Multiple molecular forms of glucose-6-phosphate dehydrogenase in normal, preneoplastic, and neoplastic mammary tissues of mice.

Multiple molecular forms of glucose-6-phosphate dehydrogenase (G6PD) in normal, preneoplastic, and neoplastic mammary tissues were separated by polyacrylamide gel electrophoresis and identified by specific straining for enzyme activity. Mammary tissue from lactating BALB/c mice showed considerable amounts (up to 50%) of a slower-migrating G6PD species, G6PD-III, which was essentially absent from glands of pregnant mice, preneoplastic nodules, and mammary carcinomas. All tissues possessed a faster-migrating species, G6PD-II, which accounted for up to 85% of the total G6PD in the glands of pregnant mice. A third species, G6PD-I, migrating more rapidly than G6PD-II, was found in both abnormal tissues (preneoplastic and neoplastic) and accounted for up to 35% of the total enzymatic activity. G6PD-I was present in moderate amounts (less than 15%) in glands from pregnant mice and was essentially absent from the lactating gland (approximately 5%). The addition of dithiothreitol did not alter the measurable G6PD activity but did increase the relative activity of G6PD-II or G6PD-I, as judged by the intensity of the bands on the gels. Mild oxidation (stirring overnight at 4 degrees in air) resulted in a loss of G6PD activity, but preparations had greater amounts of G6PD-III; presence of dithiothreitol during aeration partially prevented loss of G6PD activity and largely prvented the appearance of G6PD-III. Molecular-weight estimations with preparations from lactating mice yielded a value of 118,000 for G6PD-II and 260,000 for G6PD-III, suggesting a monomer and dimer, respectively. The addition of nicotinamide adenine dinucleotide phosphate stabilized G6PD activity by preventing heat inactivation at 47 degrees; nicotinamide adenine dinucleotide phosphate did not alter the pattern of species present. The data from heat inactivation studies suggest that G6PD-III (dimer) was the more stable species. The addition of nicotinamide adenine dinucleotide phosphate to samples after oxidation in the absence of dithiothreitol (about 70% loss of activity) resulted in no change in patterns and in recovery of full G6PD activity during heating at 47 degrees. A potential relationship between glutathione reductase activity and the pattern of G6PD species observed in the various tissues is noted.

Identity, release, and binding of mitochondrial-bound hexokinases in mammary glands and adenocarcinomas of lactating mice.

The mitochondrial-bound hexokinases (adenosine triphosphate:D-hexose 6-phosphotransferase) of mammary adenocarcinoma and of normal gland were compared in lactating C3H mice. Treatment of mitochondria isolated from both the normal and neoplastic tissue with 0.5 m NaCl or 0.1 mM glucose 0-phosphate effected the release of about 50% of the bound hexokinase. In the presence of magnesium ion, enzyme from either source attached to mitochondria from either tissue and in all combinations to the same extent. Identification of the isoenzyme complement in the mitochondrial extract by diethylaminoethylcellulose chromatography revealed only types I and II. In the tumor, the hexokinase activity in both the cytosol and the fraction solubilized from mitochondria was predominantly in the form of type I ( 60%). In contrast, the activity released from mitochondria isolated from normal gland was predominately type II, while the cytosol contained almost equivalent amounts of types I and II. While this difference does not explain differences in glucose utilization between the normal and neoplastic tissue, it may provide a means of distinguishing between the two.

In vitro transformation of mouse mammary epithelial cells grown serum-free inside collagen gels.

Mammary epithelial cells from 4-month-old virgin BALB/c mice were cultured inside collagen gels in the following serum-free media: Dulbecco's modified Eagle's medium:Hams's F-12 (1:1) supplemented with: insulin (10 micrograms/ml), bovine serum albumin (5 mg/ml), and epidermal growth factor (5 ng/ml); insulin, bovine serum albumin, progesterone (0.05 microgram/ml), and prolactin (1 microgram/ml); insulin, bovine serum albumin, progesterone, prolactin, and linoleic acid (10 micrograms/ml). Cells proliferated in all these media. The cells were treated with 0.01 micrograms/ml of 7,12-dimethylbenz(a)anthracene or 100 micrograms/ml of N-nitroso-N-methylurea on day 3 of culture and, subsequently, at 1-week intervals for 3-6 weeks. Tetradecanoylphorbol acetate (0.1 micrograms/ml) was added to selected cultures. The cultures were maintained for up to 9 weeks; the cells were then removed from the collagen gels, placed in monolayer culture for 2 days, and removed from monolayer culture, and 5 X 10(5) cells were transplanted to each of the gland-free mammary fat pads of 3-week-old female mice. Approximately 10 weeks after transplantation, the transplanted mammary fat pads were examined for outgrowths. Cells that were not treated with carcinogen and cultured for up to 9 weeks in different serum-free media and transplanted to the gland-free mammary fat pad produced only ductal outgrowths similar in morphology to the ducts of the virgin host's mammary glands. Six treatments with 7,12-dimethylbenz(a)anthracene, of cells grown in the presence of epidermal growth factor, induced 31% spindle cell tumors, 17% ductal hyperplasias, and 5% lobuloalveolar hyperplasias. Cells that were grown in epidermal growth factor and treated three times with N-nitroso-N-methylurea produced 23% ductal hyperplasias and 17% lobuloalveolar hyperplasias. Cells grown in the presence of progesterone and prolactin and treated three times with 7,12-dimethylbenz(a)anthracene produced up to 23% lobuloalveolar hyperplasias and 12% ductal hyperplasias. Three treatments with N-nitroso-N-methylurea of cells grown in progesterone- and prolactin-containing media produced a maximum of 50% lobuloalveolar hyperplasias and 33% ductal hyperplasias. The lobuloalveolar hyperplasias have the characteristics of the precancerous hyperplastic alveolar nodules found in mouse mammary tumorigenesis. The in vitro carcinogen-induced lobuloalveolar hyperplasias were transplantable, maintained their lobuloalveolar morphology in virgin hosts, and produced carcinomas.

