[CANCER RESEARCH 42, 207-218.
0008-5472/82/0042-OOOOS02.00
January 1982]
Role of Tumor Cell Membrane-bound Serine Proteases in Tumor-induced
Target Cytolysis1
John F. DiStefano,2 Gregory Beck, Bernard Lane, and Stanley Zucker
Departments of Research [J. F. D., G. B.J, Medicine [S. Z.¡,and Laboratory Service [B. L.], Veterans Administration Medical Center, Northport, New York 11768,
and Department of Medicine [J. F. D.. S. Z.¡and Pathology [B. L.J, Health Sciences Center, State University of New York, Stony Brook, New York 11 794
ABSTRACT
The tumor-induced marrow and red blood cell cytolysis as
says have been used to explore the mechanism of cancer cell
destruction of normal cells. Previously, we suggested that
tumor-induced cytolysis was caused by tumor cell membranebound serine proteases. In this study, we have shown that
concentrations of the broad-spectrum serine protease inhibitor
diisopropylfluorophosphate
that did not inhibit tumor cell DMA
and protein synthesis completely abrogated tumor-induced red
blood cell cytolysis. In addition, tumor cell membranes isolated
by differential and sucrose density gradient centrifugation and
characterized by electron microscopy and enzyme marker
analysis were cytolytic for rat 59Fe-labeled red blood cells. The
specific activity expressed as release index (%) per /¿gof
protein was 1.620 for the tumor cell membrane preparations
as compared to 0.002 for intact Walker 256 tumor cells. Tumor
cell membranes solubilized in Triton X-100 had activity in the
p-toluenesulfonyl-L-arginine
methyl ester assay for trypsin-like
enzymes and the A/-benzoyl-L-tyrosine ethyl ester assay for
chymotrypsin-like
enzymes. The enzyme activities demon
strated in these assays could be inhibited by N-o-p-tosyl-Llysine chloromethyl ketone HCI and L-1-tosylamide-a-phenylethyl chloromethyl ketone, respectively. Using [3H]diisopropylfluorophosphate affinity labeling of the tumor cell membrane
proteins followed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, we have identified membrane-bound ser
ine protease(s) that appear to be responsible for tumor-induced
marrow and red blood cell cytolysis.
INTRODUCTION
Considerable experimental evidence supports the concept
that proteolytic enzymes generated by cancer cells are of
critical importance in the pathophysiology of cancer invasion
(12, 22, 37). Most investigators have been concerned with the
effect of cancer-related enzyme activity on host extracellular
connective tissue matrix (22, 26).
The major focus of our research has been on the cell-cell
interaction occurring between cancer cells and normal host
cells during the invasive process of cancer. We have used the
tumor-induced marrow cytolysis assay and the tumor-induced
RBC cytolysis assay as in vitro model systems to explore
potential interactions between cancer cells and normal target
cells (27, 53). Based on our observations that cancer cells
readily lyse cocultured bone marrow cells in vitro, we reported
(12) the use of pharmacological agents to evaluate the mech
anism of cancer-induced target cytolysis. We concluded that
1 This research was supported by the Veterans Administration.
2 Recipient of a Veterans Administration Career Development Award. To whom
requests for reprints should be addressed.
Received February 13. 1981; accepted October 13, 1981.
JANUARY
tumor-induced target cytolysis required cell-cell contact, was
energy dependent, and appeared to be mediated by trypsinlike serine proteases located near the cancer cell membrane.
In the current study, using a more active and less toxic serine
protease inhibitor, DFP,3 and using a cell fractionation proce
dure to isolate a preparation enriched in W-256 cell mem
branes, we have provided evidence that cancer-induced target
cytolysis is caused by membrane-bound serine proteases.
MATERIALS
AND METHODS
Animals. Male Wistar rats were used throughout. W-256 carcinosarcoma cells were obtained from Arthur D. Little Co., Boston, Mass.,
under the auspices of the NIH.
Reagents. NCTC 135 was obtained from Grand Island Biological
Co., Grand Island, N. Y. TLCK, TPCK, soybean trypsin inhibitor, ptoluenesulfonyl-L-arginine
methyl ester HCI, BTEE, NPGB, DFP, phenylmethanesulfonylfluoride,
and sucrose were obtained from Sigma
Chemical Co., St. Louis, Mo. e-Aminocaproic acid was obtained from
Lederle Laboratories, Pearl River, N. Y. 59Fe, as sterile ferrous citrate,
was obtained from Mallinckrodt
Chemical Works, St. Louis, Mo.
[3H]DFP was obtained from Amersham Corp., Arlington Heights, III.
Preparation of Target Cells. The preparation of normal rat bone
marrow target cells (11,12. 52, 53) and RBC (11) labeled with 59Fe
has been described previously in detail.
Preparation of Effector Cells. The isolation and preparation of W256 effector cells from cancerous ascites fluid in rats has been de
scribed previously in detail (11, 12, 52, 53).
Preparation of Tumor Cell Fractions. W-256 carcinosarcoma cells
were found to be resistant to standard techniques for cellular disruption
(49). The cellular disruption technique of Wang et al. (48) was modified
and used as described below. The sucrose density gradient separation
described below was modified from the techniques of Warren and Click
(49).
W-256 cancer cells were collected and separated on Ficoll-Hypaque
as described previously (11, 12, 52, 53). The tumor cells were resuspended in a solution of cold 0.001 M NaHCO3 at a final concentration
of 5 x 106 cells/ml. This solution was stirred on ice for 1 hr. The
resulting suspension was centrifuged at 770 x g at 4°for 20 min in a
Servali RC5B superspeed centrifuge with an HS-4 rotor (Sorvall Instru
ments, Newtown, Conn.). The pellet that formed after this centrifugation
contained heavy participate debris and was termed the 770 x g
fraction. The supernatant suspension was then centrifuged at 9400 x
g at 4°for 20 min. The resulting pellet was resuspended in 30 ml of
cold 0.9% NaCI solution and mixed thoroughly. To this suspension, 30
ml of cold 60% sucrose were added with complete mixing for a final
sucrose concentration of 30%. Thirty ml of the resulting suspensions
were layered on a cold discontinuous sucrose gradient consisting of
15 ml of 50% sucrose, 10 ml of 48% sucrose, 25 ml of 45% sucrose,
20 ml of 43% sucrose, 10 ml of 40% sucrose, and 10 ml of 35%
3 The abbreviations
used are: DFP, diisopropylfluorophosphate;
W-256,
Walker 256; NCTC 135, National Collection Tissue Culture 135; TLCK, N-a-ptosyl-L-lysine chloromethyl ketone HCI; TPCK, L-1-tosylamide-o-phenylethyl
chlo
romethyl ketone; BTEE, W-benzoyl-L-ryrosine ethyl ester; NPGB. p-nitrophenylp'-guanidinobenzoate;
SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis.
1982
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207
J. F. DiStefano et al.
sucrose. The gradient was centrifugea
A clearly identifiable
at 1500 x g at 4°for 30 min.
membrane-enriched
layer formed at the 40 to
43% sucrose layer interface. The uppermost 30 ml of 30% sucrose
representing the residual material left behind following the centrifugation was removed carefully, diluted with 10 times its volume of cold
0.9% NaCI solution, and centrifugea at 50,000 x g at 4°for 20 min.
