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• W2042078698 abstract "12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-β in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and β-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-β antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-β. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-β in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and β-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-β antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-β. The mammalian transforming growth factor (TGF)-β 1The abbreviations used are: TGFtransforming growth factorCA-074l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-l-isoleucyl-l-prolineE-64trans-expoxysuccinyl-l-leucylamido-(4-guanidino)butaneMMPmatrix metalloproteasePABpericellular assay bufferPAI-1plasminogen activator inhibitor onePBSphosphate buffered salinePKCprotein kinase CTPA12-O-tetradecanoylphorbol-13-acetatetPAtissue plasminogen activatoruPAurokinase-type plasminogen activatorAMC7-amino-4-methylcoumarinZ-GGR-AMCbenzyloxycarbonyl-Gly-Gly-Arg-7-amido-4-methylcoumarinZ-FA-FMKbenzyloxycarbonyl-Phe-Ala-fluoromethyl ketoneand Z-RR-AMCbenzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarinPIPES1,4-piperazinediethanesulfonic acid12-S-HETE12-S-hydroxy-eicosatetraenoic acidDABCYL4-(4-dimethylaminophenylazo)benzoic acidEDANS5{(2-aminoethyl)amino}naphthalene-1-sulfonic acid family consists of three related proteins: TGF-β1, TGF-β2, and TGF-β3. In vivo studies suggest that members of this family are involved in the regulation of development, tissue remodeling, differentiation, angiogenesis, inflammation, immune regulation, and fibrosis (1.Kulkarni A.B. Huh C.G. Becker D. Geiser A. Lyght M. Flanders K.C. Roberts A.B. Sporn M.B. Ward J.M. Karlsson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 770-774Crossref PubMed Scopus (1662) Google Scholar, 2.Kulkarni A.B. Ward J.M. Yaswen L. Mackall C.L. Bauer S.R. Huh C.G. Gress R.E. Karlsson S. Am. J. Pathol. 1995; 146: 264-275PubMed Google Scholar, 3.Yaswen L. Kulkarni A.B. Fredrickson T. Mittleman B. Schiffman R. Payne S. Longenecker G. Mozes E. Karlsson S. Blood. 1996; 87: 1439-1445Crossref PubMed Google Scholar, 4.Kulkarni A.B. Karlsson S. Res. Immunol. 1997; 148: 453-456Crossref PubMed Scopus (32) Google Scholar, 5.Sanford L.P. Ormsby I. Gittenberger-de Groot A.C. Sariola H. Friedman R. Boivin G.P. Cardell E.L. Doetschman T. 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Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 13.Clark D.A. Coker R. Int. J. Biochem. Cell Biol. 1998; 30: 293-298Crossref PubMed Scopus (272) Google Scholar, 14.Li J. Foitzik K. Calautti E. Baden H. Doetschman T. Dotto G.P. J. Biol. Chem. 1999; 274: 4213-4219Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 15.Dong-Le Bourhis X. Lambrecht V. Boilly B. Br. J. Cancer. 1998; 77: 396-403Crossref PubMed Scopus (24) Google Scholar). transforming growth factor l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-l-isoleucyl-l-proline trans-expoxysuccinyl-l-leucylamido-(4-guanidino)butane matrix metalloprotease pericellular assay buffer plasminogen activator inhibitor one phosphate buffered saline protein kinase C 12-O-tetradecanoylphorbol-13-acetate tissue plasminogen activator urokinase-type plasminogen activator 7-amino-4-methylcoumarin benzyloxycarbonyl-Gly-Gly-Arg-7-amido-4-methylcoumarin benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin 1,4-piperazinediethanesulfonic acid 12-S-hydroxy-eicosatetraenoic acid 4-(4-dimethylaminophenylazo)benzoic acid 5{(2-aminoethyl)amino}naphthalene-1-sulfonic acid TGF-βs mediate their biological activities via interaction with high affinity, cell surface receptors (16.Heldin C-H. Miyazono K. Dijke P.T. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar). Several cell types synthesize and secrete TGF-βs in an inactive latent form. Latency is a consequence of intracellular processing. Specifically, after translation TGF-β1 polypeptides dimerize and subsequently undergo cleavage to yield amino-terminal latency-associated peptides and carboxyl-terminal peptides (11.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3998) Google Scholar, 12.Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 17.Lyons R.M. Gentry L.E. Purchio A.F. Moses H.L. J. Cell Biol. 1990; 110: 1361-1367Crossref PubMed Scopus (672) Google Scholar). Latency-associated peptides remain associated with the dimerized carboxyl-terminal peptides via electrostatic interactions, thus forming latent TGF-β. Latency-associated peptides must be released from the latent complex before TGF-β can activate its receptor (11.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3998) Google Scholar, 12.Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (443) Google Scholar). A variety of agents and treatments activate latent TGF-β1. Heat, acidic pH, chaotropic agents, plasmin, substilisin-like endopeptidases, and the extracellular matrix protein thrombospondin promote the release of latency-associated peptides from latent TGF-β1 in vitro(11.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3998) Google Scholar, 12.Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 17.Lyons R.M. Gentry L.E. Purchio A.F. Moses H.L. J. Cell Biol. 1990; 110: 1361-1367Crossref PubMed Scopus (672) Google Scholar, 18.Chu T.M. Kawinski E. Biochem. Biophys. Res. Commun. 1998; 253: 128-134Crossref PubMed Scopus (48) Google Scholar, 19.Ribeiro S.M. Poczatek M. Schultz-Cherry S. Villain M. Murphy-Ullrich J.E. J. Biol. Chem. 1999; 274: 13586-13593Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 20.Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). In vivo studies with thrombospondin-deficient mice support a role for thrombospondin in the physiological activation of latent TGF-β1 (21.Crawford S.E. Stellmach V. Murphy-Ullrich J.E. Ribeiro S.M. Lawler J. Hynes R.O. Boivin G.P. Bouck N. Cell. 1998; 93: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar). It has been inferred from analyses of plasminogen-deficient mice that plasminogen/plasmin do not play a major role in the in vivo processing of latent TGF-β1 (22.Ploplis V.A. Carmeliet P. Vazirzadeh S. Van Vlaenderen I. Moons L. Plow E.F. Collen D. Circulation. 1995; 92: 2585-2593Crossref PubMed Scopus (311) Google Scholar, 23.Matrat M. Lardot C. Huaux F. Broeckaert F. Lison D. J. Toxicol. Environ. Health. 1998; 55: 359-371Crossref PubMed Scopus (16) Google Scholar). Nevertheless, studies by Grainger et al.(24.Grainger D.J. Kemp P.R. Liu A.C. Lawn R.M. Metcalfe J.C. Nature. 1994; 370: 460-462Crossref PubMed Scopus (341) Google Scholar) suggest that these proteases may contribute to the in vivo activation of latent TGF-β1 in some situations, and data continue to be published documenting plasmin-mediated TGF-β activation in cell lines (25.Herbert J.M. Carmeliet P. FEBS Lett. 1997; 413: 401-404Crossref PubMed Scopus (25) Google Scholar, 26.Godár S. Horejsi V. Weidle U.H. Binder B.R. Hansmann C. Stockinger H. Eur. J. Immunol. 1999; 29: 1004-1013Crossref PubMed Scopus (150) Google Scholar). The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is cytostatic to cells of various lineages (Ref. 27.Guo M. Reiners Jr., J.J. Carcinogenesis. 2000; 21: 1303-1312PubMed Google Scholar and references therein). We recently reported that the spontaneously immortalized human breast epithelial cell line MCF10A-Neo arrests in G1following exposure to 10 nm TPA (27.Guo M. Reiners Jr., J.J. Carcinogenesis. 