0022-1554/90/$3.30 Thejournal

Histochemistry and Cytochemistry by The Histochemical Society,

of

Copyright

©

Vol. 38,

MARTIN

Heidelberg-Rohrbach, Center

D-6900

Heidelberg

D-6900

Received

for 17,

(HS),

Republic

August

24,

and

1989

WERNER

(ME,GEVE);

Heidelberg

(ME,ES,SH);

Federal

publication

1990

D-6900

Heidelberg

SPIESS,2

EBERHARD

LAH,

TAMARA

Institute

of Germany;

andj

Stefan

and in revised

form

December

doplasmic mal cells,

proteinase reticulum, delivered

a role in cellular somes

were

cathepsin

protein

turnover

by light

found

(8-11)

ofextracellular

degradation

amounts man

locations

than

of cathepsin

tumors

collagens,

are only lysosomes.

It has

B are present

(12,13).

In animal

Institute

Supported

by

Mannheim. 2 Correspondence ology, German Heidelberg,

a

grant

from

to: Eberhard

CancerResearch ERG.

ofCell

Institute,

and

Tumor

German

6111

7, 1989

Ljubljana,

and

Biology, Cancer

German Research

Yugoslavia

March

16,

1990;

Cancer Center

(ilL).

accepted

and lami-

if the enzyme been

cell systems,

found

also

that

high

in certain

hu-

high

ning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinudear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction ofthe enzyme bound to the plasma membrane in a spedded distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility ofa direct involvement ofcathepsin B in the degradation of the extracellular matrix, and thus a contribution ofthe enzyme in invasion and metastasis. (JHistothem

Cywchem WORDS:

KEY

cathepsin

the

Spiess,

Center,

Tumorzentrum

Institute

1990)

B; Tumor

cells; Plasma

Localization;

Laser

membrane;

scanning

B production has been correlated with increased Sylven et al. (16) reported that on transformation cells, and in existing tumor cells, cathepsin ized in the cell periphery. By cell fractionation in tumor

cells

are located brane

that

outside

fractions;

leads

considerable

amounts

the lysosomes

associated

it has been

B due

to an enzyme

to membrane

cathepsin

B. This

It has been

proved defect

incorporation might

suggested

that

Immu-

microscopy.

malignancy (14,15). of 3T3 fibroblast B also becomes local(17-22), it was shown

of cathepsin with

B activity

cytoplasmic

a misguided

in I-cell (23,24)

Bi-

Feld 280, D-6900

mem-

transport

disease

of

of fibroblasts

and

secretion

(24)

also

occur

in malignant

tumor

cells.

that

tumor

cell cathepsin

B can differ

of in

some molecular and enzymatic properties from cathepsin B of normal cells (7,14,21). These differences may lead to an unusual tare.g., to the plasma enzymes contribute

membrane. Membrane-localized to the malignant phenotype

cells (25). Such localization may protect enzymes from host protein inhibitors and would facilitate a concentration

Heidelberg-

of Cell and Tumor

Im Neuenheimer

38:1313-1321,

Cathepsin

nocytochemistry;

geting, radative 1

HABERMAAS,

SUSANNE

Pathology,

cathepsin

membrane

fibronectin,

or are released

tumor

Cathep-

(7) or direct

basement

conceivable

lyso-

(3-6).

in the indirect and

en-

apparatus and, in norcompartment to play

microscopy

matrix

as proteoglycans,

at the rough

Cathepsin-containing

electron

to be involved

nm. In vivo, such activities has other

(1,2).

and

sin B has also been such

B is synthesized

processed in the Golgi finally to the lysosomal

detected

components,

Lung

(9A1772).

Introduction cysteine

1990

in USA.

EBERT

ofExperimental

We demonstrated the cysteine proteinase cathepsin B two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma ofthelung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblast cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefm A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization ofthe cathepsin B was performed by conventional immunofluorescence microscopy and laser scan-

The

1313-1321,

Article

B in Two Human

TREFZ,

G#{220}NTER

SPRING,

Thoraxklinik Research

April

of Cathepsin Lines’

ERDEL,

HERBERT

pp.

9,

Printed

Original

Localization Cancer Cell

Na

Inc.

