Escherichia coli heat-stable toxin receptors in human colonic tumors

GASTROENTERGLGGY 1994$G7:1683

-

1661

Escherichia co/i Heat-Stable Toxin Receptors in Human Colonic Tumors STEPHEN L. CARRITHERS,* DONALD C. ROBERTSON,§

SCOll J. PARKINSON,* SCOTT and SCOTT A. WALDMAN*

GOLDSTEIN,*

PAULINE

PARK,t

*Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, and tDepartment of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania; and “Department of Microbiology and Biochemistry, School of Agriculture, University of Idaho, Moscow, Idaho

Background/Aims: Escherichia co/i heat-stable enterctoxins (ST) are small peptides of 18 or 19 amino acids that bind to specific cell surface receptors located on the intestinal brush border and activate guanylate cyclase, resulting in an increase in the intracellular cyclic guanosine 3’,5’-monophosphate content of the cell. The present study examined whether receptors for ST are expressed by primary and metastatic human coIonic tumors in vivo. Methods: Plasma membranes prepared from surgical tissue samples from normal colon, liver and lung, primary colonic adenocarcinomas, and colon carcinomas metastatic to lung and liver were analyzed for the structural and functional characteristics of constituent ST receptors. Results: All primary and metastatic colonic tumors examined bound ST, showing receptors of high (pmol/L) and low (nmol/L) afflnity with densities that were similar to those in normal colon. Also, affinity cross-linking of labeled ST to membranes showed similar binding proteins in primary and metastatic tumors and normal colon. ST binding and affinity-labeled proteins were not detected in normal extraintestinal tissues. Guanylate cyclase was activated by ST in membranes from all colonic tumors studied, with efficacies and potencies that were similar to those in normal colon. ST did not activate this enzyme in normal extraintestinal tissues. Conclusions: Receptors for ST are expressed by primary and metastatic human colonic tumors in vivo, with structural and functional characteristics that are similar to those in normal human colon.

In addition,

these receptors

branes facing the intestinal ing the intestine

are joined by tight

a barrier

the exchange

against

components location

isolates ST receptors

Colorectal

neoplasms

cancer-related mately

enterotoxin

(ST)

E that is a major source of infectious diarrhea worldwide. ST is an 18 - or 19 - amino acid peptide that produces diarrhea by binding to receptors on mucosal cells of the small and large intestine.’ -3 ST-receptor interaction activates particulate guanylate cyclase and elevates intracellular cyclic guanosine 3’,5’-monophosphate (cGMP), which directly mediates intestinal secretion.*-’ Functional receptors for this toxin have been identified only in intestinal mucosa cells in placental mammals.296,8

junctions

of intestinal

that form

contents

Thus,

their

and apical

from the circulation.

are the second leading

deaths in the United

160,000

mortality

to apical memMucosal cells lin-

in the circulation.‘2-‘4

cause of

States, and approxi-

new cases are expected

rate for this disease approaches

in 1994.”

The

50%, reflecting

the presence of metastases at the time of presentation.16 Although surgery is the mainstay of therapy, microscopic residual

disease results in recrudescence

in up to 50% of

patients.”

A novel approach to the diagnosis and therapy of cancer employs ligands whose receptors exist on tumor cells (see references calization

18-22

of ST receptors

for reviews).

The specific lo-

in apical membranes

of intesti-

nal mucosal cells, their isolation from the circulation, and their high affinity for toxin suggest that ST might be useful for targeting diagnostics and therapeutics to colorectal cancer cells. However, these receptors have not been shown metastatic

or characterized

colonic tumors

ST receptors

previously

in primary

in vivo. In the present

were identified

and characterized

or

report,

in normal

colon, primary colonic tumors, and adenocarcinomas of the colon metastatic to liver and lung obtained from patients

at surgery. Materials

schericbia coli secretes a heat-stable

are localized lumen.9-”

and

Methods

Clinical Specimens Human tissue was collected from resected surgical specimens under a protocol approved by the Institutional Review Board of Thomas Jefferson University. Specimens colAbbreviations used in this psper: B,, , maximum receptor binding; K,, concentration of ST that yields haif-rnaximum activation of guanylate cyclase; K.,, dissociation constant: K,, concentration of llgand that yields half-maximum displacement of radlolabsled ST; SDS, sodium dodecyl sulfate: ST, E. co/I heat-stable enterotoxin. 0 1994 by the American Gastroanterologlcal Association 001~5085/94/$3.00

1664

CARRITHERS

m

AL.

