Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

Experimental

Cell Research

172 (1987) 192-203

Casein Gene Expression in Mouse Mammary Epithelial Lines: Dependence upon Extracellular Matrix and Cell Type DANIEL

MEDINA,*,’

M. L. LI,?

C. J. OBORN,”

Cell

and M. J. BISSELL?

*Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, and fLaboratory of Cell Biology, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D line. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of /?-casein mRNA in the presence or absence of prolactin. The heterogeneous COMMA-D line, but none of the clonal lines, was induced by the presence of prolactin to produce significantly increased levels of /3-casein MRNA. The inducibility of @-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. Individual matrix components of laminin, libronectin, heparan sulfate, heparan, or hyaluronic acid were not effective as substrata for the induction of /?-casein mRNA. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types. @ 1987 Academic Press, h.

The mammary gland is an appropriate model for studying factors which regulate tissue-specific function and maintain the differentiated phenotype. Milk protein synthesis and secretion are under the control of peptide and steroid hormones and represent specific products of mammary epithelial differentiation. Recent experiments have demonstrated that the expression of most mammary epithelial functions may be regulated by the extracellular matrix (ECM). Several laboratory groups [l-9] have demonstrated that morphological differentiation of the mammary cells in culture is dependent upon the nature of the substratum. Bissell and co-workers [5, 6, 8, 91 concluded that the regulation of different milk components in mouse mammary epithelial cells is modulated to different extents by a given substratum. For instance, the floating collagen gel membrane supports synthesis and secretion of all caseins but not those of whey acidic proteins and alactalbumin. The modulation of mammary-specific function by the substratum has also been demonstrated for rat [IO] and rabbit mammary gland [ll, 121. ’ To whom reprint requests should be addressed. Copyright @ 1987 by Academic Press, Inc. AU rights of reproduction in any form reserved cn14-4827/87 $03.00

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Mammary-specific differentiation may also be dependent upon other cell types in the mammary gland, since metabolic cooperativity between epithelium and the mammary fat pad has been demonstrated in uiuo [ 131. An interaction between mammary epithelium and specific 3T3 sublines has also been shown to promote maintenance of hormone-dependent differentiation in the absence of exogenous ECM [14, 151. The possibility that this latter model is mediated by ECM deposited by one or both cell types remains an open question. The experiments described above have used primary cell cultures of mammary cells obtained from pregnant and occasionally from lactating or virgin mice. The primary cell cultures represent a large commitment in terms of animal resources and technical labor as well as restrict the feasibility of some experimental approaches such as transfection of desired gene constructs. Cell strains or permanent cell lines which retain mammary-specific functional differentiation would offer new and additional opportunities to pursue questions on regulation of differentiation. The recent description of the COMMA-D cell line 1161 has allowed an additional opportunity to examine factors which may regulate mammary cell differentiation. The COMMA-D cell line was derived from mid-pregnant mammary glands of BALB/c mice. It exhibits mammary-specific morphological differentiation upon transplantation into the mammary fat pads of syngeneic mice for at least 14 in vitro passages and exhibits mammary-specific functional differentiation in vitro for at least 22 passages under some conditions. The cell line comprises two to three cell types as characterized by antisera to different keratin proteins [ 171. It is also heterogeneous with respect to cell morphology, DNA content, and tissue morphogenesis in viva [17]. Of six mouse mammary cell lines examined, COMMA-D was the only line which exhibited mammary-specific differentiation in uiuo and in vitro [ 171. The purpose of the experiments presented here was twofold. First, since the heterogeneity of the COMMA-D line was the major characteristic distinguishing it from other mammary cell lines, we examined whether homogeneous clonal lines derived from COMMA-D exhibited similar properties of functional differentiation. Second, the effects of specific components of ECM as well as reconstituted basement membrane on expression of milk protein MRNAs were examined in COMMA-D cells. MATERIALS

AND

METHODS

Cell lines. The origin of COMMA-D was described previously [16]. The three clonal cell lines DB-1, FA-5, TA-1 were derived by limited dilution plating of the parental line COMMA-D and the sublines COMMA-F and COMMA-T [17]. The cells were seeded at a concentration to yield an average of one cell/well in a %-well dish. Epithelial islands were expanded by sequential passage into I-cm* wells, 35 mm dishes, and Falcon T-25 flasks. The clonal lines were frozen in liquid nitrogen and thawed aliquots were recovered and used in these experiments. Passage (p) numbers 5, 8, and 6 were used from DB-1, FA-5, and TA-1, respectively. Line DB-1 was isolated from COMMA-D, ~14, line FA-5 was isolated from COMMA-F, p5, and line TA-1 was isolated from COMMA-T, p5. COMMA-D, ~10-14, was used in these experiments. The origin of MOMA- was from a separate preparation of mid-pregnant BALBK mammary glands and was described in [17]. The cell line MA-4 was derived by limited dilution plating of the parental MOMAline, p8. Passage numbers 6 and 7 were used in these experiments.

