Keratin expression in human mammary epithelial cells cultured from normal

and malignant tissue: relation to in vivo phenotypes and influence of

medium

J. TAYLOR-PAPADIMITRIOU1*, M. STAMPFER2, J. BARTER1, A. LEWIS1, M. BOSHELL1,

E. B. LANE3 and I. M. LEIGH4

'imperial Cancer Research Fund Laboratories, PO Box 123, Lincoln's Inn Fields, London WC2A3PX, UKzLawrence Berkeley Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA3Imperial Cancer Research Fund, Clare Hall laboratories, Blanche Lane, South Minims, Potters Bar, Hertfordshire Ei\!6 3LD, UK4The London Hospital, 56 Ashfield Street, London El 2BL, UK

* Author for correspondence

Summary

The luminal and basal epithelial cells in the humanmammary gland can be distinguished in tissuesections on the basis of the pattern of keratins theyexpress. Moreover, the invasive cells in primarycarcinomas show a keratin profile that correspondsto that of the dominant luminal cell (7, 8, 18, 19).When homogeneous populations of luminal epi-thelial cells from milk or from breast cancer meta-stases are cultured the profile of keratin expressionseen in vivo is maintained. We have therefore usedmonospecific antibodies reactive with individualkeratins to examine the phenotype of cells culturedin three different media from reduction mammo-plasty tissue that contains both luminal and basalcells. The phenotype of cells cultured from primarybreast cancers in one of these media (MCDB170)has also been examined. In characterizing cellphenotypes, antibodies to a polymorphic epithelialmucin (PEM) expressed in vivo by luminal cells,and to smooth muscle (a) actin, expressed in vivoby basal cells, have also been used. Our resultsshow that proliferation of different cell phenotypesis selected for in different media. In milk mix (MX)developed for growth of luminal cells from milk,only the luminal cell phenotype proliferates (foronly 1 or 2 passages). In medium MCDB 170, whichwas developed for long-term growth of humanmammary epithelial cells from reduction mam-

moplasty organoids, cells from the basal layerproliferate, while in MM medium the basal pheno-type dominates, but a few cells with the luminalphenotype are found. Around passage 3, in mediumMCDB 170, most cells senesce and a subpopulationof cells proliferates on further passage. These cellsretain expression of the basal epithelial keratins butalso express some features characteristic of luminalepithelial cells, suggesting that the basal layer maycontain a stem cell that can develop along theluminal lineage. In culture, however, they do notexpress keratin 19, which in vivo is a feature of thefully differentiated luminal cell. The cells culturedfrom primary breast cancer in medium MCDB 170have a similar keratin profile to that of the normalcells cultured in this medium. They do not expresskeratin 19, even though the invasive cells in pri-mary cancers homogeneously express this keratinin vivo. The invasive phenotype, which in its kera-tin profile corresponds to the differentiated luminalcell and that of the metastatic cancer lines, cannotbe cultured from primary breast cancers using MX,which supports proliferation of the correspondingnormal cell.

Key words: keratin, mammary epithelium, malignancy.

Introduction

The mammary gland is a complex tissue containing avariety of cell types including the two major epithelialphenotypes (basal and luminal) that line the ductal tree.

Journal of Cell Science 94, 403-413 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

Breast cancer represents one of the most common carci-nomas in women in the Western world, and clearlydevelops from an epithelial cell found in the breast. Inorder to be able to study this cell and its transformation inculture, it is important to try to define the phenotypes in

403

both the normal tissue and the malignant tumours, andalso the cells that can be cultured from them. Character-ization of cell types /'// vivo can be done readily, usingimmunohistochemical techniques and monoclonal anti-bodies on tissue and tumour sections. In this context, theepithelial keratins have been found to be extremely usefulmarkers. In the human, the soft tissue keratins have beencharacterized and classified on the basis of molecularweight and charge (Moll et al. 1982) and the profile ofexpression of these components is characteristic of aparticular cell type (Sun et al. 1984). Recently, mono-specific antibodies reactive with a single keratin havebeen developed, and these are being successfully appliedin immunohistochemistry to the identification of keratinsexpressed in tissues and tumours at the single cell level(Ramaekerse/fl/. 1983«,6; Osbornetal. 1985; Morgans/al. 1987). Using such tools it is possible to distinguish thetwo major epithelial cell types in the adult mammarygland, since the basal cells express keratins typicallyexpressed by the basal cells in stratified epithelia, whilethe luminal cells express smaller keratins typical of simpleepithelia (Altmannsberger et al. 1986; Taylor-Papadimi-triou and Lane, 1987; Dairkee et al. 1988; Nagle et al.1986). Some subclassification has also been possible onthe basis of keratin expression and position in themammary tree (Bartek e? a/. 1985o, 19866). Immunohis-tochemical staining of breast cancers shows that in theirprofile of keratin expression, and in their expression of apolymorphic epithelial mucin (PEM; Gendler et al.1988), they resemble the luminal epithelial cells (Taylor-Papadimitriou and Lane, 1987).

When breast tissue or primary breast cancers are usedas starting material for culture it is difficult to ascertainwhether the in vivo markers are retained in culture,because of the heterogeneous cell population in thesample. A metastatic breast cancer on the other handrepresents a relatively homogeneous cell population, andmany cell lines have been developed from metastases,mainly from cells in pleural effusions. Similarly, milkrepresents a source of normal luminal epithelial cells,uncontaminated by fibroblasts, or epithelial cells fromthe basal epithelial layer (Buehring, 1972). Data reportedin the literature (Bartek et al. 1985«; Chang and Taylor-Papadimitriou, 1983; Taylor-Papadimitriou et al. 1987)and in this paper indicate that the profile of keratinexpression in the cultured milk cells and in breast cancercell lines is found to be remarkably consistent, corre-sponding to that of the differentiated luminal cell. Sincesome of the breast cancer cell lines have been in culturefor many years in different laboratories, it would appearthat the expression of keratins 8, 18 and 19 and sometimes7 is a stable feature of these cells that is not altered byculture. We have therefore used antibodies to thesekeratins together with other immunological markers totry to characterize cells cultured from breast tissue andtumours.

An important source of non-malignant mammary epi-thelial cells is the tissue removed at reduction mammo-plasty surgery. The epithelial organoids that are isolatedafter enzymic digestion of the tissue (Stampfer et al.1980) come from different parts of the mammary tree

(large and small ducts, and TDLU) and contain bothbasal and luminal epithelial cells. These complex orga-noids have been grown in a variety of media, andalthough the cells cultured have been clearly identified asepithelial (Bartek et al. 1985a; Curschellas et al. 1987;Dairkee et al. 1986), their detailed phenotype particularlyin terms of keratin expression has not been defined. Inthe serum-free medium MCDB 170 (Hammond et al.1984), the cells can be cultured for 10-20 passages. In thecomplex medium MM, containing some serum andconditioned medium (Stampfer, 1982), fewer passages(5-7) are obtained, while in medium MX developed forgrowth of luminal epithelial cells from milk (Taylor-Papadimitriou et al. 1980), only 1-2 passages are ob-tained. Cells cultured in these three media have beenexamined for expression of the epithelial keratins 7, 8, 14,18 and 19 (and in some cases 5), and also for expression ofthe mucin expressed in vivo by luminal cells and cv-actinexpressed //; vivo by basal cells. In addition, the keratinprofile of cells cultured in medium MCDB 170 fromprimary breast cancers has been examined. The resultsindicate that growth of different cell types is selected forby different media, and that long-term growth is shownin medium MCDB 170 by a minority population thatappears to be derived from the basal layer.

