Casein accumulation in mouse mammary epithelial cells after growth stimulated by different hormonal and nonhormonal agents.

0013-7227/90/1253-1173$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 2 Printed in U.S.A.

Casein Accumulation in Mouse Mammary Epithelial Cells After Growth Stimulated by Different Hormonal and Nonhormonal Agents* BRETT K. LEV AY-YOUNG, SUSAN HAMAMOTO, WALTER IMAGAWA, AND SATYABRATA NANDI Cancer Research Laboratory (B.K.L-Y., S.H., W.I., S.N.) and Department of Molecular and Cellular Biology (S.N.), University of California, Berkeley, CA 94720

in the progeny cell populations induced by different growthpromoting agents, all the cultures were able to accumulate aand /3-casein on subsequent stimulation by PRL and linoleic acid in the second phase of culture. Since, in vivo, luminal epithelial cells of the mammary gland are the only cell type capable of synthesizing milk products, these results indicate that all the different growth stimulants, hormonal and nonhormonal, result in the predominant proliferation of luminal-type epithelial cells. These results have important implications for studies of the mechanism of growth control in and transformation of mammary epithelial cells. (Endocrinology 126:1173-1182,1990)

ABSTRACT. Mammary epithelial cells obtained from virgin mice were cultured in collagen gel with linoleic acid-containing serum-free growth medium supplemented with hormonal (PRL and progesterone, epidermal growth factor, somatomedin-C) or nonhormonal (lithium ion, phosphatidic acid containing phospholipid liposomes) growth stimulating agents. The phenotypes of the resulting progeny cells were compared by examining the ultrastructure, immunohistochemical staining for luminal epithelial and myoepithelial cells and casein, and assessing the quantity of biochemically detectable a- and /3-casein. Although there are some differences in ultrastructure and immunostaining

M

OUSE MAMMARY epithelial cells, in vivo, grow in response to a combination of hormonal (1-3) and probably growth factor (4) influences, followed by full differentiation and the synthesis and secretion of milk (see ref. 5 for review). The absence of any of the many hormones renders the mammary epithelial cells partially or fully unable to correctly respond to the remaining influences. In seeking to understand the control of growth in mammary epithelial cells, we and others have found that in serum-free collagen gel culture mouse mammary epithelial cells show good growth responses to many different agents, including the mammogenic hormones PRL and progesterone (P; 6, 7), epidermal growth factor (EGF; 6, 8, 9), phosphatidic acid-containing phospholipid liposomes (PA-liposomes, 10), lithium ion (Li+, 11, 12), and linoleic acid (13), among others. In most cases demonstration of the growth-promoting nature of these agents was accompanied by some evidence of the mammary nature of the resultant cells. However, these characterizations are often limited (6, 8-14) and usually Received September 5,1989 * This work was supported by Public Health Service grants CA09041 and CA05388, provided by the National Cancer Institute, Department of Health and Human Services. Address all correspondence and reprint requests to: Dr. Brett K. Levay-Young, Cancer Research Laboratory, University of California, Berkeley, CA 94720

do not include characterization of the PRL responsiveness of the resultant cells or the production of milk proteins. This is of interest because production of milk protein is the primary function of mammary epithelial cells. To address the question of the effects of growth stimulators on mammary epithelial phenotype, we have attempted to recapitulate the two-phase nature of the mammary epithelial cell growth and differentiation in cell culture. This is accomplished by placing isolated mammary epithelial cells in collagen gel with serum-free medium containing different individual growth promoters. This was followed by release of the collagen gel and exposure to a medium which stimulates differentiation. In this way we hoped to assess the effect of the individual growth stimulatory agents on the ability of the progeny mammary epithelial cells to produce protein products characteristic of the differentiated state. Although we have previously examined and partially characterized mouse mammary epithelial cells after growth in culture with EGF (7, 15) and the combination of hormones PRL and P (7), these characterizations were based on one or a few criteria; and in the case of PRL and P did not involve a two-phase culture system. This previous work also leaves open the phenotype of the cells stimulated to grow by nonhormonal growth-promoting

