MCF-7 Human Mammary Carcinoma Cells Research Articles

Identification of a vitamin D3 response element in the fibronectin gene that is bound by a vitamin D3 receptor homodimer.

Fibronectin (FN) is an important adhesive noncollagenous glycoprotein involved in maintenance of the extracellular matrix and cell adhesiveness, loss of which has been implicated in the metastatic potential of cells. Regulation of FN occurs at the transcriptional level by the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Transient transfection of homologous and heterologous promoter reporter constructs into ROS 17/2.8 (rat osteosarcoma), NIH 3T3 (mouse fibroblast), and MCF-7 (human mammary carcinoma) cell lines showed a consistent two- to threefold induction of transcription when stimulated with 1,25-(OH)2D3. These heterologous promoter transfection studies with gel shift analysis locate a third, natural DR6-type vitamin D responsive element (VDRE) at nucleotide positions -171 to -154 in the murine FN promoter. Interestingly, this VDRE is also present in rat and human FN promoters. This study shows that 1,25-(OH)2D3 induces FN transcription from an existing elevated basal transcriptional activity by acting through two putative hexameric core binding motifs which bind VDR homodimers. Furthermore, the FN VDRE is the first homodimer-type VDRE that is not overlaid by a DR3-type structure.

Inhibition of ATP‐sensitive potassium channels causes reversible cell‐cycle arrest of human breast cancer cells in tissue culture

The purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF-7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration-dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3-day or 5-day exposure to drug. The IC50 values for these five drugs, quinidine (25 microM), glibenclamide (50 microM), linogliride (770 microM), 4-aminopyridine (1.6 mM), and tetraethylammonium (5.8 mM) were estimated from their respective concentration-response curves. Four other potassium channel blockers were tested at supra-maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF-7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in G0/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in G0/G1 phase and the threshold concentrations for inhibition of growth and G0/G1 arrest were similar. The G0/G1 arrest produced by quinidine and glibenclamide were reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of approximately 24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP-sensitive potassium channels in these human mammary carcinoma cells, reversibly arrests the cells in the G0/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation.

Cell type-specific desmosomal plaque proteins of the plakoglobin family: plakophilin 1 (band 6 protein)

Desmosomes represent a special type of the plaque-bearing adhering junctions, characteristic of certain pathways of cell differentiation, which compositionally are not identical in the various kinds of desmosome-forming cells. While all desmosomes contain the cytoplasmic plaque proteins desmoplakin I and plakoglobin, they can vary in their specific complement of desmosomal cadherins and by the presence of additional plaque proteins. We have raised monoclonal antibodies recognizing one such ‘accessory’ plaque protein, the cytokeratin-binding, basic protein plakophilin 1, originally introduced as ‘band 6 protein’ or ‘polypeptide D6’, which is an abundant desmosomal component in certain epithelia. Using such antibodies, we have isolated cDNA clones encoding the bovine and the human protein and determined their complete amino acid sequences. The mRNAs, which on Northern blot tests appear as two bands corresponding to approximately 4 and 2.4 kb (bovine) or 5 and 2.6 kb (human), code for 727 amino acids (calculated mol. wt. 80,180; IEP 9.25) in bovine and 726 amino acids (mol. wt. 80,496; IEP 9.34) in human plakophilin. Sequence analyses have revealed the presence of 9.2 repeated units of the arm-motif sequence, confirming our previous conclusion that this protein is a member of a larger family of proteins including, inter alia, several membrane-associated plaque proteins such as vertebrate plakoglobin and β-catenin as well as the product of the armadillo gene of Drosophila. The plakophilin antibodies and cDNA probes have also allowed us to examine its synthesis in various tissues and cell cultures. While we confirm the occurrence of the protein in cytoskeletal fractions from various stratified squamous, complex, glandular duct and bladder epithelia, where it can be localized to desmosomes, we have, surprisingly, also identified the protein, although at lower amounts, in cytoskeletal fractions from several cultured cell lines in which the protein has not been consistently localized to desmosomes by immunofluorescence microscopy. Examples include cultured cells derived from certain simple epithelia such as the kidney-derived line MDBK and cultured calf lens cells. We have also found that, in all plakophilin 1-positive cells examined, a pool of diffusible (‘soluble’) cytoplasmic plakophilin exists, including cell lines such as human mammary carcinoma MCF-7 cells in which this soluble plakophilin seems to be the only detectable form. In addition, we have identified some soluble proteins conspicuously cross-reacting with plakophilin 1. Possible functions of plakophilin and its potential value as a marker for specific states of cell differentiation are discussed, particularly with respect to tumor diagnosis.