GASTROENTEROLOGY

lected from 16 patients at the Thomas Jefferson University Hospital included 10 primary colon tumors (3 right, 2 trans-

potency

verse, and 5 sigmoid

activation.

colons),

tissue and 5 metastatic from lung,

from which

lesions,

including

were also obtained.

from 3 patients.

lung

from

were obtained

intestinal

tumors

Normal

surgical

and was used to determine as well-differentiated,

undifferentiated

liver and

specimens

for the

the classification

moderately

of the

differentiated,

and

tumors.

Cell Culture

Receptor previously

binding

with

(0.1 mg protein) mmol/L

Tris-HCl

(pH

(wt/vol)

bacitracin, binding

initiated

by the addition

1 mmol/L

metastasis

of colon

in 5% CO, at 37“C as

International

mmol/L. NaCI, 20 mmol/L

trypsin containing

ers Grove, CA). For direct

above culture medium 10 days, provided

of 3 X lo* viable cells/ml

at 37’C will increase lo-13-fold

the medium

is renewed

medium

mmol/L

within

tions of labeled

for an

10 passages. For experiments, cells were grown to in 75- or 150-cm* flasks, and at that time, the was removed.

Adhered

20 mmol/L phosphate-buffered culture

samples were incubated

once. T84 cells

were at passage no. 49 on receipt and were maintained additional confluence

acid

centrifuge

from

(SEM) in all experiments.

were prepared

from

zyme kinetics written

Tissue and Membrane Preparation

binding described

mucosa

was obtained

by scraping,

as

dissection.

Metastatic

lesions

were

separated from the surrounding normal hepatic and pulmonary parenchyma by sharp dissection. After dissection, specimens were either on ice within

quick-frozen 15 minutes

in liquid

nitrogen

after surgery.

or homogenized

Membranes

were pre-

pared from human tissues and T84 cells as described previously.**-*’ All procedures were performed at 4°C. Briefly, cells or tissue were homogenized in buffer containing 50 mmol/L. Tris-HCI (pH 7.6), 1 mmol/L dithiothreitol, 1 mmoll L EDTA, 1 mmol/L ethylene glycol-bis-(aminoethylether)-tetraacetic acid, and 0.1 mmol/L. phenylmethylsulfonyl fluoride (TEED buffer), with 250 mmol/L sucrose. The homogenate

+12%

binding

and en-

CA). Cigale was

de Pharmacologic

Cellulaire

France). Using this program, and binding

constants

were

Guanylate cyclase activity was assayed as described previously.3~24-27 Briefly, reaction mixtures (100 w total volume) contained

membranes

mmoliL

Tris-HCl

guanosine 15 mmol/L kinase.

(50- 100 pg protein)

triphosphate

regenerating

creatine phosphate

Where

indicated,

(ATP) with and without tions.

with 50

1 mmol/L

adenosine

by the addition

triphosphate

branes, incubated

for 5 minutes

was quantified

and a

that consisted

sodium

triphosphate in reac-

of substrate

(1

in 4 mmol/L MgCI,) or memat 37’C, and terminated

with

acetate (pH 4.0), followed

water for 3 minutes.

after acetylation

of

and 2.7 U of creatine phospho-

mmol/L. guanosine

in boiling

system

ST (1 l.tmolA.), was included

Assays were initiated

immersion

incubated

(pH 7.6), 10 mmol/L. theophylline,

taining 500 mmol/L (S-10 mg protein/ml)

Native STi_ia was purified from E. coli and iodinated as described previously.*~** The purified labeled peptide (iz51-

of ligand

using Cigale on an Apple Macin-

were fitted,

was centrifuged at 105,000 X g for 60 minutes. Resulting membranes were washed sequentially in TEED, TEED con-

ST Radioiodination

at least in triplicate, was less than

Inc., Cupertino,

Sophia Antipolis,

400 @ of 50 mmol/L

KCI, and TEED. Washed membranes were aliquoted and stored at -7OOC.

was esti-

Guanylate Cyclase

completely

layers by blunt

Analysis

Computer

isotherms

by sub-

binding

determined.24-27*29

previously.3V24-27 Colon adenocarcinoma tissue was from underlying muscle and overlying

separated mucosal

colonic

variability

by M. Bordes (Institute

et Moleculaire,

concentra-

moliL to 5 X

in the presence of excess (1 PrnoII

was performed

tosh IIci (Apple

experiments,

was determined

Nonspecific

ST. Assays were performed

saline, scraped into 50 mL cell

pH 7.6. Membranes

binding

mated in parallel incubations L) unlabeled

150

Co., Down-

binding

from 2 X lo-l3

binding.

and the intraexperimental

washed whole cells.

