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• W1657916616 abstract "Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor or serpin that is a major product of macrophages in response to endotoxin and inflammatory cytokines. We have explored the role of PAI-2 in apoptotic cell death initiated by tumor necrosis factor α (TNF). HeLa cells stably transfected with PAI-2 cDNA were protected from TNF-induced apoptosis, whereas cells transfected with antisense PAI-2 cDNA, a control gene, or the plasmid vector alone remained susceptible. The level of PAI-2 expressed by different HeLa cell clones was inversely correlated with their sensitivity to TNF. Loss of TNF sensitivity was not a result of loss of TNF receptor binding. In contrast, PAI-2 expression did not confer protection against apoptosis induced by ultraviolet or ionizing radiation. The serine proteinase urokinase-type plasminogen activator was not demonstrated to be the target of PAI-2 action. The P1-Arg amino acid residue of PAI-2 was determined to be required for protection, because cells expressing PAI-2 with an Ala in this position were not protected from TNF-mediated cell death. The results suggest that intracellular PAI-2 might be an important factor in regulating cell death in TNF-mediated inflammatory processes through inhibition of a proteinase involved in TNF-induced apoptosis. Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor or serpin that is a major product of macrophages in response to endotoxin and inflammatory cytokines. We have explored the role of PAI-2 in apoptotic cell death initiated by tumor necrosis factor α (TNF). HeLa cells stably transfected with PAI-2 cDNA were protected from TNF-induced apoptosis, whereas cells transfected with antisense PAI-2 cDNA, a control gene, or the plasmid vector alone remained susceptible. The level of PAI-2 expressed by different HeLa cell clones was inversely correlated with their sensitivity to TNF. Loss of TNF sensitivity was not a result of loss of TNF receptor binding. In contrast, PAI-2 expression did not confer protection against apoptosis induced by ultraviolet or ionizing radiation. The serine proteinase urokinase-type plasminogen activator was not demonstrated to be the target of PAI-2 action. The P1-Arg amino acid residue of PAI-2 was determined to be required for protection, because cells expressing PAI-2 with an Ala in this position were not protected from TNF-mediated cell death. The results suggest that intracellular PAI-2 might be an important factor in regulating cell death in TNF-mediated inflammatory processes through inhibition of a proteinase involved in TNF-induced apoptosis. Programmed cell death is conserved throughout evolution as a strategy employed by multicellular organisms to regulate a wide range of physiological processes including embryo development, tissue remodelling, immune system development and cellular responses to infection, and tumorigenesis. Cell death can be triggered by a variety of stimuli that can ultimately lead to characteristic changes in the cell, frequently resulting in chromatin fragmentation and condensation, membrane blebbing, and collapse of the nucleus, a process called apoptosis(1Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Google Scholar, 2Williams G.T. Cell. 1991; 65: 1097-1098Google Scholar). Within the immune system, tumor necrosis factor α (TNF) 1The abbreviations used are: TNFtumor necrosis factor αICEinterleukin 1β converting enzymePAI-2plasminogen activator inhibitor type 2uPAurokinase-type plasminogen activatorbpbase pairCATchloramphenicol acetyltransferasePBSphosphate-buffered salinekbkilobase pair(s)ELISAenzyme-linked immunosorbent assayMTT3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromideCMVcytomegalovirus. is an important effector of programmed cell death, playing a role in immune defense against viral, bacterial, and parasitic infections with the ability to target tumor cells and virus-infected cells(3Fiers W. FEBS Lett. 1991; 285: 199-212Google Scholar, 4Vassalli P. Annu. Rev. Immunol. 1992; 10: 411-452Google Scholar). The initial events involved in TNF-induced cell death occur through the 55-kDa TNF receptor (5Tartaglia L.A. Weber R.F. Figari I.S. Reynolds C. Palladino Jr., M.A. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9292-9296Google Scholar, 6Tartaglia L.A. Rothe M. Hu Y.-F. Goeddel D.V. Cell. 1993; 73: 213-216Google Scholar) with the subsequent signal transduction events being the subject of intense investigation(7Higuchi M. Higashi N. Nishimura Y. Toyoshima S. Osawa T. Mol. Immunol. 1991; 28: 1039-1044Google Scholar, 8Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Google Scholar). However, the specific cellular death pathway(s) triggered by TNF and their relationships to other effector-induced apoptotic cell death mechanisms are not well understood. tumor necrosis factor α interleukin 1β converting enzyme plasminogen activator inhibitor type 2 urokinase-type plasminogen activator base pair chloramphenicol acetyltransferase phosphate-buffered saline kilobase pair(s) enzyme-linked immunosorbent assay 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide cytomegalovirus. An evolutionary approach to the elucidation of mechanisms involved in cell death has led to the finding that cell death can be mediated, at least in part, by proteinases. The cell death gene, ced-3 from Caenorhabditis elegans, and its human homologue, the cysteine proteinase interleukin 1β converting enzyme (ICE), cause cell death when expressed in rodent fibroblasts(9Muira M. Zhu H. Rotello R. Hartwieg E.A. Yuan J. Cell. 1993; 75: 653-660Google Scholar). Serine proteinases are implicated in cell death induced by TNF, in that low molecular weight serine proteinase inhibitors protect tumor cells from the cytotoxic effects of TNF(10Suffys P. Beyaert R. VanRoy F. Fiers W. Eur. J. Biochem. 1988; 178: 257-265Google Scholar, 11Ruggiero V. Johnson S.E. Baglioni C. Cell Immunol. 1987; 107: 317-325Google Scholar). More recently, the serine proteinase inhibitor (serpin), plasminogen activator inhibitor type-2 (PAI-2), was reported to confer protection from TNF-mediated cytolysis when overexpressed in HT1080 fibrosarcoma cells(12Kumar S. Baglioni C. J. Biol. Chem. 1991; 266: 20960-20964Google Scholar). PAI-2 is a major product of monocytes and macrophages in response to inflammatory mediators(13Schwartz B.S. Monroe M.C. Levin E.G. Blood. 1988; 71: 734-741Google Scholar, 14Gyetko M.R. Shollenberger S.B. Sitrin R.G. J. Leukocyte Biol. 1992; 51: 256-263Google Scholar). PAI-2 was originally described as an inhibitor of the serine proteinase, urokinase-type plasminogen activator (uPA)(15Kawano T. Mormoto K. Uemura Y. J. Biochem. 1970; 67: 333-342Google Scholar, 16Golder J.P. Stephens R.W. Eur. J. Biochem. 1984; 139: 253-258Google Scholar). uPA specifically catalyzes the hydrolysis of a single Arg-Val bond in the widely distributed zymogen, plasminogen, to form plasmin, a broad spectrum serine proteinase that is capable of hydrolyzing a number of protein substrates(17Dano K. Andreason P. Grondahl-Hansen J. Kristensen P. Neilson L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Google Scholar). uPA has a well established role in cellular invasion and is involved in degradation of extracellular matrices and tissue remodelling(17Dano K. Andreason P. Grondahl-Hansen J. Kristensen P. Neilson L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Google Scholar, 18Testa J.E. Quigley J.P. Cancer Metastasis Rev. 1990; 9: 353-367Google Scholar). uPA has also been reported to function as a cellular growth factor (19Kirchheimer J.C. Wojta J. Christ G. Binder B.R. FASEB J. 1987; 1: 125-128Google Scholar, 20de Petro G. Copeta A. Barlati S. Exp. Cell Res. 1994; 213: 286-294Google Scholar) and to participate in the activation of hepatocyte growth factor(21Naldini L. Tamagnone L. Vigna E. Sachs M. Hartmann G. Birchmeier W. Daikuhara Y. Tsubouchi H. Blasi F. Comoflio P.M. EMBO J. 1992; 11: 4825-4833Google Scholar), but its potential role in cell death is not known. In HT1080 tumor cells, uPA and PAI-2 are synthesized constitutively(12Kumar S. Baglioni C. J. Biol. Chem. 1991; 266: 20960-20964Google Scholar), and it is not possible to ascertain from this model whether the presence of uPA is intrinsic to the protective effect demonstrated by overexpression of PAI-2. In the present paper, we report that expression of PAI-2 protects from TNF-induced apoptosis in HeLa cells, a cell line that does not synthesize PAI-2 or significant levels of uPA and that is sensitive to the cytotoxic effects of TNF. Protection by PAI-2 is independent of extracellular uPA activity and is likely to involve an intracellular cell death proteinase. HeLa cells (ATCC-CLL 2) and MonoMac6 cells (22Ziegler-Heitbrock H.W.L. Thiel E. Futterer A. Herzog V. Wirtz A. Reithmuller G. Int. J. Cancer. 