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Tumor Biology |
Department of Pathology, Wayne State University School of Medicine, and Karmanos Cancer Institute, Detroit, Michigan 48201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies.
Anti-human bcl-2 mAbs were purchased from DAKO (Glostrup, Denmark). Anti-human 
-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% -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 -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)
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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).
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RESULTS |
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, MCF10AneoT.TG3B (Fig. 1B)
, and MCF-7 (Fig. 1C)
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)
and mRNA (Fig. 3)
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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, 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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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. 4
, 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)
. TIMP-1 inhibition of apoptosis was further confirmed by nuclear morphological analysis (Fig. 6)
. 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)
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80% of the bcl-2- or TIMP-1-overexpressing cells remained viable under the same conditions (Fig. 8A)
. 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. 8B
, >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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, 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)
. 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. 9B
, 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. 9C
, 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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DISCUSSION |
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B through degradation of its inhibitor IB (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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. 
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.
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.
4 G. Li and H-R. C. Kim, unpublished results.
Received 6/22/99. Accepted 10/18/99.
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 A. Joiakim, P. A. Mathieu, A. A. Elliott, and J. J. Reiners Jr. Superinduction of CYP1A1 in MCF10A Cultures by Cycloheximide, Anisomycin, and Puromycin: A Process Independent of Effects on Protein Translation and Unrelated to Suppression of Aryl Hydrocarbon Receptor Proteolysis by the Proteasome Mol. Pharmacol., October 1, 2004; 66(4): 936 - 947. [Abstract] [Full Text] [PDF]
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 Q Qiu, M Yang, B K Tsang, and A Gruslin EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways Reproduction, September 1, 2004; 128(3): 355 - 363. [Abstract] [Full Text] [PDF]
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 M. Yamazaki, T. Akahane, T. Buck, H. Yoshiji, D. E. Gomez, D. J. Schoeffner, E. Okajima, S. R. Harris, O. R. Bunce, S. S. Thorgeirsson, et al. Long-term exposure to elevated levels of circulating TIMP-1 but not mammary TIMP-1 suppresses growth of mammary carcinomas in transgenic mice Carcinogenesis, September 1, 2004; 25(9): 1735 - 1746. [Abstract] [Full Text] [PDF]
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I. S. Aljada, N. Ramnath, K. Donohue, S. Harvey, J. J. Brooks, S. M. Wiseman, T. Khoury, G. Loewen, H. K. Slocum, T. M. Anderson, et al. Upregulation of the Tissue Inhibitor of Metalloproteinase-1 Protein Is Associated With Progression of Human Non-Small-Cell Lung Cancer J. Clin. Oncol., August 15, 2004; 22(16): 3218 - 3229. [Abstract] [Full Text] [PDF]
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 M. Bertrand-Philippe, R. G. Ruddell, M. J. P. Arthur, J. Thomas, N. Mungalsingh, and D. A. Mann Regulation of Tissue Inhibitor of Metalloproteinase 1 Gene Transcription by RUNX1 and RUNX2 J. Biol. Chem., June 4, 2004; 279(23): 24530 - 24539. [Abstract] [Full Text] [PDF]