2000; 21: 1303-1312PubMed Google Scholar). This arrest developed quickly and was paralleled by the production of a cytostatic medium. A variety of approaches suggested that ∼50% of the cytostatic activity of TPA was mediated by a TGF-β family member. Surprisingly, the serum used to supplement the culture medium, and not the MCF10A-Neo cells, was the source of the latent cytokine (27.Guo M. Reiners Jr., J.J. Carcinogenesis. 2000; 21: 1303-1312PubMed Google Scholar). The current study was initiated to identify the processes activated by TPA in MCF10A-Neo cultures responsible for the activation of latent TGF-β. Preliminary studies (27.Guo M. Reiners Jr., J.J. Carcinogenesis. 2000; 21: 1303-1312PubMed Google Scholar) demonstrated that the cytostatic effects of TPA could be inhibited by co-treatment with plasminogen activator inhibitor 1 (PAI-1). Consequently, we hypothesized that a proteolytic cascade involving urokinase type plasminogen activator (uPA) or tissue plasminogen activator (tPA) and plasmin was involved in this activation. This hypothesis proved to be correct. However, it involved an unexpected mechanism for the activation of pro-uPA. Specifically, TPA treatment of MCF10A-Neo cultures triggered the exocytosis of a subpopulation of lysosomes/endosomes, and released lysosomal/endosomal cathepsin B catalyzed the activation of pro-uPA. Aprotinin, cathepsin C, Me2SO, TPA, cycloheximide, l-lysine agarose, 4-methyl-umbelliferyl-N-acetyl-β-glucosaminide, control murine IgG, control rabbit IgG, and a neutralizing polyclonal pan rabbit antibody made to a mixture of TGFβ1 + TGFβ1.2 + TGFβ2 + TGFβ5 were obtained from Sigma. Recombinant-derived human PAI-1 and cathepsins L and D were purchased from Calbiochem (San Diego, CA). The fluorescent substrates Z-GGR-AMC and Z-RR-AMC were obtained from Bachem Bioscience Inc. (King of Prussia, PA). Neutralizing rabbit antibodies to tPA and uPA were purchased from American Diagnostic Inc. (Greenwich, CT). Protein G-agarose, trypsin, penicillin/streptomycin solution, and horse serum were obtained from Invitrogen. Purified human liver cathepsin B was purchased from Athens Research and Technology (Athens, GA). l-[3,4,5-3H(N)]Leucine came from PerkinElmer Life Sciences. The protease inhibitors E-64 and CA-074 were purchased from Peptides International, Inc. (Louisville, KY). The cysteine protease inhibitor Z-FA-FMK was purchased from Enzyme Systems Products (Livermore, CA). The cathepsin D substrate Ac-GE(Edans)-Z-EVNLDAEF-Z-K(Dabcyl)-G-NH2 and standard Ac-RE(Edans)-A-NH2 were synthesized by P. Richardson (Abbott Laboratories, Abbott Park, IL). The MCF10A-Neo cell line was obtained from the Cell Lines Resource (Karmanos Cancer Institute, Detroit, MI). It was derived by transfection of the spontaneously immortalized, nontumorigenic human breast epithelial MCF10A cell line with plasmid Homer 6 (pHo6) and subsequent selection for G418 resistance. The MCF10A-Neo line consists of the pooled survivors. Detailed characterizations and descriptions of the derivations of the MCF10A and MCF10A-Neo cell lines have been published (28.Soule H.D. Maloney T.M. Wolman S.R. Peterson Jr., W.D. Brenz R. McGrath C.M. Russo J. Pauley R. Jones R.F. Brooks S.C. Cancer Res. 1990; 50: 6075-6086PubMed Google Scholar, 29.Basolo F. Elliott J. Tait L. Chen X.Q. Maloney T. Russo I.H. Pauley R. Momiki L. Caamano J. Klein-Szanto A.J.P. Koszalka M. Russo J. Mol. Carcinog. 1991; 4: 25-35Crossref PubMed Scopus (159) Google Scholar). The morphological and growth properties of the two cell lines are very similar (29.Basolo F. Elliott J. Tait L. Chen X.Q. Maloney T. Russo I.H. Pauley R. Momiki L. Caamano J. Klein-Szanto A.J.P. Koszalka M. Russo J. Mol. Carcinog. 