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Cell Growth & Differentiation Vol. 11, 335-342, June 2000
© 2000 American Association for Cancer Research

The Role of p21 in Interferon {gamma}-mediated Growth Inhibition of Human Breast Cancer Cells1

Jennifer L. Gooch, Rafael E. Herrera and Douglas Yee2

Department of Medicine/Division of Oncology, University of Texas Health Science Center, San Antonio, Texas 78284-7884


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IFN-{gamma}-mediated growth inhibition requires signal transducers and activators of transcription (STAT)-1 activation and may require induction of the cyclin-dependent kinase inhibitor p21. Using an electrophoretic mobility shift assay, we identified STAT1 activation after IFN-{gamma} treatment in breast cancer cell lines. Accordingly, IFN-{gamma} inhibited proliferation of monolayer cultured MCF-7 and MDA-MB-231 breast cancer cells. Interestingly, IFN-{gamma} inhibited anchorage-independent growth of MCF-7 cells but had no effect on MDA-MB-231colony formation. Because p21 has been shown to play a role in anchorage-independent growth and is a transcriptional target of STAT1, we examined the effect of IFN-{gamma} on p21 mRNA. We found that IFN-{gamma} induced p21 mRNA in MCF-7 cells but not in MDA-MB-231 cells. Furthermore, IFN-{gamma} induced activation of a p21 promoter-luciferase reporter construct that contained the STAT1-inducible element in MCF-7 cells, but not in MDA-MB-231 cells. IFN-{gamma} treatment resulted in increased p21 protein in MCF-7 cells, whereas MDA-MB-231 cells did not appear to express detectable p21, even after IFN-{gamma} treatment. However, in MDA-MB-231 cells, p21 protein was detected only after proteosome inhibition, suggesting that degradation may be responsible for the undetectable level of p21 in these cells, despite the abundant mRNA levels. Finally, focus formation of MDA-MB-231 cells was inhibited by overexpression of p21. In conclusion, STAT1 activation does not appear to be sufficient for IFN-{gamma}-mediated growth inhibition. Furthermore, the role of p21 appears to be complex because monolayer growth inhibition occurs in the absence of p21, but anchorage-independent growth inhibition may require p21. Breast cancer cells may provide a unique model for further study of IFN-{gamma} signaling.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IFN-{gamma} is a pleiotropic immunoregulatory cytokine that functions to enhance cellular immune response by increasing T-cell cytotoxicity and natural killer cell activity. Furthermore, IFN-{gamma} increases expression of HLA genes and up-regulates expression of HLA class I and II molecules in untransformed as well as transformed cells (1 , 2) . In addition to its role as a immunoregulatory factor, IFN-{gamma} inhibits the growth of a number of nonhematopoietic cell types, including tumor cells (3, 4, 5, 6, 7) . In fact, IFN-{gamma} has been considered as an antitumor therapeutic and has been tested in the treatment of human cancer (8, 9, 10, 11) . In metastatic breast cancer, IFN-{gamma} has been shown to enhance the growth-inhibitory effect of tamoxifen (12 , 13) .

IFN-{gamma} action begins with binding of the cytokine to a heterodimeric receptor that induces activation of the JAK3 /STAT pathway (14 , 15) . JAK proteins are tyrosine kinases that associate with non-tyrosine kinase receptors and phosphorylate the receptor, other JAK proteins, and downstream signaling molecules such as STAT proteins. In the case of IFN-{gamma} signaling, JAK1 and JAK2 associate with the receptor and then recruit and phosphorylate STAT1. After phosphorylation, STAT1 dimerizes and then translocates to the nucleus to activate transcription (16) . Several cell cycle-regulatory proteins have been shown to be modulated by IFN-{gamma} treatment, including CDK inhibitor p21 (17 , 18) , retinoblastoma protein (19) , and CDK2 (17) .

