Vol. 5, 295-304, March 1994 Cell Growth & Differentiation 295

Expression of Wild-Type p53 during the CellCycle in Normal Human MammaryEpithelial Cells

Jean M. Gudas,1 Mikio Oka,2 Francesca Diella,Jane Trepel, and Kenneth H. Cowan

Medical Breast Cancer Section, Medicine Branch, Division of Cancer

Treatment lJ. M. G., M. 0., K. H. C.], Laboratory of Tumor Immunology

and Biology [F. Dl, and Clinical Pharmacology Branch [J. TI, National

Cancer Institute, Bethesda, Maryland 20892

Abstrad

In this study, we compare the expression patterns of p53mRNA and protein in normal human mammaryepithelial cells following synchronization to differentpoints in the cell cycle using two independent methods.When treated with lovastatin, the cells were blockedin G1 and appeared to express increased levels ofwild-type p53 when examined by immunostaining. Uponreversal of the metabolic block, the number of nucleithat stained positively for p53 declined dramaticallyduring mid-G1 and increased again concomitant with theentry of cells into S phase. In contrast to theimmunostaining results, Northern and Western blotanalyses revealed little change in p53 mRNA and proteinlevels in the Iovastatin-synchronized cells. When normalhuman mammary epithelial cells were made quiescentby removal of growth fadors, the mRNA for p53showed a biphasic distribution. p53 mRNA levels wereincreased during growth arrest, decreased during the G1phase, and rose again concomitant with the entry ofcells into S phase. The immunostaining pattern of p53also showed a biphasic distribution similar to the patternof mRNA expression. Despite an increase in p53 mRNAand immunostaining levels, growth fador-arrestedcells adually had less total p53 protein. Uponstimulation to proliferate, p53 protein levels remainedlow throughout G1 and increased concomitant with theentry of cells into S phase. Taken together, the resultsfrom these studies demonstrate that p53 immunostainingpatterns do not correlate with the overall levels of p53protein at different times during the cell cycle.Therefore, the distinct changes observed in p53immunostaining patterns are likely due toposttranslational modifications, conformational changes,or interadions of p53 with other cellular proteins duringthe cell cycle.

Received 9/28/93; revised 1 2/8/93; accepted 1 2/29/93.I To whom requests for reprints should be addressed, at Medical Breast Can-cer Section, Medicine Branch, National Cancer Institute, Building 10, Room

12N226, Bethesda, MD 20892.2 Present address: Second Department of Internal Medicine, Nagasaki Uni-versity School of Medicine, 7-1 Sakamoto 1 -Chome, Nagasaki 852, Japan.

Introdudion

Mammalian cell proliferation is a stringently regulated pro-

cess whose control is abrogated during the progression totumorigenesis (1 ). In somatic cells, cell cycle regulation oc-

curs at several discrete points termed control or checkpoints(2, 3). The most well-defined controls are those that occur

during the emergence of cells from quiescence into G1 (4),

at the restriction or R point, just prior to DNA synthesis (5,6), and, finally, at the transition from the G2 to M phase ofthe cell cycle (7). Although much is known about the genesinvolved in controlling the entry of cells into the cell cycleand those involved in the control of mitosis, very little isknown about the interactions of gene products at the G1-Stransition. Recently, the p53 and Rb tumor suppressor genes

(8-i i), together with the the cdc2 and cdk kinases and regu-latory G1 cyclins (1 2, 1 3), have been implicated in growth

control at the transition from G1 to S phase.Several lines of evidence indicate that the p53 protein

plays a role at the restriction point in the cell cycle. In qui-

escent mouse 3T3 and normal resting human fibroblasts and

peripheral T-lymphocytes, the levels of p53 mRNA and pro-tein are very low. When these cells are stimulated to pro-liferate, the levels of p53 mRNA and the rate of p53 proteinsynthesis increased markedly as the cells progressed from G1

into S phase (1 4, i 5). Moreover, when antibodies to the p53protein were microinjected into quiescent cells at or aroundthe time of growth stimulation, their entry into S phase wasinhibited (i 6, 1 7). These data suggested that the p53 proteinwas necessary for the � to S phase transition in normal cells.

Conversely, in tumor cells that express no p53 or a mutatedPS3, introduction of the wild-type protein arrested cellgrowth at or near the restriction point in late G1 (i 8-21).

In most cell types, p53 is detected as a nuclear phospho-protein; however, several reports indicate that its functionmay be regulated by altering subcellular distribution during

the cell cycle (19, 22). In serum-stimulated 3T3 fibroblasts,p53 protein was detected in the cytoplasm during the G1phase of the cell cycle and observed to translocate to thenucleus at the beginning of S phase (23). Furthermore, cx-periments using a temperature-sensitive mutant p53 proteindemonstrated cytoplasm ic local ization at the nonperm issivetemperature which affected the growth-inhibitory functionsof the p53 protein (1 9, 24).

