identification and characterization of a novel matrix-degrading … · the enzyme degrades gelatin...

(CANCER RESEARCH 53. 1409-1415. March 15. 1993]

Identification and Characterization of a Novel Matrix-degrading Protease fromHormone-dependent Human Breast Cancer Cells

Yuenian E. Shi,1 Jeff Torri, Lynn Yieh, Anton Wellstein, Marc E. Lippman, and Robert B. Dickson

Vincent T. Lombardi Cancer Rese arch Center, Georgetown University Medical Center. Washington. D.C. 20007

ABSTRACT

A novel matrix-degrading enzyme was identified from human breastcancer cells. This enzyme appears as major gelatinase in hormone-depen

dent breast cancer cell lines and has as an apparent molecular mass of 80kDa on gelatin zymography. The 80-kDa enzyme has a unique metal ion

specificity. In addition to calcium ions, the gelatinolytic activity can besupported by manganese and/or magnesium. Unlike 92- and 72-kDa ge-

latinases and other known members of the metalloproteinase family, the80-kDa protease is not activated by p-aminophenylmercuric acetate and

its gelatinolytic activity is not inhibited by tissue inhibitor of metalloproteinase 2. It is active over the pH range 7.5-9.5 with an optimum at pH 8.5.

The enzyme degrades gelatin and type IV collagen. The proteolytic activityof the enzyme is inhibited by EDTA and leupeptin. These unique featuresclearly distinguish the 80-kDa protease from the known 92-and 72-kDagelatinases. The expression of 80-kDa enzyme can be detected in hormone-

dependent human breast cancer cell lines in vitro and in tumors grownfrom these cells in athymic nude mice.

INTRODUCTION

MÃ©tastasesof epithelial tumors, such as breast cancer, occur whenthe epithelial cells are no longer contained by the basement membraneboundary to the underlying stromal compartment. The process ofmetastasis does not appear to be governed by precisely the samemechanisms as tumor growth, although its progressive deregulationsometimes occurs in parallel with deregulated proliferation (1,2). It isthis disconcordance which helps to make breast cancer an unpredictable disease; even very small malignant lesions at the limit of detection by mammography or palpation can already be metastatic (1, 3).

Tumor cell invasion is thought to critically depend upon proteolyticevents (4, 5). The proteases are a large family, grouped into four mainclasses: serine-proteases, such as plasminogen activators, plasmin,and elastase; cysteine proteases, principally cathepsins B and L; met-

alloproteinases, which comprise interstitial or type I collagenases,type IV collagenases or gelatinases, and stromelysins; and aspartylproteinases, such as cathepsin D (6). Although enzymes from all fourprotease classes have been implicated in the process of cancer invasion and metastasis (6-11 ), the proteolytic processes of metastasis are

thought to initially depend upon type IV collagenases which degradetype IV collagen, the major structural component of basement membrane and, presumably, one of the first barriers to mÃ©tastases(1, 12,13). At present, 72- and 92-kDa type IV collagenases are both known

to exist in breast cancer (6, 14, 15). Each is secreted in an inactiveform and requires an activational cleavage yielding 68- and 86-kDa

active enzymes, respectively. //; vivo, activational cleavage is thoughtto be either autocatalytic or dependent upon other proteases in thetumor environment (16, 17). Two natural inhibitors of these enzymeshave been characterized: TIMP-I2 and TIMP-2 (14, 15-17). The

Received 9/9/92; accepted 1/6/93.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this faci.

1To whom requests for reprints should be addressed.2 The abbreviations used are: TIMP. tissue inhibitor of metalloproteinase; Con A,

concanavalin A; PMSF. phenylmethylsulfonyl fluoride; SDS. sodium dodecyl sulfate;PAGE, polyacrylamide gel electrophoresis; APMA. /j-aininophenylmercuric acetate;IMEM. improved minimum essential medium; CM. conditioned medium; MMP. matrixmetalloproteinase(s).

inactive, undipped collagenases are generally secreted as a complexwith a TIMP (6, 19). Enzyme activity depends upon secreted ratios ofcollagenase/TIMP and activational process. Measurements of the 72-

kDa collagenase itself in breast cancer are developing as useful prognostic indicators (13, 19-22).

