In Vitro Cell. Dev. Biol.~Animal 34:747-750, November-December 1998 © 1998 Society for In Vitro Biology 1071-2690/98 $05.00 + 0.00

L e t t e r to t h e E d i t o r oL-LACTALBUMIN AS A MODULATOR OF MAMMARY CELLULAR ACTIVITY

procedures and rinsed in sterile phosphate-buffered saline solution (PBS, pH 7.4). The tissue was then blotted dry on sterile lens paper and washed in sterile Medium 199 (Cellgro, Herndon, VA). Samples were removed before culture to assay for total DNA and protein. A tissue sample was also removed for immunohistochemistry. The remaining tissue was chopped into 1-3-mm pieces and placed on siliconized lens paper. Lens paper was prepared by washing in ethyl ether 3 × , 95% alcohol 3 × , and deionized water 3 × , followed by soaking in Prosil 25 (PCR, Inc., Gainesville, FL) for 30 rain according to the methods of Topper et at. (1975). Twenty-five samples of tissue were placed in each well of a 12-we11 culture plate with 2 wells per treatment group: 1) control without bovine c~-lactalbumin (Sigma Chemical Co., St. Louis, MO) 2) 1 ng c~-LA per ml, 3) 5 ng c~-LA per ml, 4) 10 ng c~-LA per ml, 5) 20 ng c~-LA per ml, 6) 30 ng c~LA per ml, 7) 50 ng c~-LA per ml, 8) 100 ng c~-LA per ml, 9) 200 ng c~-LA per ml, and 10) 300 ng c~-LA per ml. Each group was incubated for 72 h in wells (35 mm diameter) containing 3 ml of Medium 199 with Earls sahs and L-glutamine. The pH was adjusted to 7.4 and 25 mM HEPES buffer added. Insulin and corticosterone (Sigma), and prolactin (gift, USDA-ARS, Behsville, MD) were added as supplements at a concentration of 1 ~tg/ml each. Fetal bovine serum (10%) was added along with 90 ~tl/1 of gentamicin (Sigma). Cultures were incubated in a water-jacketed incubator at 37 ° C and 5% CO2. After 48 h, media were removed, frozen, and stored at - 20 ° C for later analysis. Fresh supplemented media were added for an additional 24-h incubation period. After a total of 72 h, the cultures were terminated. Samples and media were frozen and stored for later analyses. Samples were also set aside for immunochemistry after 48 and 72 h. Following the suggestion of the manufacturer (Boehringer and Mannheim Kit #II) for cell proliferation assay, cultures terminated at 72 h were placed with 5-bromo-2'-deoxyuridine, (BrdU), for 45 min at 37 ° C in 5% CO2. Samples were rinsed in washing buffer for 30 rain at 37 ° C and 5% CO2 and then fixed in Carnoy's solution overnight at 4 ° C. The following morning, the samples were stored in 70% ethanol in paraplast and eventually embedded, blocked, and sectioned at 5 ~tm. Tissue sections were placed on gelatin-coated slides and deparaffized in Hemo-D (Fisher Scientific Co., Pittsburgh, PA) and 100% alcohol. Sections were rehydrated by washing 3 X in washing buffer followed by incubation for 30 min in anti-BrdU solution at 37 ° C. Slides were washed 3 X in washing buffer followed by incubation with anti-mouse IgG-alkaline phosphatase solution for 30 rain at 37 ° C. Slides were washed once and incubated in the chromophore plus substrate solution for 20 min at room temperature. Slides were rinsed in washing buffer, dried and mounted in permount. Evaluation of BrdU incorporation was done with a computer-driven Vanox image analysis system. Tissue was analyzed for DNA by a modified procedure of LaBarca et al. (1980). Briefly, approximately 100 mg of tissue was homoge-