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Cell culture. The cell lines maintained as stock cultures or used for the indirect immunofluorescence experiments were grown in F12 medium supplemented with 5% FBS, 15 mM Hepes buffer, gentamicin (50 ug/ml), insulin (5 ug/ml), transferrin (10 ug/ml), and epidermal growth factor (10 &ml). For examining the presence and induction of casein, NIH ovine prolactin (3 ug!ml) and cortisol (2 ug/ml) were also added to the medium. The cell cultures were incubated at 37°C in a water-saturated atmosphere containing an air-CO2 gas mixture (92.5 %: 7.5 %). Preparation of attached and jloating collagen gels. Collagen was prepared as described by Michalopoulous and Pitot [18] using rat tail tendons. Briefly, rat tails in 70% ethanol were extracted with 1: 1000 diluted acetic acid at 4°C for 48 h. Collagen was collected after centrifugation at 13,000g for 30 min at 4°C. To prepare the collagen gel in a 35-mm dish, 0.88 ml of the stock collagen solution (2-3 mg/ml) was mixed with 0.11 ml of 10x medium 199 and 0.11 ml of 0.34 N NaOH. The dish was then placed in the CO2 incubator until the gel polymerized. Cells were allowed to grow for 4 days. In the case of floating gels, the gels were released from the dish using a sterile spatula and allowed to float in the medium. Experiments were performed 24 days postflotation. Preparation of Engelbreth-Ho/m-Swarm (EHS) reconstituted matrix. The ECM from EHS tumors was prepared according to the method described by Kleinman et al. [19]. EHS tumors were initially a gift of Drs. J. D. Roll and D. M. Bissell, Jr., and subsequently were produced in our laboratory (M. J. B.) by standard procedures. All operations were performed at 4°C or on ice. Tumor tissue grown in the presence of the lathyritic agent p-aminopropionitrile was weighed and suspended in a buffer consisting of 3.4 M NaCl, 0.05 M Tris-HC1 (pH 7.4) at 1 g/ml. The tissue was then homogenized with a motorized homogenizer, followed by centrifugation in a SW 27 rotor at 25,000 rpm (112,OOOg) for 30 min. The recovered EHS material in the pellet was dissolved in 2.0 M urea overnight and clarified by centrifugation as above. The ECM-containing fractions in supematants were dialyzed against 0.5 M NaCI, 0.05 M Tiis-HCl (pH 7.4) in the presence of 0.1% chloroform overnight and then dialyzed against medium (without serum). The isolated EHS extracellular matrix was frozen until ready to use. Zsolated ECM components. ECM components, including laminin (L), fibronectin (F), heparan sulfate (HS), heparan (H), and hyaluronic acid (HA), were spread directly on plastic or collagen gel surfaces at 50-150 ug per 35-mm dish. Indirect immunofluroescence assuy (UF). The IIF assay has been described in detail in [17, 201. Cells were plated at 3~ 104/cm2 into 35-mm dishes containing glass coverslips. At 48-72 h after seeding, when the cells were 40-60% confluent, the coverslips were collected and processed as previously described [17, 201. Several controls were run to check for specificity of the reaction. These included the use of nonimmune sera; absence of the primary or secondary antibody; blocking reactions, where purified proteins were available (laminin, milk fat globule protein (MFGP)); and most important, that all five cell lines were examined concurrently with the same antibody. The cells were examined using a Leitz epifluorescence microscope. Antibodies. Both polyclonal antisera and monoclonal antibodies were utilized in these experiments. The rabbit polyclonal antisera to human epidermal keratins and bovine hoof keratins were obtained from DAKO, Inc., California and are hereafter referred to as p-keratin antibody [17]. The specific reactivities of the antisera and antibodies have been published previously [7, 21-251. We are grateful to the following individuals for providng the antiserum and antibodies listed below: Bonnie Asch for the goat anti-mouse vimentin antiserum [21]; Gordon Parry for the rabbit anti-mouse milk fat globule protein antiserum [22]; Merton Bemfeld for the rat monoclonal antibody 281-2 to mouse mammary cell surface heparan sulfate proteoglycan [23]; Jo Hilgers and Arnoud Sonnenberg for the rat monoclonal antibodies 33A10, JSE3, and 117C9 [24]; Birgit Lane and Joyce Taylor-Papadimitrious for mouse monoclonal antibodies LP-34 and LE-61 to epithelial cell keratines [25]; and Elisa Durban for rabbit anti-mouse casein antiserum [7]. Transplantarion. The tumor potentials of the mammary cell lines were assessed by the mammary fat pad transplantation method as described in [17, 261. The cells were collected by enzymatic dissociation with trypsin-EDTA and centrifuged in the presence of DMEM, 5% FBS, and insulin. The resulting pellet was resuspended in serum-free DMEM at a concentration of 1X10’ cells/ml. Aliquots (10 ul) representing 1X 10’ cells were injected into the cleared mammary fat pads of 3-weekold syngeneic BALB/c female mice with the use of a Hamilton microliter syringe with a permanent needle. At 8 weeks after transplantation, four to six of the mammary fat pads were removed from the mice and processed as whole mounts as described in [26]. The rest of the recipients were palpated every 2 weeks for mammary tumors for a period of 6 months. Each cell line was assayed two to three times except COMMA-D which was done once. Flow cytometric analysis of cellular DNA content. The analysis of cellular DNA content by flow cytometry was performed as described previously [t7]. Briefly, cells were fixed in 70% ethanol and

