changes in gene expression in established human mammary ... · len hall,2 avril henney,3 david n....

(CANCER RESEARCH 46, 5786-5794, November 1986]

Changes in Gene Expression in Established Human Mammary Tumor Cell LinesWhen Compared with Normal Breast and Breast Tumor Tissue1

Len Hall,2 Avril Henney,3 David N. L. Ralphs,4 David G. Herries,5 and Roger K. Craig6

Cancer Research Campaign Endocrine Tumour Molecular Biology Group, Counauld Institute of Biochemistry [L. H., A. H., R. K. C.J, and Department of SurgicalStudies Â¡D.N. L. R.J, The Middlesex Hospital Medical School, Mortimer Street, London W IP 7PN, and Department of Biochemistry, University of Leeds Leeds LS29JT, [D. G. H.I, United Kingdom

ABSTRACT

RNA complexity analyses of total cellular polyadenylate-containingRNA isolated from lactating human breast tissue, human breast tumortissue, and a mixture of established cell lines of mammary origin demonstrate extensive homology between the tissue RNA populations butsuggest a decrease in the complexity of cell line nuclear RNA populations,with the exception of an early-passage MCF-7 cell line. Cell-free proteinsynthesis and two-dimensional gel electrophoresis also show quantitativeand qualitative differences in gene expression between human mammarytumor tissue and reduction mammoplasty or established mammary celllines of early and late passage number.

The results (a) demonstrate a major role for transcriptional andposttranscriptional mechanisms in the regulation of gene expression inthe human mammary gland and (b) show that studies on mammary glandgene expression using established cell lines of mammary origin reflectonly in part gene expression in normal lactating human breast or breasttumor tissues.

INTRODUCTION

The biological behavior of mammary carcinoma varies greatlybetween individual patients. Consequently there is a need forreliable markers which indicate both the long term prognosisand the response to therapy. At the present time the bestprognostic indicators are the extent and involvement of axillarylymph nodes (1). Knowledge of steroid-hormone receptor statusof tumors is valuable for the prediction of response to endocrinetherapy in advanced cancer of the breast and may have prognostic significance (2). Although the application of the techniques of molecular biology should provide the means to dissectmammary cancer at the molecular level, with the subsequentidentification of gene products of diagnostic and prognosticsignificance, the application of molecular techniques has beenlimited thus far to the identification of estrogen-responsive geneproducts of unknown prognostic value from several cell linesof human mammary origin (3, 4). Recombinant techniqueshave been used to clone and characterize cDNA7 sequences

encoding proteins of potential prognostic and diagnostic value.One cDN A sequence containing the coding sequence of humantt-lactalbumin proved valuable as a hybridization probe only ina negative sense, indicating that Â«-lactalbumin was not a validmarker for estrogen responsiveness in human mammary tumors(see Refs. 5 and 6). The potential value of a second cDNA

Received 8/2/85; revised 3/21/86. 7/10/86; accepted 8/5/86.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' The Cancer Research Campaign supported the bulk of this work.2Present address: Department of Biochemistry, University of Bristol, Bristol

BS8 1TD. United Kingdom.3 Recipient of a Studentship from the Medical Research Council.4 Present address: Norfolk and Norwich Hospital, Norwich, Norfolk NR1

3SR, United Kingdom.5 Present address: Department of Biology, University of York, York YO1

5DD, United Kingdom.*To whom requests for reprints should be addressed.7The abbreviations used are: cDNA, complementary DNA; poly(A), polyade-

nylate.

sequence, an estrogen-regulated gene product of the MCF-7cell line, remains to be established (7-9).

Here we have compared gene expression in human lactatingmammary tissue, human mammary tumor tissues, human mammary primary cell cultures, and established human cell lines ofmammary origin. The results show: (a) using RNA complexityanalysis a marked reduction in the overall number of transcribedpoly(A)-containing RNA species in established cell lines, thedecrease being particularly marked in the nuclear compartment;and (b) using cell-free protein synthesis and two-dimensionalgel electrophoresis, quantitative and qualitative differences inthe abundant and moderately abundant poly(A)-containingRNA species when comparing poly(A)-containing RNA fromtumor and reduction mammoplasty, or an established mammary cell line of early and late passage number. Our observations reveal significant differences at the transcriptional andposttranscriptional level of gene expression between cells present in mammary tumors and cell lines of mammary origin inmonolayer culture.

MATERIALS AND METHODS

Materials. [5-3H]dCTP (-20 Ci/mmol) and L-[35S]methionine(-1000 Ci/mmol) were obtained from Amersham International, Pic,

Amersham, United Kingdom. Tissue culture materials were from GibcoBio-Cult, Ltd., Paisley, Scotland, except for bovine insulin which wasfrom Sigma Chemical Co., Poole, United Kingdom. Avian myeloblas-tosis virus reverse transcriptase was provided by Dr. J. W. Beard, LifeSciences Inc., St. Petersburg, FL. Hydroxyapatite was from Bio-RadLaboratories, Ltd., Watford, United Kingdom. All other chemicals andsolvents were obtained from sources described previously (10, 11).

Source of Human Mammary Tissue Samples. The tissue we describeas "normal" lactating breast (hN 1) was obtained from a nursing mother,

15 days postpartum, and consisted of a large (2-g) retention cyst withvirtually normal histology, showing lactating mammary tissue surrounded by areas of severe inflammation and abscess formation but noevidence of malignancy. Mammary tumor samples (hTl, hT2, hT9,hT33) were obtained from postmenopausal patients, none of whom hadundergone either hormone therapy or chemotherapy prior to surgery.All tissue samples were deep-frozen in liquid nitrogen within minutesof excision and then stored at â€”70Â°Cuntil required.

