Stimulation of virus-induced fusion of ehrlich ascites tumor cells by 3′,5′-cyclic AMP

Vol. 67, No. 1, 1975

STIMULATION

BIOCHEMICAL

OF VIRUS-INDUCED

AND BIOPHYSICAL

FUSION

BY 3’. 5’ -CYCLIC

RESEARCH COMMUNICATIONS

OF EHRLICH

ASCITES

TUMOR

CELLS

AMP

Kazunori Ohki, Satoshi Nakama and Akira Asano for Protein Research, Osaka University, Osaka 565, Japan and Yoshio Okada Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan

Institute

Received

September

lo,1975

SUMMARY: HVJ(Sendai virus)-induced fusion of Ehrlich ascites tumor cells was found to be stimulated by treatments which increase the intracellular level of cyclic AMP. This stimulation was optimal at an external concentration of Ca* of about 0.5 mM. During the process of cell fusion, the intracellular concentration of cyclic AMP was increased with a maximum at 2 min after the initiation of the fusion reaction. Evidence is also presented which suggests that the increase of the cyclic nucleotide is a part of control mechanism of HVJ-induced fusion of eukariotic cells. Thus, this cyclic AMP-stimulated process could be one of the step(s) requiring ATP for the overall fusion process of the tumor cells. and Gaff both of which are necessary

Membrane variety

fusion

is an important

of life phenomena

(1.2)

the mechanisms

regulating

of such control

mechanisms,

cells,

first

described

of its high efficiency, information

other

fusion

membrane

fusion

HVJ*(Sendai

the ease with which

and similar

membrane

and has recently

virus

-induced

requirements processes

cell

AMP acts as an intracellular

that in some

cases cell excitation

which

been extensively

are not yet well

fusion

is accompanied

For studies

of Ehrlich

ascites

system

and the wealth

Ca*

established, of membrane by increases

in a wide

(1). However,

tumor

because

of accumulated

has been shown to require

for ATP and/or

mediator

studied

experimental

it can be assayed,

involved

understood.

fusion

an excellent

(6). It is well

3’, 5’ -cyclic

process

virus)-induced

by Okada (3), provides

on it. This

and Ca*(4,5)

physiological

both ATP

have been reported on the other -linked

hand, events

in both cyclic

* Abbreviations: HVJ, hemagglutinating virus of Japan; PSS, balanced HAU, hemagglutination unit; &IX, 3-isobutyl-l-methylxanthine

for that (6) and

AMP

salt so!‘ltion;

Vol. 67, No. 1, 1975

synthesis

from

BIOCHEMICAL

ATP and cellular

regu!atory

function

of cellular

proteins(7).

AMP plays

a role in the process

reagents cell

known

fusion

of cyclic

AND BIOPHYSICAL

uptake

of Ca *(6).

AMP is expressed

These

to increase

findings

This level

phosphorylation

the possibility

communication

of cyclic

Ca +t is required

and that extracellular

known that the

the ATP-linked

to us to suggest

of cell fusion.

the intracellular

It is also well

through

seemed

RESEARCH COMMUNICATIONS

that cyclic

reports

AMP enhance

that HVJ-induced

for this stimulation.

Ehrlich ascites tumor cells were grown in ddo mice, MATERIALS AND METHODS: harvested, and washed as described (8). The washed cells were suspended in a balanced salt solution (BSS) consisting of 0.14 M NaCl. 54 mM KCl, 0.34 mM Na2HP04, and 0.44 mM KH2P04 and 10 mM Tris-HCI buffer (pH 7.6). HVJ, Z strain, was cultivated in embryonated eggs, purified and suspended in BSS. The dose of the virus was expressed in terms of its hemagglutination unit(HAU) which was determined by Salk’s pattern method (8). Cell fusion was measured as follows. A desired dose of the virus was added to the tumor cells suspended (to a desired density) in BSS containing 2 mM CaC12 (final volume, 1 ml) at O°C and the mixture was allowed to stand for 15 min in ice to complete the virus -induced aggregation of the cells. The fusion reaction was then started by raising the temperature to 37’C and terminated after 15 min of incubation by cooling rapidly in an ice bath. The number of cells was counted before and after the experiment by the method of Okada and Tadokoro (9). Since one fusion event results in the decrease of cell number by one, the efficiency of cell fusion was expressed as “fusion frequency”. in cell number. For determination of the intracellular level of --i. e. percent decrease cyclic AMP, the cells were sedimented from 2 ml of suspension after rapid chilling and with 3 M K2C03 and extracted with 0.4 ml of 10 % HC104. The extract was neutralized the resulting KC104 precipitate was removed by centrifugation. The cyclic AM content in the supematant was determined by competitive binding method (10) using [ BHI-cyclic AMP as competitor. A bovine adrenal extract prepared as described by Brown et -- al.(ll) was used as a cyclic AMP -binding protein preparation.

