Secretion of Casein in Cultures of Mouse Mammary Epithelial Cells on Floating Collagen Gels JUMPEI E N A M I 1 and S. N A N D I 2 Cancer Research Laboratory and Department of Zoology University of California Berkeley 94720
ABSTRACT
casein components (10, 13). The synthesis of these major casein components has been analyzed by cultured mammary explants (10). In this paper we report the effect of hormones on the secretion of all three casein components from mouse mammary epithelial ceils cultured on floating collagen gels.
To study the effect of hormones on the secretion of casein, mammary epithelial ceils were obtained from mid-pregnant mice and cultured on floating collagen gels in the presence of combinations of insulin, cortisol, and prolactin in serum-free culture medium. After labeling with phosphorus-32 labeled orthophosphate, the cells which had been cultured in the presence of all three hormones secreted hot-trichloroacetic acid precipitable radioactivity into the medium. The secreted materials were identified as three major mouse caseins by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis followed by autoradiography. The floating collagen gel culture method allows mouse mammary epithelial cells to differentiate and secrete all species of caseins in the presence of hormones. The present culture method and the phosphorus-32 labeling method will be applicable to the study of differentiation of mammary epithelial cells from a variety of mammalian species.
MATERIALS AND METHODS Cell Culture Method
INTRODUCTION
Mouse mammary epithelial cells grown in vitro on floating collagen gels differentiated morphologically (7) and secreted a large amount of casein in response to the lactogenic combination of hormones, i.e., insulin, cortisol, and prolactin (6). A radioimmunoassay for one of the components of mouse casein (9) was used in the study for the quantitation of casein. Mouse milk, however, contains two other major
Received December 30, 1977. 1Department of Physiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan. ~To whom requests for reprints should be addressed. 1978 J Dairy Sci 61:729--732
Ten- to 12-day pregnant BALB/cCrgl mice were used. Mammary glands were minced finely and dissociated with .1% collagenase (sp. act. 160 U/mg, ICN) in Hanks' balanced salt solution for 90 rain (11). Cell number was estimated by a modification of the method of Bryant et al. (2). One volume of cell suspension was mixed with 9 volumes of .02% crystal violet in .1 M citric acid and vigorously shaken in a vortex mixer for 30 s. The number of stained nuclei was counted in a hemocytometer. Cells 2 × 106 were suspended in .5 ml of Waymouth MB 752/1 medium (GIBCO) containing 10% FCS (Microbiological Associates) and 5 ug/ml insulin (bovine pancreas, Sigma), seeded into each well of 16-mm Falcon plastic multiwell plates. Each well had .3 ml of collagen gel prepared according to the method of Michalopoulos and Pitot (15). Cultures were incubated at 37 C under 5% CO2, 95% air. Two days after the seeding, the collagen gels with cells attached on top were floated by trimming the edges with a needle, and the medium was changed to serum-free medium 199 (GIBCO) containing .25% bovine serum albumin (Fraction V, Armour) and combinations of 5 ug/ml each of insulin, cortisol, and prolactin (ovine; NIH-P-S11). Medium was changed every day thereafter, and the cells were Iabeled as described below. Labeling Method
Cultured cells were washed twice with Puck's Saline A (16) and incubated with the
729
730
ENAMI AND NANDI
labeling media containing 5 ,Ci/.5 ml per well of carrier-free [32P]orthophosphate (New England Nuclear) for 24 h. Phosphorus-32 orthophosphate labeling media consisted of 1/20 medium 199 and 19/20 phosphate-free Eagle's minimum essential medium with nonessential amino acids (5), in which essential and nonessential amino acids were increased four times and two times, respectively. The labeling media also contained hormones.
U
!