1990

tive

enzyme

directed human

activity(ies)

proteolysis lung tumor

at the

focal

points

of invasion,

degof tumor endogenous of respecallowing

(26). In a previous study we have investigated cell lines for proteolytic enzyme activities (27). 1313

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

1314

ERDEL,

In the

present

work

we localized

the

cathepsin

B activity

within

30 mm

sin B. Our lysosomal in the

controls,

as they

experiments localization,

tumor

are well

revealed, plasma

known

to express

To restrict

cathep-

in rotation

in addition to the well-established membrane-associated cathepsin

and

Methods

Cell

Lines

and

Culture

HS-24

is a human

noma.

SB-3

B

Conditions

cell line derived

is a human

cell

from a bronchial

line

derived

from

squamous

a metastasis

cell carciof a primary

adenocarcinoma of the lung to the adrenal gland. Both cell lines were isolated in our laboratory and will be described in detail elsewhere. Wi-38 normal humanlung fibroblast cells(CCL 75)were purchased from the American Type Culture Collection (Bethesda, MD) and were cultivated as recommended by the supplier. Tumor cells were cultured in RPMI 1640 medium

labeling

culture

and incubated

10%

fetal

calfserum,

4 mM

L-glutamine,

25 mM

Cathepsin

B Activity

The fluorescence cytochemical used with a few modifications.

We

used

precipitate

at the site of hydrolysis

strate (30). Usually

1.5

confluence,

x i’ Miles

they

staining

solution

in a humidified

tamed EDTt

1

cells were

pH

seeded

Scientific, washed

(200

sl per chamber)without

twice

petri

dish

at 0-4’C

with

5 isM antipain

St

(31),

In inhibition

Trasylol

(Bayer;

Cell Fixation ing

with

in Hanks’ antibodies.

with

The

freshly

NSA,

and

staining

0.25

In control

experiments

In cell

lysates

nitrocellulose,

washed

with

incubated

23-32

KD

tion

product Second

after

the cells grown

HBSS,

for 2 hr with

cooled

washed second

on glass slips or

in a melting

carefully antibody,

ice bath,

three and

times washed

was done with 1% formaldehyde in PBS, cell pellets were mounted in Mowiol.

PAA

gel electrophoretic

separation

and

blotting

onto

both antibodies detect only protein bands in the range of which are characteristic for cathepsin B and its major disintegra(Figure

1).

Antibodies.

Appropriate

kD

mM

prepared

rhodamine-conjugated

a

solution DTF,

con-

0.20

experiments

second

anti-

bcd

Leverkusen,

and Immunolabeling. balanced salt solution unlimited

inhibi-

5 isM leupeptin

, .

24-

(Serva), from and

2O-l

Cathepsin

B

Cells were grown on glass slips and (HBSS) before fixation and/or labelaccessibility

45-.

mM

FRG).

of

-

Z-Arg-

500 tM PMSF(Serva),

(Serva),

66-

3629-

incubated

the following

solution:

Detection

To achieve

glass

Primary Antibodies. Antibody I was polyclonal anti-human cathepsin B IgG derived from sheep (Serotec; Camon Labor Service, Wiesbaden, ERG). Antibody H wa.s polyclonal anti-cathepsin B IgG isolated from New Zealand rabbit antiserum raised against human liver cathepsin B. The specificities ofthese antibodies were tested against cell lysates from human tumor cells.

semi-

MO), 5 MM trans-epoxysuccinyl-L(E64; Sigma), 5 tM stefin A isolate

100 MM pepstatin

Immunofluorescence washed

Louis,

covered

15 mm.

pH 6.0.

to the staining

human tongue 4000 Ku/liter

to the cell surface,

were

HBSS,

glass

the cells reached

priorfixation,

1 mM

acetate,

Arg-4MI3NA was omitted. tors were separatelyadded (Sigma; leucylamino-(4-guanidine)-butane