Normal

total

Instrument

equilibrium

in the presence of increasing

mol/L.24~25 Specific

Fil-

bound to filters was quantified

ST ranging

cells were washed twice with

tubes, and washed two more times with 50

Tris-HCl,

lo-*

tracting

Ltd., Maid-

(pH 7.2), and 1 mmol/

counter (Packard

in the

on Whatman

5 mL of buffer containing

phosphate

L EDTA at 4°C. Radioactivity

0.9 mmol/L ethylenediaminetetraacetic

was

for 2.5 hours

with 0.3% polyetheleneimine.

twice with

in a Packard gamma

NaCl

150 @). Binding

of ‘*‘I-ST and incubated

presoaked

50 0.1%

and 450 mmol/L

buffer; final volume,

described previously, grown in monolayers in polystyrene flasks (Corning, NY), and subcultured when confluent with 0.25% (EDTA).23 An inoculum

EDTA,

containing

cystamine,

by filtration

carcinoma

preparations

in a buffer

7.6), 0.67 mmol/L

were terminated

ters were washed

Cells were cultured

cyclase

as described

Membrane

GF/B glass fiber filters (Whatman stone, England)

adenocarcinoma.

full efficacy and

and guanylate

assays were performed

were incubated

(receptor

T84 cells were obtained from the American Type Culture Collection (Rockville, MD) as a transplantable human from a lung

possessed binding

modifications.**-*’

at 37°C. Reactions

cell line derived

Ci/mmol)

ST-Receptor Binding

and metastatic

comparison with diseased tissue. Histopathology confirmed the presence or absence of adenocarcinoma of the colon in each specimen

1000-2000

in assays of ST-receptor

No. 6

4 from liver and 1

Both primary

colon tumors were obtained tissue

normal

Tyrd-ST,

Vol. 107,

Generated

by radioimmunoassay.

by

cGMP All reac-

tions were performed in duplicate ot triplicate, and the data are representative of at least three experiments. Intraexperimental variability was less than 212% (SEM) in all experiments. Enzyme activity was linear with respect to protein concentration and time throughout

these experiments.

E. COLl TOXIN RECEPTORS IN HUMAN COLONIC TUMORS

December 1994

ST-Receptor Cross-linking “‘I-ST

was cross-linked

mary adenocarcinoma

to ST receptors

as described

astatic

of the colon,

colon

carcinoma

and T84 cells (Figure

to liver and lung,

1655

met-

1). Scatch-

in 20 mmol/L phospreviously.3’26’27,30 Briefly, membranes phate-buffered saline (pH 7.0; l-2 mg protein/ml) were incu-

ard analyses of these data yielded curvilinear isotherms, suggesting the presence of at least two populations of

bated for 15 minutes

binding

the presence

at 37°C with 40 nmol/L

or absence

receptor complexes midyl suberate temperature.

were cross-linked

(Pierce,

Rockford,

Reactions

buffer (0.125 Tris-HCl immersion samples

with 1 mmol/L disucciniof an

sodium dodecylsulfate

(SDS) sample

[pH 6.81, 20% glycerol,

10% 2-mer-

by SDS-polyacrylamide

on 4%-15%

blue) and

bromophenol

water bath for 5 minutes.

were analyzed

resis (PAGE)

at room

by the addition

6% SDS, and 0.004% in a boiling

ST. Ligand-

IL) for 15 minutes

were terminated

equal volume of Laemmli captoethanol,

labeled ST in

of 1 FmoUL unlabeled

linear

Cross-linked

gel electropho-

acrylamide

gradient

slab

sites for ST in all membrane

ied: high-affinity, finity, This

low-capacity

high-capacity

population

is in close agreement

viously

that showed

of ST binding

Louis, MO) as described

about

Protein Rad Laboratories,

by the method

Richmond,

Linear

CA) using bovine serum albumin in duplicate

regression

and

curve

fitting

were

analysis was performed

and results are expressed experiments. highest

or tripli-

are the mean of at least three experiments.

