Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

Vol. 122, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 16, 1984

Pages 1040-l

046

SELECTIVE ACTIVATION OF CYCLIC AMF' DEPENDENT PROTEIN RINASE BY CALCITONIN IN A CALCITONIN SECRETING LUNG CANCER CELL LINE

Zajac,S.A.

J.D. University

Livesey,

and T.J.

Martin

of Melbourne Department of Medicine Repatriation General Hospital Heidelberg, Victoria 3081 Australia

Received June 7, 1984 summary: The characteristics of the cyclic AMP-dependent protein kinase isoenzyme response to calcitonin have been studied in a calcitonin-secreting human lung cancer cell line (BEN). These cells secrete a high molecular weight calcitonin-like molecule. They have previously been shown to have calcitonin receptors In this study we demonstrate that linked to adenylate cyclase. the cells contain two cyclic AMP-dependent protein kinase isoUsing a recently reported method for studying selective enzymes. activation of these isoenzymes by hormones in intact cells,it is demonstrated that calcitonin causes selective activation of activation of isoenzyme II. isoenzyme I, with no significant Post-extraction activation was excluded by appropriate controls. The response was rapid (2 min) and efjisisted for 18 hours. Half M salmon calcitonin. maximal response occurred at 3 x 10

In mammalian cells cyclic

AMP action

protein

kinase

dependent kinase catalytic

involves

(1).

protein

isoenzymes

the major,

stimulation

kinase

isoenzymes,

are tetramers Cyclic

AMP binds

the enzyme to dissociate

subunit

(31.

if

not

Results

from several

different

functions

exclusively

the

the

catalytic

reside

regulatory the

subunit,

active

isoenzymes

that

The

and two

to the regulatory

in their suggest

(2).

AMP-

components.

isoenzymes

have

(4-7).

We have previously

reported

hormone activation

of

cells.

technique,

Using this

two cyclic

I and II

and release

between

studies

contain

types

mechanism of

AMP-dependent

of two regulatory

causing

Differences

only

of cyclic

Most mammalian cells

subunits.

predominantly

and probably

the

(8,9)

individual calcitonin

a method for isoenzymes

studying

in intact

has been shown to

Vol.

122,

No.

activate

3, 1984

exclusively

lines,

T47

that

calcitonin

a lung

adenylate high

of

isoenzyme

II

induces cell

weight

in In

(BEN). receptors

in

BIOPHYSICAL

these form

cells of

the

the

predominant

line

calcitonin

cyclase

molecular

MATERIALS

AND

D and MCF 7 (9,lO).

cancer

presence

BIOCHEMICAL

RESEARCH

human breast

present

activation We have

calcitonin

study of

which

cancer

cell

we report

isoenzyme

previously

and a calcitonin (11-131,

COMMUNICATIONS

reported

I in the

responsive also

secrete

a

(14).

AND METHODS

Hormones and chemicals: Synthetic salmon calcitonin was kindly supplied by the Armour Pharmaceutical Co., Kankakee, Illinois. Foetal calf serum and culture media were obtained from Grand Island Biological Company Ltd., stralia. Falcon plastic tissue culture bottles were used. [ y- 3s PI ATP was obtained from the Radiochemical Centre, Amersham, U.K. The synthetic peptide substrate Leu-Arg-Arq-Ala-Ser-Leu-Gly-NH2 corresponding to part of the phosphorylation site sequence in porcine hepatic pyruvate kinase, was synthesized as previously described (15) and was kindly provided by Dr B.E. Kemp. Phosphocellulose paper (P81) and microqranular DE52 ion exchange resin were products of Whatman Inc., Maidstone, U.K. All other chemicals were of reagent grade and from standard suppliers. Cell culture: Methods ror the maintenance of BEN cell cultures have bee described (11-13). BEN cells were grown to confluence in 25 cm9 tissue culture flasks in an equal mixture of Dulbecco's modification of Eagles' medium and medium 199 with 5% foetal calf serum, and penicillin and fungizone. Media were changed every 48 hours. T47 D cells were grown in RPM1 1640 with 5% foetal calf serum, 20 mM Hepes, gentamycin and minocysline (12). For protein kinase assays, cells were plated in 25 cm plastic culture dishes at least 48 hours prior to assay, each flask representing a single hormone concentration or time point. Cells were used as soon as they had reached confluence. Separation and measurement of CAMP-dependent protein kinase isoenzymes: The method used for studying the response of individual isoenzymes of CAMP-dependent protein kinase was as previously reported (E-10). Briefly, ceils growing in monolayer culture were exposed to hormone for 10 minutes, washed twice with phosphate buffered saline at 37OC and scraped into 10 ml vials containing column starting buffer (8-10). Suspensions were sonicated, centrifuged for 30 seconds at 12,000 q at and then applied to 2 ml columns of DE52, which had previously been equilibrated with starting buffer at 4OC. After loading, the columns were washed with a further 100 ml of starting buffer at 4Oc. The DE52 columns were eluted with a NaCl gradient (O-O.6 M) in starting buffer with a simultaneous NaCl gradient applied across 7 columns. 28 fractions of approximately 1 ml were collected. Kinase activity was corrected for measured fraction size. Assay for cyclic AMP dependent protein kinase activity in the presence and absence of cyclic AMP was performed (E-10). Kinase activity is calculated as pmol of ATP transferred per minute per 25 p-11aliquot of eluate. Total kinase isoenzyme activity is determined by integrating the area under the curve 1041