Hot-TCA Precipitation Method
After labeling, the medium was centrifuged at 8,000 × g for 5 min, and a .3 ml aliquot of the supernatant was mixed with an equal volume of 10% trichloroacetic acid (TEA). Sodium phosphate also was added to the mixture to a final concentration of 10 mM. The mixture was heated at 80 C for 20 rain. After cooling, 1 mg of carrier bovine serum albumin was added. The precipitate was collected on a GF/A filter paper (Whatman), washed with cold 5% TCA, dried, and transferred to a scintillation vial. Radioactivity was counted in a Packard scintillation counter after the addition of 5 ml of scintillator containing 4 g Omnifluor (New England Nuclear) in 1 liter of toluene. Electrophoresis and Autoradiography
Procedures for sodium dodecyl sulphate (SDS)-urea polyacrylamide gel electrophoresis have been described (10). Slab gel electrophoresis was in a Bio-Rad Model 220 apparatus with slab thickness of .75 mm. Width of each well was 1 cm. After electrophoresis, the slab gels were fixed in 10% T e A for 2 h, 7% acetic acid overnight, and were dried. The dried gel was exposed to Kodak RP Royal X-Omat film for 24 h. Slab gels also were stained with Coomassie brilliant blue and the phosphoprotein staining method of Cutting and Roth (4). RESULTS
Figure 1 shows the time course of accumulation of [32p]-labeled h o t - T e A precipitable materials in the culture medium. The cells which had been cultured in the presence of insulin, cortisol, and prolactin continued to secrete [32p]-labeled materials after a lag period of about 3 h. The secretion remained low when the cells had been cultured in the absence of prolactin. Journal of Dairy Science Vol. 61, No. 6, 1978
TIME (HOURS)
FIG. 1. Time course of secretion of [3 2 p] -labeled materials in cultures of mammary epithelial cells. Cells were cultured for 4 days in the presence of insulin plus cortisol (o), or insulin plus cortisol plus prolactin (o) and labeled continuously with [32p] orthophosphate. At each time point, media were collected and processed for hot-TeA precipitation. Each point represents mean from triplicate determinations.
Effect of various combinations of hormones was examined further and is shown in Table 1. Prolactin significantly increased the secretion of [32 p]-labeled materials in both insulin cultures and insulin plus cortisol cultures. The secreted radioactivity was maximum with the lactogenic combination of hormones, i.e., insulin plus cortisol plus prolactin. Figure 2 shows the results from SDS-urea polyacrylamide gel electrophoresis. Autoradiography revealed three major [32Pl-labeled bands in the medium which had contained the
TABLE 1. Effect of hormones on secretion of [3 z p] _ labeled materials in cultures of mammary epithelial cells,a
Hormones
[32 p] -CPM/ .3 ml mediumb
Insulin Insulin + prolactin Insulin + cortisol Insulin + cortisol + prolactin
901 2,532 577 9,703
223 359 206 474
aCells were cultured for 5 days in the presence of various hormones and labeled with [32p] orthophosphate for 24 h. The media were processed for hot-TeA precipitation as described in Materials and Methods. bThe data are expressed as mean +- SE determined from four cultures. Background cpm (1,313 cpm) had been subtracted.
CASEIN SECRETION IN MAMMARY CELL CULTURE
A
B
C
D
E
F :
:
".,,li---- 0 R I G I N
:,
~-'- pp44
.~: ~ ";;;~:~o ~
-,,i---pp 22
..~.__pp26
FIG. 2. The SDS-urea polyacrylamide gel electrophoresis of [32 p]-labeled materials. The same cultures as used in the experiment in Table 1 were used. Forty microliters of the labeled media were collected from each of four cultures and combined. After the addition of 40 ~g of carrier mouse milk proteins and an equal volume of 10% .TCA, the media were processed for hot-TCA •precipitation. The precipitate was collected by centrifugation, washed once with acetone and dissolved in 80 pl of SDS-urea, boiled for 2 rain, and 40 ill were applied in each well of the slab gel. For staining of the gel, 40 pg of milk protein in 40 pl SDS-urea (10) were applied in each well. Electrophoresis was done for 16 h at 40 V. A to D, autoradiographs of [32 p] -labeled media. A, cells were cultured with insulin; B, insulin plus prolactin; C, insulin plus cortisol; D, insulin plus cortisol plus prolactin; E, mouse milk proteins stained with the phosphoprotein staining method of Cutting and Roth (4); F, mouse milk proteins stained with Coomassie brilliant blue.