RESULTS ascites

AND DISCUSSION: tumor

cells

phosphodiesterase cyclic phylline

cyclic tions

was enhanced (12),

AMP -dependent

AMP.

3 -Isobutyl-1

other

effective

reagents

-methylxanthine

inhibitor

Brief

to obtain

(such as 0. 5 mM) than theophylline,

a far more

1, virus-induced a well-known

similar

events(13).

was needed

with several

in Fig.

by theophylline,

at concentrations cellular

( 5 min at 4’C)

was observed

As shown

of cyclic

inhibitor

of Ehrlich of cyclic

to those used for studies preincubation

maximal

stimulated as expected

cell

from

AMP phosphodiesterase(

with theo -

Similar

stimulation

the intracellular

content

fusion

AMP

of other

of the cells

activation.

known to elevate

332

fusion

at much lower

concentra

the fact that the former 14). Although

of

these

is

Vol. 67, No. 1,1975

BIOCHEMICAL

80

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

:/-r_ll l

60

L

I 0

2

6

Theophylline I

I

0

10

koncentrdion

&rbaAylchoiine

CmM) I

C&en&ion

;X10%4)

Effect of theophylline and carbamylcholine on HVJ-induced fusion of Ehrlich Fig. 1 ascites tumor cells, Cells suspended in ESS containing 2 mM Ca* (1.84 x lo7 cells in 1 ml) were preincubated with theophylline or carbamylcholine for 20 min then cooled before addition of 1,000 HAU of the virus. Fusion frequency was determined as described except that theophyllnie or carbamylcholine was in “MATERIALS AND METHODS”, added as indicated, M, with theophylline, @---@, with carhamylcholine.

inhibitors

have been shown to inhibit

unlikely

that the increase

because

carbamylcholine,

inhibitory

rather

i.n cyclic which

enters

the cells,

cyclase

in thyroid

The stimulatory

effects

concentration

of cyclic

course

fusion

AMP,

reaction

by raising

the content

AMP content

the temperature

(Fig.

were

again

caused

333

cyclic

the virus-induced

concentration

by an increase

stimulation

AMP,

was which

2min

cell

aggregates

fusion.

in the intracellular during

As shown in Fig. after

adenyl

dependent.

was measured

of 10 mM theophylline.

of the virus-cell

it was

has been shown to activate

of the nucleotide

was maximal

(12),

GMP content(l5),

1). Dibutyrl

E2, which

was really

in the presence

was found that the cyclic

the cyclic

also found to enhance

of these reagents

GMP as well

for the observed

to increase

to cell fusion

were

To see if the stimulation

of cell

is known

and prostaglandin

cells(l3),

of cyclic

GMP was responsible

than stimulatory

readily

the hydrolysis

the initiation to 37’C.

the 2, it

of the fusion This time

Vol. 67, No. 1, 1975

BIOCHEMICAL

01

I 0

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I 2

4

14

16

I9

20

Intracellular level of cyclic AMP during the cource of HVJ-induced fusion Fig. 2 of Ehrlich ascites tumor cells. Preincubation and agglutination with HVJ were performed as described in the legend of Fig. 1 except that 10 mM theophylline was included. The fusion reaction was started by rapidly raising the temperature to 37’C, and the intracellular level of cyclic AMP was determined as described in “MATERIALS AND METHODS”. Vertical bars in the figure indicate range of the nucleotide concentrations were also made two times for each obtained from two separate runs, determinations of the sample. Average of four determinations was shown as points. Cyclic AMP content of untreated cells in this paticular experiment was 56 picomoles/mg of cellular protein.

point

was therefore

of several cyclic

chosen

treatments

on the cyclic

AMP at a fixed

theophylline

of the virus

added,

all effective

in elevating

Since

up to 2,000

extracellular

(6), the effect

was examined.

fusion

in the absence

effect

of theophylline

AMP was almost

HAU/ml.