17-beta-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma cell line.

The MCF-7 human mammary carcinoma cell line undergoes morphological differentiation in vitro when treated with 17-beta-estradiol. A prominent feature of this process is the postconfluent development of multicellular, three-dimensional nodules that rise above the surrounding monolayer. Formation of the nodules suggests that changes in cellular adhesion occur during this cellular overgrowth. Therefore changes in the distribution of cell-matrix and cell-cell adhesion plaque proteins were examined with respect to estradiol induction of nodule development. Estradiol treatment of the carcinoma cell line had the following effects: (1) vinculin- and talin-rich cell-matrix adhesion plaques were reduced in overall number and size in confluent and postconfluent cultures. No overt change in distribution or morphology of adhesion plaques was observed in subconfluent cultures. (2) Staining for vinculin was reduced in cell-cell adhesions situated at the apical region of subconfluent, confluent and postconfluent monolayers. Staining for F-actin and plakoglobin was retained at this region in estradiol-induced cells. (3) vinculin was not detected in intercellular adhesions of nodule cells although intense labelling for both F-actin and plakoglobin was observed. In addition, in untreated monolayer cells, both F-actin and plakoglobin were concentrated in a subapical/basolateral location, as a vesicle-like pattern, which corresponded to intercellular spaces observed with phase-contrast microscopy. Treatment with estradiol caused the rearrangement of subapical/basolateral F-actin and plakoglobin staining into a more uniform pattern. The findings of this study show that estradiol induces changes in both cell-matrix and cell-cell adhesions in an estrogen-responsive carcinoma cell line. The gradual loss of vinculin from cell-matrix and cell-cell adherens junctions of the monolayer could be a potential factor in the capacity of these cells to form multilayers or nodules in postconfluent growth. Furthermore, the development of the nodules in response to estradiol may provide a useful system in which to study steroid hormone regulation of adhesion and the cytoskeleton in responsive tumor cells.

Identification and characterization of cytosolic sulfotransferase activities in MCF-7 human breast carcinoma cells

MCF-7 human mammary carcinoma cells have been reported to possess β-estradiol and dehydroepiandrosterone sulfotransferase activities. These steroid sulfotransferase activities may be important in the metabolism and activity of different steroids in these cells. This report describes and characterizes both the enzymatic activity of three cytosolic sulfotransferases found in MCF-7 cells and the corresponding immunoblot analysis of these enzymes with specific anti-sulfotransferase antibodies. Two cytosolic sulfotransferases have been purified and characterized from human tissues which are capable of sulfating estrogens. These are the phenol-sulfating form of phenol sulfotransferase (P-PST) and the hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST). The results of this study show that P-PST is the major cytosolic sulfotransferase found in MCF-7 cytosol and is responsible for most of the β-estradiol sulfation in these cells. Although DHEA-ST activity was found in MCF-7 cytosol, this activity was only about 3% of the P-PST activity. Immunoblot analysis of MCF-7 cytosol detected both P-PST and lower levels of the monoaomine-sulfating form of PST; however DHEA-ST could not be detected apparently because of low levels of expression. Human liver P-PST was expressed in Cos-7 Green monkey kidney fibroblasts and the ability of the cloned enzyme to sulfate β-estradiol was investigated. This study indicates that P-PST is the prevalent cytosolic sulfotransferase in MCF-7 cytosol and is responsible for the majority of β-estradiol sulfation in these cells.

Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro.

The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells.

Estradiol up-regulates the stimulatory GTP-binding protein expression in the MCF-7 human mammary carcinoma cell line

The effect of estradiol treatment of the human mammary carcinoma cell MCF-7 on the adenylyl cyclase system was examined. Treatment with 10 nM estradiol for 72 h increased the basal level ofcAMP, and isoproterenol-, PGE 2- or calcitonin-stimulated cAMP production. Estradiol also increased the response to cholera toxin but did not alter the response to forskolin. No significant change in growth rate was observed during the 72 h of estradiol treatment. In MCF-7 cell membranes the responsiveness to isoproterenol, PGE 2, or cholera toxin was also enhanced by estradiol treatment. The cholera toxin-catalyzed ADP-ribosylation of gsoe in MCF-7 cell membranes was significantly increased by 72 h of treatment with estradiol. Consistent with this observation, the level of Gsα immunoreactivity was increased in the estradiol-treated cell membranes. On the other hand, pertussis toxin did not change the responsiveness to isoproterenol, PGE 2 or calcitonin in either control or estradiol-treated cells. In addition, ADP-ribosylation with pertussis toxin also did not reveal any change in Gi. These results clearly indicate that Gs expression is under the control of estradiol, and that this effect may contribute to the increased sensitivity of hormone-stimulated adenylyl cyclase activities in MCF-7 cells.

Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI.

We have characterized a new selenium-dependent glutathione peroxidase, GSHPx-GI, by expressing a GSHPx-GI cDNA isolated from human hepatoma HepG2 cells in human mammary carcinoma MCF-7 cells, which have virtually undetectable expression of either the classical cellular enzyme, GSHPx-1, or GSHPx-GI at the protein level. One of the G418-resistant clones, neo-D1, expresses the transfected GSHPx-GI cDNA. This is based on 1) the presence of an additional GSHPx-GI DNA restriction fragment detected by Southern analysis; 2) the presence of a 1.9-kilobase (kb) GSHPx-GI mRNA in addition to the 1.0-kb endogenous mRNA by Northern analysis; and 3) the appearance of a 22-kDa 75Se-labeled protein which is absent in parental MCF-7 cells revealed by SDS-polyacrylamide gel electrophoresis. GSHPx-GI expressed in neo-D1 is a tetrameric protein localized in cytosol. GSHPx-GI does not cross-react with antisera against human GSHPx-1 or human plasma glutathione peroxidase (GSHPx-P). Similar substrate specificities are found for GSHPx-1 and GSHPx-GI; they both catalyze the reduction of H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide with glutathione, but not of phosphatidylcholine hydroperoxide. GSHPx-GI mRNA was readily detected in human liver and colon, and occasionally in human breast samples, but not other human tissues including kidney, heart, lung, placenta, or uterus. In rodent tissues, GSHPx-GI mRNA is only detected in the gastrointestinal tract, and not in other tissues including liver. In fact, GSHPx-GI appears to be the major glutathione-dependent peroxidase activity in rodent GI tract. This finding suggests that GSHPx-GI could play a major role in protecting mammals from the toxicity of ingested lipid hydroperoxides. In conclusion, we have demonstrated that GSHPx-GI is the fourth member in the selenium-dependent glutathione peroxidase family, in addition to GSHPx-1, GSHPx-P, and phospholipid hydroperoxide glutathione peroxidase (PHGPX).

Expression of cell adhesion molecule E-cadherin in human arachnoid villi.

