abrogation of wild-type p53-mediated transactivation is … · abrogation of wild-type p53-mediated...

[CANCKR RESKARCH 57. 5584-5589. December 15. 1997]

Abrogation of Wild-type p53-mediated Transactivation is Insufficient for Mutantp53-induced Immortalization of Normal Human Mammary Epithelial Cells1

Yu-an Cao,2 Qingshen Gao,2 David E. Wazer, and Vimla Band3

Department iif Radiatimi Oncology, Divisimi tif Radiation and Cancer Biology. Neu- England Medical Center Â¡Y-A.C.. Q. G.. D. E. W.. V. BJ. and Department of Biochemistry,

Tufts University School of Medicine Â¡V.B.Â¡.Boston. Massachusetts 02111

ABSTRACT

The p53 protein has become a subject of intense interest since thediscovery that about 50% of human cancers carry pS3 mutations. Mutations in thep5J gene are the most frequent genetic lesions in breast cancer,suggesting a critical role lor p53 protein in normal mammary epithelialcell (MEO growth control. We previously demonstrated that abrogationof the p53 function by a cancer-derived p53 mutant. del239, was sufficient

to induce immortalization of normal MECs. To further extend thesefindings and to examine the mechanism of mutant p53-induced immor

talization of MECs, we tested the immortalizing ability of four selectedp53 mutants (R175H, R248W, R249S, and R273H), which involve residuesthat cluster close to N239 in the three-dimensional structure and whichare critical for the DMA-binding function of p53. Interestingly, two of

these mutants (R175H and R249S) reproducibly immortalized 76N normal MECs, whereas the other two mutants (R248W and R273H) inducedan extension of life span but not immortalization. These results furthersubstantiate that selective ablation of p53 function with dominant-nega

tive mutants is sufficient for immortalization of MECs. To determinewhether abrogation of the transactivation function of endogenous p53 wasimportant for the differential immortalizing ability of p53 mutants, wemeasured the effects of mutant p53 on the endogenous wild-type p53-

mediated transactivation of a chloramphenicol acetyltransferase reporterlinked to a consensus p53 binding DNA sequence in transiently transfected76N MECs. All of the mutants, regardless of their immortalizing pheno-type, abrogated the endogenous wild-type p53-mediated transactivation to

a similar extent. Thus, abrogation of transactivation function is not sufficient for mutant p53-induced immortalization of normal MECs. Thep53-immortalized MECs showed substantial telomerase activity: however,induction of telomerase activity occurred at late passages and was unde-tectable in mutant p53-expressing cells prior to immortalization. We

suggest that mechanisms other than abrogation of transactivation andinduction of telomerase activity determine the differential MEC-immor-

talizing behavior of various p53 mutants.

INTRODUCTION

Nearly all breast cancers arise by oncogenic transformation ofepithelial cells that line the ducts of mammary gland. Proliferation ofnormal epithelial cells is tightly controlled, and these cells divide fora finite life span and eventually senesce. An important early step inoncogenesis involves a loss of senescence, or immortalization.

A number of in vitro studies with human cells indicate that immortalization precedes complete transformation (1-4). In these studies,

exposure of primary cells to carcinogens or their transfection withvarious oncogenes led to immortalization but not to complete transformation (1-4). Subsequently, when other oncogenes, such as mutated ras or erbB2. were introduced into these immortal cells, coin-

Received 6/20/97; accepted 10/15/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by N1H Grants CA56803 and CA64823 and the Geyerr-oundation (to V. B.).

: The first two authors contributed equally to this work.1 To whom requests for reprints should be addressed, at Department of Radialion

Oncology. Division of Radiation and Cancer Biology. New England Medical Center. 750Washington Street, Boston. MA 02111. Phone: (617)636-4776; Fax: (6I7| 636-6205;E-mail: vband(o>opal.tufls.edu.

pÃ-etetransformation was observed, as revealed by further phenotypictransformation and/or tumor formation in nude mice (5-7).4 Addi

tional evidence for immortalization as an early step in oncogenesis isprovided by spontaneous immortalization of MECs5 that are derived

from benign lesions of breast (8, 9) or from cancer-prone Li-Fraumeni

syndrome patients ( 10). Interestingly, however, normal MECs derivedfrom reduction maminoplasty never exhibit spontaneous immortalization and always senesce in culture (11, 12).

In addition to these /'/; vitro studies, studies of local recurrence

support the hypothesis that breast cancer cells in vivo are also immortal (13. 14). The incidence of recurrence within the breast afterexcisional biopsy, axillary dissection, and primary radiation therapyincreases to 10-20% at 10 years after initial diagnosis. Careful

pathological examination of these tumors indicates that recurrencesoccur predominantly in the same quadrants as the primary tumor, andtheir histolÃ³gica! types and nuclear grades were similar, if not identical, in 95 and 93% of cases, respectively (13, 14). These studiesstrongly support the existence of immortal cells in the original tumorsthat give rise to later recurrences. Thus, both in vitro and in vivostudies support that immortalization is an important step in breasttumorigenesis. Elucidating the mechanisms of MEC immortalization,therefore, is an important goal of breast cancer cell biology.