Materials and methods

Culture of human mammary epithelial cellsBreast tissues were obtained from reduction mammoplastiesand mastectomies and digested to epithelial organoids aspreviously described (Stampfer et al. 1980; Stampfer, 1985).The organoids were placed into culture in either the serum-freemedium MCDB 170 (Hammond et al. 1984), medium MM(Stampfer, 1982) or MX (milk mix; Taylor-Papadimitriou et al.1980), and subcultured as described (Stampfer, 1985). Inmedium MCDB 170 bovine pituitary extract is used as anadditive in place of serum while medium MM contains mediumconditioned by other cell lines and a low concentration of foetalcalf serum. These two media were developed for growth of cellsfrom reduction mammoplasty tissue. Medium MX was devel-oped for growing cells from human milk and contains 10%human serum as well as 10% foetal calf serum. The MMmedium and the milk mix contained cholera toxin and, whereindicated, the MCDB 170 medium contained 10~ M-isoproter-enol. For immunohistochemical analysis, cells were seeded ontocleaned glass coverslips and fixed when subconfluent with 1: 1(v/v) cold methanol/acetone for ISmin at room temperature.The coverslips were rinsed with phosphate-buffered saline(PBS), air-dried and stored at -20°C until use.

In this study, cells from a reduction mammoplasty (specimenno. 161) were examined at different passages. The phenotypesof the cells emerging after long-term culture were examined atpassage 6-9 for three other specimens (172R, 184 and 48R)from individuals with no apparent breast pathology, and forthree specimens taken from breast cancers (203T, 192T,186TL). For comparison of phenotypes cultured in differentmedium, reduction mammoplasty specimens are indicated inthe relevant experiment.

Preparation of tissue sectionsFrozen sections from tissues and tumours obtained from DrRosemary Millis at the ICRF Breast Cancer Unit, Guy's

404 jf. Taylor-Papadimitriou et al.

Table 1. Monoclonal antibodies used to detectexpression of specific keratins

Antibody

LL001C43M20C18C35CK7RCK105LE61C04BA16BA17LP2K

Target

Keratin 14Keratin 8Keratin 8Keratin 7Keratin 7Keratin 7Keratin 7Keratin 18Keratin 18Keratin 19Keratin 19Keratin 19

Reference

Leigh el at. (1988)Bartek <?/«/ (1989)van Mui]en etal. (1987)Bartekef o/. (1987)Bartekef o/. (1989)Ramaekers el a!. (1987)Ramaekers el at. (1987)Lane (1982)Bartek e/o/ (1989)Bartek el at. (1985)Bartek <?fo/. (1985)Stasiake/o/. (1989)

Hospital, were prepared and fixed in methanol/acetone (1:1,v/v) for 10 min before air drying and storing at —70°C untilused. For staining with antibody BA17, sections of methacarn-fixed paraffin-embedded sections were used (Bartek et al.19856).

Staining of sections and cultured cellsSections and cell cultures prepared as described above werestained by an indirect immunoperoxidase method using themonoclonal antibodies described in Table 1 (Bartek et al.1985a). The antibodies were used as undiluted tissue culturesupernatants.

Monoclonal antibodiesAll of the specimens were stained with the anti-keratin anti-bodies listed in Table 1. Other monospecific anti-keratin anti-bodies were used to confirm the staining patterns seen in cells inprimary cultures and third and sixth passage of reductionmammoplasty specimen 161 grown in medium MCDB 170 andin secondary cultures of specimen 184 in medium 170 and inMM with and without cholera toxin. These were: 312C8(Dairkee et al. 1986) and LL002 (Leigh et al. 1988) to keratin14; RCK105 (Ramaekers et al. 1987) and CK7 (Tolle et al.1985) to keratin 7; RCK106 (Ramaekers et al. 1987) and CK5(Tolle et al. 1983) to keratin 18, KM4.62 (Gigi-Leitner andGeiger, 1986) and A53-B/A2 (Karsten et al. 1985) to keratin 19.A polyclonal rabbit antiserum to keratin 5 (Rba5: E. B. Lane,unpublished) was also used to stain specimens cultured in MM(secondary culture of specimen 184) and medium MCDB 170(first, third and sixth passage of specimen 161).

The expression of vimentin was examined with antibodyVIM 13.2, which was obtained from ICN Immunobiologicalsand in some cultures with antibody V9 from DAKO. Fibronec-tin expression was detected using antibody FN-3, which reactswith cell-associated but not plasma fibronectin (Keen et al.1984). The expression of a polymorphic epithelial (PEM)mucin (Gendler et al. 1988) was detected with antibodiesHMFG-1 and HMFG-2 (Taylor-Papadimitriou et al. 19816;Burchell et al. 1983), and smooth muscle <r-actin was detectedusing the antibody developed by Gabbiani (Skalli et al. 1986;Gugliotta et al. 1988).

Results

Keratin expression in human mammary epithelia in situNormal resting gland. The distribution of keratins in

the human mammary gland as determined by histo-

chemical staining with the antibodies listed in Table 1 isshown diagrammatically in Fig. 1. With the exception ofthe very large ducts, where some keratin 14 expression isseen in luminal cells (Purkis et al. 1989), only basal cellsexpress keratin 14, a keratin expressed by basal cells instratified epithelia. On the other hand, all the luminalcells express keratins 7, 8 and 18, which are normallyassociated with simple epithelia. In the TDLU where theoverall level of keratin expression is lower, especially inthe basal cells, the distinction between luminal and basalcells is most clear cut; keratin 14 is not expressed in theluminal cells (see Fig. 1; also Dairkee et al. 19856) andkeratins 8 and 18 are not expressed in the basal cells. Wefind however that keratin 7, which is characteristicallyexpressed in simple epithelia, can be detected in the basalcells throughout the gland.

Keratin 19, which can be expressed in both simple andstratified epithelia is restricted to the luminal cells in theTDLU but can be found in basal cells in the larger ducts.An interesting feature of keratin 19 expression in themammary gland is that in the TDLU, a subclass ofluminal cells do not express this keratin (Bartek et al.1985a; and see Fig. 2B). Because of the increased num-ber of these cells seen in benign tumours (Bartek et al.19856, 1986a; Gusterson et al. 1987), their ability toproliferate well in culture (Bartek et al. 1985«, and thispaper) and their association with ductal branching atpuberty (Bartek et al. 1989), we have suggested (Barteket al. 1985<7,6) that these cells form a separate proliferat-ive compartment.

