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[Cancer Research 59, 6267-6275, December 1, 1999]
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[Cancer Research 59, 6267-6275, December 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Tissue Inhibitor of Metalloproteinase-1 Inhibits Apoptosis of Human Breast Epithelial Cells1

Gangyong Li, Rafael Fridman and Hyeong-Reh Choi Kim2

Department of Pathology, Wayne State University School of Medicine, and Karmanos Cancer Institute, Detroit, Michigan 48201


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The signaling pathways critical for cell survival are mediated in part by the composition and integrity of the extracellular matrix and the action of its components on specific cell adhesion receptors. Withdrawal of anchorage-dependent epithelial cells from their association with ECM results in apoptotic cell death. Consistently, the matrix metalloproteinases (MMPs) or their inhibitors (TIMPs) have been suggested to regulate apoptosis. In this report, we investigated whether bcl-2 inhibition of apoptosis involves regulation of TIMP expression. We have found that bcl-2 overexpression induces TIMP-1 expression in breast epithelial cell lines (MCF10A, MCF10AneoT.TG3B, and MCF-7), whereas it has no effect on TIMP-2 expression. We demonstrated that TIMP-1 inhibits cell death induced by hydrogen peroxide, Adriamycin, or X-ray irradiation. In addition, TIMP-1 overexpression inhibits apoptosis after the loss of cell adhesion (anoikis) in MCF10A cells, suggesting that the antiapoptotic activity of TIMP-1 does not depend on its ability to stabilize cell-matrix interactions. We also showed that TIMP-1 overexpression is associated with constitutive activation of focal adhesion kinase, a signaling molecule known to be critical for the cell survival pathway.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous cellular activities influenced by the ECM3 are mediated by signaling pathways. These pathways are regulated in part by the composition and integrity of the ECM and the action of its components on specific cell adhesion receptors (1, 2, 3, 4, 5) . Cell-matrix interactions have been shown to greatly influence cell survival, and withdrawal of anchorage-dependent cells from their association with the ECM results in apoptotic cell death (6 , 7) . Likewise, the turnover of the ECM by specific ECM-degrading enzymes has been shown to modulate cell survival. For example, apoptosis of secretory epithelial cells during involution of the mammary gland after lactation is accompanied by proteolytic degradation of the gland basement membrane (8) . Consistently, overexpression of stromelysin-1, a member of the MMP family of ECM-degrading enzymes, was shown to induce apoptosis in mammary epithelial cells in vitro and in transgenic mice (9) , possibly attributable to its effect on ECM integrity. When stromelysin-1 transgenic mice were crossed with mice overexpressing TIMP-1, a natural MMP inhibitor, apoptosis was significantly inhibited (10) , suggesting a role for MMPs and TIMPs in ECM regulation of cell survival. Although the precise mechanisms by which TIMPs control cell survival remain undefined, their effect may be mediated by their ability to regulate proteolysis of both ECM components and other biologically relevant molecules. Indeed, TIMP-3 induction of apoptosis has been suggested to be mediated by its inhibition of MMPs, resulting in stabilization of tumor necrosis factor {alpha} receptors on the cell surface (11) . However, apoptosis regulation by TIMPs may not be related only to their antiproteolytic activity. For example, although TIMP-1 and TIMP-2 inhibition of MMP enzymatic activity is interchangeable, apoptosis regulation by these inhibitors was shown to be tissue specific, suggesting that mechanisms other than inhibition of enzymatic activity may be involved (12, 13, 14) . Both TIMP-1 and TIMP-2 were also shown to regulate cell proliferation, suggesting that TIMP effects on cell survival may be mediated by yet undefined signaling pathways independent of their antiproteolytic activity (15, 16, 17, 18) . Indeed, TIMP-1 and TIMP-2 have been shown to stimulate tyrosine kinase and mitogen-activated protein kinase activity in the human osteosarcoma cell line MG-63 (19) .