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 R. Lai, G. Z. Rassidakis, L. J. Medeiros, L. Ramdas, A. H. Goy, C. Cutler, Y. Fujio, K. Kunisada, H. M. Amin, and F. Gilles Signal Transducer and Activator of Transcription-3 Activation Contributes to High Tissue Inhibitor of Metalloproteinase-1 Expression in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma Am. J. Pathol., June 1, 2004; 164(6): 2251 - 2258. [Abstract] [Full Text] [PDF]
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A.-S. Schrohl, M. N. Holten-Andersen, H. A. Peters, M. P. Look, M. E. Meijer-van Gelder, J. G. M. Klijn, N. Brunner, and J. A. Foekens Tumor Tissue Levels of Tissue Inhibitor of Metalloproteinase-1 as a Prognostic Marker in Primary Breast Cancer Clin. Cancer Res., April 1, 2004; 10(7): 2289 - 2298. [Abstract] [Full Text] [PDF]
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Y. Zhou, C. Yu, X. Miao, W. Tan, G. Liang, P. Xiong, T. Sun, and D. Lin Substantial reduction in risk of breast cancer associated with genetic polymorphisms in the promoters of the matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 genes Carcinogenesis, March 1, 2004; 25(3): 399 - 404. [Abstract] [Full Text] [PDF]
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J.-S. Rhee, R. Diaz, L. Korets, J. G. Hodgson, and L. M. Coussens TIMP-1 Alters Susceptibility to Carcinogenesis Cancer Res., February 1, 2004; 64(3): 952 - 961. [Abstract] [Full Text] [PDF]
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 M. Chromek, K. Tullus, J. Lundahl, and A. Brauner Tissue Inhibitor of Metalloproteinase 1 Activates Normal Human Granulocytes, Protects Them from Apoptosis, and Blocks Their Transmigration during Inflammation Infect. Immun., January 1, 2004; 72(1): 82 - 88. [Abstract] [Full Text] [PDF]
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H. Gakiopoulou, L. Nakopoulou, A. Siatelis, I. Mavrommatis, E. G. Panayotopoulou, I. Tsirmpa, C. Stravodimos, and A. Giannopoulos Tissue Inhibitor of Metalloproteinase-2 as a Multifunctional Molecule of Which the Expression Is Associated with Adverse Prognosis of Patients with Urothelial Bladder Carcinomas Clin. Cancer Res., November 15, 2003; 9(15): 5573 - 5581. [Abstract] [Full Text] [PDF]
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 K. Dillner, J. Kindblom, A. Flores-Morales, R. Shao, J. Tornell, G. Norstedt, and H. Wennbo Gene Expression Analysis of Prostate Hyperplasia in Mice Overexpressing the Prolactin Gene Specifically in the Prostate Endocrinology, November 1, 2003; 144(11): 4955 - 4966. [Abstract] [Full Text] [PDF]
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C. A. Fernandez, C. Butterfield, G. Jackson, and M. A. Moses Structural and Functional Uncoupling of the Enzymatic and Angiogenic Inhibitory Activities of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2): LOOP 6 IS A NOVEL ANGIOGENESIS INHIBITOR J. Biol. Chem., October 17, 2003; 278(42): 40989 - 40995. [Abstract] [Full Text] [PDF]
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X.-W. Liu, M. M. Bernardo, R. Fridman, and H.-R. C. Kim Tissue Inhibitor of Metalloproteinase-1 Protects Human Breast Epithelial Cells Against Intrinsic Apoptotic Cell Death via the Focal Adhesion Kinase/Phosphatidylinositol 3-Kinase and MAPK Signaling Pathway J. Biol. Chem., October 10, 2003; 278(41): 40364 - 40372. [Abstract] [Full Text] [PDF]
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G. Akiri, E. Sabo, H. Dafni, Z. Vadasz, Y. Kartvelishvily, N. Gan, O. Kessler, T. Cohen, M. Resnick, M. Neeman, et al. Lysyl Oxidase-related Protein-1 Promotes Tumor Fibrosis and Tumor Progression in Vivo Cancer Res., April 1, 2003; 63(7): 1657 - 1666. [Abstract] [Full Text] [PDF]
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A.-S. Schrohl, I. J. Christensen, A. N. Pedersen, V. Jensen, H. Mouridsen, G. Murphy, J. A. Foekens, N. Brunner, and M. N. Holten-Andersen Tumor Tissue Concentrations of the Proteinase Inhibitors Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and Plasminogen Activator Inhibitor Type 1 (PAI-1) Are Complementary in Determining Prognosis in Primary Breast Cancer Mol. Cell. Proteomics, March 1, 2003; 2(3): 164 - 172. [Abstract] [Full Text] [PDF]
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 W.-J. Wang, J.-C. Kuo, C.-C. Yao, and R.-H. Chen DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals J. Cell Biol., October 14, 2002; 159(1): 169 - 179. [Abstract] [Full Text] [PDF]