As many as one-halfof human epithelial tumors including

tumors of the colon, lung, breast, and bladder contain mu-tations in the p53 gene (25-27). The high frequency of p53mutations in epithelial tumors suggests an important role forthis protein in regulating the proliferation of these cells.However, the specific role of wild-type p53 in the normalcell cycle and the means by which the mutant protein trans-forms cells remain unclear. As one approach toward un-derstandingthefunction ofpS3 in human epithelial cells, wehave examined the expression and subcellular distribution

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Fig. 1. SSCP analysis of p53 in normal HMECs. Genomic DNA, isolatedfrom mammary epithehial cell cultures derived from two different individuals,was amplified by PCR reactions using primers specific for exons 5, 6, 7, 8,and 9 ofthe pS3 gene. The results from analysis ofexons 6 and 7 are presented

and are representative of the data obtained with the other exons. In each ofthe respective panels, labeled Exon 6 and Exon 7, arrows at left, pattern ofmigration of wild-type p53 alleles. Lane C contains DNA amplified from a

mammary tumor known to have a mutation in the p53 gene. The DNAs from

the mammary epithehial cells are presented in Lanes 1 and 2. Lane 3 containsDNA from a sample known to have a wild-type p53 gene by prior DNAsequencing analysis.

, The abbreviations used are: NMECs, normal human mammary epithehialcells; PCR, polymerase chain reaction; SSCP, single strand conformationalpolymorphism; MEBM, mammary epithehial basal mediuni; MEGM, mam-mary epithelial growth medium; SDS, sodium dodecyl sulfate; PBS,phosphate-buffered saline; kh, kilobase(s(; SNNTE, 5% sucrose, 1% Nonidet

P-40, 0.5 m� NaCI, 50 msa Tris (pH 7.4(, and 5 m� EDTA.

296 p53 Expression in NMECs

of p53 protein during the cell cycle of NMECs.3 These cells

are derived from reduction mammoplasties and retain manycharacteristics of normal epithelial cells when grown in cul-ture including a diploid karyotype, well-defined growth fac-

tor requirements, and a finite lifespan in vitro (28, 29). Be-cause NMECs produce a relatively stable wild-type p53protein (30) and are amenable to synchronization by severaldifferent techniques, they provide a unique model for un-derstanding how this gene is regulated at different times dur-ing the cell cycle. Furthermore, the importance of p53 in thegrowth regulation ofthese cells is underscored bythe findingthat the human papillomavirus E6 protein, which binds toand degrades p53 in vivo (3i ), is capable of immortalizingcultures of NMECs (32, 33). In the present study, we dem-onstrate that NMECs express a wild-type p53 protein thatresides in the nucleus. Moreover, we show that both p53mRNA and protein are regulated in a cell cycle-dependentmanner in synchronized populations of NMECs.

Results

NMECs Express a Wild-Type p53 Protein. An unusual fea-ture of NMECs is the finding of readily detectable levels ofp53 in these cells (32). To ascertain that the protein beingexpressed was, indeed, the wild-type form we performedPCR-SSCP analyses. Genomic DNA was isolated from mam-mary epithelial cells derived from two different individuals.Using specific oligonucleotide primers that hybridized tointron sequences on either side of p53 exons 5, 6, 7, 8, and9, genomic DNA was amplified using PCR reactions, radio-labeled, and analyzed on neutral nondenaturing polyacryl-amide gels as described previously (34-36). The sizes of thePCR-amplified DNA fragments were: 190, i84, 209, 237,and 1 57 base pairs, corresponding to exons 5, 6, 7, 8, and9, respectively. All analyses were performed in duplicatewith appropriate control DNA; they indicated that the p53gene present in these cells did not contain any mutation. Theresults obtained for exons 6 and 7 are presented in Fig. 1 andare representative of those found for all other exons exam-med. In each case, the DNA amplified from two differentNMEC samples (Fig. i , Lanes 1 and 2) migrated at the sameposition as the wild-type p53 DNA (Fig. i , Lane 3). No ano-molous bands suggesting a mutation were detected in eithercell strain. In addition to our studies, the mRNA from otherNMEC cell cultures has been sequenced along its entiretyand found to be wild-type (30, 37).

Synchronization of NMECs by Lovastatin Treatment. Todetermine whether p53 mRNA and protein were regulatedin a cell cycle-dependent manner in NMECs, we arrested thecells using two independent methods and analyzed cellcycle distribution by flow cytometry. Several groups havereported that incubation ofboth normal and tumor cells withlovastatin results in their arrest at some point in earlyG1 (38-40). Lovastatin inhibits the enzyme hydroxy-methylglutaryl-CoA reductase and thus prevents the synthe-sis of isoprenyl metabolites of the cholesterol biosyntheticpathway. The posttranslational addition of isoprenyl moi-eties to regulatory proteins is required for the progression of

Exon6 Exon7

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cells through the G1 phase. We found that treatment ofNMECs with 1 7 �M lovastatin for 30 h arrested approxi-mately 95% of the cells with a G1 DNA content as deter-mined by flow cytometry (Fig. 2C). The slightly abnormalshoulder seen in the DNA profile of these cells is likely dueto increased background fluorescence, as data from auto-radiography and Northern blot analyses of S phase-specificmRNAs indicate that none ofthe cells are in S phase at time0 (see data presented below).

p53 mRNA expression was examined in cells treated withlovastatin and in cells released from this block for the timesdesignated by the addition of fresh medium containing theend product mevalonic acid (Fig. 3). The arrested cells (time0) expressed readily detectable levels of p53 mRNA. Afterrefeeding with fresh medium containing the end productmevalonic acid, p53 mRNA levels decreased as the cellsprogressed through the G1 phase and increased again con-comitant with the entry of cells into S phase (approximately20 h after refeeding). The subtle changes in p53 mRNA levelscontrast sharply with the pattern observed for thereplication-dependent histone 3.2 mRNA. The changes ob-served were reproducible and could not be attributed to un-equal loading of the lanes, as the amount ofthe replication-independent gene 36B4 remained constant at all times.

