Altered Biochemical Properties of Mitochondria in Mouse Mammary Epithelial Cells during Primary Culture MICHAEL T. WHITE ' AND S. NAND1 Cancer Research Laboratory and Department ofZoology, University of California, Berkeley, California 94720
ABSTRACT Various biochemical properties of mitochondria isolated from primary monolayer cultures of mammary epithelial cells from mid-pregnant or hormonally stimulated mice were examined daily for seven or eight days. When compared with mitochondria from mammary glands of mid-pregnant animals, the specific activities of several mitochondrial enzymes were greatly reduced in cells after seven days in culture. There was a 5- to 6-fold reduction in the specific activities of cytochrome oxidase, succinate dehydrogenase and a glycerophosphate oxidase while malate dehydrogenase and adenylate kinase activities were 2- to 3-fold lower. The reduction in mitochondria1 enzyme activities was gradual and related to the length of time the cells were in culture. Progressive changes were also seen in the electrophoresis profiles of mitochondrial proteins in SDSurea polyacrylamide gels. Mitochondria isolated from 1-,2-, 3- and 8-day cell cultures showed a continuous reduction in the relative amounts of several mitochondrial polypeptides in the gel profiles. Addition of 35S-methionineto cell cultures for short and long periods indicated that mitochondrial protein synthesis continued throughout the 8-day culture period. However, the synthesis of several particular polypeptides was reduced progressively during the culture period. These studies indicate that mouse mammary epithelial cells have a lowered capacity for respiratory metabolism as a result of specific mitochondrial alterations which might be associated with the general loss of differentiated morphology by those cells during monolayer culture. Normal and neoplastic mammalian cells in culture commonly show high rates of aerobic glycolysis as measured by lactate production (Gregg e t al., '68; Gregg, '72). Undoubtedly, many factors, such as medium composition, culture pH, growth rate and cell density influence the level of aerobic lactate production. In cultures of normal mouse lymphocytes, Wang et al. ('76) directly linked glycolysis with cell proliferation. Glycolysis and respiration often tend to vary inversely (Paul, '65). Bissell et al. ('72) examined the relationship between lactate and C02 production in sparse, fast growing and dense, slow growing cultures of chick fibroblasts. They concluded that population density and not the growth rate determined the relative amounts of glycolysis and respiration in these cultures. Most studies on glucose metabolism in mammalian cell culture have dealt with fibroblasts, lymphocytes, established cell lines and transformed cells. Comparatively little is known about the metabolJ. CELL.
PHYSIOL., 93: 335-344.
ic properties of epithelial cells in primary culture. In culture, epithelial cells from the mammary glands of mid-late pregnant mice rapidly lose many of the cytological features which characterize the developed glands in situ (Pickett et al., '75). These cells also showed high levels of lactate production which were seemingly independent of cell density and proliferation (White and Nandi, '77). In this paper, we examined the respiratory ability of mouse mammary epithelial cells as a function of time and loss of differentiated morphology during culture. We focused on the mitochondria of these cells and analyzed the specific activities of several key respiratory enzymes, the total protein composition and the patterns of new protein synthesis in mitochondria of Received May 19, '77. Accepted July 8, '77. ' Present address: Stanford University School of Medicine, Stanford University Service (Medicine ill), V. A. Hospital, Palo Alto, California 94304.
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twice with Hank’s BSS, scraped from the dish with a teflon spatula and centrifuged a t 180 x g for five minutes. The cell pellet was resuspended in a small volume (less than 5 ml) of cold sucrose medium (SM) (0.25 M sucrose, 2 mM EDTA and 50 mM Tris-HC1, pH 7.4) containing 1%(w/v) fraction V bovine serum albumin (BSA) (Pentex, Miles Labs., Elkhart, MATERIALS AND METHODS Indiana). The cells were disrupted in a 7-ml Cell culture glass dounce (100 strokes, pestle A Bellco Mammary gland tissue was excised asep- Glass, Vineland, New Jersey). The homogetically from midpregnant BALB/cCrgl mice or nate volume was adjusted to 25 ml with BSAanimals hormonally stimulated for five weeks SM and centrifuged a t 500 X g for ten minutes with subcutaneously implanted pellets of 17p at 5°C. The supernatant was recentrifuged a t estradiol, deoxycorticosterone actetate as de- 500 x g for ten minutes and 8,700 x g for fifscribed previously (White et al., ’77). Minced teen minutes to obtain a crude mitochondrial tissue was suspended in 0.1% collagenase pellet. This fraction was resuspended in 25 ml (Type 111, 145 units/mg, Worthington Bio- BSA-SM and centrifuged a t 8,700 x g for 15 chemical, Freehold, New Jersey) in Hank’s minutes. The partially purified mitochondrial balanced salt solution (BSS) (Gibco, Santa pellet was resuspended in a small volume of Clara, California) and rotated a t moderate BSA-SM, layered onto a 15-ml linear sucrose speed in a shaking 37°C water bath for 90 gradient (1.03-1.71M sucrose in 2 mM EDTA, minutes. After centrifugation at 180 x g for 25 mM Tris- HC1, pH 7.41, and centrifuged to five minutes, the supernatant was discarded equilibrium (2 hours a t 25,000 rpm in a Beckand the cell pellet was resuspended in 0.05% man SW 27.1 rotor) a t 5°C. The mitochondrial pronase (B grade, Calbiochem, La Jolla, Cali- band was removed, resuspended in SM a t a fornia) in Hank’s BSS and agitated for 30 final sucrose concentration of 0.33 M and cenminutes a t 37°C. The suspension was cen- trifuged a t 10,000 x g for 20 minutes a t 5°C. trifuged a t 180 x g for five minutes, the cell The purified mitochondrial pellet was resuspellet was resuspended in Dulbecco’s modified pended in a small volume of SM and assayed Eagle’s medium (DMEM) (Gibco) containing for enzymatic activity or treated for analysis 10% fetal calf serum (Pacific Biologicals, of polypeptides by gel electrophoresis. Richmond, California) and filtered through Enzyme assays single, then through double layered 150-p mesh Nytex cloth (Tetko, Elmsford, New We assayed cytochrome oxidase spectrophoYork). The cells were washed twice in DMEM tometrically by following the oxidation of + 10%FCS by centrifugation a t 100 Xg for reduced cytochrome C (equine heart Type VI, five minutes to remove contaminating fibro- Sigma Chemical Co., St. Louis, Missouri) as blasts. The washed cell pellet was resuspended described by Smith (‘55). Malate dehydrogein DMEM, 10% FCS, insulin (I) (10 kg/ml), nase was assayed by the method of Ochoa Penicillin-steptomycin sulphate (PSI (100 (‘55), adenylate kinase was assayed according unitdm1 and 100 pg/ml respectively) and to Schnaitman and Greenawalt (‘68) and plated into tissue culture dishes (Corning, NADH oxidase was assayed spectrophotometCorning, New York). The cultures were main- rically observing the oxidation of NADH using tained a t 37”C, pH 7.2 in a humidified 95%air, cytochrome C as terminal electron acceptor 5%COz environment. (Green and Ziegler, ’63). The test for monoamine oxidase followed the method of Mitochondria preparation Wurtman and Axelrod (‘63) with 14C-trypMammary gland tissues - mitochondria tamine (47 mCi/mmole, New England Nuclewere prepared from mammary glands of ar, Boston, Massachusetts) as substrate. We midpregnant or 5-week hormone stimulated assayed a glycerophosphate oxidase (the mitomice as described previously (White and chondrial form of a glycerophosphate dehyNandi, ’76). drogenase) and succinate dehydrogenase as Mammary gland cells- following dissocia- described by O’Brien and Gethmann (‘731, tion cells were cultured for zero to eight days which spectrophotometrically follows the reas described above. The cells were washed duction of p-iodonitrotetrazolium violet. All
cells after various periods in culture. These studies indicate that mouse mammary epithelial cells have a lowered capacity for respiratory metabolism as a result of specific mitochondrial alterations which might be associated with the general loss of differentiated functions by these cells during monolayer culture.
ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS
enzymes were assayed for maximum activity which included whenever noted mitochondrial disruption by treatment with the nonionic detergent Lubrol WX (0.2 mglmg rnitochondrial protein, ICI America, Charlotte, North Carolina) before enzyme assay. This was of particular importance when the substrates of certain assays (ex. NADH in NADH oxidase) were not able to penetrate intact mitochondria. Protein was determined by the method of Lowry et al. (‘51) with BSA as a standard.
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periods were as follows: the first 18 hours after plating; from the second to the third day of culture and from the seventh to the eighth day of culture. In every case, the cells were harvested 18 to 24 hours after the 35S-methionine was added to the medium. Prior to harvest, the cells were washed with Hank’s BSS. Mitochondria were isolated and purified on sucrose gradients and mitochondrial polypeptides analyzed by electrophoresis on slab gels. Autoradiograms of the stained gels were made using Kodak Blue X-ray film (Eastman Kodak, Rochester, New York).
Polyacrylamide gel electrophoresis RESULTS The dissociation and electrophoresis of mitochondrial proteins were based on the methInitial experiments to determine organelle od described by Maize1 et al. (‘68).Samples of density, intactness and contamination by sucrose gradient purified mitochondria (-- 50 other cell components involved localization of Fg protein each) were dissolved in urea, SDS the mitochondrial band in a linear sucrose and /3 mercaptoethanol (5 M,1.0%and 0.2%re- gradient after centrifugation to equilibrium. spectively) by heating a t 95°C for five minutes The gradient profile of mitochondria isolated and layered onto gels after cooling. The gels, from mammary gland cells grown in culture set in either plexiglass tubes (6mm I.D.) or be- for seven days is shown in figure 1.The activtween glass plates as slabs 0.75 mm thick were ities of inner mitochondrial membrane cytocomposed of 10% acrylamide and 0.27% chrome oxidase, outer mitochondrial membisacrylamide (BioRad Labs., Richmond, Cali- brane monoamine oxidase, total protein and fornia) in 4 M urea, 0.01 M EDTA, 0.1% SDS, density along the gradient are shown. Both 0.1% N,N,N’N‘-tetramethylethylenediamine mitochondrial enzymes were found in a uniand 0.05 M Na phosphate buffer (pH 7.2). Po- form band with a mean density of 1.183 glml lymerization was catalyzed with 0.5% ammo- a t 0°C. The major protein band was coincident nium persulphate. Tube gels were electropho- with both bands of enzyme activity which resed a t 5 milliampere (MA) per gel and slab indicated that the mitochondria were relagels a t 12 MAlgel for 22 hours a t room temper- tively homogeneous in size and were not disature in 0.05 M Na phosphate buffer (pH 7.2) rupted during isolation. More than 80%of the containing 0.1% SDS and 0.01 M EDTA. The cytochrome oxidase and 75% of the monogels were fixed, stained with Coomassie blue amine oxidase activities applied to the graand destained by the method of Smith et dient were recovered in fractions 6 to 9, well al. (‘69).The gels were photographed and away from other contaminating protein in the the negatives scanned in a densitometer. Poly- gradient. No lysozyme activity was detected peptide molecular weights were determined in the mitochondrial fractions. Sucrose graby coelectrophoretic standardization using dient purification resulted in a 2- to %fold murine mammary tumor virus (MuMTV) increase in mitochondrial enzyme specific acpolypeptides (Dickson and Skehel, ’74). In all tivity compared to the organelles obtained by cases where mitochondrial polypeptide gel differential centrifugation. Mitochondria isoprofiles were compared, the samples were lated from the mammary glands of midpregcoelectrophoresed in the same chamber and nant or 5-week hormone stimulated animals approximately equal amount of protein was had similar characteristics on a sucrose graloaded into each gel. dient forming a uniform band a t a density of 1.182glml a t 0°C. By these criteria, mitochonMitochondrial protein synthesis dria isolated from mammary glands or 7-day Radioactively labelled mitochondrial pro- cultures of mammary gland cells were teins were obtained by growing mammary indistinguishable. The specific activities of seven enzymes gland cells in culture for various periods in nutrient medium (DMEM + 10%FCS I + PS) chosen for their diverse mitochondrial locacontaining 1 FCiIml 35Smethionine (250 Cil tion and their key roles in oxidative metabommole, New England Nuclear). The labelling lism were compared in gradient purified mito-
+
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MICHAEL T. WHITE AND S. NAND1
400
300 c
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500
E
\ 0
a
-
.-
C
200
400
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2 a
300
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200
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Bottom
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Fig. 1 Sucrose gradient profile of mitochondria isolated from mouse mammary epithelial cells after seven days in monolayer culture. The gradient and mitochondria were preparedand the fractions were assayed for protein, refractive index, cytochrome oxidase and monoamine oxidase activities as described in MATERIALS AND METHODS.
chondria isolated from the mammary glands of midpregnant (or 5-week hormonally stimulated) animals and their mammary cells after seven days in culture. These results are summarized in table 1. There was no difference in monoamine oxidase activities and little difference in NADH oxidase activities but adenylate kinase and malate dehydrogenase activities were 2-fold lower in cultured cell mitochondria. Most striking was the nearly 6fold reduction in specific activities of aglycerophosphate oxidase, cytochrome oxidase and succinate dehydrogenase in mitochondria from mammary cells after seven days in culture.
The comparison of mitochondrial properties was extended to structural and functional proteins analyzed by electrophoresis on SDS-urea polyacrylamide gels. Figure 2 shows the electrophoresis profiles of gradient purified mitochondria from the mammary glands of hormonally stimulated (5 weeks) animals and cells from these glands after seven days in culture. Significant differences in the relative amounts of many mitochondrial polypeptides were evident. Polypeptide 2 (84,000 daltons) was highly reduced in the mammary cell mitochondria as were the relative proportions of polypeptides 3:4, 5:6:7, 8:9:10 and 11:12. These changes in proportion indicated a de-
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ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS TABLE 1
Enzymatic actiuities of sucrose gradient-purifed mitochondria isolated in media containing 1 % BSA *Specific activity (nmoles product formediminlmg protein) ~
Mammary gland Mammary gland
Mammary gland cells
Mammary gland cells
** Cytochrorne oxidase
1365293 (7)
234226 (7)
5.83
Monoarnine oxidase (tryptamine-14C) a-glycerophosphate oxidase ** Succinate dehydrogenase Malate dehydrogenase ** NADH oxidase ** Adenylate kinase
310227 (6) 14.12.4 (4) 7.41t.15 (4) 1640k 161 (6) 180217 (4) 3481t31 (5)
247k20 (7) 2.54k0.40 (7) 1.3120.24 (7) 6302 134 (7) 269231 (7) 162k9 (7)
1.26 5.56 5.65 2.60 0.67 2.15
*
*Mean SE; numbers in parentheses = number of individual experiments. **Pretreated with Lubml WX.
