Transient increase in the c-fos mRNA level after change of culture condition from serum absence to serum presence and after cycloheximide addition in rat 3Y1 fibroblasts

BIOCHEMICAL

Vol. 159, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 501-507

March 15, 1989

TRANSIENT INCREASE IN THE c-fos mRNA LEVEL AFTER CHANGE OF CULTURE CONDITION FROIY SERUM AB%&!E TO SERUM PRESENCE AND AFTER CYCLOHEXIHIDE ADDITION IN RAT 3Yl FIBROBLASTS Atsuyuki Department

Okuda*,

Akinobu

of Virology, Kyushu

Matsuzaki

Medical

Institute

69,

Fukuoka

University

and Genki

Kimura

of Bioregulation, 812,

Japan

Received January 19, 1989

When 3Yl cells resting at a saturation density were mitotically stimulated with serum, the c-fos mRNA level markedly increased in a short period of time and then decreased rapidly to an undetectable level. Subsequent serum deprivation followed by serum re-addition or subsequent cycloheximide addition caused a transient re-increase in the c-fos mRNA level. These results can be explained by assuming that the continuous expression of the c-fos gene at a minimum level is necessary for the eventual initiation of S phase, and that the over-expression of the c-fos gene occurs when the control of the gene expression is transiently disturbed by the change of the culture condition. 0 1989Academic Press,Inc.

It

believed

is

density

are arrested

cycling

cells.

resting

cells

product

level

levels

*

cells,

the c-fos

however, cycle

(5).

as a mediator

mRNA is report,

following

from the Gl phase of transiently

with

growth

mRNA level

of the mitotic belief

that

at a saturation

shortly

factors

that

response

(6,7).

the transient from

we show that

the transient

increase

of resting

(1,2,3,4).

the c-fos

in the transition

serum stimulation

after In

is undetectable

involved

To whom correspondence

ABBREVIATIONS:

resting

There is evidence

led to the general

of c-fos

cells

increases

stimulated

functions

In this

mRNA level

are mitotically

the cell

observations

untransformed

in the "GO" phase distinct

The c-fos

proliferating throughout

that

cells

gene These

increase

in the

"GO" to Gl phase. in the c-fos

does not

link

mRNA

to the

should be addressed.

CR, cycloheximicie:

DEB, Xlbecco's

Modified

Eaqle's

medium.

0006-291x/89$1.50

501

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 159, No. 2, 1989

BIOCHEMICAL

transition

from "GO" to Gl phase.

starvation

of growth

factors

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

It

during

merely

reflects

the growth

the previous

arrest.

MATERIALS AND METHODS Cell culture. A clonal isolate (clone 1-6) of rat 3Yl-B diploid fibroblasts (8) (referred to as 3Yl) were used. Regular culture medium was Dulbecco's modified Eagle's medium (DEM) plus 10% fetal calf serum. All cultures were incubated at 37'C in a humidified atmosphere of 10% CO2/90% air. To prepare resting cultures, cells were plated at l/20 of the saturation density in plastic dishes, and incubated with the regular medium for 5 days (at this time cells became confluent) and then with DEM for another one day. Serum stimulation was performed by changing medium to DEM plus 20% serum. Total RNA was extracted from cells by the Detection of c-fos transcripts. guanidinium/cesium chloride method. Details of the procedures used were described previously (9). A sample of RNA (10 pg) was denatured and electrophoresed on a 1.5% agarose gel. After electrophoresis, RNA was transferred to a nylon filter (Hybond, Amersham, U.K.), :lybridized with nick-translated DNA probes, and exposed to an X-ray film at -8O'C. FBJ murine osteosarcoma virus v-fos (1.00 kb Pst I-Pvu II fragment) (code 7024, Takara Biochemicals, Kyoto, Japan) was used forthe detection of the c-fos mRNAs. Kinetics of entry into S phase. Cells were continuously labeled with i Hlthymidine (37 kBq/ml (1 Ci=37 GBq)) and dishes were taken out from the incubator at 2.5-h intervals and fixed for autoradiography.