Vol.

122,

No.

3, 1984

BIOCHEMICAL

using the "trapezoidal II. Percentaqe kinase follows -

AND

BIOPHYSICAL

rule" to calculate isoenzyme activation

determined determined

isoenzyme isoenzyme

RESEARCH

area is

COMMUNICATIONS

for peaks expressed

activity activity

I and as

(control) (sample) x 100

determined

isoenzyme

activity

(control)

RESULTS Both and

T47 D cells

II)

of

mammalian resulted in

cyclic

the

In

partial in

activation

of

contrasts

with

cell

T47 D,

results

in

ation

of

Fig.

2.

point

similar contrast which

in

I was half

to

the

to

effect

that

seen

activation In any

such

the

cancer

study

of

cyclic to

necessary

activation

of

the

kinase

(10). equivalent

persist

exclude

taking

place

Control

Hormone-treated

cells of

untreated

of

experiments were

1042

but line

the

is

in in

(10). kinase of rupture.

together

The prior

performed

Post-cell

first

MCF 7,

possibility

were

course

This

immediately

processed

cells.

at

protein

after.cell cells

a time

18 hours.

6 hours

the

in

salmon

noted

cell

AMP-dependent

of

illustrated

T47 D cells

beyond

by dilution

(9,lO).

number

breast

not

is

sonication

at

in

did

minimized

present in

no activ-

1 shows was

calcitonin

it

is

Table

still

cancer

The degree

3 x 10-l"M

of

activation

problem

at

result

with

as

Activation

and was

reflected

breast

II

9,lO).

by 3 x lo-'M.

most

consistently

dependent,

maximal

is

This the

isoenzyme

dose

calcitonin.

This

1,2). of

1 and Refs.

in

calcitonin

I.

treatment of

found

(I

No significant

I. (Fig.

calcitonin

(Fig.

was

peak

isoenzymes

as to

isoenzyme

seen

activation I

kinase

to calcitonin

which

(2 mins), to

was

response

isoenzyme

response

of

two

exposure

of

II

and maximal

the

time

the

Activation

calcitonin of

isoenzyme

selective

of

BEN cells,

amplitude

isoenzyme

activation

the

contain

protein

activation

reduction

line

and BEN cells

AMP-dependent

cells. in

(9)

as with

rupture

to follows an activ-

vol. 122,

3, 1984

No.

BIOCHEMICAL

AND

I

, /’ /’ I /’ ,’-I’ -I,’ 10

20

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

NaCi (MI

1D

30

1L,

Figure

of human lung cancer 1. DE52 anion exchange chromatography cells (BEN) and human breast ancer cells CT47 D) after exposure to salmon calcitonin (3 x 10 -' M). DE52 column fraction from BEN cells (A) and T47 D cells (8) were assayed for cyclic AMP dependent protein kinase activity as described in Materials and Methods. Assays w re performed on control cells (x) and cells exposed to 3 x 10 -5 M salmon calcitonin (0). The interrupted lines indicate NaCl gradients.

Figure

2. Dose response of cyclic AMP-dependent protein kinase to salmon calcitonin in BEN cells. Cells were exposed to various concentrations of salmon calcitonin for 10 minutes, processed and assayed for protein kinase activity. Percentage activation of isoenzymes I (01 and II (0) was calculated as described in Materials and Methods.

ation the

would added

appear control

concentrations II

is

not

further

cells. of

in

evidence

that

Addition

sonication

step (data

not

resulted

did

not

(Table

the

cells

post-cell of

stimulation

This

calcitonin

activated

occurred.

and II

as artefactual

in

in

complete

shown). 1043

occur

2).

The

control

rupture

5x 10m7 M cyclic

of

the

even

at

fact

that

AMP to

cells of

in

high

experiments

activation

activation

enzyme

isoenzyme is

has

not

at

the

isoenzymes

I

Vol.

122,

No.

3, 1984

Table protein

1.