PBS,

for

mM Z-Arg-Arg-4MNA,

in 0.2 M ammonium

IL). After

with

at 37’C

and

FRG)on

of the sub-

tissue culture

on two-chamber

Naperville,

were

as before

Novabiochem,

fluorescent

(Lab-tek;

removed

AG; Frankfurt,

N-benzoyloxycarbonyl-L-arginyl-

substrate for cathepsin B (29). In (NSA; Scm, Heidelberg, ERG), (4M3NA) forms an insoluble

slides

EBERT

Hepes,

L#{228}ufelfingen,Switzerland) as a specific the presence of 5-nitrososalicylaldehyde the liberated 4-methoxy-(3-naphthylamine acidic

was

LAH,

and van der Lam (28) was

(Z-Arg-Arg-4M3NA;

under

SPRING,

Test

assay ofDolbeare

L.arginyl-4-methoxy-3-naphthylamine

antibody

for 3.5 hr with first antibody,

and streptomycin/penicillin, at 37’C in water-saturated 95:5 air:CO2 atmosphere. To obtain spherical cells and cell aggregates, we transferred the cells after the trypsinization to siliconized Erlenmeyer flasks and incubated them on a gyratory shaker (70 rpm) for at least 2.5 hr (Wi-38 cells, SB-3 cells) or overnight (SB-3 cells).

Cytochemical

Excess

with Mowiol(Hoechst

again with HBSS. Cell fixation and finally slips and suspended

Materials

with

temperature.

HABERMAAS,

slides.

cells.

supplemented

at room

SP1ESS,

the slips were mounted

these tumor cell lines by cytochemical and immunocytochemical methods. In this context, we used human lung fibroblast cells (Wi38) for positive

TREFZ,

to the

cytoplasm,

the cells were fixed with either (a) methanol or (b) methanol and acetone, each for 5 mm at - 20’C, or (c) 1% formaldehyde in PBS for 5 mm at room temperature, followed by methanol and acetone each for 5 mm at - 20’C. After washing with PBS, the cells were incubated at room temperature with the first antibody for 30 mm. Excess antibody was then washed off with five changes in PBS before exposure to the second antibody for

Figure

1. Specificitytestofprimary anti-cathepsin B antibodies. Whole-cell cxSB-3 cells was separated on a 12.5% polyacrylamlde gel. (Lane a) Marker proteins with, from top to bottom, 66, 45, 36, 29, 24, 20 KD, respectively. (Lane b) SB-3 cell extract, Coomassie blue staining. The separated material was transferred to nitrocellulose (Western blot). Two lanes were individually incubated with the primary anti-cathepsin B antibodies. Subsequently, immunotract

from

complexes

were visualized

peroxidase conjugated results for antibody

with appropriate

to streptavidin with I (AB I) and antibody

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

biotinylated

second antibodies

4-chlor-1-naphthol.

(Lanes

II (AB II), respectively.

by

c,d) The

CATHEPSIN

bodies,

B LOCALIZATION

donkey

Dianova;

reduction

and

ERG),

goat

were

anti-rabbit

20 mm

at room

the

appropriate Butanol

2%

Immunoresearch;

the

cysteine

for background

This was done

antibodies

were

SB-3

1-butanol

cells

twice

diluted

to

treated

according

for 5 mm

at room

to LeGrue

temper-

(32).

Stefin

A, the

equipped

copy

was

(CLSM;

done Carl

an argon

with Zeiss,

laser

terference the

fluorescence with

overlay

Zeiss

confocal

on

image

a hard stack

a Zeiss

systems.

Laser

laser

ERG)

optical disk.

the

from

1CM 405

tumor

cells.

plete

inhibition

line

in Nomarski sections

focus),

were

the video

nm)

in-

sm

in

steps

as well

filtered

is shown is due

at high

as the

to remove

To achieve nique

Results

living

These

Cathepsin

B Activity detection of cathepsin B activity, we used living cultured on glass slides to halfconfluence. Cathepsin

B activity

demonstrated

Arg-4M3NA.

