EXPERIMENTAL
CELL
RJBEARCH
197,191-l%
(1991)
Establishment of Bovine Mammary Epithelial Cells (MAC-T): An in Vitro Model for Bovine Lactation HUNG
T. HUYNH,
Department
GILLES
of Animal
ROBITAILLE,
Science, McGill
AND JEFFREY
University,
The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 360 passages without showing any sign of senescence. They show the characteristic “cobblestone” morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in @-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) a.- and fl-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line Q, 1991 Academic Prem, Inc. unique.
INTRODUCTION
Milk represents the sole source of early postnatal nutrition in mammals. Milk synthesis occurs within clusters of differentiated mammary epithelial cells and is controlled by lactogenic hormones [l-9] and extracellular matrix (ECM) interactions [lo-171. Several mammary epithelial cell lines exist; each has detracting attributes. The most widely used in vitro mammary system is the mouse COMMA-D cell line [18] which exhibits mammary-specific functional differentiation when exposed to the appropriate ECM and lactogenic hormones [18, 191. However, this cell line is heterogeneous, being composed of at least three cell types [ 181 of which only lo-20% are capable of casein synthesis [ 191. Attempts to subclone the COMMA-D line lead to a loss
Mont&al,
D. TURNER’
Qu&ec H9X 1CO
of hormonal responsiveness [20] or a loss of ECM regulation [2]. A variety of established human breast cell lines exist and include T47D and MCF-7 lines (21) and HBL-100 [22]. These cell lines showed variable expression of differentiated phenotypes such as polarity [21]. Gaffney et al. [22] showed prolactin sensitivity and casein secretion in HBL-100 cells while Laherty et al. [23] found no prolactin binding and an absence of casein secretion. Rat mammary tumor cell lines have been described such as TMT-OSlMS, MT-lOO-TC [24] and rat Rama 25 mammary cell line [25]. The rat Rama 25 cell line, when grown on collagen gel, was able to form multicellular tubules which were similar to those observed in viuo [25]. There was no information on casein gene expression or lactose secretion reported by these workers. Established bovine mammary epithelial cell lines included the BMEC + H line [26] which are spontaneous clones which synthesize cytokeratins and desmosomal plaque proteins similar to those found in the mammary gland [27]. However, these cells do not assume a typical epithelial morphology, but instead are elongated with long slender processes extending over the surface of neighboring cells [26]. When grown on collagen gels in the presence of prolactin, these cells synthesized and secreted a very low level of casein (W. W. Franke, personal communication). The PS-BME-7 is another spontaneous bovine cell clone with hormone receptors for IGF-I [28]. However, casein secretion or prolactin sensitivity has not been reported. Expression of certain viral or oncogenes can establish primary cells causing them to circumvent senescence and crisis. These include simian virus 40 (SV40) [2834], polyomavirus [35], adenovirus Ela [36, 371, and other cellular oncogenes such as myc [38] or ~53 [3941]. We report that the expression of SV40 large T-antigen establishes primary bovine mammary epithelial cells in culture and yet allows hormonal responsiveness and secretion of milk-specific proteins and lactose. EXPERIMENTAL
’ TO whom correspondence should be addressed at Department of Animal Science, 21,111 Lakeshore Road, Ste. Anne de Bellevue, QuBbec, Canada, H9X 1CO.
PROCEDURES
Mammary tissue dissociation and culture. Primary bovine mammary gland cells were obtained by aseptic biopsy from lactating Holstein cows at slaughter and dissociated as per Burwen and Pitelka 191
0014~4827191$3.00 Copyright 0 1991 by Academic Prese, Inc. All rights of reproduction in any form reserved
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Hanks’ buffered saline solution, (HBSB, GIBCG), 11 m&f glucose, 4% BSA (Sigma, St. Louis, MO), and 200 IU/ml type II collagenase, (Sigma). The flask was rotated at 200 rpm for 60 min at 37OC. Single cells were obtained by filtration through 150 pm Nitex screens. Cells were pelleted at 800s for 5 min and then washed twice in DPBS. Cells were grown in complete DMEM [Dulbecco’s Modified Eagle’s Media (GIBCO) supplemented with 20% fetal calf serum (FCS, GIBCG), 5 rg/ml insulin (Sigma), 1 pg/ml hydrocortisone (Sigma), 50 rg/ml gentamycin (GIBCO)] on tissue culture plastic and incubated in 5% COLl and 100% humidity at 37°C. To overcome the problem of fibroblast outgrowth, cells were cloned by limiting dilution. Colonies arising from single cells with epithelial morphology and showing positive cytokeratin staining were selected for transfection. These clones and subsequent lines are mycoplasma negative. DNA transfection. Expression of the SV40 Large T-antigen was directed from the plasmid pBAPSV40TtsA58 obtained from L. Chalifour (BRI Montreal) and U. Lendahl (MIT, Boston). This plasmid includes a 4.0-kb human &actin promoter and a 0.75-kb SV40 polyadenylation sequence (Grunwald, Princeton, NJ) and a 2.6-kb BamHI fragment of SV40 large T-antigen [31]. The pSV2-neo plasmid [42] provided a dominant selectable marker and was provided by G. Matlashewski (McGill University, Montreal). Transfections were undertaken with adherent cells (1.3 X 106/cmz) using 10 pg pBAPSV40TtsA58 and 10 pg pSV2-neo per 1 X lo7 cells by the calcium phosphate procedure of Graham and van der Eb [43]. After 4 h of transfection, the cells were washed once with DPBS and glycerol shocked (15% glycerol) for 2 min, and then washed with DPBS. Cells were grown further for 24 h. Transfected cells were trypsinized with 0.25% trypsin andplatedin 6-well multiplates at a density of 5.0 X lo3 cells/cm’ in complete DMEM containing 200 pglml G418 sulfate (GIBCO). Selecting media were changed once every 3 days for 14 days. Surviving colonies were isolated with cloning rings. Evaluation of growth and temperature sensitivity. As the establishment plasmid carried a tsA mutant of SV40, it was important to determine if the G418-resistant colonies were temperature dependent. Three randomly selected colonies (MAC-T3, MAC-T4, MACT18), along with early passage JH25 cells were plated at a density of 1 X 10 cells per plate (55 cm’) in complete DMEM with 10% fetal calf serum. Cells were grown at 33 and 39’C for this experiment and at 37°C for all others. The number of cells per plate was determined daily. The mean from two plates was used to compare growth potential, doubling time, and cell density at confluency. Growth curves were constructed by plotting cell number versus the time in culture. Doubling time was determined by the slope during the log-phase. Zmmunoprecipitation of SV40 large T-antigen. To determine the presence of the large T-antigen, confluent cells were labeled with 300 &i [?S]-methionine for 5 h. Cells were washed with DPBS and then lysed with a solution of 1% NP-40,150 mMNaC1,20 mMTris, pH 8.0. Two hundred microliters of cell lysates were incubated with 15 pl of mouse anti-large T antigen kindly provided by Dr. G. Matlashewski (McGill University, Montreal) and 500 pl of net-gel buffer (50 n-&f Tris, pH 7.5,150 mMNaCl,5 mM EDTA, 0.05% NP-40,0.02% NaNs, 0.25% gelatin) and incubated overnight at 4°C. Then the staph A (immunoprecipitin) was added and the incubation was continued for an additional 30 min at 4°C. The immunoprecipitated materials were pelleted by centrifugation and washed three times with 1 ml of net-gel buffer. Radioactive precipitates were fractionated, using SDSPAGE, 12% acrylamide according to Laemmli [44]. After staining, the gel was dried under vacuum and autoradiographed using Kodak X-Omat film at -70°C. Tumorigenicity test. Seven Nu/Nu CD-l homozygous mice at 28 40 days of age (Charles River) were injected subcutaneously with 5 X lo6 cells representing clones MAC-T3, -T4, and, -T18 in free Ca2+ DPBS. As a positive control, the ~160 cell line, a v-abl-transformed NIH 3T3 cell line, was kindly provided by Dr. J. C. Bell (University of Ottawa, Canada). P160 cells cause tumors in nude mice within 4
AND
TURNER
weeks. Mice were palpated for the presence of tumor nodules at the injection site each week for 8 weeks.