Dear Editor: In addition to its nutritive value, milk is also a biological fluid. Because of the unique evolutionary functions of mammary gland epithelia, growth and inhibitory factors in milk may have developed for the control of cell proliferation. These factors include the family of transforming growth factors and tumor necrosis factors. Many polypeptides/proteins provide autocrine/paracrine regulation of growth and differentiation of the mammary gland (see Alston-Mills and Thompson, 1996, for review). In 1991, Bourtourault et al. added bovine milk whey to cultures of both human mammary cancer cells (MCF7) and human prostate cancer cells (PC3), noting significant reductions in cell growth, particularly in the mammary tumor ceils. A study by Thompson et al. (1992) provided evidence to show that a milk-whey protein, c~-lactalbumin (c~-LA), inhibited cell proliferation in both immortalized (A1N4, 80%) and transformed (MCF7, 40%) mammary cell lines, c~-LA is the modifier protein in the lactose synthase system, operating to lower the Km to allow glucose to act as an acceptor for UDP galactose by galactosyltransferase (Brodbeck and Ebner, 1966). Other reports corroborate the cell-modulating effects of c~-LA. Rejman et al. (1992) cultured the bovine mammary epithelial cell line MAC-T with concentrations of c~-LA from 0 to 625 gg/ml. In the absence of fetal bovine serum, c~-LA significantly decreased MAC-T celt proliferation. Studies were conducted by Ellis et al. (1993) using the same cell line. Concentrations of c~-LA from 0.0625 to 0.25 mg/ml resulted in a significant decrease in proliferation. Additionally, Hakansson et al. (1995) suggested that a multimeric form of c~-LA is a potent calcium-elevating and apoptosisinducing agent with cytotoxic activity on some transformed epithelial cells. To date, most of the studies describing the effects of cL-LA have been conducted by the addition of the protein to the culture media of clonally derived cell lines. Little research has been done with explants of mammary tissues which have the advantage of including the extracellular stroma that supports the population of epithelial cells. Our purpose was to determine whether c~-LA, used as a supplement, would effect total protein and DNA concentrations in mammary explants from mid-pregnant mice cultured in the presence of insulin, prolactin, and gtucocorticoids. Additionally, we wished to determine whether the effect of c~-LA in culture was concentrationdependent and whether at certain concentrations it stimulates, rather than inhibits, the proliferation of normal and transformed mammary cell lines. A fifth generation strain of mice were maintained at a constant temperature of 27 ° C with a controlled light cycle of 12:12 h (light to dark) and mouse chow and water ad libitum. Beginning at 7 to 10 wk of age, the females were paired with males and mated. Gestation was monitored from Day 0 which was the d that the vaginal plug was found. On Day 13 of gestation, the females were killed by cervical dislocation. The mammary tissue was surgically removed by sterile 747

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nized for 1 min in a polytron homogenizer in 2.5 ml of 1 × TNE buffer (100 mM Tris base, 10 mM EDTA, 1.0 M NaCL, 10 X stock solution), pH 7.4, per 100 mg tissue and incubated overnight at 4 ° C. The following morning, samples were analyzed with fresh working dye solution [90.0 ml double-distilled H20, 10.0 ml Hoechst stock solution (Sigma), and 10.0 ml 1 X TNE for 50 samples]. Samples and the standard, herring sperm DNA, were assayed with a Hoeffer model 10l TKO mini-fluorometer at 490 nm. Measurements were taken and calculated as milligrams of DNA per gram of tissue. Total protein concentrations were determined with bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL). Briefly, mammary tissue was thawed, weighed, (approximately 30 rag), and homogenized in 2.5 ml of ice-cold 0.2% NaC1 for 1 min. Ten percent trichloroacetic acid (1.25 ml) was added to each homogenized sample, vortexed, and allowed to sit on ice for 20 rain. Samples were centrifuged at 1800 rpm for 15 min at 4 ° C. The supernatant was discarded along with any fat floating on top of the homogenate. The remaining pellet was resolubilized in 50 ml of 0.2% NaC1 and vortexed. Samples were assayed directly or diluted 1:10 in sterile PBS, pH 7.4. The microtiter plate protocol of the BCA assay kit was used. Two hundred microliters of the working reagent along with 10 ~l of prepared sample were pipetted into each well of a 96-well microtiter plate. The plate was shaken on an orbital plate shaker for 30 sec and then incubated in a 37 ° C water bath for 30 min. The plate was removed and absorbance read at a single wavelength of 550 nm on a Bio-Tek plate reader. All values were analyzed for statistical significance by analysis of variance (ANOVA) and regression analysis. The transformed mammary cell line MCF-7 and the normal mammary cell line MCF-10a were obtained from the American Type Culture Collection, Rockville, MD. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), with nonessential amino acids, sodium pyruvate, and the following supplement: 10% fetal bovine serum (Sigma) for MCF-7 cells. MCF-10a cells were cultured in a 1:1 ratio of DME and Ham's F-12 nutrient mixture with 100 ng of cholera toxin per ml and 20 ng of epidermal growth factor per ml, 0.01 bovine insulin per ml, and 5% horse sernm. All cells were cultured in 75-cm tissue culture flasks and incubated at 37 ° C and 10% CO2 with media changes every 48 h. When cells reached confluency, they were split 1 to 3 following the modified procedures of Ellwood et al. (1993) until a total of 16 flasks were obtained. The remaining cells were then frozen and stored in liquid nitrogen for future use. Cells were trypsinized, washed from their flasks, and plated in 96-well tissue culture plates at a density of approximately 4.0 X 105 cells per well per 100 ~1 medium. Cells were allowed to attach to the plates for 24 h before the following treatments were added per ml c~-LA (Sigma): 0 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, and 50 ng. To verify the need for structural integrity as related to function, ct-LA was subjected to a 1-h (c~-LA-4) and a 3-h trypsinhydrolysis (a-LA-12). and used as supplements. Hydrolysates were analyzed by high performance liquid chromatography (HPLC). Cultures were terminated after 72 h and cell proliferation and viability were determined by the XTF Cell Proliferation kit (Boehringer Mannheim, Indianapolis, IN). Sodium 3-[1-phenylaminoearbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonie acid hydrate labeling reagent was added with the electron coupling reagent N-methyl dibenzopyrazine methyl sulfate to a final concentration of 0.3 mg/ml. Fifty microliters of this labeling reagent was added to each well. Cells were incubated at 37 ° C and 10% CO2 for a period of 8 h and then assessed on a Bio-Tek micro plate reader