Unlike rodent cells, human cells do not exhibit spontaneous transformation in culture (15). We demonstrated that the E6 gene of theHPV 16. naturally associated with cervical carcinomas and genitalwarts (16). was highly efficient and sufficient in inducing the immortalization of MECs (17, 18). In vitro analyses using rabbit reticulocytelysates had demonstrated that the HPV-16 E6 binds to p53 tumor

suppressor protein and induces its degradation (19. 20). Indeed, theE6-immortalized MECs showed a profound loss of the p53 proteindue to its enhanced degradation (18). Structure-function analyses ofthe HPV-16 E6 gene by mutagenesis established that degradation and

in vivo loss of p53 protein were required for the immortalization ofMECs (21). Furthermore, immortalization of normal MECs by -y-ra

diation also demonstrated that the loss of p53 protein was an earlyevent in MEC transformation (22).

These studies indicated that the loss of p53 function may represent anessential event in MEC oncogenesis, consistent with the high frequencyof mutations or loss of the p53 gene in breast cancer (23, 24). Recently,we and others have shown that the introduction of p53 mutants (del239or R273H) was sufficient to induce immortalization of MECs (2, 25).Interestingly, a number of other p53 missense mutants that were knownto have dominant-negative function in other cell types (26, 27) were

unable to induce immortalization, thereby providing a system to explorethe immortalization-related functions of p53.

The p53 protein functions as a sequence-specific DNA-binding protein

and. thereby, acts as a transcriptional activator (28). Following the exposure of cells to agents that induce DNA damage, the levels of p53 proteinincrease, resulting in the increased transcription of genes that carry ap53-binding site, such as GADD45, mdm-2, WAF1, cyclin G. Bax, and

4 Q. Gao and V. Band, unpublished data.'The abbreviations used are: MEC. mammary epithelial cell; HPV. human papillo-

mavirus; CAT, chloramphenicol acetyltransferase: mAb. monoclonal antibody; X-Gal.5-bromo-4-chloro-3-indolyl-ÃŸ-n-galactopyranoside.

5584

on July 9, 2021. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

TRANSACTIVATION AND MUTANT p53-INDUCED IMMORTALIZATION

insulin-like growth factor binding protein-3 (29-34). p53 protein has also

been shown to induce transcriptional repression, although this functionhas been mostly observed when p53 was overexpressed and does notappear to be mediated via sequence-specific DNA binding (35). It isthought that oncogenic p53 mutations either abrogate DNA-binding or

induce conformational changes that alter the transcriptional activationand/or repression functions of p53 (36). Indeed, the vast majority ofcancer-associated mutations reside within the DNA-binding domain of

p53 (35, 36). Furthermore, recent crystal structural analyses have demonstrated that the mutational hot spots correspond to residues within theDNA-binding domain that are critical for contacting the DNA or for thestability of the DNA-binding interface (35, 36). As p53 protein binds to

DNA as a tetramer, it is thought that mutant p53 proteins oligomerizewith the wild-type p53 and drive it in to a mutant, presumably inactive,

conformation, accounting for the dominant inhibitory phenotype of themutant proteins (37).

Here, on the basis of the location of earlier immortalizing mutationson or near the DNA-binding surface, we tested the immortalizing

ability of four hot spot p53 mutants (R175H, R248W, R249S, andR273H) that involve this surface. We show here that expression oftwo p53 mutants, R175H and R249S, reproducibly resulted in MECimmortalization, whereas R248W and R273H induced an extension oflife span. These results, together with our earlier results (2), demonstrate that a significant subset of cancer-associated mutations of p53 is

able to induce dominant immortalization of normal human MECs.Using transient transfection of immortalizing and nonimmortalizingmutants in normal MECs, we show that all mutants induce similarabrogation of the wild-type p53-mediated transactivation of a CATreporter linked to a consensus p53-binding site (28).

Recent studies have established a strong correlation between thetelomerase activity and cellular proliferation during oncogenesis.Nearly 90% of primary human malignancies are positive for theenzyme activity, whereas most normal tissues have either little or nodetectable activity (38). A potential role for deregulation of telomerase in mutant p53-induced MEC immortalization was suggested by astriking finding that introduction of the p53-degrading oncogeneHPV-16 E6 into primary cultures of keratinocytes and MECs tran

siently increased their telomerase activity (39). However, our resultsshow that induction of telomerase activity follows and does notprecede immortalization. Together, these analyses suggest that mechanisms other than alterations of transactivation function of endogenous p53 or induction of telomerase activity are critical for thedifferential immortalization of MECs by various p53 mutants.

MATERIALS AND METHODS

Cells and Cell Culture. Reduction mammoplasty-derived MEC strain 76Nwas grown in DFCI-1 medium, as described previously (12).

Transfection and Immortalization. Various mutant human p53 cDNAs(del239, R273L, N247I. V173L, C277F, G154V, and H179Q. obtained fromDr. Peter Howley, Harvard Medical School, Boston, MA; and V143A. R175H.R248W, R249S, and R273H, obtained from Dr. Bert Vogelstein, Johns Hopkins University School of Medicine. Baltimore. MD) were cloned into pCM-

Vneo vector (obtained from Dr. Bert Vogelstein) and transfected into 76N cells(8 /ng per IO6 cells in a 100-mm-diameter dish) using a calcium phosphate

coprecipitation method, as described previously ( 17, 18). After G418 selection(50 /j-g/ml for 10 days), cells were transferred to D2 medium (DFCI-I medium

that lacks PCS and bovine pituitary extract but is supplemented with 0.05%BSA) for selection of immortal phenotype (17, 18). Cells were passaged bysubculturing at a ratio from 1:3 to 1:10 in D2 medium.

