©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factor- Abrogates Glucocorticoid-stimulated Tight Junction Formation and Growth Suppression in Rat Mammary Epithelial Tumor Cells (*)

(Received for publication, June 10, 1994; and in revised form, December 2, 1994)

Patricia Buse Paul L. Woo David B. Alexander Helen H. Cha Avid Reza Naalla D. Sirota Gary L. Firestone (§)

From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The glucocorticoid and transforming growth factor-alpha (TGF-alpha) regulation of growth and cell-cell contact was investigated in the Con8 mammary epithelial tumor cell line derived from a 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma. In Con8 cell monolayers cultured on permeable filter supports, the synthetic glucocorticoid, dexamethasone, coordinately suppressed [^3H]thymidine incorporation, stimulated monolayer transepithelial electrical resistance (TER), and decreased the paracellular leakage of [^3H]inulin or [^14C]mannitol across the monolayer. These processes dose dependently correlated with glucocorticoid receptor occupancy and function. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevented the glucocorticoid growth suppression response and disrupted tight junction formation without affecting glucocorticoid responsiveness. Treatment with hydroxyurea or araC demonstrated that de novo DNA synthesis is not a requirement for the growth factor disruption of tight junctions. Immunofluorescence analysis revealed that the ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells and that TGF-alpha caused ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, our results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha, can override both effects of glucocorticoids. These results have uncovered a novel functional ``cross-talk'' between glucocorticoids and TGF-alpha which potentially regulates the proliferation and differentiation of mammary epithelial cells.


INTRODUCTION

The combined effects of systemic steroid and protein hormones as well as locally acting growth factors control the normal development, differentiation, and proliferation of the mammary gland(1, 2, 3, 4, 5) . Depending on their bioavailability, these extracellular signaling molecules can interdependently, and in a temporally appropriate manner, regulate the function of the three main differentiated cell types of the mammary gland (i.e. stromal, myoepithelial, and epithelial cells). The systemic humeral signals acting on mammary tissue include the ovarian steroids and lactogenic hormones, such as prolactin and glucocorticoids(6, 7, 8) . In addition, each type of mammary cell can produce and/or respond to several classes of growth factors (1, 2, 3, 4, 5) . Two important mammary-derived mitogens are transforming growth factor-alpha (TGF-alpha) (^1)and epidermal growth factor (EGF) which both act through the tyrosine kinase EGF receptor (9, 10) . TGF-alpha and EGF immunolocalize to morphologically distinct areas of the mammary gland(11) , and the expression of these growth factors is regulated at different stages of mammary gland growth and development(1, 12, 13) . Both in vitro and in vivo studies have shown that TGF-alpha can have a selective effect on the proliferation of normal mammary epithelial cells(1, 5, 12, 14, 15, 16, 17) . For example, exogenous administration of TGF-alpha stimulates ductal and terminal end bud proliferation in the virgin mouse mammary gland(11) , enhances lobuloalveolar development in mitogenically primed mice(1, 14) , and stimulates angiogenesis(15) .

Recent evidence suggests that TGF-alpha is involved in the early stages of breast tumorigenesis (1, 2, 5, 18) and acts as a potent autocrine growth regulator of mammary tumor cells(5, 12, 14, 19) . For example, constitutive expression of TGF-alpha in a nontumorigenic mouse mammary epithelial cell line induces anchorage-independent growth in vitro(20) , while introduction of TGF-alpha cDNA into the germ line of transgenic mice induces the appearance of hormone-dependent mammary adenocarcinomas(21) . Many human and rodent mammary tumors inappropriately express high levels of TGF-alpha in vivo(22) and in vitro(16) which has been proposed to account, in part, for the cellular escape from hormonal growth control of some transformed mammary epithelial cells. Consistent with the proliferative advantage of mammary tumor cells that produce this mitogen, TGF-alpha mRNA or protein expression has been found in 40-70% of primary metastatic human breast tumors(18, 23, 24) . Moreover, the expression and secretion of TGF-alpha can be coordinately regulated with the proliferative state of certain mammary tumors. In hormone-responsive human mammary tumor cell lines, ovarian steroids stimulate in vitro proliferation with a concomitant increase in the production of TGF-alpha(1, 5, 12, 14) , whereas treatment of breast cancer patients with the anti-estrogen tamoxifen often results in a significant decrease in the levels of TGF-alpha in the tumor(25) .

It is tempting to consider that TGF-alpha mediates the dysregulation of mammary tumor cell responsiveness to environmental cues by modulation or inhibition of the ability of systemic factors, such as steroid hormones, to control mammary cell differentiation and growth. To test this notion, we are utilizing Con8 mammary tumor cells, which are an epithelial cell line derived from a 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma(4, 26, 27) . These rat mammary tumor cells constitutively express cell surface glycoprotein antigens related to the human PAS-O human milk fat globule protein demonstrating that these cells retain differentiated characteristics of their mammary epithelial origin(26) . We have shown that TGF-alpha and glucocorticoid hormones have opposing actions on the proliferation of these mammary tumor cells. Glucocorticoid hormones suppress the growth(26, 27, 28) , induce c-jun expression(28) , and inhibit production of an autocrine-acting TGF-alpha(29, 30) , whereas treatment of steroid growth-suppressed Con8 cells with TGF-alpha restimulates DNA synthesis (28, 29, 30) and induces expression of c-myc and cyclin D1 transcripts(28) .

A key issue is whether particular differentiated properties usually associated with nontransformed mammary cells are also under reciprocal control by glucocorticoids and TGF-alpha in transformed Con8 cells. One such differentiated property is the regulation of tight junction permeability, since the onset of lactation in the normal mammary gland coincides with an increase in the structural development of the tight junction(31) . Thus, tight junction formation is important in establishing cell-cell interactions by the formation of ``seals'' between laterally adjacent cells(32, 33, 34) . We have recently shown in a nontransformed mouse mammary epithelial cell line of ductal origin that dexamethasone regulates tight junction permeability(35, 36) . Our results suggest that a similar ``normal-like'' differentiated property may be conferred upon Con8 mammary tumor cells by glucocorticoids. In this study, we show that glucocorticoids coordinately regulate tight junction permeability and suppress the growth of Con8 mammary tumor cells and that both steroid effects are reversed by exposure to TGF-alpha.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium/F12 (50:50) and the calf serum were supplied by BioWhittaker (Walkersville, MD), and the PBS (phosphate-buffered saline), trypsin-EDTA, and dexamethasone were obtained from Sigma. [^3H]Thymidine (5 Ci/mmol) was obtained from Amersham, and [^3H]inulin and [^14C]mannitol were purchased from ICN Flow Radiochemicals. Permeable supports manufactured by Nunc were distributed by Applied Scientific. Human recombinant TGF-alpha was purchased from Promega. The ZO-1 monoclonal antibodies (R40.76) were a generous gift of Bruce R. Stevenson (Dept. of Anatomy and Cell Biology, Univ. of Alberta, Edmonton), and fluorescein 5-isothiocyanate-conjugated goat anti-rat IgG antibodies were supplied by Cappel. All other reagents were of the highest purity available.

