Metabolic profiling reveals sphingosine-1-phosphate kinase 2 and lyase as key targets of (phyto-) estrogen action in the breast cancer cell line MCF-7 and not in MCF-12A.

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

Metformin inhibits aromatase expression in human breast adipose stromal cells via stimulation of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is recognized as a master regulator of energy homeostasis. In concert with the AMPK-kinase LKB1, it has been shown to provide a molecular link between obesity and postmenopausal breast cancer via its actions to inhibit aromatase expression, hence estrogen production, within the breast. The anti-diabetic drug metformin is known to increase the activity of AMPK and was therefore hypothesized to inhibit aromatase expression in primary human breast adipose stromal cells. Results demonstrate that metformin significantly decreases the forskolin/phorbol ester (FSK/PMA)-induced expression of aromatase at concentrations of 10 and 50 muM. Consistent with the hypothesized actions of metformin to increase AMPK activity, treatment with 50 muM metformin results in a significant increase in phosphorylation of AMPK at Thr172. Interestingly, metformin also causes a significant increase in LKB1 protein expression and promoter activity, thereby providing for the first time an additional mechanism by which metformin activates AMPK. Furthermore, metformin inhibits the nuclear translocation of CRTC2, a CREB-coactivator known to increase aromatase expression which is also a direct downstream target of AMPK. Overall, these results suggest that metformin would reduce the local production of estrogens within the breast thereby providing a new key therapeutic tool that could be used in the neoadjuvant and adjuvant settings and conceivably also as a preventative measure in obese women.

Aromatase inhibitors and breast cancer.

Oestrogens are involved in risk to, and progression of, breast cancer. Drugs that inhibit the production of oestrogens (aromatase inhibitors, AIs), are therefore extremely attractive strategies both to prevent the disease and to treat established tumours. AIs now have a central role in the treatment of established breast cancer and are being considered for prevention. Third generation agents have been derived from rational drug design. They are able to block oestrogen production and reduce oestrogen levels to a degree that has not been observed previously and without affecting levels of other hormones. Such selective reduction of endogenous oestrogen provides targeted therapy for hormone-dependent breast cancer. This has led to improved clinical benefits in patients with these tumours. Anastrozole, letrozole and exemestane all have impressive antitumour effects in postmenopausal women with breast cancer and they are at least as beneficial as or better than other established endocrine agents when used to treat hormone-sensitive cancer in the advanced setting or as an adjuvant to surgery in earlier stages of the disease; ongoing trials are exploring the use of AIs in the preventative setting. Third generation inhibitors are well tolerated, having no greater side effects than might be expected from oestrogen suppression. Important differences in endocrinological and molecular effects exist between AIs and SERMs. These have implications for the preferred drug sequence and setting in which AIs are used. Since the major obstacle to more widespread use is primary/acquired resistance, discovery of the mechanisms by which resistance occurs offers hope for the future. More detailed study of AIs will yield important information about the involvement of oestrogen on the development and progression of breast cancer. Consequently AIs offer major clinical benefits to patients with breast cancer and the promise of relatively nontoxic intervention in women at high risk of the disease.

Induction of quinone reductase by withanolides.

Thirty-seven naturally occurring withanolides (1-37), previously isolated in our laboratories, were evaluated for their potential to induce quinone reductase with cultured murine hepatoma cells (Hepa 1c1c7). Spiranoid (29, 32) and 18-functionalized withanolides (2-5, 7-9, 24) were found to be potent inducers of the enzyme, while 5alpha-substituted derivatives exhibited weak activity. Preliminary studies were performed with compound 29 to evaluate enzyme-inducing capacity in multiple organ sites of BALB/c mice. Significant induction was observed in liver and colon, but not in lung, stomach, or mammary gland.

Transcriptional regulation of aromatase expression in human breast tissue.

