Presence of VIP receptors in a human pancreatic adenocarcinoma cell line. Modulation of the cAMP response during cell proliferation

Vol. 111, No. 3, 1983 March

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

29, 1983

Pages

958-963

PRESENCE OF VIP RECEPTORS IN A HUMAN PANCREATIC ADENOCARCINOMA CELL LINE. MODULATION OF THE CAMP RESPONSE DURING CELL PROLIFERATION A. Estival,

P. Mounielou, V. Trocheris, J.L. Scemama, F. Clemente, E. Hollande and A. Ribet

INSERM U 151, Received

February

CHU Rangueil

Bat L3 - 31054

Toulouse,

France

7, 1983

It is known that the human exocrine pancreas responds to secretin stimulation more than does VIP, a structurally related peptide. We looked for the receptors for those polypeptides in a human pancreatic cancer cell line.grown in culture and in nude mice. By analysing the CAMP responses and the I25IVIP binding we found VIP receptors with a KD of 1.5 10-g M. Secretin stimulates the adenylate cyclase through the VIP receptor sites with a KD of 1.7. 10-6 M. We noted also that during cell proliferation in culture there was about a 5 fold increase of the CAMP response to VIP. It is known that pancreas

in

regulating

proliferation of the

creatic

the

to induce

pancreas cells

(2).

of

the hormone

cancer

may permit cells.

cyclic

nucleotide

We analyse

established

known for

of the cyclase

(Swiss

juice

secretion

In spite

cancer

which

is

the

wether

regulation

with

of the growth new suited

showed

that

cause

on the

pan-

human or animal. and the differen-

ways of action secretin

responses

in an adenocarcinoma

paper

VIP receptors

the

cell

frequency

the fourth

hormones

dealing

one to find

but also

increasing

presently

gastrointestinal

We previously

in this

of

on the exocrine

on

and CCK are of the

human

on the same cancer

in culture.

The results data

may act

(1).

analysed

cancer

hormones

the pancreatic

of the

has been poorly

of this

pancreatic

able

only

the action

knowledge

tiation

not

pancreatic

by cancer, cancer

A better

gastrointestinal

and differentiation

human exocrine

of death

the

the

obtained normal

response

show important human pancreas

in cultured

differences and,

cells

after

furthermore

by comparison

show a modulation

plating.

MATERIALS AND METHODS A human pancreatic adenocarcinoma was heterotransplanted strain nu+/nu+) and after 15 passages from nude to nude,

0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

958

to the

in nude mice established

BIOCHEMICAL

Vol. 111, No. 3, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

in culture (3). Cultured cells were grown in 25 cm2 Nunc flaskHam’s FI2 (Gibco) medium supplemented with 10 % foetal calf serum (Gibco) penicillin G (60 pg/ml) streptomycin (100 rig/ml) and amphotericin B (0.25 pg/ml), at 37"C, in moist air containing 5 % ~02. Medium was changed every 48 hours and the cells were subcultured weekly by EDTA treatment (0.02 %). This cell line has a doubling time of 36 hs, is tumorigen in nude mice, and does not synthesize the secretory enzymes normally exported by acinar cells. For current experiments cells were harvested 8-10 days after plating. Before each experiment culture flasks were incubated 5 min in Ham's F12 medium containing 0.02 % EDTA. Cells were then mildly harvested with a rubber policeman. Detachement with 0.1 % trypsin or collagenase leads to lower CAMP responses and higher Cells were isolated from the tumors forEDTA concentrations were unnecessary. med in nude mice as previously described (2) ; the only modification was the replacement of collagenase by EDTA (0.02 %). In brief, tumor slices were incubated 15 min in Ham's F12 medium containing EDTA in a shaking bath (120 revolution/min). The medium was then filtered through 250 n nylon mesh and centrifuged 15 min at 19 gmax on a discontinuous gradient of albumin, to separate the cancer cells from the few macrophages present in tumor. Cell viability tested by the ability to exclude trypan blue was 90 %. 1251 labelled porcine VIP was prepared by the chloramine-T technique and the radiolabelled hormone isolated by high pressure liquid chromatography on Bondapak CI8 columns (Waters) using as solvent : 0.25 M triethylamine phosphate and a step gradient of acetonitrile 28-50 %, pH 3.4. The specific activity of I25VIP was about 900 Ci/mmole. Labelled I25I-VIP was shown to stimulate the enzyme secretion from guinea pig pancreatic acini as the native hormone. Binding studies were performed by incubating the isolated cells in F12 medium complemented with 1 % albumin (fraction V Sigma), antibiotics and 25 mM Hepes, pH 7.4 at 25“ C with I25I-VIP, the incubation mixture (300 ~1) contained about 30,000 cpm and 2.105 cells in the presence or absence of unlabelled VIP or secretin (Hoffman La Roche). At the end of incubations the unbound hormone was eliminated by centrifugating 200 ul cells through I ml medium containing 4 % albumin at 4" C. Pellets were counted for radioactivity in a gamma counter. The nonspecific binding (at 10-6 M VIP) was lower than I8 % of the total binding ( -3000 cpm/2.105 cells). To examine the CAMP response at 25"C, cells were preincubated I5 min in F12 medium containing I % albumin and 5 r&l theophylline (2). The hormone was then added and the incubation stopped 30 minutes later with cold methanol (4). The plateau value of the CAMP response was reached lo-15 minute after the hormone addition and remained stable for at least60 min. After centrifugation the supernatant was collected, methanol eliminated by lyophilisation and the CAMP measured by radioimunoassay (HEN). Each experiment was performed in triplicate and repeated at least three times. RESULTS a) Characterization Fig.

