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Cell Growth & Differentiation Vol. 10, 537-544, August 1999
© 1999 American Association for Cancer Research

The Inhibitory Effects of Transforming Growth Factor ß1 on Breast Cancer Cell Proliferation Are Mediated through Regulation of Aberrant Nuclear Factor-{kappa}B/Rel Expression1

Mika A. Sovak, Marcello Arsura, Gregory Zanieski, Kathryn T. Kavanagh and Gail E. Sonenshein2

Departments of Pathology and Laboratory Medicine [M. A. S., K. T. K.] and Biochemistry [M. A., G. Z., G. E. S.], and Program in Research on Women’s Health [M. A. S., M. A., G. Z., K. T. K., G. E. S.], Boston University Medical School, Boston, Massachusetts 02118


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Nuclear factor (NF)-{kappa}B/Rel transcription factors normally exist in non-B cells, such as epithelial cells, in inactive forms sequestered in the cytoplasm with specific inhibitory proteins, termed I{kappa}Bs. Recently, however, we discovered that breast cancer is typified by aberrant constitutive expression of NF-{kappa}B/Rel factors. Because these factors control genes that regulate cell proliferation, here we analyzed the potential role of NF-{kappa}B/Rel in the ability of transforming growth factor (TGF)-ß1 to inhibit the growth of breast cancer cells. The decreased growth of Hs578T and MCF7 breast cancer cell lines on TGF-ß1 treatment correlated with a drop in NF-{kappa}B/Rel binding. This decrease was due to the stabilization of the inhibitory protein I{kappa}B-{alpha}. Ectopic expression of c-Rel in Hs578T cells led to the maintenance of NF-{kappa}B/Rel binding and resistance to TGF-ß1-mediated inhibition of proliferation. Similarly, expression of the p65 subunit ablated the inhibition of Hs578T cell growth mediated by TGF-ß1. Thus, the inhibition of the aberrantly activated, constitutive NF-{kappa}B/Rel plays an important role in the arrest of the proliferation of breast cancer cells, which suggests that NF-{kappa}B/Rel may be a useful target in the treatment of breast cancer.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGF-ß13 belongs to a family of polypeptides that plays a role in cellular proliferation, development, and extracellular matrix modeling. TGF-ß1 has been demonstrated to have significant inhibitory effects on the growth of numerous cell types (1, 2, 3) , including mammary epithelial cells. In vitro studies have revealed its inhibitory effects upon the proliferation of numerous primary human and established breast epithelial cell lines (4 , 5) . Evidence for a role of TGF-ß1 in normal mammary gland development and proliferation was provided by studies in mice. Transgenic mice expressing TGF-ß1 linked to the MMTV promoter displayed high levels of TGF-ß1 in the mammary gland (6) . These mice were demonstrated to have hypoplastic mammary duct development (6) . In addition, Silberstein and Daniel (7) demonstrated that the temporary placement of slow-release TGF-ß1 pellets in the mammary glands of virgin mice reversibly inhibited both mammary ductal growth and DNA synthesis.

The growth inhibitory actions of TGF-ß1 have also been demonstrated in breast tumor cell lines (4 , 8, 9, 10) . This effect has been observed in both ER-positive cells (MCF7) and ER-negative cells (Hs578T; Ref. 8 ), although some studies have suggested that ER-negative cells, which are often found in more advanced breast cancers, are less susceptible to the effects of TGF-ß1 (reviewed in Ref. 11 ). The effects of TGF-ß1 on breast cancer have also been seen in vivo. For example, the MMTV-TGF-ß1 transgenic mice displayed increased resistance to the breast carcinogenic effects of DMBA (6) . Furthermore, when these mice were crossed with MMTV-TGF-{alpha} transgenic mice, which show increased incidence of both spontaneous and DMBA-induced breast tumors, the offspring displayed a decreased incidence of spontaneous breast tumors as well as a resistance to the tumorigenic effects of DMBA compared with the parental TGF-{alpha} transgenics (6) . These studies suggest that TGF-ß1 significantly inhibits the development of breast cancer.