I I
234 I l l
21
20 I
19
5 6 7 8 910 I I 12 13 1 4 15 1617 18 I I I / I 1 1 1 I l l 1 1 1 1
I
I
GLAND
CULTURED MAMMARY GLAND CELLS
r
1 1 1
1 1 1 1
87 84 75 6964
I
56
I I
I
I
I
50 47 43
I
I I I
36 31.5
I
I
23
I
20.5
I
18
MOLECULAR WEIGHT x lo3 Fig. 2 Densitometer scans of photographs of Coomassie blue stained mitochondria1 polypeptides electrophoresed in tube gels. Mitochondria were isolated from hormonally stimulated mouse mammary glands or seven day monolayer cultures of these mammary gland cells. Mitochondria1 isolation, purification and electrophoresis were as described in MATERIALS AND METHODS. Fifty micrograms “Lowry” protein/aarnple were coelectrophoresed.
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MICHAEL T. WHITE AND S. NAND1
crease in the amount of several of these polypeptides in the mitochondria from mammary gland cells in culture. After seven days in culture mammary epithelial cells have mitochondria with highly diminished activities of two key electron transport enzymes, several other enzymes and reduced amounts of several undefined structurallfunctional proteins. I t is possible that these differences were the result of the method of gland dissociation andlor a selection of a specific cell type(s) during cell culture. To examine this possibility, mitochondria were isolated from mammary glands after the following treatment: undissociated glands; cells dissociated from whole glands plated and cultured for 30 minutes, l day, 2 days, 3 days and 7 days. Table 2 summarizes the enzyme activities determined in these experiments. There was no significant difference in the specific activities of monoamine oxidase, cytochrome oxidase, a glycerophosphate oxidase and succinate dehydrogenase in mitochondria isolated from undigested glands and cells from enzymatically dissociated glands plated for 30 minutes. After one day in culture monoamine oxidase activity was unchanged while cytochrome oxidase activity dropped 3-fold, and aglycerophosphate oxidase and succinate dehydrogenase were reduced nearly 2-fold. The same level of reduced activity is maintained in cells cultured for two days. By the third day, cytochrome oxidase activity was 4-fold lower while a-glycerophosphate oxidase and succinate dehydrogenase were greater than 2-fold lower. By day 7, the activity of these three enzymes were 6 fold lower while monoamine oxidase activity remained unchanged. The reduction in mitochondrial enzyme activities was gradual and related to the length of time the cells were in culture, not the method of gland
dissociation. The procedure of gland dissociation results in an almost pure (>95%)population of parenchymal cells. These cells have mitochondrial properties identical to those organelles isolated from intact glands but after just 24 hours in culture, the mitochondrial properties of these cells have changed. The gel electrophoresis profiles of mitochondrial proteins also revealed progressive changes related to the length of time these cells were cultured (fig. 3). Polypeptide 2 was present in mitochondria from cells cultured for one day but was nearly absent in cells cultured two to eight days (fig. 3). The change in the relative proportions of polypeptides 5:6:7 occurred after two days in culture as did the changes in proportion of polypeptides 8:9:10 (fig. 3). There was a gradual reduction in the amount of polypeptides 21 and 22 beginning on day 2 and continuing through day 8 (fig. 3). The change in the relative amounts of polypeptides 11:12 seen in comparisons of mammary gland and 7-day cultures of mammary gland cells (fig. 2) was evident after 24 hours of culture but became more prominent on succeeding days (fig. 3). The changes in enzyme activities and protein components of mitochondria during cell culture were not the result of mitochondrial disaggregation. The ultrastructure of mitochondria seen in thin section electron micrographs of cells after various periods of culture appeared normal and mitochondrial dissaggregation was not evident (White et al., '77; and unpublished observation). Mammary epithelial cells continued to synthesize new mitochondria and mitochondrial proteins during monolayer culture as evidenced by labelling studies and analysis of mitochondrial protein synthesis by autoradiography of SDS-urea polyacrylamide gels.
TABLE 2 Enzyme activity in mitochondria purifkd on sucrose gradients *Ratio specific activity:
Monoamine oxidase Cytochrome oxidase a-glycerophosphateoxidase Succinate dehydrogenase
Mammary Mammary gland cells Days in cuiture
Dissociated
1
2
3
I
0.84 1.21 1.02 0.97
1.20 2.98 1.79 1.71
1.25 2.90 2.31 1.92
1.39 4.42 2.51 1.91
1.16 5.87 5.64 5.79
* The average of three separate experiments;mammary glands were from mice hormonally stimulated for five weeks. In each case, mammary gland and mammary gland cell mitochondria were compared, using mitochondria isolated, purified and assayedon the same day
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ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS
I2 34 5 6 7 I I I I l l l I
8 9 10 I/ 12 13 14 15 1 6 17 I l l 1 I I l l 1 I
18 I
19 I
20 I
21 22 I I
23 I
DAY 8
l
l
l
l
l
l
l
I
1
1
1
87 79 69 61 56 5250
I
l
l
1
1
45 43 36
I
31.5
I
23
I
20.5
1
1
18 15.5
1
12.5
MOLECULAR WEIGHT x lo3 Fig. 3 Densitometer scans of photographs of Coomassie blue stained mitochondrial polypeptides electrophoresed on a slab gel. Mitochondria were isolated from cells derived from hormonally atimulated (5weeks) mammary glands. Cells were cultured for the various times indicated. Mitochondria1isolation, purification and electrophoresis were as described in MATERIALS AND METHODS. Twenty-five micrograms “Lowry” protein/samples were coelectrophoresed.
Figure 4 shows densitometer scans of autoradiographs of mitochondrial proteins from cells labelled with 35Smethionine for various periods during culture. All of the major mitochondrial polypeptides were synthesized during the first 18 hours of culture. Cells labelled from the second to the third and the seventh to the eighth day of culture also synthesized most of the mitochondrial polypeptides (fig. 4).The pattern of newly synthesized proteins mimicked the gradual changes in relative proportion of the various polypeptides seen in
figure 3. Most striking was the change in synthesis of polypeptide 8 (56,000 daltons). This polypeptide was made in significant amounts during the first 18 hours of culture, but synthesis was reduced from days 2 to 3 and very highly reduced from days 7 to 8 (fig. 4).This corresponded to the nearly complete absence of this polypeptide after day 1 as seen in stained gel profiles (fig. 3). I t is significant to note that mitochondrial protein synthesis continued in mammary cells in culture and the gradual reduction in cer-
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MICHAEL T. WHITE AND S. NAND1 I 2 3 4 5 6 7 891011 12 1 1 1 1 1 1 1 1 1 1 1 1
13 I
14
15
I
I
16 I
17 I
18 I
19 I
7th
20 I
- 8th
DAY
Fig. 4 Densitometer scans of autoradiographs of “S-methionine labelled mitochondria1 polypeptides electrophoresed on a slab gel. Cells from mammary glands of mice hormonally stimulated for five weeks were cultured with Y3-methionine in the medium for the periods indicated. Mitochondria1 isolation, purification, electrophoresis and autoradiography were as described in MATERIALS AND METHODS. Twenty-five micrograms “Lowry” protein/samples were coelectrophoresed and the autoradiograms exposed for five days at room temperature. Cells were labelledwith 3sS-methioninefor either the first 18hours after plating; or the second to the third day; or the seventh to eighth day of culture.