1988; 41: 456-461Google Scholar) (kindly provided by Prof. H. W. L. Ziegler-Heitbrock, University of Munich) were incubated in a 5% CO2 and 95% air atmosphere at 37°C and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamate, 25 mM HEPES buffer, 60 μg/ml penicillin G, and 100 μg/ml streptomycin sulfate. Cell viability was determined by trypan blue dye exclusion. All cultures were checked routinely and determined to be mycoplasma free. An 1867-bp DNA fragment encoding PAI-2 cDNA was excised from pJ7/PAI-2 (23Schleuning W.D. Medcalf R.L. Hession C. Rothenbujler R. Shaw A. Kruithof E.K.O. Mol. Cell. Biol. 1987; 12: 4564-4567Google Scholar) with EcoRI, end-filled, and inserted into the end-filled HindIII site of pRcCMV (Invitrogen). Both orientations were obtained to create sense and antisense PAI-2 expression constructs. The chloramphenicol acetyltransferase (CAT) cDNA gene was obtained by polymerase chain reaction amplification of a 785-bp region of pCATBasic (Promega) using the following polymerase chain reaction primers, and the resultant DNA fragment was cloned into the HindIII site of pRcCMV: CAT primer forward, 5′-GGCGAAGCTTCAGGCGTTTA-3′, and CAT primer reverse, 5′-TACGCCAAGCTTGCATGCCT-3′. The P1 site mutant of PAI-2 was obtained by polymerase chain reaction amplification and overlap extension following the method of Ho et al.(24Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Google Scholar). The following synthesized oligonucleotides were used to alter the nucleotides at 1186-1192, resulting in a mutation at Arg-380 (to Ala-380), and to generate a unique Nar1 restriction enzyme site (see Fig. 6A): Primer A, 5′-GGCTCAGATTCTAGAACTCC-3′; Primer B, 5′-GTATTTCTAGAAATGCACATAAC-3′; Primer C, 5′-GACAGGCGCCACTGGACATGG-3′; and Primer D, 5′-CCATGTCCAGTGGCGCCTGT-3′. Primers A and B and Primers C and D, respectively, were combined to generate DNA fragments containing overlapping mutations at nucleotides 1186-1192 in the PAI-2 cDNA gene. These DNA fragments were then combined, and Primers A and D were used to generate a 988-bp DNA fragment extending between the XbaI restriction enzyme sites at positions 790 and 1778 of PAI-2 cDNA. This DNA fragment was cloned into the corresponding XbaI sites of pJ7/PAI-2 following excision of the 988-bp DNA fragment containing the native sequence at the P1 site. The sense orientation was identified by restriction enzyme mapping, and the DNA sequence of the mutant construct was verified by DNA sequence analysis. Approximately 1 × 107 HeLa cells in log phase were electroporated with 20 μg of plasmid DNA at 250 V, 960 microfarads in RPMI 1640 containing 10% fetal calf serum. Following culture for 16 h, transfectants were selected with 0.8 mg/ml G418 (Life Technologies, Inc.). Individual clones were maintained routinely in 0.3 mg/ml G418. Expression of the transgene was determined by Northern and immunoblot analyses. Near confluent, adherent cultures were harvested by gentle scraping and washed by resuspension in phosphate-buffered saline (PBS). Total RNA was isolated from harvested cells using the guanidinium isothiocyanate method of Chomczynski and Sacchi(25Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Each RNA sample (10 μg) was electrophoresed on denaturing agarose gels containing 1.1% formaldehyde, transferred to Hybond N nylon membranes (Amersham Corp.) by capillary diffusion, and fixed by UV irradiation according to the manufacturer's instructions. The membranes were hybridized essentially as described (26Antalis T.M. Dickinson J.L. Eur. J. Biochem. 