Western Blot Analysis. Cell lysates were prepared in SDS-PAGE sample

buffer, and 50 Â¿igof each lysate (quantitated using a bicinchoninic acid proteinassay reagent kit: Pierce Chemical Co.) were resolved on 7.5% polyacrylamidegels and transferred to a polyvinylidene difluoride membrane (lmmobilon-P:

Millipore). Membranes were blocked in TBS-T [20 mM Tris base, 137 mM

sodium chloride (pH 7.6), and 0.1% Tween 2()| containing 5% nonfat dry milkand 5% BSA. Membranes were then incubated with a mAb against p53 (niAh1801; NeoMarkers Inc.), followed by goat antimouse antibody conjugated lohorseradish peroxidase (Pierce). Enhanced chemiluminescence detection wasperformed according to manufacturer's instructions (Amersham).

CAT Assays. PG,, CAT plasmid, in which 13 copies of a consensusp53-binding DNA sequence are linked to a CAT reporter gene, was obtainedfrom Dr. Bert Vogelstein (28). 76N cells (5 X 10") were plated in 100-mm-

diameter dishes for 72 h before transfection. pCMVneo vector or pCMVneocontaining the wild-type or mutant p53 cDNAs (2.5 ^tg per KXl-mm-diameter

dish) was cotransfected with PG,,-CAT (10 /j.g per 100-mm-diameter dish)using polyamine (TransIT-LTI reagent; PanVera Corporation). In initial ex

periments, the efficiency of transfection was established by cotranstection ofpcDNA3.l/His/LacZ plasmid (Invitrogen. San Diego. CA) with various p53constructs, followed by X-Gal staining using a protocol provided by themanufacturer (Invitrogen). About 1% of cells stained for X-Gal activity 48 h

after transfection in each case (data not shown). After 48 h of transfection.CAT activity was assayed according to manufacturer's protocol for the CAT

assay system (Promega) using 1-deoxy dichloroacetyl-l-'4C (Amersham Life

Sciences), as described previously (28). For CAT assays, equal amounts oftotal cellular protein (based on bicinchoninic acid protein assay reagent kit;Pierce) from lysates of cells transfected with different p53 mutants were used.

Telomerase Assay. Telomerase activity was determined using a commercially available TRAP EZE telomerase detection kit (Oncor; Ref. 40). Briefly,exponentially growing 76N cells or cells expressing various p53 mutants werelysed in lysis buffer provided with (he kit. Protein (0.5 ;ug) was added to acoupled telomerase-PCR with 5'-end-labeled telomerase-specific primers, according to the manufacturer's recommendation (Oncor). The reaction mixture

was run on 12.5% nondenaturing polyacrylamide gel to resolve the ladder oftelomerase products (with 6-bp increments), and the wet gel was used for

autoradiography.

RESULTS

Immortalization and Extension of Life Span of Normal MECsby Hot Spot p53 Mutants. Using a panel of eight p53 mutants, wehave previously shown that del239 p53 mutant was able to immortalize MECs, whereas all other mutants (R273L, N247I, V173L,C277F, G154V, H179Q, and V143A) were unable to do so (2). On thebasis of the location of the del239 mutation, close to critical residuesin the DNA-binding domain, we tested the MEC-immortalizing ability

of four p53 mutants (R175H, R248W, R249S. and R273H) thatinvolve this interface and correspond to mutational hot spots in p53found in human cancers. Mutant p53 cDNAs, cloned in pCMVneovector (28), were transfected into the 76N normal MEC strain, andG418-resistant cells were obtained. These were serially passaged at asplit ratio of 1:3 in D2 medium (see "Materials and Methods"), in

which normal MECs senesce within three passages, whereas immortalcells grow indefinitely (17, 18). An intermediate phenotype (extensionof life span) can also be observed, in which cells continue to growafter parental cells have senesced but continuous lines do not arise. Incomparison to vector-transfected cells, which senesced after three

passages in D2 medium, cells expressing R248W and R273H continued to grow for 10 passages and showed a long crisis period, followedby senescence in each of two experiments, indicating that thesemutants induced an extension of life span but did not immortalize 76NMECs (Table 1). Strikingly, 76N cells expressing R175H or R249Scontinued to grow for 10 passages and then showed a crisis period ofabout 2 weeks. After 2 weeks, cells exhibiting a transformed morphology were observed in each of two experiments. These cells weretrypsinized and passaged further. These cells have been in continuousculture in D2 medium for 50-70 passages, without any evidence of

senescence, indicating that these cells are immortal (Table 1). Thus,each of the four hot spot mutations that lie in the DNA-bindingdomain imparts an MEC growth-promoting ability on mutant p53.

5585



TRANSACTIVATION AND MUTANT pSMNDUCED IMMORTALIZATION

Table 1 /tntnorlttiiztition of 76N cellx hv p53 muittnt\ derived from human cancers

76N cells were transfected with pCMVneo vector or pCMVneo containing variousmutant pS3 cDNAs by a calcium phosphate iransfeetion method. Cells were selected inG4I8 and then passaged in D2 medium for selection of immortal cells. Cells werepassaged at 1:3 split ratio until passage 10 and at a 1:10 ratio afterwards.

p53mutantVector

RI75HR248W

R249SR273HNo.

of passages in D2medium forselection"3,3

70.''50''10,865.''

46''10.

9ImmortalizationNo

YesNo(extension of life span]

YesNo(extension of life span)

" Number of passages prior to senescence in two separate experiments.'' Cells are in continuous culture without any signs of senescence.