1991; 4: 25-35Crossref PubMed Scopus (159) Google Scholar). MCF10A cultures, like MCF10A-Neo cultures, exocytose cathepsin B following exposure to TPA. 2M. Guo and J. J. Reiners, Jr., unpublished observations. MCF10A-Neo cells were cultured in supplemented Dulbecco's modified Eagle's medium/Ham's F-12 medium as previously described (30.Basolo F. Serra C. Ciardiello F. Fiore L. Russo J. Campani D. Dolei A. Squartine F. Toniolo A. Int. J. Cancer. 1992; 51: 634-640Crossref PubMed Scopus (24) Google Scholar). The supplements consisted of human insulin (10 μg/ml), epidermal growth factor (10 ng/ml), cholera toxin (100 ng/ml), hydrocortisone (0.5 μg/ml), 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate, and 5% horse serum. Proliferation studies were performed with cultures plated at a density that ensured exponential growth for a minimum of 4 days. The cultures were routinely treated 40–48 h after plating. The details of treatment are provided in the text. TPA, cycloheximide, E-64, CA-074, Z-GGR-AMC, Z-RR-AMC, and Z-FA-FMK were all dissolved and diluted in Me2SO. Organic solvent never exceeded 0.2% of total culture/assay volume. Aprotinin and PAI-1 were diluted in sterile water. Purified human cathepsin B was diluted in a solution containing 1 mm EDTA and 20 mm sodium acetate, pH 5.0. The various antibodies used in this study were reconstituted/diluted according to the manufacturer's instructions. For estimation of cell numbers, the cultures were harvested by exposure to a solution of 0.25% trypsin, 0.1 mm EDTA. Viability was assessed by determining the ability to exclude trypan blue. Culture medium supplemented with 5% horse serum was incubated with 12 μg/ml of a rabbit neutralizing polyclonal TGF-β pan antibody or 12 μg/ml of control rabbit IgG for 2 h at 37 °C. The medium was then chromatographed on a column of protein G-agarose that had been washed and equilibrated in 10 mmsodium phosphate, pH 7.0, 0.15 m NaCl. The eluant was used as a source of TGF-β-depleted medium. Medium supplemented with horse serum was also chromatographed on a column ofl-lysine-agarose that had been washed with 0.1m NaCl, 1 mm EDTA, and 50 mmTris-HCl, pH 7.5. The eluant was used as a source of plasminogen/plasmin-depleted medium. Cells grown in 35-mm culture dishes were treated with varied concentrations of cycloheximide for 30 min. The cultures were then washed twice with PBS prior to being pulsed withl-[3,4,5-3H(N)]leucine (1 μCi/ml of culture medium) in the presence of fresh cycloheximide. The cultures were harvested at varied times thereafter for analyses of [3H]leucine incorporation into protein. The procedure used for the processing of labeled cells has been described in detail (31.Schöller A. Hong N.J. Bischer P. Reiners Jr., J.J. Mol. Pharmacol. 1994; 45: 944-954PubMed Google Scholar). Radioactivity was detected by scintillation counting and expressed as dpm/106 cells. The cells were plated in either 35-mm culture dishes or 96-well culture plates. Culture medium was also added to a parallel set of wells/dishes that lacked cells. After 1.5–2 days in culture, the cells were treated with various antibodies or protease inhibitors 1 h prior to the addition of either Me2SO or TPA (10 nm). Culture medium (200 μl) was periodically removed thereafter and transferred to 96-well plates appropriate for fluorescent assay measurements. Analyses of uPA/tPA were initiated by the addition of 100 μm Z-GGR-AMC. The release of AMC was monitored for 12–15 min on a SPECTRAmax Gemini dual scanning microplate spectrofluorometer using an excitation wavelength of 380 nm and an emission wavelength of 440 nm. Parallel analyses were performed on culture medium taken from dishes/wells lacking cells. Changes in fluorescence over a set time were determined and converted to fmol of AMC by comparison to a standard curve. Cell-derived activity was calculated as the difference in activities of media derived from dishes/wells having and lacking cells. For the calculation of specific activities, cells were released from dishes/wells by treatment with 0.25% trypsin, 0.1 mmEDTA, and counted. Z-GGR-AMC cleavage activity is reported as fmol of AMC produced per min per 103 cells. The protocol described by Linebaugh et al. (32.Linebaugh B.E. Sameni M. Day N.A. Sloane B.F. Keppler D. Eur. J. Biochem. 