grown on coverslipswere treated with a dose of UV light of 0, 4, or 20J/m2; incubated at 37’C for 2 or 8 h; and then fixedin methanol. Indirect immunofluorescence was usedto detect p53. The polyclonal Ab CM5 was incu-bated with the cells and followed by a FITC-conju-gated goat antirabbit lgG. The negative control wasa coverslip on which no CM5 Ab was placed beforethe secondary Ab (no primary Ab).

2 h (20 J/m2)

C127 (10)3

k #{149}�0 4 20 0 20

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8 h (20 J/m2)

Fig. 5. Induction of p21WAF1 is delayed after exposure to a high dose ofUv light. C127 cells were treated with a dose of UV light of 0, 4, or 20 J/m2,and total RNA was harvested 2 or 8 h later. The (1 0)3 cells, which lack p53,were treated with 0 or 20 J/m2 and harvested 8 h later. A radiolabeledprimer complementary to exon 2 of the p2iWAFl gene was hybridized toRNA and extended by reverse transcriptase.

to bind DNA (reviewed in Ref. 9); however, the regulation of

endogenous p53 function in mammalian cells is not well

understood. The specific activity of p53 as a DNA-binding

protein can be stimulated by peptides, Abs, and kinases that

bind to the regulatory region in the carboxyl terminus of the

protein, indicating that the DNA-binding activity of p53 can

be regulated allosterically (40). To determine whether the

specific activity of p53 as a DNA-binding protein determines

the activity of the P2 promoter of mdm2 after exposure ofcells to UV light, the amount of p53-specific DNA-binding

activity in nuclear extracts of Ci 27 cells was measured by

EMSA. Extracts were incubated with the consensus bindingsite for p53 (Fig. 6) or with sequences from the mdm2 gene.8No specific binding of p53 to DNA could be detected in

extracts from Ci 27 cells before or after treatment with UV

light unless the Ab 421 , which can activate the DNA-binding

function of p53 in an EMSA (41), was added to the reaction.

The addition of Ab 421 resulted in greater DNA-binding ac-

tivity by p53, such that DNA binding could be detected in

extracts from both treated and untreated cells. A comparison

of the amount of DNA bound by p53 from extracts of cells

harvested 2 h after exposure to UV light reveals that there is

more DNA-binding activity in cells after the high dose than

after the low dose. Thus, the DNA-binding activity of p53

h after UV: 0 2 8 2 8 probe

42ladded: - + - + - + - +

-,#{149}� -�

- + alone

1234

450- � �

*

*

free probe -�“

5 6 7 8 9 10

� � � �

160- � � � �.

Uv dose (i/rn2): 0 4 20

Fig. 6. Activation of p53 DNA binding by Ab 421. Cells were treated asdescribed in the Fig. 2 legend. Nuclear extracts were prepared 2 or 8 hafter exposure to UV light. Equivalent amounts of nuclear protein wereincubated with a radiolabeled double-stranded oligonucleotide with thesequence of the consensus binding site for p53. Extracts were incubatedwith the probe for 30 mm on ice in the presence (+) or absence (-) of the421 Ab, and the reactions were separated on a 5% denaturing gel. Bandscorresponding to p53, as determined by both immunodepletion and com-petition with wild-type but not mutant oligonucleotides,8 are marked (*).

controls 0 4 4 20 20 i/rn2

0 2 8 2 8h

Fig. 7. Increased DNA-binding activity by p53 from cells exposed to UVlight. The same cell lysates analyzed for the level of p53 protein (Fig. 1A)were tested for the amount of p53-specific DNA-binding activity. Lysateswere incubated with two radiolabeled DNA fragments from the mdm2gene. A 450-bp fragment contained no sequences to which p53 bindsspecifically; a 160-bp fragment contained both REsfor p53. Lanes 1-5 arecontrols. Lane 1 contains 5% of the radiolabeled probe added to eachreaction. Lane 2 shows the amount of probe that binds nonspecifically tothe protein A-Sepharose used to precipitate immune complexes from thereaction. As a positive control for p53-specific DNA binding, the probewas incubated with lysate from A1-5 cells that contain a temperature-sensitive mutant of p53 grown at the permissive temperature. After incu-bation with the probe, Al -5 cell lysate was incubated with either poly-clonal Ab CM5 that recognizes p53 (Lane 3) or with a control Ab (Lane 4).Lane 5 contains sample buffer only. Lanes 6-10 show DNA immunopre-cipitated with the CM5 polydlonal Ab after incubation with lysates of Cl 27cells. The amount of p53-specific DNA binding was calculated as the ratiobetween the amount of 160-bp fragment to the amount of 450-bp frag-ment in each lane.