tain mitochondrial proteins appeared to result from the decreased net synthesis of these components. DISCUSSION
Following enzymatic dissociation of the mouse mammary gland, parenchymal cells can be separated from fat cells, myoepithelial cells and the majority of fibroblasts by differential centrifugation. When plated at me-
dium to high densities, the parenchymal cells settle, attach, proliferate and form a continuous sheet with a polygonal morphology within 24 to 48 hours. The strict criteria for the epithelial nature of these cells are the nonrandom attachment of the cells such that the basolateral surface attaches to the substratum and the apical surface faces up with numerous microvilli projecting into the me-
ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS
dium and the occluding tight junctions that connect adjacent cells (Pickett et al., '75). If cells are derived from mid-late pregnant mammary glands, accommodation to the crowded conditions of cell culture appears to reduce the ductal and lobulo/alveolar cells to roughly similar states of cytoplasmic development so that internal distinguishing features tend to disappear. Cells in these primary cultures never approach the differentiated state of secretory mammary epithelium (Pickett et al., '75). Ultrastructural evidence suggests that loss of differentiated functions in mammary epithelial cells begins when cells are seeded onto plastic or glass substrates in tissue culture (Emerman and Pitelka, '76). Our results suggest that the mitochondria of mouse mammary epithelial cells undergo structural and functional changes in response to cell culture. I t is tempting to speculate that these mitochondrial alterations reflect a loss of differentiated function in these organelles concomitant with the general loss of developmental morphology of these mammary cells. There is compelling evidence for mitochondrial differentiation in the mouse mammary gland during pregnancy and lactation in vivo. Total mitochondrial protein and succinate oxidase and cytochrome oxidase activities increased gradually during pregnancy with a 2to %fold increase occurring during early lactation (Jones and Rosano, '72). These changes were accompanied by a corresponding increase in the buoyant density of isolated mitochondria due to an expansion of inner membrane and matrix components (Rosano and Jones, '76). This expansion was indicated by a 3- to 4fold increase in cytochrome oxidase activity and was confirmed in electron micrographic studies (Rosanoet al., '76). The major phase of mitochondrial development occurs shortly after parturition during a period subsequent to epithelial cell proliferation in the gland (Jones and Rosano, '72, '76). These studies indicate that mitochondrial differentiation in the developing mouse mammary gland is geared to increased oxidative capacity of these cells during milk production and secretion. Our results indicate that mitochondria show a markedly decreased oxidative capacity in mammary epithelial cells which have lost differentiated features as a result of cell culture. The specific activity of outer mitochondrial membrane monoamine oxidase did not change in mammary gland cells during culture. This is consistent with the observations
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of Rosano and Jones ("76) who found that monoamine oxidase activity changed very little compared to cytochrome oxidase and other enzymes in mitochondria isolated from the mammary glands of virgin, pregnant or lactating mice. Their interpretation was that the outer mitochondrial membrane was stable during mammary gland development. By analogy, it is possible that the outer mitochondrial membrane does not change during loss of differentiated functions in culture. This stability is not seen in the inner membrane and matrix components. Cell culture results in a 6-fold reduction in the specific activities of cytochrome oxidase, succinate dehydrogenase and a-glycerophosphate oxidase and a 2-fold reduction in matrix malate dehydrogenase. The metabolic implications of the lowered specific activities of these enzymes is a severe limitation on the utilization of mitochondrial oxidative pathways with electron transport impaired a t succinate dehydrogenase and cytochrome oxidase. The reduction in a-glycerophosphate oxidase and malate dehydrogenase limits two primary shuttle mechanisms whereby NADH produced in glycolysis can be transferred into and oxidized by mitochondrial electron transport. These cells are dependent upon glycolysis for energy production and lactate accumulation to regenerate NAD' . In addition to enzymatic changes, quantiative and qualitative changes were seen in various protein components of the cultured cell mitochondrion. These alterations occurred gradually over several days and were not the result of mitochondrial disintegration but were due to modulation in the synthesis of mitochondrial proteins during cell culture. Assigning a structural or enzymatic function to the various mitochondria1 polypeptides identified by gel electrophoresis would make it possible to determine which mitochondrial functions were altered during cell culture. Epithelial cells from developing mouse mammary glands respond to monolayer culture by a loss of differentiated morphology. They no longer respond to the hormones which elicit differentiation and secretion in vivo. Recently, Emerman and Pitelka ('76) reported that mouse mammary epithelial cells cultured on floating collagen gels respond to exogenous hormones and differentiate into secretory cells. I t would be of interest to examine the mitochondria of cells grown in this way to see if the changes we have observed could be
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MICHAEL T. WHITE AND S. NAND1
reversed. The ability to manipulate cellular and mitochondria1 differentiation in cell culture would facilitate the understanding of the fundamental mechanisms of cellular differentiation in vivo. ACKNOWLEDGMENTS
We thank Mr. John Underhill for photographic assistance. This investigation was supported by Grant CA 05388, awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California. LITERATURE CITED Bissell, M. J., C. Hatie and H. Rubin 1972 Patterns of glucose metabolism in normal and virus-transformed chick cells in tissue culture. J . Nat. Cancer Inst., 49: 555-565. Dickson, C., and J . J. Skehel 1974 The polypeptide eomposition of mouse mammary tumor virus. Virology, 58: 387-395. Emerman, J. T., and D. R. Pitelka 1976 Maintenance and induction of morphologic differentiation in mammary epithelial cells on floating collagen membranes. J. Cell Biol., 70: 211a (Abstract). Green, D. E.,and D. M. Ziegler 1963 Electron transport particles. In: Methods in Enzymology. Vol. 6.S. P. Colowick and N. 0. Koplan, eds. Academic Press, New York, pp. 416-424. Gregg, C. T. 1972 Some Aspects of the Energy Metabolism of Mammalian Cells. In: Growth, Nutrition and Metabolism of Cells in Culture. Vol. 1. G. H. Rothblat and V. J . Cristofalo, eds. Academic Press, New York, pp. 83-136. Gregg, C. T., J. M. Machinist and W. D. Currie 1968 Glycolytic-respiratory properties of intact mammalian cellsinhibitor studies. Arch. Biochem. Biophys., 127: 101-111. Jones, D. H., and T. G. Rosano 1972 Studies of mitochondrial development prior to lactogenesis in the mouse mammary gland. Arch. Biochem. Biophys., 153: 130-138. Lowry, D. H., N. J. Rosebrough and A. L. Farr 1951 Protein measurement with the protein phenol reagent. J. Biol. Chem., 193: 265-275. Maizel, J. V., D. 0. White and M. D. Scharff 1968 The polypeptides of adenovirus. I. Evidence for multiple protein
components in the virion and a comparison to types 2,7a and 7.Virology, 36: 115-12.5. OBrien, S. J., and R. C. Gethmann 1973 Segmental aneuploidy as a probe for structural genes in drosophila: mitochondrial membrane enzymes. Genetics, 75: 155-167. Ochoa, S. 1955 Malic dehydrogenase from pig heart. In: Methods in Enzymology. Vol. 1. S.P. Colowick and N. 0. Kaplan, eds. Academic Press, New York. pp. 735-737. Paul, J. 1965 Carbohydrate and energy metabolism. In: Cells and Tissues in Culture. Vol. 1. E. N. Willmer, ed. Academic Press, New York, pp. 239-276. Pickett, P. B., D. R. Pitelka, S. T. Hamamoto and D. S. Misfeldt 1975 Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells. J. Cell Biol., 66: 316-332. Rosano, T. G., and D. H. Jones 1976 Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. I. Behavior on isopycnic gradient centrifugation. J. Cell Biol., 69: 573-580. Rosano, T. G., S.K. Lee and D. H. Jones 1976 Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. 11. Membrane marker enzymes and membrane ultrastructure. J. Cell Biol., 69: 581-588. Schnaitman, C., and J. W. Greenawalt 1968 Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J . Cell Biol., 38: 158-174. Smith, L. 1955 Spectrophotometric assay of cytochrome C oxidase. Methods Biochem. Anal., 2: 427-434. Smith, R. E., H. J. Zweerink and W. K. Joklik 1969 Polypeptide components of virions, top component and cores of reovirus type 3.Virology, 39: 791-810. Wang, T., C. Marquardt and J. Foker 1976 Aerobic glyculysis during lymphocyte proliferation. Nature, 261: 702-705. White, M. T., A. S. L. Hu, S. T. Hamamoto and S. Nandi 1977 In uitro analysis of proliferating epithelial cell populations from the mouse mammary gland: fibroblast-free growth and serial passage. In Vitro, in press. White, M. T., and S.Nandi 1976 Biochemical studies on mitochondria isolated from normal and neoplastic tissues of the mouse mammary gland. J. Natl. Cancer Inst., 56: 65-73. (1977, submitted) Metabolic and biochemical properties of normal and neoplastic mouse mammary epithelial cells in primary monolayer culture. Cancer Res. Wurtman, R. J., and 3. A. Axelrod 1963 Sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmacol., 12: 1439-1441.
