Proc. Nati. Acad. Sci. USAVol. 91, pp. 3602-3606, April 1994Biochemistry

p53 and E2F-1 cooperate to mediate apoptosis(programmed cell death/transcription factor/tumor suppressor)

XIANGWEI WU AND ARNOLD J. LEVINE*Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014

Contributed by Arnold J. Levine, January 5, 1994

ABSTRACT The tumor-suppressor protein p53 appears tofunction at the G, phase of the cell cycle as a checkpoint inresponse to DNA damage. Mutations in the p53 gene lead to anincreased rate of genomic instability and tumorigenesis. TheE2F-1 transcription factor is a protein partner of the retino-blastoma-susceptibility gene product, RB. E2F-1 appears tofunction as a positive regulator or signal for entry into S phase.To explore possible interactions of p53 and E2F-1 in the cellcycle, a human E2F-1 expression plasmid was introduced intoa murine cell line containing a temperature-sensitive p53 allelewhich produces a p53 protein in the wild-type conformation at3rC and the mutant form at 37.5rC. Coexpression of thewild-type p53 protein and E2F-1 in these cells resulted in arapid loss of cell viability through a process of apoptosis. Thus,the cell cycle utilizes an interacting or pathwaybetween RB-E2F-1 and p53.

The p53 protein can function as a transcription factor regu-lating some genes in a positive fashion (1, 2) by interactingwith specific DNA elements (3, 4) or negatively regulatingother genes that do not have those DNA elements (5). Thelevels ofp53 protein increase in response to DNA damage (6,7) and the enhanced p53 levels can result in inhibition of celldivision (8, 9) or the killing of cells through a process ofprogrammed cell death, or apoptosis (10-15). It is thoughtthat the p53 protein may act as a checkpoint control in the cellcycle to permit the repair of damaged DNA by holding thecells in G1 (1) or to commit cells to a pathway of apoptosiswith some cell types such as thymocytes (12, 13). In additionto DNA damage, other factors that impact upon events in thecell cycle can signal the p53 protein to mediate apoptosis. Theadenovirus EMA oncogene can initiate foci formation whenexpressed in primary rat kidney cells, but most of the cellssubsequently degenerate and die (14). The cell death in thisprocess is caused by apoptosis and is accompanied byelevation of p53 protein levels due to stabilization of theprotein (15). The adenovirus E1B-19kDa protein, the humanBcl-2 protein, or mutant p53 proteins acting in a transdom-inant fashion can block apoptosis mediated by ElA in thesecells (14, 16). Thus it appears that the wild-type p53 proteinmediates or is in the pathway of ElA oncogene-inducedapoptosis.The ElA oncogene encodes a protein that binds to the

retinoblastoma-susceptibility gene product, RB (17), result-ing in liberation of an active E2F transcription factor from anRB-E2F complex (18). The E2F transcription factor can thenactivate the expression of a set of genes involved in thesynthesis ofDNA precursors, in DNA replication, and in theS phase of the cell cycle (19). The expression of ElA in cellsalso leads to increased p53 protein levels (15). Thus it appearsthat EMA could induce two conflicting signals in cells. One isa growth-promoting signal provided by activated E2F tran-scription factors, and the other is a p53-mediated block in

progression through G1. Furthermore, it is possible that suchevents could lead to p53-mediated apoptosis in these cells. Totest this hypothesis, E2F-1 and p53 proteins were coex-pressed in two different cell types. Expression of E2F-1overcame the p53-mediated cell growth arrest and led to celldeath through apoptosis.

MATERIALS AND METHODSCell Culture and Transfection. Cells were maintained in

Dulbecco's modified Eagle's medium containing 10%6 fetalbovine serum. DNA transfection was performed by a calciumphosphate precipitation procedure (2). One-tenth amount(molar) of pGK-HyG plasmid, which contains a selectablemarker for hygromycin B resistance, was cotransfected. Thecells were selected in medium containing hygromycin B (250units/ml; Calbiochem) for 2-3 weeks and individual cloneswere isolated. Cell viability was measured on duplicatedsamples by trypan blue exclusion.

