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(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
Métastasesof 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
métastases(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 4°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
4°Cin a 50 imi Tris (pH 7.4)
huiler containing 2(X) IHMNaCl. l IHMPMSF, l IHMbenzamidine, and
2
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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 ¡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
55°C
for 25 min. The activity was lost dramatically after heating the
80-kDaenzyme at 100°Cfor 5 min. No activity of 80-kDa enzyme
wasobserved after heating at 100°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 37°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. 5ßconfirm 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 37°Cfor 30 min.
Samples were then subjected to gelatin zymography (A ) for
analysis of possible auto-
cleavage and soluble gelatin degradation assay (ß|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 ±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
at37°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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IDENTIFICATION OF A NOVEL MATRIX DEGRADING PROTEASE
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-
§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 gelatìnolyticactivities 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 37°Covernight. The gelatinolytic
activity was measured as described in "Materials and Methods."
Bars, means ±SD of triplicate measurements.
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IDENTIFICATION OF A NOVEL MATRIX DEGRADING PROTEASE
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IDENTIFICATION OF A NOVEL MATRIX DEGRADING PROTEASE
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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on July 7, 2021. © 1993 American Association for Cancer
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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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