Cricket Graph (Cricket Software, Malvern, IIci. Statistical

of Bradford (Bio-

using

using Student’s

t test,

as the mean + SEM of at least three

All reagents

analytical

performed

PA) on a Macintosh

obtained

commercially

were of the

specifically

man colon (mean milligram

of the

t

SEM

normal

human

prepared

were cross-linked of 1 pmol/L labeled

showed ST-binding

(55

whereas

per

was a paucity

+

lo),

colon

carcinoma

in colon

ST displaced

adenocarcinoma

proteins.

metastatic

(110

2 40) at concentrations

near the dissociation

con-

tors specifically

stant

(&)

lop9

mol/L),

studies.23-27

In contrast,

branes

devoid

mors

obtained

noma

and

of these

on normal The

from

and

extraintestinal

binding

human

intestine

direct

equilibrium

and

colon

binding

receptors

in membranes

colon

studies.

prepared

were

saturable

from human

in normal in

specifically fashion colon,

colon tumors,

of 43, 45, 92, 96, and 160

metastatic

of the 45-kilodalton

of these pro-

in T84 cells, there binding protein, to lung,

species.

proteins

there

Unlabeled

toxin-binding

were not observed

expressed by colonic tumors

to those expressed

ST-binding

by normal

are structurcolon cells. In

proteins

identified

in these studies

to those observed

previously

in rats and hu-

mans.3,26*27X30~34-37 It is likely that the variable

quantities

of each protein band recovered in different tissues reflect differences in the degree of posttranslational processing of the parent ST receptor.3~26~27z’0X34-37 Variable recovery of different ST-binding proteins identified by affinity

and lung.

determined

ST bound

and

carci-

are not present

as liver

of ST receptors

in a concentration-dependent

tu-

adenocarcinoma but

such

tumors

are similar

that ST receptors

metastatic

characteristics

of ST

to the histopa-

showed

in vivo

tissue

addition,

carcinoma

and

(Figure

liver and lung. These data show that ST recep-

ally similar

mem-

presence

closely

which

the colon

in other

lung

and metastatic

data establish

on primary

cells derived

and

The

corresponded

specimens,

defined

liver

in all primary

in every case. These

are present

previously

normal

of ST binding.

was detected

thology

including

the relative proportion

Specific ST-binding

in normal

receptors

origin,

cells

and absence

by SDS-PAGE

‘*‘I-ST from all five labeled

cells

from

colon, pri-

and T84

by autoradiography

proteins

although

hu-

human

carcinomas,

ST, followed

visualized

of intestinal

(50 + 7), and TS4

were

from normal colon

unlabeled

in normal

is ST-

and metastatic

to those obtained

to 1251-ST in the presence

proteins

2). Tissues

1). Thus,

in primary

were comparable

and metastatic

to liver (55 + 7) and lung (ca.

sites (Table

colon.

Membranes mary

of ST bound

to receptors femtomoles

48 + 3), primary

of protein,

colon

of the total

of high-af-

of colonic origin

teins differed in each tissue. Indeed, was a paucity of the 160-kilodalton

Results ST bound

l%-5%

kilodaltons,

grade.

between

and low-affinity

abundance

for ST in membranes

colon carcinomas

as standard.31 All assays were performed cate, and results

finity receptors

receptor affinities and densities

was measured

differences

of high-

mol/L.3~‘o~20~24-27~33~34 The relative

Miscellaneous

pre-

populations

sites. Indeed, Kd values previously reported for low-affinity sites in rat intestine vary between lo-” and lo-*

Direct autoradiography of dried gels was pergels. 2,26,27Y30 formed using Kodak X-OMAT RP (XRP-5) film (Sigma, St. previously.26,27,30

obtained

1).

membranes.24-27~32

significant

for the Kd values

the tissues

results

(Table

and low-affinity

sites in rat intestinal

There were no statistically

stud-

and low-af-

of receptors

with

high-

preparations

population

to pri-

cross-linking has been observed previously in studies of the rat and human intestinal ST receptor.3*26,27,30,34-39 ST induces

intestinal

secretion

and diarrhea

presum-

ably by associating with a transmembrane protein possessing an extracellular ligand binding and cytoplasmic guanylate cyclase domain.33,36,37X40-43 ST-receptor interaction induces activation of guanylate cyclase and accu-

1656 CARRITHERS ET AL.

GASTROENTEROLOGY Vol. 107, No. 6

B (pmol/mg protein) x 10-Z Figure 1. Scatchard analyses of direct equilibrium binding of labeled ST to normal human intestine, colorectal tumors, and T84 cells. Experiments were performed as described in Materials and Methods. insets in A-E show direct equilibrium binding. Total binding (0) and nonspecific binding (m) determined in parallel incubations in the presence of 1 pmol/L unlabeled ST. Specific binding (0) is calculated as the difference between total and nonspecific binding. Results are representative of four experiments. 6, ST bound (pmol/mg of protein); B/F, bound ST/free ST ([pmol/mg protein]/nmol/L). (A) Normal colon, (B) primary carcinoma of the colon, (C) colon carcinoma metastatic to liver, (D) colon carcinoma metastatic to lung, and (E) T84 cells.

mulation alterations

of intracellular

cGMP, which directly

in fluid and electrolyte

secretion.‘-’

mediates ST has

been shown to activate guanylate cyclase in membranes obtained from human intestine and human colon carcinoma cells established studies, ST activated

in vitro.7,23,38,39943*44 In the present guanylate cyclase in membranes

isolated from normal human

colon, all primary

and meta-

models, colon carcinoma cells in vitro, and human small intestine. 2-8~20~25943~45~46 In contrast, ST did not activate guanylate

cyclase in membranes

lung (Figure

4).