To determine whether p53 protein levels and/or its sub-cellular distribution was altered during the transition from astate of growth arrest and subsequent entry into the prolif-erative phase, we stained the cells using PAb i 801 . Thisantibody recognizes an epitope in the amino terminus of p53and reliably detects p53 in the nuclei ofexponentially grow-ing NMECs. Following synchronization of NMECs with lo-vastatin, we observed dramatic differences in the p53 stain-ing patterns of lovastatin-arrested and mevalonate-restimulated cells (Fig. 4A). In the arrested cells, almost allnuclei stained positively for p53 protein. After restimulationwith fresh medium containing mevalonic acid, the numberof p53-positive nuclei decreased precipitously such that 10to 12 h after refeeding (when the cells were in mid-G1),virtually no cells stained for p53 protein. The number ofp53-positive nuclei gradually increased again concomitantwith the entry of cells into S phase.

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Cell Growth � Differentiation 297

Fi�,’. 2. Flow (ytom(’tri( analysis of DNA trorn NMECs. Exponentially grow-

ing (A). growth tactor-deprived )B(, and lovastatin-treateci )C( NMECs were

analyzed for DNA content as described in “Materials and Methods.”

The results obtai ned from immunocytochem istry werequantitated by Counting the number of p53-positive nucleiin each sample and correlating these data with the percent-age of labeled nuclei in paired samples of cells that weresynchronized and analyzed in parallel. Autoradiographicdata indicated that the cells began to incorporate radiola-beled thymicline approximately 20 h after refeeding with themevalonate-containi ng medium. U nder these conditions,we observed that the percentage of labeled nuclei in the

cultures increased concomitant with the increase in p53-positive nuclei. In fact, the curves representing the two re-suits were superimposable (Fig. 4B). These results demon-strated that p53 immunostaining was biphasic in lovastatin-synchronized cells. The intensity and number of positivelystaining nuclei were increased in the arrested cells whencompared with an exponentially growing population (Fig.8), markedly reduced upon growth stimulation, and in-creased again concomitant with entry into S phase.

To determine whether the results obtained were due toactual changes in the amount of p53 at differenttimes duringthe cell cycle, total protein extracts were prepared and cx-amined by Western blot analysis. These analyses revealedtwo fornis of p53 with most of the protein migrating in theupper, slightly higher molecular weight band. When eachlane was scanned with a densitometer and the results wereplotted in arbitrary units against time, total p53 levels wereshown to be relatively constant during the cell cycle (Fig.5B). For comparative purposes, the value for p53 in the cx-ponentially growing cells was 343 units in this particularexperiment. The ratio of p53 in each of the two bands re-mained relatively constant (approximately 90:iO) at alltimes, with the exception ofthe 36-h time point, when morep53 was detected in the lower band (80:20). In these cx-periments, actin was used as a control for equivalent loadingof lanes. The decrease in actin protein seen at time 0 doesnot reflect differences in protein loading but merely reflectsthe fact that actin synthesis is depressed in growth-arrestedcells (41). When the transfer membrane was stained withPonceau 5, all lanes were shown to be loaded equally (datanot shown).

We examined the rate of p53 synthesis at different timesduringthe cell cycle by pulse labelingthe cells with [35SJme-thionine and immunoprecipitating with PAbs 421 and 1 80i,which recognize epitopes in the amino and carboxy terminiof the protein, respectively. These antibodies have beenshown to recognize both mutant and wild-type conforma-

Fig. 3. Northern blot analysis of RNA derived from lovastatin-treated and

released NMECs. Exponentially growing HMECs were synchronized to the Gphase ofthe cell cycle by placing them in MEGM (Clonetics Corp.) containing

all required growth factors and 1 7 p� lovastatin for 27 h. At time 0, the cellssvere stimulated to proliferate by the addition of fresh MEGM containing allgrowth factors and 2 m�i mevalonic acid to reverse the metabolic blor k. At

the times designated, RNA was isolated, electrophoresed, transferred to aMagna NT membrane, and �roi�er1 sequentially for expression of p5 3. histone3.2, and 36B4.

tionsofthe protein (42, 43). Both antibodies recognized p53in cell extracts prepared at different times during the cellcycle. Because the results were similar with both antibodies,we have presented the data only for PAb 421 (Fig. 6A).

When the data were analyzed by scanning densitometryand plotted againsttime, the rate of p53 synthesis was shownto vary less than 2-fold in the lovastatin-synchronized cells(Fig. 6C, closed diamonds). The rate of p53 synthesis wasfound to be lowest in the cells pulsed 8 h after restimulationwith fresh medium. The decrease in p53 synthesis atthis timepoint coincided with the nadir of total p53 accumulation

during mid-C1 (Fig. 5). As the cells progressed through G1,the rate of p53 synthesis increased, and, by 36 h after refeed-ing, it was equivalentto that observed in exponentially grow-ing cells. Thus, the small decrease in p53 levels observed inG1 cells may be accounted for by a lower rate of p53 syn-thesis at this time during the cell cycle. As a control for thespecificity of the reactions, we performed immunoprecipa-tions using an antibody to the hsp 70 protein with extractsfrom cells obtained 24 and 32 h after reversal of the lovas-tatin block (Fig. 6A, Lane C). As expected for a wild-typeprotein, no band corresponding to p53 coprecipitated withthe hsp 70 protein in these experiments.