Prokzctin responsiueness. Clonal cells were evaluated with regard to their responsiveness to prolactin. Parameters included: (1) intracellular casein accumulation by immunohistochemistry, (2) a- and P-casein secretion by Western blotting, and (3) fl-casein mRNA abundance by Northern blotting. Calf tail collagen gels were prepared according to the method of Michalopoulos and Pitot [45]. The collagen gels were sterilized under UV light for 2 h and then equilibrated in complete DMEM for 2 h. Cells were plated at a density of 2 X ld cells/cm’. After 24 h dead cells were removed with two DPBS washes. For the control plates, cells were grown in the basal medium (DMEM, 5% FCS, 5 pglml insulin, 1 aglml hydrocortisone) and for prolactin-treated cells, 5 pgglml prolactin (USDA-GPRL-B-I) was added to basal medium. Collagen gels were released to Aoat. Cells were also plated on tissue culture plastic to serve as controls. Medium was collected at 24,36,48, and 72 h after gels were released and analyzed for cr- and @casein secretion. Collagen gels containing cells were used to measure @-casein mRNA abundance and intracellular @casein content. The (Y- and fl-casein was evaluated by immunodetection on Western blots. Medium was separated using SDS-PAGE [44] and 12% acrylamide using a mini-protean II (Bio-Rad, Mississauga, Canada), electrotransferred onto nitrocellulose filters (70 V, 3 h, 4”C), and washed briefly in water and then TBS (20 n&f Tris, pH 7.5,500 n&f NaCl). Filters were blocked with 3% gelatin in TBS for 60 min and then washed twice in TBS with 0.05% Tween-20 (TTBS). The filters were incubated overnight in rabbit anti-bovine LY-and @-caseins antiserum diluted (1:2000 v/v) with TTBS containing 1% gelatin. After three washes in TTBS, the filters were transferred to the second antibody solution, goat anti-rabbit IgG alkaline phosphatase conjugate (1:2000 v/v) for 60 min. Filters were washed twice with TTBS and then TBS, 5 min each. Enzyme activity was developed as recommended by Bio-Rad. Northern blotting. Total RNA was extracted from cells grown on plastic and collagen gels using the procedure of Towle et al. [46]. The integrity of RNA samples was verified by ethidium staining and the quantity was determined spectrophotometrically. Twenty micrograms of total RNA were glyoxylated [47] and fractionated in 1.5% agarose gel in 10 n&f phosphate buffer, pH 6.5, at 60 volts for 2 h. Northern blots were performed after capillary transfer of glyoxylated total RNA onto Zeta-probe membrane (Bio-Rad) in 0.2 M NaOH overnight. The filter was baked at 8O’C for 2 h and then prehybridized according to the procedure of Denhardt [48]. Prehybridization solution consisted of 50% formamide, 5X Denhardt’s solution, 5X SSPE (0.9 MNaCI; 50 n-&f NaH,PO,. H,O, 4 mA4EDTA). Hybridization solution was done in the above solution with the addition of 32P-labeled heat-denatured bovine @-casein cDNA insert (1150 bp from J. P. Mercier, INRA, France) or bovine j3-actin cDNA (2600 bp from Degen (49)) at 42°C for 14 to 18 h. Posthybridization washes included three washes in 2~ SSC; 0.1% SDS and a single wash in 0.5X SSC; 0.1% SDS and 0.1x SSC; 0.1% SDS at room temperature. Filters were air-dried and exposed to Kodax X-Omat with two Cronex lighting-plus intensifying screens (DuPont) at -70°C. Zmmunohistochemistry. Monoclonal antibodies which react with cytokeratin (Type II, subfamily No. 1-8; Boehringer-Mannheim) and vimentin (Boehringer-Mannheim) were used to characterize cell type as epithelial or stromal, respectively on plastic substratum. Cells were seeded in 8-chamber slides (Lab-Tex 4838, Nunc, Naperville, IL) for 24 h and then fixed and stained according to manufacturers recommendations. Immunohistochemistry also was used to define ECM and prolactin effects on intracellular casein levels. Cells on collagen gels were treated with type II collagenase (0.3 mg/ml, Sigma) for 30 min at 37”C, washed twice with DPBS, plated on collagencoated 8-chamber slides (Nunc), and allowed to attach further for 2 h, and then were fixed in cold acetone at -20°C for 10 min. Forty micro-
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BOVINE MAMMARY CELLS liters of rabbit anti-bovine b-casein in DPBS (1:5 v/v) was applied per chamber and the slides were incubated at 37°C for 1 h in a 100% humidity chamber. As a control for nonspecific secondary antibody binding, the primary antibody was omitted and DPBS was usedin its place. Slides were subjected to three 5-min wsshesin DPBS at room temperature. Following the last wash, cells were incubated with a goat anti-rabbit-IgG-FITC conjugate in DPBS (1:40 v/v, BoehringerMannheim). The incubation and washes were performed as described above. Slides were directly visualized with a Jenalunar microscope, equipped with epifluorescence optics and appropriate excitatory and barrier filters for FITC.
RESULTS
Primary cultured cells formed confluent monolayers within 4 to 5 days. The major cellular contaminants were fibroblasts. Therefore, it was necessary to select epithelial cells by cloning. Of the original 1052 cells, 63 gave rise to colonies. The number of cells per colony ranged from 3000 to 5000. During this transition stage, some clones ceased to grow, entered crisis, and sloughed off. One of the early passage epithelial cell clone, MACT3, showed a typical epithelial morphology (Fig. 1A) and intense staining of cytoplasmic meshwork of cytokeratin fibrils (Fig. 1B) but not with vimentin. These characteristics were also seen in the bovine BMGE + H cell line [24, 251. The results indicated that these cells were epithelial in origin. Cotransfection of early passage cells with pBAPSV40 Tts A58 and pSV2-neo plasmids yielded several G418resistant colonies. Among 1 X 107-transfected cells, 117 colonies were obtained. Untransfected cells were killed 100% at a concentration of 200 pg/ml G418. The transfection efficiency was 6 cells per 10’ transfected cells. Seven randomly selected clones were grown successfully in culture up to 50 passages without any sign of crisis while the primary cells entered crisis at passages 16 to 25. When MAC-T clones (-T3, -T4, -TlS) were injected into seven immunodeficient mice and allowed to grow for more than 8 weeks, there was no sign of tumor nodule formation at the injected site. The result appeared to be a true negative as the ~160 cell line initiated prompt tumor growth in control animals within 3-4 weeks postinjection. TO verify the presence of large T-antigen in transfected cells, antigen was immunoprecipited with monoclonal antibodies and fractionated on SDS-PAGE. Figure 2 shows such an immunoprecipitation with monoclonal antibodies against the large T-antigen. The 92-kDa band which is characteristic for large T-antigen was found in all SV40-transfected cells but not in nontransfected, JH25 cells. A 53-kDa protein was coprecipitated with the large T-antigen because it was precipitated only in cells expressing large T-antigen. Cells which were not transfected with SV40 large T-antigen did not show either band.
Temperature-Independent
Growth of MAC-T Cells
Although the established gene carried tsA mutant SV40 gene, three transfected cell lines MAC-T3, MACT4, MAC-TlS, grew equally well at 33 and 39°C in monolayer cultures in 10% serum (Fig. 3). Following subculture, MAC-T cell lines and JH25 progressed through a characteristic growth pattern of lag phase within the first 24 h to enter into log phase on Day 2. Cell number increased at the same rate at the two temperatures for the next 5 days. Phase contrast micrograph showed that all of the available surface was occupied and that cells were in full contact with surrounding cells. The slope calculated during the log-phase at the two temperatures revealed that MAC-T cells had a doubling time of approximately 17 h. At confluency, MACT cells reached a density 4.0 X 10’ cells/cm’ when grown at 33,37, or 39°C. Since MAC-T cells grew at the same rate as the early passage JH25 cells, showed contact inhibition, and formed a uniform pavement of closely associated cells, we could conclude that there were minimal changes in growth characteristics because of transfection.
Cellular Differentiation When MAC-T3 cells were grown on culture plastic substratum they displayed a cuboidal epithelial morphology and remained flattened (Fig. 1A). Floating collagen gels have been shown to promote functional differentiation since cells cultured on this system were able to assume a shape similar to their in uiuo tissue of origin [ll, 12, 151. When MAC-T cells were plated on attached collagen at high density, they rearranged themselves to form small organoids (Fig. 4). As time progressed, these organoids become larger. Phase contrast microscopy revealed the lumen in the center of these structures. When released to float, the collagen gels contracted and all cells became cuboidal and difficult to distinguish. Collagen gels containing cells were collected at 24-h intervals from the time gels were released to Day 3 to assay for @-casein mRNA accumulation during induction. Cells grown on plastic substratum were evaluated to determine the basal level of @-casein RNA under noninduced conditions. The patterns of @-caseinand P-a&in mRNA in MACT cells are shown in Fig. 5. fi-casein mRNA was just detectable when MAC-T cells were grown on plastic substratum. When cells were plated on collagen gels and released to float for 24 h, there was a considerable increase in /3-casein mRNA abundance which reached a maximum by 48 h. There was no further increase in P-casein mRNA on Day 3. In the presence of prolactin, the level of @-casein mRNA abundance was not different for the first 24 h when comparing cells grown on
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FIG. 1. Morphology of MAC-T3 cells grown on plastic substratum. (A) Phase contrast microscopy shows an epithelial pavement of cuboidal-shaped cells. (B) Indirect immunofluorescence analysis. Cells were fixed and then stained with anti-cytokeratin type II, NO. l-8 antibody followed by fluoresceinated goat anti-rabbit antibody. The intermediate filaments are arranged around the nucleus and extend via desmosomes between cells. Magnification 800X.