RESULTS Resting

3Yl cells

medium with is

fresh

inhibited

medium containing

(CH)-containing

stimulated

with

interrupted

for

serum for

6 h (first

Progression

Resting

stimulation),

4 or 8 h by replacing

stimulation

(total

S phase increased

interruption

(Fig.

stimulation

required

stimulation, cells

1).

less than Therefore,

to enter that

cells

during

the period

excess

serum-lacking

was

or CH-

required

to

the case of no time of serum

than the time of the first

had not retreated

of each interruption. 502

were

the time of serum

3 h compared with

S phase was less

indicating

(11) or

then the stimulation

and second stimulation)

the total

S phase

was resumed by medium

In each case,

time of the fist

the

toward

3Yl cells

the medium with

medium, and then serum stimulation (second stimulation).

resting

S phase by replacing

serum (10).

medium (12).

replacement

enter

to enter

when the medium was changed to serum-lacking

cycloheximide

containing

are stimulated

to the state

of

BlOCHEMlCAL

Vol. 159, No. 2, 1989 a

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lOOr

L

I S*

V

S-

50

r

(6 h) IO h):. (4 h):A I8 h):A

I4 h):A (8 h):A

L

* S

II S

I

5 Total

,

S+ (6 h) + s+*cll IO h):.

time

of

exposure

to

fresh

10

15

alone

serum

1/ P

20

(h)

of intervening deprivation of serum, and of intervening Fig. 1. Effects of entry into S phase after stimulation of addition of CH, on the kinetics resting 3Yl cells with serum. Resting cells were tre ted as illustrated in each graph, and the cumulative labeling index with [ s Hlthymidine was determined as a function of the total time of exposure to serum alone. S+, incubation with fresh medium containing 20% serum: S-, incubation with fresh medium not containing serum: S+*CH, incubation with fresh medium containing 20% serum and 10 PM CH. The incubation period is indicated in parentheses.

The c-fos resting

mRNA level

3Yl cells

well-known

in other

Rmc -(h) 0 05 0 05

02

123456789101112

with

serum (Fig.

cell

0 05

increased

lines

1

after

2, lanes 1,2;

(l-4).

0 '&ti 0 05

transiently

mitotic

Fig.

simulation

3, lanes l-4),

The second increase

as is

was observed

1

03

1

2

3

4

5

6

7

6

9*‘ IO 11

12

Fig. 2. The c-fos mRNAlevels after stimulation of resting 3Yl cells with serum, and after resumption of the stimulation interrupted by deprivation of serum or by addition of CH. Resting cells were treated as illustrated times after the last treatment, cells were harvested and at the indicated for mRNAblot hybridization. Abbreviations were the same as in Fig. 1. Fig. 3. Change in the c-fos mRNAlevel after stimulation of resting 3Yl cells with serum, and after resumption of the stimulation interrupted by addition of CH or by both deprivation of serum and addition of CH. For the illustration of the treatments of cells, see the legend to Fig. 2. 503

of

Vol. 159, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

6

2

3

4

5

7

6

9101112

Fig. 4. Enhanced increase in the c-fos mRNh level by exposure of resting 3Yl cells to both serum and CH, and re-increase in the c-fos mRNhlevel by exposure to CH of cells in which the transiently increased c-fos level had decreased below a detectable level. For the illustration of the treatment of cells, see the legend to Fig. 2.

after

the second stimulation

lanes

3-6),

exposure

to CH (Fig. (Fig.

2, lanes 8-11;

was performed

(Fig.

in the c-fos

has been observed gradually

in other

decreased

When resting

cells

mRNA level

and CH, the c-fos However,

after

resting

CH (Fig.

was further

zo resting

3Yl cells

cell

lines

(Fig.

mRNA level

the increased cells

6 h, followed containing

by

did not abolish

when

and CH

with

(Fig.

4, lanes l-6),

The increased

serum for

below a detectable with

again level

fresh

c-fos

level

(Fig.

transiently

was lower than

No increase

13 h (by this

as mRNA

that

observed

in the fos mRNA level

was performed

both

(Fig.

medium containing

cells

were stimulated

by replacing

serum and CH, further

replacement 504

serum

4, lanes 10shortly

both

serum and

was observed

in the absence of CR (Fig.

hand, when resting

time

4. lane 7)),

medium containing

increased

by an 8-h interruption

both

enhanced when both

4, lanes 4-6).

were exposed to fresh

4, lane 4-6).

