differential expression of multiple mdm2 messenger...

Vol. 1, 71-80, Jantiar�’ 1995 Clinical Cancer Research 71

Differential Expression of Multiple MDM2 Messenger RNAs and

Proteins in Normal and Tumorigenic Breast Epithelial Cells

Jean M. Gudas,’ Hoang Nguyen,2

Regina C. Klein, Dai Katayose, Prem Seth,

and Kenneth H. Cowan

Medicine Branch, Division of Cancer Treatment, Medical Breast

Cancer Section, National Cancer Institute, Bethesda, Maryland 20892

ABSTRACT

The MDM2 gene is a nuclear phosphoprotein that is

regulated by p53 and functions, in one capacity, to inhibit

the transcriptional activity of the wild-type p53 protein.

Multiple MDM2 transcripts were detected in human breast

epithelial cells. In estrogen receptor-negative normal, im-

mortal, and tumorigenic breast epithelial cells, we found agood correlation between MDM2 mRNA levels and expres-

sion of wild-type p53. When wild-type p53 was overex-

pressed in estrogen receptor-negative tumor cells containing

a mutant or no endogenous p53, MDM2 mRNA levels in-creased significantly, indicating that wild-type p53 positively

influences MDM2 mRNA levels in these tumor cells. Because

all estrogen receptor-positive breast tumor cells had high

MDM2 mRNA levels regardless of the status of their endo-genous p53 protein, other factors likely influence MDM2

expression in these cells Distinct MDM2 proteins (range, Mr

54,000-68,000 and 90,000-100,000, respectively) were dif-

ferentially expressed in human breast epithelial cells. The

lower molecular weight MDM2 proteins were most abun-

dant in the normal mammary cells but present at varyinglevels in many of the tumor cells examined. MDM2 was a

nuclear protein; however, nuclear staining intensity did notalways correlate with the amount of MDM2-immunoreac-

tive protein as determined by Western blot analysis. This

discrepancy suggests that MDM2 interacts with novel cellu-

lar proteins in different kinds of breast epithelial cells.

INTRODUCTION

The human homologue of the mouse MDM2 gene encodes

a zinc finger-containing protein that is a putative transcription

factor (1, 2). Overexpression of this gene in BALB/c 3T3 cells

increases their tumonigenic potential, suggesting that the MDM2

protein possesses oncogenic activity. Further evidence that

MDM2 plays a role in the process of cell transformation derives

from experiments where this protein was shown to complex

with and inhibit the transcriptional activity of wild-type p53 (3,

Received 8/16/94; accepted 10/20/94.

I To whom requests for reprints should be addressed, at MedicineBranch, Division of Cancer Treatment, National Cancer Institute, Build-ing 10, Room 12N226, Bethesda, MD 20892.

2 Supported by a Cooperative Research Development Agreement pro-vided by the Centocor Corp., Malvern, PA.

4). MDM2 interacts with the amino terminal acidic activation

domain of the p53 protein, raising the possibility that MDM2

inhibits p53 function by disrupting its interaction with the gen-

enal transcription machinery (5).

Recent evidence indicates that MDM2 is one of the down-

stream effector genes in cells that express a wild-type p53

protein (6, 7). The first intron of the MDM2 gene contains a p53

DNA binding site. This element confers wild-type p53 respon-

sivity to the endogenous MDM2 gene and to reporter genes

when placed upstream in a hetenologous promoter (7). More

recent data suggest that this p53 response element may actually

be part of an internal, cryptic p53-dependent promoter (8).

When considered together, these observations suggest that neg-

ulation of p53 and MDM2 are intimately linked and that these

two proteins may act in concert to control cell proliferation.

Our laboratory has been interested in regulatory mecha-

nisms that play a role in the genesis of breast cancer. The

importance of p53 in this process is underscored by the finding

that -40% of primary human breast tumors harbor mutations in

the coding sequences of the p53 gene (9, 10). Moreover, recent

studies indicate that expression of mutant p53 is an important

prognostic marker for node-negative breast cancers (1 1, 12).

Presumably, the remaining tumors utilize other mechanisms to

bypass the negative regulatory effects of the wild-type p.53 gene.

One possibility is that these tumor cells can up-regulate the

expression of MDM2 to a level sufficient for binding and

titrating the effects of wild-type p53. Such a mechanism has

been proposed to explain the ovenexpression of MDM2 in soft

tissue sarcomas, osteosancomas, and a subset of malignant gli-

omas (13-17). Recently, Chen et a!. (18) reported that the

amplification of MDM2 in osteosancoma cells resulted in inhi-

bition of wild-type p53-induced growth arrest following treat-

ment with ionizing radiation. These results have provided im-

portant evidence demonstrating that MDM2 overexpression can

affect p53 function in a known physiological pathway. To date,

amplification and overexpression of MDM2 has not been shown

to play a significant role in the genesis of human epithelial

tumors (19-21).

