Cell Biology lnternational Reports, Vol 16, No. 10, 1992
PROLIFERATION LINE IN THE
OF THE MAC-T BOVINE MAMMARY PRESENCE OF MAMMARY SECRETION
993
EPITHELIAL CELL WHEY PROTEINS
J.J. Rejman I, S.P. Oliver 1'3, R.A. Muenchen I, and J.D. Turner 2 Institute of Agriculture l, Department of Animal Science, University of Tennessee, Knoxville, 37901-1071 and McGill University 2, Montreal, Quebec, Canada H9X IC0, 3Corresponding author ABSTRACT
Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large Tantigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), betalactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 ~g/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation. INTRODUCTION
Little is known about specific factors and/or mechanisms associated with the transition of the bovine mammary gland from involution to lactation. Few studies have focused on the influence of mammary secretion components on bovine mammary epithelial cell proliferation. This, in part, is due to lack of a reliable in vitro bovine mammary epithelial cell line. Most of the knowledge defining
0309-1651/92/100993-91508.00/0
© 1992 Academic Press Ltd
Cell Biology lntemational Reports, Vol. 16, No. 10, 1992
994
intrinsic and extrinsic factors regulating mammary gland development, differentiation and function has been through use of laboratory animals as reviewed recently (Houdebine et al., 1985; Oka et al., 1991). However, rodent models may not accurately reflect events that occur in the bovine mammary gland. Conditions in the dairy cow during the transition from lactation to involution are generally quite distinct (Oliver and Sordillo, 1989). For example, dairy cows are pregnant at the time of cessation of milking and mammary glands at this time are still producing considerable quantities of milk, which contrasts with most other species. Recently, an established clonal cell line (MAC-T) was produced from primary bovine mammary alveolar cells by stable transfection with simian virus 40 large T-antigen (Huynh et al., 1991). With immortality of the cell line (> 350 passages without senescence), and ability of MAC-T cells to differentiate to a polar conformation and secrete milk specific components, both characteristic of lactating mammary epithelial cells, development of the MAC-T cell line has been an important contribution to the study of bovine mammary function. MAC-T cells may prove useful in delineating cellular m e c h a n i s m s a s s o c i a t e d with regulation of bovine mammary epithelial cell proliferation and function. The present study demonstrates use of MAC-T cells to determine the influence of mammary secretion whey proteins on MAC-T cell proliferation. MATERIALS
AND
METHODS
cell culture conditions. MAC-T cells were grown on plastic tissue culture flasks (Falcon; Lincoln Park, NJ) as described by Huynh et al. (1991) in growth media [Dulbecco's Modified Eagle Media (DMEM; Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 5 ~g/ml insulin, 1 ~g/ml hydrocortisone, i00 IU/ml penicillin and i00 ~g/ml streptomycin (Sigma, St. Louis, MO)]. MAC-T cells were incubated at 37 ° C in 5% CO2/balance air during culture and proliferation assays. Passage of cells included a brief wash with calcium free Dulbecco's phosphate buffered saline (DPBS; Gibco), and release with 0.5% trypsin (Sigma) in DPBS. Release of cells from plastic was achieved by rinsing flasks in trypsin solution for about 30 sec, trypsin was removed b y a s p i r a t i o n and flasks were incubated at 37 ° C until cells were released (5-15 min). Trypsinization was halted by addition of growth media plus FBS. MAC-T
Cell Biology International Reports, Vol. 16, No. 10, 1992
995
Proteins.
Serum albumin (SA), a l p h a - l a c t a l b u m i n (ALA), and beta-lactoglobulin (BLG) were purchased from Sigma. L a c t o f e r r i n (LF) and t r a n s f e r r i n (TF) w e r e p u r c h a s e d from ICN Biomedicals, Inc., Irvine, CA.