The expression of the mesenchymal intermediate fila-ment protein vimentin by basal cells of the mammarygland, has recently been reported (Guelstein et al. 1988).This report is in contrast to earlier observations onhuman mammary gland, which did not detect vimentinexpression (Dairkee et al. 1986). We have found vimentinto be undetectable in the basal cells of the mammarygland, using the commercially available antibody VIM13.2 (see Materials and methods).

Breast cancers. The profile of keratin expression byinvasive breast cancer cells, as seen by immunohisto-chemical staining of tissue sections, resembles that of thedifferentiated luminal epithelial cell (Table 2). With afew exceptions, keratin 14 is not detected in the invasivecells by antibody staining (see Fig. 2E; and Dairkee et al.1987; Wetzels et al. 1989). Where remnants of structureare visible some expression of keratin 14 can occasionallybe detected, but in contrast to the situation in the normalgland the expressing cells are lining lumens (see Fig. 2E).The keratin profile of cells in these areas resembles thatseen in benign tumours, where keratin 14 is detected andkeratin 19 is expressed heterogeneously (Bartek et al.19856).

The profile of keratin expression seen in invasive cellsin primary breast cancers and in the differentiatedluminal epithelial cell, is also seen consistently in meta-static cancers (see Table 2).

Keratin expression in cultures of luminal epithelial cellsTo determine whether the keratin expression seen in situis maintained in culture, homogeneous cultures of normal

Keratin expression in mammary epithelial cells 405

KERATIN 7Predominantly lumenalSome expression in basalcells

KERATINS 8 & 18Lumenal

KERATIN 19Predominantly lumenalSome unstained cells inend structuresSome staining of basal cells inlarge ducts

KERATIN 14Predominantly basalSome staining of lumenalcells in large ducts

Key:• • H Strong stainingt. .i *..• j Less intense stainingr •*•' •' Heterogeneous staining

Fig. 1. Diagrammatic representation of distribution of keratins in the human mammary gland.

and malignant epithelial cells showing the luminal pheno-type vivo were examined. The differentiated 19+ luminalepithelial cell is the dominant cell in the lactating breastand is shed into milk. Cultured in medium MX, thesecells continue to express keratins 7, 8, 18 and 19. All thecolonies show homogeneous expression of keratins 7, 8

and 18, and the majority (85 %) also express keratin 19homogeneously (Bartek et al. 1985a). The major differ-ence seen in the cultured milk cells compared to theluminal phenotype seen in vivo is expression of keratin14, which is seen in around 30 % of colonies (seeTable 2).

406 J. Taylor-Papadimitriou et al.

l^^^i p^y

B

v».V

Fig. 2. Immunoperoxidase staining of sections of normal tissue (A,B), fibroadenoma (C,D) and infiltrating ductal carcinoma(E,F) with antibody 001 directed to keratin 14 (A,C,E) or antibody BA17 to keratin 19 (B,D,F). A,C,E, x200; B,E,F, X300.

Table 2.

Tissue

Expression

or culture

of keratins by normal

l

luminal

8

epithelial cells

Keratin

18

and invasive

19

breast

14

cancer

PEM<

cells

Normal luminal epithelial cellsin sections of TDLU

Shed luminal epithelial cellscultured from milk inmedium MX

Invasive breast cancer cells insections of primary carcinomas

Metastatic breast cancer cells insections of L/N metastases

Cultured breast cancer celllines from metastases

MCF-7T47DZR-75.1

+t

( + )(>95 %)

()(85 %)

()(30%)

(+)*

(+)t

N.D.§ (+)*

(+)*(+)*

•Polymorphic epithelial mucin.•f +, Indicates homogeneously positive staining; (+) some cells stained, some not. The values in parenthesis indicate the proportion of

positive cells.^Proportion of cells staining varies from 50 to 100%, depending on antibody used and culture conditions.§ Not done.

Cell lines derived from metastatic cancers have beendeveloped in several laboratories. Table 2 shows that theprofile of keratins expressed in three such lines corre-sponds to the profile seen in situ. Since these lines havebeen in culture for many years, again this suggests thatthe expression of the simple epithelial keratins 8, 18 and19 is maintained in culture, and in this case, keratin 14 isnot induced.

Keratin expression in mammary epithelial cells culturedfrom reduction mammoplasty tissue

Phenotypes cultured in medium MCDB 170. MediumMCDB 170 was developed by Hammond and colleagues(1984) for the clonal and long-term culture of cells inepithelial organoids derived by enzyme digestion fromreduction mammoplasty (RM) tissue (Stampfer et al.1980). These organoids contain both basal and luminalcells from pieces of ducts and from TDLU. In mediumMCDB 170, most cells cultured from reduction mam-moplasty organoids develop a flattened irregular-edgedmorphology and cease proliferation around passage 3.

After this 'self selection' a cell population with a cobble-stone epithelial morphology can be passaged for anadditional 7-20 passages depending on the individualspecimen (Stampfer, 1985). The morphological appear-ance of the cells at different passages is illustrated inFig. 3. We first examined the primary cultures andsubsequent passages from a single specimen (161) and asummary of our findings is presented in Table 3.

In primary cultures, two dramatically different celltypes can be seen, which in their keratin expressionappear to correspond to the basal and luminal cells seen invivo, and it is the cells with the basal phenotype thatappear to proliferate. The basal-like cells, which appearto form the bottom layer, show many mitoses (seeFig. 4B), express high levels of keratins 5 and 14(Fig. 4A), no keratin 18 or 19 (Fig. 4B), moderate levelsof keratin 7 and variable but detectable levels of keratin 8.The superficial luminal-like cells on the other handexpress high levels of keratins 7, 8, 18 and 19 (Fig. 4B).These cells also stain with the antibody (HMFG-1) to thepolymorphic epithelial mucin (Gendler et al. 1988),which is a marker for luminal epithelial cells (Taylor-

Table 3. Profile of keratin expression in cells from reduction mammoplasty organoids (specimen 161) proliferatingin medium MCDB 170*

Keratin

Passage 18 19 14

Smoothmuscleo-actin

136

13 N.D.11(+)t4N.D.

( + + )*( + + + )* H—> + +

+ H—> + + + N.D.

* For composition see Hammond et al. (1984). In these experiments isoproterenol was added.•(•With antibody C43, heterogeneous staining was seen, while with antibody M20 most cells showed positive staining.t( + ) , some cells stained, some not.§ ±—* + + , ranged from weak to moderately strong staining.1[ N.D., not done.

Keratin expression in mammary epithelial cells 407

\

>vr-.\-;

4

B

Fig. 3. Morphological appearance of cells cultured from reduction mammoplasty (RM) 161 in medium MCDB 170. A. Primaryculture. B. Cells at third passage showing large senescing cells and smaller, post-selection cells. C. Cells at passage 9 (post-selection cells). D. Cells senescing at passage 16. A,B, X40; C,D, X130.

Papadimitriou et al. 19816; data not shown). Althoughsmall patches of these cells may migrate out somedistance from the original organoid they appear not toproliferate in medium MCDB 170. Interestingly, theproliferating (basal-like) cells express high levels ofvimentin (see Fig. 4D) and smooth muscle cv-actin.