Human Mammary Cell Lines. The MCF-7 mammary cell lines (passages 12 and 257) were obtained from Dr. C. M. McGrath, MichiganCancer Foundation; the Hs578T mammary cell line (late passage) wasfrom Dr. A. Hackett, University of California; and the ZR75-1 mammary cell line and reduction mammoplasty cells were from Dr. J. Easty,Ludwig Institute, Sutton, London, United Kingdom. MCF-7 andHs578T cell lines were grown in Eagle's minimal essential medium inEarle's balanced salt solution, nonessential amino acids, 1% (w/v)

glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 10% (v/v) newborncalf serum, penicillin (100 units/ml), streptomycin (100 units/ml), andbovine insulin (1 ng/ml). In the case of the ZR75-1 cell line 50% of theEagle's minimal essential medium was substituted by RPMI-1640.

Primary cultures of human reduction mammoplasty cells were grownfor 2 weeks in RPMI 1640 containing 10% (w/v) fetal calf serum,cholera toxin (50 Mg/ml), and bovine insulin (1 Â¿ig/ml).Frozen human

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CHANGES IN HUMAN MAMMARY GENE EXPRESSION

fibroblasts of mammary origin (hFlB-1; passage 4) were a gift from Â¿ig/mlto 2.5 mg/ml. Whenever possible high RNA concentrations were

Dr. J. Easty.Preparation of Total Poly(A)-containing RNA. Total cellular poly(A)-

containing RNA was isolated from frozen human lactating mammarytissue or mammary carcinoma as described previously (5). For cell linesa modified procedure was adopted. About 15 Petri dishes (9 cm indiameter) of each cell type were grown until semiconfiuent. The mediumwas then removed and cells were washed quickly in situ with phosphate-

buffered saline. Lysis was brought about by the addition to each dishof I ml of 20 mM Tris-HCl, pH 7.6, containing 1 rnw EDTA, 2% (w/v) sodium dodecyl sulfate, and proteinase K (250 Mg/ml). After incubation at 37"C for 2 h, the lysates were pooled, extracted with phenol-

chloroform, and processed as described for frozen tissue. Yields ofpoly(A)-containing RNA were typically 0.5-1% for mammary tissuesand 1-2% for established cell lines.

Preparation of Nuclear and Postnuclear Poly(A)-containing RNA.Nuclear and postnuclear supernatant fractions of human cell lines wereprepared in a manner similar to that described by Thomas et al. (12).Up to 60 dishes (9 cm in diameter) of cells were grown until semiconfiuent and then processed in batches of 15 dishes in the followingmanner. Medium was aspirated and the plates were then placed on icewater. Cells on each plate were rapidly washed with 10 ml of ice-coldhypotonie buffer (5 mM Tris-HCl, pH 7.4, containing 1.5 mM KC1 and

2.5 mM MgCh) and then lysed on ice with 1.5 ml of hypotonie buffercontaining 6 mM spermidine-HCl, 1% (v/v) Triton X-100, and 1.6%(w/v) sodium deoxycholate for 5 min. Lysates from 15 dishes werepooled after gentle pipeting to release nuclei from the plastic andcentrifuged at 2500 x g,â€žâ€žâ€žfor 5 min (0Â°C).The postnuclear super

natant was made 0.5% (w/v) with respect to sodium dodecyl sulfateand 100 Â¿ig/mlwith respect to proteinase K and incubated at 45Â°Cfor

30 min. The sedimented nuclei were resuspended in 20 ml of hypotoniebuffer containing 6 mM spermidine-HCl and 1% (v/v) Triton X-100,

centrifuged as described above, and then resuspended in 5 ml of 20 mMTris-HCl, pH 7.4, containing 1 mM EDTA. Sodium dodecyl sulfateand proteinase K were added to the lysates to concentrations of 2% (w/v) and 250 Â¿<g/ml,respectively and incubated at 45Â°Cfor 30 min.

Nuclear and postnuclear preparations were then precipitated withethanol, reprecipitated to remove traces of detergent, and incubatedwith iodoacetylated DNase (20 Â¿ig/ml)(13) for 20 min on ice in 10 mMTris-HCl, pH 7.4, containing 10 mM NaCI and 10 mM magnesiumacetate. Proteinase K and sodium dodecyl sulfate were then added toeach and the mixtures were incubated as described above, then extractedwith phenol-chloroform, and concentrated by ethanol precipitation.The poly(A)-containing RNA was then isolated from each preparationby two cycles of affinity chromatography on oligodeoxythymidylate-cellulose, as described previously (10).

Nuclear and postnuclear RNA preparations were obtained fromfrozen mammary tissue samples by a similar procedure. In this case thefrozen tissue was first ground in liquid nitrogen using a mortar andpestle and then plunged into the lysis buffer and homogenized gentlyby hand to ensure efficient lysis. The lysate was then processed asdescribed above.

Synthesis of cDNA and Fractionation into Abundance Groups. Highspecific radioactivity ['HjcDNA (1.4 x IO7cpm/^ig) complementary to

poly(A)-containing RNA isolated from normal lactating mammarytissue, breast tumors, and cell lines was prepared as described by Craiget al. (Il ). Abundant and scarce-frequency cDNA populations, transcribed from MCF-7 nuclear poly(A)-containing RNA, were prepared

by fractionation on hydroxyapatite as described by Bathurst et al. (14).A tumor tissue-specific cDNA probe (representing sequences not foundin late-passage cell lines) was prepared in the same manner.