The resulting
pellet was subjected
to electron-microscopic
analysis.
The sucrose above the membrane band was carefully pipeted off and
discarded without disturbing the membrane band. The membranes
were then removed carefully and resuspended in twice their volume of
cold 0.9% NaCI solution. This suspension was centrifugea at 10,000
x g for 20 min at 4°.The membrane pellet was washed twice with cold
0.9% NaCI solution and centrifuged at 10,000 x g for 20 min at 4°.
The enriched membrane pellet was finally resuspended in 0.9% NaCI
solution for cytotoxicity assays, a buffer suitable for membrane marker
enzyme analysis, in a Triton X-100 buffer for measurement of proteolytic enzyme activity against synthetic substrates, or in SDS for polyacrylamide gel electrophoresis.
The initial 770 x g fraction was washed twice by resuspension in 50
ml of cold 0.9% NaCI solution and centrifuged at 770 x g for 20 min
at 4°.The final pellet was treated in the same manner as the membrane
pellet.
The supernatant fluid remaining after the 9400 x g centrifugation of
the 0.001 M NaHCCvtumor
cell homogenate suspension was centri
fuged at 50,000 x g for 20 min at 4°.The pellet was washed twice by
Cell membranes were prepared as described above, and then the
isolated cell membranes were preincubated for 30 min with the appro
priate concentration of DFP. The DFP-treated cell membranes (0.1 ml)
were then added to the target RBC. Control RBC received 0.1 ml of a
DFP solution without membranes. The second technique used was to
pretreat 5 x 106 W-256 cells for 2 hr with appropriate concentration
of DFP. These cells were then subjected to the fractionation technique,
and the cell membranes were harvested. Non-DFP-treated (control) W256 tumor cells were harvested and fractionated in parallel with the
treated cells. These membrane suspensions (0.1 ml) were then added
to the target RBC.
Cytolysis was expressed as a function of the release index:
Release index
Drug-induced
radioactivity
released due to cell lysis (supernatant)
total radioactivity
(supernatant
+ cell pellet)
inhibition of cytolysis was calculated
x 100.
using the mean
of the release index.
% of inhibition of cytolysis
-
[release index (target cells + drug-treated effector cells) \
—release index (drug-treated target cells)] I
[release index (target cells + control effector cells)
—release index (control target cells)]
Requirement
i
J
x 100.
for Cell-Cell Contact. The requirement for intercellular
resuspension in cold 0.9% NaCI solution and centrifugation at 50,000
x g for 20 min at 4°. The final 50,000 x g pellet was treated and
contact between tumor cells and target RBC for cytolysis to occur was
evaluated. 59Fe-labeled RBC (5 x 10") in 2 ml of NCTC 135 were
assayed in the same manner as the membrane-enriched
placed in 17- x 100-mm friction cap tubes (Falcon) and centrifuged at
400 x g for 10 min at 4°.W-256 effector cells (5 x 106) in 1 ml of
fraction.
The supernatant suspension obtained after the 50,000 x g centrif
ugation was centrifuged at 100,000 x g for 1 hr at 4°over a 5% agar
in culture medium base in an IEC B60 ultracentrifuge with an SB283
rotor (International Equipment Corporation, Needham Heights, Mass.).
The agar-embedded pellet from this ultracentrifugation
was fixed im
mediately in 3% sodium cacodylate-buffered
glutaraldehyde and sub
jected to electron-microscopic
analysis.
NCTC 135 were placed in 10- x 75-mm friction cap tubes that had
either a 9-mm-diameter aperture in their side or a 9-mm-diameter
aperture covered by a 0.45-/im Millipore filter (Millipore Corp., Bedford,
A preparation of complete tumor cell homogenate containing all cell
fractions was prepared by taking a sample of the 0.001 M HaHCO3-
Mass.). These smaller tubes were centrifuged at 400 x g to pellet the
W-256 cells and were placed carefully in the larger tubes containing
the target RBC without disturbing the RBC pellet. As controls, 5 x 106
RBC alone and 5 x 106 RBC plus 5 x 10e W-256 cells were centri
fuged at 400 x g for 10 min at 4°in 17- x 100-mm tubes, and a 10-
tumor cell homogenate suspension and centrifuging it at 50,000 x g
for 20 min at 4°. The pellet formed, referred to as the whole-cell
x 75-mm tube containing NCTC 135 alone was placed above the cell
pellet. After 18 hr of incubation at 37° in 5% CO2 and 95% air, the
homogenate, was washed twice by resuspension in cold 0.9% NaCI
solution and centrifugation at 50,000 x g for 20 min at 4°.The final
pellet was treated and assayed in the same manner as the membrane-
smaller tubes were removed, and both the small and large tubes were
centrifuged independently at 400 x g for 10 min at 4°.The supernatant
enriched fraction.
Cytoiysis Assays. The tumor-induced marrow cytolysis assay and
the tumor-induced RBC cytolysis assay have been described previously
in detail (11, 12, 27, 53). These assays were performed as described
with the following modifications. The medium for the cytolysis assay
was NCTC 135 without fetal calf serum. All cells used in these assays
were washed 3 times with NCTC 135 to remove all serum components.
Tumor membrane-induced
RBC cytolysis was performed in an analagous fashion to tumor-induced RBC cytolysis (11 ). Tumor cell mem
branes prepared as described above were resuspended in 0.9% NaCI
solution. Protein content was determined by the method of Markwell et
al. (28), and appropriate dilutions were made. A volume of 0.1 ml of
the cell membrane suspension was added to 1 ml of culture medium
containing 5 x 10a Fe-labeled rat RBC in 10- x 75-mm friction cap
tubes (Falcon Plastics, Cockeysville, Md.). Control RBC were treated
with 0.1 ml of 0.9% NaCI solution or 0.1 ml of the final membrane wash
collected from the membrane-preparative
procedure. The cell suspen
sions were mixed thoroughly and centrifuged at 200 x g for 10 min at
4°.The tubes were then incubated for 18 hr at 37° in 5% CO2 and
95% air. The pellet and supernatant were then harvested as described
previously for the cytolysis assay (11, 12, 27, 53).
Drug-treated W-256 tumor cells and control target cells and nondrug-treated control tumor and target cells were prepared as described
previously (11).
Cell membranes
208
treated with DFP were prepared
by 2 techniques.
fluid from each small and large tube pair was then pooled in a counting
tube. The RBC and W-256 cell pellets were also pooled, and radioac
tivity was then counted. The release index was then calculated as
described previously.
Incorporation
of Isotopes Into W-256 Cells. Assays for the incor
poration of ['HJthymidine and ("C]leucine
into tumor cell DNA and
protein, respectively, were performed as described previously (11)
except that the nutrient medium was NCTC 135 without fetal calf
serum.
Significant differences were calculated by Student's f test.
Each experiment was performed at least 3 times to insure reproducibility.