Cleavage

the presence bright yellow

with of the

the

synthetic

substrate

of NSA results in the fluorescence. Cathepsin

substrate

by the

active

Z-Arg-

enzyme

in

formation of small crystals of B activity could be demon-

strated by this method in HS-24, SB-3 tumor cell lines and in human lung Wi-38 fibroblasts (Figures 2a-2d). SB-3 cells showed the strongest duced

effect about

(Figures

2a and

the same

number

mm

than

reaction

time,

cells (Figure tals.

the SB-3

Therefore,

activity sources, ofthe tion

crystal

the standard

growth

was very

to melting

the original

reaction

and material

support

are the

that this type ofactivity of all inhibitors used In control

centers

strong

shed most

without

of 15-

in the

SB-3

ofadjacent

became

crys-

indiscernible. additionally membrane-

we also

observed

come from several during the course

by the cells during prominent.

their

It should under

migra-

be noted

the influence

substrate,

fluorescence oflow intensity was detected, binding ofNSA to the cells. No crystals

were

(Figure

3f),

primary

fixed

E64,

diffuse

resulting from with the typical

which

A

of low mo-

PMSF, and Trasylol, had no inhibitory

which effects,

mM).

and

concentrated

a pattern

is likely

ofdistribution

type

confluent

and

indiscernible Several

inhibit

to reflect

tech-

to fixed

the

and control and

separated

whole-cell

cence

level,

these

experiments when

3g and

and

tumor

In such

cells

done

the

were

the Such

of the cells.

cell cultures

became

granules

became

fluorescence. As described

tested

without

against

second

in

PAGE-

antibody.

No staining

antibodies,

anti-cathepsin

Localization.

the first

was observed.

unrelated

of sheep

these

in a van-

ofSB-3 cells. They detected only bands B (Figure 1). At the immunofluores-

were

we used

3e and 3h),

appeared

compartment

when

the antibodies

no staining

Surface

nucleus

(Figures

(Figures

cells had homogeneous bright experiments were performed.

experiments

instead

fibroblasts

cells

(Figures

the lysosomal

extracts for cathepsin

characteristic

as de-

all over the cytoplasm. Within no fluorescence was observed.

packed.

Methods,

slips.

protocols,

normal

cells

studies

on glass

led to identical results. of the antigenic sites. to distinct granules

HS-24

changed

densely

Materials

rabbit

the

localization

different

type,

SB-3

able and lower concentration nucleus and at the cell surface

observed

B

we used applied

all ofwhich localization was restricted cell

around

This

three

tumor-derived

metastasis-derived

granules

of Cathepsin enzyme,

to semiconfluence

using

ofthe

lung

e.g.,

In

was also goat

anti-

B antibody.

To prove

cell surface

localization

of

cathepsin B, we used living cells which were arrested by lowering the temperature to 0-4#{176}C.At this temperature endocytosis and membrane

fluidity

are low, but cell surface

architecture

should

not

suffer severe disturbance. SB-3 cells grown to semiconfluence on glass slips (Figures 4a and 4b) and labeled as described in Materials and

Methods the

dots (Figure in all cells; contours

showed cell

appeared

fluorescence more

pattern

densely

packed

on the cell with

these

4b). This pattern is characteristic, but was not observed even when speckling of the surface was sparse the cell were

cells.

a speckled

contours

always

such

and

incom-

of stefin

inhibitors

For intracellular grown

3). Independent

3a-3d),

las weight

antipain,

to

in the

2f. The rate

immunofluorescence

Localization.

when suspended Figures 4c through

leupeptin,

not

cells.

yellow fluorescence were found. In another series of experiments inhibitors were used to block cathepsin B activity. The low molecuinhibitors

other

of the

microscopic

specimens

surface;

the specific

localization

cell monolayers

Cell specimen

disappeared completely (Figures 2e and 2f).

experiments

pro-

fluores-

conditions

together

the cells. This activity might which debris of cells damaged

experiment on the

of 5 mm

and further unspecific growth may It was not possible to identify plasma

activity by this method. non-confluent areas of the

outside among

blue-green unspecific

Under

2c). It led finally

Recrystallization blur the situation. associated In the

cells.

the

time

sites and the same

for HS-24 cells after 15 mm (Figure cells (Figure 2d) were slightly less

cence intensity as was found 2b). Wi-38 normal fibroblast reactive

2c). A reaction ofreaction

the

(0.1

scribed in Materials and Methods, Only well-spread cells allowed In these cells the fluorescence

of

For cytochemical cell monolayers was

in Figure

penetration

Localization

precise

of light

and

screen.