floating collagen vs plastic. As time progressed, @-casein mRNA accumulation was augmented by prolactin addition (threefold) and was maintained until Day 3. Subsequent hybridization on the same blot with the bovine /3-actin probe showed that relatively equal amounts of intact RNA were present in each lane. The distribution of casein secretory vesicles in MACT cells on floating collagen gels in the presence and absence of prolactin is illustrated in Figs. 6A and 6B, respectively. In prolactin-treated cells (Fig. 6A) nearly
100% of the cells immunostained. Each cell contained numerous immunoreactive secretory vesicles (Fig. 6A). However, in the absence of prolactin, cells on floating collagen gels were still synthesized casein but the number of vesicles was reduced compared to cells treated with prolactin (Fig. 6B). When primary antibodies were omitted (Fig. SC), no immunoreactive vesicles were seen. These results indicate that the secretory vesicles contained casein-like material. Time course of casein secretion in cultured MAC-T
BOVINE
kd 130, 7550,
MAMMARY
_ LT-A -PS
3927,
14, FIG. 2. Immunoprecipitation of SV40 large T-antigen in stably transfected bovine mammary epithelial cells grown at 39’C. [%IMethionine-labeled lysates from 7 MAC-T clones were immunoprecipitated with the antiSV40 large T-antigen monoclonal MA& Proteins were separated by SDS-PAGE and shown by autoradiography. All clones produced a protein of 92 kDa characteristic of the large T-antigen (LT-A). Nontransfectedcells JH25 do not produce measurable immunoreactive material. Nonimmune mouse serum (ml failed to precipitate protein. P53 was also precipitated in transfected cells as a result of its association with the viral large T-antigen.
cells maintained on floating collagen in the absence and presence of prolactin is illustrated in Fig. 7. When MAC-T cells were grown on plastic substratum, casein secretion was below the limits of detection. @casein secretion was detected within 24 h of collagen gel release. For the first 2 days, there was no difference in casein secretion whether MAC-T cells were treated with or without prolactin. However, there was an approximate threefold increase in @-caseinsecretion on Day 3 in prolactin-treated cells. In contrast, a-casein secretion was barely detectable when prolactin was absent from the media. At 48 h in the presence of prolactin, a-casein secretion was easily measured.
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ization. Immunoprecipitation analysis clearly showed the presence of a 92-kDa protein, the putative viral large T-antigen, and a coprecipitating 53-kDa protein. The specific interaction between the T-antigen and a 53-kDa protein is a characteristic of SV40-transformed cells 1501.Indeed, we speculate that the 53-kDa protein is indeed ~53, a nuclear phosphoprotein which complexes with T-antigen and other DNA tumor virus transforming proteins [51]. These results agree with workers who induced proliferation in primary cells from mice, rabbits, and pigs [29, 30, 511. Indeed, the expression of viral T-antigen has been shown to be required for the initiation and maintenance of the transformed state in vitro [52, 531. Although the DNA construct used in this study was the temperature-sensitive A mutant of SV40 gene, the three MAC-T cell lines failed to exhibit a temperaturedependent alteration in proliferation. The three clones proliferated equally well at 33 and 39°C in media containing 10% serum. Similarly, large T-antigen protein was present at the “nonpermissive” temperature, 39°C. No estimates were made at the permissive temperature. This lack of temperature sensitivity has been extensively documented [30, 31, 34, 55-571. Risser and Pollack [55] found that not all cell lines transformed by wild type displayed the standard transformed pheno[ .
MAC-T4
DISCUSSION
Bovine mammary epithelial cells transfected with SV40 large T-antigen become established in vitro but retain their ability to differentiate and secrete milk-specific products. As such, these MAC-T clones represent an in vitro model for bovine lactation. This discussion will focus on the characterization of MAC-T cells including the nature of establishment, growth potential, and differentiation. Cellular immortalization is defined experimentally by the acquisition of unlimited growth capacity in vitro [30]. To date more than 350 serial passages have been made with MAC-T3 cells with no sign of senescence. In contrast, nontransfected early passage mammary epithelial cells typically entered crisis after 16-25 passages in culture. Low level, constitutive expression of SV40 large T-antigen appears responsible for the immortal-
DAYS IN CULTURE FIG. 3. Growth curves for bovine mammary epithelial cell lines. Three transfected clones MAC-T3, 4, and 18 and early passage bovine mammary cells grew equally well at 33°C and 39% Population doubling time was estimated at 17 h.
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F‘IG. 4. Morphology of MAC-T3 cells grown on attached collagen gels. The epithelial pavement of cells grown at high density for 3 days rea:rranges i nto organoid structures. A lumen-like structure is indicated. Magnification 130x.
type. Ctsuka et al. [31] reported that heat-adapted cell line8 derived from mou8e kidney cells transfected with SV40 temperature-sensitive A gene were able to grow equally well at both temperatures. This is entirely consistent with the above results. Petit et al. [30] found that the distribution of tsA58 carrying line8 was more heterogeneous, 70% of them could be classified into temperature Sensitive class whereas 30% were not sensitive to high temperature. Brockman et al. [56] found that 1 of 16 SV4OtsA cell line8 isolated showed temperature-resistant growth properties. Graf and Beug [57] isolated transformed variant8 of rat cell8 infected by the temperature-eensitive avian 8arcoma virus mutant8 that grew at the nonpermissive temperature. Further, they emphasized that the phenotype of t8A transformants might depend on the method of clone selection. Some 3T3 cells transformed by wild type SV40 had growth properties similar to normal 3T3 cell8 or intermediate between those of normal 3T3 and SV4OtsA transformants [58, 591. Since MAC-T cell8 were selected and grown at an intermediate temperature 37°C after transfection with the SV40 temperature-sensitive mutant A gene, these three clone8 appeared to have adapted to growth at temperature8 from 33 to 39°C. MAC-T cell8 appear to be immortalized but not transformed. They did not cause tumor8 upon injection into immunodeficient mice while the control ~160 cell line caused palatable tumors. Further the MAC-T cells were 8erum dependent and anchorage dependent a8 they were not able to form colonies in soft agar. When
grown on tissue culture plastic, they showed contact inhibition and did not overgrow or form foci. MAC-T cell clone8 retained the phenotypic characteristics of bovine mammary epithelial cells, like JH25, after subculture in vitro. On plastic substratum, MAC-T cell8 displayed typically cuboidal epithelial morphology. Indirect immunofluorescence using anti-cytokeratin revealed that MAC-T cells exhibited a cla88ical epithelial array of intermediate filaments. Intercellular desmosome-containing bridges were also observed. The failure of staining with anti-vimentin indicated the absence of stromal cells. These results are consistent with those of Schmid et al. [26] who also worked with bovine mammary epithelial cell8 (BMGE + H). Direct comparison of the immunofluorescent micrographs between MACT cell8 and BMGE + H by Professor Dr. Werner W. Franke confirmed that MAC-T cell8 were indeed epithelial (personal communication). When MAC-T cell8 were grown on collagen gels, they rearranged themselves to form bhBM8 or secretory dome structures. Cell8 within these structure8 became more columnar in shape. As culture time increased, duct-like structures connecting the dome8 became numerous. These characteristics of rearrangement8 resembled the in uiuo organization of differentiated bovine mammary alveoli. Similar dome formations have been reported in the mouse mammary epithelial cell line, COMMA-D. This normal mammary epithelial cell line retained epithelial morphology and routinely formed dome8 in vitro [18]. The difference between
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than that reported by Eisenstein and Rosen [19] who showed that fi-casein mRNA levels increased within 4 h after the addition of hydrocortisone and prolactin to
FIG. 5. Extracellular matrix and prolactin induce 8-casein mRNA accumulation in MAC-T3 cells. Total RNA (20 rg), isolated from cells grown on plastic (P) substratum or on calf tail collagen in the presence of 5 pglml prolactin (+) or without prolactin (-) was Northern blotted and then probed with bovine P-casein cDNA insert (A) or bovine P-actin cDNA insert (B).