To begin to understand the complex regulation between

these two cellular proteins, we examined levels of p53 and

MDM2 mRNA and protein in human breast epithelial cells. We

determined the sizes of the mRNAs and proteins for MDM2

present in mammary epithelial cells at different stages of cell

transformation. One goal of oun study was to determine whether

a relationship existed between p53 levels and corresponding

levels of MDM2 in mammary epithelial cells that expressed

wild-type versus mutant p53 proteins. Because MDM2 levels

were high in ER�3 breast cancer cells regardless of their p53

3 The abbreviations used are: ER, estrogen receptor; NMECs, normalhuman mammary epithelial cells.

Research. on May 27, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

72 MDM2 and Breast Cancer

4 D. Katayose, submitted for publication.

status, we examined the possible influence of estrogens on

MDM2 gene expression. Finally, to determine the intracellular

localization of these proteins, we performed immunocytochem-

ical analyses of MDM2 and p53 proteins in representative breast

epithelial cell lines.

MATERIALS AND METHODS

Cells and Cell Culture. NMECs derived from reduction

mammoplasties were purchased fron Clonetics Corp. (San Di-

ego, CA) and the Corniel Institute (Camden, NJ). The immor-

talized breast epithelial cell line MCF-10 was generously pro-

vided by Dr. Sam Brooks, Michigan Cancer Foundation (22).

Immortalized 184B5 and HBL100 cells were obtained from the

ATCC. The ER� cell line MCF-7 was obtained from Dr. R.

Buick, University of Toronto (23) and the ER� BT2OT cells

were provided by Dr. K. Keyomansi, New York State Depart-

ment of Health. Other ER� cells including T47-D, ZR7S-1, and

BT474 and the ER tumor cell lines MDA-MB-157, MDA-

MB-231, MD-MBA- 453, MD-MBA-468, HS578T, and SKBr3

were all obtained from the ATCC.

NMECs and 184B5 cells were cultured in a mammary

epithelial basal medium (Clonetics Corp.) supplemented with

vitamins, 0.5% fetal bovine serum, 20 ng/ml EGF, S jxg/ml

insulin, 0.5 p.g/ml hydnocortisone, and 52 p�g/ml bovine pitu-

itary extract (24). MCF-10 cells were cultured in Dulbecco’s

modified Eagle’s medium/F12 (GIBCO) supplemented with

2.5% horse serum, 10 mM HEPES, 2 msi glutamine, 0.1 m�t

nonessential amino acids, 20 ng/ml EGF, 10 pg/ml insulin, 0.5

�xWml hydnocortisone, and 10 p.g/ml transfennin. The ER� and

ER breast cancer cells were cultured in a-MEM (GIBCO)

supplemented with 10 msi HEPES, 2 msi glutamine, 0.1 mM

nonessential amino acids, 10% fetal bovine serum, 1 ng/ml

EGF, and 2 j.g/ml insulin with the exception of MDA-MB-468,

SKBr3, and BT474 cells. These cells were grown in improved

MEM without EGF and insulin was added to a final concentra-

tion of 5 �i.g/ml. All cells were routinely screened for Myco-

plasma contamination and maintained at 37#{176}Cin an atmosphere

of 6.5% CO2.

Adenovirus Infection of Breast Tumor Cells. An Ad-

enovinus vector containing a eDNA encoding the wild-type p53

gene (AdWTp53) was constructed according to established pro-

cedunes.4 MDA-MB-157 and MDA-MB-231 cells were plated

and allowed to grow for 1 or 2 days before infection with 10

plaque-forming units ofAdWTp53 or the same virus without the

p53 gene (AdCon). Cells were harvested for protein and RNA

analyses 24 h after infection.

RNA Isolation and Northern Blot Analysis. Total

RNA was prepared by Iysing cell monolayers in guanidinium

isothiocyanate and centrifuging over a 5.7 M CsCl cushion as

described previously (25). RNA (20 pig) was electrophoresed on

denaturing formaldehyde gels, transferred to MagnaNT mem-

branes, and cross-linked with UV. The probes used for Northern

blot analyses were obtained as follows. A 2.1-kilobase BamHI-

XhoI fragment, a 750-bp BamHI-Xba I, and an 800-bp HindIll

fragment were excised from different regions of the MDM2

eDNA (a generous gift from Dr. Bert Vogelstein; Ref 13). A

2.0-kilobase BamHI fragment from the human p53 eDNA was

purchased from ATCC and an 800-bp PstI fragment was excised

from p36B4 (26). All probes were labeled with [a-32P]dCTP on

dATP to a specific activity of - 1 X i0� cpm/pg DNA using a

random-primed labeling kit (Boehninger Mannheim).

Immunoprecipitation and Western Blot Analyses. Forimmunoprecipitation studies, cells were washed 3 times in ice-cold

PBS and harvested in lysis buffer (50 m�i Tnis-HC1, pH 8.0, 5 mrvi

EDTA, 150 mM NaC1, 0.75% NP4O, 1 mist phenylmethylsulfonyl

fluoride, S �j.�ml aprotinin, S �ig/ml leupeptin, 50 msi sodium

fluoride, 10 mM sodium o-vanadate). Equal amounts of protein

were subjected to immunoprecipitation by incubating with 1 �xg

IF2 monoclonal antibody (MDM2 antibody 1; Oncogene Science)

or 3 p.1 culture supernatant from 5B10-producing hybnidoma cells

(5). Specific complexes were collected following incubation with

Protein A + G agarose (Oncogene Science). After washing three

times in SNNTE buffer (5% sucrose; 0.5 M NaC1; 1% NP-40; 50

mM Tnis, pH 7.4; 5 mM EDTA) and twice with radioirnmunopre-

cipitation assay buffer, the precipitates were electrophonesed in 8%

SDS-polyacrylamide gels, transferred to nitrocellulose, and probed

for MDM2 as described below.