bcl-2, a major gene product known to possess antiapoptotic activity, is located mostly at the outer mitochondrial membrane (20) . bcl-2 prevents cytochrome c release and inhibits the activation of caspases, a group of cysteine proteases that initiates the apoptotic process (21 , 22) . However, recent studies suggest pleiotropic roles for bcl-2 in apoptosis regulation including modulation of Ca2+ homeostasis, transcription factors, and signaling kinases (23, 24, 25) . As a model to study the antiapoptotic effects of bcl-2 in breast epithelial cells, we established stable transfectants of various breast epithelial cell lines overexpressing bcl-2. In this report, we investigated whether bcl-2 inhibition of apoptosis involves regulation of TIMPs expression. Here we report that bcl-2 overexpression results in induction of TIMP-1 expression and that TIMP-1 in the absence of bcl-2 overexpression efficiently inhibits apoptosis. We present evidence that TIMP-1 inhibition of apoptosis involves modulation of signaling pathways, including activation of FAK.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells.
Immortalized nonmalignant human breast epithelial MCF10A cells were obtained from the Barbara Ann Karmanos Cancer Institute (Detroit, MI; Refs. 26 and 27 ). MCF10AneoT.TG3B is a preneoplastic cell line generated by three rounds of implantation of T24c-Ha-ras transfected MCF10A cells into nude mice (Ref. 28 ; a generous gift from Dr. F. Miller, Karmanos Cancer Institute). MCF10A and MCF10AneoT.TG3B cells were cultured in DMEM/F-12 medium supplemented with 5% horse serum, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin, 20 ng/ml epidermal growth factor, 0.1 µg/ml cholera enterotoxin, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 0.5 µg/ml Fungizone in a 95% air and 5% CO2 incubator at 37°C. MCF-7 and bcl-2-overexpressing MCF-7 cells were kindly provided by Dr. Y. Lee (Beaumont Hospital, Royal Oak, MI) and were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum in a 95% air and 5% CO2 incubator at 37°C.

Antibodies.
Anti-human bcl-2 mAbs were purchased from DAKO (Glostrup, Denmark). Anti-human {beta}-actin mAb was purchased from Sigma (St. Louis, MO). Anti-human TIMP-1 and human MMP-9 mAbs were purchased from Oncogene (Cambridge, MA). The mAb to human PARP was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Anti-human FAK and anti-human phosphotyrosine mAbs were from Transduction Laboratories (Lexington, KY). The rabbit polyclonal antibody to TIMP-1 (that recognizes both murine and human TIMP-1) was obtained from Dr. B. Chua (East Tennessee State University, Johnson, Tennessee).

Transfection of Breast Epithelial Cells.
Establishment of bcl-2-overexpressing MCF10A clones was described previously (29) . Hereafter, the neomycin resistance vector-transfected MCF10A cells, the bcl-2-overexpressing clones, and the pooled population of bcl-2-overexpressing clones are referred to as MCF10Aneo1, bcl-2 MCF10A clone#, and bcl-2 MCF10App, respectively.

bcl-2-overexpressing MCF10AneoT.TG3B cells were established by cotransfection with 15 µg of linearized bcl-2 expression vector under the cytomegalovirus promoter (kindly provided by Dr. S. Korsmeyer, Harvard University, Boston, MA) and 5 µg of an expression vector containing the hygromycin resistance marker gene using Lipofectin as described by the manufacturer. Stable transfectants were selected in the presence of 100 µg/ml hygromycin, and individual clones were isolated. Hereafter, the hygromycin resistance vector-transfected clones and the bcl-2-overexpressing clones are referred to as TG3Bhygro and bcl-2 TG3B clone#, respectively.

TIMP-1-overexpressing MCF10A cells were established by transfection using an expression vector containing the human full-length TIMP-1 cDNA and the neomycin resistance gene under control of the long terminal repeats of the Moloney murine sarcoma virus (kindly provided by Dr. M. Johnson at Northwestern University, Chicago, IL). Control vector-transfected MCF10A cells, TIMP-1-overexpressing clones, and the pooled population of TIMP-1-overexpressing clones are referred to as MCF10Aneo2, TIMP-1 MCF10A clone# and TIMP-1 MCF10App, respectively.

Preparation of Conditioned Medium.
Cells were seeded in six-well plates (5 x 105 cells/well) and grown for 18 h in complete medium. Then, the cells were washed with PBS and incubated in serum-free DMEM/F-12 medium for an additional 24 h. The conditioned medium was collected and centrifuged to remove cell debris.