MAC-T and COMMA-D lines was that COMMA-D formed domes when they were grown on plastic substratum, whereas MAC-T cells formed domes only when grown on collagen gels. When focusing on the secretory function of MAC-T cells, (Y- and B-casein proteins were examined. Caseins are found neither in the fetal calf serum nor in the medium so the presence of these products in the conditioned medium was useful as an indication of functional differentiation of these cells. The induction of the differentiated state by floating collagen gels and prolactin was accompanied by an increase in @casein mRNA, by accumulation of the intracellular casein, and subsequent casein secretion. Simply plating the cells on collagen gels increased ,B-casein mRNA abundance. The maximal level of @casein mRNA was reached within 48 h in the presence of prolactin. This induction was slower
FIG. 6. Immunolocalization of p-casein within differentiated bovine mammary epithelial cells. Immunofluorescent staining with a polyclonal antibody against bovine @-casein showed numerous vesicles in prolactin (A)-treated MAC-T3 cells on collagen gels. In the absence of prolactin (B), the number of immunoreactive vesicles is reduced. (C) The nonspecific binding of the secondary fluorescent antibody is low. Magnification 320X.
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FIG. 7. Western blot analysis of LY-and @-casein secreted from differentiated bovine mammary epithelial cells (MAC-T3) is increased by prolactin. Media from cells grown on floating collagen gels in the presence of 5 &g/ml prolactin (solid bars) or in the absence of prolactin (open bars) were separated by SDS-PAGE and the a- and @-casein detected by rabbit anti-bovine casein polyclonal antibodies and an anti-rabbit IgG alkaline phosphatase conjugate. Cells on collagen without prolactin produced detectable p-casein at 12 h (a), 24 h (b), 36 h (d), and 48 h (f) after plating. Casein secretion from prolactin-treated cells was initially modest at 24 h (c) and 36 h (e) but increased by 48 h (g). A casein standard (s) of cow milk contained 2 pg of protein. In contrast, a-casein secretion was very low in all treatments except for the 46 h treatment with prolactin.
cells and it continued to accumulate for at least 48 h. The magnitude of induction in casein mRNA abundance observed in MAC-T cells is similar to other systems. Primary mouse mammary epithelial cells grown on floating collagen gels had 3- to lo-fold more casein mRNA than cells on plastic substratum [15]. Simiiar findings of a 5-fold increase in p-casein mRNA accumulation was noted by Blum et al. [60] when cells were grown on basement substrata. The requirement for a deformable extracellular matrix was first noted by Emerman et al. [ 121,who showed that the floating collagen membrane allowed the cells to change shape and that there was a correlation between cell shape and the degree of differentiation. Others [ 14,171 have suggested that the geometry of the cells, their interaction with extracellular matrix constituents, cell-cell interactions, and maintenance of cellular polarity played a role in the expression of milk protein genes. Bovine mammary epi-
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thelial cells share a similar requirement for an extracellular matrix and cell-cell interaction for maximum casein synthesis/secretion. cr-Casein and P-casein were synthesized and secreted when MAC-T cells were grown on floating collagen gels. Indirect immunofluorescence using anti-casein antibodies illustrated that MAC-T cells responded to prolactin in a homogenous way in terms of casein synthetic capacity since almost all of the cells produced casein secretory vesicles. In contrast, COMMA-D cells have been shown to be morphologically uniform but heterogenous in terms of casein production [ 181. Typically only lo-20% of the COMMA-D cells produced casein at only one time judged by indirect immunofluorescence. In contrast the bovine epithelial cell line BMGE + H showed epithelial morphology but produced very little casein when induced by prolactin (W. W. Franke, personal communication). Therefore, with regard to in vitro casein production the MAC-T cells represent the most uniform in vitro system available. MAC-T cells secrete large quantities of LX-and /3-casein. The presence of fl-casein can be detected in the media as early as 12 h after plating on floating collagen gels. While cz-casein secretion required a longer time course and prolactin as well as extracellular matrix. Reflective densitometry is used here to provide a semiquantitative estimate. In the absence of prolactin, total casein secretion was estimated to be between 2.5 and 10 pg/m1/24 h. Prolactin increased this secretion rate by 48 h to 50 pg/m1/24 h (7.5 pg/106 cells/24 h). This level of casein secretion is some three- to sevenfold higher than those reported for freshly dissociated mouse cells [12] rabbit cells [17], and rat cells [60]. CONCLUSION
SV40 large T antigen confers immortality to bovine mammary epithelial cells. The existence of MAC-T cells illustrates the concept of nontransformed immortalized cells. Clonal MAC-T cells have been now growing for more than two years, (>350 passages). These cells grow rapidly, are to date stable, and show no signs of crisis. MAC-T cells are responsive to manipulations either in extracellular matrix and in the lactogenic hormones. When differentiated, MAC-T cells synthesize and secrete CX-and fi-casein. As such, MAC-T cells represent an in vitro model for bovine lactation. This investigation was supported by the Natural Sciences and Engineering Council of Canada OGP 36728 to J.D.T. and a postgraduate scholarship to H.T.H. We thank Drs. J. P. Mercier, G. Matlashewski, and L. Chalifour for cDNA clones and NHPP for the bPRL.
REFERENCES 1.
Akers, R. M., Bauman, D. E., Capuco, A. V., Goodman, and Tucker, H. A. (1981) Endocrinology 109,23-30.
G. T.,
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MAMMARY
2. Bali, R. K., Fri.& R. R., Schoenenberger, C. A., Doppler, W., and Groner, B. (1988) EMBO J. ‘7,2089-2095. 3. Brett, K., Hamamoto, L-Y. S., Imagawa, W., and Nadi, S. (1990) EndacrinaZagy 126,1173-1182. 4. Cowie, A. T. (1969) in Lactogenesis: The Initiation of Milk Secretion at Parturition (Reynolds, M., and Folley, S. J., Eds.), Vol. 157, p. 157, Univ. Pennsylvania Press, Philadelphia. 5. Houdehine, L. M. (1980) in Hormone and Cell Regulation (Dumont, J., and Nunez, J., Eds.) Vol. 4, p. 175, Elsevier/NorthHolland, Amsterdam. 6. Matusik, R. J., and Rosen, J. M. (1978) J. Biol. Chem. 253, 2343-2347. 7. Mepham, T. B., Gaye, P., and Mercier, J. C. (1982) in Development in Dairy Chemistry (Fox, P. F., Ed.), Vol. 1, pp. 115-156, Applied Science Publishers, London/New York. a. Ray, D. B., Jansen, R. W., Horst, I. A., Mills, N. C., and Kowal, J. (1986) Endocrinology 118,393-407. 9. Topper, Y. J., and Freeman, C. S. (1980) Physial. Rev. 60,10491106. 10. Bisseil, M. J., Hall, G. H., and Parry, G. (1982) Theor. Biol. 99, 31-68. 11. Burwen, S. J., and Pitelka, D. R. (1980) Exp. Cell Res. 126, 249-262. 12. Emerman, J. T., Enami, J., Pitelka, D. R., and Nandi, S. (1977) Proc. Natl. Acad. Sci. USA 74,4466-4470. 13. Emerman, J. T., Burwen, S. J., and Pitelka, D. R. (1979) Tissue Cell 11, 109-119. 14. Haeuptle, M. T., Yolande, L. M. S., Bogermann, E., Reggio, H., Racine, L., and Kraehenbuhl, J. P. (1983) J. Cell Biol. 96,14251434. Lee, E. Y-H., Parry, G., and Bissell, M. J. (1984) J. Cell Biol. 98, 146-155. 