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ABSTRACT Various biochemical properties of mitochondria isolated from primary monolayer cultures of mammary epithelial cells from mid-pregnant or hormonally stimulated mice were examined daily for seven or eight days. When compared with mitochondria from mammary glands of mid-pregnant animals, the specific activities of several mitochondrial enzymes were greatly reduced in cells after seven days in culture. There was a 5- to 6-fold reduction in the specific activities of cytochrome oxidase, succinate dehydrogenase and a glycerophosphate oxidase while malate dehydrogenase and adenylate kinase activities were 2- to 3-fold lower. The reduction in mitochondria1 enzyme activities was gradual and related to the length of time the cells were in culture. Progressive changes were also seen in the electrophoresis profiles of mitochondrial proteins in SDSurea polyacrylamide gels. Mitochondria isolated from 1-,2-, 3- and 8-day cell cultures showed a continuous reduction in the relative amounts of several mitochondrial polypeptides in the gel profiles. Addition of 35S-methionineto cell cultures for short and long periods indicated that mitochondrial protein synthesis continued throughout the 8-day culture period. However, the synthesis of several particular polypeptides was reduced progressively during the culture period. These studies indicate that mouse mammary epithelial cells have a lowered capacity for respiratory metabolism as a result of specific mitochondrial alterations which might be associated with the general loss of differentiated morphology by those cells during monolayer culture. Normal and neoplastic mammalian cells in culture commonly show high rates of aerobic glycolysis as measured by lactate production (Gregg e t al., '68; Gregg, '72). Undoubtedly, many factors, such as medium composition, culture pH, growth rate and cell density influence the level of aerobic lactate production. In cultures of normal mouse lymphocytes, Wang et al. ('76) directly linked glycolysis with cell proliferation. Glycolysis and respiration often tend to vary inversely (Paul, '65). Bissell et al. ('72) examined the relationship between lactate and C02 production in sparse, fast growing and dense, slow growing cultures of chick fibroblasts. They concluded that population density and not the growth rate determined the relative amounts of glycolysis and respiration in these cultures. Most studies on glucose metabolism in mammalian cell culture have dealt with fibroblasts, lymphocytes, established cell lines and transformed cells. Comparatively little is known about the metabolJ. CELL.
PHYSIOL., 93: 335-344.
ic properties of epithelial cells in primary culture. In culture, epithelial cells from the mammary glands of mid-late pregnant mice rapidly lose many of the cytological features which characterize the developed glands in situ (Pickett et al., '75). These cells also showed high levels of lactate production which were seemingly independent of cell density and proliferation (White and Nandi, '77). In this paper, we examined the respiratory ability of mouse mammary epithelial cells as a function of time and loss of differentiated morphology during culture. We focused on the mitochondria of these cells and analyzed the specific activities of several key respiratory enzymes, the total protein composition and the patterns of new protein synthesis in mitochondria of Received May 19, '77. Accepted July 8, '77. ' Present address: Stanford University School of Medicine, Stanford University Service (Medicine ill), V. A. Hospital, Palo Alto, California 94304.
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MICHAEL T. WHITE AND S. NAND1
twice with Hank’s BSS, scraped from the dish with a teflon spatula and centrifuged a t 180 x g for five minutes. The cell pellet was resuspended in a small volume (less than 5 ml) of cold sucrose medium (SM) (0.25 M sucrose, 2 mM EDTA and 50 mM Tris-HC1, pH 7.4) containing 1%(w/v) fraction V bovine serum albumin (BSA) (Pentex, Miles Labs., Elkhart, MATERIALS AND METHODS Indiana). The cells were disrupted in a 7-ml Cell culture glass dounce (100 strokes, pestle A Bellco Mammary gland tissue was excised asep- Glass, Vineland, New Jersey). The homogetically from midpregnant BALB/cCrgl mice or nate volume was adjusted to 25 ml with BSAanimals hormonally stimulated for five weeks SM and centrifuged a t 500 X g for ten minutes with subcutaneously implanted pellets of 17p at 5°C. The supernatant was recentrifuged a t estradiol, deoxycorticosterone actetate as de- 500 x g for ten minutes and 8,700 x g for fifscribed previously (White et al., ’77). Minced teen minutes to obtain a crude mitochondrial tissue was suspended in 0.1% collagenase pellet. This fraction was resuspended in 25 ml (Type 111, 145 units/mg, Worthington Bio- BSA-SM and centrifuged a t 8,700 x g for 15 chemical, Freehold, New Jersey) in Hank’s minutes. The partially purified mitochondrial balanced salt solution (BSS) (Gibco, Santa pellet was resuspended in a small volume of Clara, California) and rotated a t moderate BSA-SM, layered onto a 15-ml linear sucrose speed in a shaking 37°C water bath for 90 gradient (1.03-1.71M sucrose in 2 mM EDTA, minutes. After centrifugation at 180 x g for 25 mM Tris- HC1, pH 7.41, and centrifuged to five minutes, the supernatant was discarded equilibrium (2 hours a t 25,000 rpm in a Beckand the cell pellet was resuspended in 0.05% man SW 27.1 rotor) a t 5°C. The mitochondrial pronase (B grade, Calbiochem, La Jolla, Cali- band was removed, resuspended in SM a t a fornia) in Hank’s BSS and agitated for 30 final sucrose concentration of 0.33 M and cenminutes a t 37°C. The suspension was cen- trifuged a t 10,000 x g for 20 minutes a t 5°C. trifuged a t 180 x g for five minutes, the cell The purified mitochondrial pellet was resuspellet was resuspended in Dulbecco’s modified pended in a small volume of SM and assayed Eagle’s medium (DMEM) (Gibco) containing for enzymatic activity or treated for analysis 10% fetal calf serum (Pacific Biologicals, of polypeptides by gel electrophoresis. Richmond, California) and filtered through Enzyme assays single, then through double layered 150-p mesh Nytex cloth (Tetko, Elmsford, New We assayed cytochrome oxidase spectrophoYork). The cells were washed twice in DMEM tometrically by following the oxidation of + 10%FCS by centrifugation a t 100 Xg for reduced cytochrome C (equine heart Type VI, five minutes to remove contaminating fibro- Sigma Chemical Co., St. Louis, Missouri) as blasts. The washed cell pellet was resuspended described by Smith (‘55). Malate dehydrogein DMEM, 10% FCS, insulin (I) (10 kg/ml), nase was assayed by the method of Ochoa Penicillin-steptomycin sulphate (PSI (100 (‘55), adenylate kinase was assayed according unitdm1 and 100 pg/ml respectively) and to Schnaitman and Greenawalt (‘68) and plated into tissue culture dishes (Corning, NADH oxidase was assayed spectrophotometCorning, New York). The cultures were main- rically observing the oxidation of NADH using tained a t 37”C, pH 7.2 in a humidified 95%air, cytochrome C as terminal electron acceptor 5%COz environment. (Green and Ziegler, ’63). The test for monoamine oxidase followed the method of Mitochondria preparation Wurtman and Axelrod (‘63) with 14C-trypMammary gland tissues - mitochondria tamine (47 mCi/mmole, New England Nuclewere prepared from mammary glands of ar, Boston, Massachusetts) as substrate. We midpregnant or 5-week hormone stimulated assayed a glycerophosphate oxidase (the mitomice as described previously (White and chondrial form of a glycerophosphate dehyNandi, ’76). drogenase) and succinate dehydrogenase as Mammary gland cells- following dissocia- described by O’Brien and Gethmann (‘731, tion cells were cultured for zero to eight days which spectrophotometrically follows the reas described above. The cells were washed duction of p-iodonitrotetrazolium violet. All
cells after various periods in culture. These studies indicate that mouse mammary epithelial cells have a lowered capacity for respiratory metabolism as a result of specific mitochondrial alterations which might be associated with the general loss of differentiated functions by these cells during monolayer culture.
ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS
enzymes were assayed for maximum activity which included whenever noted mitochondrial disruption by treatment with the nonionic detergent Lubrol WX (0.2 mglmg rnitochondrial protein, ICI America, Charlotte, North Carolina) before enzyme assay. This was of particular importance when the substrates of certain assays (ex. NADH in NADH oxidase) were not able to penetrate intact mitochondria. Protein was determined by the method of Lowry et al. (‘51) with BSA as a standard.
337
periods were as follows: the first 18 hours after plating; from the second to the third day of culture and from the seventh to the eighth day of culture. In every case, the cells were harvested 18 to 24 hours after the 35S-methionine was added to the medium. Prior to harvest, the cells were washed with Hank’s BSS. Mitochondria were isolated and purified on sucrose gradients and mitochondrial polypeptides analyzed by electrophoresis on slab gels. Autoradiograms of the stained gels were made using Kodak Blue X-ray film (Eastman Kodak, Rochester, New York).
Polyacrylamide gel electrophoresis RESULTS The dissociation and electrophoresis of mitochondrial proteins were based on the methInitial experiments to determine organelle od described by Maize1 et al. (‘68).Samples of density, intactness and contamination by sucrose gradient purified mitochondria (-- 50 other cell components involved localization of Fg protein each) were dissolved in urea, SDS the mitochondrial band in a linear sucrose and /3 mercaptoethanol (5 M,1.0%and 0.2%re- gradient after centrifugation to equilibrium. spectively) by heating a t 95°C for five minutes The gradient profile of mitochondria isolated and layered onto gels after cooling. The gels, from mammary gland cells grown in culture set in either plexiglass tubes (6mm I.D.) or be- for seven days is shown in figure 1.The activtween glass plates as slabs 0.75 mm thick were ities of inner mitochondrial membrane cytocomposed of 10% acrylamide and 0.27% chrome oxidase, outer mitochondrial membisacrylamide (BioRad Labs., Richmond, Cali- brane monoamine oxidase, total protein and fornia) in 4 M urea, 0.01 M EDTA, 0.1% SDS, density along the gradient are shown. Both 0.1% N,N,N’N‘-tetramethylethylenediamine mitochondrial enzymes were found in a uniand 0.05 M Na phosphate buffer (pH 7.2). Po- form band with a mean density of 1.183 glml lymerization was catalyzed with 0.5% ammo- a t 0°C. The major protein band was coincident nium persulphate. Tube gels were electropho- with both bands of enzyme activity which resed a t 5 milliampere (MA) per gel and slab indicated that the mitochondria were relagels a t 12 MAlgel for 22 hours a t room temper- tively homogeneous in size and were not disature in 0.05 M Na phosphate buffer (pH 7.2) rupted during isolation. More than 80%of the containing 0.1% SDS and 0.01 M EDTA. The cytochrome oxidase and 75% of the monogels were fixed, stained with Coomassie blue amine oxidase activities applied to the graand destained by the method of Smith et dient were recovered in fractions 6 to 9, well al. (‘69).The gels were photographed and away from other contaminating protein in the the negatives scanned in a densitometer. Poly- gradient. No lysozyme activity was detected peptide molecular weights were determined in the mitochondrial fractions. Sucrose graby coelectrophoretic standardization using dient purification resulted in a 2- to %fold murine mammary tumor virus (MuMTV) increase in mitochondrial enzyme specific acpolypeptides (Dickson and Skehel, ’74). In all tivity compared to the organelles obtained by cases where mitochondrial polypeptide gel differential centrifugation. Mitochondria isoprofiles were compared, the samples were lated from the mammary glands of midpregcoelectrophoresed in the same chamber and nant or 5-week hormone stimulated animals approximately equal amount of protein was had similar characteristics on a sucrose graloaded into each gel. dient forming a uniform band a t a density of 1.182glml a t 0°C. By these criteria, mitochonMitochondrial protein synthesis dria isolated from mammary glands or 7-day Radioactively labelled mitochondrial pro- cultures of mammary gland cells were teins were obtained by growing mammary indistinguishable. The specific activities of seven enzymes gland cells in culture for various periods in nutrient medium (DMEM + 10%FCS I + PS) chosen for their diverse mitochondrial locacontaining 1 FCiIml 35Smethionine (250 Cil tion and their key roles in oxidative metabommole, New England Nuclear). The labelling lism were compared in gradient purified mito-
+
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MICHAEL T. WHITE AND S. NAND1
400
300 c
0 u
-
500
E
\ 0
a
-
.-
C
200
400
a,
t
2 a
300
I00
200
I00
Bottom
Fraction number
Fig. 1 Sucrose gradient profile of mitochondria isolated from mouse mammary epithelial cells after seven days in monolayer culture. The gradient and mitochondria were preparedand the fractions were assayed for protein, refractive index, cytochrome oxidase and monoamine oxidase activities as described in MATERIALS AND METHODS.
chondria isolated from the mammary glands of midpregnant (or 5-week hormonally stimulated) animals and their mammary cells after seven days in culture. These results are summarized in table 1. There was no difference in monoamine oxidase activities and little difference in NADH oxidase activities but adenylate kinase and malate dehydrogenase activities were 2-fold lower in cultured cell mitochondria. Most striking was the nearly 6fold reduction in specific activities of aglycerophosphate oxidase, cytochrome oxidase and succinate dehydrogenase in mitochondria from mammary cells after seven days in culture.