1999; 264: 100-109Crossref PubMed Scopus (130) Google Scholar) was used for the assay of extracellular and intracellular cathepsin B. The cultures were treated as described above and washed twice with PBS at varied times after Me2SO or TPA addition before being covered with pericellular assay buffer (PAB), which consisted of Hanks' balanced salt solution lacking sodium bicarbonate but containing 0.6 mm CaCl2, 0.6 mm MgCl2, 2 mml-cysteine, and 25 mm PIPES, pH 7.0. After a 10-min incubation at 37 °C, 200 μl of the PAB solution was transferred to a 96-well plate. The assay was initiated by the addition of 100 μm Z-RR-AMC to each well in the absence or presence of 5–20 μm CA-074 and followed for 15 min on a fluorescence plate reader using an excitation wavelength of 380 nm and an emission wavelength of 440 nm. To measure intracellular cathepsin B activity, the cultures were washed twice with PBS and then lysed with PAB supplemented with 0.1% Triton X-100. The lysates were assayed as described above. Changes in fluorescence over time were determined and converted to fmol of AMC by comparison with a standard curve. Activity inhibited by CA-074 was considered to represent cathepsin B. With Z-RR-AMC as a substrate, this represented ∼90–95% of the total activity measured in Triton X-100 lysates. Duplicate cultures not used for the assay of cathepsin B were treated with trypsin/EDTA and counted. Cathepsin B specific activities are reported as fmol of AMC produced per min per 103 cells. Extracts prepared for cathepsin B analyses were also used for the assay of cathepsins L and D. The assay for cathepsin L was similar to that used for cathepsin B except for the substitution of Z-FR-AMC (100 μm) as substrate. Cleavage activity not inhibited by 10 μm CA-074 was considered to be cathepsinl-like activity. Cathepsin L specific activities are reported as fmol of AMC produced per min per 103 cells. Cathepsin D was assayed by a published procedure (33.Ladror U.S. Snyder S.W. Wang G.T. Holzman T.F. Kraft G.A. J. Biol. Chem. 1994; 269: 18422-18428Abstract Full Text PDF PubMed Google Scholar) that monitors the cleavage of Ac-GE(Edans)-Z-EVNLDAEF-Z-K(Dabcyl)-G-NH2, a highly selective substrate for cathepsin D (33.Ladror U.S. Snyder S.W. Wang G.T. Holzman T.F. Kraft G.A. J. Biol. Chem. 1994; 269: 18422-18428Abstract Full Text PDF PubMed Google Scholar, 34.Gulnik S.V. Suvorov L.I. Majer P. Collins J. Kane B.P. Johnson D.G. Erickson J.W. FEBS Letters. 1997; 413: 379-384Crossref PubMed Scopus (51) Google Scholar). This peptide corresponds to a mutant of the amyloid B protein precursor having an Asn-Leu substitution at amino acids 670–671 and contains Dabcyl and Edans groups, which act as internal quencher and fluorophore, respectively. Release of the Edans fluorophore was followed on a Shimadzu RF-540 spectrofluorophotometer using an excitation wavelength of 340 nm and an emission wavelength of 490 nm. The assays contained 40 mm sodium formate, pH 3.5, and 100 μm peptide substrate (dissolved in Me2SO). The reactions were initiated by the addition of extract. Activity inhibited by the addition of 10 μm pepstatin A represented cathepsin D. Changes in fluorescence over time were determined and converted to fmol of Edans by comparison with a standard curve made with Ac-RE(Edans)-A-NH2. Cathepsin D specific activities are reported as fmol of Edans fluorophore released per min per 103 cells. The assay described by Storrie and Madden (35.Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar) was modified slightly and used to assay β-hexosaminidase. The cells were plated in 60-mm culture dishes. After 2 days the cultures were washed, refed with 1.5 ml of growth medium, and treated with Me2SO or TPA (10 nm). Samples of 200 μl were periodically removed and incubated with 200 μl of assay mixture (0.1 mm sodium acetate, pH 4.0, 1 mm4-methyl-umbelliferyl-N-acetyl-β-glucosaminide). The reaction was terminated by the addition of 0.5 ml of 0.5 mglycine, 0.5 m Na2CO3. Samples (200 μl) of the assay mixture were transferred to a 96-well plate and analyzed with a dual scanning microplate spectrofluorometer using an excitation wavelength of 364 nm and an emission wavelength of 448 nm. The above assay represents conditions used to monitor the extracellular accumulation of β-hexosaminidase over time following the addition of TPA. To determine the rate of β-hexosaminidase secretion, 2-day-old cultures were treated with TPA or Me2SO and at varied times thereafter washed and refed with 1 ml of fresh medium. After 15 min the culture medium was removed for assay as described above. Total cellular β-hexosaminidase activity was assayed as above using lysates prepared by exposing cultures to 1 ml of 1% Nonidet P-40 in PBS for 2 min. Additional plates were exposed to a solution of 0.25% trypsin, 0.1 mm EDTA and counted. β-Hexosaminidase activity is reported as either relative fluorescent units/103 cells or relative fluorescent units/103 cells/min. Untreated/unconditioned medium (with or without Nonidet P-40 depending on the protocol) was used as a control and subtracted from each sample. MCF10A-Neo cells were plated on poly-l-lysine-coated coverslips. Two days later they were treated with either Me2SO or 10 nm TPA. At various times after treatment coverslips were washed, fixed, and processed for indirect immunolocalization of cathepsin B by confocal microscopy. The procedure used has been described in detail (36.Kessel D. Luo Y. Mathieu P. Reiners Jr., J.J. Photochem. Photobiol. 2000; 71: 196-200Crossref PubMed Scopus (133) Google Scholar). The primary antibody was a rabbit polyclonal antibody that recognizes human cathepsin B and procathepsin B (37.Moin K. Day N.A. Sameni M. Hasnain S. Hirama T. Sloane B.F. Biochem. J. 1992; 285: 427-434Crossref PubMed Scopus (111) Google Scholar). The secondary antibody was goat anti-rabbit Alexa 488 (A 11008, Molecular Probes). The images were captured using a Zeiss 310 confocal microscope and a 63×/1.4 oil immersion lens, PH3 phase condenser, and a 488-nm laser light source with a pinhole setting of 17. All of the images were captured at the same contrast and brightness settings. All enzymatic assays involved analyses of a minimum of four culture wells or three culture dishes/treatment/experiment. Cell counts were performed on three or four culture wells or culture dishes. The data were analyzed by the Tukey HSD test. The Statistica 5.0 software package (StaSoft Inc., Tulsa, OK) was used to perform these calculations. The tripeptide Z-GGR-AMC is a substrate for a limited number of serine proteases including uPA and tPA (38.Walker B. Elmore D.T. Thromb. Res. 1984; 34: 103-107Abstract Full Text PDF PubMed Scopus (5) Google Scholar, 39.Bigbee W.L. Weintraub H.B. Jensen R.H. Anal. Biochem. 