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GROWTH-PROMOTING AGENTS AND MAMMARY DIFFERENTIATION

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agents. This is an important question because there is evidence from mammary epithelial cells of the rat that growth stimulatory agents in vitro have profound effects on the phenotype of the resulting cells (16-18). For example, in rat mammary epithelial cells EGF induces proliferation of a cell type with many myoepithelial characteristics, either in collagen gel culture (17) or on plastic (18). We have a continuing interest in the potential differences between cells stimulated to grow by hormonal and nonhormonal agents, since previous work from this laboratory has shown that transformation of mammary epithelial cells in vitro by carcinogen results in tumors whose phenotype is dependent on the growth stimulator contained in the culture medium (19). In this report we describe the nature of mammary epithelial cells after growth in linoleic acid-containing serum-free medium with somatomedin-C (Sm-C, insulinlike growth factor-I), and the nonhormonal agents PAliposomes and Li+ in terms of ultrastructural morphology, immunostaining with a variety of antisera, and capability to accumulate biochemically detectable casein proteins. We have compared the cells stimulated by the nonhormonal agents to cells stimulated to grow by PRL and P or EGF, and find that the cells are functionally comparable in their ability to synthesize milk protein.

Materials and Methods Animals Adult (4-month-old) female virgin BALB/c mice were obtained from the University of California Cancer Research Laboratory breeding colony. Epithelial cell isolation The cell dissociation was essentially as described previously (20, 21). Briefly, the mammary glands were minced and dissociated with collagenase (0.05% CLS II, Worthington, Malvern, PA), and further digested with 0.01% Pronase (CalbiochemBehring, San Diego, CA). The epithelial cells were separated from stromal cells and cell fragments by centrifugation on preformed 42% Percoll (Pharmacia, Piscataway, NJ) density gradients and used directly for collagen gel culture. Collagen gel culture Collagen gel culture of mammary epithelial cells has been extensively described and reviewed elsewhere (20, 21). The serum-free medium, which has also been described (6, 13) was a 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagles's medium (both from GIBCO Laboratories, Grand Island, NY). This medium was supplemented with transferrin, soybean trypsin inhibitor, d-a-tocopherol (all from Sigma, St. Louis, MO), trace element mixture (6), and antibiotics. Fatty acid-free BSA (FAF-BSA, Armour, Tarrytown, NY) was conjugated to the sodium salt of linoleic acid (Sigma; 13), and used with the concentration of fatty acid at 10 Mg/ml and the

Endo • 1990 Voll26«No2

concentration of BSA at 160 fig/ml. This medium will be referred to as the basal medium. The medium was changed every 2 days. For growth, the cells were cultured in the basal medium with either insulin (I, Sigma) at 10 Mg/ml or Sm-C (Amgen, San Diego, CA) at 100 ng/ml. These are required in addition to growth-promoting agents, but do not promote any growth of mouse mammary epithelial cells on their own (22). They are presumably active through the type-I Sm-C receptor. Several different growth-promoting agents were used. EGF (Collaborative Research, Bedford, MA) was added at 10 ng/ml. PRL (1 Mg/ml, NIADDK oPRL PS-17) and P (50 ng/ml, Sigma) were used in combination. Lithium ion (Li+) was added as LiCl at 5 mM, previously shown to be the optimal concentration (9). Phosphatidic acid-containing liposomes (PA-liposomes) were composed of dilinoleoylphosphatidylcholine/dilinoleoylphosphatidic acid (1:1 molar ratio) and cholesterol (moles cholesterol 20% of total moles lipid phosphate) and prepared by extrusion of an aqueous lipid suspension from a French pressure cell at 18,000 psi (23). All phospholipids were from Avanti (Birmingham, AL). The liposomes were added at a concentration of 0.05 fiM lipid phosphate per ml to basal medium in which linoleate FAF-BSA complex was replaced by FAF-BSA. After 6 days of growth in these media, the cultures were terminated for analysis by the methods described below, or induced to accumulate casein as follows. The growth medium was removed, and the gels were rimmed with a small spatula to release them to float in the medium (15, 24). Release of the gel was followed by addition of the differentiation medium, containing I (10 fig/m\), PRL (1 Mg/ml), and linoleic acid. These cultures were terminated after 4-6 days of differentiation (1012 days total in culture) by the same methods as at the end of the growth phase. Termination of cultures For electron microscopy, individual collagen gels were fixed, processed, and embedded in Polybed 812 (24). For immunocytochemistry the collagen gels were processed by freeze substitution (25), as detailed previously (17). For biochemical analysis the culture was terminated by blotting the collagen gel on a stack of paper towels to remove trapped medium (26) and freezing the resulting membrane-like collagen/cell combination at -70 C for future assay (15). Electron microscopy The embedded samples were thick-sectioned at 1 /*m and stained with Mallory's methylene blue-azure II for preliminary examination. Selected samples were then thin-sectioned, stained with uranyl acetate and lead citrate, and examined in a JEOL 1005 electron microscope. Some samples were stained with ruthinium red to enhance visualization of basal lamina structure and other surface glycoproteins (27). Immunohistochemistry The immunocytochemistry techniques used have been described extensively elsewhere (7). Briefly, the freeze substituted samples were embedded in paraffin, and sections were cut at 6