Fully metastatic models of hormone-responsive breast cancer have

only recently been described, and some progress has been made in invitro invasion systems to evaluate regulatory mechanisms (23, 24).The reconstituted basement membrane extract, Matrigel, has beenutilized in assessing invasive potential of cancer cells (25). Tumor cellinvasiveness into a Matrigel barrier in a Boyden chamber is thought todepend on a proteolytic cascade involving collagenases and possibleother proteases and is also subjected to cell motility (25, 26). Invasionof hormone-dependent breast cancer cells in vitro is stimulated by

estrogen or tamoxifen (a weakly estrogenic, nonsteroidal antiestrogen)(23) but not by the steroidal pure antiestrogen ICI 164,384 (24). Thepure antiestrogen was capable of blockade of both estrogen- or tamox-ifen-induced invasion suggesting a role for the estrogen receptor in

mediating these effects. We now report the identification, partial purification, and characterization of a 80-kDa matrix metalloprotease-like enzyme from hormone-dependent human breast cancer cells. Ourdata indicate that the 80-kDa enzyme is a novel enzyme which represents a major component of gelatin-degrading proteases in hormone-

dependent human breast cancer cells.

MATERIALS AND METHODS

Reagents. The protease inhibitors phenylmethylsulfonyl fluoride, leupeptin, and pepstatin were obtained from Boehringer Mannhem. Indianapolis, IN.EDTA. benzamidine, dithiothreitol, type 1 collagen, and iodoacetamide werefrom Sigma, St. Louis, MO. Type I 'H-collagen was obtained from Dupont,

Wilmington, DE. Recombinant 72-kDa type IV collagenase, recombinuntTIMP-2, and type IV 'H-collagen were kindly provided by Dr. Roben Bird.

Molecular Oncology. Inc., Gaithersburg, MD. Transferrin, laminin. and fi-bronectin were purchased from Collaborative Research; gelatin-Sepharose andchelate-Sepharose were from Pharmacia, Piscataway, NJ.

Cell Lines and Culture Conditions. T47Dco cells were kindly providedby Dr. Dean Edwards (University of Colorado). MCF-7 cells were obtainedfrom Dr. Marvin Rich (Michigan Cancer Foundation), and MDA-MB-435 was

kindly supplied by Dr. Janet Price, The University of Texas M. D. AndersonCancer Center, Houston, TX. All other breast cancer cell lines were obtainedfrom the American Type Culture Collection (Rockville. MD). All cell lineswere maintained in Costar T75 flasks with Richters IMEM (Biofluides, Rockville. MD) supplemented with 10% fetal calf serum (GIBCO. New York, NY).

Preparation of Conditioned Medium and Plasma Membranes. In orderto remove the soluble gelatinases present in the serum, experiments wereperformed with subconfluent monolayers obtained by culturing the cells for 3days in IMEM supplemented with 10% fetal calf serum that was previouslyexposed to gelatin affinity chromatography. The medium was discarded and themonolayers were washed twice with phosphate-buffered saline. The monolay

ers were cultured in the absence of serum, in IMEM supplemented withtransferrin ( I mg/liter), fibronectin ( 1 mg/liter), and trace elements (Biofluides,Rockville. MD). After 24 h, the serum-free medium was discarded, and thecells were replenished with the fresh serum-free medium and cultured for

another 48 h. At the end of this period, the conditioned medium was collected.The medium was then centrifuged at 1200 x g. and supernatants were savedand concentrated 20-fold by Ultrafiltration using Centripreps (Amicon Division, molecular weight cutoff. 10,000) at 4Â°C.Isolation of plasma membrane

from tumors was performed as previously described (27). Briefly, the tumorsamples were homogenized with cold phosphate-buttered saline containing 1

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IDENTIFICATION Ol A NOVhl MAl'KIX DICK \DISC, I'ROIIASI

IHMPMSFand l IHMben/.amidine. After centrifugation al low speed (800 x g)for 15 min. the supernatant was further centrifuged at HX).(XX)x g for I h. Themembrane pellet was extracted overnight at 4Â°Cin a 50 imi Tris (pH 7.4)

huiler containing 2(X) IHMNaCl. l IHMPMSF, l IHMbenzamidine, and 2

IDENTIUCATION OF A NOVEL MATRIX DEGRADING PROTEASE

Fig. 2. Gelatin zymographic analysis of secreted proteases from different breast tumorcell lines. Conditioned medium was concentrated 20-fold and subjected to gelatin zymog-

raphy. The proteolytic activities were detected following overnight incubation at pH 8.5.