There are several theories as to the mechanism of action of TGF-ß1. Studies have linked its growth-inhibitory effects to the down-regulation of genes involved in cellular proliferation, such as those encoding cyclin-dependent kinases (12, 13, 14) , the retinoblastoma susceptibility product (pRB) (15 , 16) , and the c-Myc oncoprotein (16 , 17) . Recent results from our laboratory have shown that the TGF-ß1 treatment of immature B cells and hepatocytes involves a novel signaling mechanism exerted through down-regulation of the NF-{kappa}B/Rel family of transcription factors (18 , 19) . NF-{kappa}B/Rel is a family of dimeric transcription factors all of whose members contain a 300-amino-acid Rel homology domain (20 , 21) . In mammalian cells, subunit members include p50 or NF-{kappa}B1, p52 or NF-{kappa}B2, p65 or RelA, c-Rel, and RelB (20) . NF-{kappa}B/Rel factors are involved in the control of genes implicated in the regulation of cellular proliferation, cell survival, adhesion, and immune and inflammatory responses (20 , 21) . The activity of NF-{kappa}B/Rel factors is controlled posttranslationally by their subcellular localization. In most cells, other than mature B lymphocytes, NF-{kappa}B/Rel proteins are sequestered as inactive forms in the cytoplasm by association with inhibitory proteins, termed I{kappa}B’s, for which I{kappa}B-{alpha} represents the prototype (22, 23, 24, 25) . Activation involves I{kappa}B phosphorylation, which results in its ubiquitination and subsequent degradation, which then allows for the nuclear translocation of the NF-{kappa}B/Rel protein (25) . Recently, we reported that NF-{kappa}B/Rel is aberrantly activated in human breast cancer and in rat mammary tumors induced by DMBA (26) . Thus, here we have investigated the role of NF-{kappa}B/Rel in the TGF-ß1-mediated inhibition of Hs578T and MCF-7 breast cancer cell proliferation. We report that the inhibition of cell growth on TGF-ß1 treatment of these tumor cell lines correlates with a decrease in NF-{kappa}B/Rel activity due to increased stability of I{kappa}B-{alpha} protein. Furthermore, ectopic expression of c-Rel or p65 in Hs578T cells ablates the TGF-ß1-mediated decrease in NF-{kappa}B/Rel activity and inhibition of growth. These findings provide evidence for a direct role of NF-{kappa}B/Rel in the TGF-ß1-mediated decrease in the proliferation of breast cancer cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGF-ß1 Decreases Breast Tumor Cell Line Proliferation.
To confirm the effects of TGF-ß1 treatment on the proliferation of Hs578T and MCF7 breast cancer cells, the effects of this cytokine on cell numbers were monitored. Concentrations of TGF-ß1 ranging from 1 to 5 ng/ml have been reported to effectively inhibit breast cancer cell proliferation (4 , 8 , 27 , 28) . Exponentially growing Hs578T and MCF7 cells were, therefore, treated for 72 h with either TGF-ß1 dissolved in 4% BSA carrier solution or carrier solution alone as control. Cell numbers were then determined. As can be seen in Fig. 1Citation , TGF-ß1 treatment for 3 days resulted in fewer MCF7 and Hs578T cells compared with controls. Similarly, percent labeled nuclei values were largely reduced in Hs578T or MCF7 cells that had been treated with TGF-ß1 for 72 h compared with BSA-treated control cells (data not shown). The effects of TGF-ß1 were likely due to the inhibition of cellular proliferation as opposed to the induction of cell death because a visual analysis of nuclear morphology did not reveal a significant number of apoptotic cells (data not shown). Thus, as seen previously (4 , 5) , TGF-ß1 potently inhibits the growth of breast cancer cell lines.



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Fig. 1. TGF-ß1 treatment decreases proliferation of breast tumor cell lines. MCF7 and Hs578T (578T) breast tumor cell lines were plated in duplicate and treated with 1 ng/ml TGF-ß1 or carrier BSA solution as control. Cell numbers were determined after 3 days of treatment.

 
TGF-ß1 Decreases NF-{kappa}B/Rel Expression.
TGF-ß1 has been demonstrated to exert its inhibitory effects on proliferation through numerous mechanisms, including through the down-regulation of NF-{kappa}B/Rel activity (18, 19) . To determine whether TGF-ß1 affects the aberrant NF-{kappa}B/Rel activity in breast cancer cells, an EMSA analysis was performed. Nuclear extracts were prepared from Hs578T and MCF7 cells incubated in the presence of 1 ng/ml TGF-ß1 or carrier BSA for 72 h. The upstream NF-{kappa}B element from the c-myc gene (URE) was used as probe (29) . As shown in Fig. 2ACitation , TGF-ß1 treatment decreased the total levels of NF-{kappa}B/Rel binding activity in both cell lines. To determine the kinetics of the TGF-ß1-mediated down-modulation of NF-{kappa}B/Rel activity, Hs578T cells were treated for 24 or 48 h with TGF-ß1 and analyzed by EMSA. As shown in Fig. 2BCitation , TGF-ß1 treatment caused a significant decrease of NF-{kappa}B/Rel binding after 24 h, which was followed by an even more pronounced down-modulation at the 48-h time point. These effects were specific in that TGF-ß1 treatment did not alter Oct-1 binding (Fig. 2CCitation ). Thus, TGF-ß1 decreases both breast tumor cell proliferation and NF-{kappa}B/Rel binding activity.