lactogenic hormones (Fig. 2D). These bands correspond to the three major mouse casein bands, pp44 (a phosphoprotein having a molecular weight of 44,000 daltons), pp26 (a phosphoprotein having a molecular weight of 26,000 daltons) and pp22 (a phosphoprotein having a molecular weight of 22,000 daltons), identified by Coomassie brilliant blue staining (Fig. 2F) and by the p h o s p h o p r o t e i n staining m e t h o d (Fig. 2E). Labeled caseins were virtually absent in media which had c o n t a i n e d the other c o m b i n a t i o n s of h o r m o n e s (Fig. 2A, B, C).
DISCUSSION Lactogenesis in vitro has been studied with explants of m a m m a r y gland (1, 10). The major drawback for the e x p l a n t culture m e t h o d is
73 ]
that the differentiated products accumulate inside the alveolar lumen w i t h o u t being secreted into the culture medium. M a m m a r y epithelial cells in m o n o l a y e r cultures, howcver, secrete casein into the m e d i u m in response to the lactogenic c o m b i n a t i o n of h o r m o n e s although the degree of differentiation is limited (6, 8). Recently, we have succeeded in inducing secretion of a large a m o u n t of casein by culturing m a m m a r y cells on floating collagen gels (6). By using this culturc m e t h o d we have been also successful in detecting the p r o d u c t i o n of mouse m a m m a r y t u m o r virus from normal m a m m a r y epithelial cells (18). In the present study, we used the same culture system and measured the secretion of casein after labeling the cells with [32 p] orthophosphate. The lactogenic combination of h o r m o n e s significantly stimulated the secretion of [32 p]-labeled materials which were identified as mouse caseins by SDS-urea polyacrylamidc gel clcctrophoresis. In the absencc of protactin, secretion of casein was reduced greatly. Thus, casein secretion was measured quantitatively by labeling the cells with [32p] orthophosphate. Rennin-calcium (14) or antisera against casein (3, 9, 10, 12, 17) were n o t necessary for m e a s u r e m e n t of casein secreted into the m e d i u m . The floating collagen gel culture system coupled with [32plorthophosphate_labeling m e t h o d will be useful for the study of differentiation, secretion of casein, and possibly for the bioassay of prolactin for m a m m a r y epithelial cells from a variety of m a m m a l i a n spccies.
ACKNOWLEDGMENTS The authors wish to t h a n k Jason Yang for critically reading the manuscript, Kathy McCormick for excellent technical assistance, and John Underhill for photography. This investigation was supported by Grant No. 05388, awarded by the National Cancer Institute, DHEW.
REFERENCES 1 Banerjee, M. R. 1976. Responses of mammary cells to hormones. Int. Rev. Cytol. 47:1. 2 Bryant, J. C., E. L. Shilling, and W. R. Earle. 1958. Massive fluid-suspension cultures of certain mammalian tissue ceils. J. Nat. Cancer Inst. 21:331. 3 Ceriani, R. L. 1976. ttormone induction of specific protein synthesis in midpregnant mouse mammary cell culture. J. Exp. Zool. 196: 1. 4 Cutting, J. A., and T. F. Roth. 1973. Staining of Journal of Dairy Science Vol. 61, No. 6, 1978
732
5 6
7
8
9
10
11
ENAMI AND NANDI
phospho-proteins on acrylamide gel clectrophoresis. Anal. Biochem. 54:386. Eagle, It. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432. Emerman, J. T., J. Enami, D. R. Pitelka, and S. Nandi. 1977. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Nat. Aead. Sci. U.S.A. 74:4466. Emerman, J. T., and D. R. Pitelka. 1977. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen memhranes. In Vitro 13:316. Enami, J. 1977. tlormonal control of milk protein synthesis in mouse m a m m a r y epithelial cells in vitro. Ph.D. thesis, Univ. of Calif., Berkeley. Enami, J., and S. Nandi. 1977. A radioimmunoassay for a component of mouse casein. J. Immunol. Methods 18:235. Enami, J., and S. Nandi. 1977. Hormonal control of milk protein synthesis in cultured mouse mammary explants. Cell Differentiation 6:217. Enami, J., S. Nandi, and S. tiaslam. 1973. Production of three-dimensional structures - " d o m e s " from dissociated m a m m a r y epithelial cells of mice. In Vitro 8:405. (Abstr.)