Ca Mg EDTA

Ca Mg MnCa EDTA EDTA EDTA EDTA

BFig. 3. Metal Â¡onspecificity of gelatinolytic activity of 80-kDa protease. KO-kDa

protease partially purified by Zn*- chelation chromatography from T47Dco cells and

combined conditioned medium from HTI080 (92 kDa) and Hs578T (72 kDa) weresubjected to gelatin /ymography analysis for metal specificity. A. metal ion dependencyfor gelatinolytic activity. Slices of gelatin gels containing 92-, 72-, and 80-kDa gelatinaseswere prepared and incubated with Hanks' balanced salt solution at pH 7.4 with 5 m.M

EDTA or 5 mw of different metal ions. EDTA inhibited all three enzymes. The gelatinolytic activity of the 92- and 72-kDa proteases was observed only in the incubation buffercontaining calcium, but the 80-kDa protease showed activity in the preparations of eithercalcium, manganese, or magnesium. B. reversal of EDTA-induced inhibition of gelatinolytic activity by different metal ions. Prior to incubation, slices of gelatin gels containing 92-, 72-, and 80-kDa gelatinases were prepared and preincubated with 0.5 mM EDTA

in the incubation buffer for 30 min before 5 niM addition of different metal ions. All themetal ions tested restored the gelatinolytic activity of 80-kDa protease but failed to restorethe gelatinolytic activity of 92- and 72-kDa proteases.

Isolation of the 80-kDa Gelatinolytic Enzyme. To isolate andcharacterize the 80-kDa gelatinolytic enzyme, 2.5 liters of the serum-free CM of T47Dco cells were concentrated l(X)-fold by ultrafiltra-tion. The concentrated CM was first applied to gelatin-Sepharose.Unexpectedly, the 80-kDa gelatinolytic activity was detected in theflow-through fractions, unlike the 92- and 72-kDa gelatinases, whichare absorbed by gelatin-Sepharose and can be eluted with Me2SO.When gelatin-Sepharose T47Dco conditioned medium was subjectedto standard elution with Me2SO, 92- and 72-kDa gelatinase bands

were observed in gelatin /ymography (data not shown). These resultssuggest that in addition to 80-kDa as a dominant gelatinase, T47Dcocells also express low levels of 92- and 72-kDa gelatinases. Thereforewe currently use gelatin-Sepharose as a first purification step to separate 80-kDa gelatinase from other minor gelatinases by virtue of itslack of affinity for gelatin-Sepharose. Subsequent purifications wereconducted by using Con A and Zn2f chelation chromatography. The

specific activity of the recovery of enzyme activity against gelatin issummarized in Table 1. There was a decrease in the total enzymaticactivity after gelatin affinity chromatography which indicates the removal of some gelatin-binding gelatinases. After Zn2 ' chelation chro

matography, we achieved an approximate 85-fold increase in specific

activity.Catalytic Properties of 80-kDa Gelatinase: Unique Metal Ion

Specificity. The 92- and 72-kDa gelatinases are well studied metal-

loproteinases which are widely expressed in human breast cancercells. The characterization of 80-kDa gelatinase was carried out incomparison with 92- and 72-kDa gelatinases. Mammalian metallo-proteinases generally possess consensus metal ion-binding site,termed the "zinc-binding site." Operationally, calcium, as well as zinc.