Immunoblot Analysis.
Cell lysates were obtained by lysing the cell monolayer in 0.5 ml/dish of SDS lysis buffer [2% SDS, 125 mM Tris-HCl (pH 6.8), and 20% glycerol]. The lysates were boiled for 5 min and then clarified by a 20-min centrifugation at 4°C. Protein concentration was measured using BCA protein assay reagent (Pierce, Rockford, IL). Equal amount of protein samples in SDS sample buffer [1% SDS, 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 5% {beta}-mercaptoethanol, and 0.05% bromphenol blue] were boiled for 5 min and subjected to reducing SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.02% NaN3 and 0.2% Tween 20 (T-TBS) for 1 h at room temperature. The membranes were incubated with the appropriate antibodies in 5% milk in T-TBS. After three washes with T-TBS, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The antigen was detected using the ECL detection system (Pierce) according to the manufacturer’s instruction.

Northern Blot Analysis.
Total cellular RNA was isolated using the guanidinium-thiocyanate method, as described previously (29) . Ten µg of RNA samples in 50% formamide and 2.2 M formaldehyde were denatured at 68°C for 5 min and separated on a 1% agarose gel containing 2.2 M formaldehyde. The RNA was transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH) using the Turbo blotter (Schleicher & Schuell) in 20x SSC [150 mM NaCl, 15 mM sodium citrate (pH 7.0)] buffer and subsequently UV cross-linked in a Stratalinker (Stratagene, La Jolla, CA). Northern blot analysis was carried out by hybridization at 42°C in a solution containing 50% deionized formamide, 1 M NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% SDS, 10x Denhardt’s, 1 mM NaH2PO4, 1 mM Na2HPO4, and 100 µg/ml of salmon sperm DNA. The TIMP-1 mRNA was detected with 32P-labeled human TIMP-1 cDNA probe.

Purification of Recombinant TIMP-1 Protein.
Recombinant TIMP-1 was expressed in HeLa cells using a vaccinia expression system, as described (30 , 31) . TIMP-1 was purified from the serum-free medium of the infected HeLa cells by lectin Lentil Sepharose chromatography. Briefly, the medium was chromatographed on a lectin-Sepharose 4B column (Sigma) equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM CaCl2, 10% glycerol, 0.05% Brij-35, and 0.02% NaN3. TIMP-1 protein was eluted with 500 mM methyl {alpha}-D-mannopyranoside and diluted in the same buffer. The TIMP-1-containing fractions were pooled, dialyzed against HA buffer [25 mM Tris (pH 7.5), 25 mM NaCl, and 0.02% Brij-35] to an ionic equivalent of <50 mM NaCl and loaded onto a heparin-agarose column (5 ml) equilibrated with HA buffer. The column was washed with HA buffer supplemented with 100 mM NaCl, and TIMP-1 was then eluted from the column with a linear gradient of NaCl (200–400 mM) in HA buffer. The TIMP-1-containing fractions were pooled and dialyzed against PBS. The protein concentration of the recombinant TIMP-1 was determined using its molar extinction coefficient of 26,500 M-1cm-1. The purity of the recombinant TIMP-1 was determined by silver-stained SDS-polyacrylamide gel and was determined to be homogeneous, as shown previously (32) .

Nuclear Staining.
Control, bcl-2- and TIMP-1-overexpressing MCF10A cells were plated on coverslips in six-well plates. After the cells attached to the coverslips, they were treated with 500 µM H2O2 or 0.5 µg/ml of Adriamycin. After 24 h, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS overnight at 4°C. The cells were then exposed to 1 µg/ml 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride pentahydrate (bisBenzimide, Hoechst 33258; Sigma) in PBS for 30 min at room temperature and washed with PBS three times, 15 min/wash. The coverslips were mounted onto glass plates using 0.1% phenylenediamine and 90% glycerol in PBS. Nuclear morphology was examined with UV illumination on a fluorescence microscope.

SRB Assay.
Cells in 96-well plates were washed with PBS, fixed with 10% ice-cold trichloroacetic acid at 4°C for 1 h, then washed with water five times, and dried at room temperature. The cellular proteins in each well were stained with 100 µl of 0.4% SRB in 1% acetic acid at room temperature for 20 min, washed with 1% acetic acid four times, and dried at 37°C for another 30 min. To dissolve the SRB bound to cellular protein, 200 µl of 10 mM Tris were added to each well and incubated at room temperature with mechanical agitation until the color became homogenous. SRB bound to protein was measured by absorbance at 550-nm wavelength using a Benchmark Micro-Plate Reader (Bio-Rad, Richmond, CA).