Journal of Dairy Science Vol. 61, No. 6, 1978
12 l:eldman, M. K. 1974. A direct radioimmunoassay for mouse casein. Comp. Biochem. Physiol. 49A: 127. 13 Green, M. R., and J. V. Pastewka. 1976. Molecular weights of three mouse milk caseins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and K-like characteristics of a fourth casein. J. Dairy Sci. 59:1738. 14 Juergens, W. G., F. E. Stockdalc, Y. J. Topper, and J. J. Elias. 1965. Hormone-dependent differentiation of mammary gland in vitro. Proc. Nat. Acad. Sci. U.S.A. 54:629. 15 Michaelopoulos, G., and I1. C. Pitot. 1975. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94:70. 16 Puck, T. T., S. J. Gieciura, and tt. W. Fisher. 1957. Clonal growth in vitro of human cells with fihroblastic morphology. J. Exp. Med. 106:145. 17 Terry, P. M., E. M. Ball, R. Ganguly, and M. R. Banerjee. 1975. An indirect radioimmunoassay for mouse casein using [* 2sl]-labeled antigen. J. Immunol. Methods 9:123. 18 Yang, J., J. Enami, and S. Nandi. 1977. Regulation of mammary t u m o r virus production by prolactin in BALB/efC311 mouse normal m a m m a r y epithelial cells in vitro. Cancer Res. 37:3644.
ABSTRACT
casein components (10, 13). The synthesis of these major casein components has been analyzed by cultured mammary explants (10). In this paper we report the effect of hormones on the secretion of all three casein components from mouse mammary epithelial ceils cultured on floating collagen gels.
To study the effect of hormones on the secretion of casein, mammary epithelial ceils were obtained from mid-pregnant mice and cultured on floating collagen gels in the presence of combinations of insulin, cortisol, and prolactin in serum-free culture medium. After labeling with phosphorus-32 labeled orthophosphate, the cells which had been cultured in the presence of all three hormones secreted hot-trichloroacetic acid precipitable radioactivity into the medium. The secreted materials were identified as three major mouse caseins by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis followed by autoradiography. The floating collagen gel culture method allows mouse mammary epithelial cells to differentiate and secrete all species of caseins in the presence of hormones. The present culture method and the phosphorus-32 labeling method will be applicable to the study of differentiation of mammary epithelial cells from a variety of mammalian species.