Synchronization of NMECs by Growth Factor Depriva-tion. One ofthe most common methods used to achieve cellsynchrony is to remove growth factors from the culture me-dium. Cells which have been deprived ofgrowth factors exitthe cell cycle and arrest in a G0 state (i ). When epidermalgrowth factor, insulin, hydrocortisone, and bovine pituitary

extract were withdrawn from exponentially growingNMECs, the cells accumulated with a C1 DNA content as

shown in Fig. 2B. We presume that these cells are in a G0or quiescent state, as other investigators have shown thatgrowth factor-deprived NMECs fail to express the Ki-67 pro-liferation marker (40).

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Fi�,’. 5. A, Western blot analysis of p53 in lovastatin-synchronized NMECs.NMECs were synchronizer) and released as (lescribed in the legend to Fig. 3.

Alter separation on a lOX SDS-polyacrvlaniide gel, the proteins were Iran-

ferred to nitrocellulose and probed for p53 using PAb 1801 (Oncogene Sci-ence). Numbers above each lane refer to the arrested cells (time 13) and cells

that were stimulated to proliferate for the times designated. Lane Es contains

protein extract from exponentially growing NMECs; LaneMDA contains pro-tom extract from the breast cancer cell line MDA-MB-231 . Arrows at left, thetwo differentially migrating forms of p53. In lowerj)anel, the same filter wasreprol)ed for expression of the 42 kilodalton actin protein to ascertain equiva-

lent loading of lanes. SB. Quantitation of p53 levels in lovastatin-synchronized NMECs. The films depicting 5 3 were analyzed by scanning

densitometry, and the area under the peaks was summed together and plotted

against time.

298 p53 Expression in NMECs

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Fit,’. 4. A, immunocytochemical staining of p53 in lovastatin-synchronized NMECs. NMECs that were plated onto glass slides were synchronized and releasedas described in the legend to Fig. 3. The cells were washed three times in ice-cold PBS, fixed in methanol:acetone (1 :1 ), and frozen at -80’C. The slides were

stained using PAb 1 801 (p5 3 Ab-2; Oncogene Science) and specific complexes detected using a Vectastain ABC kit. B, quantitation of p53 expression inlovastatin-synchronized NMECs. The number of nuclei staining positively for p53 was counted and expressed as the percentage of total cells in a given field,

Cumulative � Hlthymidine labeling was determined in 24-well cultures that were plated and synchronized simultaneously with the chamber slides. At least 300individual cells were counted at each time point for determination of p53 staining and labeled nuclei.

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Expression of p53 mRNA was examined in the arrestedcells and in cells restimulated to proliferate by the additionof fresh medium containing a full complement of growthfactors (Fig. 7). p53 mRNA levels were elevated in the ar-

rested cells. Following stimulation to proliferate, the level ofp53 mRNA gradually decreased during mid-G1 (from 8 to 12h) and later increased again when the cells entered the S, C2,and M phases (28 to 32 h after refeeding). In these experi-ments, the amount of p53 mRNA detected at 32 h wasequivalent to the level expressed in exponentially growing

cells (data not shown).As controls for these experiments, the filters were stripped

and reprobed for the expression of the replication-dependent histone 3.2 gene (44). This mRNA was unde-tectable in the arrested cells and increased approximately 16h after refeeding the cells with fresh medium (Fig. 7). These

results correlate well with autoradiographic data indicatingthat the cells enter S phase approximately 1 2 to 1 6 h afterrestimulation with fresh growth factor-replete medium (Fig.8B). To ensure that the changes observed were not due tounequal loading of lanes, the same filters were stripped andreprobed for expression of 36B4, a gene that is not altered

during the cell cycle (38, 45).To determine whether the level of p53 protein and/or its

subcellular distribution was regulated during the cell cyclein NMECs, we performed immunocytochemical analyses ofp53 on growth factor-deprived cells and cells obtained atvarious times after stimulation with fresh medium. In growthfactor-deprived NMECs, almost every cell demonstratedprominent nuclear staining for p53 (Fig. 8A). The number

and intensity ofp53-positive nuclei decreased asthe arrestedcells were restimulated to proliferate, reaching a nadir at

approximately 8 h. At all times thereafter, the number andintensity of positively staining nuclei was observed to in-

crease. Importantly, p53 expression was always detected in

the nucleus during this transition.The results were quantitated by counting the number of

p53-positive nuclei, and these data were plotted togetherwith the percentage of labeled nuclei from paired samplesof cells that had been synchronized simultaneously andfixed for autoradiography (Fig. 8B). The results indicate thatthe cells began to incorporate radiolabeled thymidine ap-proximately 14 h after refeeding with growth factor-containing medium. By 20 h, greater than 60% of the popu-lation had entered S phase. Although the number andintensity of nuclei staining positive for p53 protein de-