Suspension Culture.
PolyHEMA (purchased from Aldrich Chemical Co., Milwaukee, WI) was solubilized in methanol (50 mg/ml) and diluted in ethanol to a final concentration of 10 mg/ml. To prepare polyHEMA-coated dishes, 4 ml of the polyHEMA solution were placed onto 100-mm Petri dishes and dried in a tissue culture hood. The polyHEMA coating was repeated twice, followed by three washes with PBS. Anoikis (apoptosis induced by loss of cell anchorage) was induced by culturing 1.5 x 106 cells on polyHEMA-coated, 100-mm dishes in a 95% air and 5% CO2 incubator.

Cell Survival in Soft Agar.
Soft agar assays were performed in six-well plates using a 3-ml base layer of 0.6% agar in MCF10A culture medium. Cells (10,000) in 0.3% top agar were plated in each well. Fresh top agar was overlaid 3 days later. After 1 week, the live cells were counted by trypan blue exclusion assay.

Immunoprecipitation of FAK.
Cells were lysed in RIPA buffer [100 mM sodium phosphate (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, and 1% SDS] containing freshly added protease inhibitors (100 µg/ml phenylmethylsulfonyl fluoride in isopropanol, 45 µg/ml aprotinin, and 1 mM sodium orthovanadate). The lysates were centrifuged for 15 min at 12,000 x g to remove debris and immunoprecipitated using an anti-FAK monoclonal antibody (Transduction Laboratories) and protein G agarose beads (Boehringer Mannheim, Indianapolis, IN). Immunoprecipitates were washed five times with RIPA buffer and resolved by reducing SDS-PAGE. Tyrosine-phosphorylated FAK proteins were detected by immunoblotting using an anti-phosphotyrosine antibody (Transduction Laboratories).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
bcl-2 Up-Regulates TIMP-1 Expression.
bcl-2 overexpression in the bcl-2-transfected MCF10A (Fig. 1A)Citation , MCF10AneoT.TG3B (Fig. 1B)Citation , and MCF-7 (Fig. 1C)Citation clones was confirmed by immunoblot analysis. Several overexpressing clones were identified and selected for additional studies. We examined whether bcl-2 modulates TIMP-1 and/or TIMP-2 expression. The bcl-2 clones exhibited higher levels of TIMP-1 protein (Fig. 2)Citation and mRNA (Fig. 3)Citation than control cells. Both the intracellular and the extracellular TIMP-1 protein levels were found to be elevated. The intracellular TIMP-1 was detected as a doublet, likely to represent the precursor and the fully glycosylated mature forms. In contrast to TIMP-1, the levels of TIMP-2 protein and mRNA expression were not altered (data not shown).



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  		Fig. 1. bcl-2 overexpression in MCF10A, MCF10AneoT.TG3B, and MCF-7 cells. Lysates (50 µg/lane) of vector-transfected (Neo or Hygro) or bcl-2-transfected clones of MCF10A (A), TG3B (B), and MCF-7 (C) cells were subjected to immunoblot analysis with an anti-bcl-2 mAb. Detection of the antigen was performed using ECL. A–C, bottom panels: {beta}-actin levels of the respective blots reprobed with an anti-human {beta}-actin antibody.

 


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  		Fig. 2. bcl-2 induces TIMP-1 protein expression in human breast epithelial cells. Lysates (cellular; 50 µg/lane) and medium (extracellular; 25 µl/lane) of parental (P), vector-transfected (Neo or Hygro), and bcl-2-overexpressing clones of MCF10A (A), TG3B (B), and MCF-7 (C) cells were subjected to immunoblot analysis with an anti-TIMP-1 antibody, followed by detection with ECL.

 


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  		Fig. 3. bcl-2 induces TIMP-1 mRNA expression in human breast epithelial cells. Northern blot analysis of total RNA (10 µg/lane) isolated from parental (P), vector-transfected (Neo or Hygro) and bcl-2-overexpressing clones of MCF10A (A), MCF-7 (B), and TG3B (C). Blots were probed with a human TIMP-1 cDNA probe as described in "Materials and Methods." Equal loading of RNAs was confirmed by staining the membranes with ethidium bromide (bottom panels).