MAC-T cell proliferation assay. MAC-T cells were c o c u l t u r e d in the p r e s e n c e of t w o - f o l d d i l u t i o n s of m a m m a r y s e c r e t i o n whey proteins; ALA, BLG, LF, TF, and SA, at final c o n c e n t r a t i o n s r a n g i n g from 0 to 625 #g/ml and o b s e r v e d for c h a n g e s in proliferation. Total p r o l i f e r a t i o n assay time was 96 hr. Initially, M A C - T cells w e r e g r o w n u n d e r serumdepleted conditions to r e m o v e serum components which stimulate optimum proliferation. Specifically, cells were p l a t e d (2 X 104 cells/well; 100 #I) in g r o w t h media minus FBS onto wells of a 96-well t i s s u e c u l t u r e p l a t e and remained serum-depleted for a total of 48 hr. After initial 24 hr of culture, m e d i a w e r e r e m o v e d and r e p l a c e d w i t h fresh m e d i a minus FBS. At 48 hr, m e d i a were r e m o v e d and r e p l a c e d with i00 #I of e i t h e r s e r u m - f r e e m e d i a or m e d i a c o n t a i n i n g 10% FBS. Subsequently, 50 #i of each whey protein, d i l u t e d in g r o w t h m e d i a m i n u s FBS, w e r e added. Assay continued for a n o t h e r 48 hr. Proliferation was e v a l u a t e d by the a d d i t i o n of 1 # C i / w e l l of [3H]-thymidine (ICN Biomedicals) in s e r u m - f r e e m e d i a for the last 20 hr of culture. M A C - T cells w e r e h a r v e s t e d onto glass fiber filters using an a u t o m a t e d cell harvester (PhD Cell Harvester; Cambridge Technology Inc., Watertown, MA). R e t a i n e d r a d i o a c t i v i t y was c o u n t e d for 2 min per vial in a liquid scintillation counter (Mark V Series Liquid Scintillation C o u n t i n g System; TM A n a l y t i c a l Inc., Elk G r o v e Village, IL). Data were e x p r e s s e d as the m e a n counts per m i n u t e (cpm) ± s t a n d a r d error of the mean (SEM) x 10 .3 of t h r e e r e p l i c a t e cultures.
Statistical analysis. M a i n e f f e c t s of each whey p r o t e i n on m e a n M A C - T cell p r o l i f e r a t i o n a c r o s s all c o n c e n t r a t i o n s evaluated were t e s t e d u s i n g a n a l y s i s of v a r i a n c e (SAS ® GLM; 1989). A n a l y s i s of cell p r o l i f e r a t i o n in the a b s e n c e and p r e s e n c e of FBS r e m a i n e d separate. If main e f f e c t s of w h e y p r o t e i n c o n c e n t r a t i o n s w e r e significant, individual protein concentrations were compared to c o n t r o l s (no protein) u s i n g S t u d e n t ' s t-test. RESULTS M A C - T cell p r o l i f e r a t i o n in the p r e s e n c e of 10% FBS was g r e a t e r (P
PROLIFERATION LINE IN THE
OF THE MAC-T BOVINE MAMMARY PRESENCE OF MAMMARY SECRETION
993
EPITHELIAL CELL WHEY PROTEINS
J.J. Rejman I, S.P. Oliver 1'3, R.A. Muenchen I, and J.D. Turner 2 Institute of Agriculture l, Department of Animal Science, University of Tennessee, Knoxville, 37901-1071 and McGill University 2, Montreal, Quebec, Canada H9X IC0, 3Corresponding author ABSTRACT
Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large Tantigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), betalactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 ~g/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation. INTRODUCTION
Little is known about specific factors and/or mechanisms associated with the transition of the bovine mammary gland from involution to lactation. Few studies have focused on the influence of mammary secretion components on bovine mammary epithelial cell proliferation. This, in part, is due to lack of a reliable in vitro bovine mammary epithelial cell line. Most of the knowledge defining
0309-1651/92/100993-91508.00/0
© 1992 Academic Press Ltd
Cell Biology lntemational Reports, Vol. 16, No. 10, 1992
994
intrinsic and extrinsic factors regulating mammary gland development, differentiation and function has been through use of laboratory animals as reviewed recently (Houdebine et al., 1985; Oka et al., 1991). However, rodent models may not accurately reflect events that occur in the bovine mammary gland. Conditions in the dairy cow during the transition from lactation to involution are generally quite distinct (Oliver and Sordillo, 1989). For example, dairy cows are pregnant at the time of cessation of milking and mammary glands at this time are still producing considerable quantities of milk, which contrasts with most other species. Recently, an established clonal cell line (MAC-T) was produced from primary bovine mammary alveolar cells by stable transfection with simian virus 40 large T-antigen (Huynh et al., 1991). With immortality of the cell line (> 350 passages without senescence), and ability of MAC-T cells to differentiate to a polar conformation and secrete milk specific components, both characteristic of lactating mammary epithelial cells, development of the MAC-T cell line has been an important contribution to the study of bovine mammary function. MAC-T cells may prove useful in delineating cellular m e c h a n i s m s a s s o c i a t e d with regulation of bovine mammary epithelial cell proliferation and function. The present study demonstrates use of MAC-T cells to determine the influence of mammary secretion whey proteins on MAC-T cell proliferation. MATERIALS
AND
METHODS
cell culture conditions. MAC-T cells were grown on plastic tissue culture flasks (Falcon; Lincoln Park, NJ) as described by Huynh et al. (1991) in growth media [Dulbecco's Modified Eagle Media (DMEM; Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 5 ~g/ml insulin, 1 ~g/ml hydrocortisone, i00 IU/ml penicillin and i00 ~g/ml streptomycin (Sigma, St. Louis, MO)]. MAC-T cells were incubated at 37 ° C in 5% CO2/balance air during culture and proliferation assays. Passage of cells included a brief wash with calcium free Dulbecco's phosphate buffered saline (DPBS; Gibco), and release with 0.5% trypsin (Sigma) in DPBS. Release of cells from plastic was achieved by rinsing flasks in trypsin solution for about 30 sec, trypsin was removed b y a s p i r a t i o n and flasks were incubated at 37 ° C until cells were released (5-15 min). Trypsinization was halted by addition of growth media plus FBS. MAC-T
Cell Biology International Reports, Vol. 16, No. 10, 1992
995
Proteins.