For Western blot analyses cells were washed three times

with ice-cold PBS, scraped into hot (80-90#{176}C) Laemmli lysis

buffer (62.5 mM Tnis-HC1, pH 6.8, 2 msi EDTA, 15% sucrose,

10% glycerol, 3% SDS, 0.7 M 2-mercaptoethanol) and the

proteins denatured by boiling for 10 mm. After centnifugation,

protein lysate (50 �iWlane) was electrophoresed on 8 or 10%

SDS/polyacnylamide gels and transferred to nitrocellulose mem-

bnanes. The filters were blocked with Tnis-buffered saline con-

taming 8% dried milk and 0.1% Tween 20 and then probed with

the respective antibody. MDM2 was detected using 2.5 p.g/ml

1F2 antibody (Oncogene Science), p53 was detected using 4

p.g/ml PAbs 1801 and DO-i antibodies (Oncogene Science) and

actin was detected with 3 p.g/ml actin antibody-i (Oncogene

Science). After washing in Tnis-buffered saline containing 0.1%

Tween 20, and subsequent incubation with horseradish peroxi-

dase-conjugated goat anti-mouse antibody (Bio-Rad), specific

complexes were detected using a chemiluminescent technique

according to the manufacturer’s recommendations (New En-

gland Nuclear).

Immunocytochemistry and Autoradiography. Cells were

grown in tissue culture chamber slides, washed three times in

ice-cold PBS, and immediately fixed in methanol:acetone (i:i) for

2 mm. After allowing the slides to air dry, they were stained for

MDM2 using the IF2 monoclonal antibody (MDM2 Ab-1; Onco-

gene Science) and PAb 1801 (Ab-2) (Oncogene Science) for de-

tection of p53. The MDM2 and p53 antibodies were diluted to a

final concentration of 4.5 �xg/ml or 1 �xg/ml, respectively. After

washing in Tnis-buffered saline containing 0.05% Tween 20, the

specific complexes were visualized using the avidin-biotin com-

plex immunoperoxidase system (Vector Laboratories, Burlingame,

CA) according to the manufacturer’s recommendations.

RESULTS

MDM2 mRNA Expression in Human Mammary Epi-

thelial Cells. In our initial experiments we sought to deter-

mine whether there were any differences in the expression of



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Clinical Cancer Research 73

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Fig. I Northern blot analysis of MDM2 and p53 RNA in human breast epithelial cells. The p53 status of the cells was obtained from several sources

and, if known, is indicated in parentheses (30, 31, 33, 36). RNA was isolated from three independently derived normal human mammary epithelialcells HMEC 1-3 (Wi’), immortalized, nontumonigenic HBL100, MCF-10 (Wi’) and 184B5 (Wi’) cells, estrogen receptor-positive MCF-7 (WT),ZR7S-1 (WT), T47-D (codon 194 mutation) and BT-474 (codon 285 mutation) tumor cells and estrogen receptor-negative MDA-MB-157 (null),

MDA-MB-231 (codon 280 mutation), MDA-MB-468 (codon 273 mutation), SKBn-3 (codon 175 mutation), HS578T (codon 157 mutation) breast

cancer cells, electrophonesed, transferred, and hybridized with probes for the MDM2, p53. and 36B4 genes, respectively. The lane designated SJSA-1contains RNA from an osteosarcoma line that has an amplification of the MDM2 gene. Arrows to the left of the top panel, sizes of the MDM2transcripts present in these cells. Single panel to the right, a shorter exposure of the lane containing RNA from the SJSA-1 cell line.

MDM2 in normal and immortal versus transformed human

mammary epithelial cells. For these experiments total RNA was

isolated from exponentially growing populations of NMECs

derived from reduction mammoplasties, immortalized but non-

tumonigenic mammary epithelial cells and estrogen receptor-

positive and -negative breast cancer cell lines. Normal and

immortalized, nontumonigenic breast cells grown in culture do

not express the estrogen receptor. This observation likely re-

fleets the biology of mammary epithelial cells as very few cells

are ER� in normal nonlactating mammary glands (27, 28). To

ascertain the estrogen receptor status of the various cells, we

probed the filter for ER mRNA (Fig.i, Panel 2). As expected,

only the ER� breast tumor cells had detectable levels of estro-

gen receptor mRNA.

Relatively high levels of MDM2 mRNA were present in all

three independently derived ER, NMEC strains (Lanes 1-3).