Detection

cells

but

B activity

KD).

concentrations

intracellular

Cytochemical

1

of cathepsin

cathepsin

lower

with

Immunofluorescence

we used

Fluorescent

inhibitor

led to reduction

for SB-3 to the

KD) in comparison (Mr

reaction

of

differential

images,

intracellular

of the intracellular

This

cytochemical

system

(514

(0.5-1.5

endogenous

weights

even

micros-

microscope

the

Other inhibitors, such as pepstatin, are not specific for cysteine proteinases,

micro-

scanning

green

Individual

(extended

and photographed

scanning

using and

Successive

stored

we used

filter

epifluorescence

(DIC).

were

noise

the

in confocal

of a whole

pixel

appropriate

Oberkochen,

contrast

z-axis)

microscopy

the

blocked

2e).

in tM concentrations,

inhibition

lecular

Microscopy scope

(Figure

B, also added

(M1 “till

For conventional

completely

types

complete were

in PBS

proteinases,

in all cell

concentration.

Extraction.

with

Finally,

1315

CELLS

pre-adsorbed

for investigation.

temperature.

working

TUMOR

(Jackson

separately

with each cell line used

for

ature

anti-sheep

Hamburg,

IN HUMAN

In Figure

pronounced.

cells were labeled 4e show different 4d a superficial

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

The

same

under optical focus

result

was obtained

the same conditions. focus planes through plane

is photographed.

1316

ERDEL,

native

Figure 2. Cytochemical detection of cathepsin B activity in adherent cells by fluorescence in (a) and 15 mm in all other experiments shown. Cathepsin B activities in (a,c) SB-3 cells, activity in SB-3 cells was brought about with (e) E64 and (f) stefin A. Bar - 25 m.

The

speckled

Figure

cence appears were performed cific

fluorescence

4e is a picture

binding It is known

distribution of the

is visible

equatorial

as a halo surrounding under the control

focus

the cell. conditions

in the focused plane.

The

area. fluores-

These experiments elaborated for spe-

in the intracellular localization experiments. that butanol treatment can extract certain

proteins

from 2%

TREFZ,

SPIESS,

SPRING,

LAH,

EBERT

photography. Reaction time was 5 mm in the experiment documented (b) HS-24 cells, (d) Wi-38 fibroblast cells. Inhibition of cathepsin B

the surface 1-butanol

ofliving in PBS.

cells (32). The

B by immunofluorescence and

HABERMAAS,

control

sin B with Confocal

cells.

This

subsequent

revealed indicates

We treated no differences

that

the association

the plasma membrane is strong. laser scanning microscopy was applied

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

the SB-3

identification

cells with

of cathepsin

between

treated

of the cathepas an additional

CATHEPSIN

B LOCALIZATION

IN HUMAN

TUMOR

CELLS

1317

3a ‘I

..

-

./

...

.

..

‘:

,:;:1f.?

3c :.-*-

‘I

.:

4

.::-&

.

.

“Lv

,,,-

.

a

I

p

I

p ;ii:

,

1IilL.)

(T

.:‘:;“-‘ -.

.:

lI’

.

.‘

tPh;l:

:t-

-

,.

-.

I

-

‘I,

#% .‘,.

.J

-

‘

..-

‘.

,

.

I

,#{149} -

‘I

4

#{149} -

11

‘

-

‘

-

...

..

ilk .

it.I.

..

A ;e

.,

p’s ‘-.‘

! ...

.-

Figure a Detection pictures of identical body I; (g,h) SB-3

.

w

,

1

‘

.

,,.

-

.‘

-j

.

;*l

of cathepsin B by Immunofluorescence. Adherent cells were fixed by methanol-acetone. (a,c,e,g) Phase-contrast and (b,d,f,h) fluorescence areas are shown. (a,b) Wl-38 cells labeled with antibody I; (c,d) Wi-38 cells labeled with antibody II; (e,f) HS-24 tumor cells labeled with antitumor cells labeled with antibody I. Bar - 25 sm.