16. Li, M. L., Aggler, J., Farson, D. A., Hatier, C., Hassell, J., and Bissell, M. J. (1987) Proc. Natl. Acad. Sci. USA 84, 136-140. Suard, Y. M. L., Haeuptle, M. T., Farinon, E., and Kraehenbuhl, J. M. (1983) J. Cell Bial. 96, 1435-1442. 18. Danielson, K. G., Oborn, C. J., Durban, E. M., Butel, J. S., and Medina, D. (1984) Proc. Natl. Acad. Sci. USA 81.3756-3760. Eisenstein, R. S., and Rosen, J. M. (1988) Mol. Cell Biol. 8,31833190. Medina, D., and Li, M. L. (1987) Exp. Cell Res. 172,192~203. Van Deurs, B., Zou, Z-Z., Briand, P., Balslev, Y., and Petersen, 0. W. (1987) J. Histochem. Cytochem. 35,461-469. GaSney, E. V., Blackburn, S. E., and Polanowski, F. P. (1976) In Vitro 12, 328-329. 23. Laherty, R. F., Goff, C., Ghosh, S., King, M., Balcavage, W. X., Alvager, T., Geib, R. W., and Personett, D. (1990) Zn Vitro Cell. Dec. Biol. 26,933-935. 24. Ghosh, S. K., Roholt, 0. A., and Kim, U. (1983) In Vitro 19, 919-927. Bennett, D. C., Peachey, L. A., and Durbin, H. (1978) CeU 15, 283-298. 26. Schmid, E., Franke, W. W., Grund, C., Schiller, D. L., Kolb, H., and Paweletz, N. (1983) Exp. CeURes. 146, 309-328. 27. Schmid, E., Schiller, D. L., Grund, C., Stadler, J., and Franke, W. W. (1983) J. Cell Bial. 96, 37-50. Received April 30,199l Revised version received August 5, 1991
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28. Hadsell, D. L., Vega, J. R., Skaar, T. C., Gibson, C. A., and Baumrucker, C. R. (1990) J. Dairy Sci. 73(Suppl. l), 215. [Abstract] 29. Black, P. H., and Rowe, W. (1963) Proc. SOC.Exp. Biol. 114, 721-721. 30. Petit, C. A., Gardes, M., and Feunteun, J. (1983) Virology 127, 74-82. 31. Otsuka, H., Dubbs, D. R., and Kit, S. (1979) J. Gen. Viral. 42, 373-386. 32. Seif, R., and Cuzin, F. (1977) J. Viral. 24, 721-728. 33. Martin, R. G., and Chou, J. Y. (1975) J. Viral. 15,599-612. 34. Jat, P. S., and Sharp, P. A. (1989) Mol. CeUBiol. 9,1672-1681. 35. Cowie, A., De Villiers, J., and Kamen, R. (1986) Mol. Cell. Btil. 6, 4344-4352. 36. Ruley, H. E. (1983) Nature 304,602-606. 37. Houweling, A., van den Elsen, P. J., and van de Eb, A. J. (1980) Virology 105,537-550. 38. Mougneau, J., Lemieux, L., Rasoulzadegan, M., and Cuzin, F. (1984) Proc. Natl. Acad. Sci. USA 81, 57585762. 39. Ellyahu, D., Raz, A., GNSS, P., Gibol, D., and Oren, M. (1984) Nature 312.646-649. 40. Jenkins, J. R., Rudge, K., and Currie, G. A. (1984) Nature 312, 651-654. 41. Parada, L. R., Land, H., Weinberg, R. A., Wolf, D., and Rotter, V. (1984) Nature 312,649-651. 42. Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Gen. 1, 327341. 43. Graham, F. L. and van der Eb, A. J. (1973) Virolagy 52.456-467. 44. Laemmli, U. K. (1970) Nature 277,680-685. 45. Michalopoulos, G., and Pitot, H. C. (1975) Exp. Cell Res. 94, 70-78. 46. Towle, H. C., Mariash, C. N., and Oppenheimer, J. H. (1980) Biochemistry 19,579-585. 47. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manuel, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 48. Denhardt, D. (1966) Biophys. Res. Commun. 23,641-646. 49. Degen, J. L., Neubauer, M. G., Degen, S. J. F., Seyfried, C. E., and Morris, D. R. (1983) J. Biot. Chem. 258,12,153-12,162. 50. Lane, D. P., and Crawford, L. (1979) Nature 278.261-263. 51. Marshall, C. J. (1991) Cell 64, 313-326. 52. Martin, G. (1981) Adu. Cancer Res. 34, l-68. 53. Tooze, J. (Ed.) (1981) DNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 54. Butel, J. S., Brugge, J. S., and Noonan, C. A. (1974) Cold Spring Harbor Symp. Quant. Bicl. 39,25-36. 55. Risser, R.; and Pollack, R. (1974) Virology 59, 477-489. 56. Brockman, W. W. (1978) J. Virol. 25,860-870. 57. Graf, T., and Beug, H. (1976) ViroZagy 72,283-286. 58. Volget, A., and Pollack, R. (1975) J. Cell Phys. 85, 151-162. 59. Pollack, R., Osborn, M., and Weber, K. (1975) Proc. Natl. Acad. Sci. USA 72,994-998. 60. Blum, J. L., Zeigler, M. E., and Wicha, M. S. (1987) Ezp. Cell Res. 173,322-340.
CELL
RJBEARCH
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(1991)
Establishment of Bovine Mammary Epithelial Cells (MAC-T): An in Vitro Model for Bovine Lactation HUNG
T. HUYNH,
Department
GILLES
of Animal
ROBITAILLE,
Science, McGill
AND JEFFREY
University,
The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 360 passages without showing any sign of senescence. They show the characteristic “cobblestone” morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in @-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) a.- and fl-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line Q, 1991 Academic Prem, Inc. unique.
INTRODUCTION
Milk represents the sole source of early postnatal nutrition in mammals. Milk synthesis occurs within clusters of differentiated mammary epithelial cells and is controlled by lactogenic hormones [l-9] and extracellular matrix (ECM) interactions [lo-171. Several mammary epithelial cell lines exist; each has detracting attributes. The most widely used in vitro mammary system is the mouse COMMA-D cell line [18] which exhibits mammary-specific functional differentiation when exposed to the appropriate ECM and lactogenic hormones [18, 191. However, this cell line is heterogeneous, being composed of at least three cell types [ 181 of which only lo-20% are capable of casein synthesis [ 191. Attempts to subclone the COMMA-D line lead to a loss
Mont&al,
D. TURNER’
Qu&ec H9X 1CO
of hormonal responsiveness [20] or a loss of ECM regulation [2]. A variety of established human breast cell lines exist and include T47D and MCF-7 lines (21) and HBL-100 [22]. These cell lines showed variable expression of differentiated phenotypes such as polarity [21]. Gaffney et al. [22] showed prolactin sensitivity and casein secretion in HBL-100 cells while Laherty et al. [23] found no prolactin binding and an absence of casein secretion. Rat mammary tumor cell lines have been described such as TMT-OSlMS, MT-lOO-TC [24] and rat Rama 25 mammary cell line [25]. The rat Rama 25 cell line, when grown on collagen gel, was able to form multicellular tubules which were similar to those observed in viuo [25]. There was no information on casein gene expression or lactose secretion reported by these workers. Established bovine mammary epithelial cell lines included the BMEC + H line [26] which are spontaneous clones which synthesize cytokeratins and desmosomal plaque proteins similar to those found in the mammary gland [27]. However, these cells do not assume a typical epithelial morphology, but instead are elongated with long slender processes extending over the surface of neighboring cells [26]. When grown on collagen gels in the presence of prolactin, these cells synthesized and secreted a very low level of casein (W. W. Franke, personal communication). The PS-BME-7 is another spontaneous bovine cell clone with hormone receptors for IGF-I [28]. However, casein secretion or prolactin sensitivity has not been reported. Expression of certain viral or oncogenes can establish primary cells causing them to circumvent senescence and crisis. These include simian virus 40 (SV40) [2834], polyomavirus [35], adenovirus Ela [36, 371, and other cellular oncogenes such as myc [38] or ~53 [3941]. We report that the expression of SV40 large T-antigen establishes primary bovine mammary epithelial cells in culture and yet allows hormonal responsiveness and secretion of milk-specific proteins and lactose. EXPERIMENTAL
’ TO whom correspondence should be addressed at Department of Animal Science, 21,111 Lakeshore Road, Ste. Anne de Bellevue, QuBbec, Canada, H9X 1CO.