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Fig. 2. TGF-ß1 treatment decreases nuclear NF-{kappa}B/Rel binding activity in breast tumor cell lines. In A, NF-{kappa}B binding. MCF7 and Hs578T (578T) breast tumor cell lines were plated at 1.1 x 104 cells/P100 or 5.6 x 104 cells/P100, respectively, and treated for 3 days with 1 ng/ml TGF-ß1 or carrier BSA solution as control. Nuclear extracts were made and subjected to EMSA using as probe the URE NF-{kappa}B oligonucleotide. B, NF-{kappa}B binding. Hs578T breast tumor cell lines were treated with carrier BSA for 48 h (C) or with 1 ng/ml TGF-ß1 for 24 or 48 h, as indicated. Nuclear extracts were prepared and processed as in A. *, a nonspecific band that did not change with TGF-ß1 treatment. C, Oct-1 binding. Hs578T breast tumor cell lines were treated with 1 ng/ml TGF-ß1 for 0, 24, or 48 hrs. Nuclear extracts were prepared and subjected to EMSA for Oct-1 binding.

 
TGF-ß1 Increases the Half-Life of I{kappa}B-{alpha} Protein.
In hepatocyte cell lines, the decrease in NF-{kappa}B activity in response to TGF-ß1 treatment was mediated through an increase in I{kappa}B-{alpha} protein specifically; the levels of I{kappa}B-ß, the other major I{kappa}B protein, were unaffected (19) . Thus, the effects of TGF-ß1 treatment of Hs578T cells on the rate of turnover of I{kappa}B proteins was assessed. Hs578T cells were incubated for 48 h in the presence of TGF-ß1 or BSA carrier solution as control, and then treated with the protein synthesis inhibitor emetine for 1, 2, or 4 h. Cytoplasmic extracts were then subjected to immunoblot analysis for the two predominant I{kappa}B proteins, I{kappa}B-{alpha} and I{kappa}B-ß (Fig. 3A)Citation . A significant decrease in I{kappa}B-{alpha} degradation was noted upon TGF-ß1 treatment. Densitometry was performed on the resulting immunoblots, and the relative levels were plotted as a function of time in Fig. 3BCitation . In the BSA-treated cells, I{kappa}B-{alpha} protein had a half-life of decay of approximately 2.75 h (Fig. 3B)Citation . TGF-ß1-treatment increased the normal half-life beyond 4 h. In contrast, I{kappa}B-ß protein had a longer half-life, and no change was detected over the 4-h time course (Fig. 3A)Citation . TGF-ß1 treatment of MCF-7 cells similarly increased the half-life of I{kappa}B-{alpha} compared with BSA-carrier-treated control cells, i.e., a 12-h half-life was determined after 48 h of TGF-ß1 treatment compared with the normal 5.3 hrs in control cells. Thus, TGF-ß1 treatment stabilizes I{kappa}B-{alpha} protein, lengthening the normal rate of decay.



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Fig. 3. TGF-ß1 treatment increases the half-life of decay of I{kappa}B-{alpha} in Hs578T breast cancer cells. In A, cultures of Hs578T cells were plated at a density of 2 x 105 cells/P100 dish the day before treatment. Cells were treated with 5 ng/ml TGF-ß1 or carrier solution for 48 h and then incubated in the presence of the protein synthesis inhibitor 20 µg/ml emetine for 0, 1, 2, or 4 h. Cytoplasmic extracts were prepared, and equal amounts (30 µg) were subjected to immunoblot analysis for I{kappa}B-{alpha} or I{kappa}B-ß. In B, the autoradiograph for a representative experiment for I{kappa}B-{alpha} was quantified by densitometric analysis, and the data were presented as a function of the amount present at time zero, set at 100% for the control and TGF-ß1-treated samples. {circ}, BSA control; •, treated with TGF-ß1.