MATERIALS AND METHODS Cell Culture Method
INTRODUCTION
Mouse mammary epithelial cells grown in vitro on floating collagen gels differentiated morphologically (7) and secreted a large amount of casein in response to the lactogenic combination of hormones, i.e., insulin, cortisol, and prolactin (6). A radioimmunoassay for one of the components of mouse casein (9) was used in the study for the quantitation of casein. Mouse milk, however, contains two other major
Received December 30, 1977. 1Department of Physiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan. ~To whom requests for reprints should be addressed. 1978 J Dairy Sci 61:729--732
Ten- to 12-day pregnant BALB/cCrgl mice were used. Mammary glands were minced finely and dissociated with .1% collagenase (sp. act. 160 U/mg, ICN) in Hanks' balanced salt solution for 90 rain (11). Cell number was estimated by a modification of the method of Bryant et al. (2). One volume of cell suspension was mixed with 9 volumes of .02% crystal violet in .1 M citric acid and vigorously shaken in a vortex mixer for 30 s. The number of stained nuclei was counted in a hemocytometer. Cells 2 × 106 were suspended in .5 ml of Waymouth MB 752/1 medium (GIBCO) containing 10% FCS (Microbiological Associates) and 5 ug/ml insulin (bovine pancreas, Sigma), seeded into each well of 16-mm Falcon plastic multiwell plates. Each well had .3 ml of collagen gel prepared according to the method of Michalopoulos and Pitot (15). Cultures were incubated at 37 C under 5% CO2, 95% air. Two days after the seeding, the collagen gels with cells attached on top were floated by trimming the edges with a needle, and the medium was changed to serum-free medium 199 (GIBCO) containing .25% bovine serum albumin (Fraction V, Armour) and combinations of 5 ug/ml each of insulin, cortisol, and prolactin (ovine; NIH-P-S11). Medium was changed every day thereafter, and the cells were Iabeled as described below. Labeling Method
Cultured cells were washed twice with Puck's Saline A (16) and incubated with the
729
730
ENAMI AND NANDI
labeling media containing 5 ,Ci/.5 ml per well of carrier-free [32P]orthophosphate (New England Nuclear) for 24 h. Phosphorus-32 orthophosphate labeling media consisted of 1/20 medium 199 and 19/20 phosphate-free Eagle's minimum essential medium with nonessential amino acids (5), in which essential and nonessential amino acids were increased four times and two times, respectively. The labeling media also contained hormones.
U
!
Hot-TCA Precipitation Method
After labeling, the medium was centrifuged at 8,000 × g for 5 min, and a .3 ml aliquot of the supernatant was mixed with an equal volume of 10% trichloroacetic acid (TEA). Sodium phosphate also was added to the mixture to a final concentration of 10 mM. The mixture was heated at 80 C for 20 rain. After cooling, 1 mg of carrier bovine serum albumin was added. The precipitate was collected on a GF/A filter paper (Whatman), washed with cold 5% TCA, dried, and transferred to a scintillation vial. Radioactivity was counted in a Packard scintillation counter after the addition of 5 ml of scintillator containing 4 g Omnifluor (New England Nuclear) in 1 liter of toluene. Electrophoresis and Autoradiography
Procedures for sodium dodecyl sulphate (SDS)-urea polyacrylamide gel electrophoresis have been described (10). Slab gel electrophoresis was in a Bio-Rad Model 220 apparatus with slab thickness of .75 mm. Width of each well was 1 cm. After electrophoresis, the slab gels were fixed in 10% T e A for 2 h, 7% acetic acid overnight, and were dried. The dried gel was exposed to Kodak RP Royal X-Omat film for 24 h. Slab gels also were stained with Coomassie brilliant blue and the phosphoprotein staining method of Cutting and Roth (4). RESULTS
Figure 1 shows the time course of accumulation of [32p]-labeled h o t - T e A precipitable materials in the culture medium. The cells which had been cultured in the presence of insulin, cortisol, and prolactin continued to secrete [32p]-labeled materials after a lag period of about 3 h. The secretion remained low when the cells had been cultured in the absence of prolactin. Journal of Dairy Science Vol. 61, No. 6, 1978
TIME (HOURS)
FIG. 1. Time course of secretion of [3 2 p] -labeled materials in cultures of mammary epithelial cells. Cells were cultured for 4 days in the presence of insulin plus cortisol (o), or insulin plus cortisol plus prolactin (o) and labeled continuously with [32p] orthophosphate. At each time point, media were collected and processed for hot-TeA precipitation. Each point represents mean from triplicate determinations.