Calcium-dependent epithelial cell adhesion molecules designated as E-cadherin (also known as uvomorulin or L-CAM) were identified in human arachnoid villi by immunoblotting and immunocytochemical analyses using a monoclonal antibody HECD-1 raised against human mammary carcinoma MCF-7 cells. Immunoblot analysis showed that HECD-1 recognizes E-cadherin with a molecular weight of 124 kD. In all arachnoid cells of an arachnoid villus, E-cadherin was detected by immunolight microscopy within the cytoplasm rather than the cellular boundaries as seen in the control group. Furthermore, the extent of expression by immunolight microscopy varied from portion to portion. The expression was usually weak in the syncytial cluster which was ultrastructurally composed of tightly juxtaposed cells characterized by few extracellular cisterns and numerous cell junctions, while it was intense in the reticular cluster and the surface layer which were ultrastructurally characterized by abundant extracellular cisterns and smaller numbers of cell junctions. The cells of the reticular cluster and the surface layer contained more free ribosomes than those of the syncytial cluster. Immunoelectron microscopy showed that E-cadherin was localized not only to the opposing plasma membranes and the cytoplasm around the free ribosomes or the rough endoplasmic reticulum but also to the extracellular cisterns. As the expression of E-cadherin was closely related to the arachnoid cells adjacent to the cerebrospinal fluid pathway, it is suggested that, instead of the cell junctions, E-cadherin may play an important role in the flexible adhesion of arachnoid cells even in the presence of the cerebrospinal fluid.

Retinoic acid modulates both invasion and plasma membrane ruffling of MCF-7 human mammary carcinoma cells in vitro.

The invasiveness of MCF-7 human mammary carcinoma cells was tested in vitro via confronting cultures with embryonic chick heart fragments. Invasive (e.g. MCF-7/6) and non-invasive (e.g. MCF-7/AZ) variants were detected. Automated image analysis of time-lapse video-microscopy recordings showed that the plasma membrane ruffling activity of the invasive MCF-7/6 variant was higher than the ruffling activity of the non-invasive MCF-7/AZ variant. Addition of all-trans-retinoic acid to the culture medium (10(-6) M) inhibited both invasion and ruffling of MCF-7/6 cells, while MCF-7/AZ cells became invasive and acquired an increased ruffling by the same type of treatment. A similar opposite effect on MCF-7 cells was not found after treatment with other ligands of the nuclear steroid/thyroid receptor superfamily. Triiodo-l-thyronine (up to 10(-5) M) and beta-oestradiol (up to 10(-6) M) did not alter the invasiveness of the cells, while dexamethasone (10(-6) M) and the pure anti-oestrogen ICI 164,384 inhibited both invasion and ruffling. Our data show that retinoic acid can modulate invasiveness in opposite directions.

Down-Regulation of Messenger Ribonucleic Acid and Protein Levels for Estrogen Receptors by Phorbol Ester and Calcium in MCF-7 Cells*

Treatment of MCF-7 cells, a human mammary carcinoma cell line, with phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] or calcium ionophore (A23187) was associated with striking effects on levels of estrogen receptor (ER) mRNA, specific binding of 17 beta-[3H]estradiol [( 3H]E2), and immunoreactive ER. TPA (10(-7) M) caused a time-dependent reduction of ER mRNA which was below the level of detection after 9 h. Similar effects of TPA appeared at levels of specific binding of [3H]E2 as well as immunoreactive ER. In contrast, TPA induced an increase in mRNA for beta-actin. Incubation of MCF-7 cells with increasing concentrations of TPA (10(-11)-10(-7) M) was associated with biphasic effects on ER mRNA and proteins. Levels of immunoreactive progesterone receptors (PR) were induced by E2 (10(-9) M) in a time-dependent manner. In the presence of TPA (10(-7) M), where ER levels were suppressed, no induction of PR was observed. Removal of TPA (10(-7) M) after 10 h (ER mRNA) or 22 h (ER proteins) of treatment was associated with a continued suppression of both mRNA and protein levels during the entire incubation period (48 h). Treatment with A23187 (2 x 10(-7) M) also caused a time-dependent down-regulation of levels of ER mRNA and proteins. These effects occurred somewhat more slowly than those of TPA. Levels of beta-actin mRNA were not changed by this treatment. These results indicate that changes in estrogen sensitivity are mediated by calcium-dependent protein kinases in human mammary carcinoma MCF-7 cells.