 
TIMP-1 Enhances Survival of Breast Epithelial Cells.
Previous studies suggested an antiapoptotic role for TIMP-1 (12 , 13) . Because bcl-2 expression results in a high level of TIMP-1 expression in breast epithelial cells, we wished to examine whether TIMP-1 plays a direct role in the survival of MCF10A cells after exposure to apoptotic agents. To this end, MCF10A cells were exposed to H2O2 in the presence or absence of exogenously added TIMP-1. Preliminary dose dependence experiments showed that H2O2 (250–500 µM) induced cell death, mostly by apoptosis, as determined by nuclear morphological analysis (29) . As shown in Table 1Citation , in the absence of TIMP-1, ~12% of cells remained viable after 48 h of H2O2 treatment. In the presence of TIMP-1 (500 ng/ml), cell survival increased to 23%. In contrast, similar amounts of TIMP-2 had no effect on H2O2-induced cell death in MCF10A cells (data not shown).


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  		Table 1 Exogenous TIMP-1 increases MCF10A cell survival after H2O2 treatment

 
To eliminate the possibility that TIMP-1-enhanced cell survival after H2O2 treatment was attributable to TIMP-1 mitogenic activity, MCF10A cells were incubated with [3H]thymidine for 48 h, followed by serum-free culture for 24 h, and then examined for survival after H2O2 treatment in the presence or absence of TIMP-1. These studies demonstrated similar results with 1.5–2-fold more [3H]thymidine associated in the cells treated in the presence of TIMP-1, compared with that in H2O2-treated cells without TIMP-1 (data not shown). Thus, the increased cell survival rate after H2O2 treatment in the presence of TIMP-1 results from an effect of TIMP-1 on cell survival and not on cell proliferation.

To further investigate the role of TIMP-1 in the regulation of apoptosis in human breast epithelial cells, we introduced a TIMP-1-expression vector into MCF10A cells. TIMP-1-transfected MCF10A clones were isolated, and the level of TIMP-1 expression was determined by immunoblot analysis. As shown in Fig. 4Citation , both intracellular and extracellular levels of TIMP-1 increased 3–6-fold in the TIMP-1-transfected MCF10A cells. The TIMP-1 expression levels in the TIMP-1-transfected MCF10A cells were comparable with those observed in the MCF10A cells overexpressing bcl-2. We next investigated whether the endogenous TIMP-1 could enhance cell survival against apoptotic stimuli including H2O2, Adriamycin, and irradiation. In addition, we compared the TIMP-1-overexpressing cells with the bcl-2-overexpressing cells. These studies demonstrated a similar rate of survival after these treatments in MCF10A cells overexpressing TIMP-1 or bcl-2 (Fig. 5)Citation . TIMP-1 inhibition of apoptosis was further confirmed by nuclear morphological analysis (Fig. 6)Citation . Whereas the control cells showed fragmented nuclei that were consistent with nuclear morphological changes in apoptotic cells (29) , no significant changes in nuclear morphology could be observed in either the TIMP-1- or the bcl-2-overexpressing cells. It should be mentioned that TIMP-1 overexpression had no effect on the basal levels of bcl-2 expression in the TIMP-1-transfected clones, suggesting that its effect on apoptosis is independent of the bcl-2 expression level (Fig. 7)Citation .



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  		Fig. 4. TIMP-1 overexpression in MCF10A cells. Medium (extracellular; 25 µl/lane; A) and lysates (cellular; 50 µg/lane; B) of vector-transfected (Neo), bcl-2- and TIMP-1-transfected clones of MCF10A cells were subjected to immunoblot analysis with an anti-TIMP-1 antibody, followed by detection with ECL. B, bottom panel, {beta}-actin levels of the respective blot reprobed with an anti-human {beta}-actin antibody.

 


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  		Fig. 5. Effect of TIMP-1 on cell survival following H2O2, irradiation, or Adriamycin treatment. MCF10A cells (vector- and bcl-2- or TIMP-1-transfected) were either treated with 500 µM H2O2 (A), irradiated at 6 Gy with a Cobalt-60 radiation unit (B), or treated with 0.5 µg/ml Adriamycin (C). At 0, 24, and 48 h after treatment, the number of live cells was determined by either trypan blue exclusion (A and C) or by SRB staining (B) as described in "Materials and Methods." The percentage of cell survival (Survival Rate) was normalized to the respective control cells (0-h treatment). All experiments were performed in triplicate; bars, SD.

 


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  		Fig. 6. TIMP-1 inhibits H2O2-induced apoptosis in MCF10A cells. MCF10Aneo1, bcl-2 MCF10A #2, MCF10Aneo2, and TIMP-1 MCF10A #3 cells were treated (48 h) with 500 µM H2O2 and analyzed for nuclear morphology using bisBenzimide staining. Arrows, apoptotic nuclei.