Ca

+t.

in table

was again

equally

elevated

for several

I, theophylline

and the stimulation

concentrations, abolished.

to assess

proportional

the effects

during

334

E2 were

the fusion

reaction.

cyclic

AMP -mediated

-induced

stimulation

of the cell

caused

no stimulation

of cell

was maximal

at a Ca

such as 5 to 10 mM,

Since the intracellular

by theophyllnie

of

to the amounts

IMX and prostaglandin

AMP content

on the theophylline

of added Ca* At higher

was almost

cyclic

IS required

designed

It was thus found that the increase

Theophylline.

the intracellular

As shown

tion of about 0.5 mM.

AMP level. concentration

concentration

of Ca*

fusion

to be used in experiments

regardless

processes

i-t

concentra

the stimulatory

concentration

of the presence

of cyclic or absence

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

Table Effect

of Ca*

concentrations

I

on theophylline

Ca+ concentration ( mM)

Cyclic (picomoles/mg. + theophy.

0. 0

RESEARCH COMMUNICATIONS

stimulation

of HVJ-induced

Fusion frequency with theophylline ( % of control)

AMP protein) - theophy. 133

250

cell fusion

95 111

0.2 0. 5

283

83

169

2. 0

251

125

131

267

133

100

10.0

Fusion was performed in Tricine buffered medium [ 135mM n’aC1 5.4 mM KC1 and 40 mM Tricine-NaOH buffer (pH 7.8)(24)] instead of IES to avoid precipitate formation at higher Ca* concentrations. The dose of HVJ was decreased to 400 HAU in these experiments, since HVJ at higher doses caused lysis of cells at low Ca* concentrations. Therefore, the effect of 10 Fusion frequency differs at different Ca* concentrations. mM theophylline on the fusion frequency was compared with that without it at the same The fusion frequency without Ca* addition was 46 g of that with Ca++ ““Y-7 ntration, . 2 mM Ca in thts particular experiment.

of Ca*(Table

I) in accordance

that extracellular

Ca

that is activated Although agglutination the overall addition

+I

participates

by cyclic

reactions

of the tumor

cells

process.

of theophylline(measured

its action

at a later

observed

that HVJ-induced

reports

in a partial

(K. Ohki,

the lack of a requirem

23).

Therefore,

it seems

step of the over

of virus-induced by the virus Since this

an increase

cell

is thought

reasonable all cell

to assume

fusion

process

unpublished

step of

step was not appreciably

influenced

by the

it is clear

process.

and fusion

ent for ATP in the fusion cyclic

335

that cyclic

In preliminary

of human

observation).

of intracellular

are not yet established, the initial

turbidometrically).

hemolysis

fusion

to represent

step or steps of the whole

with

(6,16).

AMP.

partial

cell fusion

by theophylline

with other

experiments

erythrocytes

These reaction

results

AMP exhibits

were

of human

we

was not affected in accordance erythrocytes

AMP may not be the initial

signal

(22, for

Vol. 67, No. 1, 1975

the fusion cells,

event,

BIOCHEMICAL

but might

and thus enhance Recent

studies

liliaceous

plant

cells

possess

the ability

stimulate

the overall of the fusion

(19) strongly

to fuse spontaneously

mechanism

of cell fusion

could

of the cell membrane

cytoplasmic participate findings

components,

parts

the addition

AMP action.

AMP and Gaff

(22),

bilayer

protease

are interesting

Thus,

becomes

and may indicate is to pinpoint

to a form

possible. (20,21),

Some may

In this regard,

by an inhibitor (22),

while

of

distribution

structure

and microtubules

inhibitors

bilayers

the control

molecular

of cap formation.

of

that the composition

the membrane

was inhibited

One of our next problems

’m this complex

of eukaryotic

of phospholipid

provided

that affects

and modifies

as in the c&e

and by several

of colchicine

a process

system

process

(18) and of protoplasts

are fulfilled.

of the phospholipid

that the same cell fusion D (22),

vesicles

to each other

such as microfilaments

in these processes

cytochalasin

of cyclic

of fusable

of the fusion

that bare membranes

requirements

involve

RESEARCH COMMUNICATIONS

process.

suggest

and other

contact

reaction

of phospholipid

is appropriate

in which

a partial fusion

the bilayer

on the surface

AND BIOPHYSICAL

recent

of microfilaments, it was enhanced

several

possible

the sites of action

by points

of cyclic

system.

ACKKOWLEDGEMENTS: We would like to thank Prof. Ryo Sato of our laboratory for his helpful discussions and Dr. S. Kakiuchi of Nakamiya Psychiatric Hosp’tal for his valuable suggestions on the assay of cyclic AMP and for kindly providing [ BHI-cyclic AMP and IMX.

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BIOCHEMICAL

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RESEARCH COMMUNICATIONS

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