PROCEDURES
Mammary tissue dissociation and culture. Primary bovine mammary gland cells were obtained by aseptic biopsy from lactating Holstein cows at slaughter and dissociated as per Burwen and Pitelka 191
0014~4827191$3.00 Copyright 0 1991 by Academic Prese, Inc. All rights of reproduction in any form reserved
192
HUYNH,
ROBITAILLE,
Hanks’ buffered saline solution, (HBSB, GIBCG), 11 m&f glucose, 4% BSA (Sigma, St. Louis, MO), and 200 IU/ml type II collagenase, (Sigma). The flask was rotated at 200 rpm for 60 min at 37OC. Single cells were obtained by filtration through 150 pm Nitex screens. Cells were pelleted at 800s for 5 min and then washed twice in DPBS. Cells were grown in complete DMEM [Dulbecco’s Modified Eagle’s Media (GIBCO) supplemented with 20% fetal calf serum (FCS, GIBCG), 5 rg/ml insulin (Sigma), 1 pg/ml hydrocortisone (Sigma), 50 rg/ml gentamycin (GIBCO)] on tissue culture plastic and incubated in 5% COLl and 100% humidity at 37°C. To overcome the problem of fibroblast outgrowth, cells were cloned by limiting dilution. Colonies arising from single cells with epithelial morphology and showing positive cytokeratin staining were selected for transfection. These clones and subsequent lines are mycoplasma negative. DNA transfection. Expression of the SV40 Large T-antigen was directed from the plasmid pBAPSV40TtsA58 obtained from L. Chalifour (BRI Montreal) and U. Lendahl (MIT, Boston). This plasmid includes a 4.0-kb human &actin promoter and a 0.75-kb SV40 polyadenylation sequence (Grunwald, Princeton, NJ) and a 2.6-kb BamHI fragment of SV40 large T-antigen [31]. The pSV2-neo plasmid [42] provided a dominant selectable marker and was provided by G. Matlashewski (McGill University, Montreal). Transfections were undertaken with adherent cells (1.3 X 106/cmz) using 10 pg pBAPSV40TtsA58 and 10 pg pSV2-neo per 1 X lo7 cells by the calcium phosphate procedure of Graham and van der Eb [43]. After 4 h of transfection, the cells were washed once with DPBS and glycerol shocked (15% glycerol) for 2 min, and then washed with DPBS. Cells were grown further for 24 h. Transfected cells were trypsinized with 0.25% trypsin andplatedin 6-well multiplates at a density of 5.0 X lo3 cells/cm’ in complete DMEM containing 200 pglml G418 sulfate (GIBCO). Selecting media were changed once every 3 days for 14 days. Surviving colonies were isolated with cloning rings. Evaluation of growth and temperature sensitivity. As the establishment plasmid carried a tsA mutant of SV40, it was important to determine if the G418-resistant colonies were temperature dependent. Three randomly selected colonies (MAC-T3, MAC-T4, MACT18), along with early passage JH25 cells were plated at a density of 1 X 10 cells per plate (55 cm’) in complete DMEM with 10% fetal calf serum. Cells were grown at 33 and 39’C for this experiment and at 37°C for all others. The number of cells per plate was determined daily. The mean from two plates was used to compare growth potential, doubling time, and cell density at confluency. Growth curves were constructed by plotting cell number versus the time in culture. Doubling time was determined by the slope during the log-phase. Zmmunoprecipitation of SV40 large T-antigen. To determine the presence of the large T-antigen, confluent cells were labeled with 300 &i [?S]-methionine for 5 h. Cells were washed with DPBS and then lysed with a solution of 1% NP-40,150 mMNaC1,20 mMTris, pH 8.0. Two hundred microliters of cell lysates were incubated with 15 pl of mouse anti-large T antigen kindly provided by Dr. G. Matlashewski (McGill University, Montreal) and 500 pl of net-gel buffer (50 n-&f Tris, pH 7.5,150 mMNaCl,5 mM EDTA, 0.05% NP-40,0.02% NaNs, 0.25% gelatin) and incubated overnight at 4°C. Then the staph A (immunoprecipitin) was added and the incubation was continued for an additional 30 min at 4°C. The immunoprecipitated materials were pelleted by centrifugation and washed three times with 1 ml of net-gel buffer. Radioactive precipitates were fractionated, using SDSPAGE, 12% acrylamide according to Laemmli [44]. After staining, the gel was dried under vacuum and autoradiographed using Kodak X-Omat film at -70°C. Tumorigenicity test. Seven Nu/Nu CD-l homozygous mice at 28 40 days of age (Charles River) were injected subcutaneously with 5 X lo6 cells representing clones MAC-T3, -T4, and, -T18 in free Ca2+ DPBS. As a positive control, the ~160 cell line, a v-abl-transformed NIH 3T3 cell line, was kindly provided by Dr. J. C. Bell (University of Ottawa, Canada). P160 cells cause tumors in nude mice within 4
AND
TURNER
weeks. Mice were palpated for the presence of tumor nodules at the injection site each week for 8 weeks.
Prokzctin responsiueness. Clonal cells were evaluated with regard to their responsiveness to prolactin. Parameters included: (1) intracellular casein accumulation by immunohistochemistry, (2) a- and P-casein secretion by Western blotting, and (3) fl-casein mRNA abundance by Northern blotting. Calf tail collagen gels were prepared according to the method of Michalopoulos and Pitot [45]. The collagen gels were sterilized under UV light for 2 h and then equilibrated in complete DMEM for 2 h. Cells were plated at a density of 2 X ld cells/cm’. After 24 h dead cells were removed with two DPBS washes. For the control plates, cells were grown in the basal medium (DMEM, 5% FCS, 5 pglml insulin, 1 aglml hydrocortisone) and for prolactin-treated cells, 5 pgglml prolactin (USDA-GPRL-B-I) was added to basal medium. Collagen gels were released to Aoat. Cells were also plated on tissue culture plastic to serve as controls. Medium was collected at 24,36,48, and 72 h after gels were released and analyzed for cr- and @casein secretion. Collagen gels containing cells were used to measure @-casein mRNA abundance and intracellular @casein content. The (Y- and fl-casein was evaluated by immunodetection on Western blots. Medium was separated using SDS-PAGE [44] and 12% acrylamide using a mini-protean II (Bio-Rad, Mississauga, Canada), electrotransferred onto nitrocellulose filters (70 V, 3 h, 4”C), and washed briefly in water and then TBS (20 n&f Tris, pH 7.5,500 n&f NaCl). Filters were blocked with 3% gelatin in TBS for 60 min and then washed twice in TBS with 0.05% Tween-20 (TTBS). The filters were incubated overnight in rabbit anti-bovine LY-and @-caseins antiserum diluted (1:2000 v/v) with TTBS containing 1% gelatin. After three washes in TTBS, the filters were transferred to the second antibody solution, goat anti-rabbit IgG alkaline phosphatase conjugate (1:2000 v/v) for 60 min. Filters were washed twice with TTBS and then TBS, 5 min each. Enzyme activity was developed as recommended by Bio-Rad. Northern blotting. Total RNA was extracted from cells grown on plastic and collagen gels using the procedure of Towle et al. [46]. The integrity of RNA samples was verified by ethidium staining and the quantity was determined spectrophotometrically. Twenty micrograms of total RNA were glyoxylated [47] and fractionated in 1.5% agarose gel in 10 n&f phosphate buffer, pH 6.5, at 60 volts for 2 h. Northern blots were performed after capillary transfer of glyoxylated total RNA onto Zeta-probe membrane (Bio-Rad) in 0.2 M NaOH overnight. The filter was baked at 8O’C for 2 h and then prehybridized according to the procedure of Denhardt [48]. Prehybridization solution consisted of 50% formamide, 5X Denhardt’s solution, 5X SSPE (0.9 MNaCI; 50 n-&f NaH,PO,. H,O, 4 mA4EDTA). Hybridization solution was done in the above solution with the addition of 32P-labeled heat-denatured bovine @-casein cDNA insert (1150 bp from J. P. Mercier, INRA, France) or bovine j3-actin cDNA (2600 bp from Degen (49)) at 42°C for 14 to 18 h. Posthybridization washes included three washes in 2~ SSC; 0.1% SDS and a single wash in 0.5X SSC; 0.1% SDS and 0.1x SSC; 0.1% SDS at room temperature. Filters were air-dried and exposed to Kodax X-Omat with two Cronex lighting-plus intensifying screens (DuPont) at -70°C. Zmmunohistochemistry. Monoclonal antibodies which react with cytokeratin (Type II, subfamily No. 1-8; Boehringer-Mannheim) and vimentin (Boehringer-Mannheim) were used to characterize cell type as epithelial or stromal, respectively on plastic substratum. Cells were seeded in 8-chamber slides (Lab-Tex 4838, Nunc, Naperville, IL) for 24 h and then fixed and stained according to manufacturers recommendations. Immunohistochemistry also was used to define ECM and prolactin effects on intracellular casein levels. Cells on collagen gels were treated with type II collagenase (0.3 mg/ml, Sigma) for 30 min at 37”C, washed twice with DPBS, plated on collagencoated 8-chamber slides (Nunc), and allowed to attach further for 2 h, and then were fixed in cold acetone at -20°C for 10 min. Forty micro-
193
BOVINE MAMMARY CELLS liters of rabbit anti-bovine b-casein in DPBS (1:5 v/v) was applied per chamber and the slides were incubated at 37°C for 1 h in a 100% humidity chamber. As a control for nonspecific secondary antibody binding, the primary antibody was omitted and DPBS was usedin its place. Slides were subjected to three 5-min wsshesin DPBS at room temperature. Following the last wash, cells were incubated with a goat anti-rabbit-IgG-FITC conjugate in DPBS (1:40 v/v, BoehringerMannheim). The incubation and washes were performed as described above. Slides were directly visualized with a Jenalunar microscope, equipped with epifluorescence optics and appropriate excitatory and barrier filters for FITC.