Serum albumin (SA), a l p h a - l a c t a l b u m i n (ALA), and beta-lactoglobulin (BLG) were purchased from Sigma. L a c t o f e r r i n (LF) and t r a n s f e r r i n (TF) w e r e p u r c h a s e d from ICN Biomedicals, Inc., Irvine, CA.
MAC-T cell proliferation assay. MAC-T cells were c o c u l t u r e d in the p r e s e n c e of t w o - f o l d d i l u t i o n s of m a m m a r y s e c r e t i o n whey proteins; ALA, BLG, LF, TF, and SA, at final c o n c e n t r a t i o n s r a n g i n g from 0 to 625 #g/ml and o b s e r v e d for c h a n g e s in proliferation. Total p r o l i f e r a t i o n assay time was 96 hr. Initially, M A C - T cells w e r e g r o w n u n d e r serumdepleted conditions to r e m o v e serum components which stimulate optimum proliferation. Specifically, cells were p l a t e d (2 X 104 cells/well; 100 #I) in g r o w t h media minus FBS onto wells of a 96-well t i s s u e c u l t u r e p l a t e and remained serum-depleted for a total of 48 hr. After initial 24 hr of culture, m e d i a w e r e r e m o v e d and r e p l a c e d w i t h fresh m e d i a minus FBS. At 48 hr, m e d i a were r e m o v e d and r e p l a c e d with i00 #I of e i t h e r s e r u m - f r e e m e d i a or m e d i a c o n t a i n i n g 10% FBS. Subsequently, 50 #i of each whey protein, d i l u t e d in g r o w t h m e d i a m i n u s FBS, w e r e added. Assay continued for a n o t h e r 48 hr. Proliferation was e v a l u a t e d by the a d d i t i o n of 1 # C i / w e l l of [3H]-thymidine (ICN Biomedicals) in s e r u m - f r e e m e d i a for the last 20 hr of culture. M A C - T cells w e r e h a r v e s t e d onto glass fiber filters using an a u t o m a t e d cell harvester (PhD Cell Harvester; Cambridge Technology Inc., Watertown, MA). R e t a i n e d r a d i o a c t i v i t y was c o u n t e d for 2 min per vial in a liquid scintillation counter (Mark V Series Liquid Scintillation C o u n t i n g System; TM A n a l y t i c a l Inc., Elk G r o v e Village, IL). Data were e x p r e s s e d as the m e a n counts per m i n u t e (cpm) ± s t a n d a r d error of the mean (SEM) x 10 .3 of t h r e e r e p l i c a t e cultures.
Statistical analysis. M a i n e f f e c t s of each whey p r o t e i n on m e a n M A C - T cell p r o l i f e r a t i o n a c r o s s all c o n c e n t r a t i o n s evaluated were t e s t e d u s i n g a n a l y s i s of v a r i a n c e (SAS ® GLM; 1989). A n a l y s i s of cell p r o l i f e r a t i o n in the a b s e n c e and p r e s e n c e of FBS r e m a i n e d separate. If main e f f e c t s of w h e y p r o t e i n c o n c e n t r a t i o n s w e r e significant, individual protein concentrations were compared to c o n t r o l s (no protein) u s i n g S t u d e n t ' s t-test. RESULTS M A C - T cell p r o l i f e r a t i o n in the p r e s e n c e of 10% FBS was g r e a t e r (P