 


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  		Fig. 7. Effect of TIMP-1 overexpression on bcl-2 protein levels. Lysates (50 µg/lane) of vector-transfected (Neo), bcl-2- or TIMP-1-overexpressing MCF10A clones were subjected to immunoblot analysis with an anti-bcl-2 mAb. Detection of the antigen was performed using ECL. Bottom panel, {beta}-actin levels of the respective blots reprobed with an anti-human {beta}-actin mAb.

 
TIMP-1 Inhibition of Apoptosis Does Not Depend on Its Ability to Stabilize Cell-Substrate or Cell-Cell Interactions.
Survival of epithelial cells is dependent on their interaction with the ECM (6 , 33) . After loss of cell anchorage, epithelial cells undergo anoikis, the process of apoptosis caused by loss of substrate adhesion (6 , 33) . TIMP-1 inhibition of apoptosis may result from its ability to stabilize cell-ECM interactions by inhibiting MMPs. If so, we hypothesized that TIMP-1 would not protect against apoptosis induced by loss of cell-substrate interactions. To induce anoikis, the control and the MCF10A cells overexpressing TIMP-1 or bcl-2 were cultured in dishes coated with polyHEMA to prevent cell adhesion. After 24 h, cell survival was determined by trypan blue exclusion assay. These studies showed that <20% of the control MCF10A cells remained viable in the polyHEMA-coated dishes, consistent with induction of anoikis, as described previously (6 , 33) . In contrast, ~80% of the bcl-2- or TIMP-1-overexpressing cells remained viable under the same conditions (Fig. 8A)Citation . Anchorage-independent survival of the control MCF10A, bcl-2-, or TIMP-1-overexpressing cells was also evaluated by culturing cells in soft agar. As shown in Fig. 8BCitation , >80% of bcl-2- or TIMP-1-overexpressing cells remained viable, even after 7 days of culture in soft agar, whereas <20% of the control MCF10A cells survived. Thus, both bcl-2 and TIMP-1 can prevent anoikis in MCF10A cells.



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  		Fig. 8. TIMP-1 inhibits apoptosis induced by loss of cell anchorage. MCF10Aneo1 (column 1), bcl-2 MCF10A #2 (column 2), MCF10Aneo2 (column 3), and TIMP-1 MCF10A #3 and #29 (columns 4 and 5, respectively) cells were cultured in either polyHEMA-coated dishes for 24 h (A) or in soft agar for 7 days (B). The number of live cells was then determined by trypan blue exclusion. Cell survival is expressed as a percentage of control cells (100%) at 0-h treatment. Bars, SD of the mean of triplicate samples. C, immunoblot analysis of PARP (top panel) and {beta}-actin (bottom panel) from lysates (50 µg/lane) of MCF10Aneo1, bcl-2 MCF10A #2, MCF10Aneo2, and TIMP-1 MCF10A #3 cells cultured on monolayer or in suspension (polyHEMA).

 
Cleavage of PARP is an early event in the process of apoptosis, resulting from the activation of caspase/Ced-3 family members (34) . We therefore examined the cleavage of PARP in the control, bcl-2- and TIMP-1-overexpressing cells cultured in polyHEMA-coated dishes. As shown in Fig. 8CCitation , apoptosis-specific proteolytic cleavage of PARP, to the Mr 85,000 fragment, was readily detected in suspension cultures of the control cells, whereas it was significantly inhibited in the bcl-2- or TIMP-1-overexpressing cells. Taken together, these studies suggest that TIMP-1 inhibits caspase-mediated apoptosis independent of its ability to stabilize cell-substrate or cell-cell interactions.