1992; 205: 203-209Google Scholar) and washed to a final stringency of 0.1 × SSC and 0.1% SDS at 65°C. The blots were probed with a 2.0-kb HindIII-EcoRI DNA fragment encoding PAI-2 cDNA(27Antalis T.M. Clark M.R. Barnes T. Lehrbach P.R. Devine P.L. Schevzov G. Goss N.H. Stephens R.W. Tolstoshev P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 985-989Google Scholar), the 785-bp CAT DNA fragment, or pRcCMV (for the vector alone transfectants). Plasmids containing cDNA probes for the 55- and 70-kDa TNF receptors were obtained from Dr. David Lowen (Syntex USA, Palo Alto, CA), and manganese superoxide dismutase cDNA (28Fujii J. Taniguchi N. J. Biol. Chem. 1991; 266: 23142-23146Google Scholar) was from Prof. N. Taniguchi (Osaka Medical School and Mitsui Toatsu Chemicals, Inc.). Each DNA fragment was radiolabeled by the random priming method(29Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Google Scholar). The blots were rehybridized with a labeled human 18 S rRNA oligonucleotide as a measure of total RNA loaded in each lane (26Antalis T.M. Dickinson J.L. Eur. J. Biochem. 1992; 205: 203-209Google Scholar). The blots were exposed to Kodak XK-1 film between Dupont Cronex intensifying screens at -70°C for varying times and quantitated by scanning different exposures of autoradiographs using a scanning densitometer (Molecular Dynamics) driven by ImageQuant software. For immunoblot analyses, cells were washed three times in PBS and harvested by gentle scraping into PBS. The cells were pelleted by centrifugation and lysed quickly on ice in cold PBS containing 0.5% Triton X-100, 5 mM EDTA, and 20 μg/ml of the proteinase inhibitor, 4-(2-aminoethyl)benzenesulfonylfluoride, except where indicated in the figure legends. Cellular debris was removed by centrifugation at 12,000 × g for 10 min, and the resulting cell lysate stored in aliquots at -70°C. The protein concentration of each sample was determined by Bio-Rad protein assay. The solubilized proteins (40-80 μg) were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions using a 5-15% acrylamide gradient gel, and the proteins were electrophoretically blotted onto a nitrocellulose membrane (Bio-Rad) for 16 h at 30 V. Specific antigens were detected by incubation of the membrane at room temperature for 1 h with 1 μg/ml of either the anti-catalytic, anti-uPA monoclonal antibody (American Diagnostica #394), the anti-PAI-2 monoclonal antibody (American Diagnostica #3750), the anti-CAT monoclonal antibody (5 Prime → 3 Prime Inc. #5307-310127), or the anti-bcl-2 monoclonal antibody (Oncogene Sciences #OP60). Antibody binding was visualized by ECL detection (Amersham Corp.) according to the manufacturer's instructions. Membranes were exposed to Kodak XK-1 film for various times and quantitated using a scanning densitometer (Molecular Dynamics) as described above. Quantitation of PAI-2 levels by ELISA was carried out using the Tintelize PAI-2 ELISA kit (#220220) from American Diagnostica. Cytolysis was quantitated by two methods: 1) crystal violet staining (modified from (12Kumar S. Baglioni C. J. Biol. Chem. 1991; 266: 20960-20964Google Scholar)), which measures cell survival as a function of cell adherence, and 2) the tetrazolium dye-based MTT assay(30Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Google Scholar), which provides a measure of cell viability. Cells were seeded at 104 cells/well in triplicate in 96-well plates for 16 h prior to treatment with the following agents for 8 h in the standard assay: TNF, 0-50 ng/ml as indicated in the text, and cycloheximide, 10 μg/ml. Cycloheximide was not cytotoxic at the concentrations used within the time frame of the experiments. For assay by crystal violet staining, the cells were washed in PBS and stained with 0.2% crystal violet in 10% ethanol or 10% formaldehyde. The dye was eluted with 33% acetic acid, and the absorbance was measured at 480 nm. For measurement by MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) was added to a final concentration of 0.4 μg/ml/well and incubated for 4 h. Plates were centrifuged at 800 × g for 5 min, and supernatant was removed. MTT crystals were dissolved in Me2SO, and the absorbance was measured at 570 nm. For the experiments involving addition of anti-TNF or anti-uPA antibodies, the cells were washed three times in serum-free RPMI 1640, and the antibodies were added in RPMI 1640 containing 10% acid-treated fetal calf serum at the concentrations given in the figure legends. Cells were incubated for 30 min in the