Aromatase (CYP19) is the estrogen synthetase that converts androgen to estrogen. The expression of aromatase in breast cancer cells and surrounding stromal cells is up regulated compared to non-cancerous cells. In situ estrogen synthesis is thought to stimulate breast cancer growth in both an autocrine and a paracrine manner. A complex mechanism is involved in the control of human aromatase expression, in that seven promoters have been identified and found to be utilized in a tissue-selective manner. Increased aromatase expression in breast tumors is, in part, attributed to changes in the transcriptional control of aromatase expression. While promoter I.4 is the main promoter that controls aromatase expression in non-cancer breast tissue, promoters II and I.3 are the dominant promoters that drive aromatase expression in breast cancer tissue. During the last several years, our laboratory performed a series of studies to examine the transcription regulatory mechanism of aromatase expression in breast cancer cells. We functionally characterized promoters II and I.3, and carried out DNase 1 footprinting analysis that identified two regulatory elements, S1 and CREaro. Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that four orphan/nuclear receptors, ERR alpha-1, EAR-2, COUP-TFI and RAR gamma, bind to the S1 element, and that CREB1, Snail (SnaH) and Slug proteins bind to the CREaro element. Studies from this and other laboratories have revealed that in cancer tissue versus normal tissue, several positive regulatory proteins (e.g. ERR alpha-1 and CREB1) are present at higher levels and several negative regulatory proteins (e.g. EAR-2, COUP-TFI, RAR gamma, Snail and Slug proteins) are present at lower levels. This may explain why the activity of promoters II and I.3 is up regulated in cancer tissue. An understanding of the molecular mechanisms of aromatase expression between non-cancerous and cancerous breast tissue, at the transcriptional level, may help in the design of a therapy based on the suppression of aromatase expression in breast cancer tissue.

Effects of chemopreventive and antitelomerase agents on the spontaneous immortalization of breast epithelial cells.

BACKGROUND: Activation of telomerase is an early event in the development of breast and other cancers that may lead to cell immortalization, a critical and rate-limiting step in cancer progression. Breast epithelial cells from women with Li-Fraumeni syndrome (LFS) immortalize spontaneously and reproducibly in culture. We, therefore, tested whether immortalization of these cells could be prevented by treating them with chemopreventive agents and by inhibiting telomerase activity. METHODS: Noncancerous, preimmortal breast epithelial cells derived from a patient with LFS were treated for 3 months with nontoxic concentrations of the chemopreventive agents oltipraz, difluoromethylornithine, tamoxifen, and retinoic acid or with two different telomerase inhibitors. The frequency of spontaneous immortalization of LFS-derived cells was estimated by an approach based on fluctuation analyses. Statistical analyses were two-sided. RESULTS: The frequency of spontaneous immortalization events of LFS-derived breast epithelial cells was reduced by long-term treatment with retinoic acid (P<0.001) or tamoxifen (P<0.05) compared with solvent-treated cells. The frequency of immortalization was also reduced by treating LFS-derived cells with an antitelomerase antisense oligonucleotide (P<0.001) or by inducing the cells to express a dominant negative mutant of telomerase (P<0.025) compared with cells treated with a control oligonucleotide or with empty vector, respectively. CONCLUSIONS: Treatment of preimmortal LFS breast epithelial cells with chemopreventive and antitelomerase agents decreased the frequency of spontaneous immortalization in vitro. These studies validate the application of a new cell culture model system to screen the effects of novel chemopreventive agents by use of cell immortalization as an end point. The results also suggest that the telomerase ribonucleoprotein complex may be an important molecular target for breast cancer prevention.

Modulation of aromatase expression in human breast tissue.

Aromatase plays an important role in breast cancer development through its role in the synthesis of estrogen. Aromatase expression in breast tissue can be regulated by several mechanisms. The major promoter usage for aromatase expression in breast tumors (i.e. cAMP-stimulated promoters I.3 and II) is different from that in normal breast tissue (i.e. glucocorticoid-stimulated promoter I.4). Recent characterization of transcription factors that interact with the two important regulatory elements near promoters I.3 and II, i.e. S1 and CREaro, helps us better understand the mechanism of the switch of promoter usage between normal breast tissue and cancer tissue. It is thought that in normal breast tissue, the function of promoters I.3 and II is suppressed through the binding of EAR-2, COUP-TFI, and EARgamma to S1, and through the binding of Snail/Slug proteins to their binding site that quenchs the CREaro activity. In cancer tissue, the expression levels of EAR-2, COUP-TFI, EARgamma, Snail, and Slug decrease, and aromatase expression is then up regulated through the binding of ERRalpha-1 to S1 and the binding of CREB or related factors to CREaro. Results from this and other laboratories reveal that aromatase activity in aromatase expressing cells can also be modified by treatment with aromatase inhibitors and the antiestrogen ICI 182, 780. While aromatase inhibitors are used to treat breast cancer, the treatment has been found to increase the level of aromatase in the breast tissue of some patients. The enhancement of aromatase activity by aromatase inhibitors is thought to be due to a decrease of aromatase protein degradation by enzyme-inhibitor complex formation, up-regulation of the aromatase gene transcription through a cAMP-mediated mechanism, and an induction of aromatase expression by gonadtropins that are released from the pituitary in response to a reduction of estrogen levels in circulation in premenopausal women. Antiestrogen ICI 182, 780 has been found to suppress aromatase expression, but the mechanism has not yet been determined. In addition, aromatase activity and expression can be affected by environmental chemicals. A detailed structure-function study has revealed that flavones, but not isoflavones, are inhibitors of aromatase. It was found that flavones bind to the active site of aromatase in an orientation in which their rings-A and -C mimic rings-D and -C of the androgen substrate. The modulation of aromatase expression by endocrine disrupting chemicals is exemplified by two organochlorine pesticides (i.e. toxaphene and chlordane) that have been found to be antagonists of ERRalpha-1 orphan receptor. These compounds reduce ERRalpha-1 activity, resulting in a suppression of aromatase expression.