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Fi�,’. 6. Immunoprecipitation of p53 from lovastatin- A � and growth factor-

synchronized (B) NMECs. Protein extracts were prepared from cells that were

labeled with I �SJmethionine during growth arrest (time 0) and at the limesdesignated following stiniulation to proliferate. After normalizing for the num-her ot counts incorporated, immunoprecipitation was carried out using PA))42 1 . Lane Es contains labeled p53 immunoprecipitated from exponentially

growing cells. Lane C contains protein from cells released for 1 6 h and im-munopreci�)itatec) with an isotypic antibody to hsp 70 (A) or to bromode-

oxyuridine ) Th. Arrows at left, position of p53. C. quantitation of p53 synthesisIn lovastatin- and growth tactor-synchronized NMECs. The autoradiographicfilms were analyzed ))y scanning densitometry, and the peak area correspond-ing to j5 3 was plotted against time. Open squares, results from the growthfaCtor-synchronized cells; (loser! dianionds, data from the lovastatin-

synchronized cells.

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F,�,’. 7. Northern ))lot analysis o mRNA expression in growth factor�

synchronized NMECs. Exponentially growing HMECs were synchronized to

the G, phase of the cell cycle by placing then) in MEBM (Clonetics Corp.without growth factors plus O.OS”� bovine serum albumin for 3 (lays. At time

0, thecellswerestimulated to proliferate by the addition offresh MEGM. RNA(20 pg( obtained froni cells at the times designated was electrophoresed on

a denaturing formaldehyde gel, transferred to Magna NT membrane, andprobed sequentially for the expression of pSI, histone 3.2, and the consti-

tutively expressed mRNA, 36B4.

Cell Growth ,5,Differentiation 299

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creased during G phase, the results were not as dramatic asthose observed in the lovastatin-synchronized population.However, increased nuclear p53 expression was again cor-related with entry of cells into S phase.

The differences in p53 expression detected by immuno-cytochen3istry may be a consequence of the failure of theantibody to recognize a specific p53 epitope at all timesduring the cell cycle and may not reflect actual changes inthe level of p53. To investigate this possibility, p53 protein

was quantitated in growth factor-synchronized cells usingWestern blot analysis. Our results showed that the level ofp53 was decreased in the growth factor-arrested cells and

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detected increased staining for p53 in the arrested cells, noaccumulation of protein was observed at this time. p53 pro-tein levels increased approximately 3- to 4-fold as the cellsentered S phase between 12 and 20 h after stimulation. Inthis experiment, p53 was again detected as a doublet pro-tein. The ratio of p53 in the two bands did not change sig-nificantly during the transition from G0 into S phase (ap-proximately 10% of the total pS3 was present in the fastermigrating form at all times examined).

Metabolic labeling studies were performed to determinewhether p53 was synthesized at the same rate in the growth

factor-arrested and -restimulated cells (Fig. 6B). The p53-specific band was quantitated by scanning densitometry andplotted against time after release. Synthesis of p53 was de-creased in the arrested and G1 phase cells and increasedapproximately 2.5-fold as the cells entered S phase (Fig. 6C,open squares). Thus, the changes observed in total p53 pro-tein accumulation are most likely due to small changes in therate of p53 synthesis at different times during the cell cycle.The metabolic labeling experiments have been repeated atleast three times with identical results. In both of our meta-bolic labeling experiments, the p53 monoclonal antibodiescoprecipitated bandsofapproximately49tosl and 57 to 58kilodaltons. We have subsequently found that these bands,which reproducibly come down in the synchronized cellextracts, can be eliminated if we centrifuge the extracts afterincubation with the monoclonal antibody and transfer thesupernatants to a fresh tube before adding the Protein A plusProtein C agarose beads. Thus, we believe that these bandsare polymerized proteins that form nonspecifically upon in-cubation of cell extracts with preparations of monoclonalantibodies.

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Fifl. 8. A, immunocytoheniical staining 01 p53 in growth factor-synchronized NMECs. NMECs were synchronized as described in the legend to Fig. 7. At the

times designated, the r hamber slides were washed, fixed, and frozen at -8O�C. The slides were stained using PA)) 1801 (p53 Ah-2; Oncogene Science), and specificcomplexes were detected using the ABC immunoperoxidase system as describer) in “Materials and Methods.” B, quantitation of p53 expression in growth

factor-synchronized HMECs. The number of nuclei staining positively for p53 was counted and expressed asthe percentage oftotal cells in a given field. Cumulative

I Hjthvniidine labeling was determined in 24-well cultures that were plated and synchronized simultaneously with the chamber slides. At least 300 individual

etls were (ounter) at (‘act) time l)oirlt for determination of p53 staining and labeled nuclei.

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Fit,’. 9. A, Western blot analysis of p53 from growth factor-synchronizedNMECs. Total cell protein extracts were preparer) from arrested cells (time 0),exponentially growing cells )Expo), anr) cells stimulated to proliferate for thetimes r)esignater) above each lane. Lane Con, protein extract from SAOS cellsthat r)o not express p53. After separation on a 4 to 1 2% SDS-polyacrylamidegradient gel, the proteins were transferred to nitrocellulose and probed for p53

(u,ier panel) anr) actin (lower panel). p53 was r)etecter) using a chemilu-minescent technique, and actin was rletected using the alkaline phosphatase

Procedure. B, rluanlitation of p53 levels in growth factor-synchronizedNMECs. The films depicting p53 were analyzer) by scanning densitometry,anr) the area unr)er the peaks was summer) together anr) plotter) against time.