(EL409). Average absorbance and percent stimulation or inhibition were then calculated. The role of cx-LA as a cell growth regulator in normal and transformed mammary cells and mammary explants was examined. To account for slight variation in seeding densities and the individual sample response to culture, percent change from the control (no c~-LA added) was calculated. Native c~-LA was significantly (P < 0.01) more effective in inhibiting MCF10 cells than it was on the MCF7 cells. This finding was not surprising because the regulation of transformed cells is less stringent than that of normal cells. Within each group, when compared to the control values, maximal inhibition for MCF10 cells was 39% at 40 ng/ml and minimal at 21% at 30 ng/ml. This difference was significant at P < 0.05. For the MCF7 cells, there was little deviation over all concentration ranges of c~LA (mean = 6%) (Fig. 1 a). The magnitude of our results differed from that of the findings of Thompson et al. (1992), but the comparative inhibitions between their normal immortalized cell line (80%) and their transformed cell line (40%) were similar. There were several differences between the two studies, the first being our use of a different immortalized cell line. Additionally, and probably more importantly, was the fact that our cells were initially grown and allowed to attach to 75-mm flasks. Following the harvest, cells were seeded and allowed to attach on 96-well plates before treatment rather than immediate treatment in 60-mm plates. Different methods of assessment of proliferation were also used, specifically, an automated eel1 counter rather than a ceil proliferation assay kit. The 1-h trypsin hydrolysis, designated c~-LA-4, provided a 2% digest as determined by HPLC. Maximal inhibition was again observed at 40 ng/ml (29%) and was significantly different P < 0.05) from 19% inhibition observed at 30 ng/ml. There was minimal inhibitory effect in MCF7 cells from 5 to 20 ng/ml. At 30 and 40 ng/ ml a significant (P < 0.01) stimulatory effect occurred (Fig. 1 b). This effect is further demonstrated when c~-LA is 4% hydrolyzed (e~LA12). At 30 ng/ml, which provided the least inhibition with native ct-LA and ot-LA4, the greatest stimulatory effect was observed in MCF10 cells. There was minimal change in the proliferation of MCF7 but the pattern over all concentrations of c~-LA12 suggests a slight stimulation, especially at 30 ng/ml when compared to the value at 20 ng/ml (Fig. 1 c). The data show that enzymatic modification of the protein reduced the efficacy of the protein for inhibiting cell proliferation, which is in agreement with the work of Thompson et al. (1992). In 1991, Berliner et al. showed that by selective cleavage of the calcium-binding loop, lactose synthase activity was reduced to 2.5% of the native protein. All of these observations demonstrate the importance and necessity of structural integrity for cL-LA. It is generally accepted that by using insulin, prolactin, and glucocorticoids in cultures of mammary tissue from mid-pregnant mice and rats, milk proteins, including o~-LA, will be synthesized, Our question was whether exogenous cx-LA would stimulate or inhibit cellular activity. There was variation in the response to cultm'e among explants from different mice but the overall pattern of response to c~-LA was consistent. Results were adjusted by tissue weight for total DNA and protein. The control represents values of DNA without a-LA supplement. There were no dramatic changes in total DNA over all concentrations after 72 h in culture. However, this is not to say that termination at 12, 24 or 48 h would have yielded these same results (Fig. 2). Likewise, when tissues were examined for the uptake of BrdU, equal incorporation was evident for all values of a-LA mea-