Protein andDNA Analysis. Cells were metabolically labeledwith r5S]-methionine and cell extracts were immunoprecip-itated as described (9). DNA was extracted from the cells andDNA fragmentation was analyzed by electrophoresis in 1%agarose gel (14).

Gel Shift Analysis. E2F gel shift assays were performed asdescribed (20). The sequences of the oligodeoxynucleotidesused are as follows: E2F wild type, 5'-ATTTAAGTTTCG-CGCCCTTTCTCAA-3'; E2F mutant, 5'-ATTTAAGTTTC-GATCCCTTTCTCAA-3'.Flow Cytofluorimetry. Cells were incubated at 37.50C (mu-

tant p53) or 320C (wild-type p53) for 24 hr. 5-Bromo-2'-deoxyuridine (BrdUrd) was incorporated into the DNA ofthese cells for 30 min to measure the rate ofDNA synthesis.The isolated nuclei from these cells were incubated with afluorescein-conjugated antibody that binds to BrdUrd in theDNA (Becton Dickinson). Propidium iodide was added to thenuclei and its fluorescence was employed to measure DNAcontent per nucleus.

RESULTSExpression of p53 and E2F-1 in (10.1)ValS Cells. The mouse

embryo fibroblast line (10.1)ValS contains a temperature-sensitive p53 mutant allele (and no endogenous p53 gene) (2,8, 9, 21). These cells (in the absence ofadded E2F-1 plasmid)grow exponentially at 37.50C and express a mutant p53protein, but they produce a wild-type-like p53 protein at 320Cand, because of this, they are growth-arrested in the G1 phaseof the cell cycle (2, 8, 9). The block of cell division at 320C iscompletely reversible, with >85-90%o of the cells remainingviable for >1 week at this temperature. Overexpression ofthe p53 wild-type protein at 320C activates transcription,through a p53-responsive DNA element, in several differentgenes (2). An E2F-1 cDNA expression clone (CMV-RBAP-1)

Abbreviation: CAT, chloramphenicol acetyltransferase.*To whom reprint requests should be addressed.

3602

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Proc. Natl. Acad. Sci. USA 91 (1994) 3603

(20) was introduced into the (10.1)Val5 cell line at 37.50Calong with a selectable marker for hygromycin B resistance.Drug-resistant clones of the (10.1)Val5 cells were then iso-lated and analyzed for p53 protein and E2F-1 activity.

p53 protein levels were determined by immunoprecipita-tion of soluble proteins extracted from cells labeled with[35S]-methionine at 37.50C (9). Three p53-specific monoclonalantibodies were employed: PAb421, which recognizes bothwild-type and mutant p53 proteins; PAb240, which reactswith only the mutant p53 protein; and PAb246, which bindsto the wild-type but not the mutant p53 protein (9). Theimmunoprecipitates were electrophoresed in an SDS/poly-acrylamide gel and an autoradiogram of such a gel is shownin Fig. 1A. The parental (10.1)Val5 cells with only the p53plasmid and three independent clones of cells (Cli, C12, andC18) with both p53 and E2F-1 are shown. In all four cell lines,the amount of mutant p53 protein in cells kept at 37.50C wasmuch higher than the amount of wild-type protein synthe-sized at that temperature. At 320C the wild-type protein wasin excess to the mutant p53 protein as determined by usingmonoclonal antibodies specific for the p53 mutant (PAb240)or wild-type (PAb246) protein (X.W., unpublished work).The expression of E2F-1 protein in these cell lines was