Cells and Methods of Culture

Con8 is a single cell-derived epithelial subclone obtained after collagenase digestion of the 13762NF transplantable rat mammary adenocarcinoma(26, 27) . CT93 cells are derived from Con8 mammary tumor cells by transfection of a TGF-alpha expression vector(30) . Both cell lines were routinely grown to 100% confluency on permeable tissue culture supports in Dulbecco's modified Eagle's medium/F-12 supplemented with 5% calf serum, at 37 °C in a humid atmosphere of air/CO(2) (95:5). Cell culture medium was routinely changed every 24 hours. The permeable cell culture support system by Nunc utilizes a rigid, hydrophilic, inorganic filter membrane to close one end of a short polystyrene cylinder. These inserts fit inside wells of tissue culture plates and support cell growth on the porous membrane allowing epithelial cells to import nutrients and excrete from their entire surface. In appropriate experiments, dexamethasone was added to a final concentration of 1 µM, and human recombinant TGF-alpha was added to a final concentration of 10 ng/ml. In order to inhibit DNA synthesis, cells cultures were exposed to either hydroxyurea (1 mM) or araC (cytosine beta-D-arabinofuranoside; 10 µM) at the indicated times of hormone treatment.

Assay of DNA Synthesis by [^3H]Thymidine Incorporation

DNA synthesis was quantitated by determining the incorporation of [^3H]thymidine as described previously (28) . Briefly, quadruplicate samples of Con8 or CT93 cells grown on permeable supports under the indicated hormonal conditions were incubated with 1 µCi/ml [^3H]thymidine (1 Ci/mmol) for 1 h at 37 °C in a humid atmosphere of air/CO(2) (95:5). The cells were washed four times with PBS, twice with 100% methanol, and air-dried for 12 h, and the radioactivity was quantitated by liquid scintillation counting.

Flow Cytometric Analysis of DNA Content

Cell samples containing 3 million cells were centrifuged at 800 times g for 5 min at room temperature. The supernatant fractions were removed, and resuspended cell pellets were then hypotonically lysed in 0.5 ml of ice cold DNA-staining solution (0.5 mg/ml propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100). Nuclear emitted fluorescence greater than 585 nm was measured with a Coulter Elite instrument with laser output adjusted to deliver 15 milliwatts at 488 nm. Approximately 10,000 cells were analyzed from each sample at a rate of 300-500 cells/s. The percentages of cells within the G(1), S, and G(2)/M phases of the cell cycle were determined by analysis with the computer program Multicycle provided by Phoenix Flow Systems.

Transepithelial Electrical Resistance Measurements

The transepithelial electrical resistance was measured on support-grown cells at room temperature, using the EVOM Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL) as described previously (35, 36) . Resistance measurements were taken aseptically every 8-24 h. Calculations for ohmsbulletcm^2 were determined by subtracting the resistance measurement of a blank filter and multiplying by the area of the monolayer (0.49 cm^2 for the 10-mm filters).

Paracellular Permeability

The degree of monolayer ``leakiness'' was measured by applying 400 µl of 25 µCi/ml [^3H]inulin (M(r) = 5000) or 400 µl of 25 µCi/ml [^14C]mannitol (M(r) = 182) to the apical surface of confluent cell monolayers. Six hundred microliters of cell culture medium without [^3H]inulin was applied to the basolateral reservoir of the wells. Immediately after exposing the cells to the radioactive tracer, baseline readings were taken by assaying a 4 µl aliquot of medium from the apical side and 6 µl of medium from the basolateral side of the wells. In five 1-h intervals, 4 µl from the apical surface and 6 µl from the basolateral reservoir were tested for the presence of [^3H]inulin or [^14C]mannitol by scintillation counting.

Immunofluorescence Staining for ZO-1

Con8 and CT93 cells were grown to 100% confluency on permeable 24-well permeable supports and incubated with the indicated combinations of dexamethasone and TGF-alpha. The monolayers were washed three times with Dulbecco's PBS (BioWhittaker) with 130 mg/liter CaCl(2)bullet2H(2)O and 100 mg/liter MgCl(2)bullet6H(2)O. The cells were fixed with acetone/methanol (50:50) at -20 °C for 5 min and air-dried. Fixed cells were either used immediately or stored at -20 °C. The primary and secondary antibodies for ZO-1 were diluted in TBST (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) with 3% nonfat dry milk. ZO-1-specific antibody R40.76 was used at a dilution of 1:400. Cells were incubated at room temperature for 1 h and then washed three times with TBST. The secondary antibody was fluorescein 5-isothiocyanate-conjugated goat IgG fraction to rat IgG and used at a dilution of 1:100. Cells were incubated with the antibody for 1 h in the dark at room temperature. The cell monolayers were then washed three times with TBST. The supports were punched out and mounted on glass slides in 50% glycerol, 50 mM Tris, pH 8-9 and 0.4% n-propyl gallate. Phase contrast and immunofluorescent images were photographed at 1600 ASA with Kodak Ektachrome 400ASA film.

Transfection Procedures and CAT Assays

Con8 cells from a logarithmically growing culture were incubated in Dulbecco's modified Eagle's medium/F12 medium (1:1) containing 10% calf serum and transfected by electroporation. Briefly, cells were collected after dispersion by treatment with trypsin-EDTA, washed twice with sterile calcium- and magnesium-free phosphate-buffered saline (PBS), and resuspended in sucrose buffer containing 270 mM sucrose, 7 mM sodium phosphate buffer, pH 7.4, 1 mM MgCl(2). The cells (1-2 times 10^7 cells/sample) contained in 250 µl were dispensed into sterile cuvettes and plasmid DNA (15-25 µg total including 10-15 µg of GRE-CAT) was added to cells and mixed, and 5 electric pulses (400 V square wave pulse for 99 µs) delivered to the sample using a BTX 800 Transfector apparatus (BTX Inc. San Diego, CA). Subsequently, the cells and DNA were allowed to sit for 10 min on ice, diluted with Dulbecco's modified Eagle's medium/F12 medium (1:1), plated in 100-mm Corning plastic culture dishes, and cultured at 37 °C. Twelve hours after transfection, the medium was aspirated, and the cells were washed twice in PBS and then reincubated with fresh medium. After another 12 h, cell cultures were treated for 48 h with the indicated concentrations of TGF-alpha and/or dexamethasone, and cells were harvested for CAT assays. Harvested cells were washed twice in PBS and resuspended in 0.1 M Tris-HCl, pH 7.8, and cell extracts were prepared by four cycles of freeze-thawing, alternating between ethanol-dry ice bath and 37 °C water bath, 5 min each cycle. The cell lysates were heated at 68 °C for 15 min and centrifuged at 12,000 times g for 10 min, and supernatants were recovered. The protein contents in the supernatant fractions were determined with the Bradford protein assay(37) .