Enzyme Marker Analysis. 5'-Nucleotidase,
a plasma membrane
marker, was determined by the method of Aranson and Touster (3).
The plasma membrane marker Na^-K* ATPase was determined by the
method of Quigley and Gotterer (38) and also by the methods of
Schimmel ef al. (39) and Sha'afi et al. (42). The endoplasmic reticulum
and mitochondrial
marker NADH oxidase was determined by the
method of Avruch and Wallach (4). The lysosomal marker A/-acetyl-/Sglucosaminidase was determined by the method of Sellinger ef al. (41 ).
The Golgi membrane marker thiamine pyrophosphatase
was deter
mined by the method of Meldolesi ef al. (29).
Enzymatic Analysis Using Synthetic Substrates.
Cell fractions
were solubilized by a modification of the technique of Dulaney and
Touster (13) which solubilized 70 to 100% of protein in their rat liver
cell membrane preparations. The solubilization of the cell fractions was
CANCER
RESEARCH
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VOL. 42
Tumor Cell Membrane Serine Proteases
done by suspending the pellets in 2 ml of a cold solution that was 1%
(w/v) Triton X-100, 10% (w/v) sucrose, and 0.1 M Tris-HCI (pH 8.5).
This thoroughly mixed suspension was incubated in an ice-water bath
overnight infiltration using a solution of 50% propylene oxide-50%
Epon-Araldite epoxy resin. The pellet was embedded in 100% epoxy
resin and polymerized at 28°for 16 to 20 hr. One-/im sections were
with shaking for 60 min. The suspension was then centrifuged at
50,000 x g for 20 min at 4°, and the supernatant fluid was passed
cut for light microscopy using an LKB ultramicrotome. Thin sections,
20 to 30 nm, were collected on 200-mesh copper grids and were
examined by a Hitachi H U-12 electron microscope.
Cell fractions were prepared as described previously, coded before
submission for electron-microscopic
analysis, and characterized qual
through a sterile 0.45-/im Millipore filter. This procedure solubilized
90% of the W-256 cell membrane preparation.
The proteolytic activity of the Triton X-100 extracts was assayed
using standard spectrophotometric
techniques. Trypsin-like proteolytic
activity was assayed using the synthetic substrate p-toluenesulfonylL-arginine methyl ester by the method of Walsh (45). Chymotrypsinlike proteolytic activity was assayed using the synthetic substrate BTEE
by the method of Walsh and Wilcos (46). The active-site titrant of
trypsin-like enzymes NPGB was also used to assay for proteolytic
activity by the method of Walsh (45).
SDS-PAGE. A discontinuous system for SDS-PAGE was employed
using the gel buffer and sample preparation system described by
Laemmli (24) and the stacking gel described by Dewald of al. (10). A
10% SDS separating gel and a 7% SDS stacking gel were used. Gels
were prerun and then run with sample and bromophenol blue as
tracking dye for 4 to 5 hr at 2 ma/gel.
After SDS-PAGE, the gels were fixed with 50% trichloroacetic acid
and then stained with 0.1% Coomassie Brilliant Blue R-250 stain in
50% trichloroacetic
acid for 45 min. The gels were destained in a
diffusion destainer (Bio Rad Laboratories, Richmond, Calif.) overnight
using 7% acetic acid. Gels were scanned at 560 nm in a Gilford 250
spectrophotometer and 20-cm Model 2520 gel scanner (Gilford Instru
ment Laboratories, Inc., Oberlin, Ohio).
Molecular Weight Determinations by SDS-PAGE. Molecular weight
marker proteins (Bio Rad) were run concurrently with all preparations.
Approximate molecular weights of the sample proteins were determined
by plotting the known molecular weights versus the relative mobility of
the known proteins by the method of Weber and Osborne (50).
Preparation of [3H]DFP-labeled Cell Fractions for SDS-PAGE. W-
itatively and quantitatively with reference to the predominant compo
nent and types of additional subcellular components. The pellets were
coded by one investigator and studied by a second. Each pellet was
divided into 6 blocks, and the homogeneity was evaluated by light
microscopy. Four areas from each fraction including all areas differing
from other parts of the pellet in light-microscopic
appearance were
sectioned for electron microscopy. Four random fields were photo
graphed at 3000 diameters from each section in order to assess
homogeneity. Four fields, selected at random from each section, were
photographed at 8000 diameters in order to identify organelles and
other specific structures. To characterize the structures, at least 8
fields were selected for content based upon the lower-magnification
micrograph and photographed at 17,000 to 30,000 diameters. A grid
was placed over prints with final magnifications of 24,000 diameters,
and the structures at the intercepts were scored. Intercepts without
underlying structures were not scored, nor were unrecognizable struc
tures scored. A minimum of 200 data points was obtained for each
specimen studied by electron microscopy.
RESULTS
Characterization of Tumor-induced Target Cell Lysis As
say Systems. Chart 1 presents a temporal comparison of
tumor-induced marrow cytolysis and tumor-induced RBC cytolysis. The release indexes for both marrow cells and RBC
256
tumor
cells
were
collected
as described
previously
(11, 12, 27, 53) and washed 3 times by suspension in 50 ml of NCTC
135 followed by centrifugation at 400 x g for 10 min to remove all
serum components. The tumor cells were resuspended to a concentra
tion of 1 x 10" cells/ml. This cell suspension (7.5 ml) was placed in
BSC+W-256
each of two 75-sq cm tissue culture flasks (Costar, Cambridge, Mass.).
To one flask, 0.8 ml of a 10 f M solution of NPGB was added, and the
other flask received 0.8 ml of 0.9% NaCI solution. The cells were
incubated at 37°in 5% CO;. and 95% air for 30 min. At the end of this
incubation, both flasks received 0.5 ml of [ 'H]DFP (specific activity,
6.5 Ci/mmol) containing 50 put DFP and 0.333 mCi of 3H. The flasks
were again incubated for 30 min at 37°in 5% CO2 and 95% air. At the
end of this second incubation, the cell suspensions were subjected
separately to the cell fractionation procedure described above to obtain
a [3H]DFP-labeled whole-cell homogenate, enriched membranes, 770
x g pellet, and 50,000 x g pellet for SDS-PAGE. Each pellet obtained
was dissolved in the SDS sample buffer and subjected to SDS-PAGE
as described above.
Determination of Radioactivity in [3H]DFP Gels. Polyacrylamide
gels containing the [3H]DFP-labeled samples were sliced into 2-mm
slices using a vertical gel slicer (Bio Rad). Each gel slice was dissolved
separately by incubation at 37° for 24 hr in 10 ml Omnifluor (New
England Nuclear, Boston, Mass.) containing 3% Protosol (v/v). The
vials were then equilibrated in the dark at 4°until a suitable non-'Hcontaining blank standard was free of chemoluminescence.
The gels
were then counted in a well-type ßscintillation counter (lsocap/300
liquid scintillation system; Searle Analytic Inc., Des Plaines, III.).