determined in two ways. First, the soluble proteins from thenuclei of the (10.1)Val5, C11, C12, and C18 cells were incu-bated with an oligonucleotide containing an E2F-1 binding site(22). A gel shift assay was carried out to test for binding of theE2F-1 protein to the oligonucleotide (Fig. 1B). In cell linesC11, C12, and C18, a specific factor that bound to the E2Foligonucleotide sequence was detected (Fig. 1B, lanes 4, 7,and 10), but little or no E2F-specific activity was observed inthe parental (10. 1)Val5 cells (lane 1). Unlabeled wild-type E2Foligonucleotide in 100-fold molar excess effectively competedwith the labeled probe (Fig. 1B, lanes 5, 8, and 11), whereasan equal amount of a mutant E2F oligonucleotide failed tocompete in this assay (lanes 6, 9, and 12). The parental(10.1)Val5 cell thus appears to have little or no E2F-1 proteinactivity as determined by this assay, whereas the C11, C12,and C18 clones express readily detectable E2F-1 protein.Next, the presence ofE2F-1 protein was tested in these cell

lines by a transcriptional transactivation assay (22). A DNAreporter construct containing a CAT gene regulated by apromoter element and an E2F binding site (22) was trans-fected into the (10.1)ValS, Ci, C12, and C18 cell lines at37.5°C. Forty-eight hours after transfection, cell extractswere prepared and assayed for CAT enzyme activity. Theresult of several experiments (averaged) are presented in Fig.1C. Depending upon the cell line, the Cll, C12, and C18 cellshad 2.5- to 3.5-fold more E2F transcriptional activity than theparental (10. 1)Val5 cell line. This is the expected increase inE2F activity, found previously after expression of this cDNAclone in cells (23).

Coexpression of Wild-Type p53 with E2F-1 Results in aRapid Loss of Cell Viability. The three cell lines expressingmutant p53 and E2F-1 displayed little or no difference in cellmorphology (Fig. 2 A and C) or growth rate (Fig. 3A) at37.5°C when compared with the parental (10.1)ValS cells.However, when the C11, C12, and C18 cell lines were shiftedto 32°C and wild-type p53 protein was expressed, a loss ofcell-cell contact, cell membrane blebbing, cytoplasmic con-densation, and nuclear alterations were evident within 18 hr(Fig. 2D). No such alterations could be detected with theparental cells (10.1)Val5 kept at 32°C (Fig. 2B), where theywere growth-arrested and their viability remained high for 96hr (Fig. 3B). By contrast, the viability of the C11, C12, andC18 cells, as measured by trypan blue exclusion, declinedover that 96 hr period to <5-15% of the parental (10.1)Val5cells with little or no E2F-1 (Fig. 3B).When the E2F-1 cDNA clone was expressed in the (10)1

cells [the parental cell line to (10.1)Val5 which contains no

A

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FIG. 1. Expression of p53 and human E2F-1 in (10.1)Val5 cells.(A) Immunoprecipitation of p53. The parental cell line (10.1)Val5 andthree clones of cells containing E2F-1--C11, C12, and C18-weremetabolically labeled with [35S]-methionine for 2.5 hr at 37.50C. Thecell extracts were immunoprecipitated with three monoclonal anti-bodies: PAb421, which recognizes both the wild-type and the mutantp53 (lanes 2, 5, 8, and 11); PAb240, which is specific for the mutantp53 (lanes 3, 6, 9, and 12); and PAb246, which reacts only with thewild-type p53 (lanes 4, 7, 10, and 13). Lane 1, control using PAb419,an antibody against simian virus 40 large tumor antigen. (B) Gel shiftanalysis for E2F-1 expression. 32P-end-labeled wild-type E2F oligo-nucleotide was used as a probe. Competition was performed with nocompetitor oligonucleotide (lanes 1, 4, 7, and 10) or 100-fold molarexcess of wild-type (lanes 2, 5, 8, and 11) or mutant (lanes 3, 6, 9, and12) E2F oligonucleotide. Arrowhead indicates the specific complexdetected. (C) E2F-1 transcriptional activity. An E2F reporter con-

struct, E2CAT, which contains an E2F binding site and a chloram-phenicol acetyltransferase (CAT) reporter gene (20), was transfectedinto the (10.1)ValS cells and clones C1l, C12, and C18. CAT activitywas analyzed 48 hr later. These data represent the average of threeindependent experiments.