CAT activity in the cell extracts containing 30-50 µg of lysate protein was measured by the nonchromatographic assay of Neumann and co-workers(38) . The enzyme assay was carried out in a final reaction volume of 250 µl in the presence of 1 µCi of [^3H]acetyl coenzyme A (specific activity 200 mCi/mmol, DuPont NEN), 25 µl of 1 M Tris-HCl, pH 7.8, and chloramphenicol (50 µl of a 5 mM aqueous solution). The reaction mixture was gently overlaid with 4 ml of a water-immiscible scintillation fluor (Econofluor, DuPont NEN) and incubated at 37 °C for 4 h. The CAT activity was monitored by direct measurement of radioactivity by liquid scintillation counting. Measurements of CAT activity were in the linear range of the assay as determined by a standard curve using bacterial CAT enzyme. The enzyme activity was expressed as a function of protein present in corresponding cell lysates (^3H-acetylated chloramphenicol produced, cpm/µg of protein/4 h). Pure bacterial CAT enzyme (0.01 unit; Pharmacia, Uppsala, Sweden) was utilized as a positive control for the CAT enzymatic assays, while mock-transfected cells were used to establish basal level activity. Each experiment was performed in triplicate and was repeated two or more times.


RESULTS

Glucocorticoids Stimulate Tight Junction Function and Suppress DNA Synthesis of Con8 Rat Mammary Tumor Cells

We have previously shown that the synthetic glucocorticoid, dexamethasone, stimulates tight junction formation in nontransformed mouse mammary epithelial cells in vitro by monitoring an increase in monolayer transepithelial electrical resistance (TER) and by the inhibition of apical to basolateral movement of small radiolabeled tracers(35, 36) . An intriguing issue is whether dexamethasone can induce a similar normal-like differentiated property in growth-suppressible rodent mammary tumor cells. Therefore, confluent monolayers of Con8 mammary tumor cells were grown on permeable supports, treated in the presence or absence of 1 µM dexamethasone, and assayed for the formation of tight junctions by measuring the monolayer TER over a 3-day time course. [^3H]Thymidine incorporation was used to determine rates of DNA synthesis. As shown in Fig. 1(upper panel), dexamethasone induced a striking increase in monolayer electrical resistance over a 60-h time course, while, in untreated cells, the monolayer electrical resistance remained constant and at low levels. Under these conditions, the apical to basal paracellular permeability of [^3H]inulin was inhibited 32-fold in dexamethasone-treated cells compared to untreated cells (see below) demonstrating that the increase in electrical resistance was due to the formation of a tight monolayer.


Figure 1: Dexamethasone stimulates transepithelial electrical resistance and suppresses DNA synthesis of Con8 mammary tumor cells. Upper panel, Con8 cells were cultured on permeable supports in the presence (+DEX) or absence (-DEX) of 1 µM dexamethasone. At the indicated times, the monolayer transepithelial electrical resistance was monitored, and the ohmsbulletcm^2 were calculated as described in the text. Each assay was performed in triplicate, and the results are an average from three separate experiments. Lower panel, Con8 cells were cultured on permeable supports in the presence (+DEX) or absence (-DEX) of 1 µM dexamethasone. The rate of DNA synthesis was monitored at the indicated times during a 60-h time course by determining the incorporation of [^3H]thymidine after a 1-h pulse label as described in the text.



Conceivably, dexamethasone may be stimulating monolayer tight junction-like properties by causing an overgrowth of Con8 mammary tumors on the filters. However, consistent with our previous observations using a plastic substratum(25, 26, 28) , dexamethasone significantly suppressed [^3H]thymidine incorporation of Con8 mammary tumor cells grown on permeable supports within 36 h of steroid treatment (Fig. 1, lower panel). The suppression of [^3H]thymidine incorporation using cells cultured on permeable supports is within the time frame observed for glucocorticoids to induce an early G(1) block in cell cycle progression of cells cultured on plastic substratum(28) . Thus, in the absence of glucocorticoids, Con8 mammary tumor cells rapidly proliferate and fail to produce a tight monolayer, whereas stimulation of tight junction formation was coincident with the dexamethasone inhibition of cell growth.

Glucocorticoid Stimulation of Monolayer Transepithelial Electrical Resistance and Inhibition of Paracellular Leakage Correlates with Receptor Occupancy and Function

In order to assess the functional relationship of glucocorticoid receptor occupancy and function to the steroid-dependent regulation of tight junction formation, the TER and apical to basolateral [^14C]mannitol leakage of Con8 cell monolayers were monitored after treatment of Con8 cells with different concentrations of dexamethasone. To determine the dose response of receptor function, dexamethasone inhibition of DNA synthesis (via [^3H]thymidine incorporation) was used to monitor the growth suppression response. A parallel set of mammary tumor cells was transfected with a GRE-CAT chimeric reporter gene containing three functional glucocorticoid response elements and assayed for dexamethasone-stimulated chloramphenicol acetyltransferase (CAT) activity. As shown in Fig. 2(upper panel), dexamethasone dose-dependently stimulated the monolayer TER and inhibited paracellular leakage with half-maximal responses of approximately 6 nM dexamethasone which is similar to the half-maximal binding of this steroid to Con8 cell glucocorticoid receptors(26) . Importantly, the half-maximal inhibition of apical to basolateral paracellular leakage of [^14C]mannitol (M(r) = 182) approximated the half-maximal concentration of dexamethasone required to stimulate monolayer TER (Fig. 2, upper panel). Thus, the dexamethasone-dependent increase in electrical resistance dose-dependently corresponded with a reduction in paracellular permeability confirming that the dexamethasone-induced increase in TER was indicative of a decrease in tight junction permeability or ``gate'' function(33) .


Figure 2: Dexamethasone dose-response of transepithelial electrical resistance, DNA synthesis, and receptor function. Upper panel, Con8 mammary tumor cells were cultured on permeable supports for 2 days in the presence of the indicated concentrations of dexamethasone. The monolayer transepithelial electrical resistance was determined, and the ohmsbulletcm^2 were calculated as described in the text. The apical to basolateral leakage of [^14C]mannitol (M(r) = 182) was monitored as described in the text. The results are an average of triplicate samples. Lower panel, Con8 cells were cultured on permeable supports and analyzed for DNA synthesis by incorporation of [^3H]thymidine. A parallel set of cultures were transfected with the GRE-CAT chimeric reporter gene by electroporation and assayed for dexamethasone-stimulated CAT activity as described in the text. The results are an average of triplicate samples.