 
Ectopic c-Rel Expression Ablates the Inhibitory Effects of TGF-ß1 on Hs578T Cells.
To test whether ectopic expression of a transactivating NF-{kappa}B/Rel subunit was sufficient to rescue breast cancer cells from TGF-ß1-induced cell growth arrest, populations of Hs578T cells expressing ectopic c-Rel were prepared. To this purpose, the murine c-Rel expression vector pSV-SPORT-c-Rel, which encodes a full-length c-Rel protein, was chosen. Hs578T cells were then stably transfected with the pSV-SPORT-c-Rel vector and the neomycin resistance construct pSV2neo DNA and selected for G418 resistance, as described in "Materials and Methods." Two individual clonal lines were then isolated from the mixed population of resistant cells by limiting dilution. Although studies with individual clones usually give more substantial effects, the use of mixed populations confirms that an observation is not specific to only a few individual cells within a population. The mixed population c-Rel-transfected Hs578T cells (Hs578TR) were first tested for the extent of cell growth inhibition after TGF-ß1 treatment, as compared with the response of the parental Hs578T cell line, using an MTS conversion assay. Treatment of Hs578T cells, plated at either 20 or 40% confluence, with TGF-ß1 for 24 or 48 h resulted in a significant decrease in cell proliferation (Fig. 4A)Citation . In contrast, TGF-ß1 treatment had significantly more modest effects on the cell proliferation of Hs578TR cells (Fig. 4A)Citation . The Hs578TR cells were next characterized for the effects of TGF-ß1 treatment on NF-{kappa}B/Rel binding activity (Fig. 4B)Citation . As expected, NF-{kappa}B/Rel binding was dramatically diminished after TGF-ß1 treatment of the parental Hs578T cells for 24 and 48 h. The nuclear extracts from untreated Hs578TR cells displayed a higher level of NF-{kappa}B/Rel binding activity compared with those from the parental Hs578T cells (Fig. 4B)Citation . Furthermore, the nuclear extracts from Hs578TR cells retained significantly higher levels of NF-{kappa}B/Rel binding activity even after TGF-ß1 treatment. These studies demonstrate that ectopic expression of a member of the NF-{kappa}B/Rel family prevents the drop in NF-{kappa}B/Rel and rescues cells from the growth inhibition mediated by TGF-ß1.



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Fig. 4. Ectopic c-Rel expression in the Hs578TR cell population. Cultures of Hs578T cells in exponential growth were transfected with 22.5 µg of the murine c-Rel expression vector pSPORT-c-Rel and 2.5 µg of pSV2neo DNA. G418-resistant stable transfectants were isolated. In A, parental Hs578T and Hs578TR cells were plated at 20 and 40% confluence, and treated with 2 ng/ml TGF-ß1 or BSA as control for 24 and 48 h, respectively. The effects of TGF-ß1 on growth were measured by MTS assay. Cell numbers for TGF-ß1-treated cells are given as percent values relative to BSA-treated control cells. B, transfected Hs578TR (578TR) and parental Hs578T (578T) cells were plated at 40% confluence and treated with 2 ng/ml TGF-ß1 for 24 and 48 h. Nuclear extracts were isolated and subjected to EMSA using the URE NF-{kappa}B oligonucleotide as probe. *, a nonspecific band that did not change with TGF-ß1 treatment.

 
The clones were next monitored specifically for c-Rel expression by immunoblotting. Cultures of individual clones of the Hs578TR and parental Hs578T cells were compared for expression of c-Rel (Fig. 5A)Citation . The parental Hs578T cells contained extremely low levels of c-Rel, in agreement with our previous report (26) . All of the individual clones expressed c-Rel at levels significantly higher than the parental Hs578T cells. Several of the clones (Hs578TR-C1, -C2, and -C5) expressed high levels of c-Rel, whereas Hs578TR-C3 and -C4 expressed moderately increased levels compared with the parental line. Specificity of the c-Rel protein detected was confirmed by competition with the cognate peptide (Fig. 5B)Citation . The effects of TGF-ß1 on growth were further explored using the two individual c-Rel expressing clones Hs578TR-C1 and -C2. The effects of TGF-ß1 treatment were monitored in two ways, MTS assay and percent labeled nuclei. TGF-ß1-mediated inhibition of cell proliferation was almost completely ablated in cultures plated at 20% confluence (Fig. 5C)Citation or at 70% confluence (data not shown). Similarly, the TGF-ß1 mediated inhibition of DNA synthesis was significantly ablated by expression of c-Rel (Fig. 5D)Citation . Thus, the maintenance of higher levels of NF-{kappa}B/Rel activity protects cells from TGF-ß1-induced growth arrest.