p53 protein (24)], these cells did not show a loss of viabilityat 37.5OC or 320C (X.W., unpublished work). This indicatesthat the cell death observed in C11, C12, and C18 cells resultsfrom the interaction of wild-type p53 with E2F-1.The Cell Death Is Caused by Apoptosis. The morphological

changes of the C1l, C12, and C18 cells at 320( resembledthose described in the process of apoptosis. To test for anadditional indication ofprogrammed cell death, the DNA was

isolated from cells kept at 37.50C or 320( and analyzed byelectrophoresis through an agarose gel. The DNA fromE2F-expressing clones Cli, C12, and C18 kept at 320( was

fragmented in the typical nucleosome spacing ladder indic-ative of apoptosis (Fig. 4). On the other hand, the DNA from

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3604 Biochemistry: Wu and Levine Proc. Nati. Acad. Sci. USA 91 (1994)

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FIG. 2. Morphology of the cells. The cells were grown at 37.50C and passaged into duplicate plates. One plate remained at 37.50C and theother plate was transferred to 320C. These pictures were taken 3 days later. Parental (10.1)Val5 cells grow at 37.50C (A) but arrest at 320C (B).The E2F-expressing cells are shown after incubation at 37.50C (C) and 32rC (D).

(10.1)Val5 cells at 320C or 37.50C or the DNA from C1l, C12,and C18 cells at 37.50C showed little or no indication of suchDNA fragmentation.These observations have been repeated with a rat embryo

fibroblast line expressing this same p53 temperature-sensi-tive allele (25) along with the E2F-1 cDNA clone (X.W.,unpublished work). Thus, these results are not specific to thecell line (10.1)Val5 but appear to be of more generil signif-icance.

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E2F-1 Can Overcome p53-Mediated Growth Arrest. Theexpression ofE2F in these cells should signal for the entry ofcells into S phase, whereas wild-type p53 at high levels blockscells in late G1 (8, 9). To follow the events in the cell cycle inthese cells at 320C or 37.50C, the parental cell line with atemperature-sensitive p53 gene [(10.1)Val5] or the derivedcells with the E2F clone (C18) were kept at 37.50C or 320C for24 hr and incubated with BrdUrd to measure DNA synthesisin the cells. The nuclei from these cells were isolated andincubated with a fluorescent antibody directed against Brd-Urd incorporated in the DNA. A fluorescence-activated cellsorter was employed to analyze the nuclei for DNA content(using propidium iodide fluorescence) and the level of Brd-Urd incorporation to measure recentDNA synthesis (Fig. 5).

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FIG. 3. Cell growth curve. Cells were grown in duplicate platesat either 37.50C (A) or 320C (B) and cell viability was assayed bytrypan blue exclusion. Each point is the average of duplicate plates.

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FIG. 4. DNA fragmentation. Floating cells from cells grown ateither 37.50C (lanes a) or 32TC (lanes b) were collected 18 hr after thetemperature shift. DNA was extracted and samples were run in a 1%agarose gel. Lane M, markers (1-kb ladder, GIBCO/BRL).

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Proc. Natl. Acad. Sci. USA 91 (1994) 3605

(10.1) Val5

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FIG. 5. Flow cytofluorimetric analysis of (10.1)ValS and C18 cells. Parental (10.1)Val5 cells or E2F-expressing C18 cells were measured forDNA synthesis and cell cycle analysis. The level of DNA synthesis (BrdUrd incorporation) is measured in windows 2 and 3 in each graph,whereas G1 (window 1) or G2 (window 2) cells are followed by their DNA content (propidium iodide fluorescence). The percentage of cells ineach window is as follows: (10.1)ValS at 37.50C (1, 54.8%; 2, 4.8%; 3, 21.8%; 4, 18.4%); (10.1)ValS at 320C (1, 81.6%; 2, 0.6%; 3, 3.1%; 4, 14.5%);C18 at 37.5-C (1, 46.4%; 2, 15.1%; 3, 27.90%; 4, 10.4%); and C18 at 320C (1, 72.9%o; 2, 9.6%6; 3, 11.5%; 4, 5.8%). Data from representative samplesare shown.