Overexpression of TIMP-1 Is Associated with Constitutive Activation of the FAK in an Anchorage-independent Manner.
Increasing evidence indicates that interactions of integrins with the ECM transduce biochemical signals that are mediated, in part, by the activation of FAK (33 , 35, 36, 37) . Neutralizing antibodies against integrins induce cell detachment, followed by anoikis in epithelial cells, suggesting a role for integrin signaling in the regulation of anoikis (33 , 38) . Constitutively activated forms of FAK (tyrosine phosphorylated form) play a role in protection against anoikis (39) and free radical-induced cell death (40) , suggesting that FAK activity is critical for cell survival. Therefore, we examined whether the TIMP-1 antiapoptotic activity involved the modulation of FAK activity. The expression levels of FAK were not altered by TIMP-1 overexpression, as determined by immunoblot analysis using an anti-FAK mAb (Fig. 9A)Citation . We next examined whether TIMP-1 modulates FAK activity. To this end, the FAK protein was immunoprecipitated with an anti-FAK mAb, and the active form was detected by immunoblot analysis using an antiphosphotyrosine antibody. As shown in Fig. 9BCitation , FAK is more efficiently activated in TIMP-1-overexpressing cells than in the control cells. Because previous studies showed that FAK activation requires cell anchorage (33 , 35, 36, 37) , we asked whether TIMP-1 up-regulation of FAK activation also required cell anchorage. To this end, we cultured control and TIMP-1-overexpressing cells in suspension for 12 h and examined tyrosine-phosphorylated FAK. As shown in Fig. 9CCitation , TIMP-1 constitutively activated FAK, regardless of cell anchorage. This suggests that TIMP-1 regulates apoptosis through constitutive activation of cell survival signaling pathways.



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  		Fig. 9. Constitutive activation of FAK in TIMP-1-overexpressing MCF10A cells. A, lysates (50 µg/lane) of MCF10Aneo2, TIMP-1 MCF10A #3, and TIMP-1 MCF10App cells were subjected to immunoblot analysis using an anti-FAK mAb and detection by ECL. The same blot was reprobed with anti-{beta}-actin antibody (bottom panel). B and C, MCF10Aneo2, TIMP-1 MCF10A #3, and TIMP-1 MCF10App cells were cultured (12 h) in monolayer (B) or in suspension (C) and solubilized in lysis buffer. The lysates were then immunoprecipitated with an anti-FAK mAb and protein G-Sepharose beads. The immunoprecipitates were resolved by reducing SDS-PAGE, followed by immunoblot analysis with an anti-phosphotyrosine mAb (top panels). To confirm the amount of immunoprecipitated FAK protein in each sample, the same blot was reprobed with the anti-FAK mAb (bottom panels).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The bcl-2 family consists of gene products that either promote or inhibit apoptosis (21 , 41 , 42) . One of the critical functions of the bcl-2 family members is to regulate the activation of caspases, a group of cysteine proteases that cleave substrates after aspartic acid residues and initiate the apoptotic processes (43 , 44) . Caspases exist as proenzymes in the cytosol and become activated through proteolysis after apoptotic signaling (43 , 44) . Cytochrome C and CED-4 family members, such as Apaf-1, appear to be critical for caspase activation (45) . The bcl-2 gene product at the outer mitochondrial membrane prevents cytochrome c release and inhibits CED-4 activation of caspases (21 , 22) . However, increasing evidence suggests pleiotropic roles for bcl-2 in apoptosis regulation (23, 24, 25) . In addition to the mitochondrial membrane, bcl-2 is also localized to the outer nuclear membrane and ER. We have shown previously that bcl-2 increases calcium uptake through up-regulation of the ER calcium pump (SERCA) expression, enhancing the lumenal Ca2+ concentration of ER in MCF10A cells (23) . A recent report showed that bcl-2 activates the transcription factor nuclear factor-{kappa}B through degradation of its inhibitor I{kappa}B{alpha} (25) . bcl-2 was also shown to inhibit activation of Jun-NH2-terminal kinases known to play a role in apoptosis (24) . This suggests that bcl-2 inhibition of apoptosis may involve regulation of gene expression through modulation of central signaling molecules including Ca2+ loads, transcription factors, and kinase activity. In the present study, we have shown that overexpression of bcl-2 in breast epithelial cells (nonmalignant and malignant) results in an up-regulation of TIMP-1 expression. Although the molecular mechanisms by which bcl-2 induces TIMP-1 expression remain to be defined, we have found that TIMP-1, like bcl-2, is a potent inhibitor of apoptosis induced by a variety of apoptotic insults including H2O2, Adriamycin, irradiation, and loss of cell anchorage. The ability of TIMP-1 to protect against apoptosis induced by agents working through different mechanisms suggests that TIMP-1 may act in a common pathway of the apoptotic cascade. Although TIMP-1 has no effect on the bcl-2 expression level, it may regulate the activity of antiapoptotic and/or proapoptotic members of the bcl-2 family through FAK activation. FAK was shown to be the upstream regulator of the phosphatidylinositol 3-kinase/serine/threonine kinase Akt survival pathway, and to inhibit the activation of caspases after H2O2 treatment in human glioblastoma cell line (46) . Akt mediates growth factor-induced survival pathway and inhibits apoptosis induced by a variety of stimuli (47) . Akt phosphorylates BAD, a proapoptotic member of the bcl-2 family, and prevents BAD-induced apoptosis of neuron cells (48) . The phosphorylated form of BAD no longer interacts with bcl-2, resulting in bcl-2 activation. Taken together, TIMP-1 activation of FAK may regulate activity of bcl-2 family members that are central to apoptosis regulation. The present study showed that bcl-2 up-regulates TIMP-1 expression, and TIMP-1 inhibits apoptosis in human breast epithelial cells. However, it remains to be investigated whether TIMP-1 is necessary for bcl-2 antiapoptotic activity.