does not seem to be rate-limiting for transcription 2 h after

exposure to the high dose of UV light.

The results of the EMSA could be complicated by the use

of Ab 42i , because addition of the 421 Ab to an EMSA isknown to enhance the binding of certain forms of p53 that

are thought to be inactive for DNA binding in the absence of

Ab 421 (41 , 42). Also, if exposure of Ci 27 cells to UV light

results in production of a form of p53 active for DNA binding

but not recognized by Ab 421 (41 , 43), the EMSA would not

reveal the DNA-binding activity of such a protein. Therefore,

we used a more sensitive DNA-binding assay to determine

whether changes in the specific activity of p53 in response to

UV light account for the delayed induction of the P2 promoter

of mdm2.

An assay based on the immunoprecipitation method of

Mckay (44) allowed us to measure the ability of cellular p53to bind DNA in the absence of the activating Ab 421 (Fig. 7).

Whole-cell lysates were incubated with two radiolabeled re-

striction fragments from the 5’ end of the mdm2 gene. The

smaller fragment included the two REs for p53 from the

mdm2 gene; the larger fragment lacked sequences recog-

nized by p53. Specific binding of p53 was reflected in anincreased ratio of the fragment containing the REs (160 bp)

to the fragment lacking the REs (450 bp) after immunopre-

cipitation of p53 with the polyclonal Ab CM5 (compare Lanes

3 and 4 of Fig. 7). This polyclonal Ab (CM5) recognizes

several epitopes within the p53 protein, none of which is that

recognized by Ab 421 (45). It follows that the CM5 Ab does

not enhance the DNA-binding activity of p53 in an EMSA.8

The sequence-specific DNA-binding activity of p53 was un-

detectable in extracts from cells that had not been exposed

to UV light (Fig. 7); however, the activity of p53 was increased

in cells exposed to UV light (Fig. 7). The amount of DNA-

binding activity was higher 2 h after the high dose of UV light

than 2 h after the low dose (i .9-fold, ± 0.22).

To determine whether the specific activity of p53 as a

DNA-binding protein correlated with the activity of the P2

promoter, we compared the DNA-binding activity with the

amount of p53 protein present in each lysate. The same

lysates were used to immunoprecipitate p53 in the presence

(Fig. 7) and absence (Fig. 1) of DNA. For the Mckay assay,

the radiolabeled protein was extracted from the radiolabeled

DNA before separation on the gel. The amount of DNA pre-

cipitated in the Mckay assay increased in proportion to the

amount of p53 in the extract. That is, the specific activity of

p53 as a DNA-binding protein was not different in extracts

from cells irradiated with either the low or high dose of UV

light and harvested 2 h later (P = 0.27). Thus, the differential

induction of mdm2 expression after exposure to low and highdoses of UV light must be regulated, in part, at a step

independent of the ability of p53 to bind DNA.

The Activity of the P2 Promoter Does Not Correlatewith Changes in the Immunoreactivity of p53. The activity

of the P2 promoter of mdm2 may be determined by a mod-

ification of the p53 protein or by the activity of a general

transcription factor, a repressor, or a coactivator. To attempt

to differentiate between these possibilities, we asked

whether the reactivity of p53 to several conformation-spe-

cific Abs was different 2 h after exposure to the low and high

doses of UV light.