Previous

studies

showed

from normal

that activation

liver and

of guanylate

cyclase by ST is potentiated by adenine nucleotides.37,41,45 ATP alone activated guanylate cyclase only slightly in

static colon carcinomas, and T84 cells (Figures 3 and 4). Activation was concentration dependent and saturable,

membranes from normal human colon or primary colon tumors (Figure 4). However, this adenine nucleotide acti-

and maximum

vated basal guanylate cyclase 3-5-fold in membranes from normal liver and lung, colon carcinoma metastatic

activations

of 1.5 -5-fold

were observed.

The concentration of ST yielding half-maximum activation of guanylate cyclase (K,, as determined by the method of Hanes59) was (mean X 1O-8 mol/L 2 SEM) as follows: normal colon, 1.17 + 0.30; primary adenocarcinema of the colon, 1.32 + 0.22; colon carcinoma metastatic to the liver, 0.82 2 0.33; colon carcinoma metastatic to the lung, 0.72 +- 0.18; and T84 cells, 0.88 + 0.12. These characteristics of activation are in close agreement with those reported previously for guanylate cyclase in intestinal membranes from various animal

to liver and lung, and T84 cells. Furthermore, ST activation of guanylate cyclase was potentiated by ATP. Guanylate cyclase was maximally activated lo- 15 -fold compared with basal enzyme activity by the combination of ST and ATP. Enzyme activation by ATP and ST was greater than the sum of individual activations by these with results obtained preagents, in close agreement viously with rat intestinal membranes.37*41,45 Synergistic activation of guanylate cyclase by ST and ATP was ob-

E. COLl TOXIN RECEPTORS IN HUMAN COLONIC TUMORS

December 1994

Table 1. Equilibrium in Normal Metastatic

Binding Parameters for ST Receptors Human Intestine and Primary and Colonic Adenocarcinoma Low affinity

High affinity Bmax ( fmol/mg)

Tissue Normal colon Primary colon carcinoma Colon carcinoma metastatic to liver Colon carcinoma metastatic to lung T84 cells

B

Kd (amoW)

&

(fmo%g)

7.0 f 4.3

51.6 2 18.3

3.0 + 0.9

4.7 2 2.8

87

(nmol/L)

2 40

119

4.4

t IO

15.8

2 1.5 ? 5.9

in close agreement Scatchard

3.9 ? 2.9

1.4 ? 0.5 9.4 t- 7.4

86 ? 30

1.7 It 0.4

116 + 10 138211

25.3 ? 11.8 63.9 ? 13.5

16.4 2 12.8 11.2 ? 5.3

NOTE.Equilibrium binding experiments were conducted as described in Materials and Methods. Analyses of ligand binding, including calculations of binding constants, were performed using Cigale on an Apple Macintosh llci.

nal membranes

binding

a maximum

receptor

origin.

In contrast,

nucleotides

mal liver and lung,

ATP

cyclase in these tissues, as reported studies further support the presence tors for ST in colon tumors expressed in native intestinal

(B,,)

protein. 24 Recently,

a B,, gested that two classes of binding

sites for ST may exist

cells.32 Computer-assisted binding

curve fitting

a two-site

model for binding

of ligand

that yields half-maximum ST (K,) values

4.9 nmol/L for high- and low-affinity This suggestion directly

was confirmed

measured

displace-

of 78 pmol/L

and

sites, respectively.32

of labeled ST to high- and prepared

colon, and primary

from T84 cells,

and metastatic

colon

tumors.

from nor-

activated

guanylate

that

previously.47 of functional

that are similar cells.