Hybridization Procedures. RNA excess hybridizations were performed at 68Â°Cin 50 mM 4-(2-hydroxyethyl)-l-piperazineethane-sul-

fonic acid, pH 7.4, containing 0.6 mM NaCI, 1 mM EDTA, and 0.2%(w/v) sodium dodecyl sulfate. All other procedures were as describedby Craig et al. (il ), except that glass capillaries were boiled in 1 M HCIbefore the siliconizing treatment. Hybridizations were carried out forperiods from 10 s and 116 h at RNA concentrations ranging from 2.5

used in preference to long hybridization times.Numerical Analysis of Hybridization Kinetics. Hybridization curves

resolved into a number of first-order components using a computerprogram developed for the least-squares analysis of hybridization ki

netics (15). The correction coefficient of fit for all homologous hybridization curves was better than 0.998. Attempts to fit fewer componentsthan those shown always produced significantly poorer correlationcoefficients. The nucleotide complexity of each transition was calculated, based on the hybridization kinetics of a purified globin mRNAstandard as described by Craig et al. (11). To estimate the number ofdifferent RNA species in each transition, the number average RNA sizeof each poly(A)-containing RNA preparation was determined by sucrosegradient centrifugation under denaturing conditions, followed by | '111

polyuridylate assays of gradient fractions, as described previously (11).In all cases a size distribution was obtained with a number average sizeof around 1350 nucleotides.

Cell-free Protein Synthesis and Product Analysis. Cell-free proteinsynthesis was performed in a nuclease-treated reticulocyte cell-freesystem as described by Pelham and Jackson ( 16). while two-dimensionalgel electrophoretic analysis of the in vitro synthesized [-"S]methionine-labeled polypeptides was as described by O'Farrell (17). Fluorography

was performed as described by Bonner and Laskey (18), except thatacetic acid replaced dimethyl sulfoxide (19). Protein molecular weightmarkers electrophoresed in the second dimension were phosphorylaseb (M, 92,000), bovine serum albumin (M, 69,000), ovalbumin (M,46,000), carbonic anhydrase (M, 30,000). and lysozyme (M, 14,000).

RESULTS

Comparative RNA Complexity Analysis of Total Cellular,Nuclear, and Cytoplasmic Poly(A)-containing RNA PopulationsIsolated from Human Mammary Tissues, and Human Cell Linesof Mammary Origin. Comparative analysis of the nucleotidebase sequence complexity of total cellular poly(A)-containingRNA isolated from lactating human mammary tissue, humanmammary tumor tissue, and established human cell lines ofmammary origin showed differences in terms of overall complexity. Examination of the hybridization kinetics of high specific activity pHJcDNA prepared from poly(A)-containing RNAisolated from the lactating mammary gland by hybridizationagainst an excess of its homologous RNA produced a curvewhich could be resolved into four first-order components, hybridizing over six logarithmic decades. Calculation of the basesequence complexity within each component by comparisonwith the kinetics of a highly purified rabbit globin poly(A)-containing RNA standard (Fig. 1/1), demonstrated (on the basisof a number average molecular weight of 1350 nucleotides) thatthe most rapidly hybridizing or very abundant class contained2-3 sequences, the abundant class about 40 sequences, theintermediate class about 1800 sequences, and the scarce classabout 30,000 sequences (see Table 1). Analysis of the kineticsof hybridization of poIy(A)-containing RNA populations isolated from two mammary tumors (hT2 and hT33) in the samemanner produced curves which could be resolved into threecomponents (Fig. IA; Table 1). In each instance the very abundant poly(A)-containing RNA class, which we have previouslyshown to be representative of the human milk protein mRNAspecies (5, 6, 20), was absent, but the overall complexity anddistribution of sequences within the three overall abundanceclasses were very similar to those of the abundant, intermediate,and scarce-frequency classes identified in poly(A)-containingRNA isolated from lactating mammary tissue.

In contrast to the results obtained for tissue poly(A)-contain-ing RNA populations, similar analyses of total poly(A)-contain-

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Fig. 1. Homologous and heterologous RNA complexity analyses of totalcellular, nuclear, and cytoplasmic poly(A)-containing RNA isolated from humanmammary tissue and cell lines. RNA excess hybridization procedure and dataanalysis were as described in "Materials and Methods." I. hybridization of totalcellular poly(A)-containing RNA derived from human lactating tissue (O) andhuman mammary tumor (hT2) tissue (â€¢)with their homologous cDNAs. Hybridization of highly purified rabbit globin mRNA to its cDNA (A) provided a kineticstandard. B, hybrization of total cellular poly(A)-containing RNA derived fromearly- (â€¢)and late- (O) passage MCF-7 cells to their homologous cDNAs( , globin kinetic standard). C, heterologous hybridization between humanmammary tumor (hT2) tissue (O) or late-passage MCF-7 cell line (â€¢)totalpoly(A)-containing RNA and human lactating tissue (hNl) cDNA. /'. heterologous hybridizations between human mammary fibroblast alone (â€¢)or fibroblastplus MCF-7 cell line (O) total poly(A)-containing RNA and human lactatingtissue (hN I ) cDNA. Arrows, corrected Rofwvalues for each first-order component.