presence of antibody prior to treatment with TNF and/or cycloheximide. Competitive (#042) and noncompetitive (#047) anti-TNF monoclonal antibodies were obtained from Peptide Technology Ltd. (Sydney, Australia). Cells were plated in triplicate at 2 × 105/well in 24-well culture plates (Linbro, Flow) and allowed to adhere overnight. The plates were then placed on ice, washed with binding medium (Hanks' balanced salt solution containing 5% heat-inactivated fetal calf serum), and incubated with 0.3 ml of cold binding medium containing 0.22 nM I125-labeled human recombinant TNF (105 dpm/well, 15-30 TBq/mmol, Amersham Australia). Selected wells also contained a 1000-fold excess of unlabeled TNF (Pharma Biotechnologie, Hannover). After 1 h on ice, the monolayers were washed three times with binding medium prior to extraction with 0.5 M NaOH for 48 h prior to γ scintillation counting. Cell numbers were determined by trypsinization of identically treated monolayers run in parallel with the binding studies. Cells were seeded at 103 cells/well and cultured in RPMI 1640, 10% fetal calf serum for 16 h on sterile glass coverslips in 24-well plates. The media was removed and replaced with media alone or media containing 10 ng/ml TNF and/or 10 μg/ml cycloheximide and incubated for 4 h. Staining was performed essentially as described by Chen (31) using Hoechst stain 33258 (Sigma) at 0.1 μg/ml. Coverslips were mounted in 50% glycerol in citrate phosphate buffer (0.1 M citrate and 0.2 M Na2HPO4, pH 5.5), and the nuclei were visualized by fluorescent microscopy. Cells were seeded at 2 × 106 cells/well in a 24-well plate, allowed to adhere for 16 h, and where indicated treated with TNF (10 ng/ml) and/or cycloheximide (10 μg/ml). Following incubation for 5 h, the cells were harvested and resuspended in buffer containing 20 mM Tris-HCl, pH 8.2, 800 mM NaCl, and 4 mM EDTA. SDS was added to a final concentration of 1%, proteinase K was added to a final concentration of 2 mg/ml, and the samples were incubated for 1 h at 37°C and centrifuged for 5 min at 10,000 × g. The DNA was precipitated from the supernatants with 2.5 volumes of cold ethanol. The recovered DNA was analyzed by electrophoresis on a 1% agarose gel and visualized with ethidium bromide. Binding reactions were carried out at for 10 min at 4°C with approximately 5 × 103 cpm of radiolabeled NFκB probe, 3 μg of poly(dI•dC), and 1 μg of nuclear extract under the following reaction conditions: 15 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, 75 mM NaCl, 20 μg bovine serum albumin, 7.5% glycerol, 0.3% Nonidet P-40, and 1.5 mM dithiothreitol). Synthesized single stranded oligonucleotides containing the NFκB binding site were annealed and end-filled with the Klenow fragment of DNA polymerase in the presence of [α-32P]dCTP (1000 Ci/mmol). The primers used were as follows: NFκBMain, 5′-ACTCACTTTCCGCTGCTCACTTTCC-3′, and NFκBPri: 5′-GGAAAGTGAGCAGCGG-3′. For competition experiments, excess unlabeled double stranded NFκB probe was incubated with nuclear extract in the presence of radiolabeled probe at 0°C for 10 min. DNA-protein complexes were separated on a 5% nondenaturing polyacrylamide gel run at 11 V/cm in TBE buffer (89 mM Tris borate and 2 mM EDTA, pH 8.2). ICE activity was measured essentially as described(32Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Google Scholar). Cells (2 × 107) from HeLa, S1a, A2/7, and the THP-1 monocytic cell line (positive control) were lysed in 0.5 ml of hypotonic buffer (25 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 10 μg/ml pepstatin, and 10 μg/ml leupeptin), sonicated, and centrifuged at 10,000 × g. The supernatant was brought to 10% sucrose and 0.1% Triton X-100 and stored at -20°C. For the ICE assay, 50 μl of cell extract was incubated in the presence of 25 mM HEPES, pH 7.5, 5 mM MgCl2, 10% sucrose, 0.1% Triton X-100, and 5 μM ICE fluorogenic substrate Ac-Tyr-Val-Ala-Asp-AMC (peptide 17(32Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Google Scholar), where AMC is amino-4-methylcoumarin) for 2 h. The ICE fluorogenic substrate was prepared by conventional solution-phase peptide synthesis (Auspep, Parkville, Victoria Australia). ICE activity ranged from 0.5 to 0.7 nmoles