Monoclonal Ab 421 recognizes an epitope in the carboxyl-

terminal region of p53 and has been reported to bind a form

of p53 that lacks DNA-binding activity (46), but that can be

activated for DNA binding by addition of Ab 421 to an EMSA

(41). The 246 Ab recognizes an epitope between amino acids

88-1 09 of wild-type murine p53 (42), and reactivity of p53

with Ab 246 has been proposed to indicate that p53 is

M (-) 246 421

6S�- I:� �#{149}_I._.� � � � .�4-pS3

4�- - -“. ,� ,. � �

Uv dose (i/rn2): 0 4 4 20 20 0 4 4 20 20

hiabeled: 2 2 8 2 8 2 2 8 2 8

Fig. 8. No evidence for a conformational change in p53 protein afterexposure to UV light. The same lysates analyzed in Figs. 1A and 7 weresubjected to immunoprecipitation with the conformation-specific Abs 246and 421 as described for CM5 in the Fig. 1 legend. A representative lysatewas incubated with Ab 419 that recognizes SV4O large T antigen (-).Molecular size markers are indicated (M).


functional as a DNA-binding protein (42). To determine

whether the activity of the P2 promoter correlated with a

change in the conformation of p53 from one reactive with Ab421 to one reactive with Ab 246, p53 protein was radiola-

beled and immunoprecipitated with either Ab (Fig. 8). Theamount of p53 recognized by Abs 42i and 246 was com-

pared to the amount of p53 precipitated by polyclonal Ab

CM5 (see Fig. 1). The proportion of CM5-positive p53 that

was positive for either Ab 421 or 246 did not change ontreatment of cells with UV light. For example, the percentageof CMS-positive p53 recognized by Ab 421 was 82 (±4.4)

and 88% (±6.3), on average, 2 h after exposure to the lowand high doses of UV light, respectively. For Ab 246, the

averages of the same samples were 85 (± 13) and 89%

(± 8.3), respectively. Furthermore, sequential immunopre-

cipitation of p53 with the different Abs failed to reveal spe-

cies of p53 that reacted with only Ab 246 or 421 #{149}6Finally, a

mixture of the three Abs, CM5, 421 , and 246, failed to pre-

cipitate more p53 than did CM5 alone.6 Together, these

experiments indicate that the majority of the p53 protein inCi 27 cells is reactive with both Abs 42i and 246 and that the

activity of the P2 promoter does not correlate with changes

in the conformational state of p53 as revealed by immuno-

reactivity with these Abs.

DiscussionThe p53-dependent increase in expression of the MDM2

protein after exposure of cells to UV light is regulated through

an increase in the activity of a p53-specific promoter in the

mdm2 gene. The activity of this promoter is not induced incells that lack p53 and is therefore a reflection of the ability

of p53 and its eftectors to regulate gene expression. A delay

in stimulation of P2 promoter activity relative to a rise in the

amount of p53 protein is evident after exposure to a high

dose of UV light. Two DNA binding assays showed that the

specific activity of p53 as a DNA-binding protein is not rate-

limiting for stimulation of the P2 promoter of mdm2 after

exposure of cells to the high dose of UV light. In most studies

of endogenous p53, the DNA binding activity of p53 has

been measured in the presence of Ab 421 , which is known to

increase the ability of p53 to bind DNA (6, 32, 47). Here, wehave measured the ability of cellular p53 to bind DNA in cells

that have been exposed to UV light without the use of an

activating reagent. Our assay demonstrates that the activity

of the P2 promoter of the mdm2 gene is not regulated solely

by the amount of sequence-specific DNA-binding activity of

p53 in the cell, indicating that other factors must contribute

to regulate the timing of induction of mdm2. Such factorsmay be proteins that directly modify p53 or interact with theDNA to stimulate or retard transcription from the P2 pro-

moter. Such proteins may regulate the ability of p53 to stim-

ulate several different promoters, because induction of thep53-responsive p21WAF1 and cyclin G1 genes, like that ofmdm2, is delayed relative to the increase in the level of p53.

However, cydin G expression is induced only about 2-fold,and p21WAF1 expression is induced in cells lacking p53.

Thus, the level of the transcripts from these genes is not as

reliable an indicator of the ability of p53 to stimulate tran-

scription as the ratio of transcripts from the P2 and P1promoters of mdm2.