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nm. The avidin-biotin-peroxidase complex immunoperoxidase procedure of Hsu et al. (28) was followed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The antisera used were: i) an anti-casein antiserum raised against glutaraldehyde-crosslinked acid-precipitated total mouse casein (29; diluted 1:300); ii) an antiserum raised against purified chicken smooth muscle actin (7, gift of D. Pitelka, diluted 1:300) which specifically stains myocpithelial cells in sections of mouse and rat mammary gland (17); iii) an antiserum to mainly a 50 kDa keratin which selectively stains myoepithelial cells when used on sections of mammary gland (30; gift of B. Asch, diluted 1:100); iv) an antiserum against keratin protein (Dako, Santa

FIG. 2. a) One-micron section of sample embedded in Epon and stained with methylene blue-azure II. Cross sectional view of a Li+ colony showing multilayering of the epithelial cells around a lumen (L) somewhat like that seen in the end bud. This is seen more often in Li+ colonies, but is occasionally observed with the other growth stimulants. Lumen (L). X400. b) Electron micrograph of colony showing the multilayered area of a Li+ colony. Several of the basal cells (M) are cut in cross-section and hence do not show an elongate profile. Lumen (L), collagen gel (CG). x2,800.

Barbara, CA, diluted 1:300), which stains all epithelial cell types (31). All immunocytochemistry was controlled with nonimmune or preimmune rabbit serum at equivalent dilutions. Casein assay procedures FlG. 1. a) One-micron section of sample embedded in Epon and stained with methylene blue-azure II. Cross sectional view of a PA-liposome colony and several of its branching arms. The typical arrangement of the cells in a layer 1-2 cells thick around a lumen (L) is seen. X400. b) Electron micrograph of a PA-liposome colony shows the typical 1-2 cell layer arrangement. Luminal cells have many apical microvilli and are joined by tight junctions (arrowheads); elongate myoepithelial-like cells (M) are located basally. Lateral and basal cell membranes are outlined due to ruthinium red en bloc staining. Lumen (L), collagen gel (CG). X6.300.

The effect of all culture treatments on casein accumulation was determined on triplicate cultures in each experiment. Each experiment was repeated 3-5 times. Data shown is from a typical experiment. To estimate the extent of differentiation, jS-casein was quantified by ELISA and /3-casein examined by protein blotting. Both procedures have been described extensively elsewhere (15). Prior to assay the frozen gels were homogenized in PBS with 5 mM EDTA and 1% Triton-X-100 at 4 C. The homoge-


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nate was centrifuged at 12,000 X g for 30 min at 4 C. The clear supernatant was used for the a-casein ELISA, NaDodSO4PAGE and protein assay. Protein assays were done according to the method of Bradford (32), using a kit obtained from Bio-Rad (Richmond, CA). Bovine 7-globulin was used as the standard. The ELISA was developed from the method of Engvall and Carlsson (33) and has been described previously (15). Protein blotting was done according to the method of Towbin et al. (34). Aliquots of the culture homogenates containing 25 ng protein were electrophoresed on 12.5% acrylamide gels (35), and electrophoretically blotted to nitrocellulose. The primary antibody was a rat monoclonal anti-/3-casein (36; gift of F. Stockdale). The position of the bound antibody was revealed with horseradish peroxidase labeled anti-rat IgG.