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stored at 4°C until analysis. The samples were centrifuged and the pellets were resuspended in saline plus EDTA at pH 7.4. The cell suspensions were treated with RNase (50 Kg/ml) for 30 min at room temperature and then stained with propidium iodide (PI; 50 ug/ml) for 10 min. Normal rat lymphocytes and normal mammary epithelial cells were processed along with the experimental samples. The PI-stained cells were analyzed with an Ortho 50H Cytofluorograf interfaced to an Ortho 2150 data analysis system (Data General MPi200 microcomputer). Cytoplasmic RNA isolation. Cells were washed with PBS and homogenized with 12 strokes of a Btype hand-held homogenizer in a lysis buffer containing 0.14 M NaCl, 1.5 mM MgQ, 10 mM Tris-HCI (pH 8.6), 0.5 % NP40, 10 mM vanadyl-ribonucleoside complex. Nuclei (saved for DNA determination) and cell debris were separated from cytoplasmic fraction by centrifugation at 1500s for 15 min. The cytoplasmic fraction was mixed with 3 vol of 4 M guanidinium isothiocyanate, 50 mM Tiis-HCl (pH 7.6), 10 mM EDTA, and 0. I M a-mercaptoethanol. This mixture was layered on top of 5.7 M CsCl and spun in a SW50.1 rotor at 36,000 rpm (155,OOOg) for 20 h. Cytoplasmic RNA was recovered from the pellet by dissolving in 10 mM Tiis, I mM EDTA (pH 7.6) and was stored in ethanol at -70°C until used. RNA dot b/or and hybridization. Twofold serial dilutions of RNA dotted on nitrocellulose membrane followed the method of White and Bancroft [27]. Briefly, the RNA solution in Tiis-EDTA was mixed with an equal volume of a denaturing solution (60% 20x standard saline citrate @SC), 12.8% formaldehyde) and heated at 65°C for 15 min before a sequential serial dilution and application on nitrocellulose membrane. The membrane was baked at 80°C for 2 h. Prehybridization and hybridization with denatured “P-labeled (nick-translated) probes were performed in a solution containing 50% formamide, 5x SSC, 1 x Denhardt’s solution, and 2.5 mM sodium phosphate (pH 6.5) at 42°C for 12 to 20 h. The final wash of the membrane was done in 0.1 x SSC and 0.1% SDS at 65°C. Blots were then exposed to X-ray film for an appropriate length of time. Dots on X-ray film were scanned and compared using an LKB densitometer. DNA derermination. DNA determination was done using a fluorometric assay with Hoechst dye 33258X. Purified salmon sperm DNA was used as a standard.