ing RNA from two well-established human mammary tumorcell lines (MCF-7 passage 275 and Hs578T) showed for each athree-component curve of much reduced overall base sequencecomplexity (Table 1; see also Fig. IB). This was particularlyapparent in the intermediate and scarce-frequency classes wherethe number of different poly(A)-containing RNA species appeared to be reduced by as much as 50%. However, analysis ofvery-early-passage MCF-7 cells (passage 17) revealed a distribution of sequence complexity indistinguishable from the tissuesamples (Table 1; Fig. IB), and consequently of greater overallcomplexity than that present in MCF-7 late-passage cells (Table1; Fig. IB). Analysis of total poly(A)-containing RNA from ahuman mammary fibroblast cell line (hFlB-1) in early passage

Table 1 Complexity of RNA populations derived from human lactatingmammary tissue, mammary tumors, and mammary cell lines

Calculations of the relative number of RNA species present in differentabundance groups reflect the RNA complexity (number of nucleotides) dividedby the number average molecular weight ( 1350 nucleotides); see "Materials andMethods." The standard derivatives of these values are the square roots of their

variances. Each value is based on a sufficiently large number of measurements(percentage of hybridization and Rot) to justify using a table of the normaldistribution rather than tables of the t distribution. The significance of differencesbetween pairs of values (see text) may be determined by calculating

Value I - Value 2

â€¢^Variance!+ Variancej

where variance is the square of standard deviation. The P value may then bedetermined by reference to probability distribution tables.

No. of different RNAspeciesTotal

cellularpolyaden-ylatedRNALactating

tissue(hNl)Mammarytumortissue

(hII)Mammarytumortissue

(hT33)Mammarytumortissue

(hT2)MCF-7(earlypassage)MCF-7(latepassage)Hs578ThFlB-1Reduction

mammo-plastyVery

Abun-abundantdant2Â±

1Â° 43Â±817Â±325

Â±618Â±347

Â±925Â±629Â±824Â±548

Â±9Intermediate

Scarce1,830

Â±310 30,950Â±2,8001,630Â±250 27,3 10Â±2,4301,450

Â±250 26,350Â±2,9501,750

Â±270 29,260 Â±3,8002,140

Â±360 29,860 Â±2,370890Â±160 15,420+1,070800Â±220 15,740 Â±1,2602,000+ 410 21,070Â±2,1001.700Â±

260 20.160Â± 1,980

Postnuclear polyaden-ylated RNA

Mammary tumortissue (hT33)

MCF-7 (late passage)ZR75-1Hs578T

25 Â±6 1,490 Â±530 8,780 Â±1,450

21 Â±629 + 746 Â±8

910+ 230670 Â±220

1,760+590

6,940+ 1,0605,090 Â±4206,410 Â±930

Nuclear polyadenylatedRNA

Mammary tumortissue (hT33)

MCF-7 (late passage)ZR75-1Hs578T47

Â±1242

Â±1840 Â±1947 Â±1328,160Â±

2,0701

9,980 Â±1,45017,140 Â±1,15019,090 Â±1,070

â€¢Mean + SD.

(passage 4) and reduction mammoplasty epithelial cells in primary culture also revealed a three-component curve and anucleotide base sequence complexity midway between the mammary tissues and late passage cell lines, the reduction in complexity being confined to the scarce frequency class (see Table1).

In order to determine within the limitations of the experimental procedure whether poly(A)-containing RNA sequencesexpressed in breast tumor tissue were common to those expressed in normal lactating tissue and also whether sequencespresent in the cell lines were also present in normal tissue, wehave performed a series of heterologous cross-hybridizationexperiments using cDNA prepared from the normal lactatingtissue (hNl), total cellular poly(A)-containing RNA isolatedfrom mammary tumor tissue (hT2), and late-passage MCF-7cells. Due to a scarcity of poly(A)-containing RNA originatingfrom normal lactating tissue, the reciprocal crosses were notpossible. However, the results (Fig. 1C) demonstrate that themajority of sequences present in the lactating tissue cDNAcould form stable hybrids with the breast tumor poly(A)-con-taining RNA since hybridization reached 82% (Fig. 1C), a value5-10% lower than the corresponding homologous hybridization. This difference will reflect to a very great extent the

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absence in tumor tissue of the very abundant class of poly(A)-containing RNA representing the milk protein RNA transcripts, as we have shown elsewhere that one of these, encodinga-lactalbumin, is absent from breast tumor tissue (5). Consequently, with the exception of these very abundant sequences,the normal and malignant breast tissue total cellular poly(A)-containing RNA populations exhibit a high degree of sequencehomology. Heterologous hybridization between total cellularpoly(A)-containing RNA and cDNA from two different mammary tumors also revealed extensive sequence homology, indistinguishable from the corresponding homologous hybridizations (data not shown).