STAT1 is activated by a number of ligands besides IFN-{gamma} including prolactin, platelet-derived growth factor, colony-stimulating factor 1, EGF, interleukin 10, and IFN-{alpha} (20, 21, 22) . It has been shown that EGF- and IFN-{gamma}-mediated growth inhibition requires activation of STAT1. EGF-mediated growth inhibition of A431 cells is abrogated by the expression of dominant negative STAT1 (23) . Also, the STAT1-deficient cell line U3A was found to be unaffected by IFN-{gamma} (24 , 25) . When STAT1 was transfected into this cell line, IFN-{gamma}-mediated growth inhibition was restored (18 , 26) . Chin et al. (18) demonstrated that after EGF stimulation, STAT1 and STAT3 molecules can bind to SIEs within the p21 promoter. Furthermore, up-regulation of p21 mRNA and protein has been shown to be associated with IFN-{gamma}-mediated growth inhibition (18 , 27) . As a result, it has been proposed that the mechanism of IFN-{gamma}-induced growth inhibition is STAT1-mediated transactivation of p21.

Whereas IFN-{gamma} signaling has been characterized in some systems, less is known about IFN-{gamma}-mediated growth inhibition and signal transduction in breast cancer cells. Harvat and Jetten (3) showed that IFN-{gamma} significantly inhibited the growth of normal mammary epithelial cells and induced a G1 arrest but only slightly inhibited the growth of breast cancer cells. The authors proposed that the difference in response to IFN-{gamma} was due to defects in the IFN-{gamma} signal transduction pathway in breast cancer cells. Therefore, we examined the effect of IFN-{gamma} on the growth of breast cancer cell lines and investigated IFN-{gamma} activation of STAT1 and its putative transcriptional target, p21.

We show that two breast cancer cell lines activate STAT1 in response to IFN-{gamma}. In addition, IFN-{gamma} inhibits monolayer growth of MCF-7 and MDA-MB-231 breast cancer cell lines, but anchorage-independent growth is inhibited only in MCF-7 cells. Whereas MCF-7 cells up-regulate p21 mRNA and protein levels after IFN-{gamma} treatment, MDA-MB-231 cells failed to up-regulate p21 mRNA in response to IFN-{gamma}. MDA-MB-231 cells do not express detectable p21 protein, except in the presence of specific inhibitors of the proteosome. Therefore, degradation of p21, in addition to transcriptional regulation, may be a mechanism for controlling expression. As a result, the role of p21 in IFN-{gamma}-mediated growth inhibition appears to be complex; inhibition of growth in monolayer culture occurs in the absence of p21, but inhibition of anchorage-independent growth appears to require p21. Breast cancer cell lines may provide a unique model system for further elucidation of the role of p21 in IFN-{gamma}-mediated signal transduction.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
MCF-7 and MDA-MB-231 Cells Activate STAT1 in Response to IFN-{gamma}.
IFN-{gamma}-induced growth inhibition has been shown to be dependent on activation of the transcription factor STAT1 (18 , 28) . We examined IFN-{gamma} induction of activated STAT1 in MCF-7 and MDA-MB-231 cells by electrophoretic mobility shift assay using the high affinity SIE site from the c-fos gene as a probe. The fibroblast cell line A431 has been previously shown to activate STAT1 and STAT3 after stimulation with EGF (29 , 30) and was therefore included as a positive control for STAT1 activation. After IFN-{gamma} treatment, extracts from both MCF-7 and MDA-MB-231 cells formed DNA-protein complexes that appeared to contain STAT1 (Fig. 1A)Citation . We confirmed the identity of the DNA-protein complex by incubating MCF-7 and MDA-MB-231 protein extracts with an antibody to STAT1. We observed a supershifted complex in both cell lines, whereas the addition of STAT2 antibody had no effect on the mobility of the complex (Fig. 1B)Citation .



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Fig. 1. STAT1 DNA binding is activated by IFN-{gamma} in both MCF-7 and MDA-MB-231 cells. A, MCF-7 and MDA-MB-231 whole cell lysates were treated with and without 10 ng/ml IFN-{gamma} and incubated with the labeled probe representing a STAT1 consensus binding site from the c-fos promoter. A431 cells treated with 30 ng/ml EGF were included as a positive control for STAT1 and STAT3 binding. Complexes were resolved by PAGE and autoradiography. B, MCF-7 and MDA-MB-231 whole cell lysates were incubated with labeled probe, and antibodies to STAT1 or STAT2 were added. Complexes were resolved by PAGE and visualized by autoradiography. Only STAT1 antibodies resulted in a supershift of the complex.