The dexamethasone stimulation in TER also dose-dependently correlated with glucocorticoid receptor transcriptional activation of the chimeric GRE-CAT reporter gene as well as with an inhibition of DNA synthesis (Fig. 2, upper versus lower panels). Steroids which are either neutral with respect to their glucocorticoid activity (such as beta-estradiol or testosterone) or which show glucocorticoid antagonist activity (RU38486, progesterone) failed to induce Con8 monolayer TER (data not shown). Thus, the dexamethasone stimulation of Con8 monolayer TER dose-dependently correlated with glucocorticoid receptor occupancy and function which suggests that tight junction permeability is regulated in Con8 cells by a glucocorticoid receptor-mediated process, rather than as a result of an aberrant interaction between the steroid and the plasma membrane. Furthermore, consistent with our previous results with nontransformed mammary cells (35) , the glucocorticoid-induced formation of tight junctions in Con8 mammary tumor cells required de novo protein synthesis and normal levels of extracellular calcium (data not shown).

TGF-alpha Stimulates the Cell Cycle of Glucocorticoid Growth-suppressed Con8 Cells and Inhibits Glucocorticoid-induced Transepithelial Electrical Resistance

Dexamethasone disrupts a TGF-alpha autocrine loop in Con8 mammary tumor cells(29) , while exogenously added TGF-alpha restimulates the growth of glucocorticoid suppressed cells(28, 29, 30) . The growth effects of TGF-alpha was utilized to address the question of whether the glucocorticoid-induced differentiated property of tight junction formation is related to or independent of the proliferative state of mammary tumor cells. To directly assess the cell cycle effects of TGF-alpha, nuclei isolated from Con8 cells treated with combinations of dexamethasone and TGF-alpha for 48 h were analyzed for their nuclear DNA content. Flow cytometry profiles of nuclear DNA content after propidium iodide fluorescent staining revealed that TGF-alpha reversed the dexamethasone-mediated block in cell cycle progression. As shown in Fig. 3, dexamethasone treatment altered the DNA content of the mammary tumor cell population from an asynchronous population of growing cells in all phases of the cell cycle to one in which approximately 85% of dexamethasone-treated mammary tumor cells exhibited a 2n DNA content, which is indicative of the G(1) block in cell cycle progression(28) . Exposure of Con8 cells to TGF-alpha prevented dexamethasone from inducing a cell cycle arrest since the FACS profile in the presence of both steroid and growth factor was that of a population of proliferating cells; 61% of growth factor treated cells have a 2n DNA content equivalent to the G(1) phase of the cell cycle, 29% of the cells have a S phase DNA content, and 10% have a 4n G(2)/M DNA content (Fig. 3).


Figure 3: Effects of dexamethasone and TGF-alpha on cell cycle phase distribution of Con8 mammary tumor cells. Con8 cells were treated with the indicated combinations of 1 µM dexamethasone (DEX) and 10 ng/ml human recombinant TGF-alpha, cell extracts were stained with propidium iodide, and nuclei were analyzed for DNA content by flow cytometry with a Coulter Elite Laser. Approximately 10,000 cells were analyzed from each sample. The percentages of cells within the G(1), S, and G(2)/M phases of the cell cycle were determined with the Multicycle computer program as described under ``Experimental Procedures.''



To determine whether TGF-alpha reverses the glucocorticoid-stimulated formation of tight junctions under the conditions that promote cell cycle progression, confluent mammary tumor cell monolayers grown on permeable supports were treated with 1 µM dexamethasone for 24 h to induce tight junction formation and then incubated in the presence or absence of growth factor. As shown in Fig. 4, the monolayer transepithelial electrical resistance was reduced to basal levels within 18 h of TGF-alpha treatment. We have previously shown that this time course is sufficient for TGF-alpha to stimulate the cells to move through G(1) and S phases(28) . Thus, under conditions in which TGF-alpha restimulates proliferation of glucocorticoid suppressed cells, the function of pre-formed tight junctions is coordinately disrupted.


Figure 4: TGF-alpha overrides the dexamethasone-stimulated monolayer transepithelial electrical resistance. Con8 mammary tumor cells were cultured on permeable supports in the presence (+DEX) of 1 µM dexamethasone, and one control culture was incubated in the absence of steroid (-DEX). After 24 h in dexamethasone, cells were treated in the presence (+DEX/+TGFalpha) or absence (+DEX) of 10 ng/ml of human recombinant TGF-alpha. Throughout the 42-h time course, the monolayer transepithelial electrical resistance was determined at the indicated times, and the ohmsbulletcm^2 were calculated. The results are an average from triplicate samples.



De Novo DNA Synthesis Is Not Required for TGF-alpha to Disrupt Tight Junction Formation

Given the mitogenic effects of TGF-alpha on Con8 mammary tumor cells, it was important to test whether the disruption of tight junction formation by this growth factor is an indirect consequence of the onset of cell proliferation or a more direct target of TGF-alpha-mediated receptor signaling. To test this notion, the effects of TGF-alpha on monolayer TER was monitored in the presence or absence of either of two DNA synthesis inhibitors, hydroxyurea or cytosine beta-D-arabinofuranoside (araC). After 48 h of dexamethasone treatment, Con8 cells were treated with combinations of TGF-alpha and hydroxyurea or araC and assayed for monolayer TER over the next 48 h. As shown in Fig. 5, TGF-alpha reduced the monolayer TER in the presence of either araC (upper left panel) or hydroxyurea (lower left panel), although this effect was slightly delayed compared to cells treated with TGF-alpha only. Neither of these DNA synthesis inhibitors prevented dexamethasone from further stimulating the monolayer TER in the absence of TGF-alpha. Under the conditions of this experiment, araC (upper right panel) or hydroxyurea (lower right panel) abolished the incorporation of [^3H]thymidine in the presence or absence of TGF-alpha, demonstrating that these metabolic agents effectively inhibited DNA synthesis in cells cultured on permeable supports. These results demonstrate that the TGF-alpha inhibition of tight junction formation does not require de novo DNA synthesis and, since these inhibitors act at or near the G(1)/S border(39, 40) , cell cycle progression into and past S phase is not needed in order for TGF-alpha to disrupt the function of intercellular junctional complexes.


Figure 5: TGF-alpha disrupts the glucocorticoid-stimulated formation of tight junctions in the presence of DNA synthesis inhibitors. Con8 mammary tumor cells were cultured on permeable supports in the presence of 1 µM dexamethasone (DEX), while one set of control cultures were not treated with hormone (No Additions). After a 48-h steroid treatment (arrow), dexamethasone-treated cells were incubated with the indicated combinations of 10 ng/ml TGF-alpha (TGF-alpha) or either 10 µM cytosine beta-D-arabinofuranoside (top panels: araC) or 1 mM hydroxyurea (lower panels: HU). At the indicated times, the monolayer transepithelial electrical resistance was determined, and the ohmsbulletcm^2 were calculated (left panels). At the final time point, DNA synthesis was monitored by the incorporation of [^3H]thymidine as described in the text (right panels). The results are an average from triplicate samples.