Consistent with results obtained from nucleotide complexityanalysis (Table 1), total poly(A)-containing RNA from late-passage MCF-7 (Fig. 1C) or Hs578T cell lines (data not shown)hybridized to only 60-65% of those sequences present in cDNAsynthesized from the lactating tissue poly(A)-containing RNApopulation. Thus a large proportion of the poly(A)-containingRNA sequences present in the normal lactating tissue, and byinference tumor tissue, appear not to be expressed in theequivalent poly(A)-containing RNA populations in late-passagecell lines. Similar analyses using poly(A)-containing RNA fromthe mammary fibroblast cell line (hFlB-1) resulted in hybridization to 72% of the lactating tissue cDNA, a value increasedby only 1-2% when an equal mixture of late-passage MCF-7and hFlB-1 poly(A)-containing RNA was used (Fig. ID). Thisresult, in keeping with the complexity of the early-passageMCF-7 cell line, suggests that the greater complexity of themammary tissue poly(A)-containing RNA population cannotbe explained simply by the presence of different cell types withinmammary tissues, each with qualitatively different patterns ofexpression thereby accounting for the increased RNA complexity observed. Additional heterologous cross-hybridizations between poly(A)-containing RNA and cDNA from late-passageMCF-7 cells and Hs578T cells revealed extensive homology; inthis instance hybridizations were only 5% less than the corresponding homologous hybridizations (data not shown). Thustwo independently established mammary tumor cell lines showreduced complexity when compared with tissue poly(A)-con-taining RNA populations and express in qualitative terms similar poly(A)-containing RNA populations.

Nuclear and Cytoplasmic Distribution of Poly(A)-containingRNA in Mammary Tumors and Cell Lines of Mammary Origin.Poly(A)-containing RNA was isolated from nuclear and post-nuclear preparations of a breast tumor (hT33) and several celllines of mammary origin (late-passage MCF-7, Hs578T, ZR75-1), cDNA was prepared from each, and the kinetics of hybridization of each was determined against their homologous RNApopulations. Analysis of the kinetics of hybridization of post-nuclear preparations from the late passage MCF-7 cell line andtumor tissue (hT33) revealed three-component hybridizationcurves (Fig. 2). In each instance the first two transitions weresimilar in RNA complexity to those observed in the corresponding analyses of total cellular poly(A)-containing RNA (see Table1). The postnuclear scarce-frequency class, however, showed asignificantly reduced complexity (P < 0.001) when comparedwith the total cellular population for both tissue (8,800 against26,400) sequences) and cell line (7,000 against 15,700) sequences. Similar analyses of nuclear poly(A)-containing RNApopulations when hybridized against their homologous cDNApreparations exhibited, as expected, markedly different kineticsof hybridization to either total or postnuclear poly(A)-contain-ing RNA preparations. In this instance, two component curves

Fig. 2. Distribution of poly(A)-containing RNA species between the nucleusand cytoplasm of mammary tumor tissues and cell lines. Hybridizations wereperformed between nuclear (O) or postnuclear (â€¢)poly(A)-containing RNApreparations derived from MCF-7 mammary cell line (A) or mammary tumor(HT33) tissue (B), and their corresponding homologous cDNAs. Similar analyseswith total cellular poly(A)-containing RNA and homologous cDNA are shownfor comparison ( ).

were obtained by analysis of tumor tissue and MCF-7 cell linenuclear poly(A)-containing RNA populations (Fig. 2). The

abundant class in tissue and cell lines had similar complexitiesrepresentative of 40-50 poly(A)-containing RNA species (Table1), but the scarce class of nuclear sequences differed markedlyin complexity. Thus there were about 20,000 nuclear scarce-frequency poly(A)-containing RNA species present in the late-passage MCF-7 cell line compared with 28,000 in the equivalenttumor population. Analysis of nuclear and postnuclear poly(A)-containing RNA populations obtained from two other late-passage mammary tumor cell lines, Hs578T and ZR75-1, produced a distribution of nuclear and postnuclear poly(A)-con-taining RNA sequences very similar to that observed for thelate-passage MCF-7 cells (see Table 1).

To examine in greater detail the relative distribution of thecell line nuclear poly(A)-containing RNA abundance classeswithin the postnuclear poly(A)-containing RNA population,cDNA was synthesized from late-passage MCF-7 nuclearpoly(A)-containing RNA, then fractionated into the two nuclearabundance groups by hybridization to the homologous RNApopulation to a R0t of 5.6 mol â€¢liter"'-s~' followed by separation

of the single-stranded scarce-frequency cDNA from the hybridized abundant cDNA species on hydroxyapatite. Hybridizationof the individual purified cDNA populations to MCF-7 nuclearpoly(A)-containing RNA demonstrated (Fig. 3) that both hybridized with the expected kinetics, with little evidence of cross-contamination between the abundance groups. Examination ofthe distribution of each nuclear cDNA population within thepostnuclear poly(A)-containing RNA population by heterologous cross-hybridization produced contrasting results. Theabundant nuclear cDNA hybridized essentially to completionover 2.5 logarithmic decades, with kinetics similar to that ofthe abundant postnuclear poly(A)-containing RNA population.The scarce-frequency nuclear population, however, did not hybridize to completion, hybridization occurring over 3.5 logarithmic decades and reaching a value of about 60% relative tothe corresponding homologous hybridization at high Rot values(see Fig. 3). Although hybridization occurred predominantlywith the expected kinetics of scarce-frequency postnuclearpoly(A)-containing RNA species, low yet significant hybridi-

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Fig. 3. Distribution of abundant and scarce nuclear poly(A)-containing RNAtranscripts within the nucleus and cytoplasm of MCF-7 cells. cDNA synthesizedfrom late-passage MCF-7 nuclear poly(A)-containing RNA was fractionated intoabundant and scarce populations. These were then hybridized to either nuclearor postnuclear poly(A)-containing RNA derived from MCF-7 cells, as follows:abundant nuclear cDNA versus nuclear (O) or postnuclear (â€¢)RNA; scarce nuclearcDNA versus nuclear (A) or postnuclear (A) RNA. Hybridizations between totalnuclear cDNA and postnuclear cDNA and their corresponding RNAs (see Fig.2/0 are shown ( ) for comparison.

zation also occurred with kinetics similar to the intermediatepostnuclear poly(A)-containing RNA population. However, in

spite of this observation, the results clearly demonstrate that asmuch as 40% of the scarce nuclear poly(A)-containing RNAspecies were specifically confined to the nucleus and that theremainder constituted predominantly the scarce frequency post-nuclear poly(A)-containing RNA class. A series of heterologoushybridizations using the MCF-7 abundant- and scarce nuclearcDNA probes driven with nuclear and postnuclear poly(A)-containing RNA isolated from Hs578T and ZR75-1 cell lineswere also performed, producing kinetics of hybridization essentially identical to those described for the late-passage MCF-7cells (results not shown).