is usually used to study the proteolytic activity of enzymes. While the80-kDa enzyme can also utilize 5 ITIMCa2 ' to yield maximal activity,

closer examination (Fig. 3/4) demonstrated a unique metal ion specificity. EDTA. at 5 mM. inhibited the activity of all three gelatinases.In contrast to 92- and 72-kDa gelatinases which were able only toutilize calcium, the 80-kDa gelatinase showed a broader range of

divalent cation dependence. In addition to calcium ions, the gelatinolytic activity of 80-kDa protease can be supported by manganese

and/or magnesium ions.To further confirm that the blocking effect of EDTA on the gelat

inolytic activity of 80-kDa gelatinase is due specifically to its metal-

chelating activity, a reversibility assay was carried out. As demonstrated in Fig. 3fi, as little as 0.5 ITIMEDTA totally blocked thegelatinolytic activity of all three gelatinases. This inhibitory effect ofEDTA on the gelatinolytic activity of 80-kDa gelatinase was com

pletely reversed by 5 mM of calcium, magnesium, and manganese.Unexpectedly, under the same conditions, these metal ions failed torestore the gelatinolytic activity of 92- and 72-kDa gelatinases. One

likely possibility for this failure is that besides calcium, zinc is alsorequired for enzymatic activity of 92- and 72-kDa gelatinases.

Table I Partial purification of XU-kDiiproteaseThe activity of the enzyme was determined in the gelatin degradation assay as described in "Materials and Methods." Protein present in the different steps of purification was

estimated from the absorbance at 280 nm using bovine serum albumin as a standard. Each point represents the value for a single measurement. Similar results were detected in twoseparate experiments.

PurificationstepConcentrated

mediumGelatin-Sepharoseflow-throughConA-SepharoseZn2*

chelationTotalprotein.Aim

(mg)84793.90.136Totalactivity(units")11,2341,522412221Specificactivity'1(units/mg)133191051,625Purification(-fold)15.585Recovery(%)KKC2716

" One unit activity is defined as KXX)dpm of soluble gelatin (total soluble count subtracted with background count) in 20 ul of pooled active fraction.h Specific activity was calculated by dividing total activity by total protein.'" 92- and 72-kDa collagenases and other gelatinase were removed by gelatin-Sepharose; thus the activity of the gelatin-Sepharose flow-through sample represents the 80-kDa

enzyme.

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IDENTIFICATION OF A NOVEL MATRIX DEGRADING PROTEASE

Optimal pH for Gelatinolytic Activity of 80-kDa Protease.Since the 92- and 72-kDa gelatinases are neutral metalloproteinases,we determined the optimal pH of 80-kDa gelatinase compared to 92-and 72-kDa gelatinases. As shown in Fig. 4, the 80-kDa gelatinase

from T47Dco cells was active in the range from pH 7.5 to pH 9.5, withits optimal activity around pH 8.5-9. In contrast, the optimal pH for92- and 72-kDa gelatinases was 7.5, and gelatinolytic activity of 92-and 72-kDa gelatinase was dramatically diminished at pH 8.5-9. None

of the gelatinases had acidic pH optima, suggesting that they were notlysosomal enzymes released from dying cells.

Stability. The enzymatic activity of the 80-kDa protease was active when the samples were incubated at room temperature or at 55Â°C

for 25 min. The activity was lost dramatically after heating the 80-kDaenzyme at 100Â°Cfor 5 min. No activity of 80-kDa enzyme wasobserved after heating at 100Â°Cfor 15 min (data not shown).

Effect of AI'MA on the Activation of 80-kDa Gelatinase. Mam

malian metalloproteinases are usually secreted as latent proenzymes(zymogen) and require activation for their enzymatic activity. In vitro,the zymogen forms of metalloproteinases can usually be activated byproteolytic cleavage induced either by serine proteases such as trypsinand plasmin or by autocatalytic cleavage induced by organomercurialcompounds such as APMA. To test the effects of such an activator, the80-kDa enzyme was incubated in the presence or absence of 1 mmAPMA at 37Â°Cfor 30 min. The gelatinolytic activity was measured

both by gelatin substrate zymography and by soluble gelatin degradation assay. As shown in Fig. 5/4, incubation of 92- and 72-kDa

gelatinases with APMA resulted in conversion of high molecular massof proenzymes to low molecular mass enzyme, as determined byzymography. The intensities of the zymographic bands of the 72- and92-kDa enzymes were not significantly affected by the presence of