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Fig. 5. Ectopic c-Rel expression in individual clones ablates TGF-ß1-mediated growth inhibition. Individual clones (C1–C5) were isolated from the Hs578TR mixed population by limiting dilution. A and B, immunoblot analysis. Expression of c-Rel in the individual clones was determined by immunoblot analysis. Nuclear extracts were isolated from exponentially growing clones and from parental Hs578T cells, and equal samples (80 µg) were subjected to immunoblot analysis using an anti-c-Rel antibody (SC-070, Santa Cruz Biotechnology) in the absence (A) or presence (B) of a 1:1 molar ratio of cognate peptide. *, position of a nonspecific band. C, MTS proliferation assay. Parental Hs578T and clonal Hs578TR-C1 and Hs578TR-C2 cells were plated at 20% confluence and treated with 5 ng/ml TGF-ß1 or BSA as control for 24 and 48 h. The effects of TGF-ß1 on growth were measured by MTS assay. Cell numbers for TGF-ß1-treated cells are given as percent values relative to BSA-treated control cells. D, DNA synthesis. Parental Hs578T and clonal Hs578TR-C1 and Hs578TR-C2 cells were treated in duplicate with 5 ng/ml TGF-ß1 for 48 h or with BSA as control. Cells were then incubated in media containing 2 µCi of [3H]thymidine per ml for 6 h, fixed, and exposed for autoradiography. Percent labeled nuclei was determined by visual counting. Mean and SD were determined in two different experiments. Black columns, parental Hs578T cells; grey columns, clone 1; white columns, clone 2.

 
Ectopic Expression of p65 Ablates the Growth Inhibitory Effects of TGF-ß1.
To determine whether expression of the p65 subunit would similarly alter the response of Hs578T cells to TGF-ß1, these cells were transiently transfected using FUGENE (Roche Diagnostics Corp., Indianapolis, IN) reagent, which permits a much higher efficiency of transfection. Cells were transfected either with a human p65 pMT2T expression vector or with the parental pMT2T vector DNA. A GFP expression vector was added to estimate transfection efficiency, which was estimated at approximately 70% based on GFP staining. After a 48-h treatment with TGF-ß1, cell proliferation was assessed by MTS assay, and the average of two experiments, carried out in triplicate, are shown in Fig. 6Citation . The reduction of Hs578T cell proliferation normally seen on TGF-ß1 treatment was greatly ablated on expression of p65. These results, taken together with the findings presented above, indicate that expression of either RelA or c-Rel can significantly reduce the growth-inhibitory activity of TGF-ß1 on Hs578T breast cancer cells.



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Fig. 6. Ectopic expression of p65 ablates TGF-ß1-mediated growth arrest of Hs578T cells. Hs578T cells were plated in triplicate at 70% confluence in 96-well dishes. After removal of the media, cells were incubated according to the manufacturer’s directions for 24 h in a 4-µl solution of DNA in FUGENE [either 130 ng pMT2T parental + 20 ng GFP/well, or 130 ng of human p65 pMT2T + 20 ng GFP/well (+p65)]. After 24 h, the cells were treated with carrier BSA (B) or with 5 ng/ml TGF-ß1 (T), and the effects of TGF-ß1 on growth were measured by MTS assay. The average of two experiments are shown; values are given as percent cell proliferation relative to BSA carrier-treated control cells that had been transfected with the parental pMT2T vector DNA. Transfection efficiency was estimated to be approximately 70% based on GFP staining.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Here we provide evidence that the drop in NF-{kappa}B/Rel plays an important role in TGF-ß1-mediated inhibition of breast cancer cell growth. TGF-ß1 treatment of Hs578T and MCF7 human breast cancer cell lines decreased their rate of proliferation and concomitantly decreased the overall levels of NF-{kappa}B/Rel binding activity in these cells. Ectopic expression of either c-Rel or p65 led to resistance to the growth inhibitory effects of TGF-ß1, demonstrating a direct role of NF-{kappa}B/Rel factors in control of proliferation. Stabilization of I{kappa}B-{alpha} specifically was implicated in the observed decrease in NF-{kappa}B/Rel activity. Overall, these studies indicate that NF-{kappa}B/Rel activity is important in the control of breast tumor cell proliferation. Previously, activation or constitutive NF-{kappa}B/Rel expression had been reported to promote growth of various cell types, including B and T lymphocytes, fibroblasts, and smooth muscle and liver cells (20 , 21 , 30, 31, 32, 33, 34, 35) . Our results extend the growth-promoting role of NF-{kappa}B/Rel factors to neoplastically transformed breast epithelial cells; furthermore, they suggest that targeting this activity may be useful in the treatment of breast cancer.

The inhibitory effects of TGF-ß1 on breast cancer have been demonstrated in vivo with transgenic mouse studies. MMTV-TGF-ß1 transgenic mice were highly resistant to DMBA-induced tumorigenesis, suggesting that overexpression of TGF-ß1 has profound inhibitory effects on breast cancer development (6) . The resistance of the MMTV-TGF-ß1 mice to DMBA suggests that TGF-ß1 counteracts the actions of DMBA. It is possible that developmental effects of TGF-ß1 expression decrease the mammary gland susceptibility to DMBA-induced tumorigenesis. However, the glands in these animals were still able to differentiate normally during pregnancy, which indicated that development of the gland was not severely impaired. Interestingly, we have recently shown that mammary tumors induced in female Sprague-Dawley rats treated with DMBA are typified by aberrant induction of NF-{kappa}B/Rel activity (26) . Specifically, we observed that over 85% of tumors expressed high levels of nuclear NF-{kappa}B/Rel. On the basis of the work presented here, it is tempting to speculate that inhibition of NF-{kappa}B/Rel activity may play an important role in the ability of TGF-ß1 to interfere with the DMBA-induced tumorigenic process.