Together with our previous results, these data support a role for thealterations of the DNA-binding interface in mutant p53-induced MEC

immortalization.Expression of p53 Mutants in 76N Cells. To rule out the possi

bility that nonimmortulizing p53 mutants may have failed to expressa stable protein, we used Western blot analysis of lysates obtainedfrom 76N cells following mutant p53-transfection and G418 selection

but prior to selection for immortal phenotype. Similar to our previousfindings (2), all transtectants revealed an increase in the level of p53protein compared to parental 76N cells, which express much lowerlevels of wild-type p53. reflecting a substantial expression of the

transfected mutant p53 genes (Fig. 1). In addition, immunoperoxidasestaining using anti-p53 antibody mAb 1801 revealed intense p53

signals in mutant p53 transfectants compared to relatively low levelsof staining in parental cells (data not shown). Together, these resultsindicate that the inability of certain p53 mutants to induce MECimmortalization is not related to their inability to be expressed.

Transactivation of a Reporter Linked to a Consensus p53-

binding DNA Sequence Does Not Distinguish Immortalizing andNonimmortalizing p53 Mutants. One of the most important functions of the wild-type p53 that is presently known is its ability toinduce transactivation of a number of genes that bear a p53-binding

site in their promoter regions (36). In view of the clustering ofimmortalizing mutations near or on the DNA-binding interface, it

appeared plausible that immortalizing mutants may have a selectivealteration of the transactivation function in MECs. To investigate thispotential mechanism of mutant p53-induced MEC immortalization,

we transfected the immortalizing or nonimmortalizing p53 mutants innormal MECs. together with a reporter construct pG,,-CAT. pGLi-CAT plasmid contains 13 copies of a consensus p53-binding DNAsequence linked to a CAT reporter gene (28). Binding of a transcrip-tionally functional p53 to its cognate site in pG,,-CAT leads to CAT

gene transcription, which is measured as CAT activity. Plasmid DNAswere transfected into 76N cells, cell extracts were prepared after 48 h,and equal amounts of protein was assayed for CAT activity.

As expected from the expression of relatively high levels of wild-

type endogenous p53 in MECs, a substantial level of CAT activitywas observed in vector-transfected cells (Fig. 2). Transfection of anexogenous wild-type p53 led to a 2-3-fold increase in CAT activity

(Fig. 2). Surprisingly, transtection of the immortalizing p53 mutantsdel239, RI75H, and R249S, as well as several nonimmortalizingmutants, led to a comparable and essentially complete inhibition of theendogenous wild-type p53-induced pG,,CAT transactivation (Fig. 2).

The transfection efficiencies, as measured by cotransfection of theLacZ plasmid. followed by X-Gal activity, showed all constructs were

transfected at similar efficiencies (data not shown). Further analyses,using decreasing amounts of plasmid DNA for the transtection, didnot reveal any significant quantitative differences between immortalizing and nonimmortalizing mutants in their ability to abrogate PGL1-

CAT reporter transactivation (data not shown). These results indicate

that abrogation of the transactivation function of p53, as assessed byPG13-CAT reporter assay, is insufficient for MEC immortalization.

Induction of Telomerase Activity Is a Late Event in Immortalization of Normal MECs by p53 Mutants. It has been shown thatthe introduction of HPV-16 E6 gene into MECs induces telomeraseactivity (39). Because HPV-16 E6 induction of MEC immortalization

is accompanied by abrogation of p53 function (21 ), we considered thepossibility that immortalizing p53 mutants may selectively activatetelomerase at an early stage during immortalization. We, therefore,compared the telomerase activity of cells transfected with nonimmortalizing and immortalizing p53 mutants. The cells expressing nonimmortalizing mutants could only be assessed at early passages, whereascells expressing the immortalizing mutants were tested before, as wellas after, immortalization. As seen in Fig. 3, no telomerase activity wasdetected in negative controls (Lane I, negative control provided withthe kit; and Lane 4, heat-inactivated 76N cell lysate), whereas telom

erase activity was observed in the positive control cell lysates (Lane2, provided with the kit). When examined at the early passages, MECsexpressing del239 (Lane 5), R175H (Lane 7). and R249S (Lane 9) didnot show any detectable telomerase activity. However, cells expressing the immortalizing p53 mutants showed high levels of telomeraseactivity, when examined soon after crisis (when cells with transformed morphology could be observed), as well as at late passages.No detectable telomerase activity was observed in 76N cells expressing R248W and R273H p53 mutants, which only induce extension oflife span, when examined at passage 8 after transfection. In anadditional experiment, neither cells expressing the immortalizingdel239 mutant nor those expressing seven nonimmortalizing p53mutants reported previously (2) showed detectable telomerase activityat early passages (data not shown). Collectively, these data indicatethat the immortalizing p53 mutants do not induce the telomeraseactivity in MECs prior to selection for the immortal phenotype.However, once cells were fully immortalized, a clear increase intelomerase activity was seen, indicating that the induction of telomerase activity is a relatively late event.

DISCUSSION

The p53 protein has emerged as a key regulator of the cell cycle eventswhen cells are exposed to DNA-damaging agents (41,42). Cells that lack

a functional p53 protein exhibit an unstable genome, predisposing themto further genetic alterations en route to full oncogenic transformation(43). Consistent with this view, about 50% of human tumors contain p53mutations (35). Our previous studies with the HPV E6- and radiation-

DNA:

p53t

IM

Iv

Fig. I. Western blot analysis of mutant p53 proteins in 76N cells transfected withvarious p53 mutants. Normal 76N MECs were transfecled with either pCMVneovector or the indicated p53 mutants cloned in the same vector (8 ^tg each). Afterselection in G4I8. whole-cell lysates (50 /ng protein) of transfected cells wereimmunohlotled with anli-p53 antibody mAb 1801. Note that all mutant p53 transfec

tants express levels of p53 proteins that are significantly higher than the level ofendogenous wild-type p53 in the vector Iransfectant.