amino-4-methylcoumarin released per hr/mg of protein for both PAI-2-expressing and -nonexpressing cells. To generate sense and antisense PAI-2 cDNA, a DNA fragment containing the entire PAI-2 coding sequence and 3′-untranslated region was inserted in two orientations into the expression vector, pRcCMV, under control of the constitutive cytomegalovirus (CMV) promoter. As a positive control for an irrelevant gene, the coding sequence for the CAT gene was inserted into the same vector. HeLa cells were transfected with each of these constructs, as well as vector alone as a negative control, and stable transfectants were selected by resistance to G418. A range of stable clones were isolated from each transfection and analyzed for expression of the transgene by Northern and immunoblot analyses. Each clone was found to express varying levels of transgene mRNA. Northern blots of a selection of sense PAI-2, antisense PAI-2, and CAT clones are shown in Fig. 1A. In the PAI-2 sense clones, two mRNA transcripts are detected that correspond to the mobilities predicted as a result of alternate use of 3′-untranslated sequences, one being the PAI-2 3′-untranslated region (1.9 kb) and the second being the bovine growth hormone 3′-untranslated sequence contained within pRcCMV (2.1 kb). The CAT mRNA transcript is detected at approximately 1.0 kb. Analysis of PAI-2 expression by immunoblot analysis shows that PAI-2 mRNA levels correlate with the synthesis of immunoreactive PAI-2 protein in the sense clones, with none detected in the antisense PAI-2 or vector alone control cell lines (Fig. 1B). The PAI-2 protein synthesized has a molecular mass of 47 kDa and migrates with the same mobility as native PAI-2 induced by lipopolysaccharide in the macrophage-like MonoMac6 cell line. A second minor band is detected beneath the 47-kDa PAI-2 band in both the sense PAI-2 clones and in the MonoMac6 cell lysates, which likely represents a PAI-2 proteolytic cleavage product. The amount of PAI-2 protein synthesized by each of the cell lines was quantitated by ELISA and was found to correlate with the levels detected by immunoblot analysis (Fig. 1B). Stable cell lines that had been transfected with the CAT gene produced the 80-kDa CAT protein(33Shaw W.V. Methods Enzymol. 1975; 43: 737-755Google Scholar), as shown in Fig. 1C. The PAI-2 synthesized by the cells was tested for biological activity demonstrated by the ability to react with uPA, the serine proteinase target of PAI-2. Interaction of uPA and PAI-2 results in the formation of an SDS-resistant complex that demonstrates altered mobility on SDS-polyacrylamide gel electrophoresis or a characteristic “band shift” from 47 kDa to approximately 92 kDa(34Ragno P. Montuori N. Vassalli J.-D. Rossi G. FEBS Lett. 1993; 323: 279-284Google Scholar). As demonstrated in Fig. 1D, addition of purified uPA to cell lysates derived from PAI-2 transfectants results in such a band shift, demonstrating that the recombinant PAI-2 is biologically active. As has been observed with a number of cell types in vitro, HeLa cells are susceptible to cell death by TNF when protein synthesis is inhibited(35Wallach D. J. Immunol. 1984; 132: 2464-2469Google Scholar). Each of the HeLa cell transfectants was treated with TNF in the presence of cycloheximide for 8 h, after which time the parental HeLa cell line showed evidence of extensive cell death. The amount of cell death was quantitated by two independent assays: 1) crystal violet staining, which monitors cell survival as a function of cell adherence, and 2) MTT assay, which provides a measure of viable cells. When assayed by either method, transfectants expressing PAI-2 (S1a, S1b) showed greater than 80% cell survival relative to antisense PAI-2 transfectants (A2/7, A2/17), cells transfected with vector alone (CMV), or cells transfected with CAT, each of which showed less than 30% survival over the 8-h time period (Fig. 2A). The cell death observed was not due to an effect of inhibition of protein synthesis, as treatment with cycloheximide alone had minimal effects on cell survival. The effect of TNF concentration on the sensitivity of the HeLa transfectants to cell death is shown in Fig. 2C. The PAI-2-expressing cell lines show enhanced