Around passage 3, most of the cells grown in medium170 appear to enlarge and senesce, and the phenotype ofthe large senescing cells is complex. These cells continueto express keratin 7, 8, 5 and 14, but also stain positively,but less intensely, with antibodies to keratin 18; they donot however, express keratin 19. The cells also expressfibronectin, sometimes in the form of a network, high

levels of vimentin, and reduced but detectable levels ofsmooth muscle <r-actin.

The cell type that emerges after passage 3 appears toexpress keratins 5 and 14 as well as the simple epithelialkeratins, 7, 8 and 18, but keratin 19 is not detected.There is considerable variation in the level of individualkeratin expression between cells, and this is most obviouswith keratin 18 and sometimes with 8; keratins 7 and 14however are expressed more homogeneously. Expressionof keratin 5, which generally is co-expressed with keratin14, was examined in cells in primary cultures and at thirdand sixth passage, and strong positive staining was seenwith the polyclonal antibody

408 jf. Taylor-Papadiniitriou et al.

Fig. 4. Phenotype of cells in primary and secondary cultures of reduction mammoplasty organoids grown in different media.Cultures were prepared as described in Materials and methods and stained in an indirect immunoperoxidase assay, using anti-keratin antibodies (A-C,E,F) or an antibody to vimentin (D). A,B- Primary cultures of RM 161 in medium MCDB 170 stainedwith antibodies monospecific for keratin 14 (A), and keratin 18 (B). C. Primary culture of RM 17N grown in milk mix andstained with antibody to keratin 19. D. Secondary culture of RM 184 grown in medium MCDB 170 and stained with anantibody to vimentin. E,F. Secondary cultures of RM 184 grown in MM and stained with antibodies to keratin 14 (E) or keratin18 (F). A-C, X200; D-F , X300.

BffiFig. 5. Phenotvpe of cells selected from reduction niammoplasty (A,B) o r primary breast cancers (C,D) for long-termproliferation in medium MCDB 170. Cultures were prepared and passaged in medium MCDB 170, fixed at the passagesindicated and stained with the indirect immunoperoxidase method using antibodies monospecific for keratins. A. RM 172, ninthpassage, stained with antibody LL001 to keratin 14. B. RM 48, eighth passage, stained with antibody C04 to keratin 18.C. Primary cancer 203, seventh passage stained with antibody LL-001 to keratin 14. D. Primary cancer 203, seventh passage,stained with antibody C35 to keratin 7. X200.

Table 4. Keratin expression in human mammary epithelial cells cultured in different media

Medium CulturePattern of

growth

Keratin expression

18 19 14 PEM*

MCDB 170

MM§

MM§

Milk MixU(MX)

RM184Passage 2RM184Passage 2RM184Passage 2RM17NPrimaryCulture

Open

Closed

(+)t4

( + )t

* Polymorphic epithelial mucin, detected by staining with antibody HMFG-1.t ( + )i some cells stained, some not.% Staining weaker and more heterogeneous with antibody C43, than with M20.§ For composition, see Stampfer (1982).H For composition, see Taylor-Papadimitriou et at. (1980).

Three other reduction mammoplasties from indi-viduals with no apparent epithelial cell pathology wereexamined at passages 6-8 and the pattern of staining seenwith 161 was confirmed, i.e. all cells stained withantibodies to keratins 7 and 14 and positively but variablyand heterogeneously with antibodies to 8 and to 18 (seeFig. 5A,B). No evidence for expression of keratin 19 wasseen, using three different antibodies. Vimentin con-tinued to be expressed in the post-selection cells; how-ever, the intensity of staining was generally lower, butappeared to vary with both the antibody and the tissuespecimen, the strongest staining being observed withspecimen 161 (passage 6) and the antibody V9.Interestingly, the fibronectin in these cells shows thepunctate pattern previously reported for epithelial cellsgrown from milk (Taylor-Papadimitriou et al. 1981a),and smooth muscle cr-actm was not detectable. In laterpassages (passage 13, specimen 161), when the cells werebeginning to senesce, cr-actm could again be detected andlow levels of the mucin could be detected with theantibody HMFG-1.

The above results are consistent with the interpretationthat medium MCDB 170 supports the proliferation ofbasal cells for two to three passages, at which point thesecells senesce and a cell type expressing some properties ofboth basal and luminal cells is selected and continues toproliferate.

Keratin expression in mammary epithelial cells culturedfrom reduction mammoplasty tissue

Phenotypes cultured in other media. Medium MCDB170 was developed to obtain the maximum number of celldivisions in cells cultured from reduction mammoplastytissue. Before the development of MCDB 170 epithelialcells from reduction mammoplasty tissue were culturedin a medium (MM) containing serum and mediumconditioned by other cell types (Stampfer et al. 1980). InMM medium the cells can be subcultured for fewerpassages. Medium MX (milk mix), which was developedto culture human mammary epithelial cells from milk(Taylor-Papadimitriou et al. 1980), can also be used toculture cells from reduction mammoplasty tissue. This

medium contains human serum as well as foetal calfserum, and allows the milk cells to go through approxi-mately 15-20 divisions (1-2 passages). All of the mediacontain cyclic AMP-elevating agents (see Materials andmethods).

Table 4 lists the phenotypes seen in primary or second-ary cultures made from reduction mammoplasty tissueusing medium MM, and the MX medium and comparesthem with those seen in cultures grown in mediumMCDB 170. Fig. 4 illustrates some of the stainingpatterns. In early passages of cells grown in mediumMM, there appear to be two rather well-defined morpho-logical phenotypes. The minority phenotype forms com-pact colonies (closed) and is surrounded by the dominantphenotype, which has looser packing and gives an 'open'appearance (see Fig. 4E,F). The closed colonies stainstrongly with antibodies to keratins 7, 8 and 18 (seeFig. 4F), and heterogeneously with keratin 19 anti-bodies. Keratin 14 may or may not be expressed(Fig. 4E); some mucin is detected with antibodyHMFG-1 and vimentin is not detected. Thus, thesesmall groups of cells resemble the /'// vivo luminalphenotype. The dominant phenotype on the other handresembles the basal cells in its expression of keratin 5 and14 (Fig. 4E) and although keratin 7 is homogeneouslyexpressed and keratin 8 somewhat more heterogeneously,18 and 19 are not detected (Fig. 4F). Vimentin, theintermediate filament protein normally found in mes-enchymal cells, is however expressed at a high level, aswas noted by Dairkee and colleagues (1985«). Subculturein the same medium does not result in a selection foranother cell type as is observed with MCDB 170, butrather both phenotypes seem to be maintained until thecultures senesce, although the small colonies of cells ofthe luminal phenotype formed only 1-5% of the popu-lation after the second passage of reduction mammoplasty184.