The results presented above implicate posttranscriptionalevents (see "Discussion") and provide further evidence that the

disparity in total complexity between tissue and late-passagecell line poly(A)-containing RNA populations resides in the

nuclear fraction, since subcellular fractionation did not revealmajor qualitative disparities between the tumor or cell linepostnuclear poly(A)-containing RNA populations. In order toestablish that a significant subset of the nuclear polyadenylatedRNA sequences expressed in tumor tissue were not expressedin the corresponding late-passage cell line populations, a seriesof heterologous cross-hybridizations were performed betweencDNA populations synthesized from tumor (hT33) postnuclearand nuclear poly(A)-containing RNA, and late-passage MCF-7postnuclear and nuclear poly(A)-containing RNA populations.This demonstrated (Fig. 4A) that the kinetics and degree ofhybridization of MCF-7 postnuclear poly(A)-containing RNAto the equivalent tumor cDNA preparation were strikinglysimilar to those of the tumor cDNA with its homologouspostnuclear poly(A)-containing RNA population but that theanalogous nuclear cross-hybridization resulted in only 70%protection of the tumor nuclear cDNA population by the MCF-7 nuclear poly(A)-containing RNA population, when comparedwith the tumor nuclear homologous hybridization (see also Fig.2B). To further establish whether the differences reflect theabsence of tumor scarce-frequency nuclear sequences from thelate-passage MCF-7 nuclear poly(A)-containing RNA population, a tumor tissue-specific cDNA population was generatedby hybridization of late-passage MCF-7 total cellular poly(A)-containing RNA with cDNA derived from breast tumor (hT33)total poly(A)-containing RNA to a Rot of 316 mol â€¢liter"'-s~',followed by recovery of the "tumor-specific" unhybridized sin

gle-stranded cDNA on hydroxyapatite. Hybridization analyseswith the tumor tissue-specific cDNA probe revealed (Fig. 4B)

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Fig. 4. Distribution of mammary tumor tissue-specific cDNA sequences, notexpressed in tumor cell lines. In A, heterologous hybridizations were performedbetween either mammary tumor (hT33) tissue nuclear (O) or postnuclear (â€¢)cDNA and the equivalent nuclear or postnuclear poly(A)-containing RNA preparation derived from late-passage MCF-7 cells. The corresponding homologoushybridizations ( ) are shown for comparison. In B, a mammary tumortissue-specific cDNA population containing sequences not expressed in MCF-7cells was prepared as described in the text. This cDNA was then hybridized tomammary tumor (hT33) tissue nuclear (O), postnuclear (â€¢),or late-passage MCF-7 total cellular (A) po!y(A)-containing RNA preparations.

negligible hybridization to the late-passage MCF-7 total cellularpoly(A)-containing RNA, slight hybridization to tumor tissuepostnuclear poly(A)-containing RNA, but substantial hybridization to tumor tissue nuclear poly(A)-containing RNA, withthe expected kinetics of the scarce-frequency class of nuclear

sequences. These results demonstrate that a subset of the leastabundant "tumor tissue" and by analogy "normal tissue" nu

clear poly(A)-containing RNA sequences (see Table 1 and Fig.

1C) are not expressed (or expressed at a very much reducedlevel) in mammary tumor cell lines after prolonged culture invitro.

Differential Expression of PoIy(A)-containing RNA Sequencesin Mammary Tissues and Cell Lines as Determined by mRNA-directed Cell-free Translation Systems. Quantitative and quali

tative changes in individual gene expression were investigatedin tissue and cell line poly(A)-containing RNA preparationsusing a mRNA-directed reticulocyte lysate cell-free translationsystem and the distribution of in vitro synthesized ["SJmethio-nine-labeled proteins examined by two-dimensional gel electro-

phoresis and fluorography. The results revealed contrastingdistributions of 35S-labeled proteins when translation products

directed by tumor tissue and reduction mammoplasty, or MCF-7 early- and late-passage poly(A)-containing RNA preparationswere compared. In all instances (Fig. 5) a wide spectrum of 35S-

labeled proteins were synthesized ranging in molecular weightfrom 14,000 to in excess of 100,000, with the majority of theproteins focusing within a pi range of 4.5-7.3. Comparison oftumor (hT9) and reduction mammoplasty cell-free productsrevealed a very different pattern of predominant 35S-labeled

proteins (Fig. 5, A and B). Some of the differences observedbetween the two populations were quantitative in nature, but ingeneral significant seemingly qualitative differences were alsoapparent, within the abundant and less abundant sequence