Results Morphology after growth phase The ultrastructural features of freshly isolated mouse mammary epithelial cells and cells grown in serum-free medium with I, P and PRL, and I and EGF have been described previously (7). In those media, the cells proliferate and form branching duct-like structures, also seen in the present study with the growth stimulants PAliposomes or Li+ in basal medium containing I or Sm-C. These three-dimensional colonies are composed of one to two layers of cells around a lumen (Fig. 1, a and b), although multilayering is occasionally noted, especially in Li+ colonies (Fig. 2, a and b). The multilayering and cell ultrastructure of cells grown in Li+ is reminiscent of the cells of the neck of the growing prepubertal end bud


(37). Immunostaining with antibody to keratin, a general marker for epithelial cells (31), showed mild to strong staining of most cells cultured with the various growth agents (data not shown). This confirms the epithelial nature of the progeny cells regardless of the growth stimulant used. Scattered cells positioned basally to the luminal cells and adjacent to the collagen substrate often have some of the features ascribed to the myoepithelial phenotype. These include dense fibril bundles, caveolae, hemidesmosomes, and abundant microfilaments (38). These basal cells were seen in all media conditions (Figs, lb, 3, and 3 inset) but were more often and consistently seen in Li+ colonies (Fig. 4). These cells are stained by actin antiserum (Fig. 4) or by antiserum to a 50 kDa myoepithelial keratin (30) (Fig. 5), further establishing the myoepithelial-like phenotype. They generally show an elongated profile, although when cut in cross-section can appear more rounded (Figs, lb, 2b, and 3). When the various growth factors were employed with Sm-C instead of I, the overall results were generally consistent with cells grown with I (Table 1), and the morphological features were similar to cells cultured in I (Fig. 6). There was little apparent secretory differentiation in cells cultured in Sm-C (Fig. 6). In all of the media combinations, no basal lamina formation was observed beneath the cell layer, as reported previously (7). En bloc staining with ruthinium red outlined the lateral and basal plasma membranes but did not reveal basal lamina fragments or structure (Fig. 3 with inset). Immunostaining for casein permitted an evaluation of the state of differentiation of the cells after growth. At the end of the growth phase, no staining for casein was seen in cultures grown with PA-liposomes ((—), see Table 2), occasional mild staining in EGF or Li+ cultures (— to —/+), and mild to moderate staining in PRL and P cultures (+ to ++). This scoring was based on the proportion of cells stained and the intensity of staining. An example of mild to moderate staining after growth with PRL and P is shown (Fig. 7). Morphology after differentiation phase

FIG. 3. Electron micrograph of a Li+ colony stained en bloc with ruthinium red shows lateral and basal cell membranes distinctly, but no basal lamina fragments or structure below the cell layer adjacent to the collagen gel. Lumen (L), collagen gel (CG). X4.500. Inset: higher magnification of an area from the same sample showing the interface between the basal cell membrane and collagen gel matrix and no lamina structure and myoepithelial features such as pinocytic caveolae (arrows), and filament bundles (arrowheads). xl5,000.

After the gels were released and medium changed to the differentiation medium of I, PRL, and linoleic acid, good secretory differentiation was seen in cultures grown with PRL and P, EGF, Li+, or PA-liposomes. A representative example is shown in Fig. 8. The luminal cells showed evidence of secretory differentiation such as increased cytoplasmic volume and organelles, many secretory vesicles containing dark casein-like granules and fat droplets, much like that seen in the gland of a pregnant mouse (24). Immunocytochemistry with casein antiserum showed that all cultures exhibited moderate (++)



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FIG. 4. Light micrographs of immunostained paraffin sections of cells cultured in Li+. a) Antiserum to actin. Basal myoepithelial-like cells are darkly stained. X150. b) Normal rabbit serum control. X150.

FIG. 5. Light micrographs of immunostained paraffin sections of PA-liposome colonies, a) Antiserum to actin. X200. b) Antiserum to 50 kDa keratin. Basal myoepithelial-like cells are stained giving similar pattern as seen with actin antiserum. X175.

to strong (+++) positive staining for casein in some of the luminal cells and colony lumens (Fig. 9). The amount of immunostaining was assessed as above and was in good agreement with the biochemical detection of casein production by ELISA results (Table 2).