Local endocrine effects of aromatase inhibitors within the breast.

To determine the effects of aromatase inhibitors on oestrogen uptake, in situ aromatase activity and endogenous oestrogens in the breast, postmenopausal women with large primary ER-rich breast cancers have been treated neoadjuvantly for 3 months with either letrozole (2.5 or 10mg daily) or anastrozole (1 or 10mg daily) or exemestane (25mg daily). Patients were given an infusion of 3H-androstenedione and 14C-oestrone for 18h before and at the end of the study period. Blood, tumour and non-malignant breast were taken immediately after each infusion; oestrogens were extracted and purified. Tumour volume was measured before and during treatment at monthly intervals so that endocrinological changes could be related to clinical response. Treatment with each of the aromatase inhibitors was associated with a profound reduction in peripheral aromatase (as monitored by the level of plasma 3H-oestrone). There was no consistent effect on uptake of radioactively labelled oestrogen into breast tumours but a tendency for levels to increase after treatment in non-malignant breast. Conversely, therapy was associated with a marked inhibition of in situ oestrogen synthesis in both tumour and non-malignant breast (in occasional tissues, inhibitors appeared to be less effective but the effect was not related to clinical or pathological responses). Similar decreases were apparent in endogenous levels of oestrone and oestradiol. The absence of in situ aromatase activity tended to be associated with lack of clinical response to aromatase inhibition but the relationship was not absolute, limiting the utility of measurements of tumour aromatase as a predictive indices. Ex vivo studies of tissue aromatase indicated that such measurements consistently underestimate the inhibitory potential of reversible non-steroidal agents (and occasionally paradoxical in vitro increases in aromatase activity were seen with treatment). However, in situ assays demonstrate that new aromatase inhibitors such as anastrozole, exemestane and letrozole have profound effects on the local endocrinology within the postmenopausal breast, these being compatible with the clinico-pathological changes which occur with treatment.

SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells.

In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

The lactose analog GalNAcbeta1-->4Glc is present in bovine colostrum. Enzymatic basis for its occurrence.

We have isolated from bovine colostrum the lactose analog GalNAcbeta1-->4Glc. The enzymatic basis for its occurrence was studied by assaying the activities of GlcNAcbeta-R beta4-N-acetylgalactosaminyltransferase (beta4-GalNAcT) and GlcNAcbeta-R beta4-galactosyltransferase (beta4-GalT) in primary milk and several lactating bovine mammary gland fractions. As the beta4-GalNAcT, which appears to be tightly membrane bound, is induced by the milk protein alpha-lactalbumin (alpha-LA) to act on Glc, it is concluded that beta4-GalNAcT is responsible for the synthesis of GalNAcbeta1-->4Glc in the gland. The comparatively low level (15-20 mg/l) at which this disaccharide is produced may be due to the relatively poor interaction of beta4-GalNAcT with alpha-LA as well as to the fact that alpha-LA does not inhibit the action of the enzyme on N-acetylglucosaminides.

Xenobiotic metabolizing and antioxidant enzymes in normal and neoplastic human breast tissue.

An investigation was made of ethoxyresorufin O-deethylase (EROD) activity, a cytochrome P450 (CYP) dependent enzyme mainly catalyzed by CYP1A1, glutathione S-transferase (GST) activity toward the substrates 1-chloro-2,4- dinitrobenzene (CDNB) and ethacrynic acid (EAA), reduced glutathione (GSH) levels, and antioxidant enzyme (AOE) activity namely catalase (CAT) and selenium- dependent glutathione peroxidase (Se-GPx) in tumor and surrounding tumor-free (normal) tissues in female breast cancer patients. Wide interindividual variations were found in the enzyme activities in both tumor and normal breast tissues. No significant differences were noted between mean EROD and CAT activities in tumor and normal breast tissues. The mean activities of CDNB GST, EAA GST and Se-GPx and GSH levels in tumor tissue were significantly higher than those in normal breast tissue. These results show that CYP, GST and AOE behave differentially in breast tumors.