Electron Microscopy. Cell fractions were prepared as a layer over
5% agar culture medium, and then 3% sodium cacodylate-buffered
glutaraldehyde was added as fixative. A 0.2 M cacodylate buffer wash
was followed by postfixation with 1% buffered osmium tetroxide, block
staining with uranyl acetate, and subsequent dehydration in 50 to
100% alcohol solutions. Propylene oxide treatment was replaced by
JANUARY
1982
01234567
9
10 II
12 13 14 15 16 17 18 19 20
Time in Hours
Chart 1. A comparison of tumor-induced cytolysis of RBC and marrow cells.
Marrow cells (1 x 10') were prelabeled in vitro with transferrin-bound S9Fe(2
/iCi/ml) as described previously. ''''Fe-labelRd RBC were obtained from a rat
given [9°Fe]ferrouscitrate (200 pCi/animal l.p.) as described previously. ''"Re
labeled marrow cells (5 x 10°)or RBC (5 x 10e) in 0.1 ml were added to 5 x
10eW-256 cancer cells in 1 ml NCTC 135 or to control tubes containing only 1
ml of NCTC 135. The cell suspensions were mixed, centrifuged at 200 x g for
10 min at 4°to insure cell-cell contact, and then incubated for the varying time
intervals as indicated at 37°in 5% CO., and 95% air. The cells were then
centrifuged at 400 x g for 10 min at 4°,and the cell pellet and supernatant were
placed in counting tubes. Cytolysis was expressed as a function of the release
index. Point, mean; oars, S.D. Prior to 6 hr, there were no statistically significant
differences in results between W-256 and marrow cells and marrow controls or
between W-256 and RBC and RBC controls (Student's t test). After 6 hr, these
comparisons are statistically significant at the p < 0.001 level.
209
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J. F. DiStefano et al.
parallel each other over time. There is no demonstrable cytolysis prior to 6 hr of coincubation of tumor cells and target cells.
At 8 hr, cytolysis was evident as a marked increase in the
release index for both the marrow cells and the RBC. The
release index then continued to rise for the duration of coin
cubation of tumor and target cells. Both control marrow and
RBC target cells did not have an increase in the spontaneous
release index of more than 10% in contrast to the marked
increase in release index due to tumor-induced cytolysis. The
kinetics of onset of cytolysis for both marrow cells and RBC
were similar, with the RBC having a lower spontaneous release
index and a greater degree of cytolysis noted after 6 hr of
incubation.
The necessity for tumor cell-target cell contact is demon
strated by the following cell separation experiment. The release
index of RBC alone [12.1 ±1.4% (S.D.)] and RBC separated
from tumor cells by a Millipore filter (14.3 ± 1.1%) were not
significantly different. In contrast, when the RBC were in direct
contact with the tumor cells, a marked degree of cytolysis was
noted (release index of 91.4 ±0.4%; p < 0.001 compared to
RBC alone). When the tumor cell pellet in the inner tube was
separated from the RBC pellet in the outer tube by an aperture
which permitted fluid access, a moderate degree of target cell
lysis was noted (release index of 29.9 ± 3.0%; p < 0.001
compared to RBC alone). Using [3H]thymidine-labeled tumor
cells, it was determined that 0.25% of the tumor cells present
in the inner chamber (1.25 x 104 tumor cells) passed through
the aperture in the absence of the Millipore filter. These cells
were recovered from a nonradioactively labeled RBC pellet in
the outer chamber, indicating that they were making direct
contact with the target cells. The effect of the Millipore filter in
eliminating cytolysis is to prevent small numbers of tumor cells
or membrane vesicles that have detached from the tumor cell
pellet from making contact with the RBC pellet.
Table 1 shows the effect of protease inhibitors on tumorinduced marrow cytolysis after pretreating the tumor cells with
TLCK or DFP for 2 hr. TLCK is a substrate analog that reacts
covalently and irreversibly with trypsin-like proteases (40). DFP
is a broad-spectrum serine protease inhibitor that inhibits all
Table 1
Effect of DFP and TLCK on tumor-induced marrow cytolysis
W-256 cells were suspended at 5 x 108 cells/ml ¡nNCTC 135 containing
either no drug or the drug indicated at the appropriate concentrations. At the end
of a 2-hr preincubation, 5 x 10" marrow cells in 0.1 ml were added to the
cultures. Non-drug-treated and drug-treated marrow cell cultures without tumor
cells were also established as controls. The cells were mixed thoroughly and
centrifuged at 200 x g for 10 min at 4°to insure cell-cell contact. After 18 hr of
incubation, the cells were centrifuged at 400 x g for 10 min at 4°, and the
supernatant and cell pellet were placed in counting tubes. The release index and
the percentage of inhibition of cytotoxicity were then determined as described
previously. DFP was prepared from a 1 M stock solution in anhydrous isopropanol
by making suitable dilutions in NCTC 135. TLCK was dissolved directly in NCTC
135. All reagents were sterilized by Millipore filtration prior to use. Isopropanol
at the concentrations used in the assay had no significant effect on marrow cell
controls or on tumor-induced marrow cytotoxicity.
Drug concentration
Qig/ml)DFP
DFP
DFPTLCK1841.510
TLCK
100
TLCK18.415184.15
200%
* Mean ±S.D. of 3 determinations.
6 Significantly different from non-drug-treated
»«
test).
210
of inhibition of
cytotoxicity27.2
±4.9a- "
51.4 ±4.96
80.6
3.2b14.8
±
cytolysis. The trypsin specificity protease inhibitors phenylmethanesulfonylfluoride
(14) and TLCK were also effective
inhibitors of tumor-induced RBC cytolysis. TPCK, p-toluenesulfonyl-L-arginine methyl ester, and NPGB had modest effects
on RBC cytolysis. e-Aminocaproic acid was an ineffective in
hibitor of cytolysis.
Chart 2 shows the effect of increasing concentrations of DFP
on the percentage of inhibition of tumor-induced RBC cytolysis
as a function of the length of time the 59Fe-labeled RBC target
cells were in contact with the W-256 cells prior to adding the
DFP. At the highest concentration, 10~2 M, the DFP caused
complete inhibition of RBC cytolysis even if it was added to the
cultures 2 hr after the target cells were added. After 6 hr of
tumor cell-target cell contact, 10~2 M DFP did not significantly
inhibit RBC cytolysis. The greater than 100% inhibition of
cytolysis demonstrated at 10~2 M DFP may be a function of the
enhanced efficiency of pelleting the target RBC in the presence
of DFP-inactivated tumor cells. At lower DFP concentrations,
the degree of inhibition of RBC cytolysis was less.
Tumor Cell Membranes. Tumor cell fractionation was ac
complished by hypotonie cell lysis followed by sucrose density
gradient centrifugation. Table 3 shows the distribution of the
membrane marker enzymes in the enriched membrane fraction,
the 770 x g fraction containing heavy cellular debris, and the
whole-cell homogenate. The plasma membrane marker 5'-nucleotidase was 9 times enriched in the membrane fraction.