Biochemical Differentiation During the first 6 days of culture (growth phase), mouse mammary epithelial cells grown with EGF, Li+, or PA-liposomes do not accumulate large amounts of a-



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TABLE 1. Accumulation of a-casein in cells grown in Sm-C containing medium, then switched into I containing differentiation medium Growth medium


TABLE 2. Accumulation of a-casein in cells grown with different growth-promoting agents characterized by immunocytochemistry and ELISA

ng a-casein per million cells Sm-C°

Sm-C->IC

EGF 0.002 ± 0.001 0.015 ± 0.007 1.21 ± 0.11 1.11 ± 0.29 PRL + P 0.037 ±0.010 0.58 ±0.17 0.42 ± 0.018 1.30 ± 0.32

Growth medium PRL + P EGF PA-Lpc

Growth 0

Cytochem +/+++ -/+

Differentiation 6

ELISA 0.126 ± 0.012 ± 0.003 ± 0.024 ±

Cytochem

ELISA

0.023 +++/++++ 5.39 ± 1.76 0.008 +++/++++ 2.84 ± 0.59 0.0006 ++ 5.44 ± 1.95 0.001 +++/++++ 8.94 ± 1.72

"Cells grown with Sm-C and the agents listed under "Growth Medium," then terminated after the growth phase and assayed for acasein content. 6 Cells grown with I and the agents listed under "Growth Medium," then terminated after the growth phase and assayed for a-casein content. c Cells grown with Sm-C and the agent listed under "Growth Medium," then switched into differentiation medium containing I, PRL, and Lin. After the differentiation phase, the cultures were terminated and assayed for a-casein content. d Cells grown with I and the agent listed under "Growth Medium," then switched into differentiation medium containing I, PRL, and Lin. After the differentiation phase, the cultures were terminated and assayed for a-casein content. Cells obtained from virgin mice were cultured in the media listed. Each medium was used in triplicate cultures, the figures listed are the mean with SD from a representative experiment.

° Immunocytochemistry, scored visually based on intensity of staining and proportion of cells stained with casein antiserum. * ELISA for a-casein, values are ng casein per >g protein. c Phosphatidic acid containing liposomes. Cells obtained from virgin mice were cultured during the growth phase with basal medium, I, and the specified agent for 6 days. Groups of cultures were terminated and processed for immunocytochemistry or ELISA, while similar cultures were released and the medium switched to I, PRL, and linoleic acid for 6 more days. These cultures were then terminated and processed as above. Figures are the mean of triplicate cultures with SD from a representative experiment.

FIG. 6. Electron micrograph of a colony after growth in medium with Sm-C, PRL and P. Note relative absence of indications of secretory activity and casein granules. Lumen (L). x6,000

Discussion

casein as measured by ELISA. This is not surprising, since there is no PRL in these media. Cells grown in medium with PRL+P do accumulate some casein but less than is accumulated during the differentiation phase of the culture (Table 2). When growth promoted by EGF, PRL and P, Li+ or PA-liposomes, the ability of the cells to accumulate casein on subsequent exposure to I, PRL, and Lin-BSA during the differentiation phase was not impaired (Table 2). As seen in our previous work (15), the cells after these different growth stimuli respond to PRL in the absence of F when linoleic acid is present. Thus, although there were some minor morphological differences at the end of

-/+

the growth phase there were no apparent differences in the ability of the progeny cells to accumulate casein resulting from the different growth stimulants. When cells were cultured with Sm-C instead of I, they accumulated very little casein at the end of the growth phase (Table 1). Although there was more variability in experiments with Sm-C, subsequent addition of I, PRL, and linoleic acid was able to stimulate casein accumulation in these cells (Table 1). The data presented above using the a-casein ELISA is essentially duplicated if the accumulation of /9-casein is examined by protein blotting after SDS-PAGE using a monoclonal anti-/3 casein antibody (36). Again, after growth in any medium without PRL, there is no detectable jfl-casein accumulation. After growth with PRL+P, a /?-casein band is barely visible. However, after the differentiation phase /3-casein is clearly detectable in all cases (Fig. 10).