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

1318

ERDEL,

TREFZ,

SPIESS,

HABERMAAS,

SPRING,

LAH,

EBERT

Figure 4. Detection of surface cathepsin B by immunofluorescence in (a,b) adherent and (c-a) suspended SB-3 cells using primary antibody I. Cells were immobilized at 0-4#{176}C and labeled at this temperature before fixation in 1% formaldehyde. (a,c) Phase-contrast pictures cover the same field as (b,d,e) fluorescence pictures. Pictures in d and e are a superficial and an equatorial focus plain, respectively, of the cells shown in c. Bars: a,b = 25 tm; c-c = 10 tm.

tool

to demonstrate

new

type

the

cell

of microscopy

Therefore,

we used

surface

cell aggregates

ticularly

useful

gregates

is low; only small,

for such

a study more

are formed even after extended laser scanning microscopy, cells about 1 pm thick. 5. The first section cell. It provides offluorescence. deeper

than

Pictures (Figure

useful

for thick

for immunolabeling. because

its ability

or less two-dimensional time were

Sd are from

distant

from

SB-3 is par-

the cell surface,

and

cell ag-

and

the lower

clusters

tures

B. This

to form cell

under rotation. By confocal optically sectioned in slices

oftwo ofthese cells are shown in Figure 5a) is from the upper pole of the larger

a view of the cell surface, with In the following section (Figure the first,

Sc and

specimens.

ofcathepsin

localization

is particularly

fluorescence

is found

a speckled Sb), which

to form

a ring.

pattern is 2 tm

the

Sf are from

in all respects.

structed

from

surface-related

the

first

equatorial

section. appears Figure

a total

of both

cell pole

and

cells,

is bright,

also in a speckled

It clearly

B fluorescence.

6-7

Sh

Se

two pic-

picture

demonstrates Figure

to

Figures

the first focus

tm

confined

pattern.

resemble

Sg is the extended

of 13 sections.

cathepsin

regions

Fluorescence

reconthe cell

shows

the

differential equatorial

interference contrast picture of the cell group in the plane. This series of pictures clearly proves the cell surface-

associated

and

cence

in the

unequally

SB-3

tumor

Figures

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

distributed

cells.

cathepsin

B-related

fluores-

CATHEPSIN

B LOCALIZATION

IN

HUMAN

TUMOR

1319

CELLS

minogen

In

activators,

particular,

collagenases,

cathepsin

proteoglycanases,

and

been

with

B has

correlated

cathepsins. increased

metastatic potential of tumor cells (14,15 ,18). Our recent studies on the cathepsin B activity in human lung tumor tissue also corroborate this hypothesis (34). Therefore, we have investigated the cathepsin recently

B activity and localization in human isolated from a primary squamous cell

metastasis (27).

from

HS-24

an adenocarcinoma

a cytochemical

By

cells.

assay

Considerably

ofthe

lung

we detected

higher

demonstrated

the enzyme

membrane

of the

dular

tissues

likely

that

sion

from

of the

By the

from

reaction:

prising,

because

has

B was localized we also clearly

B (35).

acquired

Therefore,

cathepsin

the

they

we ab-

different

degrees

of

of malig-

are derived.

we could those

it is

B expres-

At the moment,

between

which

only

tipain, stefm A)known the reaction. Stefin cathepsin B, showed

in

was found

the two cell lines and the degree

of inhibitors

cytochemical

cathepsin In addition,

to malignancy.

within

tumor

use

B activity

cathepsin

lines

a correlation

B activity

nancy

cell

leading

gland

B activity

on the outer surface of the cytoplasmic Normal cells of epithelial and glan-

to express

of these

drawing

cathepsin

cells.

are able none

in the process

stain

.

SB-3

to the adrenal

cathepsin

cathepsin

in SB-3 cells. By immunocytochemistry, in lysosomes of HS-24 and SB-3 cells.

lung tumor cells carcinoma and a

verify

the

inhibitors

specificity

(E64,

of the

leupeptin,

an-

to be specific forcysteine proteinases blocked A, the most potent intracellular inhibitor of the weakest effect in this assay. This is not sur-

stefin

A, as compared

with

the

other

inhibitors,

is a larger molecule (Mr 11 KD); its uptake into living cells must be considerably slower compared with that of the low molecular weight inhibitors. It has been demonstrated (36) that cystatins and stefins

of this

perimental the cells

size

are able

molecular

indirect

evidence

of a larger

cell surface. localization therefore,

studies

the plasma

fraction

is rarely

of active

achievable

we applied

tisera were performed labeling or targeting

This observation performed with Biochemical organdIes

using

two different

of evidence

supports

the

involvement

of proteolytic

in the cascade of events leading to the formation of (33). Most frequently discussed in this context are plas-

cytological

studies

obviously

respect

identified

and

mature

represent

cell. It is well known that ing the cell cycle. Sylven

the ct

active

and Of

becomes

particular

B an-

either whole-cell Both antibodies revealed a patthe cell nucleus.