The amount of MDM2 mRNA detected in spontaneously arising

MCF1O (Lane 5) and carcinogen-induced i84B5 (Lane 6) ER,

immortalized, nontumonigenic cells was similar to the amount

present in NMECs. In contrast, HBL100 mammary epithelial

cells, that were immortalized by SV4O large T antigen, ex-

pressed a much lower level of MDM2 mRNA (Lane 4). This is

an important observation as SV4O T antigen binds to and mac-

tivates the function of wild-type p53. The four estrogen receptor

positive breast cancer cell lines MCF-7, ZR7S-1, T47-D and

BT474 (Lanes 7-10) expressed similar MDM2 mRNA levels to

those found in normal mammary epithelial cells. In contrast, the

five estrogen receptor negative breast cancer cell lines exam-

ined, MDA-MB-157, MDA-MB-231, MDA-MB-468, SKBr-3,

and HSS78T, all expressed MDM2 RNA levels that were S to iO

fold lower than in any of the other cells analyzed (Lanes Il-is).

Although all human mammary epithelial cells expressed readily

detectable MDM2 transcripts, the abundance of this mRNA was

well below the amount present in the overexpressing human

osteosarcoma cell line, SJSA-1 (16).

MDM2 mRNAs having several different molecular

weights were detected in our panel of mammary epithelial cells.

To determine the sizes of the various transcripts, identical




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Fig. 2 Immunoprecipitation of MDM2 protein from human breast epithelial cells. Protein extracts were prepared from representative breast epithelial

cells and immunoprecipitation was performed by incubating 2 mg protein extract with the lF2 (Oncogene Science) or 5B10 antibody at 4#{176}C.Specific

protein complexes were separated in an 8% SDS-polyacrylamide gel, electrophoretically transferred to a nitrocellulose membrane, and subsequently

probed for MDM2 using the lF2 antibody. Numbers, migration of prestained molecular weight markers that were coelectrophoresed and transferred

with the samples. The lane labeled Control contains 2 mg protein extract from MCF-7 cells precipitated with an isotypic nonspecific antibody. Arrows,

positions and intensities of the MDM2 proteins.

amounts of RNA standards were coeleetnophoresed and their

migration was compared with MDM2 mRNAs from mammary

epithelial and SJSA-l osteosarcoma cells. The major form of

MDM2 mRNA in human mammary epithelial cells migrated as

a 6.7-kilobase mRNA. We often saw higher molecular weight

transcripts of -8.0 and 12.5 kilobase in normal and immortal

mammary epithelial cells hut rarely in tumonigenie mammary

epithelial cells. In the MDM2 ovenexpressing SJSA-l cells,

another band of -4.5 kilobase could be readily detected. This

band was detected occassionally in RNA samples from all

mammary epithelial cells.

Since MDM2 is known to lie downstream of p53 in a

nuclear signal transduction pathway (6, 7), we examined the

relationship between p53 and MDM2 mRNA levels in the same

mammary epithelial cells. When the filter shown in the upper

panel was stripped and hybridized with a probe for p53, most of

the cell lines showed readily detectable levels of p53 mRNA

regardless of their state of transformation. The two exceptions

were ZR75-l cells which expressed significantly lower levels of

p53 mRNA and MDA-MB-157 cells which had no detectable

p53 mRNA at all. The different patterns of p53 and MDM2

mRNA expression observed among the mammary epithelial

cells could not be attributed to unequal loading of the lanes as all

cells expressed similar levels of the control p36B4 mRNA.

MDM2 Protein Levels in Mammary Epithelial Cells.

MDM2 is expressed as a series of phosphoproteins ranging in

molecular weight from 55,000 to 90,000 in munine 3T3DM cells

that contain multiple copies of the MDM2 gene (1, 29). To

determine the molecular weight(s) of the MDM2 proteins cx-

pressed in human mammary epithelial cells, immunoprecipita-

tion studies were performed using the IF2 and 5BlO antibodies

which recognize epitopes in the amino and carboxy terminal

regions of MDM2, respectively (5, 14). The results presented in

Fig. 2 demonstrate that the IF2 antibody recognized at least four

distinct forms of MDM2 in breast epithelial cells. The Mr 90,000

form of MDM2 was the predominant protein present in most of

the cell lines examined. Two additional MDM2 proteins with

molecular weights of around 75,000 and 85,000 were detected at

lower levels from all mammary epithelial and SJSA-i osteosan-

comas cells. In the normal and immortalized 184B5 cells, a

higher Mr 95,00�) MDM2 protein was also detected.

Immunoprecipitations performed with the 5B10 antibody,

which recognizes an epitope in the carboxy terminus of MDM2,

yielded slightly different results. This antibody efficiently pre-

cipitated the Mr 90,000 and 95,000, and, to a lesser extent, the

Mr 75,000 forms of MDM2. The relative differences in inten-

sities of the various bands likely reflect bonafide differences in

protein levels as equivalent amounts of actin were detected from

each sample (data not presented). As a control for these exper-

iments we demonstrate that numerous protein bands with mo-

leculan weights ranging from 75,000 to 90,000 are also precip-

itated from SJSA-1 cells that contain an amplified MDM2 gene.