300 53 Expression in NMECs

Discussion

As one approach toward understanding the role of p53 in

cell cycle regulation, we undertook a study ofthe expressionofthis protein in synchronized populationsofnormal humanmammary epithelial cells. We reasoned that, if wild-type

p53 played a role in the growth arrestor proliferation of thesecells, then we may detect qualitative and/or quantitativechanges in the expression of this protein following differentmetabolic conditions that result in inhibition of cell prolif-eration. Because the p53 gene is frequently mutated in tumor

samples and in cultured cells (25, 46, 47), we first sought todetermine that the protein expressed in normal human mam-mary epithelial cells was indeed wild-type. PCR-SSCP analy-

sis of the conserved regions of p53, wherein greater than950/() of all mutations have been detected (25, 46), failed to

show any differences in mobility from a wild-type DNA se-quence. These results are not surprising given the fact thatNMECs are primary cells derived from normal individualsand they have a finite lifespan in vitro. Moreover, despite theincreased half-life of p53 expressed in these cells, it displaysmany features characteristic of the wild-type protein (32,33). The recent description of strong immunohistochemicalstaining for p53 in normal epithelial and mesenchymal cellsfrom two related individuals, together with the finding ofincreased wild-type p53 expression in a significant numberof benign colon adenomas, suggests that some independentcellular mechanism is involved in determining p53 stability(48, 49).

One of the most striking results from this study were thedifferences in the patterns of p53 expression observed byimmunocytochemical staining for p53 in cells obtained at

different points during the cell cycle. Both growth factor-deprived and lovastatin-arrested cells demonstrated an in-crease in the intensity and number of nuclei that stainedpositively for p53 when compared with exponentially grow-ing cells. The monoclonal antibody PAb 1 801 used for thesestudies recognizes a determinant located between aminoacids 32 and 79 ofthe p53 protein. This region contains twopotential sites for serine phosphorylation at amino acid po-sitions 33 and 46. Baunoch et a!. (50) have reported that

Cell Growth & Differentiation 301

4 J. M. Gudas, unpublished data.

phosphorylation at one or both ofthe sites within the regionrecognized by this antibody blocks antibody-ligand detec-tion in fixed cells. Thus, the possibility exists that the dif-ferent immunostaining patterns observed may be due to dif-ferential phosphorylation of p53 during the cell cycle.Alternatively, p53 may bind to another cellular protein suchas the mdm2 protein, and this interaction may render p53undetectable by PAb 1801 (51-53).

When the arrested cells were stimulated to proliferate, theintensity and number of nuclei staining positively for p53declined as the cells progressed through C1 . This effect wasmost pronounced for the lovastatin -treated and releasedcells where virtually none of the cells in mid-C1 stained forthe p53 antigen. The more pronounced difference in thestaining pattern observed in the lovastatin-synchronizedcells likely reflects the prolongation of the C1 period byabout 4 to 5 h when cells are synchronized by this method(compare Fig. 48 with Fig. 88). The increased time spent inthe G1 phase may permit a more uniform modification ofp53 to occur during this period in the lovastatin-synchronized cells.

Significantly, p53 was detected in the nucleus at all timesduring the cell cycle of NMECs. This is an important findingin viewofthefactthatnuclearlocalization isessentialfortheactivity of p53 (54). We have tried staining NMECs with avariety of p53 antibodies including PAbs 421, 1620, and240, which recognize both mutant- and wild-type-specificforms of the protein; however, none of these antibodies re-producibly recognized p53 in NMECs at any point duringthe cell cycle. These results are in agreement with otherimmunohistochemical studies of p53 in human cells wherethe protein has been localized to the nucleus in both normaland tumor cells. The finding of cytoplasmic p53 staining ina subset of inflammatory breast cancer patients (55) suggeststhat mechanisms distinct from those operative during thenormal cell cycle may also function to control the subcel-lular distribution of p53 in human mammary cells.

A distinct feature of the p53 expressed in NMECs is therecognition of the protein by both wild-type- and “mutant-specific” antibodies in immunoprecipitation reactions (30).�We were surprised to find only subtle differences in the abil-ity of different p53 antibodies to recognize the protein fromcell extracts prepared at different points during the cell cycle.These results contrast with other studies using mouse 313and neuroblastoma cells, where significant changes in therecognition of p53 by specific antibodies are observed fol-lowing release of cells from a growth factor-deprived state(56, 57). Because the latter studies were performed with im-mortalized ortransformed cells, other physiological changesthat occur during the process of tumor progression may ac-count for the differences observed in our studies.