The comparison of mitochondrial properties was extended to structural and functional proteins analyzed by electrophoresis on SDS-urea polyacrylamide gels. Figure 2 shows the electrophoresis profiles of gradient purified mitochondria from the mammary glands of hormonally stimulated (5 weeks) animals and cells from these glands after seven days in culture. Significant differences in the relative amounts of many mitochondrial polypeptides were evident. Polypeptide 2 (84,000 daltons) was highly reduced in the mammary cell mitochondria as were the relative proportions of polypeptides 3:4, 5:6:7, 8:9:10 and 11:12. These changes in proportion indicated a de-
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ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS TABLE 1
Enzymatic actiuities of sucrose gradient-purifed mitochondria isolated in media containing 1 % BSA *Specific activity (nmoles product formediminlmg protein) ~
Mammary gland Mammary gland
Mammary gland cells
Mammary gland cells
** Cytochrorne oxidase
1365293 (7)
234226 (7)
5.83
Monoarnine oxidase (tryptamine-14C) a-glycerophosphate oxidase ** Succinate dehydrogenase Malate dehydrogenase ** NADH oxidase ** Adenylate kinase
310227 (6) 14.12.4 (4) 7.41t.15 (4) 1640k 161 (6) 180217 (4) 3481t31 (5)
247k20 (7) 2.54k0.40 (7) 1.3120.24 (7) 6302 134 (7) 269231 (7) 162k9 (7)
1.26 5.56 5.65 2.60 0.67 2.15
*
*Mean SE; numbers in parentheses = number of individual experiments. **Pretreated with Lubml WX.
I I
234 I l l
21
20 I
19
5 6 7 8 910 I I 12 13 1 4 15 1617 18 I I I / I 1 1 1 I l l 1 1 1 1
I
I
GLAND
CULTURED MAMMARY GLAND CELLS
r
1 1 1
1 1 1 1
87 84 75 6964
I
56
I I
I
I
I
50 47 43
I
I I I
36 31.5
I
I
23
I
20.5
I
18
MOLECULAR WEIGHT x lo3 Fig. 2 Densitometer scans of photographs of Coomassie blue stained mitochondria1 polypeptides electrophoresed in tube gels. Mitochondria were isolated from hormonally stimulated mouse mammary glands or seven day monolayer cultures of these mammary gland cells. Mitochondria1 isolation, purification and electrophoresis were as described in MATERIALS AND METHODS. Fifty micrograms “Lowry” protein/aarnple were coelectrophoresed.
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MICHAEL T. WHITE AND S. NAND1
crease in the amount of several of these polypeptides in the mitochondria from mammary gland cells in culture. After seven days in culture mammary epithelial cells have mitochondria with highly diminished activities of two key electron transport enzymes, several other enzymes and reduced amounts of several undefined structurallfunctional proteins. I t is possible that these differences were the result of the method of gland dissociation andlor a selection of a specific cell type(s) during cell culture. To examine this possibility, mitochondria were isolated from mammary glands after the following treatment: undissociated glands; cells dissociated from whole glands plated and cultured for 30 minutes, l day, 2 days, 3 days and 7 days. Table 2 summarizes the enzyme activities determined in these experiments. There was no significant difference in the specific activities of monoamine oxidase, cytochrome oxidase, a glycerophosphate oxidase and succinate dehydrogenase in mitochondria isolated from undigested glands and cells from enzymatically dissociated glands plated for 30 minutes. After one day in culture monoamine oxidase activity was unchanged while cytochrome oxidase activity dropped 3-fold, and aglycerophosphate oxidase and succinate dehydrogenase were reduced nearly 2-fold. The same level of reduced activity is maintained in cells cultured for two days. By the third day, cytochrome oxidase activity was 4-fold lower while a-glycerophosphate oxidase and succinate dehydrogenase were greater than 2-fold lower. By day 7, the activity of these three enzymes were 6 fold lower while monoamine oxidase activity remained unchanged. The reduction in mitochondrial enzyme activities was gradual and related to the length of time the cells were in culture, not the method of gland
dissociation. The procedure of gland dissociation results in an almost pure (>95%)population of parenchymal cells. These cells have mitochondrial properties identical to those organelles isolated from intact glands but after just 24 hours in culture, the mitochondrial properties of these cells have changed. The gel electrophoresis profiles of mitochondrial proteins also revealed progressive changes related to the length of time these cells were cultured (fig. 3). Polypeptide 2 was present in mitochondria from cells cultured for one day but was nearly absent in cells cultured two to eight days (fig. 3). The change in the relative proportions of polypeptides 5:6:7 occurred after two days in culture as did the changes in proportion of polypeptides 8:9:10 (fig. 3). There was a gradual reduction in the amount of polypeptides 21 and 22 beginning on day 2 and continuing through day 8 (fig. 3). The change in the relative amounts of polypeptides 11:12 seen in comparisons of mammary gland and 7-day cultures of mammary gland cells (fig. 2) was evident after 24 hours of culture but became more prominent on succeeding days (fig. 3). The changes in enzyme activities and protein components of mitochondria during cell culture were not the result of mitochondrial disaggregation. The ultrastructure of mitochondria seen in thin section electron micrographs of cells after various periods of culture appeared normal and mitochondrial dissaggregation was not evident (White et al., '77; and unpublished observation). Mammary epithelial cells continued to synthesize new mitochondria and mitochondrial proteins during monolayer culture as evidenced by labelling studies and analysis of mitochondrial protein synthesis by autoradiography of SDS-urea polyacrylamide gels.
TABLE 2 Enzyme activity in mitochondria purifkd on sucrose gradients *Ratio specific activity:
Monoamine oxidase Cytochrome oxidase a-glycerophosphateoxidase Succinate dehydrogenase
Mammary Mammary gland cells Days in cuiture
Dissociated
1
2
3
I
0.84 1.21 1.02 0.97
1.20 2.98 1.79 1.71
1.25 2.90 2.31 1.92
1.39 4.42 2.51 1.91
1.16 5.87 5.64 5.79
* The average of three separate experiments;mammary glands were from mice hormonally stimulated for five weeks. In each case, mammary gland and mammary gland cell mitochondria were compared, using mitochondria isolated, purified and assayedon the same day
341
ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS
I2 34 5 6 7 I I I I l l l I
8 9 10 I/ 12 13 14 15 1 6 17 I l l 1 I I l l 1 I
18 I
19 I
20 I
21 22 I I
23 I
DAY 8
l
l
l
l
l
l
l
I
1
1
1
87 79 69 61 56 5250
I
l
l
1
1
45 43 36
I
31.5
I
23
I
20.5
1
1
18 15.5
1
12.5
MOLECULAR WEIGHT x lo3 Fig. 3 Densitometer scans of photographs of Coomassie blue stained mitochondrial polypeptides electrophoresed on a slab gel. Mitochondria were isolated from cells derived from hormonally atimulated (5weeks) mammary glands. Cells were cultured for the various times indicated. Mitochondria1isolation, purification and electrophoresis were as described in MATERIALS AND METHODS. Twenty-five micrograms “Lowry” protein/samples were coelectrophoresed.