The level of many cell cycle-regulated proteins does not

always correlate with the abundance of their mRNAs. To deter-

mine quantitative differences in the levels of MDM2 protein in

our panel of mammary epithelial cells, we prepared total cell

protein extracts and performed Western blot analyses. The pre-

dominant form of MDM2 detectable by Western blot was the Mr

90,000 protein (Fig. 3). We found a good correlation between

the level of MDM2 mRNA and the amount of the Mr 90,000

MDM2 protein in the different cells. The normal (Fig. 3, Lanes

I and 2), immortal, nontumonigenic (Fig. 3, Lanes 3 and 4) and

the ER� breast cancer cells (Fig. 3, Lanes 5-8) all had higher

levels of the Mr 90,000 MDM2 protein than the ER� breast

cancer cells (Fig. 3, Lanes 9-13). An additional MDM2 protein

having a Mr 57,000 was present in the SJSA-i osteosarcoma

cells and many of the breast epithelial cells examined. This band

was not detectable in the immunoblotting experiments because

it comignates on gels with the mouse monoclonal antibodies

used for immunoprecipitation. The bands corresponding to the

major MDM2 proteins detected in the SJSA-l osteosancoma line

are indicated (Fig. 3, arrows on the right). To ensure that the

differences in protein levels detected in the various mammary

epithelial cells were not due to unequal loading and transfer of

proteins, the lower portion of the same membrane was subse-

quently probed for actin (Fig. 3, bottom).

Several groups have described a binding site for wild-type

p53 within the first intron of the MDM2 gene (7, 8). This DNA

sequence is apparently involved in the regulation of MDM2

gene transcription by wild-type p53. Thus, in the presence of

wild-type p53, MDM2 mRNA and protein levels were concom-

itantly increased presumably due to the influence of wild-type



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Fig. 3 Western blot analysis of MDM2 and p53 proteins in human breast epithelial cells. Total protein extracts from exponentially growing normal

mammary epithelial cells (Lanes 1 and 2), 184B5 cells (Lane 3), immortalized MCF-l0 (Lane 4), estrogen receptor-positive MCF-7 (Lane 5), T47-D(Lane 6), ZR7S-l (Lane 7), and BT-474 (Lane 8) tumor cells, and estrogen receptor-negative SKBr3 (Lane 9), HS578T (Lane 10), MDA-MB-157(Lane 11), MDA-MB-231 (Lane 12), and MDA-MB-468 (Lane 13) tumor cells were prepared in Laemmli lysis buffer. Protein (50 �xWlane) was

separated in an 8% SDS polyacylamide gel, electroblotted onto nitrocellulose, and the membranes were reacted with antibodies corresponding to

MDM2, PS3, or actin. Protein molecular weight markers that were coelectrophoresed and transferred are indicated on the left of each panel. Lanedesignated SJSA-l contains protein extract from osteosarcoma cells that ovenexpress the MDM2 protein.

p53 in the cells (6, 7). To determine whether there was a

correlation between MDM2 and p53 protein levels in different

mammary epithelial cells, we performed p53 Western blot anal-

yses on the same extracts. These results showed no apparent

correlation between the absolute levels of p53 and MDM2

proteins in these cells.

A more critical consideration in these comparisons is

whether the p53 expressed in the various cells is mutant or wild

type. Normal and immortalized, nontumonigenic breast cells

grown in culture do not express the estrogen receptor (Fig. 1).

This finding likely reflects the biology of mammary epithelial

cells since very few cells are ER� in normal nonlactating

mammary glands (27, 28). The normal and immortal 184B5

mammary epithelial cells used in these studies express a wild-

type p53 protein that has increased stability (30-32). The p.53

gene in the immortalized MCF1O cells has been sequenced

along its entirety and shown to be wild type.5 All of these cells

expressed relatively high levels of MDM2 protein. In contrast,

the ER tumor cells (Lanes 10-14) expressed either a mutant on

no p53 and had relatively low levels of MDM2 mRNA and

protein (33). Thus, when examining ER mammary epithelial

cells, we found a good correlation between wild-type p53 ex-

pression and corresponding levels of MDM2 mRNA and the Mr

90,000 MDM2 protein.

The same positive correlation between wild-type p53 and

MDM2 protein expression did not hold true for the ER� breast

cancer cells. All of the ER� breast cancer cells expressed

relatively high levels of full length MDM2 protein when com-

pared to the ER cells. MCF-7 and ZR75-l cells express a

wild-type p53 protein (34, 35); however, T47-D and BT-474

cells both express mutant p53 (33, 36). Thus, factors other than

p53 are likely responsible for the high levels of MDM2 mRNA

and protein in ER� breast tumor cells. Among the various cell

types examined, MDM2 mRNA levels showed proportionally

greater variations than did MDM2 protein levels. These findings

suggest additional regulation of MDM2 protein accumulation at

some posttnanscniptional level in human breast epithelial cells.