These recepto those

possesses

a ligand-binding

and a guanylate ST-receptor

cyclase catalytic

interaction

lytic activity

activates

and accumulation

which directly

mediates

domain

extracellularly

domain

in the cytosol.

guanylate

cyclase cata-

of intracellular

alterations

cGMP,

in fluid and electroguanylate

cyclase in

Discus8ion

membranes

This report describes

colonic adenocarcinoma cells established in vitro, including T84, Caco-2, and HT29glucell lines.7,23z39*44,46In

acterization colon,

of functional

primary

colonic

the identification

ST receptors tumors,

and char-

in normal

and

colon

human

carcinomas

the present

metastatic to the liver and lung. This is the first report of the presence of ST receptors in human colon carcinomas in vivo and high-affinity intestine.

Affinity showed

and metastatic obtained with

ST receptors

cross-linking

specific ST-binding

tide-dependent

of labeled

fashion

and metastatic

in primary

and adenine

colon carcinomas

prepared

nucleofrom

with an efficacy

and potency similar to those in normal intestine. In contrast, normal liver and lung did not show ST binding, specific ST-cross-linked proteins or ST-activated guanylate cyclase. These data indicate that ST receptors that are functionally and structurally similar to those in native intestinal cells are present in primary and metastatic colonic tumors tissues.

but not in normal

human

studies,

from human

ST-activated

A

B

C

-t

-t

-t

D

intestine

and human

guanylate

cyclase

in

E

F

G

-t

-t

-t

ST to mem-

proteins

in membranes

obtained

in human

colon tumors that were similar to those normal intestine. ST activated guanylate

cyclase in a concentration-dependent primary

of ST, with con-

in the present studies that

the binding

sites in membranes

human

of data

assays with these cells in vitro

centration

of radiolabeled

and

it was sug-

suggested ment

pro-

sites with a Kd of 1.9 nmol/L

lytes. ST has been shown to activate

branes

of 75 fmol/mg

and

obtained

in membranes although

suggesting

ST induces intestinal secretion and diarrhea presumably by binding to a transmembrane protein-receptor

ST had no effect on this enzyme in the presence or absence of adenine

binding

of 0.75 pmol/mg

in T84

in rat intesti-

isotherms,

sites with a Kd of 21 pmol/L

tein and low-affinity

normal of intestinal

results.1’3X23-27332,36V43 binding

yielded curvilinear

high-affinity

low-affinity

served in all specimens

previous

analyses of equilibrium

from competitive 1.2 ? 0.2

with

1657

extraintestinal

Identification of high- and low-affinity binding sites for ST in normal human colon and colon tumors are

116 : 49 37

ST

-+

figure 2. Cross-linking of 1251-STto membranes from normal human colon, colorectal tumors, and T84 cells. Cross-linking was performed using 1 mmol/L disuccinimidyl suberate in the absence (-) or presence (t) of 1 pmol/L unlabeled ST as described in Materials and Methods. Samples were analyzed by SDS-PAGE on a 4% - 15% gradient polyacrylamide gel in the presence of reducing agent. Similar amounts of membrane protein were placed in each well (loo- 125 pg of protein). The resulting gel was dried and exposed for autoradiography. (A) Normal colon, (B) primary carcinoma of the colon, (C) normal liver, (D) colon carcinoma metastatic to liver, (E) normal lung, (F) colon carcinoma metastatic to lung, and (G) T84 cells. Molecular weights (kilodaltons) are indicated at the far left of the figure. Arrows identify the position of the bands of interest.

1656 CARRITHERS ET AL.

GASTROENTEROLOGY Vol. 107, No. 6

40

i2 x 2 >

30

21 U/I

: F 2

n

JK 20 00

12

10

0

6

!

!I

00

4

-0.2 0

1

12

10

8

0.4 ST

6

1.

ii;"

4

240C

60

E 0

1200

WI 0 00

10

8

6

4

00

10

0

6

4

[,

-II 00

10

8

6

4

Figure 3. Guanylate cyclase activation by ST in membranes of normal human intestine, colorectal tumors, and T84 cells. Experiments were performed as described in Materials and Methods. Membranes were prepared from nomlal and colorectal tumors, aliquots (50- 100 ug of protein) were incubated with the indicated concentrations of ST, and guanylate cyclase activity was determined (pmol of cGMP produced per of ST activation of guanylate cyclase (0) analyzed by minute per milligram of protein). insets in A and 8 show the concentration-dependence the method of Hanes to determine K, values. 5g Results are representative of four or more experiments (0). (A) Normal colon, (B) primary carcinoma of the colon, (C) colon carcinoma metastatic to liver, (D) colon carcinoma metastatic to lung, and (E) T84 cells.