RESULTS Biological Characteristics of Cell Lines

The cell lines were characterized with respect to morphology by phase contrast microscopy, to relative DNA content by flow cytofluorometry, to tumor potential by transplantation into syngeneic mice, and to immunoreactivity of mammary cell-specific proteins by polyclonal and monoclonal antibodies. Some of these characteristics are illustrated in Tables 1 and 2. The morphology of COMMA-D cells has been described previously [16, 171. The cell line was heterogeneous and

TABLE

1

Characteristics of mammary cell lines Cell line

Parent

COMMA-D DB-1 FA-5 TA-1 MA-4

COMMA-D COMMA-D COMMA-D/F COMMA-D/T MOMA- 1

Relative DNA content’ 2.1c 1.8C 2.6C 2.8C 2.1C

(70) (74) (92) (81) (88)

Tumorigenicityb 4112 0124 O/36 O/36 0124

’ Numbers in parentheses refer to percentage of cells containing this DNA content. Diploid DNA content for normal mammary cells is 2C. b The number of tumors per total number of mammary fat pads injected.

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contained both polygonal and angular (slightly elongated) cells. Its relative DNA content of 2.1C was compatible with the original karyotype analysis 116, 171. The COMMA-D cell line was weakly tumorigenic and gave rise to four tumors by 6 months after transplantation. The tumors were “sarcomatous” in morphology, similar to the results of earlier experiments [ 171. The frequency of preneoplastic outgrowth in the fat pads was low and only 25% of the outgrowths produced alveolar outgrowths with a mean percentage of 31% of the fat pad filled. The outer four cell lines exhibited various degress of epithelial morphology from cuboidal (MA-4) to polygonal (FA-5, TA-1) to angular (DB-1). In contrast to the COMMAD cell line, these lines were more homogeneous in confluent cultures. The relative DNA content varied with each cell line (i.e., DB-1, 1-W; FA-5, 2.6C; TA-I, 2.K; and MA-4, 2.1C). The four cell lines were nontumorigenic and did not produce any type of normal or preneoplastic outgrowths in the mammary fat pads. Antigenic

Patterns

of Cell Lines

The cell lines were examined for the expression of mammary epithelial cellspecific proteins by an indirect immunofluorescence assay using a variety of polyclonal antisera and monoclonal antibodies (Table 2). The reactivity of most of these reagents have been described elsewhere [7, 21-251. The COMMA-D cell line exhibited epithelial (p-keratin, LE-61, laminin, 33A10, and JSE3)- and mammary epithelial (MFGP, 281-2)-specific proteins and was negative for a mesenchymal-specific (117~9) protein. The partial staining with vimentin antisera was seen previously [17]. The differential response of normal mammary cells to 33AlO and JSE3 (Table 2) was noted in the original description of these antibodies [24]. Of interest was the positive staining of the COMMA-D cells to the monoclonal antibodies LP34 and LE61 which react with rodent myoepithelial and mammary epithelial cells, respectively [25]. These results supported the interpretation of earlier experiments which indicated that the COMMA-D cell line was cytologically heterogeneous [ 171. Cell line DB-1 was unique since it expressed very few of the specified proteins; however, it reacted with mammary epithelial-specific antisera MFGP and laminin. Unlike COMMA-D cells, the DB-1 cells expressed the protein recognized by JSE3 and not 33AlO. Interestingly, DB-1 cells did not react with antibodies to keratins or vimentin. Cell lines FA-5 and TA-1 exhibited almost identical reactivity. The cell lines stained positively with p-keratin and LE-61 in a homogeneous fashion and negatively for LP-34, indicating a relatively pure epithelial population. This interpretation was supported by positive staining in a homogeneous fashion with antisera to MFGP and antibody to 281-2. As with cell line DB-I, both cell lines reacted positively with JSE3 and negatively with 33A10 antibodies. Interestingly, neither of these two cell lines exhibited laminin staining. Cell line MA-4 stained in a homogeneous fashion for epithelial markers (pkeratin, LE-61, MFGP, 28 1-2, and laminin), very weakly for a myoepithelial marker (LP-34), and heterogeneously for both 33AlO and JSE3. It is interesting to

(~10-12)

COMMA-D COMMA-D COMMA-D/F COMMA-D/T MOMA- 1

3+b 3+ 4+ 4+

2+ 2+ 1+ (+I

Vimentin (1 : 40)

p-Keratin (1: loo)