Na*-K+ ATPase, also a plasma membrane marker, was 32
±7.9
30.5 ±10.86
35.1 ±0.76
controls; p < 0.01 (Student's
serine proteases (36) including trypsin (21 ), chymotrypsin (21 ),
and plasmin and plasminogen activator (36). As demonstrated
previously (11,12), TLCK at 100 jug/ml inhibited cytolysis 31 %
without significantly affecting tumor cell protein synthesis (Ta
ble 2). TLCK at 200 fig/ml inhibited cytolysis by 35% but was
associated with 41 % inhibition of tumor cell protein synthesis
(Table 2). The broad-spectrum organophosphorous serine pro
tease inhibitor DFP caused dose-dependent inhibition of tumorinduced marrow cytolysis. As the dose of DFP was increased
from 18.42 jig/ml, cytolysis was inhibited increasingly from 27
to 81 %. This inhibition of cytolysis was not accompanied by
significant inhibition of tumor cell DMA or protein synthesis or
trypan blue viability despite the very high concentrations of
DFP used (Table 2). In order to demonstrate that the inhibition
of tumor-induced marrow and RBC cytotoxicity caused by DFP
was not due to possible contaminants that might be present in
commercially prepared DFP, in an independent experiment,
the DFP was distilled under vacuum by the method of Gould
and Liener (19). The distilled DFP was found to retain 100% of
the inhibitory activity present in the undistilled sample, indicat
ing that inhibition of tumor-induced cytotoxicity was due to the
presence of active DFP and not a contaminating inhibitor (19).
In addition, DFP that had been hydrolyzed by the techniques
of Ferluga et al. (16) did not cause any inhibition of tumorinduced marrow or RBC cytotoxicity (data not shown).
Table 2 shows the effects of a variety of protease inhibitors
on tumor-induced RBC cytolysis after a 2-hr preincubation of
the tumor cells with these drugs. The broad-spectrum serine
protease inhibitor DFP caused dose-dependent inhibition of
cytolysis. DFP at the highest concentration, 1841.5 /ig/ml or
10~2 M, caused complete inhibition of tumor-induced RBC
t
times enriched in the plasma membrane fraction. The lysosomal
marker /V-acetyl-ß-glucosaminidase and the mitochondrial and
endoplasmic reticulum marker NADH oxidase were enriched
CANCER
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VOL. 42
Tumor Cell Membrane Sehne Proteases
Table 2
Effect of protease inhibitors on tumor-induced RBC cytolysis and on W-256 cancer cell DNA and protein
synthesis
Assay conditions are the same as for Table 1 except that 5 x 10e MFe-labeled RBC are added to the
cultures as target cells after the initial 2-hr preincubation of tumor cells with the drugs. Inhibition of tumor
cell DNA or protein synthesis is obtained by incubating 5X10° control or 5 x 10' drug-treated tumor cells
with either 0.1 ml (1 ¿iCi)
of [3H]thymidine or 0.1 ml (0.25 fiCi) of [14C]leucine for 18 hr at 37°in 5% CO2 and
95% air. Cold 0.9% NaCI solution (1 ml) was then added. The contents of each tube were poured onto glass
fiber filters, and the nucleic acids and protein were extracted with cold trichloroacetic acid. The filters were
dried and counted in Omnifluor in a ßscintillation counter.
% of inhibition ••
mean cpm of trichloroacetic acid precipitates '
from drug-treated cells
mean cpm of trichloroacetic acid precipitates
from untreated control cells / _
x 100.
DFP and TLCK were prepared as In Table 1. p-Toluenesulfonyl-L-arginine methyl ester was dissolved
directly in NCTC 135. Phenylmethanesulfonylfluoride was dissolved in anhydrous isopropanol at a concen
tration of 50 mg/ml and diluted with NCTC 135. TPCK was dissolved in 50 mw phosphate buffer. pH 7.4,
at a concentration of 2000 pg/ml and diluted in NCTC 135. NPG6 was dissolved in dimethyl sulfoxide at a
concentration of 34 mg/ml and diluted in NCTC 135. All reagents were sterilized by Millipore filtration prior
to use. Isopropanol and dimethyl sulfoxide at the concentration used in the assay had no significant effect
on RBC controls or on tumor-induced RBC cytotoxicity.
of stimulation ( + ) or in
of stimulation (+) or in
hibition (-) of DNA syn
hibition (-) of protein
Drug concentration
% of inhibition of cytotoxicityDFPDFPDFPDFPPMSF0NPGBTLCKTLCKTPCKTMETMEEACAEACA1841.5184.1518.4151.8415503.420010020050010020001000105.3
(/jg/ml)
thesis±8.4
synthesis-8.5
3.0°60.5
±
27.4-1.1
±
10.3±1.5
±
20.6-1.4
±
7.551.±
15.1-1.7
±
24.2-11.8±
±
19.8-1-1.7
±
3.324.4
2 ±
8.9-4.8
25.2+ ±
12.650.1
±
13.1619.9±
±
84.4d-17.2
100.5 ±
12.9-33.5
±
IS.S6-41.8
±
13.3-40.8
±
9.621.4
9.96-6.3
±
14.0-3.8
±
10.120.5
±
8.9619.6±
±
16.3+ ±
20.0-47.1
±
8.0-9.6±
8.319.7±
18.4+
11.4 ±
23.5-9.6
±
23.3-5.8
1.5 ±
6.64.4
12.2-40.6±
21.6-2.3
±
7.12.4
±
19.2"-22.6
±
17.5-3.7
±
8.812.1
±
±28.0%
±16.6
±12.7%
Mean ±S.D.
Significantly different from non-drug-treated controls; p < 0.01.
c PMSF, Phenylmethanesulfonylfluoride; TME, p-toluenesulfonyl-L-arginine methyl ester; EACA, €-aminocaproic acid.
d Significantly different from non-drug-treated controls; p < 0.05.
approximately 5 times and 7 times, respectively, in the plasma
membrane preparation. A/-Acetyl-/?-glucosaminidase (18) and
NADH oxidase (7) have been described on cell membranes.
The Golgi apparatus marker thiamine pyrophosphatase was
not detectable in the plasma membrane fraction.
Electron micrographs of the Golgi fraction (Fig. 1) and the
plasma membrane-enriched fraction (Fig. 2) demonstrate the
marked enrichment of the plasma membrane-enriched fraction.
The tumor cell plasma membrane fraction (Fig. 2) was analyzed
by counting the structures underlying a 360-intercept grid
placed over 4 representative micrographs. Five hundred sixtyone intercepts were scored. Five hundred thirty-nine intercepts
were over plasma membrane processes, 14 were over rough
endoplasmic reticulum, 6 were over disrupted mitochondria,
and 2 were over Golgi saccules. This analysis demonstrates
that the final tumor cell membrane fraction was 96.0% plasma
membranes with less than 2.5% contamination by rough en
doplasmic reticulum, 1.1% contamination by mitochondria, and
0.4% contamination by Golgi saccules. The uppermost 30 ml
of 30% sucrose recovered after isolation and removal of the
plasma membrane fraction revealed that this fraction included
plasma membrane segments, rough endoplasmic reticulum,
contracted and disrupted mitochondria, and small numbers of
lysosomes and Golgi components. Overlay of a grid on repre
sentative micrographs and scoring of recognizable structures
underlying intercepts revealed that, of 459 structures, 202
JANUARY
1982
were plasma membrane or cell surface, 184 were rough en
doplasmic reticulum, 46 were mitochondria, and 18 were Golgi
or lysosomes. The Golgi membrane fraction (Fig. 1) formed by
the 100,000 x g ultracentrifugation
demonstrates that this
fraction contains the majority of the Golgi membranes. Scoring
of a 360-intercept grid placed over each of 3 representative
micrographs revealed that 330 intercepts overlayed membra
nous or other recognizable structures. Two hundred seven
were Golgi components, 111 were ribosomes on or free of
membranes, and 12 were other membrane profiles. The 770
x g fraction was found to contain mitochondria, lysosomes,
fragments of cytoplasm, and a few intact cells.