TGF-alpha Does Not Impair Glucocorticoid Receptor Function

Conceivably, one indirect mechanism by which TGF-alpha disrupts tight junction formation is by inhibiting glucocorticoid receptor function. To test this possibility, Con8 cells were transfected with the GRE-CAT chimeric reporter gene and assayed for CAT activity after treatment with combinations of dexamethasone and TGF-alpha during a 48-h time course. The dexamethasone strongly stimulates transcriptional activation of the chimeric GRE-CAT reporter gene after 48 h of continuous treatment in steroid in the presence or absence of TGF-alpha (Fig. 6, upper four bar graphs). Since TGF-alpha stimulates cell cycle progression of dexamethasone-treated cells, the CAT enzymatic specific activity of dexamethasone/TGF-alpha-treated cells is approximately 50% lower than dexamethasone-treated cells due to the increase in total cell protein with no corresponding change in CAT plasmid levels. Consistent with this concept, if TGF-alpha is added during the last 24 h of a 48-h dexamethasone time course, the dexamethasone-induced CAT specific activity is intermediate between cells treated with TGF-alpha for 48 h or without any added TGF-alpha (Fig. 6, bottom bar graph versus 2nd and 4th bar graphs). Finally, when dexamethasone is added during the last 24 h of a 48-h time course in TGF-alpha, the dexamethasone-induced CAT specific activity is essentially the same as that observed in cells treated with only dexamethasone for 24 h (Fig. 6, 6th versus 7th bar graph). Taken together, these results demonstrate that TGF-alpha does not impair glucocorticoid receptor function under conditions in which tight junction formation is rapidly disrupted.


Figure 6: TGF-alpha does not inhibit glucocorticoid receptor function. Con8 mammary tumor cells were transfected with the GRE-CAT chimeric reporter gene by electroporation and then cultured on permeable supports for 48 h with the indicated combinations and time of incubation with 1 µM dexamethasone (DEX) and 10 ng/ml TGF-alpha (TGF-alpha). Cell extracts were assayed for CAT specific activity as described in the text. The results are an average of two independent sets of triplicate samples.



Constitutive Expression of TGF-alpha Prevents the Glucocorticoid-stimulated Formation of Tight Junctions

Compared to their normal cell counterparts, many mammary tumor cells constitutively produce autocrine acting growth factors which cause the cells to proliferate in an uncontrolled manner. Therefore, as a complementary approach to examining the relationship between the continuous presence of TGF-alpha and disruption of cell-cell contact, the glucocorticoid-regulated formation of tight junctions was monitored in mammary tumor cells which constitutively overexpress TGF-alpha. Con8 cells were transfected with a cytomegalovirus promoter-driven TGF-alpha expression vector; one subclone of transfected Con8 cells (designated CT93) was found to constitutively produce high levels of secreted TGF-alpha(30) . The ability of dexamethasone to induce tight junction formation and suppress proliferation of Con8 and CT93 cells was examined in cells cultured on permeable filter supports. Monolayer transepithelial electrical resistance was measured over a 48-h time course. Dexamethasone failed to stimulate the TER of mammary tumor cell monolayers which constitutively express TGF-alpha (Fig. 7). In contrast, glucocorticoids regulated the monolayer tight junction permeability of nontransfected Con8 cells (Fig. 7) as well as vector-transfected controls (data not shown). To determine whether constitutive expression of TGF-alpha also prevents the glucocorticoid-mediated inhibition of paracellular transport, the apical to basolateral movement of [^3H]inulin (M(r) = 5,000) was monitored in TGF-alpha-transfected and nontransfected Con8 cells. As shown in Fig. 8, dexamethasone induced a 32-fold reduction of [^3H]inulin paracellular transport in nontransfected Con8 cells, while steroid treatment failed to reduce [^3H]inulin leakage in CT93 cells which constitutively express TGF-alpha.


Figure 7: Constitutive expression of transforming growth factor-alpha blocks the glucocorticoid stimulation of monolayer transepithelial electrical resistance. Con8 mammary tumor cells and CT93 cells, which constitutively express TGF-alpha, were cultured on permeable supports in the presence (+DEX) or absence (-DEX) of 1 µM dexamethasone. The transepithelial electrical resistance was monitored over a 48-h time course, and the ohmsbulletcm^2 were determined as described in the text.




Figure 8: Effects of glucocorticoids and constitutive expression of transforming growth factor-alpha on paracellular transport of [^3H]inulin. Con8 mammary tumor cells and CT93 cells, which constitutively express TGF-alpha, were cultured on permeable supports in the presence or absence of 1 µM dexamethasone for 3 days. [^3H]Inulin (M(r) = 5000) was added apically and assayed in the basolateral media after 4 h. The paracellular transport was calculated as the amount of radiolabeled [^3H]inulin detected in the basolateral media, divided by the total amount of [^3H]inulin added to the apical media compartment. The baseline used to determine the -fold induction is defined by the amount of [^3H]inulin which diffused through the support membrane of cell-free filters.



To confirm that constitutive expression of TGF-alpha overrides the glucocorticoid-mediated growth suppression response, nontransfected Con8 cells and TGF-alpha-transfected CT93 cells were cultured on permeable supports, and DNA synthesis was examined in cells treated with or without dexamethasone. Dexamethasone inhibited [^3H]thymidine incorporation in nontransfected Con8 cells but not in the CT93 cells (Fig. 9). Thus, constitutive expression of TGF-alpha prevents glucocorticoids from suppressing the growth and regulating tight junction permeability. Conceivably, the failure of glucocorticoids to stimulate TER and reduce paracellular transport in CT93 cells could be due to clonal variation of transfected cells and not due to the effects of TGF-alpha per se. Two lines of evidence argue against this possibility. First, several independently isolated subclones of transfected Con8 cells which constitutively express TGF-alpha (30) show the same phenotype as CT93 cells (data not shown). Secondly, the direct addition of TGF-alpha to dexamethasone-treated Con8 cells reduced the monolayer TER back to basal levels within 24 h of growth factor treatment, concomitantly with a stimulation in [^3H]thymidine incorporation (Fig. 9). TGF-alpha addition to transfected CT93 cells did not further reduce the TER or stimulate DNA synthesis (Fig. 9).


Figure 9: Effects of glucocorticoids and transforming growth factor-alpha on DNA synthesis and transepithelial electrical resistance of mammary tumor cells. Con8 mammary tumor cells and CT93 cells, which constitutively express TGF-alpha, were cultured on permeable supports in the presence or absence of 1 µM dexamethasone for 3 days. Cells which had been treated with 1 µM dexamethasone for 48 h were then exposed to medium containing dexamethasone and 10 ng/ml human recombinant TGF-alpha. The cells were then incubated with both factors for 2 days. The incorporation of [^3H]thymidine and transepithelial electrical resistance were monitored as described in the text.