survival at concentrations of TNF below 10 ng/ml but demonstrate less resistance to the cytolytic effects of TNF at higher concentrations of TNF. At 10 ng/ml the clones expressing the highest levels of PAI-2, S1a and S1b, were essentially resistant to TNF-induced cell death, as compared with antisense PAI-2, CAT, or CMV transfectants; whereas at 50 ng/ml TNF, S1a and S1b show approximately 50% survival compared with 25-30% for the control transfectants. Furthermore the resistance to TNF-induced cell death appears to correlate with the relative level of PAI-2 expressed by the transfected cells. Indeed, when the concentration of TNF required for 50% cell death for each of the sense PAI-2-expressing cell lines was plotted against the log of the concentration of PAI-2 expressed by each cell line (Fig. 2D), a linear relationship was obtained. That the cell death observed was a direct consequence of TNF action was demonstrated by comparing the effects of noncompetitive and competitive anti-TNF antibodies (which interfere with TNF binding to its receptor) on cell death (Fig. 3A). The PAI-2-expressing clone, S1a, and the antisense clone, A2/7, were incubated with increasing concentrations of each of the antibodies and then subjected to challenge with TNF and cycloheximide. Incubation with increasing concentrations of the competitive anti-TNF antibody resulted in increased survival of both the sense and the antisense clones, whereas the noncompetitive antibody had no significant effect on the relative survival of either clone. Therefore, PAI-2 demonstrates protection against cell death mediated directly through TNF. The inflammatory cytokine, interferon-γ, synergizes with TNF in the absence of cycloheximide to promote cell death(36Sugarman B.J. Aggarwal B.B. Hass P.E. Figari I.S. Palladino M.A. Shepard H.M. Science. 1985; 230: 943-945Google Scholar, 37Sugarman B.J. Lewis G.D. Eessalu T.E. Aggarwal B.B. Shepard H.M. Cancer Res. 1987; 47: 780-786Google Scholar). Treatment of HeLa cells with interferon-γ alone has a cytostatic effect on HeLa cells, whereas treatment with TNF in the presence of interferon-γ results in approximately 90% cell death after 72 h. However, the PAI-2-expressing clones, S1a and S1b, were protected from TNF- and interferon-γ-mediated cell death, demonstrating 70-80% cell survival, relative to the control clones, A2/7, A2/17, CMV, and CAT transfectants (10-30% survival) as determined by both MTT (Fig. 2B) and crystal violet assays (data not shown). Interferon-γ treatment alone had a cytostatic effect on antisense and control transfectants similar to that observed in HeLa cells, whereas interferon-γ-treated PAI-2-expressing cells continued to grow during this period. A mechanism by which cells can become resistant to cytolysis mediated by TNF is through shedding of TNF receptors. Soluble TNF-binding proteins resulting from the proteolytic cleavage of TNF receptors can compete with the cell-associated receptors for TNF binding(38Olsson L. Gatanaga T. Gullberg U. Lantz M. Granger G.A. Eur. Cyotkine Netw. 1993; 4: 169-180Google Scholar). Therefore, we examined whether the reduced sensitivity of PAI-2-expressing clones to TNF-induced cell death may be attributed to altered TNF receptor expression. Examination of TNF receptor mRNA levels in the sense and antisense transfectants by Northern blot analysis (Fig. 3B) showed that although each transfectant expressed variable levels of 55-kDa TNF receptor mRNA, TNF receptor expression could not be correlated with the sensitivity of the clones to cell death. No 70-kDa TNF receptor mRNA was detected in any of the transfectants or the parental HeLa cell line (data not shown). The TNF receptors present on the transfected clones were" @default.
• W1657916616 created "2016-06-24" @default.
• W1657916616 creator A5014095375 @default.
• W1657916616 creator A5027779533 @default.
• W1657916616 creator A5050047400 @default.
• W1657916616 creator A5051169622 @default.
• W1657916616 date "1995-11-01" @default.
• W1657916616 modified "2023-10-12" @default.
• W1657916616 title "Plasminogen Activator Inhibitor Type 2 Inhibits Tumor Necrosis Factor α-induced Apoptosis" @default.
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