The timing of induction ofthe P2 promoter ofmdm2 seems

to be specifically regulated in response to different doses of

UV light, because it does not correspond to the recovery of

the rate of total cellular RNA synthesis. Furthermore, ourassays indicate that transcription from the P2 promoter is

increased, whereas transcription from the Pi promoter is not

increased after exposure to UV light. The high dose of UV

light causes a delay in induction of the P2 promoter with no

change in the level of transcription from the P1 promoter.This delay does not seem to be the result of direct DNA

damage, because the template for transcription from the P1promoter encompasses the template for transcription fromP2 and includes more than 600 additional bp (34). The P1template would be expected to suffer more DNA damagethan the P2 template, resulting in an increased chance of

inhibition of transcriptional elongation (48). Thus, exposure of

cells to the high dose of UV light seems to affect the activityof the P2 promoter through a transacting factor that may be

p53 itself or an unidentified protein that influences the effect

of p53. The factor(s) influencing the ability of p53 to induce

expression of mdm2 does not affect all UV-responsive genes

similarly, because the c-jun gene, unlike mdm2, is induced to

a greater extent 2 h after exposure to the high dose than 2 h

after the low dose.

The factor(s) influencing the ability of p53 to induce ex-

pression of mdm2 is either inhibiting or rate-limiting at early

times after exposure to the high dose of UV light. Severalproteins, such as cyclin E and Ref-i , stimulate the ability of

p53 to bind DNA and to activate transcription (49, 50). The

activities of these proteins are unlikely to account for the

delay in expression of mdm2 after exposure of cells to a highdose of UV light, because our data indicate that mdm2

expression is likely to be regulated at a step distinct from

regulation of the ability of p53 to bind DNA. Some proteins

stimulate the ability of p53 to induce transcription throughmechanisms that are apparently independent of stimulatingthe DNA-binding activity of p53. For example, exogenous

expression of either the p300 or CBP coactivator proteins

increases the ability of p53 to induce the expression of

reporter genes (12, 51, 52), including one regulated by themdm2 promoter (1 2). It is unknown whether any of theseproteins are required for p53 function in the response to UV


light. Recently, a repressor of mdm2 transcription has beenproposed to be induced by high doses of UV light (53). Thismodel is based on a transient decrease in total mdm2 RNAlevels after exposure of primary cells to high doses of UVlight (53). Evidence suggests that this effect is independentof p53 and therefore may be due to a decrease in the level ofthe P1 transcript, not an inhibition of induction of the P2

transcript. Because we did not detect a significant decreasein RNAS from either the P1 or P2 RNAs, we cannot distin-guish between an induced repressor and an inactive (co)ac-tivator. Our data indicate that the delay in mdm2 expressionat the high dose of UV light is due to a delayed induction,because the amount of transcript from the P1 and P2 pro-moters is not decreased.

The factors coordinating with p53 to regulate the activity ofthe P2 promoter may be intimately connected to the tran-scriptional machinery. In fact, several components of themultisubunit general transcription factor TFIIH bind to p53,

including two helicases mutated in the repair disorder xero-derma pigmentosum (54). Different forms of TFIIH are activein transcription and in repair (55). The subunit composition ofTFIIH changes in response to UV light, and the activity of thekinase associated with TFIIH is reduced (55, 56). This kinaseis required for transcription of some genes by RNA polym-erase II (57). Thus, it has been proposed that the repair-competent form of TFIIH may down-regulate transcription incells that have suffered large amounts of damage (58). Thetranscriptionally active form of TFIIH may be required for p53to efficiently stimulate transcription from the P2 promoter ofmdm2. Perhaps the higher levels of p53 protein seen 2 h after

exposure to the high dose of UV light can compensate for adecrease in transcription-competent TFIIH. The signals reg-ulating the conversion of repair-competent TFIIH to tran-scriptionally active TFIIH after repair of DNA damage areunknown. These may be the same signals that regulate therecovery of the rate of total RNA synthesis; however, it isclear that the recovery of the rate of total RNA synthesis isnot required for induction of P2 activity, because mdm2 isinduced before the recovery of the rate of RNA synthesis.These observations indicate that factors independent of therecovery of total RNA synthesis may be rate-limiting for P2function after exposure to the high dose of UV light. Althoughlittle is understood about the mechanisms governing therecovery of the rates of RNA synthesis in response to UVlight, the amount of repair of UV-induced DNA damage is notthe only factor, because cells derived from patients withCockayne’s syndrome, which have normal global repair, failto recover normal rates of general RNA synthesis after ex-posure to UV light (36).