APMA, as expected, since the latent proenzyme is known to beactivated by a conformational change induced during SDS-PAGE. Nosuch molecular mass conversion was observed in the 80-kDa enzyme,indicating that the 80-kDa enzyme was not responsive to APMAtreatment. This lack of modification of 80-kDa gelatinase by APMA

was further confirmed by a soluble gelatin degradation assay. Theresults in Fig. 5ÃŸconfirm that the gelatinolytic activity of the 80-kDa

gelatinase was not changed after incubation with APMA. As a positivecontrol, the gelatinolytic activity of purified recombinant 72-kDa en

zyme was dramatically increased upon APMA treatment.In order to rule out the possibility that the activation of the 80-kDa

enzyme by APMA may take place during the longer time period ofincubation, the enzyme was incubated with 1 miviAPMA overnight atroom temperature. Neither change of gelatinolytic activity in soluble

20000

BFig. 5. Analysis of effect of APMA on activation of 80-kDa gelatinase. Partially

purified 80-kDa protease and combined conditioned medium containing 92- and 72-kDaproteases (see Fig. 3) were incubated with or without 1 mv APMA at 37Â°Cfor 30 min.

Samples were then subjected to gelatin zymography (A ) for analysis of possible auto-

cleavage and soluble gelatin degradation assay (ÃŸ|for determining gelatinolytic activity.A. Lane 1. control 92- and 72-kDa gelatinases; Lane 2, APMA-treated 92- and 72-kDagelatinases; Lane 3, control 80-kDa gelatinase; Lane 4. APMA-treated 80-kDa gelatinase.In B. soluble gelatin degradation assay was conducted as described in "Materials andMethods." Bars, means Â±SD of triplicate measurements.

Fig. 4. pH dependence of the 80-kDa gelatinase. Zn2* chelation partially purified

80-kDa protease and combined conditioned medium containing 92- and 72-kDa gelati

nases (see Fig. 3) was subjected to gelatin zymography. After electrophoresis, slices ofgelatin gel containing the 92-. 72-, and 80-kDa proteases were incubated in Hanks'

balanced salt solution containing 5 mvi calcium at the indicated pH. The 80-kDa gelatinase

was active in the range from pH 7.5 to pH 9.5. with its optimal pH at 8.5.

gelatin degradation assay nor induced autocleavage as determined ingelatin zymography of the 80-kDa enzyme was observed (data not

shown).Substrate Specificity of 80-kDa Protease. We next investigated

the ability of the 80-kDa protease to degrade substrates other thangelatin. The substrate specificity of the 80-kDa protease when tested

against the type IV collagen (native type IV collagen purified from theEngelbreth-Holm-Swarm tumor) was examined in the soluble assay.

As shown in Fig. 6, when the reaction mixtures were incubated at37Â°Covernight, the a-chain of type IV collagen was completely

digested by both recombinated 72-kDa type IV collagenase and 80-

kDa protease; no specific degradation fragment pattern was observed,presumably due to the excess of enzymes over the substrate. When the80-kDa sample was diluted 20-fold, partial digestion was observed.

Inhibitors for 80-kDa Protease. We examined the effects of var

ious protease inhibitors specific for serine, aspartate, cysteine. and

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DISCUSSION

A general aspect of malignant neoplasia is the capacity to invadeand degrade tissue barriers, such as basement membrane, by enhancedproteolysis. Evidence has accumulated that important proteases in themetastatic process include metalloproteinases, serine, cysteine, andaspartyl proteases ( 11). Of these, most of the evidence to date in earlystages of breast cancer metastasis implicates metalloproteinases; both

Fig. 6. Degradation of type IV coligan by 80-kDa gclatinasc. Partially purified 80-kDawas subjected to type IV 'H-collagen soluble assay as described in "Materials andMethods." After incubation of type IV 'H-collagen with proteases, 20 pi of reaction

mixture were subjected to SDS-PAGE followed by autoradiography. Lane I, nontreatedtype IV collage; Lane 2. incubated with 35 ng of recomhinant APMA-activaled 72-kDaprotease; Lane 3, incubated with partially purified SO-kDa protease; Lane 4. incubatedwith 20-fold diluted 80-kDa protease.