At 37.50C, (10.1)Val5 cells in G1, S, and G2 were detected,with the S-phase cells containing most of the BrdUrd incor-porated (windows 2 and 3 in Fig. 5). In (10.1)Val5 cells at320C, most cells are in G1 (window 1) and very little BrdUrdincorporation is detected. In the C18 cells at 32°C withwild-type p53 and E2F, a significant increase in cells enteringS phase and incorporating BrdUrd (windows 2 and 3) wasobserved relative to cells'without the E2F factor. Thus thebiological function of E2F-1 in these cells is to overcome thewild-type p53 G1 block (with time, more C18 cells enter Sphase), but the conflicting nature of these two signals (p53and E2F) in the cell cycle results in a program of apoptosis.Expression of E2F-1 can induce quiescent cells to enter Sphase (23). The ability of E2F in overcoming the p53-mediated growth arrest indicates that E2F transcription fac-tor plays a general and important role in entrance intoS-phase. It is not clear whether the C18 cells must enter Sphase with a wild-type p53 to initiate apoptosis or whether theconflicting signals could result in cell death in the absence ofDNA replication.

DISCUSSIONCoexpression of E2F-1 and wild-type p53 leads to cell

death through apoptosis. These results help to explain anumber of observations reported in the literature over thepast 14 years. The DNA tumor viruses simian virus 40,adenovirus, and human papilloma virus (17, 26) all encode aseries of oncogene products that bind to the RB protein andliberate an active E2F transcription factor (18, 27). As aresult, these viral oncogene products signal the cell to enterthe replicative phase of the cell cycle (18). A second proteinor function encoded by each of these viruses has been shown

to bind to the p53 protein (28-32) and inactivate its functionas a transcription factor (33, 34). It was thought that thissecond function may inhibit the tumor-suppressor propertiesofp53 and facilitate viral transformation. Since activated E2Fand wild-type p53 cooperate to mediate apoptosis, it couldwell be that the important function of binding to the p53protein by the viral oncogene products is to block p53-mediated apoptosis as shown with the adenovirus ElA geneproduct (14). In a second example of this, it is possible thatthe extensive cell death in the central nervous system ofmicewith homozygous insertion mutations in both the RB alleles(the so-called knockout mice for RB) could be due to apop-tosis mediated by E2F-1 and p53 (35-37). This hypothesis,however, does not explain all the facts and observations.First, the adenovirus type 5 E1B 55-kDa protein binds to p53protein (31) and blocks its ability to act as a transcriptionfactor (34). However, it is the adenovirus E1B 19-kDa proteinthat most efficiently blocks the ElA/p53-mediated apoptosisin these cells (14), suggesting that the EMA 19-kDa proteinmust act upon the p53 protein or an event downstream of thep53 protein (in the program of apoptosis) in some differentmanner. Second, some viable cancerous cells contain thewild-type p53 protein and mutant RB alleles which are thenexpected to liberate E2F. Whether there are alternativepathways to turn off p53-mediated apoptosis in such cells orother mechanisms involved remains to be seen.

In summary, the results reported here demonstrate thatexpression of transcription factor E2F-1 overcomes the p53G1 growth arrest, but the conflicting signals result in activa-tion of an apoptotic pathway. Thus, two important tumor-suppressor gene products, RB and p53, can communicatewith each other in checkpoint control of the cell cycle. Themolecular mechanisms that mediate this communication re-main to be elucidated.

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3606 Biochemistry: Wu and Levine

We acknowledge Drs. W. G. Kaelin, Jr., and D. M. Livingston forproviding the E2F-1 expression plasmid CMV-RBAP-1 and Drs.M. E. Perry, J. A. Jerome, and D. Dittmer for their help in flowcytofluorimetry. We thank Drs. M. E. Perry, J. Lin, C. A. Finlay,and R. Quartin for helpful discussion. We thank M. E. Perry forcritical reading of the manuscript and K. James for help in prepa-ration of the manuscript. This research is supported by grants fromMerck (X.W.) and the National Institutes of Health (A.J.L.).

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