5586



TRANSACTIVATION AND MUTANT p5.VINDUCED IMMORTALIZATION

Bma,

DNA:Â»N

Z

rÂ»

S esZ

-nrÂ»

r-r-

S

&9ir*

XF00

DNA: z sr-

a

10

Oer.

OÃ¼LLO

LUÃ¼tuLUQ_

S-u. >t~- â€¢¿�*r- inCM T-

O O

Vector WT R175H R248W R249S R273H

Fig. 2. /l and H. inhibition of transactivation by p53 mutants in 76N cells. A and B. 76N cells (5 X 10*) were plated 72 h prior lo transleclion. Cells were transfected with pCMVneo

vector, alone or with same vector containing wild-type p53 or various mutant p53 (2.5 fig each) genes, together with pii,,CAT (III ng). using polyamine reagent according tomanufacturer's protocol (PanVera). Equal amounts of protein from extracts of cells transfected with various constructs were analyzed for CAT activity 4X h after transleclion. Top,

acetylated chloramphenicol. Note that all immortali/.ing and nonimmortali/ing p53 mutants abrogate endogenous wild-type p53-mediated transactivation in 76N cells. C and I), a

quantitative comparison of the relative transactivation of pG,,CAT in 76N cells transfected with various mutants. The acetylated chluramphenicol spots were quantified by densitometryof an auloradiogram. Columns (C and DÃ¬.means of three separate experiments carried out as in A and B. respectively: bars. SD.

immortalized MECs indicated a critical role for the loss of p53 functionin the immortalization of MECs (21, 22). More importantly, introductionof certain p53 mutants but not others was sufficient to induce theimmortalization of normal MECs (2, 25).

The vast majority of cancer-associated p53 mutations represent

missense mutations, primarily clustered around six hot spots (44).Recent crystal structural studies have shown that the mutational hotspots correspond to the residues that are most critical for the DNAbinding activity of the wild-type p53. and these mutations are expected to interfere with DNA binding and, thus, lead to a transcrip-

tionally nonfunctional p53 protein (45). Oligomerization of the mutantprotein with the wild-type protein drives the latter into a mutantnonfunctional conformation, resulting in a dominant-negative pheno-

type of the mutants (37).Although our initial studies used eight different mutants expected to

behave as dominant-negative forms, only one (del239) was able to

immortalize MECs. Here, we tested four hot spot mutants that lie in

or near the DNA-binding face of p53 protein for their ability to induce

MEC immortalization. We show that two of these (R175H andR249S) are able to induce immortalization, whereas two others(R273H and R248W) induced a partial growth promotion, as indicatedby a significant extension of life span. Another independent study byGollahon and Shay (25) showed immortalization of normal MECswith one p53 mutant. R273H. whereas the other three mutants(V143A. R175H. and R248W) failed to do so. Taken together, theseresults demonstrate that the selective abrogation of wild-type p53

function is sufficient for immortalization of MECs. Furthermore.MEC immortalization with several distinct p53 mutants supports thelikelihood that immortalization is a consequence of the loss of p53function rather than of unrelated secondary events.

Our present results differ from those of Gollahon and Shay (25) intwo respects. Whereas they showed MEC immortalization withR273H mutant but not with R175H, our analyses showed R175H to beimmortalizing and R273H to induce only an extension of life span.

5587



TRANSACTIVATION AND MUTANT pSMNDUCED IMMORTALIZATION

z

sinr-

â€¢¿�/-.asTT

3

-:Â«e

123456789101112

Fig. 3. Telomerase activity in 76N cells transfected with immortalizing or nonimmor-(alizing p53 nun,mi 76N cells were translccted with the indicated p53 mutants andselected in G4I8 selection. Cells were then shifted to D2 selection medium and analyzedfor telomerase activity as suggested by the manufacturer's protocol (Oncor). Cells were

analyzed at early passages (Â£)or upon full immortalization (/) as follows. Lanes I and 2.negative and positive control lysates, respectively provided in the kit: Laut- .1. 76N cells

grown in DFCI medium and used at passage 10; Lane 4. heat inactivated 76N lysate;Lanes 5 and 6. N239 del. E (passage Si and / (passage 50): Lanes 7 and A. R175H. Â£(passage 6) and / (passage 60): Lanes Vand IO. R249S. Â£(passage 7) and / (passage 60):Lane II. R248W, Â£(passage 8); Lane 12. R273H. Â£(passage 8).

We have confirmed the expression of only the R175H in our immortalcells, using PCR cloning and sequencing (data not shown). In addition, the R273H cDNA construct was confirmed by sequence analysis(data not shown). We have also confirmed the expression of R273Hmutant in immortal cells derived by Collation and Shay/' One possible

explanation for the discrepancy between the two studies is the use oftwo different normal MEC strains. In this regard, it is noteworthy thatearly passages of mammary tissue-derived cell strains contain distinct

MEC subpopulations with markedly different susceptibility to thep53-inactivuting oncogene HPV-16 E6 and the Rb-inactivating oncogene HPV-16 Â£7(1). A more detailed analysis using several distinct

MEC strains at early versus late passages will be required to furtheraddress this issue.