Parent line 3+ 4+ 4+ 4+ 4+

MFGP (1 : 250) (+I -

Casein (1 : 50)

Polyclonal antisera”

4+ 4+ 4+

3+ 3+ 4+ 4+

3+ 2+

33AlO

cell-specific

281-2’ (1: 100)

epithelial

Laminin (1 : 20)

assay for mammary

2

(+I 2+ 3+ 1+ 1+

JSE3

Monoclonal

markers

-

117c9

antibodies

2+ (+I

LP34

4+ 4+ 4+ 4+

LE61

’ Numbers in parentheses refer to dilutions of antisera and antibodies. The monoclonal antibodies were used undiluted except 281-2. b Grading scale (% positive): -, 0%; (+), l-S%; l+, 6-25%; 2+, 26-50%; 3+, 51-75%; 4+, >75%. ’ The monoclonal antibodies recognize specific molecules on mammary cells. Antibody 281-2 recognizes a mammary epithelial cell surface heparan sulfate-rich proteoglycan [23], antibodies 33AlO and JSE3 recognize glycoproteins expressed predominately by differentiated (luminal and alveolar) and undifferentiated (basal and stem) mammary epithelial cells, respectively [24, 291, antibody 117C9 recognizes an epitope on the Forssman glycolipid expressed on tibroblasts [24, 331, and antibodies LP34 and LE61 recognize keratins expressed preferentially by mouse myoepithelial and all epithelial cells, respectively [25].

COMMA-D DB-1 FA-5 TA-1 MA-4

Cell line

immunojluorescent

Indirect

TABLE

ii 2 z. B

B a 4 ; 2 9 3

g

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Fig. 1. Quantification of /3-casein mRNA in COMMA-D cells. RNA was extracted and processed from cultures grown for 4 days on plastic (I and 2) on attached gels (3 and 4), and on floating gels (5 and 6). Lanes 2, 4, and 6 contained prolactin as described under Materials and Methods. All cultures contained insulin and cortisol. RNA equivalent to 15 ug of DNA was dotted (top). The second and third lane dots are l/2 dilutions.

point out that only COMMA-D contained a minor expressed casein in the presence of prolactin.

population

of cells which

Modulation of @-Casein

The influence of substrata and hormones on the expression of tissue-specific mRNA synthesis was monitored using hybridization with 32P-labeled p-casein and whey acidic protein (WAP) probes. The levels of /3-casein mRNA produced in COMMA-D cells grown on plastic that attached and released collagen type I gels in the presence and absence of prolactin were compared (Fig. 1, Table 3). pCasein expression in COMMA-D cells was found to be greatly modulated by both prolactin and substrata. A 50-fold increase in /?-casein message was detected in cells grown on a floating collagen gel in the presence of prolactin as opposed to cells grown on plastic in the absence of prolactin. Cells grown on an attached collagen substrate exhibited a live-fold increase compared to cells grown on plastic (Fig. 1). Three of the other four cell lines (DB-1, FA-5, and TA-1) were not inducible for fl-casein mRNA under any of the culture conditions. The fourth cell line (MA-4) exhibited a sevenfold increase in p-casein mRNA when grown on floating collagen gels in the presence of prolactin; however, the absolute amount of casein message was very low compared to the amount found in COMMA-D cells (Table 3). Whey acidic protein message was not detected under any conditions (data not shown). The levels of @casein mRNA were inconsistent in late passage COMMA-D cells (3 passage 12). In one experiment, MRNA was detectable in cells grown on attached collagen gels but in the absence of prolactin. In another experiment, Bcasein mRNA was not detectable under any conditions. The basis for this inconsistency was not evident; however, it might be related to selection of unresponsive cells with culture passage. It is important to note, however, that late passage COMMA-D (~16-22) cells have been shown to be inducible for /L casein by other investigators (J. Rosen, personal communication).

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MG

C

EHS

MG EHS

HA

HA

L

L

H

HSH

F

P

HS

F CI

Fig. 2. #I-Casein mRNA levels in COMMA-D cells grown on different substrata. Cells were grown and harvested as described under Materials and Methods. Top dots are RNA equivalent to 5 ug DNA with l/2 dilutions for second and third dots. (A) Cells grown on either plastic (P) or plastic coated with different substrata. MG is equivalent RNA isolated from late pregnant gland. Other abbreviations are as described in text. (B) Sister cultures were plated on top of flat thick collagen gels coated with the specific substrate as described under Materials and Methods. Two days after plating, the gels were floated and cells were harvested 2 days later. After collagenase treatment (Worthington, Type III, 0.3 %) cytoplasmic RNA was prepared as described. Note there is an appreciable (fourfold) increase in mRNA levels over plastic in all cultures, including those grown on EHS-coated collagen gels.