Effects of Glucocorticoids and TGF-alpha on ZO-1 Distribution

ZO-1 is an intracellular peripheral membrane protein that is associated with tight junctions in epithelial cells(32, 41) . Immunofluorescence studies using monoclonal ZO-1 specific antibodies demonstrated that after 48 h of glucocorticoid treatment, during which monolayers of Con8 cells established significant electrical resistance, ZO-1 protein is localized to the tight junction at cell peripheries in a sharp continuous band of immunostaining surrounding each cell (Fig. 10B). In the absence of steroid treatment, a much higher proportion of ZO-1 immunofluorescence was distributed throughout the cytoplasm (Fig. 10A). Treatment with (C) or constitutive expression of (D) TGF-alpha disrupted the distribution of ZO-1 at the cell periphery that is typically observed in glucocorticoid growth suppressed Con8 mammary tumor cells. In the presence of TGF-alpha, a high proportion of ZO-1 was detected in the cytoplasmic compartment as indicated by the diffuse staining throughout the cell. In addition, the residual ZO-1 immunostaining at the cell periphery exhibited a spotty and discontinuous pattern (Fig. 10, C and D). Thus, the differential distribution patterns of the ZO-1 tight junction protein reflect the ability of TGF-alpha to override the glucocorticoid stimulation of tight junction formation in mammary tumor cells.


Figure 10: Effects of glucocorticoids and transforming growth factor-alpha on ZO-1 localization. Con8 mammary tumor cells were treated with no hormones (panel A: Con8 - DEX), 1 µM dexamethasone (panel B: Con8 + DEX), or dexamethasone and 10 ng/ml human recombinant TGF-alpha (panel C: Con 8 + DEX/+ TGFalpha) for 2 days. CT93 cells, which constitutively express TGF-alpha, were treated with 1 µM dexamethasone (CT93 + DEX) for 2 days. The cells were fixed and analyzed for ZO-1 localization by indirect immunofluorescence as described in the text. Cell pictures were originally photographed at times 430 magnification.




DISCUSSION

Our results with Con8 mammary tumor cells have demonstrated that glucocorticoids can coordinately suppress cell proliferation and stimulate tight junction formation and thereby confer to this transformed cell type normal-like growth and differentiation characteristics. Exposure of glucocorticoid growth-suppressed cells to the mammary mitogen TGF-alpha rapidly stimulated cell proliferation and caused the dysregulation of tight junction permeability, resulting in a loss of monolayer tightness. Moreover, constitutive expression of TGF-alpha precluded glucocorticoids from mediating either the growth suppression or tight junction responses. The TGF-alpha disruption of tight junctions is based on the observed reduction in monolayer transepithelial electrical resistance, stimulation of paracellular transport, and redistribution of the ZO-1 tight junction protein. Malignantly transformed mammary cells can often display a loss of responsiveness to particular sets of extracellular signals. One mechanism of dysregulation is the inappropriate production of growth factors and/or function of their cognate receptors, which alter proliferative and/or differentiated properties(1, 2, 3, 4, 5) . It is therefore tempting to consider that TGF-alpha may exert many of its tumorigenic effects on mammary epithelial cells by not only providing a proliferative advantage to transformed cells expressing EGF receptors, but also by altering the way in which the cells respond to steroid-induced signals, which are normally responsible for maintaining critical cell-cell interactions at junctional complexes.

The glucocorticoid stimulation of transepithelial electrical resistance is a receptor-dependent process which occurs under conditions in which the mammary tumor cells are growth-suppressed, whereas TGF-alpha simultaneously stimulates DNA synthesis and reverses the steroid effects on tight junction permeability. This inverse relationship between cell proliferation and regulation of cell-cell interactions may have important implications for understanding mechanisms of invasiveness and metastasis of mammary tumors. The unrestricted growth of tumors is dependent upon vascularization of the tumor(42) . It is conceivable that autocrine or paracrine growth factors with angiogenic activities may regulate this process, in part, by causing the dissolution of epithelial cell tight junctions to allow invasion of new blood vessels. A number of other studies have shown that other types of cell-cell interactions are altered in transformed cells. For example, the formation of desmosomes, which are patches of intercellular contacts(43) , is inversely related to the stage of lung cancers and their ability to metastasize(44) . Similarly, it was found that certain connexins, which are gap junction proteins that regulate the formation of intercellular channels(45, 46) , are transcriptionally down-regulated in human mammary tumor cell lines but not in primary normal or nontransformed cells(47) . It has been proposed that the connexin-mediated channels help to transmit growth-controlling signals between cells(47, 48) . Several connexins have been shown to be selectively produced in nontumorigenic cells and when overexpressed slow the growth of transformed cells(48, 49) . Thus, an alteration in cell-cell communication may protect transformed cells from particular types of growth inhibition, or alternatively, growth-inhibited cells may be more capable of forming particular intercellular junctional complexes. In this regard, we have previously shown that glucocorticoids induce a G(1) block in cell cycle progression of Con8 mammary tumor cells(28) , suggesting that this growth suppression is a prerequisite for the assembly of functional tight junctions.

The TGF-alpha disruption of tight junction formation did not require the mammary tumor cells to be actively cycling since the growth factor-mediated reduction in monolayer TER occurs in the presence of two different inhibitors of DNA synthesis. Both araC and hydroxyurea block cell cycle progression at the G(1)/S boundary(39, 40) , whereas, as discussed above, TGF-alpha overrides the glucocorticoid-mediated block in cell cycle progression early in the G(1) phase(28) . These results suggest that the regulation of tight junction functionality may either be directly linked to the control of the G(1) phase of the cell cycle up to the S phase boundary or that tight junction formation may not be a cell cycle-regulated process per se. Regardless of the precise connection between cell cycle control and tight junction formation, the disruption of monolayer TER by TGF-alpha in the presence of DNA synthesis inhibitors implicates the tight junction machinery as a selective target of EGF receptor signaling and not just an indirect consequence of cell cycle progression after growth factor treatment.

Components of intercellular junctional complexes have recently been implicated in growth regulation, such as the genes which encode certain tight junction-associated proteins and adhesion molecules(50) . One such gene product is the tight junction-associated ZO-1 protein which is homologous to a class of tumor suppressor genes. The amino-terminal half of ZO-1 displays significant sequence homology to the product of the lethal discs large (dlg) gene of Drosophila(50, 51) . The dlg gene product is localized in the undercoat of the septate junction in Drosophila which is considered to be analogous to the tight junction of vertebrate epithelial cells. Mutations in dlg result in a loss of apical-basolateral epithelial cell polarity and in neoplastic growth which implicates this gene as a tumor suppressor gene(50) . It is conceivable that junctional plaque proteins play a role in suppression of the malignant phenotype by orchestrating the interactions of junctional adhesion receptors and cytoplasmic signal transducers which are involved in the negative regulation of cell growth. Consistent with this idea, we have shown that under conditions in which TGF-alpha mediates a dysregulation of Con8 cell growth and tight junction permeability, ZO-1 is redistributed from a pericellular location to a more cytoplasmic compartment. The regions of ZO-1 most homologous to the tumor suppressor genes are the filamentous domain, an SH3 domain, and a guanylate kinase domain(50, 51) . These domains represent interesting potential targets for steroid or growth factor control of the localization and/or function of this tight junction-associated protein, since similar structural features are fundamentally involved in receptor-mediated signal transduction (52) .

The maintenance of tight junction function is a normal-like differentiated property which prevents the mixing of molecules from the apical and basolateral membranes and which precludes paracellular permeability(34, 53, 54, 55) . Tight junction permeability of mammary epithelia is regulated during the onset of lactation in which milk components are strictly secreted into the lumen of the ducts via apical-directed secretory pathways(31) . Lactogenic steroid and protein hormones and a variety of growth factors are known to be involved in regulating the temporal and tissue-specific development of the lactogenic state. Our in vitro work with nontransformed mammary epithelial cells (35, 36) suggests that glucocorticoids are likely to be the lactogenic hormones responsible for regulation of tight junction permeability. Glucocorticoids can exert their effects on gene expression by specific binding of the steroid receptor complex to DNA transcriptional enhancer elements which are present in promoters of steroid controlled genes, or by interfering with the action of other transcription factors, such as the JunbulletFos AP-1 transcription complex, via protein-protein interactions(56, 57, 58, 59) . Given this mechanism of glucocorticoid hormone action, is it likely that dexamethasone regulates the transcription of key genes encoding protein components or regulatory factors which modulates tight junction formation. The timing of glucocorticoid-induced gene expression is critical in that it may initiate a transcriptional cascade in which early regulated gene products initiate the growth suppression response, whereas later-acting response genes may maintain the growth-inhibited state and regulate tight junction permeability.

TGF-alpha overrides glucocorticoid growth suppression and coordinately causes a dysregulation of tight junction permeability and ZO-1 localization which suggests a novel role for TGF-alpha in disrupting the differentiated function of mammary tumor cells. The degree of permeability of the tight junctions is known to be regulated by intracellular signals initiated by protein kinase C, phospholipase C, adenylate cyclase, and GTP-binding proteins, as well as calcium (60, 61, 62, 63, 64) . Any of these signaling components may be downstream targets of TGF-alpha-mediated cascades initiated by activation of the EGF receptor tyrosine kinase. We are currently attempting to elucidate the transcriptional and secondary signaling events underlying TGF-alpha control that operate in mammary tumor cells and allow this growth factor to override the effects of glucocorticoids. Conceivably, such pathways represent an important ``cross-talk'' between growth factor and steroid receptor signal transduction cascades that are necessary to guide the functional relationships between particular sets of environmental cues acting on mammary epithelial cells.


FOOTNOTES

*
This research was supported by a grant from the National Institutes of Health (DK-42799). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Molecular and Cell Biology, Box 591 LSA, University of California, Berkeley, CA 94720.

(^1)
The abbreviations used are: TGF-alpha, transforming growth factor-alpha; EGF, epidermal growth factor; TER, transepithelial electrical resistance; PBS, phosphate-buffered saline; araC, cytosine beta-D-arabinofuranoside; CAT, chloramphenicol acetyltransferase; GRE, glucocorticoid response element.


ACKNOWLEDGEMENTS

We thank Carolyn Cover, Anita C. Maiyar, and Ross Ramos for their constructive comments during the course of the work and for their critical reading of this manuscript. We also express our appreciation to John Underhill and Jerry Kapler for their skillful photography, Peter Schow for his assistance with the flow cytometry, and Anna Fung for her preparation of this manuscript as well as Charles Jackson, William Meilandt, Marina Chin, Ritu Patel, Vinh Trinh, and Thai Truong for their technical support.


REFERENCES

  1. Imagawa, W., Bandyopadhyay, G. K., and Nandi, S. (1990) Endocrine Rev. 11, 494-523 [Medline] [Order article via Infotrieve]
  2. Topper, Y. J., and Freeman, C. S. (1980) Physiol. Rev. 60, 1049-1106 [Free Full Text]
  3. Welsch, C. W. (1985) Cancer Res. 45, 3415-3443 [Abstract]
  4. Segaloff, A. (1966) Recent Prog. Horm. Res. 22, 351-379 [Medline] [Order article via Infotrieve]
  5. Dickson, R. B., and Lippman, M. E. (1987) Endocrine Rev. 8, 29-43 [Medline] [Order article via Infotrieve]
  6. Oka, T., and Perry, J. (1974) J. Biol. Chem. 249, 3586-3591 [Abstract/Free Full Text]
  7. Dembinski, T. C., and Shiu, R. P. C. (1987) in The Mammary Gland Development, Regulation, and Function (Neville, M. C., and Daniel, C. W., eds) pp. 355-381, Plenum Publishing Corp., New York
  8. Haslam, S. Z. (1987) in The Mammary Gland: Development, Regulation, and Function (Neville, M. C., and Daniel, C. W., eds) pp. 499-533, Plenum Publishing Corp., New York
  9. Derynck, R. (1988) Cell 54, 593-595 [Medline] [Order article via Infotrieve]
  10. Carpenter, G., and Cohen, S. (1990) J. Biol. Chem. 265, 7709-7712 [Free Full Text]
  11. Snedeker, S. M., Brown, C. F., and DiAugustine, R. P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 276-280 [Abstract]
  12. Vonderhaar, B. K. (1988) Cancer Treat. Res. 40, 252-266
  13. Salomon, D. S., Dickson, R. B., Normanno, N., Saeki, T., Kim, N., Kenney, N., and Ciardello, F. (1992) Curr. Perspect. Mol. Cell. Oncol. 1, 211-260
  14. Lippman, M. E., and Dickson, R. B. (1989) Recent Prog. Horm. Res. 45, 383-440 [Medline] [Order article via Infotrieve]
  15. Schreiber, A. B., Winkler, M. E., and Derynck, R. (1986) Science 232, 1250-1253 [Medline] [Order article via Infotrieve]
  16. Bates, S. E., Davidson, N. E., Valverius, E. M., Freter, C. E., Dickson, R. B., Tam, J. P., Kudlow, J. E., Lippman, M. E., and Salomon, D. S. (1988) Mol. Endocrinol. 2, 543-555 [Abstract]
  17. Arteaga, C. L., Coronado, E., and Osborne, C. K. (1988) Mol. Endocrinol. 2, 1064-1069 [Abstract]
  18. Klijn, J. G. M., Berns P. M. J. J., Schmitz, P. I. M., and Foekens, J. A. (1992) Endocrine Rev. 13, 3-17 [Abstract]
  19. Clarke, R., Brunner, N., Katz, D., Glanz, P., Dickson, R. B., Lippman, M. E., and Kern, F. G. (1989) Mol. Endocrinol. 3, 372-380 [Abstract]
  20. Ciardello, F., McGeady, M. L., Kim, N., Basolo, F., Hynes, N., Langton, B. C., Yokozaki, H., Saeki, T., Elliot, J. W., and Masui, H. (1990) Cell Growth Differ. 1, 407-420 [Abstract]
  21. Matsui, Y., Halter, S. A., Holt, J. T., Hogan, B. L. M., and Coffey, R. J. (1990) Cell 61, 1147-1155 [Medline] [Order article via Infotrieve]
  22. Liu, S. C., Sanfilippo, B., Perroteau, I., Derynck, R., Salomon, D. S., and Kidwell, W. R. (1987) Mol. Endocrinol. 1, 683-692 [Abstract]
  23. Murray, P. A., Barrett-Lee, P., Travers, M., Luqmani, Y., Powles, T., and Coombes, R. C. (1993) Br. J. Cancer 67, 1408-1412 [Medline] [Order article via Infotrieve]
  24. Ciardello, F., Kim, N., McGeady, M. L., Liscia, D. S., Saeki, T., Bianco, C., and Salomon, D. S. (1991) Ann. Oncol. 2, 169-182 [Abstract]
  25. Noguchi, S., Motomura, K., Inaji H., Imaoka, S., and Koyama, H. (1993) Cancer 72, 131-136 [Medline] [Order article via Infotrieve]
  26. Webster, M. K., Guthrie, J., and Firestone, G. L. (1990) J. Biol. Chem. 265, 4831-4838 [Abstract/Free Full Text]
  27. Webster, M. K., Guthrie, J., and Firestone, G. L. (1991) Cancer Res. 51, 6031-6038 [Abstract]
  28. Goya, L., Maiyar, A. C., Ge, Y., and Firestone, G. L. (1993) Mol. Endocrinol. 7, 1121-1132 [Abstract]
  29. Alexander, D. B., Goya, L., Webster, M. K., Haraguchi, T., and Firestone, G. L. (1993) Cancer Res. 53, 1808-1815 [Abstract]
  30. Goya, L., Alexander, D. B., Webster, M. K., Kern, F. G., Guzman, R. C., Nandi, S., and Firestone, G. L. (1993) Cancer Res. 53, 1816-1822 [Abstract]
  31. Pitelka, D. R. (1978) in Lactation: A Comprehensive Treatise (Larson, B. L., and Smith, V. R., eds) pp. 41-66, Academic Press, New York
  32. Citi, S. (1993) J. Cell Biol. 121, 485-489 [Medline] [Order article via Infotrieve]
  33. Mandel, L. J., Bacallao, R., and Zampighi, G. (1993) Nature 361, 552-555 [CrossRef][Medline] [Order article via Infotrieve]
  34. Gumbiner, B. M. (1993) J. Cell Biol. 123, 1631-1633 [Medline] [Order article via Infotrieve]
  35. Zettl, K. S., Sjaastad, M. D., Riskin, P. M., Parry, G., Machen, T. E., and Firestone, G. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9069-9073 [Abstract]
  36. Singer, K. L., Stevenson, B. R., Woo, P. L., and Firestone, G. L. (1994) J. Biol. Chem. 269, 16108-16115 [Abstract/Free Full Text]
  37. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  38. Neumann, J. R., Morency, C. A., and Russian, K. O. (1987) BioTechniques 5, 444-447
  39. Rius, C., and Aller, P. (1992) J. Cell Sci. 101, 395-401 [Abstract]
  40. Tomita, K., and Plager, J. E. (1979) Cancer Res. 39, 4407-4411 [Abstract]
  41. Madara, J. L., Carlson, S., and Anderson, J. M. (1993) Am. J. Physiol. 264, C1096-C1101
  42. Denekamp, J. (1993) Br. J. Radiol. 66, 181-196 [Abstract]
  43. Jones, J. C., and Green, K. J. (1991) Curr. Opin. Cell Biol. 3, 127-132 [Medline] [Order article via Infotrieve]
  44. McDonagh, D., Vollmer, R. T., and Shelburne, J. D. (1991) Modern Pathol. 4, 436-439 [Medline] [Order article via Infotrieve]
  45. Bräuner, T., Schmid, A., and Hülser, D. F. (1990) Invasion & Metastasis 10, 18-30
  46. Bräuner, A., and Hülser, D. F. (1990) Invasion & Metastasis 10, 31-48
  47. Lee, S. W., Tomasetto, C., Paul, D., Keyomarsi, K., and Sager, R. (1992) J. Cell Biol. 118, 1213-1221 [Abstract]
  48. Mehta, P. P., Hotz-Wagenblatt, A., Rose, B., Shalloway, D., and Loewenstein, W. R. (1991) J. Membr. Biol. 124, 207-225 [Medline] [Order article via Infotrieve]
  49. Lee, S. W., Tomasetto, C., and Sager, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2825-2829 [Abstract]
  50. Tsukita, S., Itoh, M., Nagafuchi, A., Yonemura, S., and Tsukita, S. (1993) J. Cell Biol. 123, 1049-1053 [Medline] [Order article via Infotrieve]
  51. Willott, E., Balda, M. S., Fanning, A., Jameson, B., Van Itallie, C., and Anderson, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7834-7838 [Abstract/Free Full Text]
  52. Pawson, T., Olivier, P., Roxakis-Adcock, M., McGlade, J., and Henkemeyer, M. (1993) Proc. R. Soc. Lond. Ser. B Biol. Sci. 340, 279-285
  53. Ren, J., Hamada, J., Takeichi, N., Fujikawa, S., and Kobayashi, H. (1990) Cancer Res. 50, 358-362 [Abstract]
  54. Schneeberger, E. E., and Lynch, R. D. (1992) Am. J. Physiol. 262, L647-L661
  55. Schoenenberger, C. A., Zuk, A., Kendall, D., and Matlin, K. S. (1991) J. Cell Biol. 112, 873-879 [Abstract]
  56. Wahli, W., and Martinez, E. (1991) FASEB J. 5, 2243-2249 [Abstract/Free Full Text]
  57. Fuller, P. J. (1991) FASEB J. 5, 3092-3099 [Abstract/Free Full Text]
  58. Gronemeyer, H. (1992) FASEB J. 6, 2524-2529 [Abstract/Free Full Text]
  59. Diamond, M. I., Miner, J. N., Yoshinaga, S. K., and Yamamoto, K. R. (1990) Science 249, 1266-1272 [Medline] [Order article via Infotrieve]
  60. Citi, S. (1992) J. Cell Biol. 117, 169-178 [Abstract]
  61. Balda, M. S., Gonzalez-Mariscal, L., Contreras, R. G., Macias-Silva, M., Torres-Marquez, M. E., Garcia Sainz, J. A., and Cereijido, M. (1991) J. Membr. Biol. 122, 193-202 [Medline] [Order article via Infotrieve]
  62. Ellis, B., Schneeberger, E. E., and Rabito, C. A. (1992) Am. J. Physiol. 263, F293-F300
  63. Janecki, A., Jakubowiak, A., and Steinberger, A. (1991) Mol. Cell. Endocrinol. 82, 61-69 [CrossRef][Medline] [Order article via Infotrieve]
  64. Nigam, S. K., Rodriguez-Boulan, E., and Silver, R. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6162-6166 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.