Wild-Type p53 Induces MDM2 Expression in ER Tu-

mor Cells. To obtain independent evidence that wild-type p53

influences MDM2 expression in human breast tumor cells, two

ER tumor cell lines, MDA-MB-157 and MDA-231, were

infected with an adenovirus vector carrying a wild-type p.53

gene at 10 plaque-forming units/cell. The relative levels of

wild-type p53 protein present in the cells 24 h after infection are

shown in Fig. 4A. We observed no difference in p513 expression

between uninfected cells (Fig. 4A, Lanes 1 and 4) and cells



MDA-1 57

123

MDA-231

456

A

B

p

I I

ences MDM2 mRNA expression in ER breast tumor cells that. express mutant on no endogenous p53 protein.

Effect of Estrogen on MDM2 Expression. Because all

of the ER� tumor lines contained high levels of MDM2 protein,

regardless of the status of the p.53 gene, we examined whether

estrogen could influence MDM2 expression in these cells.

- 49 5 MCF-7 and ZR75-1 cells were grown in phenol red-free me-

U dium containing charcoal-stripped serum for 5 days. These

conditions have been shown previously to slow the growth of

the cells and down-modulate the expression of estrogen-depen-

dent genes (37, 38). After scanning the autoradiograms and

correcting for slight loading differences, a 2-fold increase in

MDM2 mRNA levels was observed in MCF-7 cells after refeed-

ing the cells with estrogen-containing medium for 4 to 8 h (Fig.

5A). The slight increase in MDM2 mRNA detected in ZR75-l

cells at 4 and 8 h after estrogen repletion could be attributed to

minor loading differences as the level of the control mRNA,

36B4, was also increased slightly in these samples (bottom, Fig.

SB). We examined MDM2 protein levels in the same samples

and found that they were not affected by estrogen levels in the

M DM2 culture medium!’ In contrast to the minor changes observed in

MDM2 mRNA levels, the estrogen inducible gene, pS2, was

down-regulated in estrogen-depleted cells and increased -20-

fold 4 to 8 h after the addition of �3-estradiol to the culture

medium (middle). When considered together these data suggest

that estrogen levels may not directly influence MDM2 mRNA

expression in cultured breast epithelial cells.

Immunocytochemical Localization of MDM2 and p53

in Human Mammary Epithelial Cells. To determine the

intracellular location of the MDM2 protein, we stained repre-

sentative breast cell lines using the IF2 antibody. After evalu-

ating different fixatives, methanol:acetone (1:1) was found to

yield the most reproducible staining results. All of the mammary

epithelial cells studied contained cleanly detectable MDM2 pro-

tein using Western blot analysis (Fig. 3); however, considerable

variation in immunocytochemical staining patterns was ob-

served. The NMECs and immortalized l84B5 mammary epi-

thelial cells showed distinct nuclear staining for MDM2 with

some variation in intensity from one cell to another (Fig. 6A).

When the same cells were stained for p53 using the PAb 1801

antibody, a similar pattern of heterogenous nuclear staining was

observed. In contrast another immortalized, mammary epithelial

cell line MCF-10 showed less intense and more variable staining

for both MDM2 and p53 proteins. The decreased staining in-

tensity observed in these cells may be due to the slightly lower

levels of protein accumulation as determined by Western blot-

ting (Fig. 3).

The estrogen receptor-positive breast epithelial lines all

contained relatively high levels of MDM2 protein when exam-

med using Western blots (Fig. 3). However, when analyzed

immunocytochemically, the ER� tumor cells stained poorly, if

at all, for MDM2. In MCF-7 cells the small amount of peninu-

clear staining obtained with the MDM2 antibody is likely non-

specific because we see similar results using isotypic monoclo-

nal antibodies for other cellular proteins that are not expressed

36B4

Fig. 4 A, infection of ER� tumor cells with an adenovirus vector

containing a wild-type gene. Protein lysate prepared from exponentially

growing MDA-MB-157 and MDA-MB-231 ER tumor cells (Lanes 1

and 4), cells infected with a control adenovirus vector (Lanes 2 and 5),

and cells infected with AdWTpS3 virus (Lanes 3 and 6) was examined

for p53 expression using Western blot analysis. B. RNA was isolatedfrom cells treated as described in the legend to A and examined forMDM2 expression using Northern blot analysis. Lower panel, 36B4

expression from the same filter.

infected with a control virus lacking the p53 gene (Fig. 4A,

Lanes 2 and 5). In contrast, cells infected with a virus carrying

the wild-type p53 gene expressed significantly increased levels

of p53 protein (Fig. 4A, Lanes 3 and 6).

MDM2 mRNA levels were examined from cells indepen-

dently infected with the same viral constructs (Fig. 4B). MDM2

mRNA levels were relatively low in the uninfected cells (Fig.