Our cell cycle analyses of p53 expression in NMECs dif-fer markedly from those reported for immortalized mouse313 fibroblasts. We would like to emphasize that all ofthese experiments have been carried out with NMECs de-rived from at least two (and, in some cases, three) differentindividuals, and, therefore, the results cannot be attributedto some peculiarity of the cells obtained from a specificmammoplasty culture. In mouse cells, no p53 mRNA orprotein was detectable in the serum-starved quiescentstate (23, 24). Following stimulation of 313 cells with se-

rum, the amount of p53 mRNA and protein increasedduring the C1 phase and peaked prior to entry into Sphase. Strikingly, in murine cells, the p53 protein re-mained cytoplasmic until cells reached the G1-S bound-ary, whereupon the protein became detectable in thenucleus (23). In contrast, we found readily detectable 1ev-els of p53 in both lovastatin-arrested and growth factor-deprived NMECs. Although the total amount of p53 variedless than 2-fold in the Iovastatin-synchronized cells, weobserved a notable increase in p53 protein when thegrowth factor-deprived and restimulated cells entered intoS phase. The failure of mouse 3T3 cells to express signifi-cant levels of p53 in the quiescent state might reflect a lossof some function of the protein during the process of

immortalization.Our finding of novel p53 immunostaining patterns in ar-

rested and late G1 -S phase cells may reflect distinct functionsof p53 at different times during the cell cycle. In this regard,the wild-type p53 protein has been shown to be a transcrip-tion factor capable of activating (58-60) or repressing (61,62) transcription, depending upon the promoter examined.In addition, wild-type p53 has features of a DNA-bindingprotein and has been reported to inhibit the replication ofSV4O in vitro (61 -64). Lovastatin-treated and growth factor-arrested NMECs represent useful models to study the regu-lation of p53 gene expression and possible changes in p53function at distinct times during the cell cycle. An under-standing of the role of this protein at different points duringthe cell cycle in normal epithelial cells should, in turn, shedlight on the role of p53 in the progression to tumorigenesisin mammary and other epithelial cells.

Materials and Methods

Cell Culture and Cell Synchronization. Normal humanmammary epithelial cells derived from reduction mammo-plasties were obtained from Clonetics Corp. (San Diego,CA). Cells were cultured in MEBM (Clonetics Corp.) supple-mented with vitamins, 20 ng/ml epidermal growth factor, 10pg/mI insulin, 0.5 pg/mI hydrocortisone, and 1 3 mg/mI bo-vine pituitary extract. The complete medium is referred to asMEGM. For growth factor synchronization, 7 X i0� cells/1 50-mm tissue culture dish or 10� cells/4-well tissue culture

chamber slide (Lab Tek) were plated and allowed to grow inMEGM for 2 to 3 days. The MEGM was removed, the cellswere washed once with MEBM, and incubation was con-tinued for an additional 3 days in MEBM supplemented with0.05% bovine serum albumin (Sigma). At this time, fewerthan 5% ofthe cells were cycling, as determined by [3HJthy-midine incorporation and flow cytometric analysis of DNAcontent. The cells were restimulated to proliferate by theaddition of fresh MEGM containing the full complement ofgrowth factors.

Synchronization of cells to the C1 phase with lovastatinwas achieved by plating 8 x 1 0� cells/i 50-mm tissue culturedish or 2 X 10� cells/4-well tissue culture chamber slide andallowing them to grow for 2 to 3 days. At this time, themedium was removed and replaced with MEGM containing1 7 �M lovastatin (a generous gift from Dr. Albert Alberts,Merck Sharpe & Dohme). Conversion of the lactone ring tothe active form was performed as described previously (65).The cells were restimulated to proliferate by the addition offresh MEGM containing 2 mr�i mevalonic acid.

PCR-SSCP. DNA was extracted from subconfluent cul-tures and resuspended in 1 ml oflysis buffer(1 0 msi Tris-HCI,400 msi NaCI, and 2 m� EDTA, pH 8.2), treated for 30 mm

302 p53 Expression in NMECs

at 37#{176}Cwith 20 p1 of 10% SDS and 10 p1 of RNase (10mg/mI), and digested for 10 mm at 60#{176}Cwith 20 uI of Pro-teinase K (1 0 mg/mI). Saturated NaCI (0.67 ml) was added,and the tubes were mixed and centrifuged for 1 5 mm at 2500rpm. The supernatant was precipitated with 1 00% ethanol,washed and dissolved in 200 pl Tris-EDTA buffer, and stored

at -20#{176}C.DNA fragments corresponding to exons 5 through 9 of the

p53 gene were amplified by polymerase chain reaction, andmutations were detected as described previously (35). Spe-

cific oligonucleotide primers for the PCR reaction corre-sponding to intronic p53 sequences were synthesized witha 8700 DNA synthesizer (Milligen/Bioresearch) and purifiedusing standard techniques (34, 66). The PCR reactions wereperformed using 100 ng of genomic DNA and 0.5 p1 of[32PIdCTP in 1 O-pl reaction volumes, as described by Os-borne etal. (36). The samples were diluted in loading bufferand run at 35 W in a neutral 6% acrylamide gel at roomtemperature or at 4#{176}Cin the presence or absence of glycerol,respectively. The gels were then dried and exposed to anX-ray film for 5 to 12 h.

Flow Cytometry. Approximately 1 06 cells, synchronizedby the conditions described above, were trypsinized,washed three times in ice-cold PBS, and fixed in ice-cold

40% ethanol. The cells were centrifuged to remove the etha-nol, washed once with PBS, and resuspended in 0.5 ml PBScontaining 1 mg/mI chromomycin. After staining for 1 h at

4#{176}C,cytofluorometry was performed in a Becton DickinsonFacstar using an argon laser at 457 nm.