 
IFN-{gamma} Inhibits the Growth of Breast Cancer Cell Lines in Monolayer Culture and Increases the G0-G1 Fraction of the Cell Cycle.
IFN-{gamma} has been reported to inhibit the growth of a number of different cell types, including normal mammary epithelial cells and breast cancer cell lines (3 , 4 , 7 , 8) . We first examined the monolayer growth of two breast cancer cell lines treated with IFN-{gamma} over a period of 4 days and found that growth of MCF-7 cells and MDA-MB-231 cells was significantly inhibited by IFN-{gamma} (Fig. 2Citation ; P < 0.002 and P < 0.001 for MCF-7 and MDA-MB-231 cells, respectively). However, MCF-7 cells, which are hormone and growth factor dependent, experienced a lag in proliferation when incubated with IFN-{gamma}. This inhibition was seen only in the presence of a low amount of serum (0.25%) or estradiol (data not shown) and not when cells were cultured in the absence of mitogenic stimulation. Thus, cell proliferation was required to see the inhibitory effects of IFN-{gamma}, although the effect was weak because IFN-{gamma} was unable to inhibit the growth effects of 5% serum. This supports previous findings that IFN-{gamma}-mediated growth inhibition is related to control of the cell cycle.



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Fig. 2. IFN-{gamma} inhibits the growth of MCF-7 and MDA-MB-231 cells in monolayer culture. MCF-7 cells were cultured in SFM plus 0.25% serum, and MDA-MB-231 cells were cultured in SFM alone. IFN-{gamma} (10 ng/ml) was added, and growth was determined by MTT assay at days 0, 2, and 4. Each point represents the mean of triplicate samples ± SE. Asterisks represent statistically significant differences due to IFN-{gamma}. *, P < 0.002; **, P < 0.001.

 
IFN-{gamma}-mediated growth inhibition has previously been attributed to an arrest of the cell cycle in G1 (17 , 19) . Therefore, we examined the cell cycle distribution of MCF-7 and MDA-MB-231 cells after IFN-{gamma} treatment by flow cytometry. Table 1Citation shows cell cycle distributions of MCF-7 and MDA-MB-231 cells treated with IFN-{gamma}. Both cell lines demonstrated a statistically significant increase in G0-G1 cells (P < 0.05 for MCF-7 cells and P < 0.04 for MDA-MB-231 cells) and decreases in S-phase and G2-M-phase cells after IFN-{gamma} treatment, consistent with the decrease observed in monolayer growth.


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Table 1 IFN-{gamma}-mediated growth inhibition is associated with an increase in G0-G1 and a decrease in S phase and G2-M phase of the cell cycle

MCF-7 and MDA-MB-231 cells were cultured in IMEM plus 5% FCS and treated with 10 ng/ml IFN-{gamma} for 24 h. Cells were fixed in ethanol, stained with propidium iodide, and sorted by DNA content. Data shown are the mean of duplicate samples ± SD. Data shown are representative of three separate experiments.

 
Because growth in soft agar is one hallmark of the transformed phenotype, we also examined the response of breast cancer cell lines to IFN-{gamma} in an anchorage-independent growth assay. Colony formation of MCF-7 cells in soft agar was significantly inhibited by the addition of IFN-{gamma} (P < 0.007). However, IFN-{gamma} had no effect on the colony formation of MDA-MB-231 cells (Fig. 3)Citation . It has been reported previously that growth in soft agar has different requirements than proliferation in monolayer culture. Growth under anchorage-independent conditions has been associated with changes in cell cycle-regulatory proteins (31 , 32) . Furthermore, anchorage-independent growth can be inhibited by overexpression of p21 (33 , 34) . Because p21 is also a putative STAT1 transcriptional target, we next examined the effect of IFN-{gamma} on p21 protein levels.