Zugmaier et al. (8) studied the effects of TGF-ß1 on numerous breast cancer cell lines and were able to inhibit the growth of most cell lines independent of ER status. However, there was a discrepancy in the response of MCF7 cells depending on passage number. Early passage (<100) MCF7 cells were inhibited by TGF-ß1, while late passage (>500) cells were resistant to its effects (data not shown), which suggests that biological changes that occur with continuous passage in vitro are in part responsible for the variable phenotype of this cell line. The MCF7 cells used in these experiments were above passage number 154 (as provided by the ATCC) and were still sensitive to the growth-inhibitory effects of this agent.

Although there are published reports describing MCF7 cells undergoing apoptosis after treatment with TGF-ß1 (10 , 36) , several other studies did not observe apoptosis of breast cancer cells on TGF-ß1 treatment (8) . In our experiments, TGF-ß1 did not appear to induce apoptosis in either MCF7 or Hs578T cells, as judged by propidium iodide staining of chromatin, DNA laddering, or terminal deoxynucleotidyl transferase mediated nick end labeling assay. In our previous study (26) , apoptosis was induced in 40% of Hs578T cells microinjected with I{kappa}B-{alpha}. There are several explanations of the apparent lack of cell death in the present studies. It is possible that partial inhibition of NF-{kappa}B/Rel slows cellular growth, and that complete and rapid inhibition is necessary to induce apoptosis. Interestingly, residual NF-{kappa}B/Rel binding levels was observed in both of the cell types after TGF-ß1 treatment. Also, similar to differential sensitivity to growth inhibitors (8) , sensitivity to the apoptotic effects of TGF-ß1 may change with passage number. Alternatively, cells may be differentially sensitive to apoptotic stimuli depending on the stage of the cell cycle.

The studies presented here indicate that the inhibition of constitutive NF-{kappa}B/Rel activity may not be sufficient to induce apoptosis in all breast cancer cells; however, down-regulating this activity may decrease proliferation of breast tumor cells. Several studies have found an association between response to TGF-ß1 and poor prognosis in patients (36, 37, 38, 39, 40) . The findings raised the question of whether the loss of responsiveness to TGF-ß1 is associated with a progression to more aggressive breast tumors. Our findings raise the intriguing possibility that resistance to TGF-ß1, which is most often due to the loss of TGF-ß1 receptor (27 , 41, 42, 43) , can be circumvented inasmuch as the same inhibitory effects on tumor cell proliferation may be achieved via the use of inhibitors of NF-{kappa}B. Thus, inhibition of NF-{kappa}B may provide a new method to sensitize breast cancer cells to chemotherapeutic treatments.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines Culture Conditions and Treatments.
MCF7 cells were kindly supplied by F. Foss (Boston University Medical School, Boston, MA) and C. Sonnenschein (Tufts University Medical School, Boston, MA), or purchased from the ATCC. Human breast cancer Hs578T cells were kindly supplied by M. Sobel (National Cancer Institute, Bethesda, MD), or purchased from the ATCC. MCF7 cells were maintained in DMEM supplemented with 10% heat-inactivated FCS (Life Technologies, Inc., Gaithersburg, MD), 100 µ g/ml streptomycin (Life Technologies, Inc.), and 100 units/ml penicillin (Life Technologies, Inc.). Hs578T cells were propagated in DMEM with 10% heat-inactivated FCS, 4.5 g/liter glucose, 10 µg/ml of insulin (Sigma Chemical Co.), and antibiotics as above. Cells were plated the day before treatment: Hs578T cells at densities ranging from 2,600–4,300 cells/cm2 and MCF7 cells at 7,100–14,000 cells/cm2 depending on the length of treatment. A sterile stock solution of TGF-ß1 (R&D Systems, Minneapolis, MN) was made to 5 ng/µl dissolved in 0.1% carrier BSA solution. The final concentration of TGF-ß1 in the media culture was 1–5 ng/ml as indicated; controls were treated with equal amounts of BSA carrier solution.