Quantitative HPLC analysis of human plaque proteins in coronary and thoracic aorta arteries.

Quantitative HPLC analysis of saline-soluble proteins obtained from human coronary and thoracic aorta plaque and from whole internal mammary artery were performed. Protein extracts were characterized by anion exchange and reverse-phase HPLC and the integrated chromatographs revealed significant differences in both peak retention times and areas for protein species from coronary artery compared to thoracic aorta artery plaque. Coronary artery plaque proteins possessed a high degree of cationic charge and polarity compared to those present in thoracic aorta plaque and normal mammary artery. This suggests that specific protein markers may be expressed in plaque of different anatomical origin, and that the processing of protein may be distinct to plaque sites. In contrast, characterization of molecular weight by gel electrophoresis resolved no major differences between plaque types. These findings indicate that proteins in human plaque lesions of different anatomical origin can be resolved by HPLC methodology and that they exhibit different charge and polarity. Such an HPLC approach may prove useful in the quantitative identification and ultimate isolation of specific protein markers present in plaque during atherogenesis, and in the study of mechanisms of protein involvement in plaque formation.

Metabolism of 7,12-dimethylbenz[a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels.

Mammary epithelial cells from 3-4-month-old BALB/c virgin mice were cultured inside collagen gels in the following serum-free media: Dulbecco's Modified Eagle's Medium/Ham's F-12 (1:1) supplemented with (A) insulin, bovine serum albumin, epidermal growth factor; (B) insulin, bovine serum albumin, progesterone, prolactin; (C) insulin, bovine serum albumin, progesterone, prolactin, linoleic acid. Cell number increased with all media used. At day 7 of culture, [3H]dimethylbenz[a]anthracene (DMBA) was added to the cultures and its metabolism to water soluble and organic soluble compounds was determined. Mouse mammary epithelial cells were able to metabolize [3H]DMBA to water and organosoluble metabolites. By 72 h, 77-94% of the added DMBA had been metabolized by the epithelial cells in the three media to water and organosoluble metabolites in equivalent amounts. The distribution between water soluble and organosoluble metabolites was approximately equivalent. The high pressure liquid chromatography profiles of organosoluble fractions from the media indicated that the major products appeared to be the phenols, 2-,3-, or 4-hydroxydimethylbenz[a]anthracene, the hydroxymethyl derivatives, 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene, trans-3,4-dihydro-3,4-dihydroxydimethylbenz[a]anthracene and one or two major fractions eluting just behind the marker cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene. The major fraction eluting just ahead of the cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene was most likely trans-8,9-dihydro 8,9-dihydroxydimethylbenz[a]anthracene. The profiles were similar for the cells cultured in all three serum-free media. The results demonstrate that mouse mammary epithelial cells cultured inside collagen gels with serum-free media can metabolize DMBA to putative carcinogenic forms.

Human mammary epithelial cells in culture: differentiation and transformation.

Large quantities of normal and malignant human mammary epithelial tissues are readily available as surgical discard material. We have developed culture conditions that permit long term, active proliferation of these HMEC in a serum-free medium. Thus, large pools HMEC can be stored frozen for repetition of experiments from the same individual's cell population, and for use of the same cell pool by multiple investigators. Of all the specimens that we have thus far examined, we have observed no instances of spontaneous transformation to immortality, nor any karyotypic abnormalities in the cells derived from reduction mammoplasties. However, exposure of normal HMEC to the chemical carcinogen, benzo(a)pyrene did lead to expression of an extended life in culture, and two instances of transformation to immortality. These two established cell lines contain some chromosomal abnormalities, yet retain a relatively stable karyotype upon continued passage in culture. Transformation to malignancy was achieved by exposing these cell lines to tumor viruses and oncogenes. Both the normal HMEC, and the HMEC transformed in vitro, are now being utilized to understand the factors controlling expression of mammary specific properties, response to and production of various growth factors, and the nature of the progressive events leading to malignancy. The maximal usefulness of this, and other human epithelial cell systems, for elucidating the mechanisms of normal and diseased human cellular physiology will require continued efforts to optimize the culture conditions so that they resemble as closely as possible the processes occurring in humans in vivo.