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Fig. 3. IFN-{gamma} inhibits anchorage-independent growth of MCF-7 cells but not MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were plated in soft agar with or without 10 ng/ml IFN-{gamma}. Colonies were allowed to form and were counted after 10 days. Error bars, the mean of duplicate samples ± SE.

 
MCF-7 Cells, but not MDA-MB-231 Cells, Up-Regulate p21 mRNA in Response to IFN-{gamma}.
It has been reported that IFN-{gamma} may induce growth inhibition by increasing transcription of the CDK inhibitor p21 (17 , 35) . It has also been reported that p21 may be transcriptionally up-regulated by STAT binding to a SIE within the p21 promoter (18) . Moreover, IFN-{gamma}-mediated up-regulation of p21 appears to be concomitant with activation of STAT1 DNA binding (27) . Using a RNase protection assay, we examined the effect of IFN-{gamma} on p21 mRNA levels in MCF-7 and MDA-MB-231 cells. RNA from A431 cells treated with EGF was included as a positive control for p21 mRNA up-regulation. Eight h after exposure, the levels of p21 were quantitated by densitometric analysis and then normalized by the levels of 36B4 (included as a loading control). In MCF-7 cells, a modest increase in p21 mRNA was detected after IFN-{gamma} treatment. Whereas p21 mRNA was detectable in MDA-MB-231 cells, IFN-{gamma} treatment did not appear to increase the level of mRNA (Fig. 4)Citation . Also, labeled probe hybridized with tRNA, which did not result in a protected band, was included as a negative control (data not shown).



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Fig. 4. IFN-{gamma} induces increased p21 transcription in MCF-7 cells but not in MDA-MB-231 cells. RNA from MCF-7 and MDA-MB-231 cells treated with and without 10 ng/ml IFN-{gamma} was collected, and p21 mRNA expression was detected by RNase protection assay. RNA from A431 cells treated with and without 30 ng/ml EGF was included as a positive control. Hybridization to labeled probe representing 36B4 was used as a loading control. p21 message was quantitated, and values for p21 divided by 36B4 are depicted as a bar graph. Data shown are representative of three independent experiments.

 
A Region of the p21 Promoter Containing the STAT-inducible Element Is Required for IFN-{gamma}-mediated Transcriptional Activation.
We next examined IFN-{gamma}-mediated transcriptional activity of a p21 promoter-luciferase construct. It has been shown that STAT1 binds to a SIE element located at -603 bp and induces transcription of the p21 gene (18) . Therefore, we looked at activation of the p21 promoter by IFN-{gamma} in MCF-7 and MDA-MB-231 cells using a series of deletion constructs. MCF-7 cells showed significant (P < 0.05) inducible activation of the -837-bp promoter fragment when treated with IFN-{gamma}. When MCF-7 cells were transfected with a promoter fragment that was -567 bp in length and therefore lacked the SIE, no induction by IFN-{gamma} was seen (Fig. 5)Citation . Also, activity on a promoter fragment that was -143 bp in length was comparable with activation of vector alone. In contrast, when compared with raw luciferase values in MCF-7 cells, IFN-{gamma} treatment resulted in only minimal induction of the -843-bp fragment in MDA-MB-231 cells. ß-gal values, determined as a control for transfection efficiency, were similar in MCF-7 and MDA-MB-231 cells and did not change with transfection of different constructs or IFN-{gamma} treatment (data not shown). Therefore, IFN-{gamma} appears to induce activation of the p21 gene primarily through a region containing the SIE in MCF-7 cells, but in MDA-MB-231 cells, IFN-{gamma} did not significantly induce the activity of any p21 promoter fragments.



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Fig. 5. STAT1 transactivates a p21 promoter-luciferase reporter construct in MCF-7 cells but not in MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were transfected with constructs containing a luciferase gene downstream of various p21 promoter constructs. The cells were treated with 10 ng/ml IFN-{gamma} for 24 h, and then luciferase was measured. Error bars, the mean of triplicate samples ± SE. The asterisk represents a statistically significant difference between control and IFN-{gamma} (P < 0.05). Data shown are representative of three separate experiments.