On the other

(2-4,13,14).

were stimulated

decreased

the medium replacement 8,9).

2,

and in those not

of serum removal

mRNA level

and then the medium was replaced

12).

interrupted

3, lanes 5-8)

by a combination

serum and CH were administered

the c-fos

in cells

(Fig.

3, lane 9-12).

The increase

level

Fig.

by serum removal

The second increase

4, lanes 7-9).

interruption

addition

interrupted

but was not or was not so markedly

interrupted the

in cells

with

the medium with

when

1

4, lanes serum for medium

of the medium with

medium.

BIOCHEMICAL

Vol. 159, No. 2, 1989

containing

both

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

serum and CH did not cause such a marked increase

fos mRNA level

(Fig.

2, lanes 10,12).

performed

only

in the absence of serum, the c-fos

increased

after

replacing

Naturally,

the medium with

in the c-

when the interruption mRNA level

was

markedly

medium containing

both serum and

2, lane 7).

CH (Fig.

DISCUSSION The c-fos transiently

mRNA level

increased

absence to presence

proliferating

state.

(S),

gene following

of the c-fos phase (6,7)

or in cell

proliferation

According

S phase occurs Gl phase,

process

to their

the trans:ent

leading

to the next

continuous

expression

model,

the process

throughout with

detectable

level)

initiation

of S phase,

is necessary

initiate

DNA synthesis

external

stimulation

with

cycle,

a Gl-DNA content

of S phase. gene at a very

to continue

because the c-fos

Then, we can interpret

the expression of S

the model of the (17-20)

factors

seems

to the initiation

the transiently

of

not restricted because they which

low level

in are

inhibit

the

(below a

leading

to the

to be necessary

continuously

is absent,

and Cooper

We assume that

the process gene

(7) or to proliferate by growth

specific

to the initiation

leading

the cell

initiation

of the c-fos

cycle

(16). are in compatible

are arrested

expression

that

in S, G2 and M phases under the conditions

leading

expressed.

of 12-O-

proposed by Okuda & Kimura

accumulatively

and cells

to stop

proliferation

by change from serum

suggesting

in the process

the

to

Lhas been

is not a cell

are findings

mentioned-above

of cell

(21-24).

there

mRNA level

Therefore,

was not

state

by addition

serum stimulation

and

increase

restin

in NIH 3T3 cells

(15).

gene is involved

The findings control

hand,

This

from the

and in WI-38 cells

level,

was changer; from serum

in the c-fos

cycle,

phorbol-13-acetate

On the other

unable

the transition

The increase

absence to presence

event.

with

in any phases of cell

of the c-fos

condition

or from CH absence to presence.

in parallel

tetradecanoyl

below a detectable

when the culture

necessarily

observed

was usually

(16).

to

When the

the gene is not increased

expression

of

BIOCHEMICAL

Vol. 159, No. 2, 1989

the c-fos

gene following

factors,

as the result

leading

to the initiation

mitotic that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

stimulation

accompanies of S ;hase,

Thus, when we consider

initiation

of S phase and the e:rpression

Similar

the concept

increase

can be explained responding

since

cells

transcription observed.

It

feedback

short

(1) results

regulation (l-3),

(1).

by the fos protein

negative

Just

gene

feedback-,

serum

the life

elevation

accumulation

itself

arc

mechanism does

after

Since

the c-fos

that

the transcriptional

operates.

that

gene (24).

of the c-fos

and then

in the transient

cells.

cells

the feedback

the transient

of

serum addition

by a hypothesized

condition,

has been demonstrated

regulation

after

In proliferating

the gene rapidly

as the feedback

mRNA is very

of the c-c

mRNA level

the gene is not transcribed

transcribe

declines

the c-fos

levei

in a series

and non-cycling

in serum, the transcription

below a detectable

not operate,

level

factors

event

gene, we need not

cycling

as follows.

by growth

of the process

of the c-fos

in tne c-fos

cells

between the control

the expression

Under the serum-deprived

mechanism.

addition,

for

in more detail

to growth

is controlled

the relationship

of "GO" to distinguish

view has been presented

The transient

the resumption

but not as an early

of events.

introduce

of the resting

time of

of the

of the mRNA as

mRNA is under negative

(25),

supporting

this

explanation. The increase cultures the

after

inhibitory

exposure.

of c-fos (14).