4B, Lanes I and 4) and in the cells infected with a control virus

(Fig. 4B, Lanes 2 and 5). MDA-MB-157 and MDA-MB-231

cells expressing a wild-type p53 protein showed 6- and 10-fold

respective increases in MDM2 mRNA levels (Fig. 4B, Lanes 3

and 6). These increases in MDM2 mRNA levels are in accon-

dance with the finding of slightly higher levels of wild-type p53

protein in the MDA-MB-231 cells. When considered together

these data support the idea that wild-type p53 positively influ-


6 J� M. Gudas, unpublished data.

MDA-157 MDA-231

1 2 34 5 6



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A MCF-7

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B ZR75i

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Fig. 5 Northern blot analysis of MDM2 mRNA in ER� cells following estrogen stimulation. MCF-7 and ZR75-l cells were depleted of estrogen

for S days and subsequently refed with medium containing charcoal-stripped serum and 10 nM �3-estradiol. At the times indicated above each lanetotal RNA was prepared, electrophoretically separated, transferred to filters, and probed for expression of MDM2 (upper panels), p52 (middle panels),

and 36B4 (lower panels). Large and small arrows, migration of the 6.7-kilobase and 4.5-kilobase MDM2 transcripts. respectively.

in these cells.6 Very few nuclei from ER� ZR7S-1 cells stained

for MDM2 protein and some cells may show some cytoplasmic

localization. Interestingly, staining of the ZR75-i cells for p53

revealed a distinct cytoplasmic staining that was highly local-

ized within discrete foci. To our knowledge, a similar staining

pattern for p53 has not been described in other cell types.

The ER, MDA-MB-231 breast cancer cells had very low

levels of MDM2 when examined usingWestern blot analysis

(Fig. 3) and did not stain at all for MDM2. Two other ER

breast cancer cell lines that expressed very low levels of MDM2

protein by immunoprecipitation and Western blotting, MDA-

MB-157 and MDA-MB-453, were also negative for MDM2

when examined immunocytochemically (data not shown). We

believe that the level of MDM2 protein may be below the

detection limit by immunocytochemistry in the ER breast

cancer cells. As expected for cells that express a mutant p53, all

nuclei in MDA-MB-231 cells stained intensely for p53. As

controls for the immunocytochemistry, we show that SJSA-1

cells, which overexpress MDM2, stained intensely for this an-

tigen while very few nuclei from these cells were positive for

p53 (Fig. 6).

DISCUSSION

In this study we report two independent observations con-

cerning MDM2 expression in human breast epithelial cells. The

first finding is that a good correlation exists between expression

of wild-type p53 in normal and immortalized mammary epithe-

hal cells with accumulation of relatively high levels of MDM2

mRNA and protein in the same cells. Because the magnitude of

the difference in relative MDM2 mRNA levels was greaten than

that observed for corresponding MDM2 protein levels, other

factors, including protein stability and translational efficiency,

likely play a role in determining the total amount of MDM2

protein in a cell. The normal and immortalized human mammary

epithelial cells used for these studies are estrogen receptor

negative and express a wild-type p53 protein. The wild-type p53

is unusual in these cells because it has a half-life of -3 h (30r

compared to a half-life of -20 mm in many other cell types (30,

39, 40).

In contrast, all of the estrogen receptor-negative breast

tumor cell lines studied had either a mutant on no p53 protein

and expressed significantly less MDM2 mRNA and protein.

Because the ER cell lines we examined all had mutations in the

conserved DNA binding domain of the protein (41), they are all

likely defective in their ability to transactivate cellular target

genes. Previous studies from other laboratories have demon-

strated a consensus p53 DNA binding site in the MDM2 gene

which is necessary for regulation by wild-type p53 (6, 7). To

determine whether wild-type p53 could, indeed, affect MDM2

expression, we introduced a wild-type p53 gene into ER breast

tumor cells that contained a mutant on no p53 protein. In both

cell types wild-type p53 expression resulted in an increase in

MDM2 mRNA levels. These data suggest that wild-type p53

functions to increase the expression of MDM2 mRNA and

protein in estrogen receptor-negative breast epithelial cells and

this regulatory circuit becomes disrupted when the cells

progress and gain mutations in the p.53 gene. ER� breast tumors

are usually more differentiated than ER tumor cells (42, 43).

Because MDM2 mRNA and protein levels are lower in ER

breast cancer cells, our data suggest a diminution rather than an

increase in MDM2 expression during the progression of mam-

many tumors.

The second major finding from this study is that MDM2

mRNA and protein levels are significantly higher in ER� breast



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Fig. 6 A, immunocytochemical localization of MDM2 and p53 proteins in normal and immortal human breast epithelial cells. Cells were grown on

glass tissue culture chamber slides, fixed in methanol:acetone (1:1), and subsequently stained for MDM2 and p53 using the 1F2 or PAb 1801

antibodies, respectively. After washing, specific complexes were detected using the avidin-biotin complex immunoperoxidase detection system as

recommended by the manufacturer (Vectastain). B, MDM2 and p53 immunostaining in tumorigenic breast epithelial cells.


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cancer cells independent of their p53 status. Only MCF-7 and

ZR75-1 cells have a wild-type p53 gene (35). The other estro-

gen ER� breast cell lines examined, T47-D and BT474, both

expressed mutant p53 proteins (33, 36). Despite the presence of

a mutant p53 gene, these cells had relatively high levels of

MDM2 protein. Thus, other cellular factors, apart from p53,

must account for the high levels of MDM2 mRNA and protein

in these cells.