membranes isolated from normal human colon, T84 cells,

the suggestion

and primary and metastatic colon carcinomas (Figures 3 and 4). Activation was concentration-dependent and

coupled to guanylate cyclase activation in a manner similar to those expressed in native intestinal cells are present

saturable, and half-maximum activation was achieved with approximately 1 O-’ mol/L ST. Also, adenine nucle-

in primary and metastatic colonic tumors in vivo. The presence of two functionally distinct populations of binding sites for ST and the heterogeneity of affinity-

otides have been shown to regulate basal and ligandstimulated particulate guanylate cyclase.37,41 In the present studies, ATP potentiated ST-activated guanylate cyclase in normal human colon, primary and metastatic colon tumors, and T84 cells. Although ATP stimulates guanylate cyclase activity in normal liver and lung membranes, ST has no effect on enzyme activity in these tissues. These characteristics of ST activation and adenine nucleotide potentiation are in close agreement with those published previously for guanylate cyclase in intestinal membranes prepared from animals, humans, and colon carcinoma cells in vitro. 2-'.23.25&39,*5 These data support

that ST receptors that are functionally

labeled ST binding proteins suggest the presence of different receptors for ST in intestinal cell membranes. However, a single complimentary DNA-encoding STreceptor binding activity (guanylate cyclase C), which is a member of the peptide receptor guanylate cyclase family of proteins, has been identified in rat and human Recombinant ST receptors exintestinal ce11s.33~36Y37~40-42 pressed in COS-7 or human kidney 293 cells yield multiple toxin-binding proteins when they are affinity-labeled with 1251-ST.36,37The molecular weights of these affinitylabeled proteins closely compare with those obtained

5. COLI TOXIN RECEPTORS IN HUMAN COLONIC TUMORS

Deoember 1994

with

receptors

from

native

intestinal

membranes

as

organs

in the North

American

1659

opossum. 49,50 Northern

shown by the present study and others.3B24-27*30These data support the suggestion that a single receptor for ST

blot analyses showed steady-state levels of ST-receptor guanylate cyclase messenger RNA in rat and bovine small

is expressed

in normal

cells, which

yields

intestine, T84 cells, and rat colon’9*33,51 but absence from rat adrenal, brain, heart, kidney, liver, lung, and testis.33

intestinal

ST-binding

neous structure

and function.

ST receptors

seem ideally

for the diagnosis

and colon

and

proteins

carcinoma

with

heteroge-

In contrast, suited

treatment

as molecular

targets

of colorectal

tumors.

studies

complementary

using

DNA

cyclase C messenger

polymerase

cloning

chain

suggested

reaction

RNA may exist in rat adrenal,

and olfactory

tine

Polymerase chain reaction and cloning are sensitive techniques for detecting the expression of proteins in tissues;

are joined

against nents

by tight

the exchange

junctions

of intestinal

in the circulation.‘2-‘4

on mucosal tion.

cells isolates

Introduction

should

that contents

Thus,

of ST into

and compo-

their apical location

ST receptors

not affect intestinal

form a barrier

from the circula-

the systemic

receptors

they are

isolated from this toxin by the mucosal barrier. Therefore, ST receptors cell-surface

seem to fulfill determinant

ficity. In addition, nicity

that

contrast

ST is a small peptide

elicits

only

to murine

nogenicity

should

over protracted geting. Although

the criteria

a weak

monoclonal permit

periods

functional

tumor

antibodies.48

in patients ST receptors origin

requiring

response

in

Low immuand

such tat-

have been detected

in placental

in epithelial

speci-

with low antige-

immune

ST to be used repeatedly

only in cells of intestinal they have been detected

for a targeted

with near-absolute

however,

mammals,

cells from various

that in placental

evidence

functional

ST recep-

(Waldman, could

not

with

5 X

for 90 minutes

by 40%.52 However,

in this tissue

cells

to decrease proximal

of guanylate

ST receptors

mucosal

tissues.

of rat kidneys

reabsorption

by amplifi-

by intestinal

lo-’ mol/L ST was reported toxin activation

cells

mucosa.”

biochemical

mammals,

primarily

rather than by extraintestinal

sodium

epithelium tracheal

can be complicated

The overwhelming

tors are expressed Perfusion

airway

cells; and bovine

this sensitivity

cation artifacts. suggests

circulation

because

and T84

human

brain,

These receptors are localized to apical membranes, facing the intestinal lumen.9-11 Mucosal cells lining the intes-

in vitro

mucosa;

or

that guanylate

ST binding

cyclase cannot unpublished

be detected

Northern

analysis, polymerase

mentary

DNA

or

be detected

data).2,6p’0 Also, in this

chain reaction,

cloning. 19*33These

tubular

results

tissue

by

or complesuggest

that

the observed effects of ST on renal tubular sodium reabsorption may not have been mediated by receptors for ST similar to those localized in normal colon or colorectal tumors. The existence