Figure 4 shows densitometer scans of autoradiographs of mitochondrial proteins from cells labelled with 35Smethionine for various periods during culture. All of the major mitochondrial polypeptides were synthesized during the first 18 hours of culture. Cells labelled from the second to the third and the seventh to the eighth day of culture also synthesized most of the mitochondrial polypeptides (fig. 4).The pattern of newly synthesized proteins mimicked the gradual changes in relative proportion of the various polypeptides seen in
figure 3. Most striking was the change in synthesis of polypeptide 8 (56,000 daltons). This polypeptide was made in significant amounts during the first 18 hours of culture, but synthesis was reduced from days 2 to 3 and very highly reduced from days 7 to 8 (fig. 4).This corresponded to the nearly complete absence of this polypeptide after day 1 as seen in stained gel profiles (fig. 3). I t is significant to note that mitochondrial protein synthesis continued in mammary cells in culture and the gradual reduction in cer-
342
MICHAEL T. WHITE AND S. NAND1 I 2 3 4 5 6 7 891011 12 1 1 1 1 1 1 1 1 1 1 1 1
13 I
14
15
I
I
16 I
17 I
18 I
19 I
7th
20 I
- 8th
DAY
Fig. 4 Densitometer scans of autoradiographs of “S-methionine labelled mitochondria1 polypeptides electrophoresed on a slab gel. Cells from mammary glands of mice hormonally stimulated for five weeks were cultured with Y3-methionine in the medium for the periods indicated. Mitochondria1 isolation, purification, electrophoresis and autoradiography were as described in MATERIALS AND METHODS. Twenty-five micrograms “Lowry” protein/samples were coelectrophoresed and the autoradiograms exposed for five days at room temperature. Cells were labelledwith 3sS-methioninefor either the first 18hours after plating; or the second to the third day; or the seventh to eighth day of culture.
tain mitochondrial proteins appeared to result from the decreased net synthesis of these components. DISCUSSION
Following enzymatic dissociation of the mouse mammary gland, parenchymal cells can be separated from fat cells, myoepithelial cells and the majority of fibroblasts by differential centrifugation. When plated at me-
dium to high densities, the parenchymal cells settle, attach, proliferate and form a continuous sheet with a polygonal morphology within 24 to 48 hours. The strict criteria for the epithelial nature of these cells are the nonrandom attachment of the cells such that the basolateral surface attaches to the substratum and the apical surface faces up with numerous microvilli projecting into the me-
ALTERED MITOCHONDRIA IN MAMMARY EPITHELIAL CELLS
dium and the occluding tight junctions that connect adjacent cells (Pickett et al., '75). If cells are derived from mid-late pregnant mammary glands, accommodation to the crowded conditions of cell culture appears to reduce the ductal and lobulo/alveolar cells to roughly similar states of cytoplasmic development so that internal distinguishing features tend to disappear. Cells in these primary cultures never approach the differentiated state of secretory mammary epithelium (Pickett et al., '75). Ultrastructural evidence suggests that loss of differentiated functions in mammary epithelial cells begins when cells are seeded onto plastic or glass substrates in tissue culture (Emerman and Pitelka, '76). Our results suggest that the mitochondria of mouse mammary epithelial cells undergo structural and functional changes in response to cell culture. I t is tempting to speculate that these mitochondrial alterations reflect a loss of differentiated function in these organelles concomitant with the general loss of developmental morphology of these mammary cells. There is compelling evidence for mitochondrial differentiation in the mouse mammary gland during pregnancy and lactation in vivo. Total mitochondrial protein and succinate oxidase and cytochrome oxidase activities increased gradually during pregnancy with a 2to %fold increase occurring during early lactation (Jones and Rosano, '72). These changes were accompanied by a corresponding increase in the buoyant density of isolated mitochondria due to an expansion of inner membrane and matrix components (Rosano and Jones, '76). This expansion was indicated by a 3- to 4fold increase in cytochrome oxidase activity and was confirmed in electron micrographic studies (Rosanoet al., '76). The major phase of mitochondrial development occurs shortly after parturition during a period subsequent to epithelial cell proliferation in the gland (Jones and Rosano, '72, '76). These studies indicate that mitochondrial differentiation in the developing mouse mammary gland is geared to increased oxidative capacity of these cells during milk production and secretion. Our results indicate that mitochondria show a markedly decreased oxidative capacity in mammary epithelial cells which have lost differentiated features as a result of cell culture. The specific activity of outer mitochondrial membrane monoamine oxidase did not change in mammary gland cells during culture. This is consistent with the observations
343
of Rosano and Jones ("76) who found that monoamine oxidase activity changed very little compared to cytochrome oxidase and other enzymes in mitochondria isolated from the mammary glands of virgin, pregnant or lactating mice. Their interpretation was that the outer mitochondrial membrane was stable during mammary gland development. By analogy, it is possible that the outer mitochondrial membrane does not change during loss of differentiated functions in culture. This stability is not seen in the inner membrane and matrix components. Cell culture results in a 6-fold reduction in the specific activities of cytochrome oxidase, succinate dehydrogenase and a-glycerophosphate oxidase and a 2-fold reduction in matrix malate dehydrogenase. The metabolic implications of the lowered specific activities of these enzymes is a severe limitation on the utilization of mitochondrial oxidative pathways with electron transport impaired a t succinate dehydrogenase and cytochrome oxidase. The reduction in a-glycerophosphate oxidase and malate dehydrogenase limits two primary shuttle mechanisms whereby NADH produced in glycolysis can be transferred into and oxidized by mitochondrial electron transport. These cells are dependent upon glycolysis for energy production and lactate accumulation to regenerate NAD' . In addition to enzymatic changes, quantiative and qualitative changes were seen in various protein components of the cultured cell mitochondrion. These alterations occurred gradually over several days and were not the result of mitochondrial disintegration but were due to modulation in the synthesis of mitochondrial proteins during cell culture. Assigning a structural or enzymatic function to the various mitochondria1 polypeptides identified by gel electrophoresis would make it possible to determine which mitochondrial functions were altered during cell culture. Epithelial cells from developing mouse mammary glands respond to monolayer culture by a loss of differentiated morphology. They no longer respond to the hormones which elicit differentiation and secretion in vivo. Recently, Emerman and Pitelka ('76) reported that mouse mammary epithelial cells cultured on floating collagen gels respond to exogenous hormones and differentiate into secretory cells. I t would be of interest to examine the mitochondria of cells grown in this way to see if the changes we have observed could be
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MICHAEL T. WHITE AND S. NAND1
reversed. The ability to manipulate cellular and mitochondria1 differentiation in cell culture would facilitate the understanding of the fundamental mechanisms of cellular differentiation in vivo. ACKNOWLEDGMENTS
We thank Mr. John Underhill for photographic assistance. This investigation was supported by Grant CA 05388, awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California. LITERATURE CITED Bissell, M. J., C. Hatie and H. Rubin 1972 Patterns of glucose metabolism in normal and virus-transformed chick cells in tissue culture. J . Nat. Cancer Inst., 49: 555-565. Dickson, C., and J . J. Skehel 1974 The polypeptide eomposition of mouse mammary tumor virus. Virology, 58: 387-395. Emerman, J. T., and D. R. Pitelka 1976 Maintenance and induction of morphologic differentiation in mammary epithelial cells on floating collagen membranes. J. Cell Biol., 70: 211a (Abstract). Green, D. E.,and D. M. Ziegler 1963 Electron transport particles. In: Methods in Enzymology. Vol. 6.S. P. Colowick and N. 0. Koplan, eds. Academic Press, New York, pp. 416-424. Gregg, C. T. 1972 Some Aspects of the Energy Metabolism of Mammalian Cells. In: Growth, Nutrition and Metabolism of Cells in Culture. Vol. 1. G. H. Rothblat and V. J . Cristofalo, eds. Academic Press, New York, pp. 83-136. Gregg, C. T., J. M. Machinist and W. D. Currie 1968 Glycolytic-respiratory properties of intact mammalian cellsinhibitor studies. Arch. Biochem. Biophys., 127: 101-111. Jones, D. H., and T. G. Rosano 1972 Studies of mitochondrial development prior to lactogenesis in the mouse mammary gland. Arch. Biochem. Biophys., 153: 130-138. Lowry, D. H., N. J. Rosebrough and A. L. Farr 1951 Protein measurement with the protein phenol reagent. J. Biol. Chem., 193: 265-275. Maizel, J. V., D. 0. White and M. D. Scharff 1968 The polypeptides of adenovirus. I. Evidence for multiple protein
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