For the analysis of cells entering DNA synthesis, the percentage of cells incorporating [3H]thymidine was measured. Briefly, cells were labeled for 6 h with 2 µCi/ml and fixed, and the percentage of labeled nuclei was assessed, as we have described previously (44) . For the Non-radioactive Cell Proliferation assay (Promega), cells were seeded at the indicated confluence in 96-well tissue culture dishes. TGF-ß1- and BSA-treated cultures were incubated in triplicate for 4–6 h in the presence of MTS tetrazolium salt compound solution (333 µg/ml) and 25 µM phenazine methosulfate according to the manufacturer’s directions. The A490 was measured using an ELISA plate reader. For studies on the half-life of I{kappa}B-{alpha}, cells were plated the previous day to achieve 50% confluency on the day of treatment (Hs578T cells, 2 x 105/p100; MCF7, 5.2 x 105/p60). A stock solution of 20 mg/ml of emetine (Sigma) was made in water, and cells were treated with 20 µg/ml for various lengths of time as indicated.

EMSA.
Nuclear extracts were prepared from breast cancer cells by a modification of the method of Dignam et al. (45) Cells were washed twice with ice-cold PBS (Ca2+- and Mg2+-free) containing protease inhibitors (0.5 mM DTT, 0.5 mM PMSF, and 10 µg/ml LP). They were then resuspended in 1 ml of cold hypotonic RSB buffer [10 mM NaCl, 3 mM MgCl2, and 10 mM Tris (pH 7.4)] containing 0.5% NP40 detergent plus protease inhibitors as above. After a 15 min incubation on ice, the cells were dounce-homogenized until cell lysis occurred. Nuclei were resuspended in two packed nuclear volumes of extraction buffer C plus protease inhibitors as above and incubated on ice for 30 min. Protein concentration was determined using the Bio-Rad protein assay, following the manufacturer’s directions. For the labeling of the NF-{kappa}B URE or Oct-1 oligonucleotides, a 150–300-ng sample was incubated for 30 min at 37°C in a solution adjusted to a final concentration of 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 10 mM ß-mercaptoethanol, 20 µM each dATP and dTTP, 50 µCi each of [32P]dCTP and [32P]dGTP, and 5 units of Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA). The URE oligonucleotide, described previously (29) , has the following sequence: 5'-GATCCAAGTCCGGGTTTTCCCCAA CC-3'. The underlined sequences indicate the core binding elements. The Oct-1 oligonucleotide has the following sequence: 5'-TGTCGAATGCAAATCACTAGAA-3'. For the binding reaction, 32P-oligonucleotide (20,000–25,000 cpm) was incubated with 5 µg of nuclear extract, 5 µl sample buffer (10 mM HEPES, 4 mM DTT, 0.5% Triton X-100, and 2.5% glycerol), 2.5 µg poly dI-dC as nonspecific competitor, and the salt concentration adjusted to 100 mM using buffer C. The reaction was carried out at room temperature for 30 min. DNA/protein complexes were subjected to electrophoresis at 11 V/cm and resolved on a 4.5% polyacrylamide gel (using 30% acrylamide/0.8% bisacrylamide) with 0.5x TBE running buffer [90 mM Tris, 90 mM boric acid, and 2 mM EDTA (pH 8.0)].

Immunoblot Analysis.
For cytoplasmic and nuclear extracts, cells were washed twice with PBS containing DTT, PMSF, and LP, as described above; resuspended in 200–400 µl lysis buffer [10 mM Tris (pH 7.6), 10 mM KCl, and 5 mM MgCl2] containing the above protease inhibitors and 1% NP40; and incubated in ice for 5 min. Nuclei were pelleted for 4 min at 2,500 rpm at 4°C. The supernatant containing cytoplasmic proteins was stored at -80°C. The nuclear pellet was washed once in lysis buffer without detergent, centrifuged, and the nuclear proteins extracted using 100–300 µl radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% sodium lauryl sarcosine, 1% NP40, 0.1% SDS, and 1 mM EDTA] plus DTT, PMSF, and LP. The DNA was sheared by pulling the solution 20 times, first through a 23G and then a 25G7/8 needle. After microcentrifugation for 30 min at 14,000 x g at 4°C, the supernatant containing the nuclear proteins was removed and stored at -80°C. Protein concentrations were determined using the Bio-Rad Dc protein assay. Proteins samples (20–40 µg) were resolved in a 10% polyacrylamide-SDS gel, transferred to PVDF membrane (Millipore, Bedford, MA), and subjected to immunoblotting, as described previously (26) . The antibodies preparation for I{kappa}B-{alpha} (SC-371), I{kappa}B-ß (SC-945), and c-Rel (SC-070) were purchased from Santa Cruz Biotechnology Inc.