Effect of various combinations of hormones was examined further and is shown in Table 1. Prolactin significantly increased the secretion of [32 p]-labeled materials in both insulin cultures and insulin plus cortisol cultures. The secreted radioactivity was maximum with the lactogenic combination of hormones, i.e., insulin plus cortisol plus prolactin. Figure 2 shows the results from SDS-urea polyacrylamide gel electrophoresis. Autoradiography revealed three major [32Pl-labeled bands in the medium which had contained the
TABLE 1. Effect of hormones on secretion of [3 z p] _ labeled materials in cultures of mammary epithelial cells,a
Hormones
[32 p] -CPM/ .3 ml mediumb
Insulin Insulin + prolactin Insulin + cortisol Insulin + cortisol + prolactin
901 2,532 577 9,703
223 359 206 474
aCells were cultured for 5 days in the presence of various hormones and labeled with [32p] orthophosphate for 24 h. The media were processed for hot-TeA precipitation as described in Materials and Methods. bThe data are expressed as mean +- SE determined from four cultures. Background cpm (1,313 cpm) had been subtracted.
CASEIN SECRETION IN MAMMARY CELL CULTURE
A
B
C
D
E
F :
:
".,,li---- 0 R I G I N
:,
~-'- pp44
.~: ~ ";;;~:~o ~
-,,i---pp 22
..~.__pp26
FIG. 2. The SDS-urea polyacrylamide gel electrophoresis of [32 p]-labeled materials. The same cultures as used in the experiment in Table 1 were used. Forty microliters of the labeled media were collected from each of four cultures and combined. After the addition of 40 ~g of carrier mouse milk proteins and an equal volume of 10% .TCA, the media were processed for hot-TCA •precipitation. The precipitate was collected by centrifugation, washed once with acetone and dissolved in 80 pl of SDS-urea, boiled for 2 rain, and 40 ill were applied in each well of the slab gel. For staining of the gel, 40 pg of milk protein in 40 pl SDS-urea (10) were applied in each well. Electrophoresis was done for 16 h at 40 V. A to D, autoradiographs of [32 p] -labeled media. A, cells were cultured with insulin; B, insulin plus prolactin; C, insulin plus cortisol; D, insulin plus cortisol plus prolactin; E, mouse milk proteins stained with the phosphoprotein staining method of Cutting and Roth (4); F, mouse milk proteins stained with Coomassie brilliant blue.
lactogenic hormones (Fig. 2D). These bands correspond to the three major mouse casein bands, pp44 (a phosphoprotein having a molecular weight of 44,000 daltons), pp26 (a phosphoprotein having a molecular weight of 26,000 daltons) and pp22 (a phosphoprotein having a molecular weight of 22,000 daltons), identified by Coomassie brilliant blue staining (Fig. 2F) and by the p h o s p h o p r o t e i n staining m e t h o d (Fig. 2E). Labeled caseins were virtually absent in media which had c o n t a i n e d the other c o m b i n a t i o n s of h o r m o n e s (Fig. 2A, B, C).
DISCUSSION Lactogenesis in vitro has been studied with explants of m a m m a r y gland (1, 10). The major drawback for the e x p l a n t culture m e t h o d is
73 ]
that the differentiated products accumulate inside the alveolar lumen w i t h o u t being secreted into the culture medium. M a m m a r y epithelial cells in m o n o l a y e r cultures, howcver, secrete casein into the m e d i u m in response to the lactogenic c o m b i n a t i o n of h o r m o n e s although the degree of differentiation is limited (6, 8). Recently, we have succeeded in inducing secretion of a large a m o u n t of casein by culturing m a m m a r y cells on floating collagen gels (6). By using this culturc m e t h o d we have been also successful in detecting the p r o d u c t i o n of mouse m a m m a r y t u m o r virus from normal m a m m a r y epithelial cells (18). In the present study, we used the same culture system and measured the secretion of casein after labeling the cells with [32 p] orthophosphate. The lactogenic combination of h o r m o n e s significantly stimulated the secretion of [32 p]-labeled materials which were identified as mouse caseins by SDS-urea polyacrylamidc gel clcctrophoresis. In the absencc of protactin, secretion of casein was reduced greatly. Thus, casein secretion was measured quantitatively by labeling the cells with [32p] orthophosphate. Rennin-calcium (14) or antisera against casein (3, 9, 10, 12, 17) were n o t necessary for m e a s u r e m e n t of casein secreted into the m e d i u m . The floating collagen gel culture system coupled with [32plorthophosphate_labeling m e t h o d will be useful for the study of differentiation, secretion of casein, and possibly for the bioassay of prolactin for m a m m a r y epithelial cells from a variety of m a m m a l i a n spccies.