Effect of Components of the Extracellular

Matrix

on B-Casein Modulation

Since the COMMA-D cell line in early passage was readily inducible for bcasein mRNA synthesis, we examined the influence of specific components of

TABLE

3

Relative concentration of /3-casein mRNA in presence and absence of prolactin in mammary epithelial cell lines Plastic

Floating gels

Cell line

Prl _a

Prl +

Prl -

Prl +

COMMA-D DB-1 FA-5 TA- 1 MA4

lb 0.5 1 1 0.2

2 0.4 1 1 0.5

5 0.4 1 1 1

50 0.4 1 1 1.5

a Prl, prolactin; -, absence; +, presence. b The level of /I-casein mRNA in COMMA-D on plastic in the absence of prolactin was considered the baseline and was assigned the value of 1, The levels of fi-casein mRNA in the other groups are expressed as the multiple of the baseline value.

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Fig. 3. Morphology of COMMA-D on different substrata. COMMA-D cells were seeded at 1x106/35-mm plate coated with different substrata as in Fig. 2A. Phase contrast micrographs were taken 3 days later (x40).

the extracellular matrix as well as a reconstituted basement membrane (EHS) on this process. The results of these experiments are shown in Fig. 2. RNA dot-blot analysis indicated that cells grown on individual ECM-components-coated dishes were about equal or slightly better than on plastic surface alone; however, none were as successful at inducing fi-casein gene expression as the EHS extract (Fig. 2A). When thin coats of each of the same ECM material were applied to the top of thick type I rat tail collagen gels and floated, a fourfold increase in the level of /3-casein mRNA was observed in all cases (Fig. 2B). COMMA-D cells grown on EHS-coated floating gels made the greatest amount of #&casein mRNA which was 40-70% of /?-casein mRNA levels seen in the pregnant gland controls based on a DNA equivalent. The EHS reconstituted matrix also exerted a profound effect on the light microscope morphology of subconfluent cultures. Whereas the COMMA-D cells grown in the presence of individual components grew as polygonally shaped cells in a loose sheet, the cells exposed to EHS matrix grew as dense clusters (domes) separated by bands of more flattened cells (Fig. 3). Morphological examination of COMMA-D cells in dense confluent cultures revealed dome formation on all surfaces coated with the various extracellular materials, including fibronectin, laminin, heparan hyaluronic acid, and EHS. The domes were more frequent and occurred earlier on EHS.

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DISCUSSION The first aim of these experiments was to determine if homogeneous cell populations derived from the COMMA-D cell line were inducible for /3-casein. It was evident that cloned cell populations derived from COMMA-D were not inducible for /I-casein under our culture conditions. This is despite the fact that all lines were mammary epithelial and represented three different cell phenotypes as determined by expression of specific mammary proteins. In contrast, the modulation of /3-casein by hormones in the parental COMMA-D cell line was evident and marked. The possibility that the clonal lines lost the capacity for functional differentiation with passage in vitro needs to be considered. We intentionally examined the clonal lines as early as feasible, i.e., within 5-8 passages of their establishment as lines. It is for this reason that a second cloning was not performed. The single cloning was a compromise between achieving cell homogeneity and avoiding late passage. Recent experiments have demonstrated that careful trypsinization to recover all of the COMMA-D cells from the flask during serial passage results in retention of the property of /I-casein inducibility for up to 22 passages (Rosen and Medina, unpublished). These results support the earlier interpretation that the unique growth and functional characteristics of COMMAD may be due to the cellular heterogeneity of the cell line. Whether this heterogeneity was generated by a stem cell or several stable subpopulations was not resolved by the experiments. The second aim of these experiments was to examine the effect of substrata on induction of B-casein. As with normal mammary epithelial cells, the expression of B-casein was regulated by both the presence of hormones and the nature of the substrata [S, 8, 281. The present experiments have examined single components of the ECM, i.e., hyaluronic acid, heparan, heparan sulfate, fibronectin, and laminin, which were shown not to induce fi-casein gene expression by themselves. However, each component in combination with floating collagen I gels slightly enhanced p-casein expression. In contrast, a complex extracellular material (i.e., the ECM from EHS tumor) was very effective in allowing an appreciable hormonal induction of @-casein and did not require a collagen gel. The ECM from EHS is high in laminin, collagen, and proteoglycans [19], indicating the importance of complex interactions between the responsive cells and the ECM molecules. The data suggest that the response of inducible cell populations is a result of interactions among specific cell types, multiple ECM components, and hormones. The roles of different cell types might extend to the ability to synthesize and secrete specific macromolecules (i.e., laminin and proteoglycans) which are necessary for the alveolar-type epithelial cells to respond to hormones. Alternatively, one cell type may influence in a direct manner the response of a second cell type. The importance of cell-cell interactions in maintenance of the fully differentiated mammary cell in culture was recently documented by Levine and Stockdale [14, 151. The response to lactogenic hormones of normal mammary epithelial cells grown in monolayer cell culture was maintained if the cells were grown on feeder layers of 3T3-Ll or 3T3-C2 cells, but not on parental 3T3 cells.