Identification and characterization of a stereospecific human enzyme that catalyzes 9-cis-retinol oxidation. A possible role in 9-cis-retinoic acid formation.

All-trans- and 9-cis-retinoic acid are active retinoids for regulating expression of retinoid responsive genes, serving as ligands for two classes of ligand-dependent transcription factors, the retinoic acid receptors and retinoid X receptors. Little is known, however, regarding 9-cis-retinoic acid formation. We have obtained a 1.4-kilobase cDNA clone from a normalized human breast tissue library, which when expressed in CHO cells encodes a protein that avidly catalyzes oxidation of 9-cis-retinol to 9-cis-retinaldehyde. This protein also catalyzes oxidation of 13-cis-retinol at a rate approximately 10% of that of the 9-cis isomer but does not catalyze all-trans-retinol oxidation. NAD+ was the preferred electron acceptor for oxidation of 9-cis-retinol, although NADP+ supported low rates of 9-cis-retinol oxidation. The rate of 9-cis-retinol oxidation was optimal at pHs between 7.5 and 8. Sequence analysis indicates that the cDNA encodes a protein of 319 amino acids that resembles members of the short chain alcohol dehydrogenase protein family. mRNA for the protein is most abundant in human mammary tissue followed by kidney and testis, with lower levels of expression in liver, adrenals, lung, pancreas, and skeletal muscle. We propose that this cDNA encodes a previously unknown stereospecific enzyme, 9-cis-retinol dehydrogenase, which probably plays a role in 9-cis-retinoic acid formation.

Purification and characterization of cathepsin D from normal human breast tissue.

The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad area of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.

Ornithine decarboxylase over-expression stimulates mitogen-activated protein kinase and anchorage-independent growth of human breast epithelial cells.

In these experiments we tested the hypothesis that constitutive activation of polyamine(PA) biosynthesis may contribute to mammary carcinogenesis. Spontaneously immortalized normal human MCF-10A breast epithelial cells were infected with the retroviral vector pLOSN containing a cDNA which codes for a truncated and more stable ornithine decarboxylase (ODC), the rate-limiting enzyme in PA synthesis. Upon chronic selective pressure with alpha-difluoromethyl-ornithine (DFMO) (an irreversible inhibitor of ODC), infected MCF-10A cells exhibited an approximately 250-fold increase in ODC activity, which persisted despite discontinuation of DFMO. ODC-over-expressing MCF-10A cells showed a modest decrease in S-adenosylmethionine decarboxylase and an increase in spermidine/spermineN1-acetyltransferase. Analysis of cellular PA profile revealed a selective accumulation of putrescine without alterations in spermidine and spermine contents. Lesser degrees of increased ODC activity were obtained reproducibly by re-exposing the cells to incremental small doses of DFMO. We observed a bell-shaped dose-related positive effect of ODC activity on clonogenicity in soft agar of MCF-10A cells. Since anchorage-dependent growth was actually reduced, such positive influence on this feature of transformation was not a non-specific consequence of a growth advantage provided by ODC over-expression. In addition, we observed a close parallelism between the dose-dependent effects of ODC expression on clonogenicity and activity of the ERK-2 kinase, a central element of the MAPK cascade. Our data demonstrate an interaction between PA and the MAPK signalling pathway and suggest that the latter may be involved in ODC-induced transformation of mammary epithelial cells.

A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription.

C19 steroids are converted to estrogens in a number of tissues by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom). Adipose tissue is the principal site of estrogen formation in postmenopausal women. Aromatase activity as well as P450arom transcripts primarily reside in the stromal cell component of the adipose tissue. Studies designed to investigate whether increased local aromatase activity in breast adipose tissue influences the growth of breast cancers have yielded discrepant results. In an attempt to clarify this controversy, adipose tissue was obtained from the four breast quadrants at the time of mastectomy (n = 13) performed for removal of a tumor. Breast fat P450arom messenger RNA levels were quantified and compared between the four quadrants within each specimen using competitive polymerase chain reaction after reverse transcription in which 10 micrograms human adipose total RNA together with 1 pg rat complementary RNA (internal standard) were reverse transcribed and coamplified. In 9 out of 13 patients (69%), highest P450arom transcript levels colocalized to the quadrants bearing tumors. This correlation was statistically significant (P < 0.001). The regional distribution of P450arom transcripts in breast adipose tissue of disease-free individuals, obtained during reduction mammoplasty (control group, n = 9), did not favor any particular region of the breast. We also quantified by morphometry the histological components of the adipose tissue samples from each quadrant in mastectomy specimens. The distribution of stromal cells significantly correlated with the distribution of P450arom transcript levels, in that quadrants containing highest proportions of stromal cells matched to highest transcript levels (P < 0.01). Although the quadrants bearing tumors contained the highest percentage of stromal cells, this correlation was not statistically significant. The adipose tissue surrounding a breast tumor displays increased estrogen biosynthesis, which may promote tumor growth. It is further suggested that the distribution of stromal cell components in breast adipose tissue gives rise to locally elevated P450arom expression, which in turn may favor neoplastic development and growth in these predisposed areas of the breast. The correlation between the presence of a tumor and elevated P450arom levels in the proximal adipose tissue is independent of tumor size, node involvement, histological type or grade, estrogen/progesterone receptor status, DNA index, or S-phase fraction.