Table 4 shows the RBC-cytolytic activity of the cell fractions
compared to whole tumor cells. The plasma membrane fraction
was 810 times more cytolytic than the intact tumor cells and
12 times more cytolytic than the W-256 whole-cell homogenate. In a separate experiment, increasing the quantity of
membrane protein from 26 to 103 fig resulted in an increase
index from 35.0 ±5.1 % to 66.2 ±9.4% (p < 0.001 compared
to RBC alone). Cell membranes prepared from normal rat
spleen cells using the same technique used to prepare tumor
cell membranes did not cause RBC cytolysis. The RBC release
index in the presence of 104.7 ±5.5 fig of lymphocyte mem
brane protein was 3.2 ±0.4%.
Table 5 demonstrates the effects of pretreating the cancer
cell membrane preparations for 30 min with protease inhibitors
211
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J. F. DiStefano et al.
prior to adding 59Fe-labeled RBC. DFP inhibited tumor cell
membrane-induced RBC cytotoxicity by 76% at 184 /ig/ml
(10~3 M). TLCK at 200 /tg/ml (5.4 x 10~4 M) inhibited cytolysis
by 69%. TPCK and soybean trypsin inhibitor did not signifi
cantly inhibit RBC cytolysis. In a parallel experiment, intact W256 cells were pretreated for 2 hr with 10~3 M DFP, and their
membranes were then prepared as described above. These
DFP-treated cell membranes were not cytolytic for 59Fe-labeled
RBC target cells as compared to control tumor cell membranes
(percentage of inhibition of cytolysis by DFP, 98.1 ±1.8%; p
< 0.001).
Enzymatic activity, demonstrated with 2 synthetic substrates,
of the Triton X-100 extracts of tumor cell membrane fractions
and the whole-cell homogenate is shown in Table 6. Using the
substrate for trypsin-like enzymes, p-toluenesulfonyl-L-arginine
methyl ester, the enriched tumor cell plasma membrane prep120
no
100
-5
s
u
W
relation to the protein bands for each fraction is shown. The
enriched cell membrane preparation contained 2 peaks of
[3H]DFP uptake. The first of these [3H]DFP-labeled protein
bands was almost completely blocked from [3H]DFP uptake by
the active-site titrant of serine proteases NPGB. This protein
70
60
I
X
JÌ"
&S
The site of uptake of DFP in the tumor cells was investigated
in 3 separate experiments through the use of [3H]DFP. Chart 3
shows the typical distribution of the [3H]DFP in the tumor
proteins for the cell fractions and the effect of blocking [3H]DFP
uptake by serine proteases with the active-site titrant NPGB.
Tumor cells were pretreated with either 0.9% NaCI solution or
10~3 M NPGB and were then treated with [3H]DFP. The cells
were then fractionated, and the cell fractions obtained were
subjected to SDS-PAGE. The distribution of the [3H]DFP in
80
9
1
aration was found to have 0.7 unit activity/mg protein. This
was 11 times greater than the activity of the whole-cell homog
enate in this assay. In addition, the trypsin inhibitor, TLCK,
inhibited the trypsin-like membrane activity 85.6 ±6.2%. Using
the substrate for chymotrypsin-like
enzymes, BTEE, the en
riched tumor cell plasma membrane preparation was found to
have 0.3 BTEE unit/mg protein. This chymotrypsin-like activity
was inhibited 49.5 ± 22.6% by the chymotrypsin inhibitor
TPCK. The plasma membranes were only minimally enriched
in chymotrypsin-like activity when compared to the whole-cell
homogenate.
The active-site titrant of serine proteases, NPGB, was also
used to assay the Triton X-100 membrane extract. Using this
assay, 1 mg of membrane protein was equivalent to 1.06 ±
0.45 x 10~3 mg of trypsin.
may therefore be regarded as a probable serine protease (9)
present on the cell membrane. The molecular weight of this
protein is approximately 102,000. The 770 x g fraction con
taining the cellular debris had the second peak of [3H]DFP
20
uptake seen in the membrane fraction but had a greatly dimin
ished first peak. Since the [3H]DFP uptake of the second peak
-2
*l
*2
was not blocked by NPGB, its identity is not clear. The wholecell homogenate and the 50,000 x g fraction were not found
to be significantly labeled by the [3H]DFP. This probably reflects
*3
Time in Hours
Chart 2. Effect of DFP on tumor-induced RBC cytolysis. W-256 tumor cells
and RBC were cocultured with appropriate controls as described previously. The
RBC were added to all cultures at the zero time point with thorough mixing
followed by centrifugation at 200 X g for 10 min at 4°.At the times designated,
DFP, at the indicated concentrations, was added to the cultures with thorough
mixing followed by centrifugation at 200 x g for 10 min if RBC were present in
the mixture. Point, mean of 3 separate determinations; bars, S.D. RBC cytolysis
was measured after 18 hr of incubation as described earlier.
dilution of the membrane proteins by other cellular proteins in
the whole-cell homogenate and the absence of labeled proteins
in the 50,000 x g fraction. However, the possibility of com
peting substrates or inhibitory substances cannot be ruled out
as an explanation for the absence of [3H]DFP uptake by the
whole-cell homogenate or 50,000 x g fraction.
Table 3
Marker enzyme characterization of tumor cell fractions
Enzyme assays were performed using standard techniques. The relative specific activity is calculated
formula:
from the
specific activity of cell fraction
Relative specific activity - specific activity of the whole-cell homogenate
Standard deviations were calculated where necessary by the rules for the propagation
protein)Marker
Specific activity (/iM/rnin/mg
ho
enzyme5'-Nucleotidase mogenate0.053
±0.003a
Na*-K* ATPase
0.006 ±0.001
N-Acetyl-0-glu0.0440.218
1.084 ±
cosaminidase
± 0.037770
NADH oxidaseWhole-cell
! Mean ± S.D.
' Significantly
different
212
from whole-cell
X0.039
fraction±
of indeterminate
activity770
x0.736
errors.
specific
fraction±
0.070
0.027*
0.189 ±
0.833 ± 0.324
0.005 ±0.001
0.059*0.117*Relative
0.4290.477g
±
0.024±
5.2161.432±
0.4650.104g
±±0.003
0.0180.037Membranes0.482
±±0.026*
0.711
31.500 ± 6.915
4.8126.569±
±
0.203±
0.188Membranes9.094 1.237
homogenate;
p < 0.001.