 
IFN-{gamma} Treatment Results in Increased p21 Protein in MCF-7 Cells; no p21 Protein Is Detectable in MDA-MB-231 Cells, Even after IFN-{gamma} Treatment.
We next determined whether the increase in p21 mRNA due to IFN-{gamma} resulted in increased p21 protein in MCF-7 cells. The fibroblast cell line A431 which has also been shown to up-regulate p21 in response to EGF, was included as a positive control. After 8 h of treatment, both A431 and MCF-7 cells responded with an increase in p21 protein. Interestingly, MDA-MB-231 cells did not appear to express detectable p21 protein, even after IFN-{gamma} treatment (Fig. 6)Citation .



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Fig. 6. p21 protein is induced by IFN-{gamma} in MCF-7 cells but not in MDA-MB-231 cells. Total protein was collected from MCF-7 and MDA-MB-231 cells treated with and without 10 ng/ml IFN-{gamma}. Lysates were then separated by SDS-PAGE and immunoblotted with p21 antibody. Total protein from A431 cells treated with EGF was included as a positive control for the induction of p21 protein.

 
We considered that the absence of p21 protein in the presence of detectable p21 mRNA could be due to a mutation in the coding region of the gene. Although mutations of p21 are rare, several have been described previously (36 , 37) . After sequencing the complete coding region of the p21 gene in MDA-MB-231 cells, we determined that the gene contained a previously described Ser-Arg polymorphism at codon 31 (38) , but no mutations (data not shown). An alternative explanation for the lack of detectable protein could therefore be constitutive degradation of p21 protein in MDA-MB-231 cells. Therefore, we treated MDA-MB-231 and MCF-7 cells with lactacystin, an inhibitor of the p26 subunit of the proteosome (39 , 40) , for increasing lengths of time and examined p21 protein expression. In Fig. 7Citation , we show that lactacystin treatment increased p21 protein levels in MCF-7 cells after about 4 h. Interestingly, we also found that inhibition of the proteosome resulted in rescue of p21 protein in MDA-MB-231 cells as well.



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Fig. 7. Inhibition of the proteosome rescues p21 protein in MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with lactacystin (10 µM), a specific inhibitor of the proteosome, for increasing lengths of time. p21 protein was then detected by Western blotting.

 
To further investigate the loss of p21 protein in MDA-MB-231 cells and the role of p21 in growth inhibition, we transiently transfected MDA-MB-231 cells with p21 and then examined the ability of MDA-MB-231 cells to form foci. MCF-7 cells were also transfected as a positive control. Fig. 8ACitation shows that p21 protein was expressed in pooled MDA-MB-231 cells transiently transfected with a copy of the p21 gene, but not when cells were transfected with vector alone. Colony formation was significantly (P < 0.05) inhibited in both MCF-7 and MDA-MB-231 cells after transfection with p21 (Fig. 8B)Citation .



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Fig. 8. Transfected p21 is expressed in MDA-MB-231 cells and inhibits foci formation. A, MDA-MB-231 and MCF-7 cells were transfected with p21 or vector alone (pCDNA3.1), and lysates were collected, separated by SDS-PAGE, and immunoblotted for p21. B, MDA-MB-231 and MCF-7 cells were transfected with p21 or vector alone and then selected with neomycin for 3 weeks. The resulting foci were stained with 1% crystal violet and counted. Asterisks represent statistically significant differences due to p21 (P < 0.05).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In MCF-7 cells, IFN-{gamma} treatment results in the activation of STAT1, up-regulation of p21 mRNA and protein, and activation of a p21 promoter construct containing a SIE. These data, along with the finding that IFN-{gamma} inhibits the growth of MCF-7 cells in both monolayer and anchorage-independent growth, strongly support the idea that IFN-{gamma} inhibits growth via activation of STAT1, which subsequently transcriptionally up-regulates p21. MDA-MB-231 cells, however, are inhibited in monolayer culture by IFN-{gamma} and show a statistically significant increase in G0-G1 phase but do not up-regulate p21 mRNA in response to IFN-{gamma} or express detectable p21 protein. This suggests that IFN-{gamma} may inhibit monolayer growth via a p21-independent signaling mechanism. Interestingly, MDA-MB-231 cells were not inhibited by IFN-{gamma} in an anchorage-independent growth assay, but foci formation is inhibited when p21 is ectopically expressed, suggesting a possible role for p21 in IFN-{gamma}-mediated inhibition of colony formation.