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M. N. Holten-Andersen, I. J. Christensen, H. J. Nielsen, H. Lilja, G. Murphy, V. Jensen, N. Brunner, and T. Piironen Measurement of the Noncomplexed Free Fraction of Tissue Inhibitor of Metalloproteinases 1 in Plasma by Immunoassay Clin. Chem., August 1, 2002; 48(8): 1305 - 1313. [Abstract] [Full Text] [PDF]
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F. R. Murphy, R. Issa, X. Zhou, S. Ratnarajah, H. Nagase, M. J. P. Arthur, C. Benyon, and J. P. Iredale Inhibition of Apoptosis of Activated Hepatic Stellate Cells by Tissue Inhibitor of Metalloproteinase-1 Is Mediated via Effects on Matrix Metalloproteinase Inhibition. IMPLICATIONS FOR REVERSIBILITY OF LIVER FIBROSIS J. Biol. Chem., March 22, 2002; 277(13): 11069 - 11076. [Abstract] [Full Text] [PDF]
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M. N. Holten-Andersen, I. J. Christensen, H. J. Nielsen, R. W. Stephens, V. Jensen, O. H. Nielsen, S. Sorensen, J. Overgaard, H. Lilja, A. Harris, et al. Total Levels of Tissue Inhibitor of Metalloproteinases 1 in Plasma Yield High Diagnostic Sensitivity and Specificity in Patients with Colon Cancer Clin. Cancer Res., January 1, 2002; 8(1): 156 - 164. [Abstract] [Full Text] [PDF]
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 X. Han, Y. Sun, S. Scott, and D. Bleich Tissue Inhibitor of Metalloproteinase-1 Prevents Cytokine-Mediated Dysfunction and Cytotoxicity in Pancreatic Islets and {beta}-cells Diabetes, May 1, 2001; 50(5): 1047 - 1055. [Abstract] [Full Text]
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L. Guedez, A. J. McMarlin, D. W. Kingma, T. A. Bennett, M. Stetler-Stevenson, and W. G. Stetler-Stevenson Tissue Inhibitor of Metalloproteinase-1 Alters the Tumorigenicity of Burkitt's Lymphoma via Divergent Effects on Tumor Growth and Angiogenesis Am. J. Pathol., April 1, 2001; 158(4): 1207 - 1215. [Abstract] [Full Text] [PDF]
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L. Guedez, A. Mansoor, B. Birkedal-Hansen, M. S. Lim, P. Fukushima, D. Venzon, W. G. Stetler-Stevenson, and M. Stetler-Stevenson Tissue inhibitor of metalloproteinases 1 regulation of interleukin-10 in B-cell differentiation and lymphomagenesis Blood, March 15, 2001; 97(6): 1796 - 1802. [Abstract] [Full Text] [PDF]
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Y. Jiang, M. Wang, M. Y. Çeliker, Y. E. Liu, Q. X. Amy Sang, I. D. Goldberg, and Y. E. Shi Stimulation of Mammary Tumorigenesis by Systemic Tissue Inhibitor of Matrix Metalloproteinase 4 Gene Delivery Cancer Res., March 1, 2001; 61(6): 2365 - 2370. [Abstract] [Full Text]
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L. Wei, Y. Yang, and Q. Yu Tyrosine Kinase-dependent, Phosphatidylinositol 3'-Kinase, and Mitogen-activated Protein Kinase-independent Signaling Pathways Prevent Lung Adenocarcinoma Cells from Anoikis Cancer Res., March 1, 2001; 61(6): 2439 - 2444. [Abstract] [Full Text]
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 Z. Kadri, E. Petitfrère, C. Boudot, J.-M. Freyssinier, S. Fichelson, P. Mayeux, H. Emonard, W. Hornebeck, B. Haye, and C. Billat Erythropoietin Induction of Tissue Inhibitors of Metalloproteinase-1 Expression and Secretion Is Mediated by Mitogen-activated Protein Kinase and Phosphatidylinositol 3-kinase Pathways Cell Growth Differ., November 1, 2000; 11(11): 573 - 580. [Abstract] [Full Text]
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M. N. Holten-Andersen, R. W. Stephens, H. J. Nielsen, G. Murphy, I. J. Christensen, W. Stetler-Stevenson, and N. Brunner High Preoperative Plasma Tissue Inhibitor of Metalloproteinase-1 Levels Are Associated with Short Survival of Patients with Colorectal Cancer Clin. Cancer Res., November 1, 2000; 6(11): 4292 - 4299. [Abstract] [Full Text] [PDF]
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 M. J. P. Arthur Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis Am J Physiol Gastrointest Liver Physiol, August 1, 2000; 279(2): G245 - G249. [Abstract] [Full Text] [PDF]
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S. E. Hoegy, H.-R. Oh, M. L. Corcoran, and W. G. Stetler-Stevenson Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Suppresses TKR-Growth Factor Signaling Independent of Metalloproteinase Inhibition J. Biol. Chem., January 26, 2001; 276(5): 3203 - 3214. [Abstract] [Full Text] [PDF]
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