In the medium MX, developed for growth of luminalcells shed into milk, fewer cells attach from the orga-noids, and although considerable cell division occurs inprimary culture, giving rise to large colonies, furtherpassage is difficult. In primary culture, the dominant

Keratin expression in mammary epithelial cells 409

phenotype resembles that of the differentiated luminalcell. These cells form closed colonies that stain stronglywith antibodies to the simple epithelial keratins 7, 8 and18 and to the mucin; most of these colonies also expresskeratin 19 (see Fig. 4C). MX medium therefore appearsto select for the differentiated luminal phenotype that hasa limited life span. As with the luminal cells culturedfrom milk, however, some expression of keratin 14 isseen. A similar phenotype can be grown in MX mediumfrom fibroadenomas, but we have been unsuccessful inculturing any epithelial cells from primary carcinomas inthis medium.

Keratin expression in cells cultured in medium MCDB170 from primary breast carcinomasTo see whether there was any difference in the phenotypeof cells cultured in MCDB 170 from malignant and non-malignant breast tissue, cultures at passages 7-8 fromthree tumour specimens were also examined. Again ahomogeneous positive staining was seen with antibodiesto 7 and 14 (Fig. 5C,D), the cells stained variably andheterogeneously with antibodies to keratins 8 and 18, andno staining could be detected with antibodies to keratin19. These studies suggest that in medium MCDB 170,the keratin profile of the cells cultured from breastcancers is similar to that of the cells cultured from thenormal breast. The phenotype of this cell differs fromthat of the invasive cancer cell identifiable in tissuesections (Fig. 2) and in cell lines derived from metastaticbreast cancers (Table 2), which express keratins 8, 18 and19 (and sometimes 7), but do not usually express keratin14 (Vojtesek and Bartek, unpublished data; Curschellasetal. 1987).

Effect of cyclic AMP-elevating agents on keratinexpressionIn medium MCDB 170 some reduction mammoplastyspecimens require isoproterenol to move through the'self-selection' period, which occurs around third pass-age. Other specimens (e.g. specimen 161), however,grow equally well in medium MCDB 170 in the absenceof an added cyclic AMP-elevating agent. We thereforeexamined the effect of isoproterenol on keratin expressionin specimen 161. We observed that expression of all thekeratins was lower in cells grown in the absence of cyclicAMP-elevating agents and keratin 14 expression wasdramatically increased by addition of isoproterenol. Wealso examined secondary cultures of reduction mammo-plasty 184 grown in MM with and without cholera toxin.The intensity of staining of the majority of cells withantibody LL001 to keratin 14, and with antibodies CK7and RCK105 to keratin 7, was greatly increased in cellscultured in medium containing cholera toxin. Moreover,most of the compact colonies showed stronger stainingwith antibodies to keratins 18 and 19. There was also anapparent increase in the numbers of cells stained withthese antibodies, including several more-dispersed cellsseen as 'strings' of cells maintaining contact with eachother or as single cells around the islands. In the MXmedium, cell proliferation is much reduced in the ab-sence of the cyclic AMP-elevating agent (cholera toxin),

so that the effect of this agent on keratin expression incells expressing the luminal phenotype in culture couldnot be examined.

Discussion

In this paper, we have examined the expression ofkeratins by mammary epithelial cells cultured in differentmedia, using antibodies that are monospecific for a singlekeratin and compared the phenotypes with those seen inbasal and luminal cells in vivo. Antibodies directed toother markers of basal and luminal epithelial cells werealso used to define the cultured cells. It is very clear fromthis study that the phenotype of the cultured cells variesdramatically with the medium used, and can, wheresubculture is possible, also change with passage.

We chose to use the epithelial keratins as markers, sincethe profile of keratin expression is tissue specific, and canbe used to define subpopulations of cells in complextissues such as the mammary gland. Moreover, whererelatively homogeneous populations of cells have beencultured, as with metastatic breast cancer cell lines, andluminal epithelial cells from milk, the observed keratinexpression is similar to that observed in vivo. In the caseof the cell lines, the phenotype is identical to that of theinvasive breast cancer cell in vivo (8, 18, 19 andsometimes 7). There is one difference in the culturedmilk cells in that, in addition to the simple epithelialkeratins 7, 8, 18 and 19, some cells express keratin 14. Invivo, although keratin 14 is associated only with basalcells in the smaller ducts, some expression is seen also inluminal cells in the large ducts. Thus the 14+ cells maycome from the larger ducts. It is possible however thatthe cholera toxin present in the MX medium used forculturing milk cells induces keratin 14 expression. Animportant conclusion that can be drawn, however, is thatexpression of keratin 7, 8, 18 and 19 is not lost in cultureusing the widely different media used for growing breastcancer cell lines and in medium MX used for growingnormal cells from milk. Therefore the absence of any ofthese keratins in cultured mammary epithelial cellssuggests that the cells came from a cell that was notexpressing the keratin in vivo.

Unlike the cells in milk, the organoids derived byenzymic digestion of reduction mammoplasty tissue con-tain both basal and luminal cells, and the total digestcontains structures coming from all parts of the gland.Using the medium MX developed for luminal cellculture, the luminal cell phenotype selectively prolifer-ates, and the basal cells appear to die. As was seen withthe luminal cells cultured from milk, some expression ofkeratin 14 is seen in addition to keratins 7, 8, 18 and 19.With the two other media used in this study (MCDB 170and medium MM), the basal cells proliferate and initiallyexpress keratins 5, 7 and 14 homogeneously (in a few cellskeratin 8 can be detected) as well as high levels ofvimentin and smooth muscle actin. In this context it is ofinterest that medium MCDB 170 does not support theproliferation of the epithelial cells found in milk (Ham-mond, S. and Taylor-Papadimitriou, J., personal com-

410 jf. Taylor-Papadimitriou et al.

munication). Small colonies of cells with the luminalphenotype can be seen in early passages of MM culturedcells but their numbers are reduced with passage. Both ofthese media therefore favour the proliferation of the basalcell phenotype. It seems likely that basal cells areexpressing low levels of vimentin in vivo as reported byGuelstein and colleagues (1988) and that the levels areincreased by culture (Franke et al. 1979).

Of great interest is the observation that the cells thatemerge from self-selection in medium MCDB 170 afterpassage 3 show a different phenotype to early passagecultures, in that they show increased expression ofkeratins 8 and 18 (more heterogeneously expressed thankeratins 7, 5 and 14). They also show a reduced ex-pression of fibronectin, O"-actin and vimentin. They donot however express keratin 19, and generally produceonly small or undetectable levels of the mucin recognisedby antibody HMFG-1 (although mucin and cv-actin levelscan increase when the cells eventually senesce). Theobservations suggest that a minority cell type with highproliferative potential is selected for, with passage inmedium MCDB 170. This cell appears to acquire somefeatures of the luminal cell (expression of keratin 8 and18) but does not express keratin 19 and does not lose thebasal cell-related keratins that were detected in earlypassage. Transferring MCDB 170 grown cells (passage 8)to medium MM or to the same medium supplementedwith serum, resulted in some increase in the level ofexpression of keratins 7, 8 and 18, and in a low expressionof the PEM mucin, but keratin 19 expression was notinduced in any of the cells (data not shown).