cl-LACTALBUMIN MODULATES CELLULAR ACTIVITY

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FIG. 2. The effects of specific concentrations of native c~-LAon explants from mid-pregnant mice cultured for 72 h. Media were changed after 48 h. Total DNA and total protein (TPRO) were measured. There were no significant differences in total DNA. Each diamond represents the mean of six replicates. There was a significant increase in protein at 30 ng c~-LA per ml and a significant decrease at 50 ng/ml when compared to the control (no a-LA added). Each circle represents a mean of eight replicates + SE.

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FIG. 1. The effects of specific concentrations of (a) native; (b) 1-h trypsm hydrolysis; (c) 3-h trypsin on mammary epithelial cell proliferation. Overall, for MCF10, note the most effective inhibitory concentration at 40 ng/ml and least effective at 30 ng/ml. The percent inhibitions for a and b are plotted with each point representing eight replicates of the mean percent from the control (no cL-LA) + SE.

sured 1 h before termination at 72 h. The effect of c~-LA on DNA may be acute rather than chronic. In contrast, changes were observed with protein concentrations. There was an increased dose-response for total protein between 5 and 30 ng/ml of c~-LA, with the maximal response at 30 ng/ml. At 50 ng/ml or more, there was a precipitous decline in total protein (Fig. 2). The medium was assayed for protein released as a result of c~-LA addition. From our results, there were no significant differences in media protein after correcting for all initial protein additions (to the media). In the mouse mammary gland, the content of c~-LA is low ( < 5 pg/g) between Days 10-14 of gestation and rises to - 10 mg/g on Day 18 (Thordarson et al., 1989). Our results do not differentiate between endogenous and exogenous ~x-LA. However, the evidence appears to indicate modulation of activity by c~-LA, showing both stimulatory and inhibitory effects on total protein. Similar actions for c~-LA of stimulation or inhibition have been observed for rat parotid acinar cell growth and differentiation, depending on the stage of development (Humphreys-Beher et al., 1987). Other examples of peptides having both bifunctional actions are EGF for c~- and K-caseins on gene expression in rats and mice (Vonderhaar and Nakhasi, 1986) and TGF~ (Roberts et al., 1985) that inhibits or stimulates epithelial cell growth. It is difficult to determine whether the effects of c~-LA on total protein concentrations in explants is direct or indirect. However, direct modulation by c~-LA on cells is clearly demonstrated. Thompson et al. (1992) demonstrated inhibition of 3H-lys uptake into protein by NRK cells at a concentration of 10 ng c~-LA per ml, but no results were given beyond that concentration. Our results with the cell lines also provide evidence for direct modulation. Precise mechanisms by which c~-LA effects cell activity remain unclear. The mammary gland undergoes involution once suckling is discontinued (Pitelka, 1988) and the gland becomes engorged with milk. It is during this stage that a "high dosage" inhibition may be evident, resulting in a loss of secretory cells as well as dedifferentiation. Programmed