Because this phenotype was only observed in the ER�

breast cancer cells, we asked whether some function or genes

regulated by the estrogen receptor could be involved in the

increased expression of MDM2 in these cells. When ER� cells

were cultured in estrogen-depleted medium, the mRNA for the

estrogen-regulated gene, pS2, was decreased and increased sig-

nificantly on nefeeding the cells with medium containing �3-es-

tradiol. Under the same conditions only a 2-fold change in

MDM2 mRNA levels in MCF-7 cells and no change in ZR75-l

cells was detected. Therefore, the elevated levels of MDM2

mRNA and protein in these cells may not involve a direct effect

of the estrogen receptor itself. Many other genes including

glutathione-S-transferase in and gluathione penoxidase are dif-

ferentially expressed in ER� versus ER breast cancer cells

(44-46). Presumably, differences in the posttranscniptional pro-

cessing of these mRNAs differences and/on differences in their

chnomatin structure accounts for preferential mRNA expression

in one cell type versus another. These results are similar but not

identical to those reported by Saeed et al. (47) who found that

estrogen increased MDM2 expression in ER cells transfected

with a gene encoding the estrogen receptor. Because the estro-

gen receptor does not always induce estrogen-dependent pro-

moters when introduced into ER cells (48), the two results are

not directly comparable.




Several different MDM2 transcripts, ranging in size from

4.5 to 12.5 kilobases, were detected in mammary epithelial cells.

At present we do not know if these various mRNA species have

functional significance or if some of the higher molecular

weight transcripts represent partially on alternatively processed

MDM2 mRNAs. Interestingly, evidence from DNA sequencing

studies indicates that both the human and mouse MDM2

mRNAs can be alternatively spliced (1, 49). Because all mRNA

bands hybridized with probes obtained from both the 5’ and 3’

end of the coding region of the human MDM2 eDNA, we

believe that they are all bona fide MDM2 transcripts.

Results from immunoprecipitation and Western blot anal-

yses showed a complex pattern of many different MDM2 pro-

teins in human breast epithelial cells. The predominant protein

detected from all mammary epithelial cells migrated as a Mr

90,0000 band. This form likely represents the full-length

MDM2 protein while the smaller, -57,000 form, may be an

alternatively spliced variant as described in mouse and rat cells

(I, 29). Haines et al. (29) have necently reported that the

different molecular weights of the MDM2 proteins in mouse

cells represent alternatively spliced variants. Moreover, they

have demonstrated that these proteins differ in their ability to

physically associate with, and thus inhibit, the function of wild-

type p53 (29). When considered along with the finding of

multiple mRNA species, we believe that the different forms

of MDM2 present in breast epithelial cells likely represent

distinct MDM2 proteins that may have both overlapping and

unique biological functions.

Immunocytochemical staining for MDM2 showed that the

protein was nuclear in breast epithelial cells; however, the

intensity and number of positive cells did not always correlate

with the level of protein detectable by biochemical methods.

Some of the variation in nuclear MDM2 staining seen in the

NMECs and l84B5 cells may reflect differences in cell cycle

distribution as we have previously shown that these cells stain

variably for p53, depending upon their position in the cell cycle

(32). ER� MCF-7, ZR75-1, and BT474 (data not shown) cells

showed little or no staining for MDM2 although they had

comparable amounts of the Mr 90,000 MDM2 when assayed

using Western blot analysis (Fig. 3). One possibile explanation

for the anomolous immunocytochemical results in ER� cells is

that MDM2 is somehow sequestered in a larger complex that is

only dissociated on lysis of the cells. We are presently perform-

ing more detailed experiments to test this hypothesis. With the

exception of a unique cytoplasmic focal staining pattern for p53

observed in ZR7S-1 cells, our results for p53 staining in breast

epithelial cells are similar to those reported by other investiga-

tons (30, 33, 50).

Increasing evidence suggests that p53 and its downstream

effector proteins MDM2 and CIP/Waf-1 play a role in deter-

mining cellular responses to endogenous and exogenous signals.

The differential expression of multiple MDM2 mRNAs and

proteins raises the possibility that these products may perform

distinct functions in mammary epithelial cells at different stages

of tumor development. We believe that further study of p53 and

its downstream effector proteins MDM2 and CIP-1 (51, 52)

should enhance our understanding of the processes of cell trans-

formation and the means by which cells modulate their response

to DNA damage and environmental stress. A better understand-

ing of these cellular processes should in turn translate into more

specific, and, hopefully, more effective therapeutic approaches

for the treatment of breast cancers.

ACKNOWLEDGMENTS

We thank Drs. Arnold Levine and Vincent Marachel for providing

us with the 5BlO monoclonal antibody. We are also grateful to Dr. Bert

Vogelstein for providing the human MDM2 eDNA clone, Dr. Thomas

Look for the SJSA-1 osteosarcoma cells, and Dr. Erasmus Schneider for

helpful comments on the manuscript.

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1995;1:71-80. Clin Cancer Res J M Gudas, H Nguyen, R C Klein, et al. proteins in normal and tumorigenic breast epithelial cells.Differential expression of multiple MDM2 messenger RNAs and

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