Isolation of Hs578T Stable Transfectants.
Activity of the murine c-Rel expression vector pSV-SPORT-c-Rel (kindly provided by T. Gilmore, Boston University, Boston, MA), which encodes a full-length c-Rel protein, was confirmed by transient transfection analysis in 3T3 cells, which do not express constitutive NF-{kappa}B/Rel factors (46) .4 The Hs578T c-Rel stable transfectants were prepared using 38 µg of pSV-SPORT-c-Rel and 2 µg of pSV2neo DNA. Cells were transfected by calcium phosphate as described previously (26) . After 24 h, 1.2 mg/ml G418 (Life Technologies, Inc.) were added to the medium, and selective growth conditions were maintained for approximately 2 weeks. Clones were isolated by limiting dilution.

Transient Transfection of Hs578T Cells.
Hs578T cells were plated in triplicate at 70% confluence in 96-well dishes. After removal of the media, cells were incubated for 24 h in a 4-µl solution of DNA in FUGENE Transfection reagent (Boerhinger/Mannheim), according to the manufacturer’s directions. DNA used per well was either 130 ng of human p65 pMT2T (kindly provided by U. Siebenlist, NIH, Bethesda, MD) or parental pMT2T DNA, plus 20 ng of GFP expression plasmid (kindly provided by C. Gelinas, Robert Wood Johnson Medical School, Piscataway, NJ). After 24 h, the cells were treated either with carrier BSA or with 5 ng/ml TGF-ß1, and the effects on growth were measured by MTS assay after 48 h. An approximate 70% transfection efficiency was estimated using GFP staining.


    Acknowledgments
 
We thank T. Gilmore, U. Siebenlist, and C. Gelinas for generously providing the cloned Rel, p65, or GFP expression vector DNAs, and F. Foss, C. Sonnenschein, and M. Sobel for kindly providing breast cancer cells. The advice of M. Wu in preparation of the c-Rel stable cell lines is gratefully acknowledged. We thank L. Gazourian for technical assistance and D. Sloneker for assistance in preparation of the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Commonwealth of Massachusetts Department of Public Health Grant SC-DPH-3408-699D018 (to M. A. S.), Department of Army Research Grants DAMD17-94-J-4468 and DAMD17-98-1 (to G. E. S.), and Training Grant DAMD 17-94-J-442194 (to K. T. K.), and the Karin Gruenbaum Cancer Research Fellowship (to K. T. K.). Back

2 To whom requests for reprints should be addressed, at Department of Biochemistry, Boston University Medical School, 715 Albany Street, Boston, MA 02118. Phone: (617) 638-4120; Fax: (617) 638-5339; E-mail: gsonensh{at}bu.edu Back

3 The abbreviations used are: TGF-ß1, transforming growth factor ß1; MMTV, mouse mammary tumor virus; ER, estrogen receptor; DMBA, 7,12-dimethylbenz({alpha})anthracene; NF, nuclear factor; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium inner salt; Oct-1, Octomer-1; ATCC, American Type Culture Collection; LP, leupeptin; buffer C, 420 mM KCl, 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.2 mM EDTA, and 20% glycerol; PMSF, phenylmethylsulfonyl fluoride; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay. Back

4 D. W. Kim and G. E. Sonenshein, unpublished observations. Back

Received for publication 3/15/99. Revision received 6/23/99. Accepted for publication 6/28/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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D. M. Brantley, C.-L. Chen, R. S. Muraoka, P. B. Bushdid, J. L. Bradberry, F. Kittrell, D. Medina, L. M. Matrisian, L. D. Kerr, and F. E. Yull
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Mol. Biol. Cell, May 1, 2001; 12(5): 1445 - 1455.
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Clin. Cancer Res.Home page
A. W. Tong, M. H. Papayoti, G. Netto, D. T. Armstrong, G. Ordonez, J. M. Lawson, and M. J. Stone
Growth-inhibitory Effects of CD40 Ligand (CD154) and Its Endogenous Expression in Human Breast Cancer
Clin. Cancer Res., March 1, 2001; 7(3): 691 - 703.
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J Biol ChemHome page
E. Cocolakis, S. Lemay, S. Ali, and J.-J. Lebrun
The p38 MAPK Pathway Is Required for Cell Growth Inhibition of Human Breast Cancer Cells in Response to Activin
J. Biol. Chem., May 25, 2001; 276(21): 18430 - 18436.
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Proc. Natl. Acad. Sci. USAHome page
D. K. Biswas, S.-C. Dai, A. Cruz, B. Weiser, E. Graner, and A. B. Pardee
The nuclear factor kappa B (NF-{kappa}B): A potential therapeutic target for estrogen receptor negative breast cancers
PNAS, August 28, 2001; 98(18): 10386 - 10391.
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