the

the

because diffuse

interest

Downloaded from jhc.sagepub.com at East Tennessee State University on June 19, 2015

lysosomes

cathepsin

lysosome

as the

B. Therefore,

distribution

in the

lysosome distribution changes dural. (16) observed different cathepsin

to interphase

on the cell morphology cells

apIm-

is consistent with similar localization experiments tumor material or tumor cell lines (3,4,16,20,37,38).

and

with

B on

methods. anti-cathepsin

and

pre-mitotic

also found varying cathepsin B distribution, correlate it with the phase of the cell cycle.

body

ex-

by the histochemical

under conditions that permit restricted to the cell surface.

containing

the granules

cathepsin

immunofluorecence

revealed matching localizations. The first protocol tern ofgranules particularly concentrated around

B patterns

enzymes metastases

However,

our

frac-

tumor Precise

inhibitors.

munofluorescence

A large

Under

ofstefin A into that of the low A. Beconsti-

proach;

Discussion

cells.

membrane

the

weight

the

37#{176}C), the uptake slower compared with

lion cathepsin B was accessible to and inhibited by stefin cause this effect was observed only in tumor cells, it might tute

Figure 5. Confocal laser scanning microscopy; immunofluorescence detection of surface cathepsin B using antibody I in suspended cells. SB-3 tumor cells were immobilized at 0-4#{176}C and labeled. Six optical sections from a series of 13 are shown. (a) “Upper” cell pole; reference (z 0 tm) for the consecutive optical sections in the z-axis with a thickness of 1 run. (b) Section at z - 2 tm below the reference. (c,d) Sections atthe cell equator at z - 6 and 7 tm below the reference in c and d, respectively. (e,f) Sections at the lower” cell pole at z - 11 and 12 tm below the reference in a and f, respectively. (g) Extended focus image composed of all 13 sections. (h) Differential interference contrast image of the equatorial plane. Bar - 5 tm.

to penetrate

mm,

conditions (15 was considerably

it is best

in densely is the

question

cells.

We have

but it is difficult to It definitely depends

visualized

packed

in well-spread

cells.

of plasma

membrane-

ERDEL,

1320

associated in the

cathepsin

cell

cortex

membrane made

B. Sylven but

were

of the adjacent

a similar

able

cathepsin by labeling

for membrane

fluidity

ments

were

SB-3

cells.

outer

cell surface.

clearly,

cells.

4 and cence

living

with

the

The

more

the

with

particularly

contact

proteolysis

(25,26).

role

minogen

to shift areas,

and

related

with

with

and

further

to certain

supports

enzymes,

such

metalloproteinases,

as urokinase

and

is not disputed.

secretion.

cept of surface-localized viously and remarkably tunes

more

containing

electron

type

cathepsin

However,

However,

in conclusion,

An even

zyme

plas-

10.

Reckiies in rumor

11.

Lah U,

12.

Poole

there

have

experimental

been

strong

emphasis

proteinases. corroborate

precise

studies

only

con-

13.

discussed

cell surface under

Dri Taijana Institute)for

(antibody

copy,

Popovic the ge/i

II in this

study),

andJoza

Babnik

ofhuman

liver

respectively.

andDrH.-R

discussions.

Boston:

GK Hall,

Bi.

J

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en-

1974;137:387

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Buck MR. Honn KV, CrissmanJD, Rao NC, Liotta AL, Sloane BE Degradation of laminin by human tumor cathepsin B. Clin Exp 1989;7:461

AR,

Tikman

K), Recklies

Mort)S, proteinase

Leduc from

AD,

Stoker

TAM.

MS.

Recklies

AD.

Characterization

Differences

P, Baici A, Keist R, Matzku as a marker for metastatic

in secre-

breast

carci-

of a latent cysteine form of cathepsin

ascitic fluid as a high molecularweight Biophys Acta 1983;755:369

B, Snellman

0,

Taylor)D. marker for

5, Keller R. Cathepsin B-like tumor cell variants. Exp Cell

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Pietras

RJ, Roberts

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to the

Division

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proBiol

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B like enzymes.