Modulation by ketone bodies of the rate of fatty acid synthesis in mammary gland slices from lactating rats.

The absolute rate of fatty acid synthesis was measured in slices of mammary gland from lactating rats by incubation in [3H]2O-labeled medium containing 10 mM D-3-hydroxybutyrate or acetoacetate alone and paired in combination with 10 mM glucose, lactate, or pyruvate. When compared with our previous studies, the ketone bodies alone supported significant fatty acid synthesis; the rate of synthesis from either ketone body and lactate was higher than that from pyruvate and lactate, and that from pyruvate and glucose; the rate of synthesis from D-3-hydroxybutyrate and lactate was the highest we have observed in the absence of an exogenous substrate for the hexose monophosphate pathway. This study confirms our previous contention that, in rat mammary gland, substrates formed in the mitochondria can be utilized in the cytosol to provide some of the NADPH necessary for fatty acid synthesis.

Factors influencing benzo[a]pyrene metabolism in human mammary epithelial cells in culture.

We have examined the human mammary epithelial cell (HMEC) culture system developed in our laboratory for factors that might influence the metabolism of chemical carcinogens, specifically, the impact of interindividual variation and the effect of different culture conditions. Benzo[a]pyrene metabolism and DNA adduct formation in HMEC from 13 normal reduction mammoplasty specimens, from five nontumorous mastectomy tissues and from eight primary carcinomas were investigated. The interindividual variation in formation of water and organosoluble metabolites was similar in all of the three categories of HMEC. A similar range of variation was found for DNA adduct formation among HMEC from reduction mammoplasty specimens. However, when the individual results of DNA adduct formation in the three categories were examined, HMEC from some specimens from non-tumorous mastectomy tissue and primary carcinomas had significantly increased DNA modification. We also measured the effects of BaP concentration, different culture media, the length of time in culture, the culture density, and the frequency of feeding on the conversion of tritiated BaP to various water and organosoluble metabolites. The BaP metabolite pattern by HMEC was generally stable in the face of these variables. However, suboptimal feeding regimens and lengthy passaging in culture reduced the capacity of the cells to metabolize BaP. The yield of BaP conjugates was reduced 10-fold with lengthy passaging in culture whereas the organosoluble metabolite yield was halved and DNA adduct formation was not affected. Increasing the concentration of BaP decreased the yield of water soluble metabolites relative to that of organosoluble, indicating differences in the capacity of the enzyme systems involved. The HMEC culture system offers several advantages for studies into the biochemical and molecular basis of chemical carcinogenesis in human epithelial cells: the large numbers of cells required can be easily generated; cells at all stages can be frozen for future experiments with no loss in activity; and a high capacity for carcinogen activation is retained during long-term culture.

The absolute rate of fatty acid synthesis by mammary gland slices from lactating rats.

Mammary gland utilizes a combination of substrates in vivo. To determine if and how the substrates presented to the gland alter milk fat synthesis, slices of mammary gland of lactating rats were incubated with combinations of glucose, L-lactate, and pyruvate. Uptakes and conversions of uniformly and specifically labeled substrates to CO2 and fatty acid were measured. The absolute rate of fatty acid synthesis was measured by incoporation of tritium from tritiated water into fatty acid. The extent, but not the type, of fatty acids synthesized was affected by the substrates utilized. Glucose stimulated uptake and conversion to fatty acid of l-lactate and, to a smaller extent, of pyruvate. Analysis of CO2 and fatty acid yields revealed that (a) the major stimulatory effect of glucose was on the conversion of acetyl coenyzme A from other substrates to fatty acid; (b) the rate of fatty acid synthesis paralleled the activity of the hexose monophosphate pathway up to a point. A likely source of the NADPH required beyond this point is provided by oxidation of malate to pyruvate. Reduction of NADH of oxaloacetate, produced during citrate cleavage, would yield malate. Hence, maximal synthesis would require production of NADPH and NADH, a condition met when glucose and L-lactate were substrates. Decreased fatty acids synthesis in pyruvate's presence supports this suggested requirement for cytosolic NADH in rat mammary gland.

Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene.

Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to 1 microgram of benzo[a]pyrene (B[a]P) per ml for two or three 24-hr periods. The B[a]P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heterogeneity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epithelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignantly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties characteristic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Additionally, analysis of the multiple steps occurring in the El cultures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during human epithelial cell carcinogenesis and escape from senescence.

Interrelationship of glycogen metabolism and lactose synthesis in mammary epithelial cells of mice.

Glycogen metabolism in mammary epithelial cells was investigated (i) by studying the conversion of glucose into glycogen and other cellular products in these cells from virgin, pregnant and lactating mice and (ii) by assaying the enzymes directly involved with glycogen metabolism. We find that: (1) mammary epithelial cells synthesized glycogen at rates up to over 60% that of the whole gland; (2) the rate of this synthesis was modulated greatly during the reproductive cycle, reaching a peak in late pregnancy and decreasing rapidly at parturition, when abundant synthesis of lactose was initiated; (3) glycogen synthase and phosphorylase activities reflected this modulation in glycogen metabolism; (4) lactose synthesis reached a plateau during late pregnancy, even though lactose synthase is reported to increase in the mouse mammary gland at this time. We propose that glycogen synthesis restricts lactose synthesis during late pregnancy by competing successfully for the shared UDP-glucose pool. The physiological advantage of glycogen accumulation during late pregnancy is discussed.

Metabolic cooperativity between epithelial cells and adipocytes of mice.

We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from [14C]glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy, We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations.

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol in human mammary epithelial cells: feedback inhibition by 7-hydroxybenzo[a]pyrene.

The metabolism of benzo[a]pyrene (B[a]P) and (-)-transbenzo[a]pyrene-7,8-dihydrodiol (B[a]P-diol) was compared in human mammary epithelial cells (HMEC) grown in serum-free medium, MCDB-170. Conversion of B[a]P-diol to the carcinogen (+)-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide (BPDE), as measured by analysis of their tetraol hydrolysis products, occurred much more efficiently in cultures incubated with [3H]B[a]P-diol than in cultures incubated with [3H]B[a]P. In cultures pretreated with unlabeled B[a]P (24 h, 400 nM), the conversion of [3H]-B[a]P-diol to [3H]tetraols is inhibited 49%, while the conversion of [3H]B[a]P to [3H]B[a]P-diol- is not affected. These observations led to the identification of a major B[a]P-derived metabolite as 7-hydroxybenzo[a]pyrene (B[a]P-7-ol), which was found to be an extremely potent and selective inhibitor of the conversion of B[a]P-diol to BPDE, with a KI estimated at 3-12 nM. Thus B[a]P activation in HMEC appears to be significantly limited by a feedback inhibition pathway induced by B[a]P-7-ol. The potency and selectivity of the B[a]P-7-ol-induced inhibition suggests that the diol to diolepoxide conversion is affected by a selective oxygenase in HMEC, rather than a non-enzymatic, peroxy radical-induced mechanism. B[a]P-7-ol should prove to be a valuable tool in the study of B[a]P carcinogenesis.

Metabolism of benzo[a]pyrene by human mammary epithelial cells: toxicity and DNA adduct formation.

Pure cultures of human breast epithelial cells and of fibroblastic cells in early passage provided the opportunity to ask whether either cell type had the capability for metabolizing chemical carcinogens and, if so, was the fate of the metabolic products compatible with chemical carcinogens being a factor in the initiation of breast cancer in women. For this purpose, cells were exposed to benzo[a]pyrene (BaP), and (i) the influence on growth potential and (ii) the extent, type, and persistence of adducts between the metabolites of BaP and DNA were measured. Compared with fibroblasts, inhibition of growth by epithelial cells was 50-100 times more sensitive to BaP. Because of this differential sensitivity, epithelial cells were exposed to 0.4 microM BaP and fibroblasts were exposed to 4.0 microM BaP in the studies of DNA adduct formation. Separation by high-pressure liquid chromatography of adducts between (+/-)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide) and nucleosides from purified DNA revealed that epithelial cells contained modified DNA within 6 hr after adding BaP. Adducts between the 7R anti stereoisomer of BaP diol epoxide and deoxyguanosine predominated at all times. syn BaP diol epoxide adducts with deoxyguanosine and what appeared to be BaP diol epoxide adducts with deoxycytidine were consistently present but at much lower frequency. All three types of BaP diol epoxide--DNA adducts persisted in epithelial cells for 72 hr in BaP-free medium. No adducts were detected in fibroblastic cultures until 96 hr after first exposure to BaP. At this time, the type and extent of BaP diol epoxide--DNA adduct formation was similar to that in epithelial cells exposed to one-tenth the dose of BaP. The type, extent, rate of formation, and persistence of the adducts in human breast epithelial cells was similar to that in cells transformable by exposure to BaP, an indication that they may be targets for chemically induced carcinogenesis.