Our data show that the inability of IFN-{gamma} to induce p21 is due to at least two defects in MDA-MB-231 cells. First, whereas STAT1 is activated after IFN-{gamma} treatment and can bind to a SIE construct, no increase in p21 mRNA or promoter activity can be detected. The lack of p21 mRNA induction by IFN-{gamma} and the lack of promoter activation associated with STAT1 suggest that there may be transcriptional defects in this pathway in MDA-MB-231 cells. Recent work by Zhang et al. (41) suggests that STAT1 may cooperate with other transcription factors to activate transcription at discrete sites within a promoter. In MDA-MB-231 cells, STAT1 activation alone is insufficient to enhance transcription of STAT1-regulated genes.

Second, the lack of detectable protein despite the production of mRNA suggests that regulation of p21 expression in MDA-MB-231 cells could also occur posttranscriptionally. Our data indicated that p21 was regulated by degradation of the protein. Inhibition of the proteosome with lactacystin restored a detectable level of p21 in MDA-MB-231 cells. The p21 protein level could also be enhanced by inhibition of degradation in MCF-7 cells, indicating that degradation may be a common means of regulating p21 protein. In addition to degradation of the protein, it is also possible that p21 is regulated by other posttranscriptional mechanisms. Esposito et al. (42) reported that induction of p21 protein in cells exposed to oxidative stress (a p53-independent mechanism) occurs via a posttranscriptional mechanism of mRNA stabilization. The authors suggest that manipulation of mRNA stability might be a way to rapidly control levels of p21 protein in the absence of p53. In the case of MDA-MB-231 cells, it is interesting to speculate that in addition to mutation of the p53 gene (43) , the hormone- and growth factor-independent cell line has developed a mechanism that enhances protein degradation in addition to a means that prevents the transcriptional induction of p21 by inhibitory cytokines such as IFN-{gamma}.

Our data show that whereas IFN-{gamma} inhibits the growth of both MCF-7 and MDA-MB-231 cells in monolayer culture, only MCF-7 cells are inhibited in anchorage-independent growth. There is ample evidence in the literature to suggest that there are different requirements for adherent growth such as monolayer culture and anchorage-independent growth in soft agar (31 , 32) . Transformation releases cells from dependence on an adherent matrix for growth and allows the formation of colonies in soft agar. This release from anchorage dependence has been characterized by alterations in cell cycle proteins and activities. Therefore, it is particularly interesting that IFN-{gamma}-mediated growth inhibition of MCF-7 cells represents a reversion of the transformed phenotype, as evidenced by decreased growth in soft agar, whereas IFN-{gamma} fails to change MDA-MB-231 anchorage-independent growth. One explanation for this difference may be the lack of p21 expression by MDA-MB-231 cells because increased p21 expression has been shown to be associated with decreased colony formation in soft agar (33 , 34) .