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JS,

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Fisher

HD.

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B and

phosphomannosyl

oflysosomal

AR.

enzymes.

Decker

RS.

D in human

recognition

Immunofluorescent

fibroblasts.

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J Cell Biochem

for in-

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avi

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Van Dongen)M, Willemsen R, Ginns El, Sips H), Tager)M, Barranger )A, Reuser A)). The subcellular localization ofsoluble and membrane

bound

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tivity on murine

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p.’.-’

Hatfield ), Crissman ID, cysteine proteinase in human

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script,

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collagen.

Sylvn

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way in our

Biochem

256:8536

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ful

A), Lazarus

native

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ob-

18.

Willy

activation.

Koppel teinase

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spontaneous

15.

laboratory.

j

and

Sloane BF, Honn KV, Sadler)G, Turner WA, KimpsonJ), Cathepsin B activity in B16 melanoma cells: a possible metastatic potential. Cancer Res 1982;42:980

by use of immuno-

are presently

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degrades

B. Biochim

on the conhere

kallikrein,

14.

A number are

is placed

of the

B is possible

Such

(39).

data presented view.

demonstration

cathepsin

microscopy.

Our this

many

tissues.

of cathepsin immunoen-

non of the proteinase cathepsin B at the edges of human noma and fibroadenomas. Nature 1980;273:545

B, in cancer

most

and

MC,

that

metastasis.

areas,

approaches were based on the assumption that these to be secreted, either by tumor or by host cells, and that increasing malignancy must be positively cor-

enzyme

review;

9. Burleigh

sup-

surface

and

RIG, Barrett A), Dingle JT Cathepsin Bi and D action cartilage proteoglycans. Biochim Biophys Acta 1973;302:411

human

the view of directed

troversial results, as recently reviewed by Zucker of variables, systematic, technical, and conceptual, in this

sin B, plasmin

8. Morrison

cells, the

B in cells

EBERT

J 1977;166:21

fluores-

In adherent

of contact

cathepsin

LAH,

Y, Vaes G. Further studies on the activation of procollagenase, precursor of bone collagenase. Effects of the lysosomal cathep-

the latent

Metastasis

metastasis

and theoretical activities have it was assumed

is not even.

in areas

7. Eeckhout

very

(Figures

3). The

SPRING,

5. Yokota S, ‘Buji H, Kato K. Immunocytochemical localization B in rat kidney. I. Light microscopic study using indirect zyme technique. J Histochem Cytochem 1986;34:891

for sus-

is weak

labeling(Figure

the cathepsin

of proteolytic

activator,

invasion

cells and

In suspended cells, which are unpolarized, fluoresall over the cell surface concentrated into irreguplaques. This observation might be an indication

cell is able

The

staining

on the

microscope,

HABERMAAS,

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experi-

metastasis-derived

fluorescence

lysosomal

We

blocked

fluorescence

scanning

on the cell surface intense

porting substrate. cence appeared larly distributed

B-rich,

membrane-linked

cells. The

this for adherent laser

plasma

were

shock.

cathepsin-related

We demonstrated

et al. (37)

in tumor which

by cold

cathepsin

plasma

(15,18,19),

SPIESS,

method for the localization of rabbit J Histochem Cytochem 1983;31:803

B also

to the

Maciewicz

cells

endocytosis

cells show

5)as compared distribution

we found

area.

B was found

by the use of a confocal

pended

that

and

performed

These

cathepsin

it clearly

By cell fractionation

tackled

problem

located

to assign

cytoplasmic

statement.

membrane-associated this

al. (16)

ct

not

TREFZ,

Hanewinkel

tured

normal

enzymes study.

H, Gossl

in I-cell fibroblasts: a comparative Biol 1985;39:179

immu-

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CELLS

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Le Grue 5). 1-Butanol

extraction

and subsequent

L.

with den-

reconstitution

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from

which

mediate

metastatic

phenotype.

K, Hothy#{228}M, Salo

matrix

in tumor

T Proteolytic

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Biochim

degradation

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Neoplasma ofthe

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role

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surface

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enzymes

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in can-

Invest