Calmodulin and cell proliferation

Vol. 104, No. 2, 1982 January 29, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 451-456

CALMOOULIN AND CELL PROLIFERATION Yasuharu Department

Received

Sasaki

and Hiroyoshi

Hidaka*

of Pharmacology, Mie University Tsu 514, Japan

December

4,

School

of Medicine

1981

The calmodulin content of synchronized Chinese hamster ovary (CHO-Kl) The calmodulin content cells was determined at each phase of the cell cycle. was minimum in the Gl phase, increased after the cells entered S phase and reached the maximum level at the late G2 or early M phase. When 30 pM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G2 or early M phase. The addition of W-7 also prevented the These results suggest that morphological changes caused by cholera toxin. calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis. INTRODUCTION Calcium

ion

cell

processes

cell

adhesion

glants

(6,

(4),

with

(5),

and change

versatile

Ca

in the

In order in cell

division

* To whom all The abbreviations

of DNA synthesis to further cycle

define and change

correspondence used are:

secretion (8,

in endocrine 9).

All

(2,

or some these

Means and collaborators

found

that

a ubiquitous

calmodulin

we found

that

cells

levels

requests

in culture

(10,

may be involved

(12).

between

in calmodulin

using

the assemblyldis-

calmodulin

cells

relationship

and reprint

of

and

may be associated

and may regulate apparatous

3),

and exocrine

protein.

in cultured the

of DNA synthesis

calmodulin,

in the mitotic antagonist,

of various

involving

microtubules

a calmodulin

initiation

-binding

or modulation

initiation

states

technique

of microtubules

Using

2+

regulation

exocytotic

mechanisms

the chromosome-to-pole

11).

(l),

in metabolic

immunofluorescence

assembly

in the

division

motility

may be regulatory

extraordinarily indirect

implicated

such as cell

7),

processes

has been

should

the cell of cultured

progression cells,

be addressed.

W-7, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide CHO, Chinese hamster ovary 0006-291X/82/020451-06$01.00/0 451

Cop.vrrghf 0 1982 b-v Academic Press, Inc. .A Il rrqh Is of reproducrion m anv form reserved.

Vol. 104, No. 2, 1982 we determined effect

BIOCHEMICAL

the

calmodulin

of calmodulin

hamster

ovary

AND BIOPHYSICAL

content

antagonist,

during

W-7 (13)

the

RESEARCH COMMUNICATIONS

cell

on the

cycle

cell

and examined

progression

the

of Chinese

(CHO-K,). MATERIALS

AND METHODS

Materials : N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide synthesized according to the methods of Hidaka et al. from Rikaken Co., Ltd. (Nagoya, Japan).

(W-7) was or was purchased

(13)

Cells and cell culture : CHO-Kl cells were cultured in Ham's F-12 medium supplemented with 5% fetal bovine serum at 37°C in a 5% CO2 humidified atmosphere. Synchronized cells were prepared in the followinq two ways. Mitotic cells were prepared by the minor modification of mitotic shake procedure by Terashima and Tolmach (14). Cells were cultured in F-12 medium supplemented with 5% fetal bovine serum until reaching a semi-confluent monolayer, and then the medium was replaced with F-12 medium supplemented with 10% - 15% fetal bovine serum. At an appropriate period, the semi-confluent monolayer was vigorously shaken to separate the mitotic cells. Higher concentrations of fetal bovine serum, up to 20% in the medium, were effective for mitotic cells to release and to float from the monolayer (15). In order to prepare the cells in S phase, the mitotic cells were treated with 2.5 mM thymidine for 14 hr and then incubated in a fresh medium without drugs for another 3 hr. The plating efficiency for CHO-Kl cells was more than 92%. Generation time for CHO-Kl cells in this experimental condition was ascertained to be about 13 hr. These cultures were also ascertained to be in the Gl phase of 6 hr, S + G2 phases of 6 hr, and M phase of 1 hr, using the combination method of pulse labeling-mitosis procedure and excess thymidine block-mitosis procedure, described previously (12). Cell number and cell size were monitored at appropriate periods with a Coulter Counter and Channelyzer, respectively. The concentration of W-7 required to suppress the cell progression was dependent on the cell density and the cell density was maintained constant in all of the experiments. Assay for the calmodulin : Cells were gently rinsed 4 times with cold phosphate buffered saline. detached with rubber policeman after addition of 2.0 ml of hypo-osmotic buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgC12, and 1 mM ethylene glycol bis(8-aminoethyl ether)-N,N,N',N'-tetraacetic acid and then sonicated. The sonicate was centrifuged after addition of 500 uq of bovine serum albumin and trichloroacetate (final 5.0%), and the resulting precipitate was dissolved in 1.0 ml of 0.15 M Tris-HCl (pH 7.5). An aliquot was subjected to calmodulin assay. Calmodulin was assayed by its ability to activate calmodulin-dependent phosphodiesterase partially purified from hog brain cortex

(16). RESULTS AND DISCUSSION The calmodulin was determined modulin

content

using

content

synchronized

was maintained

the mitotic

cells

and r‘eached

a maximum level

modulin

entered

are considered

DNA synthesis Recently,

in CHO-Kl cells cells at the

S phase, in late

to play

and the cell we reported

that

(Fig. lowest

the

1). level

phases calmodulin 452

of the cell

the Gl phase,

of 250 ng/106 content

G2 or M phase. role

phases

During

calmodulin

an important

division

at various

began

Calcium

ion

in requlatinq

of the cell antagonist

cycle

calWhen

cells. to increase or/and t!le

(5,

cycle

cal-

early 17).