Although TIMP-1 and TIMP-2 share a high percentage of amino acid homology and their MMP inhibitory activities are mostly interchangeable, their regulation of apoptosis appears to be different. TIMP-2 inhibits apoptosis in melanoma cell lines but not in B lymphocytes, whereas TIMP-1 prevents apoptosis in B lymphocytes (12, 13, 14) . TIMP-1- and TIMP-2-specific regulation of apoptosis may result from the differences between TIMP-1- and TIMP-2-mediated signaling pathways (15, 16, 17 , 49) . The present study also showed that TIMP-1, but not TIMP-2, is a downstream mediator of bcl-2 in human breast epithelial cells. TIMP-1 effectively inhibits anoikis, suggesting that TIMP-1 inhibition of apoptosis does not depend on its ability to stabilize cell-substrate interactions through inhibition of matrix-degrading enzymes. We have shown that TIMP-1 constitutively activates FAK activity known to be crucial for cell survival and cell cycle progression (39 , 50) . This is in agreement with recent reports that TIMP-1 inhibition of apoptosis in B lymphocytes occurs independent of its ability to inhibit the enzymatic activities of MMPs (12 , 13) . Analysis of gelatinase (MMP-2 and MMP-9) expression in the bcl-2- and TIMP-1-overexpressing MCF10A cells showed no correlation with bcl-2 or TIMP-1 expression; therefore, gelatinase expression could not be associated with the antiapoptotic effects of either bcl-2 or TIMP-1.4 Despite the various reported effects of TIMP-1 on cellular behavior, the identification of surface TIMP-1 binding proteins remains elusive and warrants further investigation.

In vitro and in vivo studies clearly suggest a role for ECM-degrading enzymes on tumor cell invasion and metastasis formation, especially MMP-2 and MMP-9, which degrade type IV collagen, the major structural collagen of basement membranes (51, 52, 53, 54, 55) . TIMP-1 and TIMP-2 were shown to reduce tumor cell invasion through MMP inhibition (14 , 56 , 57) . However, immunohistochemical studies showed that increased TIMP-1 expression is often associated with negative prognosis in many human solid tumors, including metastatic breast cancer (58, 59, 60) , colorectal cancer (61) , gastric carcinoma (62) , lymphoma (63) , and non-small cell lung carcinoma (64) . The present study may provide an explanation for the unexpected results of these clinical studies. TIMP-1 inhibition of anoikis may be critical for anchorage-independent viability of disseminating cells during tumor cell metastasis. TIMP-1 inhibition of apoptosis independent of its inhibition of MMPs enzymatic activities may contribute to cancer progression.


   ACKNOWLEDGMENTS
 
We thank Dr. Mark Johnson for providing the TIMP-1 expression vector, Dr. Yong J. Lee for providing the bcl-2-transfected MCF-7 cells, David C. Gervasi for technical assistance, and Mary Ann Krug for helping with the 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 NIH/National Cancer Institute Grant CA-64139 and Department of Defense Contract DAMD17-96-1-6181 (to H-R. C. K.) and NIH/National Cancer Institute Grant CA-61986 (to R. F.) G. L. was supported by Predoctoral Fellowship DAMD17-97-1-7200 from the Department of Defense Breast Cancer Program. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. Phone: (313) 577-2407; Fax: (313) 577-0057; E-mail: hrckim{at}med wayne.edu. Back

3 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; FAK, focal adhesion kinase; PARP, poly(ADP-ribose) polymerase; SRB, sulforhodamine B; PolyHEMA, polyhydroxyethylmethacrylate; ER, endoplasmic reticulum; mAb, monoclonal antibody; ECL, enhanced chemiluminescence. Back

4 G. Li and H-R. C. Kim, unpublished results. Back

Received 6/22/99. Accepted 10/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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