Determination of aromatase cytochrome P450 messenger ribonucleic acid in human breast tissue by competitive polymerase chain reaction amplification.

Local production of estrogen in breast tissue may influence the growth of breast cancers. Peripheral conversion of C19 steroids to estrogens is catalyzed by the aromatase enzyme complex which is comprised of a specific form of cytochrome P450, aromatase cytochrome P450 (P450AROM) and the flavoprotein, NADPH-cytochrome P450 reductase. To evaluate P450AROM mRNA levels in breast tissue, a specific competitive polymerase chain reaction amplification procedure was devised. In this method, a rat P450AROM complementary RNA is coamplified as an internal standard in order to compare amplification reactions. The amplification products are recognized by hybridization with 32P-labeled oligonucleotides specific for each species. Densitometry is used to quantitate autoradiographs. Initial studies using RNA from whole breast tissue obtained from reduction mammoplasty revealed linearity of the relationship between the densitometer signal from the human amplification product and total RNA concentration. Breast tissue was then separated into a floating adipocyte fraction and a pelleted fraction containing the other cellular elements by collagenase digestion and centrifugation. Comparison of specific content of aromatase amplification product per unit weight of RNA extracted from adipocytes and pelleted cells revealed considerably higher levels in the RNA from the nonadipocyte fraction. Immunocytochemical characterization of this fraction revealed the presence of several cell types including macrophages, ductal epithelial cells, and endothelial cells, but primary cells of stromal origin.

Glutathione peroxidase, glutathione S-transferase and glutathione reductase activities in normal and neoplastic human breast tissue.

Glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-Tr) and glutathione reductase (GSSG-Rx) activities have been determined in normal and neoplastic human breast tissues. Large interindividual variations in the activities of all enzymes tested were found in both tumor and non-tumor specimens. In general a significant increase in the activities of the 3 enzymes was found in tumors, whereas in fibroadenoma they were as high as in healthy tissues. When a comparison was made between normal and neoplastic tissues of the same individual, GSH-Tr and GSSG-Rx activities were found to be higher in 15 and 11 cases, respectively, out of 17. GSG-Px activity was higher in all cases. From measurement of GSG-Px activity with both H202 and cumene hydroperoxide, it was deduced that human breast contains only the selenium-dependent form.

Cholesterol esterification by mammary gland microsomes from the lactating rat.

Cholesterol esterification by fatty acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) has been demonstrated in microsomes prepared from breast tissue of the lactating rat, employing the incorporation of [1-14C]oleoyl coenzyme-A into cholesteryl[14C]oleate. The regulation of this activity in vitro was studied by supplementing microsomes with unesterified cholesterol supplied either as a dispersion in acetone or by incubating microsomes with cholesterol-enriched serum lipoproteins prior to enzyme assay. ACAT activity was increased by more than 80% when the ratio of unesterified cholesterol to microsomal protein was increased from 29 to 48 micrograms/mg protein, indicating that the normal cholesterol content of mammary gland microsomes does not saturate this enzyme. Cholesterol esterification could be inhibited nearly completely in vitro by addition of the polar steroid progesterone (93% decrease in ACAT activity with 75 microM progesterone) and, to lesser extents, by estradiol and retinol. Enzyme assays were also performed after incubating microsomes with N-acylamides that inhibit cholesteryl ester synthesis in other systems. Under conditions where the mammary gland ACAT reaction was inhibited by more than 82%, the esterification of retinol was reduced by less than 30% and incorporation of [14C]oleoyl-CoA into triglycerides was not inhibited at all. These studies indicate that the lactating mammary gland contains ACAT activity having properties similar to ACAT in other organs. The presence of ACAT activity in the lactating mammary gland provides a possible mechanism for the synthesis of cholesteryl esters found in milk. It can also be inferred that ACAT in mammary gland microsomes is likely to be distinct from the microsomal acyltransferases that catalyze the esterification of retinol and glycerides.