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One or both of the above-mentioned mechanisms may be important in the response of normal mammary cells to the feeder layers. The immunocytochemical staining experiments utilizing polyclonal antisera and monoclonal antibodies to epithelial- and mammary-specific proteins were an attempt to delineate specific mammary cell phenotypes. This approach was recently reported by Sonnenberg et al. [24, 291 using similar antibodies. Some of the results were reminiscent of those reported by Sonnenberg et al. [24, 291 and Dulbecco et al. [30]. For instance, the absence of staining in line DB-1 with keratin and 281-2 antibodies in the presence of positive staining to MFGP and laminin antisera reflects the keratin-negative mammary epithelial cell type reported by these authors [24, 301. Similarly, Sonnenberg et al. [24] noted that differentiated mammary cells (luminal/alveolar) stained positive for 33AlO and negative for JSE3 antibodies; in contrast, JSE3 stained the basal cells in end buds, alveoli, and ducts. It is tempting to categorize the three clonal cell lines as representing specific cell phenotypes as cataloged by Sonnenberg et al. [241. For instance cell lines MA-4 and DB-1 had staining characteristics similar to those of the luminal type 1 cell (strong keratin and 33AlO staining) and the alveolar cell (keratinnegative but MFGP-positive), respectively. However, the correlations break down with respect to the staining with JSE3 which should be expressed only in basal cells and with laminin which is thought to be expressed predominately by myoepithelial cells. Furthermore, it is well documented that mouse mammary epithelial cells will exhibit altered expression of keratin proteins [31] and cellsurface proteins [29] when grown in monolayer cell culture; thus, a delineation based on expression of multiple cell-stage-specific proteins is hazardous. Nevertheless, it is significant that the clonal cell lines represented the subpopulations found in the parental COMMA-D line with one exception. So far, we have not isolated a clonal line which expresses myoepithelial cell characteristics (i.e., LP34-positive, MFGP-negative). Our current approach is to examine the functional capabilities of the clonal lines using different combinations much as we did using a reconsituted basement membrane. If such a reconstituted cell population included a myoepithelial-like cell, then such an approach might make it possible to group cell types according to function and interrelatedness. Alternatively, the continued analysis of new clones of COMMA-D may yield a multipotent cell which yields several cell types. In summary, these experiments support the hypothesis that both extracellular matrix @-lo, 28, 321 and cell interactions 114, 151 are important in the modulation of functional differentiation of the mammary gland. In addition, the experiments illustrated the interactions of several macromolecules in a reconstituted ECM which duplicated the effect of a floating collagen gel. Such preparations should allow a finer delineation of cellular-ECM interactions in the hormonal induction of milk proteins. This investigation was supported by the Health Effects Research Division, Offuze of Health and Environmental Research, US DOE Contract DE-ACO3-76SFOOO98, research grants from the NIH, BSRG RR05918 (M.J.B.) and CA-39017 (D.M.), and a gift for research from Monsanto Company to M.J.B. We thank Drs. Jeffrey Rosen and Lothar Henninghausen for cDNA clones to B-casein and Dr.

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Robert Ulhich for the flow cytometric analysis. We gratefully acknowledge the technical assistance of Frances S. Kittrell and Carroll Hatier.

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