RESULTS
Primary cultured cells formed confluent monolayers within 4 to 5 days. The major cellular contaminants were fibroblasts. Therefore, it was necessary to select epithelial cells by cloning. Of the original 1052 cells, 63 gave rise to colonies. The number of cells per colony ranged from 3000 to 5000. During this transition stage, some clones ceased to grow, entered crisis, and sloughed off. One of the early passage epithelial cell clone, MACT3, showed a typical epithelial morphology (Fig. 1A) and intense staining of cytoplasmic meshwork of cytokeratin fibrils (Fig. 1B) but not with vimentin. These characteristics were also seen in the bovine BMGE + H cell line [24, 251. The results indicated that these cells were epithelial in origin. Cotransfection of early passage cells with pBAPSV40 Tts A58 and pSV2-neo plasmids yielded several G418resistant colonies. Among 1 X 107-transfected cells, 117 colonies were obtained. Untransfected cells were killed 100% at a concentration of 200 pg/ml G418. The transfection efficiency was 6 cells per 10’ transfected cells. Seven randomly selected clones were grown successfully in culture up to 50 passages without any sign of crisis while the primary cells entered crisis at passages 16 to 25. When MAC-T clones (-T3, -T4, -TlS) were injected into seven immunodeficient mice and allowed to grow for more than 8 weeks, there was no sign of tumor nodule formation at the injected site. The result appeared to be a true negative as the ~160 cell line initiated prompt tumor growth in control animals within 3-4 weeks postinjection. TO verify the presence of large T-antigen in transfected cells, antigen was immunoprecipited with monoclonal antibodies and fractionated on SDS-PAGE. Figure 2 shows such an immunoprecipitation with monoclonal antibodies against the large T-antigen. The 92-kDa band which is characteristic for large T-antigen was found in all SV40-transfected cells but not in nontransfected, JH25 cells. A 53-kDa protein was coprecipitated with the large T-antigen because it was precipitated only in cells expressing large T-antigen. Cells which were not transfected with SV40 large T-antigen did not show either band.
Temperature-Independent
Growth of MAC-T Cells
Although the established gene carried tsA mutant SV40 gene, three transfected cell lines MAC-T3, MACT4, MAC-TlS, grew equally well at 33 and 39°C in monolayer cultures in 10% serum (Fig. 3). Following subculture, MAC-T cell lines and JH25 progressed through a characteristic growth pattern of lag phase within the first 24 h to enter into log phase on Day 2. Cell number increased at the same rate at the two temperatures for the next 5 days. Phase contrast micrograph showed that all of the available surface was occupied and that cells were in full contact with surrounding cells. The slope calculated during the log-phase at the two temperatures revealed that MAC-T cells had a doubling time of approximately 17 h. At confluency, MACT cells reached a density 4.0 X 10’ cells/cm’ when grown at 33,37, or 39°C. Since MAC-T cells grew at the same rate as the early passage JH25 cells, showed contact inhibition, and formed a uniform pavement of closely associated cells, we could conclude that there were minimal changes in growth characteristics because of transfection.
Cellular Differentiation When MAC-T3 cells were grown on culture plastic substratum they displayed a cuboidal epithelial morphology and remained flattened (Fig. 1A). Floating collagen gels have been shown to promote functional differentiation since cells cultured on this system were able to assume a shape similar to their in uiuo tissue of origin [ll, 12, 151. When MAC-T cells were plated on attached collagen at high density, they rearranged themselves to form small organoids (Fig. 4). As time progressed, these organoids become larger. Phase contrast microscopy revealed the lumen in the center of these structures. When released to float, the collagen gels contracted and all cells became cuboidal and difficult to distinguish. Collagen gels containing cells were collected at 24-h intervals from the time gels were released to Day 3 to assay for @-casein mRNA accumulation during induction. Cells grown on plastic substratum were evaluated to determine the basal level of @-casein RNA under noninduced conditions. The patterns of @-caseinand P-a&in mRNA in MACT cells are shown in Fig. 5. fi-casein mRNA was just detectable when MAC-T cells were grown on plastic substratum. When cells were plated on collagen gels and released to float for 24 h, there was a considerable increase in /3-casein mRNA abundance which reached a maximum by 48 h. There was no further increase in P-casein mRNA on Day 3. In the presence of prolactin, the level of @-casein mRNA abundance was not different for the first 24 h when comparing cells grown on
194
HUYNH,
ROBITAILLE,
AND
TURNER
FIG. 1. Morphology of MAC-T3 cells grown on plastic substratum. (A) Phase contrast microscopy shows an epithelial pavement of cuboidal-shaped cells. (B) Indirect immunofluorescence analysis. Cells were fixed and then stained with anti-cytokeratin type II, NO. l-8 antibody followed by fluoresceinated goat anti-rabbit antibody. The intermediate filaments are arranged around the nucleus and extend via desmosomes between cells. Magnification 800X.
floating collagen vs plastic. As time progressed, @-casein mRNA accumulation was augmented by prolactin addition (threefold) and was maintained until Day 3. Subsequent hybridization on the same blot with the bovine /3-actin probe showed that relatively equal amounts of intact RNA were present in each lane. The distribution of casein secretory vesicles in MACT cells on floating collagen gels in the presence and absence of prolactin is illustrated in Figs. 6A and 6B, respectively. In prolactin-treated cells (Fig. 6A) nearly
100% of the cells immunostained. Each cell contained numerous immunoreactive secretory vesicles (Fig. 6A). However, in the absence of prolactin, cells on floating collagen gels were still synthesized casein but the number of vesicles was reduced compared to cells treated with prolactin (Fig. 6B). When primary antibodies were omitted (Fig. SC), no immunoreactive vesicles were seen. These results indicate that the secretory vesicles contained casein-like material. Time course of casein secretion in cultured MAC-T
BOVINE
kd 130, 7550,
MAMMARY
_ LT-A -PS
3927,
14, FIG. 2. Immunoprecipitation of SV40 large T-antigen in stably transfected bovine mammary epithelial cells grown at 39’C. [%IMethionine-labeled lysates from 7 MAC-T clones were immunoprecipitated with the antiSV40 large T-antigen monoclonal MA& Proteins were separated by SDS-PAGE and shown by autoradiography. All clones produced a protein of 92 kDa characteristic of the large T-antigen (LT-A). Nontransfectedcells JH25 do not produce measurable immunoreactive material. Nonimmune mouse serum (ml failed to precipitate protein. P53 was also precipitated in transfected cells as a result of its association with the viral large T-antigen.
cells maintained on floating collagen in the absence and presence of prolactin is illustrated in Fig. 7. When MAC-T cells were grown on plastic substratum, casein secretion was below the limits of detection. @casein secretion was detected within 24 h of collagen gel release. For the first 2 days, there was no difference in casein secretion whether MAC-T cells were treated with or without prolactin. However, there was an approximate threefold increase in @-caseinsecretion on Day 3 in prolactin-treated cells. In contrast, a-casein secretion was barely detectable when prolactin was absent from the media. At 48 h in the presence of prolactin, a-casein secretion was easily measured.
195
CELLS
ization. Immunoprecipitation analysis clearly showed the presence of a 92-kDa protein, the putative viral large T-antigen, and a coprecipitating 53-kDa protein. The specific interaction between the T-antigen and a 53-kDa protein is a characteristic of SV40-transformed cells 1501.Indeed, we speculate that the 53-kDa protein is indeed ~53, a nuclear phosphoprotein which complexes with T-antigen and other DNA tumor virus transforming proteins [51]. These results agree with workers who induced proliferation in primary cells from mice, rabbits, and pigs [29, 30, 511. Indeed, the expression of viral T-antigen has been shown to be required for the initiation and maintenance of the transformed state in vitro [52, 531. Although the DNA construct used in this study was the temperature-sensitive A mutant of SV40 gene, the three MAC-T cell lines failed to exhibit a temperaturedependent alteration in proliferation. The three clones proliferated equally well at 33 and 39°C in media containing 10% serum. Similarly, large T-antigen protein was present at the “nonpermissive” temperature, 39°C. No estimates were made at the permissive temperature. This lack of temperature sensitivity has been extensively documented [30, 31, 34, 55-571. Risser and Pollack [55] found that not all cell lines transformed by wild type displayed the standard transformed pheno[ .
MAC-T4
DISCUSSION
Bovine mammary epithelial cells transfected with SV40 large T-antigen become established in vitro but retain their ability to differentiate and secrete milk-specific products. As such, these MAC-T clones represent an in vitro model for bovine lactation. This discussion will focus on the characterization of MAC-T cells including the nature of establishment, growth potential, and differentiation. Cellular immortalization is defined experimentally by the acquisition of unlimited growth capacity in vitro [30]. To date more than 350 serial passages have been made with MAC-T3 cells with no sign of senescence. In contrast, nontransfected early passage mammary epithelial cells typically entered crisis after 16-25 passages in culture. Low level, constitutive expression of SV40 large T-antigen appears responsible for the immortal-
DAYS IN CULTURE FIG. 3. Growth curves for bovine mammary epithelial cell lines. Three transfected clones MAC-T3, 4, and 18 and early passage bovine mammary cells grew equally well at 33°C and 39% Population doubling time was estimated at 17 h.