Estrogen Stimulates Transcription of c-junProtooncogene

Estrogen is a mitogen for the rat uterus, where it induces transient activation of c-fos and c-myc protooncogene expression, followed by increases in DNA synthesis and cell proliferation. JUN-C, the product of the c-jun protooncogene, is a nuclear protein that can interact with FOS to modulate the activity of AP-1-responsive promoters. To test whether c-jun is a target for estrogen regulation, we measured the effects of 17 beta-estradiol on the expression of this gene in rat uterus. A human c-jun cDNA probe detects in rat uterus two mRNA species of 2.5 and 3.2 kilobases. Treatment of the animals with estrogen results in a rapid transient increase in the concentrations of these mRNAs; a 4- to 5-fold increase over the prestimulation level was detected starting 30 min after estrogen injection and lasting for 2 h, with a return to the prestimulation level after 4 h. In accordance with the results obtained by analysis of the mRNA, we found that estrogen increases 3- to 4-fold c-jun gene transcription in the uterus, at the same time it induces its mRNA accumulation. The ability of estrogen to induce c-jun gene expression was not abolished by the protein synthesis inhibitor cycloheximide, suggesting that transcriptional activation of this protooncogene is a primary response to the hormone. Furthermore, we found that in the estrogen-responsive MCF-7 human mammary carcinoma cells, estrogen stimulates transcription of a reporter gene containing four copies of a jun/AP-1 response element. These data demonstrate that c-jun gene expression is regulated by estrogen and suggest that JUN-C could play a role in the activation of cell proliferation by estrogen.

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata.

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.

Catechol estrogen formation in MCF-7 cell culture and effects of bromoestrogen inhibitors

Agonist and antagonist activities have been reported for several catechol estrogens given exogenously. Since the metabolic clearance rate for catechol estrogens in the body is very rapid, catechol estrogens produced at other tissues will have minimal effect on breast tissue. Information of the extent of catechol estrogen formation within cells is critical in assessing the overall importance of these estrogen metabolites. Investigations of the conversion of estrogens to catechol estrogens were performed in the MCF-7 human mammary carcinoma cell culture system. Reverse-phase high-performance liquid chromatography (HPLC) analysis demonstrated that very little metabolism of estradiol occurs after 48 h, with only small amounts of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, and estriol being observed. The total amount of 2-hydroxyestrogen products formed from 1 μM estradiol was 406.2 pmol (SD = 60.9) per 3 × 10 7 cells in 48 h. Similar results were obtained using the simpler radiometric assay for estrogen 2-hydroxylase, which measures the release of 3H 2O from [2- 3H]estradiol. The effects of inhibitors of estrogen 2-hydroxylase were also examined in MCF-7 cells. 2-Bromoestradiol, 4-bromoestradiol, and 2,4-dibromoestradiol effectively block estrogen 2-hydroxylase in a dose-dependent manner in MCF-7 cultures, with ED 50 of approximately 1 μM for each inhibitor. Furthermore, these bromoestrogens bind poorly to estrogen receptors in MCF-7 cells and do not alter cell growth. Thus, in MCF-7 mammary cell cultures, metabolism of estradiol occurs to only a minor degree, and it is unlikely that the levels of catechol estrogens would reach physiologically relevant concentrations in the intact breast cancer cells. In addition, the bromoestrogens are effective inhibitors of estrogen 2-hydroxylase in cell culture systems, bind poorly to the estrogen receptor, and do not exhibit estrogenic effects at concentrations up to 1 μM.