The data presented here show that mouse mammary epithelial cells respond to different individual growthpromoting agents, including nonhormonal agents, by proliferation of predominantly luminal epithelial-like progeny cells which are similar by several criteria, including their ability to produce casein. Thus, the nonhormonal agents we used in this work and the hormones and growth factor we used here and in our previous work (7) do not induce a major shift of phenotype in the mouse mammary epithelial cells. Cells which have many characteristics of myoepithelial cells are maintained in this system by all agents and, except when Li+ is the growthpromoting agent, at roughly the same proportion as seen for myoepithelial cells in vivo. Even in this latter case,



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FIG. 7. Light micrographs of immunostained parafiin sections of PRL and P colonies before differentiation phase, a) Antiserum to casein. Moderate staining of some cells, b) Normal rabbit serum control. X200

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FlG. 8. Electron micrograph of a colony after growth in Li+ and SmC and differentiation in I, PPL, and linoleic acid. The lumen (L) is filled with darkly staining secretory product, and the lumihal cells show a high level of secretory differentiation: increased cytoplasmic volume, many mitochondria, RER strands, active golgi (arrows), numerous secretory vesicles (arrowheads), and fat droplets (FD). x8,000. Inset: Several secretory vesicles showing micellar pattern of the casein granule. X49.000

where the myoepithelial-like cells are seen in greater abundance, the topological relationship between the myoepithelial-like cells and the ductal/alveolar cells was maintained; the myoepithelial-like cells remain as scattered cells basally located in the colonies immediately adjacent to the collagen gel. In no case do the myoepithelial-like cells make up more than a small proportion

of the total cell number. This is in contrast to rat mammary epithelial cells cultured in EGF containing medium (17), where many myoepithelial-like cells proliferate and then migrate out into the collagen gel forming large, distinguishable colonies composed predominantly of myoepithelial-like cells. In some instances it appears that the myoepithelial-like cells stain for casein by the immunocytochemistry (cf. Fig. 9), and we have no ready explanation for this. Perhaps these cells actually represent an intermediate cell type, with features of both luminal and myoepithelial cells. It is also possible that the staining is an artifact, either due to the culture methods or due to spreading of abundant casein or immunoperoxidase reaction product. Regardless of the growth stimulating agent used, hormonal or nonhormonal, high levels of casein accumulation only occur in this culture system during the differentiation phase in the simultaneous presence of I, PRL, and linoleic acid. The only serum-free growth stimulator previously shown to produce progeny cells capable of casein accumulation in this two-stage culture system was EGF (15, 39). Thus, the PRL mediated regulation of casein accumulation is maintained in this serum-free system irrespective of the growth stimulant used. Although PRL is required for casein accumulation, F is not, replicating our previous observation (15). In a related observation, casein accumulation in cells grown with PRL and P is reduced until the P is removed from the medium. This aspect of hormonal regulation was not examined in our previous work (7). Previously, some investigators (40, 41) suggested that the inhibition of casein accumulation by P is mediated via an antago-


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FIG. 9. Light micrographs of immunostained paraffin sections of PRL and P colonies after differentiation phase in I, PRL, and linoleic acid, a) Antiserum to casein. Heavy staining of all cells and lumen, which may obscure visibility of myoepithelial-type cells, b) Normal rabbit serum control. X200.

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FlG. 10. Protein blot after SDS-PAGE and exposure to monoclonal anti-0-casein antibody. Cells were cultured as in the methods section in basal medium with various growth-promoting agents for 6 days of growth and then terminated. Duplicate cultures were released and exposed to basal medium with I and PRL for 6 days of differentiation, a) Mouse milk standard; b) MW markers, top to bottom kDa 130, 75, 50, 39, 27, 17; C-F, end of growth; G-I, after differentiation; c) I and EGF; d) I, PRL, and P; e) I and Li+; f) I and PA-liposomes; g) I and EGF to I, PRL, and linoleic acid; h) I, PRL, and P to I, PRL and linoleic acid; i) I and Li + 1, PRL, and linoleic acid; j) I and PA-liposomes to I, PRL, and linoleic acid. 25 fig protein loaded for each lane.