Breast cancer cells suggest a complex role for p21 in IFN-{gamma}-mediated growth inhibition. Monolayer growth of breast cancer cells is inhibited by IFN-{gamma}, although p21 protein is not detectable in MDA-MB-231 cells, suggesting that IFN-{gamma}-mediated growth inhibition may be p21 independent. However, anchorage-independent growth of MDA-MB-231 cells is not inhibited by IFN-{gamma}, and this is associated with a lack of p21 protein. Therefore, whereas monolayer growth inhibition by IFN-{gamma} may occur in the absence of p21, anchorage-independent growth appears to require p21 expression. The inability of IFN-{gamma} to inhibit tumor growth could be due to multiple post receptor defects, and human breast cancer cell lines appear to provide a unique model system for further investigation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials
MCF-7 cells were provided by C. Kent Osborne (University of Texas Health Science Center, San Antonio, TX) and maintained in IMEM (Life Technologies, Inc., Bethesda, MD) plus phenol red supplemented with 5% fetal bovine serum (Summit, Ft. Collins, CO). MDA-MB-231 and A431 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in IMEM without phenol red plus 5% fetal bovine serum. Insulin-like growth factor I was obtained from GroPep (Adelaide, Australia), and IFN-{gamma} was obtained from Sigma (St. Louis, MO). STAT1, STAT2, and p21 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Electrophoretic Mobility Shift Assay
STAT1 was detected with the high-affinity STAT-binding site from the c-fos gene promoter (GTGACATTTCCCGTAAATC; Ref. 44 ). Extracts were made as follows: cells were treated with 10 ng/ml IFN-{gamma} for 45 min and then washed once with 1x PBS and harvested with trypsin-EDTA. Cells were centrifuged, and pellets were resuspended in high-salt homogenization buffer [20 mM Tris-HCl (pH 7.5), 2 mM DTT, 20% glycerol, 0.4 M KCl, 10 mg/ml pepstatin, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml antipain, and 100 mM phenylmethylsulfonyl fluoride]. Protein (10 µg) was incubated with end-labeled probe at room temperature for 30 min. For the supershift reaction, 1 µg of antibody was added to protein extracts and incubated at room temperature for 30 min before the addition of labeled probe. Reactions were run on nondenaturing 4% acrylamide, 0.5% Tris-borate EDTA gels. Gels were dried and exposed to film. Data shown are representative of three separate experiments.

Cell Growth Assays
All growth assays were performed at least three separate times.

Monolayer Growth.
Growth assays were performed by MTT assay as described previously (45) . MCF-7 cells were plated in triplicate at a density of 18,000 cells/well, and MDA-MB-231 cells were plated at 12,000 cells/well in 24-well cell culture plates. Cells were allowed to adhere overnight and then washed once in 1x PBS; culture medium was replaced with SFM overnight. Cells were then treated with IFN-{gamma} in SFM supplemented with 1% FCS. After treatment, 60 µl of MTT (5 mg/ml in PBS) were added to the medium for 4 h. Medium and MTT were then removed, DMSO and 2.5% DMEM were added, and absorbance was measured at 540 nm.

Anchorage-independent Growth.
MCF-7 cells were plated at a density of 15,000 cells/plate, and MDA-MB-231 cells were plated at a density of 7,500 cells/plate. Cultures were prepared with a base layer of IMEM supplemented with 20% FCS and containing 0.5% low-melting point agarose (Sea Plaque; FMC Bioproducts, Rockland ME). Cells were then plated over the base layer in duplicate in IMEM supplemented with 10% FCS and containing 0.5% low-melting point agarose. IFN-{gamma} was added at a concentration of 10 ng/ml, and cells were allowed to grow for 7–10 days before colonies of at least 20 cells were counted.

Flow Cytometry.
MCF-7 and MDA-MB-231 cells were plated at 0.5 x 106 cells/60-mm dish. Cells were allowed to adhere overnight and then washed once in 1x PBS; culture medium was replaced with SFM overnight. Cells were then treated with IFN-{gamma} in SFM supplemented with 1% FCS. Treatments continued for 48 h, and cells were then washed with 1x PBS, harvested with trypsin-EDTA, pelleted, washed with 1x PBS, pelleted again, and resuspended in 100 µl of PBS. Ice-cold 70% ethanol (200 µl) was added dropwise while vortexing. Cells were fixed overnight at -20°C, and then 0.5 ng/ml propidium iodide and 0.5 mg/ml RNase A were added. Cells were analyzed using a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA) and gated on forward light scatter, pulse height, and pulse width for analysis of cell cycle fractions. Resulting histograms were evaluated using Modfit LT software (Verity House, Topsham, ME).