It is difficult to relate the cells proliferating at laterpassages in medium MCDB 170 to the basal and luminalepithelial cells seen in vivo. It may be related to thekeratin 19~ luminal cell that is associated with ductalbranching at puberty, and which shows a less differen-tiated phenotype than the 19+ luminal cell (does notproduce secretory component) in the adult breast (Barteket al. 1989). The keratin 19~ luminal cell does nothowever, express the basal epithelial keratins in vivo.Conceivably, a stem cell found in the basal layer, isprecursor to the 19~ luminal cell in vivo, and it is thisprecursor that proliferates and shows partial differen-tiation in long-term culture in medium MCDB 170. Thiswould imply that not all the cells in the basal layer thatexpress keratins 5 and 14 are committed myoepithelialcells. If this is the case, as has also been suggested for therat mammary gland (Allen et al. 1984), it should bepossible to identify markers that would distinguish thesmall populations of putative stem cells from the majorityof basal cells.

The observations of keratin expression in cells culturedfrom primary tumours are rather surprising. As indi-cated, the invasive cancer cell seen in primary andmetastatic lesions has a keratin profile identical to that ofthe differentiated luminal epithelial cell. Using mediumMX, which can be used to grow this cell type from thenormal gland, no cells can be cultured from primarycarcinomas. The phenotype of the cells that are culturedfrom cancers in medium MCDB 170, however, is thesame in its keratin profile as the phenotype of the cells

cultured from reduction mammoplasty in this medium.The tumour-derived cells could be normal or dysplasticbasal cells associated with but unrelated to the malignantcomponent. In support of this idea is the observation thatcells cultured in a similar medium or in medium MM arediploid, even when cultured from aneuploid tumours(Wolman et al. 1985; Zhang et al. 1989). Alternatively,they may represent more primitive neoplastic cells, whichcan differentiate in vivo to give the luminal phenotype ofthe invasive breast cancer cell. This would suggest thatthe original carcinogenic event occurred in a cell with ahigh proliferative potential, but that the invasive functionis expressed by the more differentiated phenotype, whichin malignancy acquires proliferative potential. Why it isso difficult to culture the invasive cancer cells fromprimary breast carcinomas is not clear. Only a smallfraction of these tumours grow as xenografts in the nudemouse. Certainly, immortality appears to be a late eventonly seen in late-stage patients, generally in pleuraleffusion metastases (Smith et al. 1987). It seems quitelikely that the phenotype that will become the invasivecell can initially go through only a limited number of celldivisions, many of which will have been exhausted invivo.

It will be of great importance to examine the behav-ioural properties of the cells that can be cultured fromprimary breast cancers in medium MCDB 170 to see ifthey show any malignant features. Some studies havebeen done with cells grown from primary cancers inmedium MM and although these are diploid even whengrown from tumours exhibiting aneuploidy they do showinvasive features (Smith et al. 1985) and are moresensitive to tumour necrosis factor (TNF) than thecorresponding normal cells (Dollbaum et al. 1988). Itwill also be important to study the effect of oncogenictransformation on the different phenotypes that can becultured from the normal gland. Some immortalized lineshave been developed using chemical carcinogens andretroviruses to transform MCDB 170-grown cells(Stampfer and Bartley, 1985; Clark et al. 1988) andsimian virus 40 to transform luminal epithelial cellsgrown from milk (Chang et al. 1982). We are nowdeveloping immortalized lines that accurately maintainthe phenotype and keratin profile of the luminal epithelialcell, in order to examine the effect of a variety ofoncogenes on this cell type.

The authors are grateful to Ms P. Purkis and Ms J. Steel fortheir contribution to the development of the antibody LL001directed to keratin 14. They also thank Linda Hayashi fortechnical assistance and Dr Rosemary Millis for supplies oftissues and tumours. Part of this work vas supported by NIHgrant CA-24844 and by the Office of Health and EnvironmentalResearch, US Department of Energy, under contract DE-AC03SF00098.

References

ALLEN, R., DULBECCO, R., SYKA, P., BOWMAN, M. AND ARMSTRONG,B. (1984). Developmental regulation of cytokeratins in cells of therat mammary gland studied with monoclonal antibodies. Ptvc.natn.Acad. Sci. U.S.A. 81, 1203-1207.

Keratin expression in mammary epithelial cells 411

ALTMANNSBERGER, M., DIRK, T., DROESE, M., WEBER, K. AND

OSBORN, M. (1986). Keratin polypeptide distribution in benignand malignant breast tumours: subdivision of ductal carcinomasusing monoclonal antibodies. Yirchoivs Arch. Zellpath. 51,265-275.

BARTEK, J., BARTKOVA, J., SCHNEIDER, J., TAYLOR-PAPADIMITRIOU,

J., KOVARIK, J. AND REJTHAR, A. (1986a). Expression ofmonoclonal antibody-defined epitopes of keratin 19 in humantumours and cultured cells. Eur.J. Cancer din. Oncol. 22,1441-1452.

BARTEK, J., BARTKOVA, J., TAYLOR-PAPADIMITRIOU, J., REJTHAR, A.,

KOVARIK, J., LUKAS, Z. AND VOJTESEK, B. (19866). Differential

expression of keratin 19 in normal human epithelial tissues revealedby monospecific monoclonal antibodies. Histochem. J. 18, 565-575.

BARTEK, J., DURBAN, E. M., HALLOWES, R. C. AND TAYLOR-

PAPADIMITRIOU, J. (1985fl). A subclass of luminal epithelial cells inthe human mammary gland, defined by antibodies to cytokeratins.jf. Cell Sci. 75, 17-33.

BARTEK, J. , KOVARIK, J. , BURCHELL, J. , TAYLOR-PAPADIMITRIOU, J. ,

BARTKOVA, J. , VOJTESEK, B., REJTHAR, A., SCHNEIDER, J. ,

PETREK, M. , STASKOVA, Z. AND M I L L I S , R. (1987). Monoclonal

antibodies to breast epithelial antigens in the study of

differentiation and malignancy. In Molecular Biology and

Differentiation of Cancer Cells (Oncogenes, Growth Factors,Receptors) (Lapis, K. and Eckhardt, S., eds), Akademiai Kiado,Budapest.

BARTEK, J., KOVARIK, J., VOJTESEK, B., BARTKOVA, J., STASKOVA,

Z., REJTHAR, A. AND LAUEROVA, L. (1989). Subclassification ofhuman tumours by monoclonal antibodies to keratins. InMonoclonal Antibodies to Tumour Associated Antigens and theirClinical Application (Abelev, G. I., ed.), Akademai Kiado,Budapest (in press).

BARTEK, J., TAYLOR-PAPADIMITRIOU, J., MILLER, N. AND MILLIS, R.

(19856). Patterns of expression of keratin 19 as detected withmonoclonal antibodies in human breast tissues and tumours. Int. J.Cancer 36, 299-306.

BUEHRINO, G. C. (1972). Culture of human mammary epithelialcells: Keeping abreast of a new method. J. natn. Cancer Inst. 49,1433-1434.

BURCHELL, J., DURBIN, H. AND TAYLOR-PAPADIMITRIOU, J. (1983).

Complexity of expression of antigenic determinants recognised bymonoclonal antibodies HMFG-1 and HMFG-2, in normal andmalignant human mammary epithelial cells. J. Immiin. 131,508-513.

CHANG, S. E., KEEN, J., LANE, E. B. AND TAYLOR-PAPADIMITRIOU,

J. (1982). Establishment and characterisation of SV40-transformedhuman breast epithelial cell lines. Cancer Res. 42, 2040-2053.

CHANG, S. E. AND TAYLOR-PAPADIMITRIOU, J. (1983). Modulation ofphenotype in cultures of human milk epithelial cells and its relationto the expression of a membrane antigen. Cell Differ. 12, 143-154.

CLARK, R., STAMPFER, M. R., MILLEY, R., O'ROURKE, E., WALEN,

K. H., KRIEGLER, M., KOPPLIN, J. AND MCCORMICK, F. (1988).

Transformation of human mammary epithelial cells by oncogenicretroviruses. Cancer Res. 48, 4689-4694.

CURSCHELLAS, E., MATTER, A. AND REGENASS, U. (1987).Immunolocahzation of cytoskeletal elements in human mammaryepithelial cells. Eur.J. Cancer din. Oncol. 23, 1517-1527.

DAIRKEE, S. H., BLAYNEY, C. M., ASARNOW, D., SMITH, H. S. AND

HACKETT, A. J. (1985o). Early expression of vimentin in humanmammary cultures. /// Vitro 21, 321—327.

DAIRKEE, S. H., BLAYNEY, C , SMITH, H. S. AND HACKETT, A. J.

(19856). Monoclonal antibody that defines human myoepithelium.Proc. natn. Acacl. Sci. U.S.A'82, 7409-7413.

DAIRKEE, S. H., BLAYNEY-MOORE, C. M., SMITH, H. S. AND

HACKETT, A. J. (1986). Concurrent expression of basal and luminalepithelial markers in cultures of normal human breast analyzedusing monoclonal antibodies. Differentiation 32, 93-100.

DAIRKEE, S. H., LJUNG, B. M., SMITH, H. AND HACKETT, A. (1987).

Immunolocahzation of a human basal epithelium specific keratin inbenign and malignant breast disease. Breast Cancer Res.Treatment 10, 11-20.

DAIRKEE, S. H., PUETT, L. AND HACKETT, A. J. (1988). Expression

of basal and luminal epithelium-specific keratins in normal, benignand malignant breast tissue. J. natn. Cancer Inst. 80, 691-695.

DOLLBAUM, C , CREASEY, A. A., DAIRKEE, S. H., HILLER, A. J.,

RUDOLPH, A. R., LIN, L., VITT, C. AND SMITH, H. S. (1988).

Specificity of tumor necrosis factor toxicity for human mammarycarcinomas relative to normal mammary epithelium and correlationwith response to doxorubicin. Proc. natn. Acad. Sci. U.S.A. 85,4740-4744.

FRANKE, W. W., SCHMID, E., WINTER, S., OSBORN, M. AND WEBER,

K. (1979). Widespread occurrence of intermediate-sized filamentsof the vimentin type in cultured cells from diverse vertebrates.Expl Cell Res. 132, 25-46.

GENDLER, S., TAYLOR-PAPADIMITRIOU, J., DUHIG, T., ROTHBARD, J.

AND BURCHELL, J. (1988). A highly immunogenic region of ahuman polymorphic epithelial mucin expressed by carcinomas ismade up of tandem repeats. J. biol Chein. 263, 12820-12823.

GIGI-LEITNER, O. AND GEIGER, B. (1986). Antigenic interrelationshipbetween the 40-kilodalton cytokeratin polypeptide anddesmoplakins. Cell Motil. Cytoskel. 6, 628-639.

GUELSTEIN, V. I., TCHYPYSHEVA, T. A., ERMILOVA, V. D.,LITVINOVA, L. V., TROYANOVSKY, S. M. AND BANNIKOV, G. A.

(1988). Monoclonal antibody mapping of keratins 8 and 17 and ofvimentin in normal human mammary gland, benign tumours,dysplasias and breast cancers. Int.J Cancer 42, 147-153.

GUGLIOTTA, P., SAPINO, A., MACRI, L., SKALLI, O., GABBIANI, G.

AND BUSSOLATI, G. (1988). Specific demonstration ofmyoepithehal cells by anti-alpha smooth muscle actin antibody. J'Histochem. Cytochem. 36, 659-663.

GUSTERSON, B. A., SLOANE, J. P., MIDDWOOD, C , GAZET, J. C ,

TROTT, P., TAYLOR-PAPADIMITRIOU, J. AND BARTEK, J. (1987).

Ductal adenoma of the breast - a lesion exhibiting a myoepithelial/epithelial phenotype. Histopathology 11, 103-110.

HAMMOND, S. L., HAM, R. G. AND STAMPFER, M. R. (1984).

Serum-free growth of human mammary epithelial cells: Rapidclonal growth in defined medium and extended serial passage withpituitary extract. Proc. natn. Acad. Sci. U.S.A. 81, 5435-5439.

KARSTEN, U., PAPSDORF, G., ROLOFF, G., STOLLEY, P., ABEL, H.,

WALTHER, I. AND WEISS, H. (1985). Monoclonal anti-cytokeratinantibody from a hybndoma clone generated by electrofusion. Eur.J. Cancer din. Oncol. 21, 733-740.

KEEN, J., CHANG, S. E. AND TAYLOR-PAPADIMITRIOU, J. (1984).

Monoclonal antibodies that distinguish between human cellular andplasma fibronectin. Molec. Biol. Med. 2, 15-27.

LANE, E. B. (1982). Monoclonal antibodies provide specificintramolecular markers for the study of epithelial tonofilamentorganization. J Cell Biol. 92, 665-673.

LANE, E. B., BARTER, J., PURKIS, P. E. AND LEIGH, 1. M. (1985).

Keratin antigens in differentiating skin. In Intermediate Filaments(E. Wang, D. Fischman, R. H. K. Liem and T.-T. Sun, eds),vol. 455, pp. 241-258, New York Academy of Science, New York.

LEIGH, I. M., LANE, E. B. AND PURKISS, P. E. (1988). Monoclonal

antibodies to keratin 14 as a marker of keratinocyte lineage. J.invest. Demi. 91, 415.

MOLL, R., FRANKE, W. W., SCHILLER, D. L., GEIGER, B. AND

KREPLER, R. (1982). The catalog of human cytokeratins: Patternsof expression in normal epitheha, tumors and cultured cell. Cell31, 11-24.

MORGAN, P. R., SHIRLAW, P. J., JOHNSON, N. W., LEIGH, I. M.

AND LANE, E. B. (1987). Potential applications of anti-keratinantibodies in oral diagnosis. J . Oral Path. 16, 212-222.

NAGLE, R. B., BOECKER, W., DAVIS, J. R., HEID, H. W.,

KAUFMANN, M., LUCAS, D. O. AND JARASCH, E.-D. (1986).

Characterisation of breast carcinomas by two monoclonalantibodies distinguishing myoepithelial from luminal epithelialcells. J. Histochem. Cytochem. 34, 869-881.

OSBORN, M., ALTMANNSBERGER, M., DEBUS, E. AND WEBER, K

(1985). Differentiation of the major human tumour groups usingconventional and monoclonal antibodies specific for individualintermediate filament proteins. Ann. N.Y. Acad. Sci. 455, 649-668.

RAMAEKERS, F. C. S., HAAG, D., KANT, A., MOESKER, O., JAP, P.

H. K. AND Voous, G. P. (19836). Coexpression of keratin andvimentin-type intermediate filaments in human metastaticcarcinoma cells. Proc. natn. Acad. Sci. U.S.A. 80, 2618-2622.

RAMAEKERS, F. C. S., HUYSMANS, A., SCHAART, G., MOESKER, O.

AND Voous, P. (1987). Tissue distribution of keratin 7 asmonitored by a monoclonal antibody. Expl Cell Res. 170, 235-249.

412 J. Taylor-Papadimitriou et al.

RAMAEKERS, F. C. S., PUTS, J. J. G., MOESKER, O., KANT, A.,HUYSMANS, A., HAAG, D., JAP, P. H. K., HERMAN, C. J. ANDVoous, G. P. (1983o)- Antibodies to intermediate filamentproteins in the immunohistochemical identification of humantumours: An overview. Histochem. jf. 15, 691—713.

SCHAEFER, F. V., CUSTER, R. P. AND SOROF, S. (1983). Squamousmetaplasia in human breast culture: Induction by cyclic adeninenucleotide and prostaglandins and influence of menstrual cycle.Cancer Res. 43, 279-286.

SKALLI, D., ROPRAZ, P., TRZECIAK, A., BENZONANA, G., GILLESEN,D. AND GABBIANI, G. (1986). A monoclonal antibody against a-smooth muscle actin: a new probe for smooth muscledifferentiation. J. Cell Biol. 103, 2787-2796.

SMITH, H. S., LIOTTA, L. A., HANCOCK, M. C , WOLMAN, S. R. ANDHACKETT, A. J. (1985). Invasiveness and ploidy of humancarcinomas in short term culture. Proc. natii. Acad. Sci. U.S.A. 82,1805-1809.

SMITH, H. S., WOLMAN, S. R., DAIRKEE, S. H., HANCOCK, M. C ,LIPPMAN, M., LEFF, A. AND HACKETT, A. J. (1987).Immortalization in culture: occurrence at a late stage in theprogression of breast cancer. jf. natn. Cancer hist. 78, 611-615.

STAMPFER, M. R. (1982). Cholera toxin stimulation of humanmammary epithelial cells in culture. //; Vitro 18, 531-537.

STAMPFER, M. R. (1985). Isolation and growth of human mammaryepithelial cells. J . Tiss. Culture Meth. 9, 107-116.

STAMPFER, M. R. AND BARTLEY, J. C. (1985). Induction oftransformation and continuous cell lines from normal humanmammary epithelial cells after exposure to benzo(a) pyrene. Proc.natn. Acad. Sci. U.S.A. 82, 2394-2398.

STAMPFER, M., HALLOWES, R. C. AND HACKETT, A. J. (1980).Growth of normal human mammary cells in culture. In Vitro 16,415-425.

STASIAK, P. C , PURKIS, P., LEIGH, I. M. AND LANE, E. B. (1989).Keratin 19: Predicted amino acid sequence and broad tissuedistribution suggest it evolved from keratinocyte keratins. J. invest.Derm. 92, 707-716.

SUN, T.-T., EICHNER, R., SCHERMER, A., COOPER, D., NELSON, W.G. AND WiESS, R. A. (1984). Classification, expression andpossible mechanisms of evolution of mammalian epithelial keratins:A unifying model. In Cancer Cells: I. The Transformed Phenotype(A.J. Levine, G.F. Vande Woude, W.C. Topp and J.D. Watson,eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,NY.

TAYLOR-PAPADIMITRIOU, J., BURCHELL, J., CHANG, S., BARTER, J.,GENDLER, S. AND DURBAN, E. M. (1987). Use of monoclonal

antibodies to define the phenotype of cells cultured from thehuman mammary gland. In Gmwth and Differentiation ofMammary Epithelial Cells in Culture (J. Enami and R.G. Ham,eds), pp. 1-38, Japan Scientific Societies Press, Tokyo.

TAYLOR-PAPADIMITRIOU, J., BURCHELL, J. AND HURST, J. (1981o).

Production of fibronectin by normal and malignant humanmammary epithelial cells. Cancer Res. 41, 2491-2500.

TAYLOR-PAPADIMITRIOU, J. AND LANE, E. B. (1987). Keratinexpression in the mammary gland. In The Mammary Gland:Development, Regulation and Function (M.C. Neville and C.Daniel, eds), pp. 181-215, Plenum NY.

TAYLOR-PAPADIMITRIOU, J., PETERSON, J. A., ARKLIE, J., BURCHELL,

J., CERIANI, R. L. AND BODMER, W. F. (1981/;). Monoclonalantibodies to epithelium-specific components of the human milk fatglobule membrane: production and reaction with cells in culture.int.J. Cancer 28, 17-21.

TAYLOR-PAPADIMITRIOU, J., PURKIS, P. AND FENTIMAN, I. S. (1980).

Cholera toxia and analogues of cyclic AMP stimulate the growth ofcultured mammary epithelial cells. J. cell. Physwl. 102, 317-321.

TOLLE, H.-G., WEBER, K. AND OSBORN, M. (1985). Microinjectionof monoclonal antibodies specific for one intermediate filamentprotein in cells containing multiple keratins allows insight into thecomposition of particular 10 nm filaments. Eur.J. Biol. 38,234-244.

VAN MUIJEN, G. N. P., WARNAAR, S. O. AND PONEC, M. (1987).Differentiation-related changes of cytokeratin expression incultured keratinocytes and in fetal, newborn, and adult epidermis.Expl Cell Res. 171, 331-345.

WETZELS, R. H. W., HOLLAND, R., VAN HAELST, U. J. G. M.,LANE, E. B., LEIGH, I. M. AND RAMAEKERS, F. C. S. (1989).Detection of basement membrane components and basal cellkeratin 14 in noninvasive and invasive carcinomas of the breast.Am. J Path. 134, 571-579.

WOLMAN, S. R., SMITH, H. S., STAMPFER, M. AND HACKETT, A. J.

(1985). Growth of diploid cells from breast cancers. Cancer Genet.Cytogenet. 16, 49-64.

ZHANG, R., WILEY, J., HOWARD, S. P., MEISNER, L. F. AND

GOULD, M. N. (1989). Rare clonal karyotypic variants in primarycultures of human breast carcinoma. Cancer Res. 49, 444-449.

(Received 6 January 1989 —Accepted, in revised form,11 August 1989)

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