nistic effect on the glucocorticoid receptor, reducing the ability of glucocorticoid to synergize with I and PRL to cause casein accumulation in vivo or in organ culture. We showed earlier that in this culture system glucocor-

ticoid is not required for casein accumulation, and that at least some of the effects of F may be mediated via the stroma (15). Thus, in our collagen gel culture system P probably does not reduce casein accumulation via an antagonistic interaction with the glucocorticoid receptor. We cannot suggest an alternate mechanism based on our data, but it may implicate a direct action of progesterone via its own receptor. Recently, similar results have been reported in organ culture using tissue from pregnant rabbits (42). The growth stimulator Li+ produced cell colonies where the majority of the progeny cells superficially resembled the epithelial body cells seen in the "neck" of the growing prepubertal end bud (37). This observation is based on ultrastructural comparison with the in vivo tissue, since we do not have specific markers for this cell type. If this characterization can be substantiated by other means it may give some insight into the growth mechanisms which distinguish the end bud cells, which grow in prepubertal and pubertal mice in the absence of pregnancy hormones, from the ductal cells which reinitiate growth in the presence of pregnancy hormones. In summary, the current study provides unequivocal demonstration that mouse mammary epithelial cells, after responding to either hormonal or nonhormonal growth promoting agents, give rise to progeny cells which most closely resemble the luminal-type of mammary epithelial cells. Although these conclusions may seem unsurprising, they are important for several reasons. Recently, investigators in this laboratory have succeeded in transforming mouse mammary epithelial cells in vitro using carcinogens. The type of preneoplastic lesion, the numbers of tumors, (19) and the nature of oncogene activations (S. Miyamoto, personal communication) are



all dependent on the type of growth-promoting agent used. This might have been thought to be due to large phenotypic shifts in the in vitro cellular population, which is clearly not the case. Some of us (W.I., S.N.) are currently investigating the mechanisms of growth promotion by PA-liposomes in mouse mammary epithelial cells. These investigations are obviously of more import if the cells resulting from the growth stimulation retain their mammary nature.

Acknowledgments The authors gratefully acknowledge Dr. F. Stockdale, Dr. B. Asch, and Dr. D. Pitelka for providing antibodies and/or antisera for this work. Ovine PRL was the gift of the National Hormone and Pituitary Program of the NIADDK. We thank Dr. R. C. Guzman for editorial advice, and Mr. J. Underhill for photographic support.

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of casein gene expression. J Biol Chem 257:2182 41. Matusik RJ, Rosen JM 1978 Prolactin induction of casein mRNA in organ culture. J Biol Chem 253:2343 42. Iahn GA, Djiane J, Houdebine L-M 1989 Inhibition of casein synthesis by progestagens in vitro: modulation in relation to concentration of hormones that synergize with prolactin. J Steroid Biochem 32:373

The Endocrine Society's 72nd Annual Meeting Atlanta June 2 0 - 2 3 , 1990 Georgia World Congress Center For program and registration information please contact: The Endocrine Society 9650 Rockville Pike Bethesda, MD 20814 (301) 571-1802 FAX (301) 571-1869 Meet Us On Peachtree, June 20-23, 1990.


Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones.

Secretion of casein in cultures of mouse mammary epithelial cells on floating collagen gels.

Simultaneous production of casein and mammary tumor virus in mouse mammary epithelial cells grown on floating collagen gels.

Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.

Osmotic shock of cultured primary mammary cells amplifies the hormonal induction of casein gene expression.

Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells.

Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

Arginine Relieves the Inflammatory Response and Enhances the Casein Expression in Bovine Mammary Epithelial Cells Induced by Lipopolysaccharide.

Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells.

Serotonin regulates β-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors.

Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model.

Emodin inhibits LPS-induced inflammatory response by activating PPAR-γ in mouse mammary epithelial cells.

Selenium retention and inhibition of cell growth in mouse mammary epithelial cell lines in vitro.

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro.

The anti-inflammatory and antioxidant effects of melatonin on LPS-stimulated bovine mammary epithelial cells.

Binding of mouse and rabbit iron-59 transferrins to lactating mouse mammary epithelial cells.

Mouse mammary tumor virus production stimulated by hormones and polyamines in cells grown in semi-synthetic in vitro conditions.

C3H strain of mouse mammary tumour virus, like GR strain, infects human mammary epithelial cells, albeit less efficiently than murine mammary epithelial cells.

Mammary stroma modulates hormonal responsiveness of mammary epithelium in vivo in the mouse.

Unique growth pattern of human mammary epithelial cells induced by polymeric nanoparticles.

Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro.