2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits growth factor withdrawal-induced apoptosis in the human mammary epithelial cell line, MCF-10A

John W. Davis, II, Karla Melendez, Virginia M. Salas, Fredine T. Lauer and Scott W. Burchiel1

Toxicology Program, The University of New Mexico College of Pharmacy, Albuquerque, NM 87131, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases cell recovery in the human mammary epithelial cell line MCF-10A grown under growth factor-restricted conditions. TCDD was also found to mimic growth factor signaling pathways by stimulating the tyrosine phosphorylation of numerous effector molecules, and increased phosphatidylinositol 3-kinase (PI3K) activity in the absence of exogenously added growth factors. In the present studies, we have expanded on these initial results to show that TCDD (3–30 nM) increases cell recovery on days 2–6 by as much as 80% when insulin or epidermal growth factor (EGF) was removed from the media. The mechanism for this effect appears to be complex as TCDD inhibited apoptosis stimulated by EGF, or EGF and insulin, withdrawal by almost 80% as determined by Annexin V binding. However, withdrawal of insulin alone did not induce apoptosis even though TCDD did increase cell number in its absence. These results were corroborated by immunoblot analysis of poly(ADP-ribose) polymerase cleavage. Since TCDD stimulates PI3K activity, the phosphorylation status of Akt, a serine/threonine kinase that mediates PI3K-dependent inhibition of apoptosis, was examined. Immunoblot analysis revealed that TCDD causes a transient increase in the phosphorylated form of Akt that peaks at 6 h and disappears by 12 h. It appears that EGF stimulates an anti-apoptotic pathway, while insulin signals a pro-mitogenic pathway. By stimulating or mimicking one or both of these pathways TCDD may alter tightly regulated growth pathways in the MCF-10A cell line.

Abbreviations: AhR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; IGF-I, insulin-like growth factor-I; IGF-IR, IGF-I receptor; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PI3K, phosphatidylinsitol 3-kinase; PS, phosphatidylserine; SFH, EGF and insulin-deficient media; SFHE, insulin-deficient media; SFIH, EGF-deficient media; SFIHE, complete growth media; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cancer incidence has increased in the Western world during the latter half of the twentieth century, with the most notable increase in breast cancer. One in 10 women in the USA develop breast cancer and over 40 000 per year are expected to die from the disease (13). Breast cancer is a complex disease and its etiology remains a mystery. It has been postulated that environmental pollutants may be involved. The exposure of women to ubiquitous environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatic hydrocarbons have been well documented (4,5). Moreover, these chemicals are resistant to metabolic breakdown, extremely lipophilic in nature, and can be stored in human breast fat and milk (5,6). However, the link between exposure to environmental contaminants and breast cancer has yet to be established. Exposure of women to TCDD from an industrial accident in Seveso, Italy did not result in increased incidence of breast cancer (4), while other reports suggest that exposure to environmental contaminants may increase risk of breast cancer (7).

Activation of growth factor receptors and their cognate signaling pathways is a potential mechanism of mammary tumor promotion and progression (reviewed in ref. 8). Previous studies have demonstrated that TCDD mimics growth factor stimulation and cell growth in the human mammary epithelial cell line, MCF-10A (9). In the absence of insulin, TCDD increased total tyrosine phosphorylation, as well as tyrosine phosphorylation of the insulin-like growth factor-I receptor ß (IGF-IRß), insulin receptor substrate-1 and Shc. In addition, TCDD treatment led to an increase in phosphatidylinositol 3-kinase (PI3K) activity, and TCDD's growth stimulatory effects upon co-treatment with the PI3K inhibitor LY29004 were attenuated. Finally, Shc tyrosine phosphorylation is not unique to IGF signaling. Shc can also bind to phosphotyrosine residues on the epidermal growth factor receptor (EGFR) resulting in Ras activation through Grb2–Sos interactions and stimulation of the mitogen activated protein kinase pathways (10). These results argue that TCDD could act as a mammary tumor promoter by over-stimulating epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) signal transduction pathways.

The inhibition of apoptosis is widely accepted as one possible mechanism of tumor promotion/progression (11 and references therein) and PI3K activation has been demonstrated to provide a powerful anti-apoptotic stimuli (12). PI3K in turn activates another serine/threonine kinase, Akt (13,14). Akt, also known as protein kinase B, is activated by many growth factors, including EGF and IGF-I (15), and has been observed to be up-regulated in ovarian and breast carcinomas (16). Activated Akt protects against apoptosis by phosphorylating Bad, a member of the Bcl-2 family of proteins (17), which in turn prevents Bad from heterodimerizing with Bcl-2 or Bcl-XL (18).

The present studies were initiated to characterize the ability of TCDD to increase cell number of the human mammary epithelial cell line, MCF-10A, in the absence of exogenously added growth factors. Previous studies examining the effects of TCDD on mammary epithelial cell growth have used tumor-derived cell lines that over-express the estrogen receptor, a complicating factor given TCDD's anti-estrogenic activity (19). MCF-10A cells are an estrogen receptor-negative cell line, and make an attractive model for studying growth factor pathways of human mammary epithelial cells because they exhibit a near normal phenotype. Similar to primary cultures of normal human mammary epithelial cells (HMECs), the MCF-10A cell line does not grow tumors when injected in nude mice, and has a strict requirement for EGF and IGF-I for growth in serum-free media (20). We hypothesized that removal of growth factors would induce apoptosis in MCF-10A cells and that TCDD would act to mimic growth factor signaling and inhibit apoptosis. Treatment with 30 nM TCDD was able to increase cell recovery in the absence of EGF and/or insulin for up to 6 days. Removal of EGF induced apoptosis and TCDD was able to attenuate the induction of apoptosis. Interestingly, withdrawal of insulin did not induce apoptosis. In addition, TCDD treatment resulted in a transient increase in phosphorylated Akt under conditions that lead to decreased apoptosis. These data suggest that TCDD is able to mimic multiple growth factor pathways in the MCF-10A cell line. EGF appears to inhibit apoptosis while IGF signaling produces a mitogenic response in MCF-10A cells. Therefore, it appears as though TCDD possesses weak mitogenic activity and is also able to protect cells from death. These pleiotropic effects of TCDD on MCF-10A cells under growth factor-defined conditions may have consequences in human mammary tumor promotion and progression.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
All chemicals were purchased from Sigma (St Louis, MO), unless otherwise indicated. TCDD was obtained from Cambridge Isotopes Laboratories (Andover, MD) at >99% purity. TCDD was maintained as a stock solution (300 µM) in anhydrous tissue culture grade dimethyl sulfoxide (DMSO). The final concentration of DMSO in all experiments was 0.1%.

MCF-10A cell culture
MCF-10A cells, a non-transforming, estrogen receptor-negative human mammary epithelial cell line that exhibits anchorage- and growth factor-dependent growth were a gift from Dr Stephen P.Ethier (University of Michigan, Ann Arbor, MI). Cells were grown on Vitrogen-coated (Collagen Corp., Palo Alto, CA) 100x20 mm dishes (Corning Glass, Corning, NY) in a 10% CO2 incubator and passed every 4–5 days (~75% confluent). Cells were maintained in a serum-free media developed by Ethier et al. (21,22). Complete media consisted of Ham's F-12 (JRH Biosciences, Lenexa, KS) supplemented with 1 mg/ml bovine serum albumin (JRH Biosciences), 1 µg/ml hydrocortisone, 10 ng/ml epidermal growth factor, 5 µg/ml insulin, 5 µg/ml gentamycin (Gibco BRL, Grand Island, NY), 5 µg/ml transferrin, 50 µM sodium selenite, 10 µM 3,3',5-triiodo-L-thryonine, 5 mM ethanolamine, 10 mM HEPES (Gibco BRL) and 5 µg/ml fungizone (Gibco BRL).

Cell counting assay
MCF-10A cells were plated on Vitrogen-coated 6-well plates (Corning Glass) at 1x105 cells/well in complete media plus 2% fetal bovine serum to allow for attachment. After 1 day, plating media was removed and cells were allowed to equilibrate for 1 day in a serum-free (SF) media supplemented with insulin (I), hydrocortisone (H) and EGF (E) for complete media (SFIHE). Twenty-four hours later, cells were switched to one of four growth factor-defined conditions: SFIHE, growth factor-free (SFH), insulin-deficient (SFHE) and EGF-deficient (SFIH) media. The next day media was removed and cells were treated with TCDD in the specified media. Cells were grown for 6 days with media and treatment changed every 48 h, harvested and total nuclei determined using a Coulter Counter (23).

Detection of apoptosis
MCF-10A cells were plated and treated as detailed above. Apoptosis was determined by flow cytometry using a kit that employs Annexin V conjugated to FITC (PharMingen, San Diego, CA). One of the early changes occurring during apoptosis is a change in the plasma membrane whereby phosphatidylserine (PS) is transposed from the inner to the outer surface of the plasma membrane (24,25). Annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for PS. To distinguish between apoptosis and necrosis, cells that stained for propidium iodide (PI) or PI and Annexin V were determined to be necrotic and not counted as apoptotic.

Detection of poly(ADP-ribose) polymerase (PARP) cleavage
Cells were treated under growth factor-defined conditions with 30 nM TCDD (or 0.1% DMSO) for 18 h. Following treatment, cells were rinsed twice with cold PBS and lysed on ice for 10 min in RIPA++ buffer [50 mM Tris pH 8.0; 150 mM NaCl; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 200 µM phenylmethylsulfonyl fluoride; protease inhibitor cocktail purchased from Boehringer Mannheim (Indianapolis, IN) and 200 µM sodium orthovanadate]. Cell debris was pelleted for 10 min at 12 000 g in a refrigerated microcentrifuge, and protein content determined by micro BCA assay (Pierce, Rockford, IL). SDS–PAGE was performed according to Laemmli (26). Cell lysates (30 µg) were diluted with 5x sample buffer, boiled, separated on 12% Tris–glycine gels and transferred overnight to PolyScreen® PVDF membrane (NEN Life Sciences, Boston, MA). Intact and cleaved PARP were detected with a monoclonal antibody that recognizes both forms (PharmMingen) and visualized using NEN's Renaissance® western blot chemiluminescence reagent.

Detection of serine phosphorylated Akt
Cells were treated and harvested as described above for PARP determination. Cell lysates (100 µg) were diluted with 5x sample buffer, boiled, separated on 12% Tris–glycine gels and transferred overnight to PolyScreen® PVDF membrane (NEN Life Sciences). Total and Akt phosphorylated at serine 473 were detected using New England BioLabs' (Beverly, MA) PhosphoPlus® Akt (Ser473) antibody kit as per supplied directions and visualized using chemiluminescence. To quantitate the extent of phosphorylation, chemiluminescent films were scanned and band densities determined using Kodak Digital Science Image System 440 scanner and software (Rochester, NY). Phospho-Akt band densities were normalized to the density of the total Akt band.

Statistical analysis
Data were analyzed for statistical difference (P < 0.05) between control and treated groups using SigmaStat statistical software (Jandel Scientific, San Rafael, CA). ANOVA followed by Dunnett's t-tests were performed on sample means.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
TCDD increases MCF-10A cell number
Previously published results in our laboratory have demonstrated that TCDD increases MCF-10A cell number in the absence of exogenously added growth factors (9). The present studies were undertaken to further characterize these initial results. MCF-10A cells were plated at 105 cells/well on Vitrogen-coated 6-well plates in a serum-free (SF) media developed by Ethier et al. (21,22) supplemented with insulin (I), hydrocortisone (H) and EGF (E). Cells were treated with 30 nM TCDD (or 0.1% DMSO) for 6 days under one of four growth factor-defined conditions: SFIHE, SFH, SFIH or SFHE, harvested and counted on days 2, 4 and 6 as described in Materials and methods. Treatment of MCF-10A cells grown in SFIHE with TCDD had no affect on cell number (Figure 1Go, upper left panel). Removal of insulin and EGF from the growth media resulted in an initial suppression of cell growth followed by accelerated cell death. TCDD appeared to decrease the rate of cell death at 2 and 4 days (Figure 1Go, upper right panel). Removal of EGF or insulin from the media gave similar results. However, in both cases TCDD appeared to stimulate cell growth at later time points (Figure 1Go, lower panels). Concentration–response analyses yielded similar observations (Figure 2Go). While TCDD had no affect when cells were grown for 6 days in SFIHE or SFH media, concentrations as low as 3 nM TCDD increased cell recovery in the absence of EGF or insulin. The results suggest that insulin and EGF are required for MCF-10A growth and/or suppression of cell death. TCDD appears to mimic the effects of these growth factors. More importantly, TCDD was able to alter cell growth at 3 nM, which is in the range of aryl hydrocarbon receptor (AhR) saturation reported for other tissues (27), suggesting a role for AhR for mediating the effects of TCDD on growth regulation in MCF-10A cells.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. TCDD increases MCF-10A cell number. MCF-10A cells were plated at 1x105 cells/well in collagen-coated 6-well plates. One day after plating media was removed and cells were allowed to equilibrate for 1 day in a serum-free media supplemented with insulin, hydrocortisone and EGF for complete media (SFIHE). Twenty-four hours later, cells were switched to one of four growth factor-defined conditions: SFIHE, growth factor-free (SFH), insulin-deficient (SFHE) and EGF-deficient (SFIH) media. The next day media was removed and cells were treated with 30 nM TCDD (or 0.1% DMSO) in the specified media (Day 0). Cells were treated for 6 days, re-fed with fresh treatment every 2 days and total nuclei determined at 48 h intervals using a Coulter Counter. Results shown are the means ± SE for cell counts obtained in triplicate cultures from a representative experiment in which significant differences were observed in at least three different experiments. #Significantly different from DMSO control (P < 0.05).

 


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Concentration–response analysis of TCDD-mediated MCF-10A cell recovery. MCF-10A cells were plated and cultured as indicated in Figure 1Go. Cells were treated with TCDD (0–300 nM) in DMSO for 6 days, re-fed with fresh treatment every 2 days and total nuclei determined by Coulter Counting. Results shown are the means ± SE for cell counts obtained in triplicate cultures from a representative experiment in which significant differences were observed in at least three different experiments. #Significantly different from DMSO control (P < 0.05).

 
TCDD inhibits EGF withdrawal-induced apoptosis
We have shown that TCDD is able to partially reverse MCF-10A cell loss due to growth factor withdrawal (Figures 1 and 2GoGo). In addition, when MCF-10A cells were examined under a light microscope after 48 h without EGF or insulin, there was a substantial amount of cell loss and irregular shaped cells, whereas 30 nM TCDD appeared to protect in the absence of EGF and/or insulin (data not shown). To confirm our previous observation that TCDD inhibits growth factor withdrawal-induced apoptosis, MCF-10A cells were cultured and grown as indicated in Figure 1Go. At the indicated times cells were analyzed for apoptosis, as determined by Annexin V binding using flow cytometry. One of the early changes occurring during apoptosis is a change in the plasma membrane whereby PS is transposed from the inner to the outer side of the plasma membrane (24,25). Annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for PS. To distinguish between apoptosis and necrosis, cells that stained for PI, or PI and Annexin V, were determined to be necrotic and not counted as apoptotic. Withdrawal of EGF (SFH and SFIH media) resulted in the induction of apoptosis in ~20% of the cells, and treatment with 30 nM TCDD inhibited apoptosis by as much as 66% when compared with DMSO controls (Figure 3Go, upper right and lower left panel). Interestingly, withdrawal of insulin did not induce apoptosis (Figure 3Go, lower right panel) even though it did decrease cell number (Figure 1Go). These results correlated with the observation that TCDD increased cell viability in the absence of EGF (as determined by those cells that did not stain positive for Annexin V or PI, data not shown). The results of a concentration–response analysis for the inhibition of apoptosis yielded results similar to those observed for cell recovery. TCDD suppresses apoptosis induced by EGF at concentrations as low as 3 nM (Figure 4Go). These results demonstrate that EGF signaling produces an anti-apoptotic signal in MCF-10A cells, and that TCDD is able to mimic EGF and protect against EGF withdrawal-induced apoptosis. Furthermore, it appears as though insulin (used as a surrogate for IGF-I) produces a pro-mitogenic signal as its withdrawal results in a decrease in cell recovery, without an induction of apoptosis, and TCDD mimics insulin's effects.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. TCDD inhibits epidermal growth factor withdrawal-induced apoptosis in MCF-10A cells. MCF-10A cells were plated, cultured and treated with 30 nM TCDD for 4 days as indicated in Figure 1Go. Induction of apoptosis was determined on days 2, 3 and 4 using flow cytometry to detect FITC-conjugated Annexin V binding to cells. Cells stained with PI were gated out so that only viable cells were analyzed. Results shown are the means ± SE for cell stained for Annexin V but not PI. Data were obtained in triplicate cultures from a representative experiment repeated on at least three occasions. #Significantly different from DMSO control (P < 0.05).

 


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4. Concentration–response analysis of TCDD-mediated inhibition of apoptosis in MCF-10A cells. MCF-10A cells were plated and cultured as indicated in Figure 1Go. Cells were treated with TCDD (0–300 nM) in DMSO for 3 days and apoptosis determined as in Figure 3Go. Results shown are the means ± SE for cell stained for Annexin V but not PI. Data were obtained in triplicate cultures from a representative experiment repeated on at least three occasions. #Significantly different from DMSO control (P < 0.05).

 
TCDD inhibits EGF withdrawal-induced PARP cleavage
Treatment of MCF-10A cells with TCDD suppresses apoptosis induced by EGF withdrawal (Figures 3 and 4GoGo). To further characterize these observations cells were analyzed for PARP cleavage by western blot as another marker of apoptosis. PARP is a 116 kDa nuclear chromatin-associated enzyme that catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins. During early apoptosis, caspases become active and cleave PARP from the full-length form to an 85 kDa fragment (28). Although the role of PARP in apoptosis is unknown it is considered a marker for the induction of apoptosis.

MCF-10A cells were grown in the absence of EGF (SFIH) or insulin (SFHE) for 24 h, followed by treatment with 30 nM TCDD (or 0.1% DMSO) for 18 h. Cell lysates were then analyzed for PARP cleavage using an antibody that recognizes both full-length and cleaved PARP (Figure 5Go). As expected, cleaved PARP was not detected in cells grown in SFIHE (lane 1). However, the 85 kDa fragment was detected in DMSO-treated cells grown in the absence of EGF (SFIH) and TCDD suppressed PARP cleavage (Figure 5Go, lanes 2 and 3, respectively). Finally, removal of insulin had no effect on PARP cleavage (Figure 5Go, lanes 4 and 5). These results confirm that TCDD reverses EGF withdrawal-induced apoptosis in MCF-10A cells.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5. TCDD inhibits EGF withdrawal-induced PARP cleavage in MCF-10A cells. Sub-confluent cultures were grown for 24 h in SFIHE, SFIH or SFHE (as indicated) and then treated for 18 h with 30 nM TCDD (or 0.1% DMSO) as described in Materials and methods. Total cell lysate proteins (30 µg) were resolved on a 12% polyacrylamide gel, transferred to a PVDF membrane and probed with an anti-PARP antibody. The numbers and arrows on the left of the gel indicate full-length (116 kDa) and cleaved (85 kDa) PARP. The blot is representative of three separate experiments.

 
TCDD-mediated Akt phosphorylation correlates with inhibition of apoptosis
We have demonstrated by Annexin V staining and PARP cleavage that removal of EGF stimulates apoptosis in MCF-10A cells and that TCDD protects against apoptosis (Figures 3–5GoGoGo). In addition, previously published results have indicated that TCDD stimulates PI3K under insulin-deficient conditions (9). PI3K signaling can activate other downstream kinases such as Akt through PDK1-dependent phosphorylation (14), which in turn may result in an inhibition of apoptosis (29). The ability of TCDD to increase phosphorylation of Akt was examined in the absence of EGF or insulin using an antibody that recognizes Akt phosphorylated at serine 473. MCF-10A cells were grown and treated as described previously. Treatment of cells with 30 nM TCDD transiently increased Akt phosphorylation, the maximal effect was observed at 6 h and disappeared by 12 h (Figure 6AGo), this is similar to EGF which increased Akt phosphorylation after 15 min. To confirm this observation, cells were cultured overnight in SFIH media and then treated in triplicate with 30 nM TCDD (or 0.1% DMSO) for 6 or 8 h. Total and phospho-Akt bands were quantitated by densitometric analysis as described in Materials and methods. As expected by visual examination of the blot in Figure 6AGo, TCDD significantly increased Akt phosphorylation at 6 h (Figure 6BGo). These results suggest that TCDD inhibits apoptosis in an EGF-like manner through the phosphorylation of Akt.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6. TCDD-mediated Akt phosphorylation in MCF-10A cells. (A) Sub-confluent cultures were grown for 18 h in SFIH, followed by treatment with 30 nM TCDD (T) or 0.1% DMSO (D) for the indicated times. Also included were untreated cells (unt) and cells treated with 10 ng/ml EGF (E) for 15 min. Total cell lysate proteins (100 µg) were resolved on a 12% polyacrylamide gel, transferred to a PVDF membrane and probed with an anti-phospho Ser473-Akt antibody (upper panel) or anti-total Akt antibody (lower panel). (B) In a separate experiment, triplicate cultures were treated with 30 nM TCDD (or 0.1% DMSO) for 6 or 8 h. Total cell lysates were analyzed as in (A) for phospho-Ser473 Akt and total Akt. Chemiluminescent films were scanned and band densities determined using Kodak Digital Science Image System 440 scanner and software. Phosph-Akt band densities were normalized to the density of the total Akt band. Results shown are the means ± SE and the P-values were obtained as indicated in Materials and methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent observations indicate that TCDD increases cell recovery and alters tightly controlled growth regulatory pathways in the human mammary epithelial cell line, MCF-10A (9). Activation of growth factor receptors, particularly EGFR and IGF-IR, and their cognate signaling pathways, is a potential mechanism of mammary tumor promotion and progression (8). The inhibition of apoptosis by growth factor signaling pathways is one possible mechanism of tumor promotion/progression (11). TCDD is able to act as a rodent tumor promoter by inhibiting apoptosis in initiated livers (30,31). Therefore, we attempted to characterize the ability of TCDD to alter cell growth in the human mammary epithelial cell line MCF10A, with the hypothesis that TCDD increases cell number by inhibiting apoptosis in this cell line.

TCDD (30 nM) increased MCF-10A recovery in the absence of EGF and/or insulin (SFIH, SFHE and SFH media) for up to 6 days (Figure 1Go). The ability of TCDD to positively regulate cell growth in human mammary epithelial cells differs from previously published reports. TCDD was found to inhibit IGF signaling exerting an anti-proliferative effect in MCF-7 cells a malignant human mammary epithelial cell line (32,33). However, MCF-7 is a malignant cell line that grows independently of added growth factors in culture, and forms tumors when injected into nude mice. The MCF-10A cell line is an attractive model because it exhibits a normal phenotype and, under serum-free conditions, its proliferation is dependent upon addition of EGF and insulin (20). Furthermore, preliminary data demonstrate that TCDD increases cell number in primary cultures of HMECs under similar growth factor-defined conditions (S.L.Tannheimer and S.W.Burchiel, unpublished data).

The mechanism by which TCDD exerts its effects in MCF-10A cells was further investigated by concentration–response analysis (Figure 2Go). After 6 days of treatment, TCDD was able to replace EGF or insulin (SFIH and SFHE media, respectively) at concentrations as low as 3 nM. In addition, 0.3 nM TCDD also increased cell number, although it was not significantly different from control. These results are interesting because TCDD-mediated cell growth occurs at a concentration range (0.3–3.0 nM) that would suggest AhR occupation (34,35). Moreover, a recent report from our lab demonstrates that MCF-10A cells express AhR and its heterodimerization partner, AhR nuclear translocator (36). These observations suggest that TCDD could exert its growth regulatory effects in MCF-10A cells in an AhR-dependent manner. Further experiments are underway in an effort to address this question.

In these studies, we have clearly demonstrated that TCDD is able to mimic EGF or insulin and support MCF-10A growth. Since TCDD is able to induce an insulin-like signaling pathway in these cells resulting in an increase in PI3K activity (9), we hypothesized that TCDD would inhibit the induction of apoptosis that was due to growth factor withdrawal. As expected, removal of EGF from the growth media resulted in an induction of apoptosis (Figure 3Go, SFH and SFIH media), while 30 nM TCDD suppressed apoptosis on days 2–4. However, removal of insulin did not result in apoptosis (Figure 3Go, SFHE media), even though it is required for optimal cell growth. EGF and IGF signaling work in concert to drive proliferation of human mammary epithelial cells. It would appear that EGF delivers an anti-apoptotic signal while insulin provides pro-mitogenic stimuli in this cell line. TCDD is seemingly able to mimic either pathway in a manner that is similar to neu differentiation factor/heregulin (NDF/HRG), which has been demonstrated to be a dual specificity growth factor in human mammary epithelial cells (20). Similar to the cell recovery data, TCDD at concentrations as low as 3 nM was able to protect MCF-10A cells from cell death (Figure 4Go).

TCDD is able to increase PI3K activity in MCF-10A, which is a potential route for delivery of an anti-apoptotic signal (12). Since PI3K is known to phosphorylate Akt, thereby propagating the anti-apoptotic signal (13,14) the ability of TCDD to increase Akt phosphorylation was investigated. TCDD treatment transiently increased Akt phosphorylation under conditions that lead to suppression of cell death (Figure 6Go). More importantly, TCDD appears to deliver an EGF-like signal in MCF-10A cells as EGF treatment also resulted in an increase in Akt phosphorylation (Figure 6Go) (15). Phosphorylated Akt is thought to mediate an EGF-dependent anti-apoptotic stimulus by phosphorylating Bad (17), preventing it from dimerizing with Bcl-2 or Bcl-XL (18).

In summary, TCDD is able to mimic both EGF and insulin signaling in MCF-10A cells. The end result of this is an increase in cell number with EGF producing an anti-apoptotic signal while insulin yields a pro-mitogenic stimulus. The mechanism by which TCDD exerts an anti-apoptotic effect (and possibly a pro-mitogenic stimulus) was not resolved in these studies. TCDD's ability to suppress apoptosis occurred at concentrations that suggest an AhR involvement. It is possible that TCDD is able to regulate MCF-10A growth by altering the expression of target genes involved in these pathways. There are numerous examples of TCDD-dependent alteration of growth regulatory genes, although the exact role of AhR in mediating the expression of these genes is unknown (3740). In MCF-10A cells, TCDD could produce an autocrine effect by up-regulating the expression of growth factors such as NDF/HRG, which could deliver a dual signal through both an EGFR and an IGF-IR pathway, the end result being an inhibition of apoptosis and a stimulation of mitogenesis. Further studies are required to fully delineate the mechanism by which TCDD regulates MCF-10A cell growth, and to explore signaling pathways by which it may act as a tumor promoter.


    Acknowledgments
 
The authors would like to thank Dr Laurie G.Hudson for critical evaluation of the data and her invaluable input. This work was supported by NIEHS RO1-ES-07259. J.W.D.II was supported by NIEHS 1-F32-ES-05895-01


    Notes
 
1 To whom correspondence should be addressed Email: burchiel{at}unm.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Lancaster,J.M. and Wiseman,R.W. (1997) Recent advances in the molecular genetics of hereditary breast and ovarian cancer. Prog. Clin. Biol. Res., 396, 31–51.[ISI][Medline]
  2. Rahman,N. and Stratton,M.R. (1998) The genetics of breast cancer susceptibility. Annu. Rev. Genet., 32, 95–121.[ISI][Medline]
  3. Hortobagyi,G.N. (1998) Treatment of breast cancer. N. Engl. J. Med., 339, 974–984.[Free Full Text]
  4. Bertazzi,P.A., Pesatori,A.C., Consonni,D., Tironi,A., Landi,M.T. and Zocchetti,C. (1993) Cancer incidence in a population accidentally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Epidemiology, 4, 398–406.[ISI][Medline]
  5. Hooper,K., Petreas,M.X., Chuvakova,T., Kazbekova,G., Druz,N., Seminova,G., Sharmanov,T., Hayward,D., She,J., Visita,P., Winkler,J., McKinney,M., Wade,T.J., Grassman,J. and Stephens,R.D. (1998) Analysis of breast milk to assess exposure to chlorinated contaminants in Kazakstan: high levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in agricultural villages of southern Kazakstan. Environ. Health Perspect., 106, 797–806.[ISI][Medline]
  6. Obana,H., Hori,S., Kahimoto,L. and Kunita,N. (1981) Polycyclic aromatic hydrocarbons in human fat and liver. Bull. Environ. Contam. Toxicol., 27, 23–27.[ISI][Medline]
  7. Hunter,D.J., Hankinson,S.E., Laden,F., Colditz,G.A., Manson,J.E., Willett,W.C., Speizer,F.E. and Wolff,M.S. (1997) Plasma organochlorine levels and the risk of breast cancer. N. Engl. J. Med., 337, 1253–1258.[Abstract/Free Full Text]
  8. Bièche,I. and Lidereau,R. (1995) Genetic alterations in breast cancer. Genes Chromosomes Cancer, 14, 227–251.[ISI][Medline]
  9. Tannheimer,S.L., Ethier,S.P., Caldwell,K.K. and Burchiel,S.W. (1998) Benzo[a]pyrene- and TCDD-induced alterations in tyrosine phosphorylation and insulin-like growth factor signaling pathways in the MCF-10A human mammary epithelial cell line. Carcinogenesis, 19, 1291–1297.[Abstract]
  10. Sasaoka,T., Draznin,B., Leitner,J.W., Langlois,W.J. and Olefsky,J.M. (1994) Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21 ras-GTP formation. J. Biol. Chem., 269, 10734–10738.[Abstract/Free Full Text]
  11. Meyn,R.E., Milas,L. and Stephens,L.C. (1997) Apoptosis in tumor biology and therapy. Adv. Exp. Med. Biol., 400B, 657–667.[ISI]
  12. Kennedy,S.G., Wagner,A.J., Conzen,S.D., Jordan,J., Bellacosa,A., Tsichlis,P.N. and Hay,N. (1997) The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev., 11, 7701–7713.
  13. Datta,K., Bellacosa,A., Chan,T.O. and Tsichilis,P.N. (1996) Akt is a direct target of the phosphatidylinositol 3-kinase. J. Biol. Chem., 271, 30835–30839.[Abstract/Free Full Text]
  14. Stephens,L., Anderson,K., Stokoe,D., Erdjument-Bromage,H., Painter,G.F., Holmes,A.B., Gaffney,P.R., Reese,C.B., McCormick,F., Tempst,P., Coadwell,J. and Hawkins,P.T. (1998) Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science, 279, 710–714.[Abstract/Free Full Text]
  15. Marte,B.M. and Downward,J. (1997) PKB/Akt: connecting phosphatidylinositide 3-kinase to cell survival and beyond. Trends Biol. Sci., 22, 355–358.
  16. Bellacosa,A., Feo,D.D., Godwin,A.K., Bell,D.W., Cheng,J.Q., Altomare,D.A., Wan,M., Dubeau,L., Scambia,G., Masciullo,V., Ferrandina,G., Panici,P.B., Mancuso,S., Neri,G. and Testa,J.R. (1995) Molecular alterations of the Akt2 oncogene in ovarian and breast carcinomas. Int. J. Cancer, 64, 280–285.[ISI][Medline]
  17. Peso,L., Gonsalez-Garcia,M., Page,C., Herrera,R. and Nunez,G. (1997) Interleukin-3 induced phosphorylation of Bad through the protein kinase Akt. Science, 278, 687–689.[Abstract/Free Full Text]
  18. Duronio,V., Schied,M.P. and Ettinger,S. (1998) Downstream signaling events regulated by phosphatidylinositol 3-kinase activity. Cell. Signal., 10, 233–239.[ISI][Medline]
  19. Safe,S. (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol. Ther., 67, 247–281.[ISI][Medline]
  20. Ram,T.G., Kokeny,K.E., Dilts,C.A. and Ethier,S.P. (1995) Mitogenic activity of neu differentiation factor/heregulin mimics that of epidermal growth factor and insulin-like growth factor-I in human mammary epithelial cells. J. Cell. Physiol., 163, 589–596.[ISI][Medline]
  21. Ethier,S.P., Moorthy,R. and Dilts,C.A. (1991) Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cell. Cell Growth Differ., 2, 593–602.[Abstract]
  22. Ethier,S.P. and Moorthy,R. (1991) Multiple growth factor independence in rat mammary carcinoma cells. Breast Cancer Res. Treat., 18, 73–81.[ISI][Medline]
  23. Ethier,S.P., Kudula,A. and Cundiff,K.C. (1987). Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cell in vitro. J. Cell,. Physiol., 132, 161–167.[ISI][Medline]
  24. van Engeland,M., Ramaekers,F.C., Schutte,B. and Reutelingsperger,C.P. (1996) A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry, 24, 131–139.[ISI][Medline]
  25. van Engeland,M., Nieland,L.J., Ramaekers,F.C., Schutte,B. and Reutelingsperger,C.P. (1997) Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry, 31, 1–9.[ISI]
  26. Laemmli,U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]
  27. Poland,A. and Knutson,J.C. (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examinations of the mechanism of toxicity. Annu. Rev. Pharmacol., 22, 517–554.[ISI][Medline]
  28. Patel,T., Gores,G.J. and Kaufmann,S.H. (1996) The role of proteases during apoptosis. FASEB J, 10, 587–597.[Abstract/Free Full Text]
  29. Burgering,B.M.T. and Coffer,P.J. (1995) Protein kinase B (c-akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature, 376, 599–602.[ISI][Medline]
  30. Stinchcombe,S., Buchmann,A., Bock,K.W. and Schwarz,M. (1995) Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumor promotion in rat liver. Carcinogenesis, 16, 1271–1275.[Abstract]
  31. Worner,W. and Schrenk,D. (1996) Influence of liver tumor promoters on apoptosis in rat hepatocytes induced by 2-acetylaminofluorene, ultraviolet light, or transforming growth factor beta 1. Cancer Res., 56, 1272–1278.[Abstract]
  32. Liu,H., Biegel,L., Narasimhan,T.R., Rowlands,C. and Safe,S. (1992) Inhibition of insulin-like growth factor-I responses in MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Mol. Cell. Endocrinol., 87, 19–28.[ISI][Medline]
  33. Gierthy,J.F., Bennett,J.A., Bradley,L.M. and Cutler,D.S. (1993) Correlation of in vitro and in vivo growth suppression of MCF-7 breast cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Res., 53, 3149–3153.[Abstract]
  34. Bradfield,C.A. and Poland,A. (1988) A competitive binding assay for 2,3,7,8-tetrachlorodibenzo-p-dioxin and related ligands of the Ah receptor. Mol. Pharmacol., 34, 682–686.[Abstract]
  35. Gasiewicz,T.A. and Rucci,G. (1991) Alpha-naphthoflavone acts as an antagonist of 2,3,7,8-tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor. Mol. Pharmacol., 40, 607–612.[Abstract]
  36. Tannheimer,S.L., Lauer,F.T., Lane,J. and Burchiel,S.W. (1999) Factors influencing elevation of intracellular Ca2+ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons. Mol. Carcinog., 25, 48–54.[ISI][Medline]
  37. Hudson,L.G., Toscano,W.A.Jr and Greenlee,W.F. (1985) Regulation of epidermal growth factor binding in a human kerotinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol., 77, 251–259.[ISI][Medline]
  38. Sutter,T.R., Guzman,K., Dold,K.M. and Greenlee,W.F. (1991) Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science, 254, 415–418.[ISI][Medline]
  39. Gaido,K.W., Maness,S.C., Leonard,L.S. and Greenlee,W.F. (1992) 2,3,7,8-Tetrachlorodibenzo-p-dioxin-dependent regulation of transforming growth factors-{alpha} and ß2 expression in a human kerotinocyte cell line involves both transcriptional and post-transcriptional control. J. Biol. Chem., 267, 24591–24595.[Abstract/Free Full Text]
  40. Wang,W.L., Porter,W., Burghardt,R. and Safe,S.H. (1997) Mechanism of inhibition of MDA-MB-468 breast cancer cell growth by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Carcinogenesis, 18, 925–933.[Abstract]
Received June 17, 1999; revised January 12, 2000; accepted January 21, 2000.