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VOL. 42
Tumor Cell Membrane Serine Proteases
Table 4
Cytolytic effect of tumor cell fractions on RBC
Tumor cell fractions were prepared by hypotonie lysis of W-256 cells in cold 0.001 M NaHCOa. An aliquot of this suspension was centrifugea
at 50.000 X g for 20 min at 4°and is termed the whole cell homogenate. The remainder of the 0.001 M NaHCO:1 suspension was centrifugea
at 770 x g for 20 min at 4°. and the precipitate formed, termed the 770 x g fragments, was resuspended in 0.9% NaCI solution and
represents heavy cellular debris. The supernatant solution was centrifugea at 9400 x g for 20 min at 4°.The precipitate was resuspended in
a final solution of 30% sucrose and placed on a sucrose density gradient. The gradient was centrifugea at 1500 x g for 30 min at 4°,and a
membrane layer formed at the 40 to 43% sucrose interface. The membranes were carefully pipeted off, washed twice, and resuspended in
0.9% NaCI solutions. The supernatant solution from the 9400 x g centrifugation was centrifuged at 50,000 x g for 20 min at 4°. The
precipitate was resuspended in 0.9% NaCI solution and is termed the 50.000 x g pellet and represents the light cellular material. Protein
concentrations were determined.
The cytolytic assay was performed with the same technique as described previously. Each suspension of ''"Fe-labelod RBC received either
0.1 ml of the indicated cell fraction, 0.1 ml suspension of whole tumor cells, 0.1 ml of 0.9% NaCI solution buffer, or 0.1 ml of membrane wash.
The cell mixtures were agitated thoroughly and then centrifuged at 200 x g for 10 min at 4°.The mixtures were incubated at 37°in 5% CO2
and 95% air for 18 hr followed by centrifugation at 400 x g for 10 min at 4°.The cell pellet and supernatant were placed in counting tubes,
and the release indices were calculated.
The specific activity was expressed as the percentage of RBC cytolysis per /ig of protein according to the formula:
release index (RBC + tumor cell fraction) - release index (RBC + buffer)
Specific activity
/<g of protein per tumor cell fraction
The relative specific activity was calculated
according to the formula.
in relation to the intact tumor cells and the whole-cell
homogenate
(number in parentheses)
specific activity of fraction assayed
Relative specific activity - specific activity of comparative fraction'
protein/ml
sample3.141
(%)4.0Index
±0.2*
64.0 ±^.6b
SampleRBC
-H buffer
RBC + intact W-256 cells
RBC -I- W-256 cell homogenate
11.1 ±2.2
76.6 ±7.0b
RBC + W-256 770 x g fragments
RBC t W-256 enriched plasma membranes
45.8 ±4.3
RBC + 50,000 x g pellet
4.1 ±2.5
RBC
wash"
+ 0.1 ml membrane
±0.1p 1.2
Mean ±S.D.
b Significantly different from RBC controls;Release
< 0.001./ig
Table 5
Effect of protease inhibitors on tumor cell membrane-induced
x 104
5.421 x 10'
3.714 ±103
2.580 X 10'
1.472 X 10*
0Specific
RBC cytolysis
Tumor cell membranes were prepared as in Table 4. To 1 ml of medium
containing the indicated drug was added 0.1 ml of membrane suspension in
0.9% NaCI solutions. These suspensions were incubated at 37°for 30 min in 5%
CO2 and 95% air. At the end of the incubation period, 5x10° MFe-labeled RBC
were added to each tube and to control tubes containing drug alone. The tubes
were mixed thoroughly and centrifuged at 200 x g for 10 min at 4°.The mixtures
were incubated for 18 hr at 37° in 5% CO; and 95% air. The cells were then
centrifuged at 400 x g for 10 min at 4°,and the supernatant and cell pellet were
placed in counting tubes. The release index and percentage
cytotoxicity were then determined.
Drug concentration
(/«g/ml)DFPDFP
TLCK
200
TPCK
200
SBTIC184.1518.415
500%
Mean ±S.D.
6 Significantly different from non-drug-treated
c SBTI, soybean trypsin inhibitor.
of inhibition
of
of inhibition of
cytolysis75.7
±10.9s'6
17.2 ±33.5
68.8 ± 9.5b
18.3 ±10.0
7.5 ± 2.4
membrane controls; p < 0.001.
DISCUSSION
The importance of proteolytic enzymes and specifically ser
ine proteases in various aspects of cancer cell biology is under
intensive investigation. Serine proteases have also been iden
tified as secretory products or as cell constituents of many
invasive but physiologically normal cells (1, 2, 5, 8, 20) and
have been implicated as mediators of target cell lysis and tissue
remodeling (1, 2, 20, 31). A particulate cell fraction of human
polymorphonuclear leukocytes contains a protease that can be
inhibited by DFP (8). A DFP-inhibited neutral proteinase has
JANUARY
1982
activity0.002
±0.000
0.131 ±0.030
0.020 ±0.002
1.620 ±0.167Relative
specific
activity65.5
10
810.00
(0.15)
(12.37)
been extracted from human lymphocytes and has been shown
to be cytotoxic for tumor target cells in vitro (20). Lymphocyteinduced tumor cell cytotoxicity has been shown to be inhibited
by organophosphorous
serine protease inhibitors (16). Cell
fractionation revealed that the cytotoxic activity of lymphocytes
could be localized to the plasma membrane (15). Activated
macrophages also secrete DFP-inhibited serine proteases that
have been implicated in tumor cell cytolysis (1, 2).
Transformation of embryonic cells by oncogenic viruses has
also been associated with increased production of serine pro
teases identified as plasminogen activators (43, 44). Trans
formed cell lines contain serine proteases in membrane-en
riched fractions (35) and release these enzymes into the me
dium in tissue culture. These secreted proteases have been
studied by active-site labeling with [3H]DFP in the presence of
other competing active-site reagents (9).
Cancer cell serine proteases have been postulated to affect
cancer cell proliferation (40), adhesion to substrate (33, 51),
motility (32), chemotaxis (23), invasion (6, 12, 25), and metas
tasis (6, 17, 37, 47). The importance of the plasma membrane
in determining arrest and metastasis of tumor cells has been
supported by experiments in which the fusion of membrane
vesicles from a melanoma variant of high metastatic potential
to another melanoma variant of low metastatic potential re
sulted in the transfer of increased metastatic capacity (34).
The ability of W-256 tumor cells to cause cytolysis of normal
bone marrow and RBC in vitro has been postulated to be due
to cell-bound serine proteases (11,12). The ability of DFP, a
broad-spectrum serine protease inhibitor, to cause complete
inhibition of tumor-induced RBC cytolysis without significantly
213
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J. F. DiStefano et al.