It is possible that the induction of the P2 promoter isspecifically delayed, because transcripts from this promoter

may be more efficient than transcripts from the P1 promoterat directing translation of MDM2 (30). Because MDM2 inhib-its p53 function (1 6) and stimulates p53 degradation (59, 60),cells severely damaged by UV light may forestall increasingthe amount of MDM2 until p53 has carried out its requiredfunctions. Sunburned epidermal cells undergo p53-stimu-

lated apoptosis (61), and it may be in the best interest of theorganism to delay mdm2 expression until such time as cells

have committed to apoptosis, because MDM2 can inhibitp53-dependent apoptosis (62). Studies of the requirementsfor induction of the p53-responsive P2 promoter of mdm2may reveal unknown mechanisms of regulation between

these two proteins after exposure to agents that damageDNA.

Materials and MethodsCell Culture. Ci 27 is a nontransformed murine cell line expressing wild-type p53 (63). Cells lacking p53 [(10)3 and (10)1] were immortalized from14-day-old embryos from BALB/c mice (64). Va15 is a derivative of (10)1and expresses a temperature-sensitive allele of p53 (aIal3SvaI) that actsas a transcriptional activator at 32#{176}Cbut not at 37#{176}C(7). Al-S cells are ratembryo fibroblasts transformed with Ha-ras and the temperature-sensi-

tive allele of p53 (65). Cells were cultured in DMEM with 10% FCS(Hyclone) in a humidified environment of 5% CO2.

Exposure to UV Ught. C127 or (10)3 cells were plated at a density of2.1 x 106 cells/iS-cm dish and exposed 24-36 h later to a Blak-Ray Uvlamp with a wavelength of254 nm (UVC). The distance between the platesand the lamp was adjusted so that the fluence of the light was 2 J/m2/s,as measured by a Blak-Ray J-225 UV meter (UV Photoproducts, San

Gabriel, CA).MetabolIc LabelIng and Immunoprecipftation. For determination of

the levels of p53 protein after stabilization by UV light, C127 cells wereradiolabeled for 1 h (three p53 half-lives) before treatment with UV lightand for 2 or 8 h after exposure to a dose of 0, 4, or 20 J/m2. Cells wereincubated for the length of time indicated with 100 �CVmI 35S-Express (amixture of r5Sjmethionine and [�S]cysteine; DuPont New England Nu-

clear) in normal growth medium (DMEM plus 10% FCS). After lysis (66),the concentration of protein in each sample was determined using the

Bio-Rad protein assay (67), and equivalent amounts of total protein (200�Lg) were incubated with the CM5 Ab (described below). For determinationof the rate of synthesis of MDM2 protein, the cells were labeled for 30 mmwith 100 �CVml �S-Express in DMEM lacking methionine supplemented

with 2% dialyzed FCS. The amount of radiolabeled protein in each samplewas quantified by trichloroacetic acid precipitation and used to normalizethe volume of lysate added to the immunoprecipitation reactions such that

each had S x 106 cpm. Quantitation was performed on a Molecular

Dynamics Phosphorimager.Abs. Polyclonal CM5 is a rabbit antiserum specific for p53 (Novocas-

tra); monoclonalAb 421 recognizesthe carboxyltermmnus ofwild-type andmutant p53 (45), and monoclonalAb 246 recognizes wild-type murine p53(45). The Ab used as a negative control was monoclonal Ab 419, whichrecognizes SV4O large T antigen (68), a protein not expressed in any of thecells used in this study. Hybridomas for Abs 421 , 246, and 419 were fromArnold J. Levine(Princeton University). The polyclonalAb 628 was used to

immunoprecipitate MDM2. This Ab was raised in our laboratory against ahistidmne-tagged fusion protein of murine MDM2 purified from Escherichiacell (as first described in Ref. 69).