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30

14

Fig. 5. Differential gene expression in human mammary lumor tissue, reduction mammoplasty, and cell lines of mammary origin. Total poly(A)-containing RNAwas translated in a nuclease-treated reticulocyte-lysate cell-free system in the presence of Â¡"S]methionine, and the individual translation products (20,000-30,000cpm) were identified by two-dimensional polyacrylamide gel electrophoresis and fluorography. Arrows, position of the major endogenous reticulocyte protein:molecular weight markers were as described in "Materials and Methods." I. mammary tumor hi'): B. reduction mammoplasty; C, MCF-7 cell line early passage; D,

MCF-7 cell line lale passage.

classes within the two poIy(A)-containing RNA populations

examined.The comparison of MCF-7 early- and MCF-7 late-passage

cell-free translation products also revealed differences in themajor ["SJmethionine-labeled protein products (Fig. 5, C and

D). In particular a number of abundant proteins (M, 60,000, pi6.0; A/r 44,000, pi 5.3; M, 18,000, pi 5.6; M, 13,000, pi 6.0)encoded by MCF-7eariy poly(A)-containing RNA were not directed in detectable amounts by the MCF-7iale poly(A)-contain-ing RNA preparation. Conversely a number of predominantgene products (M, 64,000, pi 5.3; M, 47,000, pi 6.9; M, 44,000,pi 4.6; M, 43,000, pi 4.6; M, 24,000, pi 4.6) were directed bypoly(A)-containing RNA from MCF-7|ale as opposed to MCF-7eariycells. Differential gene expression was also apparent in

early- and late-passage MCF-7 cell-free translation productswithin the putative actin gene cluster (M, 46,000, pi 5.6-5.8).

DISCUSSION

The advent of recombinant DNA technology and the subsequent use of sequence-specific DNA hybridization probes hasin recent years eclipsed the value of RNA complexity analysesusing liquid hybridization techniques. To a large extent this hasbeen accelerated by an increased awareness of the limitationsof complexity analysis, particularly in terms of quantitation.Hybridizations in solution rarely go to completion and cDNAprobes, if not prepared under optimum conditions and carefullycharacterized, tend to be more representative of the 3' ends of

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the RNA templates. Both of these technical drawbacks lead touncertainty if accurate quantitation is required. In addition,problems can arise through numerical presentation of data.Thus whereas nucleotide complexity of RNA populations maybe considered as absolute values when compared with a common standard (globin mRNA), calculations of the numbers ofRNA species present in different RNA populations illustraterelative differences only. This is because the calculation of thenumber of different RNA species is based on the numberaverage weight of each RNA population, a value which maydiffer from tissue to tissue and which is inevitably biased by thepresence of a small number of relatively abundant RNA species.Taking into account such variables, it is hardly surprising thatstudies with cloned, sequence-specific probes, which tend notto suffer from these limitations, are proving more popular.However, it is rather naive to think that careful analyses withjust a handful of these specific, cloned probes alone will allowus to make general statements about the overall control of themany thousands of RNA transcripts found in a mammalian cellat any given time. This led us to reappraise the value of RNAcomplexity analysis and apply the technique in such a way thatany conclusions we made were not solely dependent on theaccuracy of quantitation. Consequently, whenever a differencewas found between cell types or cell compartments, in terms ofcalculated RNA complexity (or in this instance RNA species,since a number average molecular weight of 1350 nucleotideswas used in all calculations), these differences were furtherconfirmed by appropriate heterologous hybridizations, or hybridization with individually purified components. In this waymost of the above uncertainties can be overcome or minimized.

We have previously used hybridization analysis to investigatethe relative complexity of nuclear and cytoplasmic poly(A)-containing RNA populations in lactating guinea pig tissues.Such studies have demonstrated a major role for posttranscrip-tional events in the preferential accumulation of transcribedsequences in the cytoplasm, and the tissue-specific distributionof transcribed nuclear poly(A)-containing RNA sequences between nucleus and cytoplasm (14, 21). Our studies on nuclearand cytoplasmic poly(A)-containing RNA populations fromhuman mammary tumor tissue and a number of establishedmammary cell lines of tumor origin extend these observations.In common with the animal model system, in normal lactatinghuman breast tissue, in breast tumor tissues, and in establishedcell lines of mammary origin, posttranscriptional mechanismsoperate which determine the preferential accumulation andrelative abundance of transcribed sequences in the cytoplasm.Such studies are clearly dependent on the purity of nuclear andpostnuclear preparations, and for this reason the isolation ofmRNA from purified polysomes may seem preferable. However, while this is technically possible for the established celllines, preparation of polysomes in reasonable yield from humantumor tissue of this type is notoriously difficult and could leadto selective losses of particular sequences. We therefore choseto adopt a method based on that of Thomas et al. (12) whichresults in a nuclear and postnuclear RNA preparation in goodyield and which, on the basis of homologous and heterologoushybridization experiments, results in minimal cross-contamination. However, even slight contamination of the less complexpostnuclear population with the more complex nuclear population could significantly affect the observed complexities ofthese two fractions, as given in Table 1, and such values shouldbe taken as an indication only. Nevertheless, such uncertaintywould not affect our overall conclusion that postranscriptionalcontrol mechanisms play an important part in mammary cell

lines and tissues since (a) the complexity of profiles for nuclearand postnuclear populations are clearly very different, with adifferent number of components, and (b) the observation thatas much as 40% of the scarce nuclear sequences are not expressed in the cytoplasm must be taken as a minimum estimatesince nuclear contamination of the postnuclear fraction wouldreduce this value. In general, therefore, cross-contaminationbetween nuclear and postnuclear fractions would tend to decrease the observed differences between these two cell compartments and hence underestimate the role played by posttransla-tional mechanisms.