metalloproteinases on gelatin-degrading activity of 80-kDa enzyme(Fig. 7). Although the activity of the 80-kDa gelatinase was totally

inhibited by the metal chelator EDTA. the specific metalloproteinaseinhibitors TIMP-2 and phosphoramidon failed to inhibit the activity of80-kDa enzyme (Fig. 1A). The inability of TIMP-2 to inhibit the80-kDa gelatinolytic activity was further confirmed in the soluble

gelatin degradation assay. As demonstrated in Fig. IB, although thegelatinolytic activity of 72-kDa enzyme was completely inhibited byTIMP-2 at the molar ratio of 1 to 3.5, the gelatinolytic activity of80-kDa enzyme was not inhibited by TIMP-2 at the concentrations

tested. These results did not rule out the possibility that the failure ofTIMP-2 to inhibit 80-kDa protease is due to an insufficient molar ratio

of inhibitor to enzyme, because we did not know the molar ratiobetween the TIMP-2 and the 80-kDa protease in our test. However,this possibility is unlikely based on the fact that no TIMP-2-mediatedconcentration-dependent inhibition of the 80-kDa enzyme was observed within the range of 10-fold of the tested concentrations ofTIMP-2. Inhibitors of the aspartate, serine, and cysteine classes of

protease, including pepstatin, benzamidine, PMSF, and iodoacetamidehad no discernable effect on the activity of the 80-kDa enzyme. Theproteolytic activity of the 80-kDa enzyme was completely blocked bythe cysteine protease inhibitor leupeptin and the reducing agent dithio-

threitol. Although these inhibition data do not directly demonstrate towhich protease family this 80-kDa enzyme belongs, the inability ofTIMP-2 to inhibit the 80-kDa protease together with the data on theinability of APMA to activate the enzyme suggest that this metal-dependent, matrix-degrading protease does not belong to the previ

ously defined matrix metalloproteinase family.Detection of 80-kDa Protease in Breast Tumors. As a prelimi

nary study to develop the methodology for evaluation of levels of the80-kDa enzyme in primary human breast tumor tissues, we havecarried out studies of its expression in MCF-7 cells grown as tumors

in nude mice. Tumor membrane fractions were prepared and subjectedto gelatin zymography analysis. As shown in Fig. 8, the 80-kDaenzyme was clearly visible in detergent-solubilized plasma membranefraction. The lack of doublet of the MCF-7 tumor derived 80-kDa

protease may be due to the degradation of the lower and usuallyweaker gelatinase band during the preparation of tumor membranefractions.

20000

8 10000-

IT

T

8SÃ-

Â§D- S

CL

BFig. 7. Effects of various protease inhibitors on gelatinolytic activity of 80-kDa pro

tease. A, gelatin zymographic analysis of protease inhibitors specific for serine, cysteine,asparatic, and metalloproteinases. After electrophoresis. the 80-kDa protease was subjected to the incubations with different protease inhibitors as described in "Materials andMethods." The inhibitors used are: dithiothreitol, 2 HIM;leupeptin, O.I HIM;phosphora

midon. UK)ug/ml; iodoacetamide. 10 HIM:TIMP-2. 8 ug/ml; ETDA, 5 HIM;PMSF, 1 HIM;benzamidine. 2 HIM;pepstatin. O.I HIM.R, analysis of effects of TIMP-2 on gelatÃ¬nolyticactivities of 72- and 80-kDa proteases in soluble gelatin degradation assay. After titration.the submaximal dose of 80-kDa protease and 20 ng of recombinant APMA-activated72-kDa gelatinase were incubated with or without TIMP-2 at various amounts at 37Â°Covernight. The gelatinolytic activity was measured as described in "Materials and Methods." Bars, means Â±SD of triplicate measurements.

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ACKNOWLEDGMENTS

We are most grateful to Dr. Robert Bird for helpful advice and for providingrecombinant 72-kDa type IV collagenase and TIMP-2. We wish to thank Drs.William Stetler-Stevenson and Erik Thompson for critical discussion and re

view of this work. We thank Dr. Rafi Fridman for excellent technical assis

tance.

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1993;53:1409-1415. Cancer Res Yuenian E. Shi, Jeff Torri, Lynn Yieh, et al. Protease from Hormone-dependent Human Breast Cancer CellsIdentification and Characterization of a Novel Matrix-degrading

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