A new monoclonal antibody (A46-B/B10) highly specific for the blood group H type 2 epitope: Generation, epitope analysis, serological and histological evaluation

A monoclonal antibody recognizing the blood group H type 2 antigen has been obtained from a BALB/c mouse immunized with MCF-7 (human mammary carcinoma) cells. The specificity of this antibody (A46-B/B10, IgM, kappa) has been identified by haemagglutination tests, immunohistochemistry, binding inhibition studies, and absorption experiments performed with synthetic oligosaccharides. The antibody is virtually nonreactive with H type 1 antigen or with closely related type 2 structures (e.g., Y antigen). A46-B/B10 strongly agglutinates human erythrocytes according to the amount of H substance expressed and can, therefore, easily discriminate between blood groups A1 and A2 as well as A1B and A2B (A1 and A1B are not or only weakly agglutinated). In immunohistochemistry, this antibody seems to provide a highly specific reagent for a restricted number of carcinomas and epithelial lineages in tissue sections and in vitro.

7 α -Substituted androstenediones as effective in vttro and invivo inhibitors of aromatase

Research efforts over the past several years have focused on the synthesis of competitive and irreversible aromatase inhibitors and examination of these inhibitors in microsomal preparations, in cell culture, and in vivo . Several 7α-substituted androstenediones have demonstrated high affinity for placental aromatase, with apparent K i's ranging from 1 to 30 nM. Inactivation of aromatase occurred following incubation with alkylating and enzyme-activated irreversible inhibitors. 7α-(4'-Amino)phenylthio-4-androstene-3,17-dione (7α-APTA) exhibits potent inhibitory activity of aromatase in the MCF-7 human mammary carcinoma cell line with an ED 50 of approximately 25 nM. The inhibitor did not bind to the estrogen receptor of the cells in vitro nor induce levels of progesterone receptors in intact cells. In vivo studies of 7α-APTA in the DMBA-induced rat mammary carcinoma model resulted in 80% of the tumors responding completely or partially at doses of 25 and 50 mg/kg body wt/day. Thus, these 7α-substituted steroidal aromatase inhibitors are effective medicinal agents and may be useful for the treatment of estrogen-dependent breast cancer.

Modulation of lactogenic receptors by progestins in cultured human breast cancer cells.

Progesterone receptors (PgR) are present in many breast cancers, but few specific actions of progestins in breast cancer cells have been reported. We now report that progestins specifically modulate lactogenic receptor expression in cultured T-47D and MCF-7 human mammary carcinoma cells. When T-47D cells were preincubated for 24 h with 1 nM medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-4-pregnene-3,20-dione), specific binding of [125I]human GH ([125I]hGH) and [125I]human PRL was increased to 205 +/- 22% (+/- SE) (P less than 0.01) and 175 +/- 32% (P less than 0.05), respectively, of that in control cultures. There was no significant effect on cell number and no significant enhancement of specific binding of [125I]porcine insulin, [125I]salmon calcitonin, [125I]human transferrin, or [3H] Concanavalin A. Lactogenic receptor number was increased from 6,490 +/- 500 (n = 12) to 13,180 +/- 3,270 (n = 7; P less than 0.01) sites/cell, with no significant change in affinity for hGH. Progesterone, which is readily metabolized by these cells, was less potent than the synthetic progestins (medroxyprogesterone acetate, R 5020 (17 alpha, 21-dimethyl-19-norpregn-4,9-diene-3,20-dione), and ORG 2058 (16 alpha-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione), but physiological concentrations of progesterone (1 nM) significantly enhanced specific binding of [125I]hGH to 153 +/- 23% of the control value (n = 6; P less than 0.05). Physiological concentrations of androgens, estrogens, and glucocorticoids had no significant effect. MCF-7 cells were considerably less sensitive to these effects of progestins than T-47D cells, probably due to the lower PgR concentration in MCF-7 cells. These observations, which indicate that lactogenic receptor expression is controlled, at least in part, by progestins in these mammary carcinoma cell lines, may have important implications in the management of human breast cancer, where high levels of this receptor may reflect a functional PgR and a highly hormone-dependent phenotype.

Transformation of NIH 3T3 cells by DNA of the MCF-7 human mammary carcinoma cell line induces expression of an endogenous murine leukemia provirus.

Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.

Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is ∼80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.