W-7 suppresses

the cell

Vol.

104,

No.

2. 1982

BIOCHEMICAL

c

’

AND

Cl

*

*

2

.

-

/h’

RESEARCH

*

.

COMMUNICATIONS

GI

14

10 Plating

Calmodulin content of CHO-Kl cells at various phases of the cell division cycle. The method for the determination of calmodulin was described in the The cell size of CHO-KT cells increased according to the cell text. progression in the cell cycle, and two peaks in cell size population were observed at 14 hr after the cell release. Cell division was observed at 1.5 hr and at 14 hr after cell release.

Hour

Fig.

S+Gz

after

Hours

1

*

/

6

2

Fig.

BIOPHYSICAL

Effect of W-7 on the cells were incubated with 2.5 mM thymidine and reincubated in a 0 time (a,). with 30 uM W-7 (b.tt). of 30 PM W-7.

after

the

removal

of

TdR

cell cycle of CHO-Kl cells. 13.0 x lo4 Mitotic in 60 m!l plastic dishes containing F-12 medium without W-7 for 14 hr. The cells were washed fresh conditioned medium containing no drug at After 3 hr incubation, the cells were treated "c" indicates the time for the removal

453

Fig.

3

praoliferatio

Effect of W-7 on the morphology of CHO-K cells. All micrographs are 200x under phase-con 1 rast microscope. (A) No treatment; (8) treated with 30 PM W-7 for 26 hr; with 100 rig/ml cholera toxin for 26 hr; (D) treated with and 100 rig/ml cholera toxin for 26 hr.

In of

bo undary

or

Th lerefore,

early t .he

ex amined

in

CHO-K,

the

cells

S phase, effect

of

CHO-Kl

and a time

calmodulin cell

c ell

division

was

me dium

and

c ell

division

began

Hi gher

conce ntrations

of

W-7

the

cell

cycle

when

the

DNA

synthesis

antagonist

cycle.

3c 1 PM W-7,

that

When

not at were

2 hr

cells

until after

necessary 454

(W-7)

the

apparent

is

on in

W-7 this

to

blocked initiated

the

G2 or

of the

the

M phase treated

from W-7

late

G,

-s

(12)

were

removed

removal suppress

at

is

S phase

was

(C) treated 30 PM W-7

was I wi th

the

(Fiq.

2).

G2 or

M T)has e,

Fig.

compared suggest

with that

the case of the W-7 blocks

as at the GJ-S boundary CHO-Kl

cells

Mitotic plastic

cells

spread

substratum.

26 hr.

the

of the early

cycle

at the

Moreover, Gl phase

late

These

G2 or the M phase,

calmodulin

but is

S phase.

is not

involved

as well

essential

both

in the

results

for initiation

and in the mitosis.

30 JIM W-7 was added another

phase.

Continued.

suppression

the cell

to traverse

of DNA synthesis

3--

out

in the

first

3 hr following

attachment

At 3 hr after

plating,

100 rig/ml

cholera

to the medium,

and then

the

were

The control

CHO-Kl

cells 455

cells

(no drug)

assumed

toxin

to the or/and

incubated an epithelial

for form

Vol. 104, No. 2. 1982 with

some knobs

W-7, the

CHO-Kl

Similar

results

to 1.0 ,uM. the shape

BIOCHEMICAL on the cell cells were

obtained

with

of CHO-Kl

also

seen with

that

the

of CHO-Kl

3).

cells

from

cholera

affect cells

Following

with

PM W-7 abolished

toxin

(Fig.

3). not

of W-7 on the

This shown).

initiation

by disruption

form,

the

with

some knobs

cholera

to fibroblastic

Thirty

is mediated

treatment

at concentrations

and Puch (18),

epithelial

(data

RESEARCH COMMUNICATIONS

and compact

by Hsie

1.0 pM colchicine

inhibitory

(Fig.

colchicine

As has been reported

by 100 rig/ml

mitosis

surface

bacame more circular

as reverse-transformation. caused

AND BIOPHYSICAL

30 uM (Fig.

from

0.5 pM

toxin

alters

referred

3).

to

reverse-transformation

effect

induced

These

by W-7 was

results

of DNA synthesis or rearrangement

suggest and of micro-

tubules. ACKNOWLEDGMENTS We thank M. Ohara, Kyushu University for critical This work was supported in part by a Grant-in-Aid the Ministry of Education, Science and Culture, Japan

reading of the manuscript for Cancer Research trOm (1981).

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