1 reports

cultured

cells

maximal

response

which

is

nucleotide

of the

the dose-response

after

stimulation

to VIP is

a gastrointestinal response

maxima1

response

response

curves

only

obtained

at high

obtained

curves

with

2.1O'g

the

CAMP accumulation

structurally

with cells

10-5

959

Secretin,

analogue

to VIP,

starting

from

M secretin.

isolated

from

in

at 25" C. The half-

M concentration.

concentrations,

reached with

for

VIP and secretin, with

hormone

is not yet were

VIP receptors.

10-8

Identical the

tumor

evokes

a

M. The dose-

grown

in

Vol. 111, No. 3, 1983

01

BIOCHEMICALAND BIOPHYSICAL RESEARCHCOMMUNICATIONS

11 10 9 8 7 _ Log M

6

0r2

5

12 11 10 9

8

7 6

,LogM

w-

* Effect of VIP and secretin on CAMP accumulation in cultured cells. arves ed cells were incubated for 30 min at 25'C in the presence of theophylline (5 mM) and VIP or secretin. Values from three separete experiments, each one performed in triplicate.

: Competitive inhibition of 1251-VIP binding to cultured cells by iiF an secretin. Cells were incubated for 45 min at 25°C in the presence of 125I-VIP and increasing concentrations of VIP and secretin. Results are expressed as the percentage of radioactivity specifically bound in absence of unlabelled peptide. All values are means of 4 separate experiments each one performed in triplicate.

nude mice, with

from which

cultured

obtained binding

cells

well

with

VIP competes inhibition for

is

very

to the 1.7.

During were

variable.

the

plateau

Binding

requirements

binding

values

1.2.

1). Fig.

for

10-g M VIP, 2 also

properly

a value

reports

For secretin

by secretin.

by the measure

performed

and reversibility

1251-VIP (Fig.

of

studies

125 I-VIP binding of

The saturability

one obtained

10-6

b) Modulations

the with

(Fig,

provoked

KD is

the basic

obtained

VIP binding

the

incubation.

with

CAMP formation

closed

was established.

at 25°C show that

a 1 hour

after fit

the culture

the also,

speak

2),

are of this

of receptors.

and the

half-maximal

comparable

to that

found

inhibition

of the

1251-

the competition

curve

of the CAMP accumulation,

is where

M. of the

course

We then

cyclic

of these looked

for

nucleotide studies

response

we noted

the responsible

960

that

maximal

parameters

CAMP responses and observed

5

BIOCHEMICAL

Vol. 111, No. 3, 1983

Table

AND BIOPHYSICAL

I : CAMP modulation CAMP

during

pmoles/106

growth.

cells

Stimulated

6

days

38.41 (S/B

15 days

that

VIP stimulations plating

(Table

after

plating,

the

levels

as high

(Table

I) there This

either

the

is

increasing

CAMP accumulation those

of the

kinetics

is very

6 days

in the nucleotide

; this

of a new type

Stimulations the presence

responses

basal

fact

the days

since

15 days

to basal

ratios)

reaches

In the same time

plating.

concentration

increase

does

during

important

after

is

of the CAMP accumulation

VIP and secretin

+ 0.96

(stimulated

found

no difference

modulation

appearance

M) evoke

4.18

basal ratios. 10-T M VIP in (n = 6).

Such an increase

as 5 times

of the

ves for

I).

3.80 t 1.27

212 t 12.50 (S/B : 50)

(10-T

after

Basal

t 11.90 f 10)

to S/B : stimulated were performed with of 5 mM theophylline

RESEARCH COMMUNICATIONS

of CAMP.

unrelated

or of the

not agree

with

to modifications dose-response

the

cur-

hypothesis

of

of receptor.