ACKNOWLEDGMENTS The authors wish to t h a n k Jason Yang for critically reading the manuscript, Kathy McCormick for excellent technical assistance, and John Underhill for photography. This investigation was supported by Grant No. 05388, awarded by the National Cancer Institute, DHEW.
REFERENCES 1 Banerjee, M. R. 1976. Responses of mammary cells to hormones. Int. Rev. Cytol. 47:1. 2 Bryant, J. C., E. L. Shilling, and W. R. Earle. 1958. Massive fluid-suspension cultures of certain mammalian tissue ceils. J. Nat. Cancer Inst. 21:331. 3 Ceriani, R. L. 1976. ttormone induction of specific protein synthesis in midpregnant mouse mammary cell culture. J. Exp. Zool. 196: 1. 4 Cutting, J. A., and T. F. Roth. 1973. Staining of Journal of Dairy Science Vol. 61, No. 6, 1978
732
5 6
7
8
9
10
11
ENAMI AND NANDI
phospho-proteins on acrylamide gel clectrophoresis. Anal. Biochem. 54:386. Eagle, It. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432. Emerman, J. T., J. Enami, D. R. Pitelka, and S. Nandi. 1977. Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Nat. Aead. Sci. U.S.A. 74:4466. Emerman, J. T., and D. R. Pitelka. 1977. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen memhranes. In Vitro 13:316. Enami, J. 1977. tlormonal control of milk protein synthesis in mouse m a m m a r y epithelial cells in vitro. Ph.D. thesis, Univ. of Calif., Berkeley. Enami, J., and S. Nandi. 1977. A radioimmunoassay for a component of mouse casein. J. Immunol. Methods 18:235. Enami, J., and S. Nandi. 1977. Hormonal control of milk protein synthesis in cultured mouse mammary explants. Cell Differentiation 6:217. Enami, J., S. Nandi, and S. tiaslam. 1973. Production of three-dimensional structures - " d o m e s " from dissociated m a m m a r y epithelial cells of mice. In Vitro 8:405. (Abstr.)
Journal of Dairy Science Vol. 61, No. 6, 1978
12 l:eldman, M. K. 1974. A direct radioimmunoassay for mouse casein. Comp. Biochem. Physiol. 49A: 127. 13 Green, M. R., and J. V. Pastewka. 1976. Molecular weights of three mouse milk caseins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and K-like characteristics of a fourth casein. J. Dairy Sci. 59:1738. 14 Juergens, W. G., F. E. Stockdalc, Y. J. Topper, and J. J. Elias. 1965. Hormone-dependent differentiation of mammary gland in vitro. Proc. Nat. Acad. Sci. U.S.A. 54:629. 15 Michaelopoulos, G., and I1. C. Pitot. 1975. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94:70. 16 Puck, T. T., S. J. Gieciura, and tt. W. Fisher. 1957. Clonal growth in vitro of human cells with fihroblastic morphology. J. Exp. Med. 106:145. 17 Terry, P. M., E. M. Ball, R. Ganguly, and M. R. Banerjee. 1975. An indirect radioimmunoassay for mouse casein using [* 2sl]-labeled antigen. J. Immunol. Methods 9:123. 18 Yang, J., J. Enami, and S. Nandi. 1977. Regulation of mammary t u m o r virus production by prolactin in BALB/efC311 mouse normal m a m m a r y epithelial cells in vitro. Cancer Res. 37:3644.