BIOCHEMICAL

Time course of kinase isoenzymes

AND

activation by salmon

Time

I

Isoenzyme

10 mins

89

9

60 mins

69

8

6 hr

81

18

18 hr

58

11

24 hr

23

0

II

M salmon in monolayer culture were exposed to 3x10-' Cyclic AMPfor periods of 2 minutes to 24 hours. protein kinase activity was measured and percentage in comparison to control cells was calculated as in Materials and Methods.

2.

Assessment Added control cells

of post-extraction Added test cells

Determined kinase activity pmol/min

activation. Predicted kinase activity pmol/min

+

2.08

+

+

4.22

+

+ +

2.14 4.26

4.28

+

+ +

0.68 3.11

2.82

+

+ +

1.32 3.66

3.46

+

+ +

1.10 3.35

3.24

3 x 10-g

3 x lo-7

Activation

0

0

3 x 10-8

AMP-dependent in BEN cells

69

Table

3 x lo-l1

COMMUNICATIONS

2 mins

BEN cells calcitonin dependent activation described

0

RESEARCH

of cyclic calcitonin

Percentaqe Isoenzyme

Salmon calcitonin (Ml

BIOPHYSICAL

To assesspost-cell rupture activation, equivalent numbers of untreated BENcells were added to buffer used to dilute the test cells, and Cyclic AMP-dependent protein kinase sonicated simultaneously. activity was measured in the test cells in the presence and absence of control cells after exposure to several concentrations of calcitonin. Pretreated kinase activity was calculated by the sum of the activity of added control cells, and activity of test cells alone. Significant post-extraction activation occurs if the predicted value is greater than the determined value. Data for isoenzyme I only is shown, but results for isoenzyme II also exclude post-extraction activation. 1044

Vol.

122,

No.

3,

BIOCHEMICAL

1984

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Discussion Previous cells of

of

reports

a calcitonin

a secreted

(11-14).

extends

to

for

selective

breast suggests

that

mechanisms

of

may imply

of

it

may occur

T47

selective

system, results

suggest

to

hormonal

on both

the

hormone

response

with

In

the

BEN lung

isoenzyme protein the

I. kinase

diversification

In the

mediating several

AMP-dependent

have protein

the

can

the

cytoskeleton. isoenzymes

in

is

dependent

appears

that

the

pattern

isoenzyme

I or

cancer

cells,

breast

isoenzyme

cyclic

the

which

II,

AMP-dependent early

step

in

may be important on cells.

isoenzymes

influence

II,

predominantly

a crucial

hormones

that

of

cell

of

of

but

intact

activates

effects

kinase

of

hormone

action,

indicated

the

exclusively

hormone

the

components

of

may represent

in

and evidence

of

human

activation

differential studies

responses

cells

of

and

be determined,

of

activation

activates

isoenzymes

to

cell

It

kinase

The molecular

the

type.

cell.

Selective

that

(8),

activation in

i.e.

cancer

the

i.e.

activation

protein

participation

and cell

T47 D and MCF 7, calcitonin

demonstrated

remain

stimulation

target

this

selective

to calcitonin,

on the

Furthermore

(16-18).

in

by

AMP action,

D and MCF 7 (Q,lO),

widely

that

calcitonin

cyclic

osteoblasts

activation

perhaps

response

depends

in

BEN and

activation

AMP-dependent

occurs

lines

of

the

cyclase,

to

that

We have

cyclic

cell

related

kinase. I.

in

to adenylate

step

compartmentalisation

response

of

next

isoenzyme

by hormones

cancer

These

the

coexistence

demonstrates

protein

activation

isoenzymes

linked

study

AMP-dependent

selective

the

immunologically

The present

cyclic

described

receptor

product

calcitonin

is

have

Certainly

I and II

different

in

of

cyclic

cellular

(4-7).

The coexistence calcitonin-responsive

in

BEN cells

adenylate

of cylase

1045

calcitonin and cyclic

receptor, AMP-dependent

Vol.

122,

No.

3, 1984

BIOCHEMICAL

protein

kinase

has

been

documented.

but

investigation

now

related, the

genetic

synthesis the

with

the

disturbance of

components

calcitonin-like

related

areas

resolve

this

secretion It

the

BIOPHYSICAL

of

is

not

RESEARCH

clear

how

provide

useful

in

these

cells.

It

of

the

genome.

calcitonin

are

molecule

these

is

of

phenomena

information not

response

determined

Study

COMMUNICATIONS

a calcitonin-like

might

molecule of

AND

known

BEN cell

about

whether

mechanism

genetically cDNA will

are

and

from help

question.

ACKNOWLELXXHENTS

This work was supported by grants from the National Health and Medical Research Council and the Anti Cancer Council ot Victoria. The excellent technical assistance of Mrs Sharyn Omond is gratefully acknowledged.

REFERENCES

1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18.

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