196
HUYNH,
ROBITAILLE,
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F‘IG. 4. Morphology of MAC-T3 cells grown on attached collagen gels. The epithelial pavement of cells grown at high density for 3 days rea:rranges i nto organoid structures. A lumen-like structure is indicated. Magnification 130x.
type. Ctsuka et al. [31] reported that heat-adapted cell line8 derived from mou8e kidney cells transfected with SV40 temperature-sensitive A gene were able to grow equally well at both temperatures. This is entirely consistent with the above results. Petit et al. [30] found that the distribution of tsA58 carrying line8 was more heterogeneous, 70% of them could be classified into temperature Sensitive class whereas 30% were not sensitive to high temperature. Brockman et al. [56] found that 1 of 16 SV4OtsA cell line8 isolated showed temperature-resistant growth properties. Graf and Beug [57] isolated transformed variant8 of rat cell8 infected by the temperature-eensitive avian 8arcoma virus mutant8 that grew at the nonpermissive temperature. Further, they emphasized that the phenotype of t8A transformants might depend on the method of clone selection. Some 3T3 cells transformed by wild type SV40 had growth properties similar to normal 3T3 cell8 or intermediate between those of normal 3T3 and SV4OtsA transformants [58, 591. Since MAC-T cell8 were selected and grown at an intermediate temperature 37°C after transfection with the SV40 temperature-sensitive mutant A gene, these three clone8 appeared to have adapted to growth at temperature8 from 33 to 39°C. MAC-T cell8 appear to be immortalized but not transformed. They did not cause tumor8 upon injection into immunodeficient mice while the control ~160 cell line caused palatable tumors. Further the MAC-T cells were 8erum dependent and anchorage dependent a8 they were not able to form colonies in soft agar. When
grown on tissue culture plastic, they showed contact inhibition and did not overgrow or form foci. MAC-T cell clone8 retained the phenotypic characteristics of bovine mammary epithelial cells, like JH25, after subculture in vitro. On plastic substratum, MAC-T cell8 displayed typically cuboidal epithelial morphology. Indirect immunofluorescence using anti-cytokeratin revealed that MAC-T cells exhibited a cla88ical epithelial array of intermediate filaments. Intercellular desmosome-containing bridges were also observed. The failure of staining with anti-vimentin indicated the absence of stromal cells. These results are consistent with those of Schmid et al. [26] who also worked with bovine mammary epithelial cell8 (BMGE + H). Direct comparison of the immunofluorescent micrographs between MACT cell8 and BMGE + H by Professor Dr. Werner W. Franke confirmed that MAC-T cell8 were indeed epithelial (personal communication). When MAC-T cell8 were grown on collagen gels, they rearranged themselves to form bhBM8 or secretory dome structures. Cell8 within these structure8 became more columnar in shape. As culture time increased, duct-like structures connecting the dome8 became numerous. These characteristics of rearrangement8 resembled the in uiuo organization of differentiated bovine mammary alveoli. Similar dome formations have been reported in the mouse mammary epithelial cell line, COMMA-D. This normal mammary epithelial cell line retained epithelial morphology and routinely formed dome8 in vitro [18]. The difference between
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than that reported by Eisenstein and Rosen [19] who showed that fi-casein mRNA levels increased within 4 h after the addition of hydrocortisone and prolactin to
FIG. 5. Extracellular matrix and prolactin induce 8-casein mRNA accumulation in MAC-T3 cells. Total RNA (20 rg), isolated from cells grown on plastic (P) substratum or on calf tail collagen in the presence of 5 pglml prolactin (+) or without prolactin (-) was Northern blotted and then probed with bovine P-casein cDNA insert (A) or bovine P-actin cDNA insert (B).
MAC-T and COMMA-D lines was that COMMA-D formed domes when they were grown on plastic substratum, whereas MAC-T cells formed domes only when grown on collagen gels. When focusing on the secretory function of MAC-T cells, (Y- and B-casein proteins were examined. Caseins are found neither in the fetal calf serum nor in the medium so the presence of these products in the conditioned medium was useful as an indication of functional differentiation of these cells. The induction of the differentiated state by floating collagen gels and prolactin was accompanied by an increase in @casein mRNA, by accumulation of the intracellular casein, and subsequent casein secretion. Simply plating the cells on collagen gels increased ,B-casein mRNA abundance. The maximal level of @casein mRNA was reached within 48 h in the presence of prolactin. This induction was slower
FIG. 6. Immunolocalization of p-casein within differentiated bovine mammary epithelial cells. Immunofluorescent staining with a polyclonal antibody against bovine @-casein showed numerous vesicles in prolactin (A)-treated MAC-T3 cells on collagen gels. In the absence of prolactin (B), the number of immunoreactive vesicles is reduced. (C) The nonspecific binding of the secondary fluorescent antibody is low. Magnification 320X.
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FIG. 7. Western blot analysis of LY-and @-casein secreted from differentiated bovine mammary epithelial cells (MAC-T3) is increased by prolactin. Media from cells grown on floating collagen gels in the presence of 5 &g/ml prolactin (solid bars) or in the absence of prolactin (open bars) were separated by SDS-PAGE and the a- and @-casein detected by rabbit anti-bovine casein polyclonal antibodies and an anti-rabbit IgG alkaline phosphatase conjugate. Cells on collagen without prolactin produced detectable p-casein at 12 h (a), 24 h (b), 36 h (d), and 48 h (f) after plating. Casein secretion from prolactin-treated cells was initially modest at 24 h (c) and 36 h (e) but increased by 48 h (g). A casein standard (s) of cow milk contained 2 pg of protein. In contrast, a-casein secretion was very low in all treatments except for the 46 h treatment with prolactin.
cells and it continued to accumulate for at least 48 h. The magnitude of induction in casein mRNA abundance observed in MAC-T cells is similar to other systems. Primary mouse mammary epithelial cells grown on floating collagen gels had 3- to lo-fold more casein mRNA than cells on plastic substratum [15]. Simiiar findings of a 5-fold increase in p-casein mRNA accumulation was noted by Blum et al. [60] when cells were grown on basement substrata. The requirement for a deformable extracellular matrix was first noted by Emerman et al. [ 121,who showed that the floating collagen membrane allowed the cells to change shape and that there was a correlation between cell shape and the degree of differentiation. Others [ 14,171 have suggested that the geometry of the cells, their interaction with extracellular matrix constituents, cell-cell interactions, and maintenance of cellular polarity played a role in the expression of milk protein genes. Bovine mammary epi-
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thelial cells share a similar requirement for an extracellular matrix and cell-cell interaction for maximum casein synthesis/secretion. cr-Casein and P-casein were synthesized and secreted when MAC-T cells were grown on floating collagen gels. Indirect immunofluorescence using anti-casein antibodies illustrated that MAC-T cells responded to prolactin in a homogenous way in terms of casein synthetic capacity since almost all of the cells produced casein secretory vesicles. In contrast, COMMA-D cells have been shown to be morphologically uniform but heterogenous in terms of casein production [ 181. Typically only lo-20% of the COMMA-D cells produced casein at only one time judged by indirect immunofluorescence. In contrast the bovine epithelial cell line BMGE + H showed epithelial morphology but produced very little casein when induced by prolactin (W. W. Franke, personal communication). Therefore, with regard to in vitro casein production the MAC-T cells represent the most uniform in vitro system available. MAC-T cells secrete large quantities of LX-and /3-casein. The presence of fl-casein can be detected in the media as early as 12 h after plating on floating collagen gels. While cz-casein secretion required a longer time course and prolactin as well as extracellular matrix. Reflective densitometry is used here to provide a semiquantitative estimate. In the absence of prolactin, total casein secretion was estimated to be between 2.5 and 10 pg/m1/24 h. Prolactin increased this secretion rate by 48 h to 50 pg/m1/24 h (7.5 pg/106 cells/24 h). This level of casein secretion is some three- to sevenfold higher than those reported for freshly dissociated mouse cells [12] rabbit cells [17], and rat cells [60]. CONCLUSION
SV40 large T antigen confers immortality to bovine mammary epithelial cells. The existence of MAC-T cells illustrates the concept of nontransformed immortalized cells. Clonal MAC-T cells have been now growing for more than two years, (>350 passages). These cells grow rapidly, are to date stable, and show no signs of crisis. MAC-T cells are responsive to manipulations either in extracellular matrix and in the lactogenic hormones. When differentiated, MAC-T cells synthesize and secrete CX-and fi-casein. As such, MAC-T cells represent an in vitro model for bovine lactation. This investigation was supported by the Natural Sciences and Engineering Council of Canada OGP 36728 to J.D.T. and a postgraduate scholarship to H.T.H. We thank Drs. J. P. Mercier, G. Matlashewski, and L. Chalifour for cDNA clones and NHPP for the bPRL.
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