Plasmids. The plasmid encoding histidine-tagged MDM2 was pro-vided by Donna George (University of Pennsylvania). Plasmids containing

cDNAs from mdm2 (pGEM1 F and pGEM1X2) were gifts from Moshe Oren(Welzmann Institute). pGEM1 F contains a cDNA from a transcript includ-

ing exons 1-12 of mdm2, whereas pGEM1X2 contalns a cDNA from a

transcript including exons 2-1 2 (30). To generate a source of a probe forthe Si nuclease protection assay, an EcoRl fragment from pGEM1 F

containing the entire cDNA for mdm2 was excised and cloned into the

EcoRI site of Bluescript (Stratagene) to generate pBSF.Primer ExtensIon. An oligonucleotide primer(iOO ng)ofthe sequence

5’-TCGAAGCCUGTrCTCTGAGACCi�A-3’, complementary to exon 3 ofmdm2 (34), or an oligonucleotide (100 ng) ofthe sequence 5’-TCACCAG-

GATTGGACATG-3’ (5), complementary to exon 2 of p21WAF1, was end-labeled with [‘y-32PJATP using T4 kinase (New England Biolabs) and pre-cipitated with ethanol in the presence of RNA homopolymer (Pharmacia).Total RNA was harvested according to Sambrook et a!. (70), and theprimer extension reaction was modified from that described by Treizen-berg (71). Radiolabeled primer (3 x iO� cpm) was annealed to 20 �g oftotal RNA in a buffer containing 300 m� NaCI, 10 m�i Tris (pH 7.5), 12.5pg/mI actinomycin D (Calbiochern), and 1 mp�i EDTA by denaturation at80#{176}Cfor 1 mm, followed by incubation at 62#{176}Cfor 15 mm. After annealing,

128 UV Ught Induces a p53-Specific Transcript of MDM2

the buffer was adjusted to contain 10 m�.i Tris(pH 8.0), 8 m� MgCl2, 10 m�DTT, 1 mM deoxynucleotide triphosphates, and 25 units of avian myelo-

blastosis virus reverse transcriptase (Boehringer Mannheim). The reac-tions were incubated at 42#{176}Cfor 90 mm and then at 52#{176}Cfor 30 mm. Thesamples were subsequently treated with RNase A (Bcehringer Mann-heim), precipitated with ethanol, and separated on a 5% denaturing

polyacrjlamide gel. To generate a marker for mdm2, pGEM1 F was so-quenced with the primer described above using Sequenase version 2.0

(United States Biochemical) according to the manufacturer’s instructions.Products migrating more slowly than that from pGEM1 F RNA were ex-

cised from the gel after electrophoresis and purified using the Wizard PCRPurification Kit (Promega). The eluted cDNA was then amplified in a 5’

rapid amplification of cDNA ends (Life Technologies, Inc.) reaction usinga nested primer(5’-ATCTGTGAGGTGClTGCAG) for the PCR. The result-

ant PCR products of expected size were then gel purified, reamplified, anddirectly cloned into a TA vector [pT7BIue(R), Novagen]. Fourteen clones

were sequenced as described above.SI Nuclease ProtectIon Assays. The probe was a 254-bp AccI re-

striction fragment from pBSF that includes sequences from exons 1 , 2,

and 3 of mdm2 as well as 27 bp from the plasmid vector at the 5’ end.Twenty �L9 of total RNA were analyzed in each reaction. End-labeling,

hybridization, and Auclease treatment were performed as described bySambrook at al. (70) with a hybridization temperature of 4&C. The pro-tected products were separated on a 5% denaturing polyacrylamide gel.