The similarity between the total complexity of poly(A)-con-taining RNA in normal lactating breast and breast tumor tissueis in agreement with studies on a number of experimentalanimal model systems, in particular liver, and cell lines transformed by viruses or carcinogens using total, cytoplasmic, ornuclear polyadenylated RNA (22-25). These studies also demonstrated little variation in the overall complexity of poly(A)-containing RNA from normal and transformed tissues and celllines, although, where analyzed, differences in the relative abundance of sequences or groups of sequences were observed (24,25). Our preliminary studies using mRNA-directed cell-freeprotein synthesis and two-dimensional sodium dodecyl sulfate-polyacrylamide gel analysis of the labeled products support theview that differences in the relative abundance of some sequences exist between tumor tissue and "normal" reduction

mammoplasty cells, as well as between early- and late-passageMCF-7 cells.

It is apparent that the complexity of total cellular poly(A)-containing RNA in an early-passage clonal cell line (MCF-7eariy)is within experimental error the same as the total cellularpoly(A)-containing RNA present in normal lactating breast ortumor tissues, each containing many cell types, the formerpredominantly epithelial (see Ref. 20), but the latter a polyglotof cell types. This suggests that within mammary tissue aremarkably similar spectrum of poly(A)-containing sequencesis transcribed in all cell types and that the greater complexityof total sequences in tissues, when compared with establishedcell lines, cannot be explained simply by the expression ofdifferent sets of genes in the different cell types. This conclusionis supported by additive experiments where no significant increase in overall complexity was observed using mixtures ofpoly(A)-containing RNA isolated from primary mammary fi-broblast cultures and late-passage MCF-7 cells. Thus, withinexperimental limits, in human mammary tissue a similar spectrum of genes (excluding the major lactation-specific milk proteins; see Refs. 6 and 20) is expressed, irrespective of cell typeand irrespective of whether the RNA is derived from normal ortumor tissue. Such observations argue that in mammary tissue,posttranscriptional mechanisms play a major role in the determination of cell phenotype, a conclusion in keeping with earlycomparative work using sea urchins, which showed that transcribed single-copy nuclear sequences are common to embryoand adult tissues but that cytoplasmic mRNA populations differ(26-28).

The apparent decrease in the number of transcribed nuclearpolyadenylated sequence during long-term culture and, in thecase of the early and late MCF-7 cell lines, an associated changein relative abundance of the major mRNA species, may alsohave important implications. Numerous comparisons have beenmade between the relative complexities of polysomal poly(A)-containing RNA species from cell lines and normal and tumor-derived tissues, generally resulting in reports of qualitative aswell as significant quantitative differences, particularly in the

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abundant mRNA species (29-32). Relatively few studies havecompared nuclear and cytoplasmic poly(A)-containing RNAfrom normal and tumor tissue, as well as cell lines of similarorigin. Fausto et al. (25) working with normal, regenerating,and neoplastic rat liver reported quantitative, as opposed toqualitative, changes in nuclear polyadenylated RNA sequences,while others, comparing normal rat liver with transplantablehepatoma preparations, reported the loss of a substantial proportion of nuclear sequences in the hepatoma when comparedwith normal liver (33, 34).

It is our opinion that the difference in transcribed nuclearsequences, seen in the cell lines and tissues in question, mayreflect conditions of cell culture and hence cell shape. Recentstudies on the growth and differentiation of normal mammaryepithelial cells in culture have demonstrated the importance ofthe extracellular matrix and the effect of cell shape on themaintenance of their differentiated state (35-38). The extracellular matrix has also been shown to be crucial for the sustainedgrowth and three-dimensional organization of primary mammary epithelial cells (39), and for the reconstitution of branching tubule structures from tumor cell lines in culture (40). Fromour data it is apparent that even after short-term culture asignificant loss of scarce-frequency transcribed sequences occurs (compare reduction mammoplasty or mammary' fibroblasts

in primary culture with breast tissue preparations). Observations similar to ours have been made using poly(A)-containingRNA isolated from various regions of the brain. These showedan equivalence of complexity, whereas a reduced complexitywas observed in primary glial cell cultures, glioma, and neuroblastoma preparations (41). By analogy with our own data wewould predict that this reduced complexity reflects, the preferential loss of nuclear sequences.

In conclusion, our studies (a) raise interesting questions asto the role of posttranscriptional events in the regulation ofgene expression and (/>)show that studies on mammary glandgene expression using established cell lines may only partlyreflect expression in breast tissues. Such observations shouldbe borne in mind when using human mammary tumor cell linesgrown in monolayer culture, as model systems for the study ofhuman breast cancer, particularly when designing strategies forthe identification and isolation of gene probes of potentialdiagnostic or prognostic value. A better approach might be toidentify genes expressed only in tumors as opposed to normalbreast tissue, when using defined probes return to establishedcell lines to determine whether or not they are expressed andultimately what factors modulate their expression.

ACKNOWLEDGMENTS

We are indebted to Dr. Jerry Easty, Ludwig Institute of CancerResearch, Sutton, United Kingdom, for preparing and providing thehuman mammary fibroblast and reduction mammoplasty primary cellcultures.

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1986;46:5786-5794. Cancer Res Len Hall, Avril Henney, David N. L. Ralphs, et al. Breast Tumor TissueTumor Cell Lines When Compared with Normal Breast and Changes in Gene Expression in Established Human Mammary

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