The availability of several immortalizing and nonimmortalizingp53 mutants provided an opportunity to address the potential mechanisms of MEC immortalization induced by the abrogation of p53function. The p53 protein is a transcription factor that transactivates anumber of known and unknown genes that possess a consensus p53binding site in the promoter region (35. 36, 44). Although the p53mutants used here have been shown to abrogate transactivation mediated by p53 in other cell types (26, 27), it appeared possible that

4L. S. Gollahon and V. Band, unpublished data.

immortalizing p53 mutants may selectively abrogate transactivationfunction of endogenous wild-type p53 in the milieu of the MECs used

for the assessment of immortalization. Alternatively, immortalizingmutants could produce a quantitatively more severe defect of trans-

activation. It has been demonstrated previously that a single p53mutant can function differently in two different cell types (46). We,therefore, introduced the immortalizing or nonimmortalizing p53 mutants into 76N cells and analyzed the ability of these mutants toabrogate the endogenous wild-type p53-mediated transactivation ofpG,,-CAT reporter containing 13 consensus p53-binding sites (28).Our data show that all mutants were able to abrogate the wild-typep53-mediated transactivation to a similar extent. Thus, although theabrogation of wild-type p53-mediated transactivation is likely to be

critical for immortalization, our results show that it is not sufficient.Consistent with our findings, another study has also reported thatabrogation of the transactivation function did not correlate with ratembryo fibroblast transformation with p53 mutants in conjunctionwith HPV-16 E7 and activated Ras (47). It remains possible that the

immortalizing p53 mutants may selectively abrogate the transactivation of certain specific genes that do not possess a consensus p53-

binding sequence. It is also possible that the consequence of the lossof p53 function on the transactivation of p53 target genes in vivo is notuniform, given that each gene is regulated by a combinatorial interplay of transcriptional regulatory proteins. Thus, immortalizing mutants may have a more profound effect on a particular target or set oftarget genes regulated by p53. Analyses of the transcriptional activityof individual p53 target genes in MECs made to express the immortalizing or nonimmortalizing p53 mutants will be required to addressthese possibilities.

p53 is also known to induce repression of a number of cellular genesthat lack a consensus DNA-binding site, although most of the targets have

been identified only under relatively nonphysiological conditions of p53overexpression (48). In initial analyses, using two distinct CAT reporterplasmids that contain the SV40 21-bp repeats and either the terminaltransferase gene "initiator" element plnr-CAT or the adenovirus majorlate promoter 'TATA box" pTATA-CAT (49-51), no correlation be

tween immortalizing ability and abrogation of p53-mediated repression

was observed in 76N cells (data not shown). It has been reported thatcertain p53 mutants exhibit a gain of function (52). For example, a p53mutant, W281G, induced an increase in soft agar growth when introduced into p53-null SAOS-2 cells (53). Further studies are needed toascertain if the MEC-immortalizing ability of a subset of p53 mutants

might reflect a gain of function.Recent studies have shown a close correlation between the induc

tion of telomerase activity and cellular proliferation during oncogen-esis (38). Notably, introduction of the p53-degrading oncogeneHPV-16 E6 into primary keratinocytes and MECs resulted in a tran

siently increased telomerase activity, suggesting a potential role forderegulation of telomerase in mutant p53-induced MEC immortaliza

tion (39). However, our analyses demonstrate that little telomeraseactivity was observed in mutant p53-expressing cells prior to immor

talization and that such activity was seen only after the crisis period,once transformed morphology was observed. Although we do notobserve a prolonged crisis period prior to immortalization, there is alag period of about a week or two for cells to exhibit immortalmorphology. Our results suggest that induction of telomerase activityis likely to be a consequence rather than a cause for immortalization.

In summary, the use of selected p53 mutants, corresponding tocancer-associated mutational hot spots, has allowed us to generalize

the conclusion that selective abrogation of the endogenous p53 function is sufficient to induce MEC immortalization. Surprisingly, abrogation of the transactivation function of the wild-type p53 in the

milieu of normal MECs is not sufficient for immortalization. The

5588



TRANSACTIVATION AND MUTANT pS.VINDUCED IMMORTALISATION

23.

present system provides an experimental tool to delineate the p53functions that are critical for maintenance of the untransformed phe-

notype of MECs. Abrogation of such functions may be a critical eventin breast cancer, in which p53 mutations are frequent. -4

ACKNOWLEDGMENTS

REFERENCES

25.

26.We thank Drs. Peter Howley (Harvard Medical School. Boston, MA). John

Minna (Simmons Cancer Center. Dallas, TX), and Bert Vogelstein (JohnsHopkins University School of Medicine, Baltimore. MD) for providing various 27constructs and Hamid Band (Harvard Medical School. Boston. MA) for critical

reading of the manuscript. 28.

29.

1. Wa/er, D. E.. Liu. X-L.. Chu. Q.. Gat). Q.. and Band. V. Immortali/ation of disiine!human mammary epithelial cell types hy human papilloma virus-16 E6 or E7. Proc.Nati. Acad. Sci. USA, 92: 3687-3691. 1995.

2. Gao. Q.. Hauser. S. H.. Liu, X-L.. Wazer. D. E.. Madoc-Jones. H.. and Band. V.Mutant p53-induccd immortalization of primary human mammary epithelial cells.Cancer Res.. 56: 3129-3133, 1996.