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cell death is a suggested mechanism which has been demonstrated in both ruminants such as the goat (Quarrie et al., 1994) and rodents (Walker et al., 1989; Strange et al., 1992; Quarrie et al., 1995). What is of interest is that the 30 ng/ml concentration had similar effects on proliferation of cell lines (i.e., decreased inhibition) and increased total protein in explants. This observation may be coincidental but does warrant further study. Whatever the mechanisms, there is convincing evidence of an autocrine/paracrine modulation by c~-LA on the mammary gland in addition to its recognized function in lactose synthesis. ACKNOWLEDGMENTS The authors thank Dr. Marvin E Thompson, USDA retired/St. Augustine's College, Raleigh, for the preparation of trypsin hydrolyzed c~-LA and Dr. Xiaolin Huang, Department of Food Science, North Carolina State University, for the hydrolysis analysis by HPLC. Funding was provided by the North Carolina Agricultural Experiment Station and the North Carolina Institute of Nutrition. REFERENCES Alston-Mills, B.; Thompson, M. P. A theoretical approach to possible biological functions of the milk-whey proteins c~-lactalbumin and [3-1actoglobulin. Comments Agric. Food Chem. 3:175-208; 1996. Berliner, L. J.; Meinholtz, D. C.: Hirai, Y.: Mosci, G.; Thompson, M. P. Functional implications resulting from disruption of the calcium-binding loop in bovine c~-lactalbumin. J. Dairy Sci. 74:2394-2402; 1991. Bourtourault, M.; Buldon, R.; Sampdrez, S.; Jouam J. Effet des protdines du lactosdrum bovin sur la multiplication de cellules cancdreuses humaines. C. R. Soc. Biol. 185:319-323; 1991. Brodbeck, U.; Ebner, K. E. Resolution of a soluble lactose synthetase into two protein components and solubilization of microsomal lactose synthetase. 1. Biol. Chem. 241:762-764; 1966. Ellis, S.; Akers, R. M. The effect of purified whey protein on in vitro cell proliferation. J. Dairy Sci. 76(Suppl.):170; 1993. Ellwood, K. C.; Chatzidakis, C.; Failla, M. L. Fructose utilization by the human intestinal epithelial cell line, Caco-2. Proc. Soc. Exp. Biol. Med. 202:440--446; 1993. Hakansson, A.; Zhivotovsky, B.~ On'enius, S.; Sabharwal, H.; Svanborg, C. Apoptosis induced by a human milk protein. Proc. Natl. Acad. Sci. USA 92:8064-8068; 1995. Humphreys-Beher, M. G.; Schneyer, C. A.; Zelles, T. Alpha-lactalbumin acts as a bimodal regulator of rat parotid acinar cell growth. Biochem. Biophys. Res. Comm. 17:174-181; 1987. LaBarca, C.; Paiger, K. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352; 1980.

Pitelka, D. R.; The mammary gland. In: Weiss L. (ed) Cell and Tissue Biology. Urban and Schwartzenberg. Baltimore; 1998:877-898. Quarrie, L. H.; Addey, C. V. P.; Wilde, C. J. Local regulation of mammary apoptosis in the lactating goat. Biochem. Soc. Trans. 22:178S; 1994. Quarrie, L. H.; Addey, C. V. P.; Wilde, C. J. Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling. Cell Tissue Res. 281:413--419; 1995. Rejman, J. J.; Oliver, S. R; Muenchen, R. A.; Tuner, J. D. Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretory whey proteins. Cell Biol. Int. Rep. 16:993-1001; 1992. Roberts, A. B.; Anzano, M. A.; Wakefield, L. M.; Roche, N. S.; Stern, D. E; Spem, M. B. Type B transforming growth factor: a bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. USA 82:119-123; 1985. Strang, R.; Li, F.; Saurer, S.; Burkhardt, A.: Friis, R. R. Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115:49-58; 1992. Thompson, M. P.; Fan'ell, Jr., H. M.; Mohanam, S.; Liu, S.; Kidwelk W. R.; Basal, M. P.; Cook, R. G.; Medina, D.; Kotts, C. E.; Bano, M. Identification of human milk cL-lactalbumin as a cell growth inhibitor. Protoplasma 167:134-144; 1992. Thordarson, G.; Ogron, L.; Day, J. R.; Bowens, K.; Fielder, P.; Talamantes, E Mammary gland development and c~-lactalbumin production in hypophysectomized, pregnant mice. Biol. Reprod. 40:517-524; 1989. Topper, Y. J.; Oka, T.; Vonderhaar, B. K. Techniques for studying development of normal mammary epithelial cells in organ culture. In: B. W. O'Malley and J. G. Hardman (ed.) Methods In Enzymology. Vol. 39. Academic Press, Inc., New York; 1975:443454. Vonderhaar, B. K.; Nakhasi, H. L. Bifunctional activity of epidermal growth factor on a- and K-casein gene expression in rodent mammary glands in vitro. Endocrinology 119:1178-1184; 1986. Walker, N. I.; Bennett, R. E.; Kerr, J. E R. Cell death by apoptosis during involution of the lactating breast in mice and rats. Amer. J. Anat. 185:19-32; 1989. Brenda Alston-Mills ] Christopher D. Hepler Lisa Sternhagen

Jonathan C. Allen K. Alan Meshaw

Department of Animal Science (B. A.-M., C. D. H., K. A. M.) and Department of Food Science (L. S., J. C. A.) North Carolina State University Raleigh, North Carolina 27695-7621 (Received 15 April 1998) tTo whom correspondence should be addressed.