15

nal tissues localizing

E I

of receptors that bind ST in extraintesti-

of placental

mammals

sites of production

may be suggested

of endogenous

peptides

by for

these receptors. Recently, two newly discovered endogenous peptides, guanylin and uroguanylin, were extracted from rat jejunums3 and opossum urine,54 respectively. These low-molecular-weight peptides mimic the action of ST, and together

they

form

a peptide

family

that

activates intestinal particulate guanylate cyclase and stimulates cGMP accumulation, inducing Cl- secretion.32VJ3-5*

3

0

A

B

C

D

B

Pigum 4. Effect of ATP and ST on guanyiate cyclase activity in membranes prepared from normal human tissue, colon tumors, and T84 cells. Guanylate cyclase activity was determined in the presence or absence of maximally activating concentrations of ATP (1 mmol/L) with and without 1 umol/L ST, as described in Materials and Methods. Basal activities in these experiments were (pmol of cGMP produced par minute per milligram of protein): (A) normal colon, 12.8; (6) primary carcinoma of the colon, 21.8; (C) normal liver, 25.2; (D) colon carcinoma rnetastatic to liver, 30.1; (5) normal lung, 3.8; (F) colon carcinoma metastatic to lung, 5.1; (G) T84 cells, 312.2. Fold activation of guanylate cyclase was calculated as the ratio of enzyme activity in the presence or absence of ATP and ST. Results are expressed as the mean -C SE of three or more experiments.

Messenger RNA encoding guanylin has been detected in humans only in the intestine.55 Also, receptor binding, stimulation by guanylin

of guanylate

cyclase, and increases in cGMP

have been observed

in placental

mammals

only in rat intestinal brush border membranes and cultured human colonic cells.32~53-s5~57~5s Furthermore, guanylin and uroguanylin are lo- 1 OOO-fold less potent than ST in binding receptors and stimulating cGMP accumulation.32353-S5,57,58Limited expression of guanylin only in the intestine supports the suggestion that expression of functional ST receptors is limited to intestinal mucosal cells in humans.

1660

CARRITHERS ET AL.

In summary,

GASTROENTEROLOGY Vol. 107, No. 6

the present

studies

show that

human

II. Almenoff JS, Williams SI, Scheving LA, Judd AK, Schoolnik GK. Ligand-based histochemical localization and capture of cells exPressing heat-stable enterotoxin receptors. Mol Microbial 1993;8:865-873.

colonic tumors express ST receptors with structural and functional characteristics that are similar to those receptors expressed

in normal

mary and metastatic

colonic epithelial

tumors

examined

ing receptors of high (pmoliL)for this toxin

with

receptor

to those in normal not detected

colon.

In contrast,

ST binding

tumors

were not detected

in extraintestinal

pro-

that were similar

to those in normal colon, whereas toxin-binding guanylate examined

proteins

tissues. ST activated

cyclase in membranes from all colon tumors with an efficacy and potency that were similar

to those in normal this enzyme characteristics

colon. However,

in normal

extraintestinal

ST did not activate tissue. Functional

of ST receptors in colonic tumors

lar to those reported

previously

including

ligands

peptide

for targeting and

monoclonal

are simimolecules, antibo-

dies.‘8-21 These data suggest that ST may be useful for targeting diagnostic and therapeutic agents to colorectal tumors.

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Received January 31, 1994. Accepted July 15, 1994. Address requests for reprints to: Scott A. Waldman, M.D., Ph.D., Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Thomas Jefferson University, IIOD Walnut Street, MOB-813, Philadelphia, Pennsylvania 19107. Fax: (215) 9555681. Supported in part by grants from the W. W. Smith Charitable Trust, the National Science Foundation (IBN-9205717), and the National Institutes of Health (1 R55 DK43805). Stephen L. Carrithers was the recipient of a National Institutes of Health Postdoctoral Fellowship (1 F32 CA6376401). Scott A. Waldman was the recipient of a Pharma ceutical Manufacturers’ Association Faculty Development Award in pharmacology and toxicology. The authors thank Andrew Patrinellis for expert technical assistance and statistical analysis, Dr. Ray Urbanski for helpful comments, and the members of the Department of Surgery of Thomas Jefferson University for their cooperation and surgical specimens. Surgical specimens were kindly provided by Drs. Francis E. Rosato, Herbert E. Cohn, Jerome J. Vernick, Donna J. Barbot, Vincent T. Armenti, and Frederick R. Armenti.