3. Bartek. J.. Bartkova. I.. Kyprianou, N., Lalani. E. N.. Sta.skova. 7... Shearer. M,Chang. S.. and Taylor-Papadimitriou. J. Efficient Â¡mmortali/ation of luminal epithe

lial cells front human mammary gland hy introduction of simian virus 40 large tumorantigen with recombinant retroviru.s. Proc. Nati. Acad. Sci. USA. SS: 3520-3524,

1991.4. Stampfer. M. R.. and Bartley. J. C. Induction of transformation and continuous cell

lines from normal human mammary epithelial cells after exposure to hen/od;lpyrene.Proc. Nati. Acad. Sci. USA. 82: 2394-2398. 1985.

5. Clark. R.. Stampfer. M. R.. Milley. R.. O'Rourke. E., Walen, K. H.. Kriegler, M.,

Kopplin. J.. and McCormick. F. Transformation of human mammary epithelial cellsby oncogenic retroviruses. Cancer Res.. 48: 4689-4694. 1988.

6. Pierce. J. H.. Arnstein. P.. DiMarco. E.. Artrip. J.. Kraus. M. H.. Lonardo. F.. DiFiore. P. P.. and Aaronson. S. A. Oncogenic potential of erbB-2 in human mammaryepithelial cells. Oncogene, ft: 1189-1194, 1991.

7. Zhai. Y. F.. Beittenmiller. H.. Wang. B.. Gould. M. N.. Oakley. C., Esselman. W. J..and Welsch, C. W. Increased expression of specific protein tyrosine phosphatases inhuman breast epithelial cells neoplastically transformed by the neu oncogene. CancerRes.. 53: 2272-2278, 1993.

8. Soule. H. D.. Maloney. T. M.. Wolman. S. R.. Pelerson. W. D.. Brenz. R.. McGrath.C. M.. Russo. J.. Pauley. R. J., Jones. R. F.. and Brooks. S. C. Isolation andcharacteri/.ation of a spontaneously immonali/-ed human breast epithelial cell line.MCF-IO. Cancer Res.. 50: 6075-6086. 1990.

9. Briand. P.. Petersen, O. W.. and Van Deurs. B. A new diploid nontumorigenic humanbreast epithelial cell line isolated and propagated in chemically defined medium. InVitro Cell. Dev. Biol.. 23: 181-188, 1987.

10. Shay, J. W., Tomlinson, G., Piatys/ek. M. A., and Gollahon. L. S. Spontaneous invitro immortalization of breast epithelial cells from a patient with Li-Fraumenisyndrome. Mol. Cell. Biol.. 15: 425-432. 1995.

11. Hammond. S. 1... Ham. R. G.. and Stampfer, M. R. Serum-free growth of human

mammary epithelial cells: rapid clonal growth in defined medium and extended serialpassage with pituitary extract. Proc. Nail. Acad. Sci. USA, SI: 5435-5439. 1984.

12. Band. V.. and Sager. R. Distinctive traits of normal and tumor-derived humanmammary epithelial cells expressed in a medium that supports long-term growth ofboth cell types. Proc. Nati. Acad. Sci. USA. 86: 1249-1253. 1989.

13. Kennedy, M. J.. and Abeloff. M. D. Management of locally recurrent breast cancer.Cancer (Phila.). 71: 2395-2409. 1993.

14. Fisher, E. R., Anderson, S., Redmond. C.. and Fisher. B. Ipsilaterial breast tumorrecurrence and survival following luinpectomy and irradiation: pathological findingsfrom NSABP protocol B-06. Semin. Surg. Oncol., 8: 161-166. 1992.

15. Barrett. J. C. Comparison of human irrv/i.v rodent cell transformation: importance ofcell aging. In: i. S. Rhim and A. Dilischilo (eds.). Neoplaslic Transformation inHuman Cell Culture, pp. 3-13. New York: Humana Press. 1991.

16. Zur Hausen. H. Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus types. Curr. Top. Microbiol. Immunol.. 186: 131-156. 1994.

17. Band, V.. DeCaprio. J. A.. Delmolino. L.. Kulesa. V.. and Sager. R. Loss of p53protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J. Virol.. 65: 6671-6676, 1991.

IS. Band. V.. Dalai. S., Delmolino. L.. and Androphy. E. J. Enhanced degradation of p53protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.EMBO J., 12: 1847-1852. 1993.

19. Scheffner. M.. Werness. B. A.. Huibregtse. J. M.. Levine. A. J., and Howley. P. M.The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes thedegradation of P53. Cell, 63: 1129-1136, 1990.

20. Crook. T.. Tidy. J. A., and Vousden. K. H. Degradation of p53 can be targeted byHPV-E6 sequences distinct from those required for p53 binding and transactivation.Cell. 67: 547-556. 1991.

21. Dalai, S., Gao. Q.. Androphy. E. J., and Band. V. Mutational analysis of humanpapillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells. J. Virol., 70: 683-688. 1996.

22. Wazer. D. E., Chu. Q., Liu. X-L., Gao, Q., Safaii. H.. and Band, V. Loss of p53protein during radiation transformation of primary human mammary epithelial cells.

5589

30.

31.

32.

33.

34.

Mol. Cell. Biol.. l-l: 2468-2478. 1994.

Jego. N.. Thomas. G.. and Hamelin. R. Short direct repeats Hanking deletions, andduplicating insertions in p53 gene in human cancers. Oncogene, fi: 209-213.1993.Runnebaum. I. B.. Nagarajan. M.. Bowman. M.. Solo. D.. and Sukumar. S. Mutationsin p53 as potential molecular markers for human breast cancer. Proc. Nati. Acad. Sci.USA, 88: 10657-10661. 1991.