(25,26)

this

reduction

the c-fos

The c-fos

explained

gradually

by assuming that (26,27)

c-fos

in continuously as follows.

in the presence decreased

utilized

increase,

of CH. However,

the synthesis

during

506

of CH.

if

of c-fos inhibition

of unstable

in the presence

CH inhibits

control

In addition,

in stabilization

would continuously

of

reduces by CH

the negative

increases.

serum-exposed The synthesis

transcription

suppresses

transcription

mRNA level

factor

for

by CH results

mechanism operated

fos mRNA level

mRNA levels

can be explained

factor

translation

control

initiat:ng

CH addition

Since

transcription,

in the c-fos

c-fos

no other the increased This can be

of the transcription-

the transcription.

mRNA

c-

BIOCHEMICAL

Vol. 159, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS We wish to thank c-myc

expression

His vzew stimulated

Dr.

in relation

S. Cooper for to his

us to perform

informing

"continuum

this

us his

model"

view

before

(24) on the publication.

study.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

E.B. (1984) Nature 311, 433-438. Greenberg, M-E., and Ziff, Kruijer, W., Cooper, J-A., Hunter, T., and Verma, 1-M. (1984) Nature 312, 711-716. Burckhardt, J., and Curran. T. (1984) Nature Mtlller, R., Bravo, R., 312, 716-720. Cochran, B.H., 2~110, J., Verma, I.M., and Stiles, C.D. (1984) Science 226, 1080-1082. Bravo, R., Burckhardt, J., Curran, T., and Milller, R-(1986) EMBO J. 5, 695-700. Nishikura, K., and Murray, J.M. (1987) Mol. Cell. Biol. 7, 639-649. Riabowol, K. T., Vosatka, R.J., Ziff, E.B., Lamb, N.J., and Feramisco, J.R. (1988) Mol. Cell. Biol. 8, 1670-1676. Kimura, G., Itagaki, A., and Summers, J. (1975) Int. J. Cancer 15, 694706. Matsuzaki, A., Shiroki, K., and Kimura, G. (1987) Virology 160, 227235. Okuda, A., and Kimura, G. (1978) Exp. Cell Res. 111, 55-62. Okuda, A., and Kimura, G. (1983) Exp. Cell Res. 145, 155-165. Okuda, A., and Kimura, G. (1988) J. Cell Sci. 91, 296-302. Greenberg, M-E., Hermanowski, A-L., and Ziff, E.B. (1986) Mol. Cell. Biol. 6, 1050-1057. Wilson, T., and Treisman, R. (1988) Nature 336, 396-399. Calabretla, B., and Marcer, W. E. (1987) Biochem. Biophys. Res. Commun. 147, 716-723. Holt, J.T., Gopal, T.V., Moulton, A.D., and Nienhuis, A.W. (1986) Proc. Natl. Acad. Sci. USA 83, 4794-4798. Okuda, A., and Kimura, G. (1982) J. Cell. Physiol. 110, 267-270. Okuda, A., and Kimura, G. (1984) Exp. Cell Res. 155, 24-32. Okuda, A., and Kimura, G. (1986) Exp. Cell Res. 163, 127-134. Okuda, A., and Kimura, G. (1988) J. Cell Sci. 89, 379-386. Cooper, S. (1979) Nature 280, 17-19. Cooper, S. (1982) In Cell Growth (C. Nicolini, Ed.), pp.315-336. Plenum Press, New York. Cooper, S. (1982) J. Theor. Biol. 94, 783-800. Cooper, S. J. Theor. Biol. in press. Sassone-Corsi, P., Sisson, J.C., and Verma, I.M. (1988) Nature 334, 314-319. Sassone-Corsi, P., and Verma, I. M. (1987) Nature 326, 507-510. Treisman, R. (1987) EMBO J. 6, 2711-2717.

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