For controls, the probe was digested after annealing to RNAS transcribed

in vitro from pGEM1F and pGEM1X2 (Stratagene).Total RNA Synthesis. RNA synthesis was measured 0, 2, 5, 8, and

1 0 h after exposure to a UV dose of 4 or 20 J/m2. Cells were incubated

with 10 �tCVmI rH]uridine(29 CVmmol; Arnersham)for 1 h after treatment,

washed twice in PBS, and lysed in SDS. After reduction ofviscosity of the

samples by passage 10 times through an 18-gauge needle, duplicate

samples were spotted onto Whatman 3 MM paper and precipitated withtrichloroacetic acid as described previously (36). As a control for cell loss,the cells were prelabeled with 0.02 �xCi/mI[14C]thymidine(56.9 mCVmmol;Arnersham) for 24 h before exposure to UV light; the results indicated no

loss of cells during the time course of this assay.IndIrect Immunofluorescence. Cl 27 cells were grown on coverslips,

irradiated, and then incubated for the times indicated. The coverslips were

then rinsed in PBS and fixed for 10 mm in 100% methanol at roomtemperature. Coverslips were rinsed with PBSBT, incubated for 1 h in thepresence or absence of a polyclonal Ab specific for p53 (CM5), and then

washed three times for 5 mm each with PBSBT. The coverslips were thenincubated for 45 mm with a FITC-conjugated goat antirabbit IgG (VectorLaboratories). After extensive washing with PBSBT, the coverslips were

mounted on slides and viewed and photographed with a Zeiss Axiophotphotomicroscope.

EMSA. Nuclear extracts were prepared, and binding assays wereconducted as described by Price and Calderwood (47) with the followingmodifications: the binding assays were performed with 0.2 �g of Ab and1 0 �g of nuclear extract and incubated at room temperature for 30 mm. An

oligonucleotide identical to the consensus binding site for p53 defined byEl-Dairy et al. (Ref. 72; S’-GCTGGGCATGCCCGGGCATGCCCGGC-3’)was end-labeled with [�y-32P1ATP, and 1 ng of the radiolabeled oligonu-cleotide (3 x 1o� cpm) was incubated with each sample. Protein-DNAcomplexes were separated on a 5% nondenaturing polyacrylamide gel.Loading dye was added only to the sample containing probe alone.

Mckay Assay. C127 cells were lysed by the same method used forimmunoprecipitation, aliquoted, and stored at -80#{176}C.Binding reactionswere based on the method of McKay (44) and contained two fragments

from the mdm2 gene. The larger fragment contained sequences fromupstream of the two REs for p53 (nonspecific probe), and the othercontained sequences that include the two REs for p53 (specific probe). To

generate these fragments, a 610-bp BglIl-AvaII fragment from plasmid

CosxlCat (7) was digested with Hincll. The resulting fragments, a 450-bpBg!lI-Hincll fragment and a 160-bp HincII-Avall fragment, were treatedwith calf intestinal phosphatase (Boehringer Mannheim) and end-labeledwith T4 polynudleotide kinase (New England Biolabs). The i60-bp frag-ment contains both 20-bp REs for p53, whereas the 450-bp fragmentcontains no sequences recognized specifically by p53 (7). The immuno-

precipitation reaction was modified from Kern et al. (73) to contain: 2 x

io� cpm of 32P-Iabeled probe, 20 �g of lysate, 1.6 g�g of CM5 An, 60 �dof binding buffer, and sufficient lysis buffer to bring the total volume to 75

�tl. Binding reactions were then incubated with protein A-Sepharose (5mg-ma)and poly(deoxymnosinic-deoxycytidylic acid)(Pharrnacia)as described

by Kern et a!. (73). DNA was purified from proteins by digestion with 50�ig/ml proteinase K (Boebringer Mannheim) in 10 m� Tris (pH 7.8), 5 m�EDTA, and 0.5% SDS, followed by extraction with phenol and chloroformand precipitation with ethanol. The labeled DNA fragments were sepa-rated on a 5% nondenaturing polyacrylamide gel and quantified on aPhosphorimager.

Statistical Analysis. Data are expressed as means ± SE. The un-paired Student t test was used to determine the significance of differencesbetween data.

AcknowledgmentsWe thank Donna George and Moshe Oren for providing plasmids andArnie Levine for providing Abs. The encouragement of Moshe Oren and

Arnie Levine is appreciated, and we are grateful to Bill Sugden and PaulLambert for advice, stimulating discussions, and critical comments on the

manuscript.

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