Gollahon. L. S.. and Shay. J. W. Immortalization of human mammary epithelial cellsiransfected with mutant p53 (273His). Oncogene. 12: 715-725. 1996.Baker. S. J.. Markowilz, S., Fearon, E. R., Willson. J. K. V.. and Vogelstein. B.Suppression of human colorectal carcinoma cell growth hy wild-type p53. Science(Washington DC). 249: 912-915. 1990.Chen. J-Y.. Funk. W. D.. Wright. W. E.. Shay. J. W., and Minna, J. D. Heterogeneity

of transcriplional activity of mutant p53 proteins and p53 DNA target sequences.Oncogene. 8: 2159-2166. 1993.Kern. S. E. p53: tumor suppression through control of the cell cycle. Gastroenterol-ogy. 106: 1708-1711, 1994.Kastan, M. B.. Zhan. Q.. EI-Deiry, W. S., Carrier, F., Jacks. T.. Walsh. W. V..Plunkelt. B. S.. Vogelstein. B., and Fornace. A. J.. Jr. A mammalian cell cyclecheckpoint utilizing p53 and GADD45 is defective in ataxia telangiectasia. Cell, 71:587-597. 1992.Momand. J . /.ambctti. G. P.. Olson. D. C.. George. D.. and Levine. A. J. The mtlm-2oncogene product forms a complex with the p53 protein and inhibits p53-mediatedtransactivation. Cell. 69: 1237-1245. 1992.EI-Deiry. W. S., Tokino. T., Velculescu. V. E.. Levy, D. B.. Parsons, R., Treni. J. M.,Lin, D.. Mercer. W. E.. Kinzler. K. W.. and Vogelstein. B. WAFI. a potentialmediator of p53 Iunior suppression. Cell, 75: 817-825, 1993.

Okamoto. K.. and Beach. D. Cyclin G is a Iranscriptional target of the p53 tumorsuppressor protein. EMBO J.. 13: 4816-4822. 1994.

Miyashita. T.. and Reed. J. C. Tumor suppressor p53 is a direct transcriptionalactivator of the human />53 is a

cell cycle checkpoint determinant following irradiation. Proc. Nail. Acad. Sci. USA,89: 7491-7495, 1992.

Paulovich. A. G., Toczyski, D. P., and Hartwell. !.. H. When checkpoints fail. Cell.8Â«:315-321, 1997.Prives. C. How loops, ÃŸsheets, and Â«helices help us to understand p53. Cell. 78:543-546. 1994.

Cho, Y.. Gorina, Sâ€žJeffrey, P. D.. and Pavletich, N. P. Crystal structure of a p53tumor suppressor-DNA complex: understanding tumorigenic mutations. Science(Washington DC), 265: 346-356, 1994.Forrester. K.. Lupold. S. E., Oil, V. L., Chay, C. H., Band, V., Wang, X. W., andHarris, C. C. Effects of p53 mulanls on wild-type p53-mediated Iransaclivalion arecell type dependent. Oncogene. IO: 2103-2111, 1995.Crook. T.. Marston. N. J.. Sara. E. A., and Vousden. K. H. Transcriptional activationby p53 correlates with suppression of growth but noi transformation. Cell. 79:817-828. 1994.Murphy. M.. Hinman. A., and Levine. A. J. Wild-type p53 negatively regulates theexpression of a microtubule-associated protein. Genes Dev., 10: 297I-29KO. 1996.Mack. D. H.. Vartikar. J.. Pipas. J. M.. and Laimonis. L. A. Specific repression ofTATA-medialed but not initiator-mediated transcription by wild-type p53. Nature(Lond.). 363: 281-283. 1993.Sabbatini. P.. Chiou, S-K.. Rao. L., and White. E. Modulation of p53-McdiatedIranscriptional repression and apoptosis by adenovirus E1B 19K prolein. Mol. Cell.Biol., 15: 1060-1070. 1995.

Mercer. W. E., Shields, M. T., Lin. D.. Appella. E., and Ullrich, S. J. Growthsuppression induced hy wild-type p53 protein is accompanied hy selective down-regulation of proliferating-cell nuclear antigen expression. Proc. Nati. Acad. Sci.USA, SÃ„:1958-1962. 1991.Dittmer, D.. Pali. S.. Zambetti, G., Chu, S.. Teresky. A. K.. Moore. M.. Finlay, C., andLevine. A. J. Gain of function mutations in p53. Nat. Genet., 4: 42-46, 1993.

Lin, J.. Teresky. A. K.. and Levine. A. J. Two critical hydrophobic amino acids in theN-terminal domain of the p53 protein are required for the gain of function phenotypesof human p53 mutants. Oncogene, IO: 2387-2390. 1995.



1997;57:5584-5589. Cancer Res Yu-an Cao, Qingshen Gao, David E. Wazer, et al. Human Mammary Epithelial CellsInsufficient for Mutant p53-induced Immortalization of Normal Abrogation of Wild-type p53-mediated Transactivation is

Updated version

http://cancerres.aacrjournals.org/content/57/24/5584

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/57/24/5584To request permission to re-use all or part of this article, use this link


http://cancerres.aacrjournals.org/content/57/24/5584http://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/57/24/5584http://cancerres.aacrjournals.org/

abrogation of wild-type p53-mediated transactivation is … · abrogation of wild-type p53-mediated...

Documents