1978; 88: 114-122Crossref PubMed Scopus (15) Google Scholar, 40.Goretzki L. Schmitt M. Mann K. Calvete J. Chucholowski N. Kramer M. Günzler W.A. Jänicke F. Graeff H. FEBS Lett. 1992; 297: 112-118Crossref PubMed Scopus (144) Google Scholar). MCF10A-Neo cultures constitutively expressed a non-cell-associated, extracellular activity capable of cleaving Z-GGR-AMC (Fig. 1 A). This activity was not affected by exposure to Me2SO but was elevated after exposure to TPA (Fig. 1 A). Maximal increases were noted 1–2 h after TPA treatment. Thereafter, cleavage activity declined and returned to control levels within 8 h of TPA treatment. The assay employed in Fig. 1 A was not designed to measure extracellular, cell-associated Z-GGR-AMC cleavage activity. To measure such activity, cleavage assays were performed directly in the culture wells (cell-associated + non-cell-associated activities) and compared with the activities in medium assayed after removal from the culture wells (non-cell-associated activity). No additional activity was detected when the assays were performed in the presence of cells (Fig. 1 B). Hence, the TPA-induced extracellular Z-GGR-AMC cleavage activity was not cell-associated. Exposure of cultures to the serine protease inhibitor aprotinin prior to Me2SO treatment strongly inhibited extracellular Z-GGR-AMC cleavage activity (Fig. 2 A). Aprotinin co-treatment also suppressed TPA-induced extracellular cleavage activity. Indeed, the extracellular Z-GGR-AMC cleavage activity of cultures co-treated with TPA and aprotinin was similar to the activity measured in cultures co-treated with Me2SO and aprotinin (Fig. 2 A). Treatment of cultures with the tPA/uPA inhibitor PAI-1 had no effect on basal extracellular Z-GGR-AMC cleavage activity (Fig. 2 B). However, co-treatment of TPA cultures with PAI-1 completely suppressed the TPA-dependent induction of extracellular Z-GGR-AMC cleavage activity (Fig. 2 B). Neutralizing antibodies to tPA and uPA were used to determine whether either protease was responsible for extracellular Z-GGR-AMC hydrolysis (Fig. 3). Antibodies to tPA affected neither basal nor TPA-induced Z-GGR-AMC cleavage activity. Neutralizing uPA antibody had no effect on basal Z-GGR-AMC cleavage activity. However, the uPA antibody inhibited completely the extracellular Z-GGR-AMC cleavage activity induced by TPA (Fig. 3). Hence, the extracellular Z-GGR-AMC cleavage activity induced by TPA appeared to be uPA. Co-treatment of cultures with uPA antibody suppressed the cytostatic effects of TPA by ∼50% (Table I). In contrast, uPA antibody had no effect on the growth of solvent-treated cultures. Control IgG had no effect on the growth of either solvent- or TPA-treated cultures (Table I). The magnitude of protection (∼50%) afforded by co-treatment with uPA antibody is significant because TGF-β was shown previously to mediate ∼50% of the cytostatic effects of TPA on MCF10A-Neo proliferation (27.Guo M. Reiners Jr., J.J. Carcinogenesis. 2000; 21: 1303-1312PubMed Google Scholar).Table ICytostatic effects of TPA on MCF10A-Neo cells cultured in the presence of uPA antibody or CA-074Hours after treatmentTreatmentCell number (×10−3)Percentage of control response1-aThe difference in cell counts between the 23-h Me2SO treatment group and the time zero group was set as the 100% control response. The effects of CA-074 and TPA on proliferation were calculated by subtracting the numbers of cells counted in" @default.
• W2042078698 created "2016-06-24" @default.
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• W2042078698 date "2002-04-01" @default.
• W2042078698 modified "2023-10-18" @default.
• W2042078698 title "Phorbol Ester Activation of a Proteolytic Cascade Capable of Activating Latent Transforming Growth Factor-β" @default.
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• W2042078698 doi "https://doi.org/10.1074/jbc.m108180200" @default.
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