DISCUSSION Detailed

available

from

agonist

"in for

mal secretory secretin

event

if

viva"

response

and

moreover

the

responses

are qualitatively

data

lead

one to believe

from

the

it

is

cells

provoked

identical that

human pancreatic

(5)

lower

only

by

polypeptides : secretion VIP interacts adenocarcinoma 961

than

which

doses

not

show that

analysed

VIP is a

obtained

with

of

In

VIP.

receptors. here,

any

identical,

and bicarbonate.

secretin

are

The maxi-

quantitatively

of water with

Some data

to secretin.

that

higher

are

a structural

lacking.

in comparison

by VIP is

to both

are

investigations secretion

evoked

VIP and secretin,

for

pancreatic

clinical

the pancreatic

they

issued

on the receptors

of VIP, on human normal

analogue

poor

studies

resoond

These Cells to

Vol. 111, No. 3, 1983

VIP and secretin higher

; the binding

affinity

These

for

affinity

Or cancer favour

the

are

possessing

hypothesis

the dose-response

for

concentrations (6).

1 nM (8-9).

Besides,

cases

the

sites

explored

CAMP accumulation

after

there

are

red.

Preliminary

should

us to find

results

are

finding

of identical

not

disagrees

with

of cells

lacking

report

on the

results

an isolated

secretin presence

another lead

curves

during

of VIP receptors

for

VIP.

with

cells

knowledge,

in pancreatic

to that

in both

Moreover

maximal

doses

not

the

of VIP

additive

but

It seems then,

shown).

cell

line

explo-

cancer

one to think to that

To our

corresponds

cancer

the establishment

receptors.

secretin-receptor

human pancreatic

peculiar

dose-response

not

pancreatic

that

finding

selection

(data

unphysiological

have a KD of about

(10m6 M) is

in the

high

since

and therefore

with

do not

receptors

secretin

to those

stimulation

with

at

M.

human normal

Our results

secretin

by secretin

VIP alone

obtained

identical

the

(6,7).

for

dose of secretin

VIP receptors

in other

generally

curve

have a

: KD 1.7 10-6

non specific

receptors

correspond

with

found

begins

for

hormones

secretin

of specific

found

a combined

results

allowed

existence

of 12SI-VIP

obtained

only

for

to those

RESEARCH COMMUNICATIONS

these

VIP receptors

dose-response

M) and a non maximal

that

M than

close

secretin

the

the displacement

the values

very

lo-'

with

the CAMP formation

Specific

for

equals

explored

as generally

found

(lo-7

sites

specific

of the

curve

interactions

AND BIOPHYSICAL

VIP : KD 1.2-2

constants

cells

hormone

BIOCHEMICAL

that

pancreatic

the

from

in culture is

cancer

present

cancer.

isolated

this

line

The

tumors

of a clone the first

either

in human

or in animal. The VIP receptors AMP response, that

the

lated. the

amplitude

the

et al with

VIP.

nucleotide

only

response

when cells

response

(10) showed

at least

we analysed.

nucleotide

density,

of the

functional

parameter

of the

At low cell

Morera tion

level

the

are

is

then

response

to VIP is

to the cyclic

The experimental

are growing

in the

data

inhibition

be of interest

962

of the

to know wether

a phenotypic

cell cell

show is modu-

exponential

In a mouse adrenal

lower.

only

regard

due to the VIP stimulation

a dose-dependant

It would

with

line

phase, (Y - 1)

prolifera-

the modulation

expression

during

of cell

BIOCHEMICAL

Vol. 111, No. 3, 1983

proliferation

or if

the

receptors

AND BIOPHYSICAL

for

VIP are

somehow

RESEARCH COMMUNICATIONS

involved

in cell

proli-

feration. Acknowledgments We thank iodinated

Dr L. Pradayrol

for

labelling

and purification

of the

VIP.

REFERENCES F. and Bastie, M.J. (1981) Reprod. Nutr. 1. Balas, D., Senegas-galas, Develop. 21, 783-803. F. and Ribet, A. (1981) Biochem. Biophys. Res. 2. Estival, A., Clemente, Commun. 102, 1336-1341. 3. Hollande, E. In press. 4. Rosselin, G.,Freychet, P., Fouchereau, M., Rancon, F. and Broer, Y. (1974) Hormone Metab. Res. 5, 78-86. 5. Domschke, S., Domschke, W., Rosch, W., Konturek, S.J., Sprugel, W., Mitznegg, P. and Demling, L. (1977) Gastroenterology 73, 478-480. 6. Laburthe, M., Rousset, M., Chevalier, G., Boissard, C., DuPont, C., Zweibaum, A. and Rosselin, G. (1980) Cancer Res. 40, 2529-2533. 7. DuPont, G., Broyart, J.-P., Broer, Y., Chenut, B., Laburthe, M. and Rosselin, G. (1981) J. Clin. Invest. 67, 742-752. 8. Robberecht, P., Deschodt-Lankman, M., Lammens, M., De Neef, P. and Cristophe, J. (1977) Gastroenterol. Clin. Biol. 1, 519-525. 9. Jensen, R.T. and Gardner, J.D. (1981) Federation Proc., 40, 2486-2496. 10. Morera, A.M., Cathiard, A.M., Laburthe, M. and Saez, J.M. (1979) Biochem. Biophys. Res. Corrmun. 90, 78-85.

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