Western Blots
Cells were treated with 30 ng/ml EGF or 10 ng/ml IFN-{gamma} for 8 h, harvested with trypsin-EDTA, pelleted, and washed with 1x PBS. Protein was extracted using a buffer containing 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1% NP40, 100 mM NaCl, 100 mM sodium orthovanadate, 100 µg/ml leupeptin, 20 µg/ml aprotinin, and 10-7 M phenylmethylsulfonyl fluoride. Protein (50 µg) was analyzed by 12% SDS-PAGE, and after transfer of the proteins to nitrocellulose, the membrane was incubated in 5% milk-Tris-buffered saline Tween 20 [.15 M NaCl, .01 Tris-HCl (pH 7.4), .05% Tween 20] and then immunoblotted with a 1:1000 dilution of anti-p21 antibody. Horseradish peroxidase-conjugated goat antimouse secondary antibody was added at a 1:2000 dilution, and proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL). Data shown are representative of repeated experiments.

RNase Protection Assay
RNA from A431, MCF-7, and MDA-MB-231 cells treated with SFM or SFM plus 10 ng/ml IFN-{gamma} for 8 h were isolated using the guanidinium thiocyanate method (46) , measured by spectrophotometry, and checked for integrity by separation on a 1% formaldehyde-agarose gel. RNase protection was performed according to our previously published method (47) , and RNA loading was corrected with the ribosomal protein 36B4 (48) . Briefly, 20 µg of RNA were hybridized with radiolabeled antisense complementary RNAs (cRNAs) transcribed from p21 and 36B4 cDNAs. The p21 RNase protection probe was generated by PstI restriction digestion. The resulting 300-bp fragment was subcloned into pGEM4Z. pGEM4Z-p21 was linearized with Xho, and transcription with T7 RNA polymerase was carried out in the presence of [32P]UTP to produce labeled antisense cRNA. For 36B4, a 145-bp PstI-PstI fragment was cloned into pGEM4Z, linearized with EcoRI, and transcribed with T7 RNA polymerase. After hybridization of RNA with radiolabeled probe, single-stranded RNA was digested with RNase A, and samples were separated on 8 M urea/6% SDS-PAGE. tRNA was hybridized as a negative control. The gel was dried and exposed to X-ray film. Data shown are representative of repeated experiments.

Promoter Assays
p21 promoter-luciferase constructs were made as described previously (49) and were a gift from Dr. L. P. Freedman (Memorial Sloan Kettering Cancer Center, New York, NY). Cells (2.5 x 105 ) were plated in triplicate in DMEM + 5% FCS in 6-well plates and transfected the next day. Briefly, cells were washed once with PBS and transiently cotransfected with 1.0 µg of each promoter construct plus 0.1 µg of pSVß-gal using Lipofectin transfection reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Transfected DNA was left on the cells overnight. The next morning, media were changed to control or 10 ng/ml IFN-{gamma} for 24 h. Cells were harvested, and luciferase was measured using the Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. ß-Gal activity was measured as described by Rouet et al. (50) . Luciferase values were divided by the appropriate ß-gal value. Resulting values for each p21-promoter-luciferase construct were then normalized by values for vector alone and are therefore expressed as arbitrary units. Data shown are representative of repeated experiments.

Statistics
Statistical analyses were calculated on representative experiments. For MCF-7 and MDA-MB-231 monolayer growth, two-way ANOVA was used. For analysis of anchorage-independent growth, reporter assays, and foci formation assays, Student’s t test was used.


    Acknowledgments
 
We thank Drs. John Ludes-Meyer and Phang-Lang Chen for helpful discussion of this work and Drs. Adrian Lee and Anthony Valente for critical review 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 USPHS Grant R01CA74282 and USPHS Cancer Center Support Grant P30CA54174 (to D. Y.) and Department of Defense Grant DMAD1798188339 (to J. L. G.). Back

2 To whom requests for reprints should be addressed. Present address: University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware Street SE, Minneapolis, MN 55455. Phone: (612) 624-8484; Fax: (612) 626-4842; E-mail: yeexx006{at}tc.umn.edu Back

3 The abbreviations used are: JAK, Janus-activated kinase; STAT, signal transducers and activators of transcription; CDK, cyclin-dependent kinase; EGF, epidermal growth factor; SIE, c-sis-inducible element; ß-gal, ß-galactosidase; IMEM, improved MEM; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SFM, serum-free medium. Back

Received for publication 3/ 1/99. Revision received 1/11/00. Accepted for publication 5/ 2/00.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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