Table 6
Enzymatic activity of Triton X-100 extracts of the whole-tumor cell homogenstes and the tumor cell
plasma membranes
The enzymatic activities of the whole-tumor cell homogenate and the tumor cell plasma membrane
preparation were assayed after solubilization of the fractions in 2 ml of a cold solution that was 1% (w/v)
Triton X-100, 10% (w/v) sucrose, and 0.1 M Tris-HCI (pH 8.5). This procedure solubilized 90% of the W256 cell membrane preparation. The proteolytic activities were assayed according to the standard spectrophotometric techniques for trypsin-like enzymes in the p-toluenesulfonyl-L-arginine
methyl ester assay and
for chymotrypsin-like enzymes in the BTEE assay.
Cell fraction
Whole-cell homogenate
Activity in units/
mg Protein
Substrate
p-Toluene-sulfonyl-L-arginine methyl ester
Enriched plasma membranes
Enriched membranes + 200 fig
TLCK
Whole-cell homogenate
Enriched plasma membranes
Enriched membranes + 200 fig
TPCK
BTEE
Specific activity
relative to wholecell homogenate
0.063 ±0.024
0.702 ±0.1716
0.101 ±0.028C
11.149 ±4.992
0.181 ±0.026
0.273 ±0.0606
0.138 ±0.006e
1.508 ±0.393
Mean ±S.D.
6 p < 0.01 compared to whole-cell homogenate.
c p < 0.01 compared to enriched plasma membranes.
10
I4
.75
O
o
.50
X
Z
O.
u
.25
! îî
i i i i r
i i i i i i r
75
o
o
50
X
*
l
.25
t
t
20 11.6 9.4
t
t
t t
20 116 94
t
6.8 4.3
3.0
Molecular Weight 110000
I I I I I I I I I I TT~T V [ r~TT
10
20
10
GEL SLICE NUMBER
214
20
30
GEL SLICE NUMBER
Chart 3. Distribution of [3H]DFP in W-256 cell fractions and the effect of
NPGB on [3H]DFP labeling. W-256 cells were treated for 30 min at 37° in 5%
CO, and 95% air with either 10~3 M NPGB or 0.9% NaCI solution. Each flask
then received 0.5 ml of [3H]DFP containing 50 fiM DFP and 0.33 mCi of 3H
followed by another incubation for 30 min. The cells were then fractionated,
6.8 4.3
3.0
2.1
Molecular Weight «10000
the fractions obtained were dissolved in the SDS buffer and subsequently
subjected to SDS-PAGE. A, whole-cell homogenate; 8, 770 x g fraction; C,
50,000 x g fraction; D, membrane-enriched
fraction.
, absorbance of
Coomassie blue-stained gels at 560 nM; •.[3H]DFP; A. [3H]DFP -I- NPGB.
and
CANCER
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VOL.
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Tumor Cell Membrane Serine Proteases
affecting tumor cell DNA synthesis and protein synthesis or
colony formation" strongly supports this hypothesis. Chloromethyl ketone serine protease inhibitors have also been shown
to inhibit cytolysis but at drug concentrations that also inhibit
tumor cell DNA and protein synthesis.
The requirement for 4 to 6 hr of tumor cell contact with
marrow target cells for cytolysis to occur has been demon
strated previously (53) by adding a large volume of high-mo
lecular-weight dextran to the mixed cell cultures at various time
intervals to minimize tumor cell-target cell interactions. In the
present study, we used a 0.45-/im Millipore filter which physi
cally separated tumor cells from target RBC to demonstrate
that tumor-induced RBC cytolysis required tumor cell-target
cell contact. The effect of adding the serine protease inhibitor
DFP to tumor cells at various times before or after the addition
of target cells in the cytolytic assay was also studied (Chart 2).
The addition of 1CT2 M DFP to cell cultures even 2 hr after
mixing the cancer cells and target RBC together led to complete
inhibition of target cell lysis. After 6 hr of contact between
effector and target cells, DFP has no inhibitory effect, indicating
that the serine protease-induced target cell insult was com
pleted in 4 to 6 hr even though the actual release of 59Fe was
not observed until 8 to 10 hr after the initiation of the mixed
cell cultures. Tumor-induced bone marrow cytolysis showed a
similar time course for lysis as tumor-induced RBC lysis (Chart
1).
In this study, fractionation of W-256 cells by hypotonie cell
lysis followed by differential and sucrose density gradient centrifugation resulted in the isolation of a highly purified tumor
cell membrane fraction that was highly enriched in cytolytic
activity for 59Fe-labeled rat RBC. Tumor cell membrane-in
duced cytolysis was inhibited effectively by DFP and TLCK,
suggesting that a membrane-bound trypsin-like serine protease
was responsible for cytolysis. To confirm the presence of serine
proteases in the cell membrane fractions, we assayed for
enzyme activity using substrates commonly used to identify
trypsin and chymotrypsin-like enzymes. The Triton X-100 ex
tracts of the tumor cell plasma membrane preparation had
activity in the p-toluenesulfonyl-L-arginine
methyl ester assay
for trypsin-like activity and in the BTEE assay for chymotrypsinlike enzymes.
The results with [3H]DFP active-site labeling of cellular pro
teins suggest that cell membrane-bound proteases play a piv
otal role in tumor-induced RBC and marrow cell lysis. The
second [3H]DFP-labeled band in the membranes may reflect
either [3H]DFP reacting with proteins that are not serine pro
teases (9, 30) or an unexplained inability of NPGB to block the
serine site on these proteases. This will require further study.
In conclusion, we have demonstrated that W-256 cancer cell
membrane bound-serine protease(s) with trypsin-like activity
are responsible for the in vitro cytolytic activity of cancer cells.
Four other transplantable cancer cell lines (27) and the B16
melanoma cell variants" also have erythroid cytolytic activity,
suggesting that cancer cell membrane proteolytic activity may
be found commonly in transplantable tumors. The isolation and
identity of cancer cell enzyme(s) with target-cytolytic capacity,
their relationship to other cancer cell proteases described
previously, (26, 37) and most importantly, the role of these
membrane-bound enzymes in cancer cell invasion in vivo re
mains to be clarified. Currently, we are attempting to isolate
and purify the W-256 cancer cell membrane serine proteases
by gel and affinity column-chromatographic
techniques and to
further identify these proteases using autoradiography.
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CANCER
RESEARCH
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VOL. 42
Tumor Cell Membrane Sehne Proteases
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Fig. 1. Electron micrograph
icrograph of the 100,000 x g orr Golgi
Gol{ membrane
rane fraction which contained Golgi saccules and rough endoplasmic reticulum
:omponents were present,
4,000. insert, representative field including Golgi saccules and ribosomes. x 60,000.
or recognizable cell components
present x 24,000
JANUARY
1982
217
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Fig. 2. Electron micrograph of the tumor cell membrane-enriched fraction. The membrane-enriched fraction was composed of segments of plasma membranes
with few organelles. Among these, rough endoplasmic reticulum was most common and represented less than 2.5% of the total membranous elements present. There
were rare Golgi components and no lysosomes. x 12,000. Insert demonstrates that membranes were uniform in thickness and had attached filaments, x 60,000.
218
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Role of Tumor Cell Membrane-bound Serine Proteases in
Tumor-induced Target Cytolysis
John F. DiStefano, Gregory Beck, Bernard Lane, et al.
Cancer Res 1982;42:207-218.
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