Immunocytochemistry and Autoradiography. Cellsgrowing in tissue culture chamber slides were washed three

times in ice-cold PBS and immediately fixed in methano-l:acetone (1 :1) for 3 mm. After allowing the slides to air dry,they were stored at -80#{176}C.Thefixed cells were stained usingthe anti-p53 monoclonal antibody PAb 1 801 (Ab-2) (On-cogene Science, Manhasset, NY), which had been diluted toa final concentration of 500 ng/mI. PAb 1801 reacts spe-cifically with a denaturation-stable epitope between aminoacids 32 and 79 in the amino terminus of p53 (67). Afterwashing in PBS containing 0.05% Tween 20, the specificcomplexes were visualized using the ABC immunoperoxi-dase system (Vector Laboratories, Burlingame, CA) accord-ing to the manufacturer’s recommendations.

Cumulative labeling of nuclei was performed by incubat-ing the cells in medium containing 1 �iCi/ml [3Hlthymidine(New England Nuclear). Incorporation of thymidmne wasstopped at the designated times by the addition of 1 M ascor-bate. The cells were washed three times with PBS and fixedin methanol:acetone (1 :1), and autoradiography was per-formed as described previously (68).

RNA and Northern Blot Analysis. Cells were washedthree times in ice-cold PBS and dissolved in a solution ofguanidine isothiocyanate, and the RNA was harvested aftercentrifugation over a 5.7 si cesium chloride cushion (69).Electrophoresis in denaturing formaldehyde gels and transferof RNA to Magna NT filters were performed as describedpreviously (70). The filters were hybridized with a 2.0-kbBamHl fragment derived from the human p53 cDNA, a0.8-kb Xbal-Pstl fragmentofthe mouse histone3.2gene, anda 0.8-kb PstI fragment from the cell cycle-independent ri-bosomal acidic protein gene, p36B4.

Immunoprecipitation of p53. Medium was removedfrom synchronized cells at the designated times and incu-bation continued in Dulbecco’s modified Eagle’s mediumwithout methionine for 1 h. The cells were labeled with

[35Sjmethionine (150 pCi/mI) for 2 h, and protein lysateswere prepared as described (71 ). Briefly, cells were washedthree times in ice-cold PBS and harvested in lysis buffer [50mM Tris-HCI (pH 8.0), 5 mtsi EDTA, 150 mM NaCI, 0.5%Nonidet P-40, 1 m� phenylmethylsulfonyl fluoride, 5 pg/mIaprotinin, 5 pg/mI leupeptin, 50 mt�i sodium fluoride, 10 mt�isodium orthovanadate, 1 0 mta sodium pyrophosphate, and1 m� �-glycerophosphate]. The lysates were precleared byaddition of anti-HCG monoclonal antibody followed by ab-sorption with Protein A plus Protein C agarose (OncogeneScience). Equivalent amounts of incorporated label (2 X 10�cpm) were subject to specific immunoprecipitation by in-cubating with 1 pg of monoclonal antibody (PAb 421 , PAb1801 , PAb 240, or PAb 1620), which corresponded to Ab-1,Ab-2, Ab-3, and Ab-5, respectively (Oncogene Science).After washing three times in SNNTE buffer and two timeswith radioimmunoprecipitation assay buffer, the immuno-precipitates were electrophoresed on 7.5% or 10%SDS-polyacrylamide gels, fixed, and subjected to fluorog-raphy (Enlightening, Dupont/NEN) prior to drying andautoradiography.

Western Blot Analysis. The cells were washed three timesand then scraped into ice-cold PBS. After centrifugation, thecell pellet was resuspended in 1 to 2 ml of 1 x SDS-polyacrylamide gel electrophoresis buffer [62 mt�i Tris-HCI(pH 6.8), 2 m� EDTA, 1 5% sucrose, 10% glycerol, 3% SDS,0.7 M 2-mercaptoethanoll, and the proteins were denaturedby boilingforlo mm. Protein(50 pg/lane) was electrophore-

sed on SDS-polyacrylamide gels and transferred to nitrocel-lulose. For p53 detection, the filters were blocked with Tris-buffered saline containing 5% dried milk and 0.1% Tween20 and then probed with 5 pg/mI pAB 1801 (Ab-2; Onco-gene Science). After washing in Tris-buffered saline contain-ing 0.1 % Tween 20, specific complexes were detected usingthe enhanced chemiluminescence technique according to

the manufacturer’s recommendations (ECL; Amersham). Ac-tin levels were examined by probing with 5 pg/mI actin an-tibody (Ab-1 ; Oncogene Science) and detecting specificcomplexes using the alkaline phosphatase method. The au-toradiographic films ofthe Western blots were analyzed witha Hoefer Scientific GS300 scanning densitometer.

Acknowledgments

We would like to thank Drs. Moshe Oren and William Marzluff for their giftsof the p53 and histone 3.2 probes, respectively. We are also grateful to Dr.

Lucy Gilbert for advice and encouragement with the immunocytochemistry,to Megan McCabe for assistance in quantitating the autoradiographic data,

and to Kristin Kelley and Hoang Nguyen for help with the synchronization

experiments. We appreciate the helpful discussions and comments on themanuscript from Drs. Erasmus Schneider and Vimla Band and are grateful to

Vimla Band for sharing her data prior to publication.

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