Vol. 178, No. 2, 1991 July 31, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 480-485

BIOCHEMICAL

Cell-density Dependent Expression of the c-w Gene in Primary Cultured Rat Hepatocytes* Atsushi

Kumatori,

Toshikazu

Nakamural

and Akira

Ichihara

Institute for Enzyme Research, University Tokushima, Tokushima 770 and lDepartment of Biology, Faculty of Science, Kyusyu University, Fukuoka 812, Japan

of

Received June 7, 1991 SUMMARY: During culture of mature rat hepatocytes as monolayers, c-myc mRNA was found to be expressed transiently within 2 h, decreasing rapidly to the basal level at 10 h. Then its level increased again to over lo-fold the basal level at 24 h, and remained at this high level during culture. The increase of c-myc mRNA in the second phase was shovm by nuclear run-off experiments tobeduetoanincrease of its transcription. The second, but not the first, increase in c-m c expression was inversely proportional to the cell density in cu 35ture. The expression of c-myc mRNA was not affected by various hormones including growth factors. These results indicate that hepatocytes in culture at lower cell density tend to move from the Go phase to the Gl phase, but remain in the Go phase when cultured at high cell density. 0 1991 Academic Press, Inc.

Mature specific partial of

liver

hepatocytes functions

fully,

hepatectomy. functions

cultures

of

insulin,

epidermal

to the

in vivo

medium

rat

but

are

quiescent

they

The complex

proliferate mechanisms

and regeneration

markedly

Moreover

we found

dependent

on cell

that

actively

Additions

factor(EGF)

hepatocyte

growth

in

DNA synthesis

expression

in primary

or hepatocyte DNA synthesis

density[5-81:

in

of mitogens

stimulated

liver after

involved

can be studied

hepatocytes[l-41. growth

and express

of

culture

such as

growth the

cells.

was strictly

was stimulated

*This work was supported by a Grant for Cancer Research from Ministry of Education, Science and Culture of Japan. Abbreviations:EGF, epidermal growth factor;TGF-8, transforming growth factor-% ;IL-18, interleukin-18. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

480

factor

more the

Vol.

178, No. 2, 1991

BIOCHEMICAL

at

lower

than

cell

expression

density

of

density

than

mature at

liver

low cell

cell

contact

controls

that

mature

hepatocytes

cell

density,

but

cent

cultures

of

Gl arrest, culture the

rather are

Gl

expression gene liver

is

is

expressed

strictly

in

regulated

in rat

this gene

the

in

early

lower

shift,

stage

cell

of the

by cell

second

phase

of

the

the

expression

high

state

of

primary

the

Go to

changes

in

because

Gl phase[ll

of its

in

from

we show that

hepatocytes,expression the

in

shift

and

As quies-

hepatocytes of

cell-

at

density.

hepatocytes,

Here

that

reciprocally,

we measured

cultured

vivot9,lOl.

suggest

are usually

study

cell

when cultured

mature

for

at high

functions

lines

Go state,

Conversely,

results

Go state at

useful

at

and that

the

cell

the

c-m=

of mature

These

Gl state

than

density.

and mature

established

regeneration

biphasic

was greater

To investigate of the

cultures

functions

are

particularly

state.

cell

density.

the

RESEARCH COMMUNICATIONS

at higher

growth

in

AND BIOPHYSICAL

this

and during in primary c-=

gene is

density.

MATERIALS

AND METHODS

used for isolation and culture of Materials --- The materials adult rat hepatocytes were as described before[ll]. The materials used for measurement of transcription were also as reported[l2]. exon 3 of the rat c-w gene(pSmBH; Plasmid pSP65, which contains 830 bp insert), was a gift from Dr. Kenshi Hayashi, National Cancer Center, Tokyo. Isolation and Primary Culture of Adult Rat Hepatocytes --- Adult male Wistar rats, weighing 200 g, were used. Their liver was perfused with collagenase and isolated hepatocytes were cultured in Corning plastic dishes(l0 cm diameter) as described[8]. A few hours after plating the medium supplemented with serum was replaced by medium supplemented with 5 ug/ml aprotinin and 1 nM insulin. Northern Blot Analysis of c-myc --- Samples of about 200 ug of RNA were extracted from lo7 hepatocytes, aliquots of 10 pg were denatured, electrophoresed and hybridiz&d ~~fae;~~~]l~~e;~~~r~~~b~=~~~r~~ describedI81, by the multiprime DNA labeling method[l3], The membrane was then washed, dried in air, and subjected to autoradiography and densitometric scanning for quantification of RNA. Assay of Transcription by the Nuclear, Run-off Method --- Nuclei (lo71 were isolated from cultured hepatocytes and incubated with 0.5 mM NTPS containing 100 uCi of [a-32P]UTP at 25OC for 40 min as described[l2]. The transcribed RNA was extracted by the hot phenol method and hybridized with linearized pSmBH on a nitroThe filter was washed, dried and radioautocellulose filter. The average efficiency of hybridization was 22%. graphed. 481

Vol.

178,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS AND DISCUSSION When hepatocytes

were isolated

culture

dishes,

ntially

undetectable(Fig.

5 x lo4

cells/cm’

over

This

the at

l), 2 h,

basal

until

gene at

at

40 h of

Next

cultured

lo4

with

2 ;ri

0

2

4

6

8

C-E

Time 10

markedly

at

the

for density

either

of hepatocyte

after of

isolated

2).

c-myc 3,

gene in

culture

expression

of

range

with

of

15 - 2.5 X

previous

results

growth[5-71.

(h ) 12

14

16

20

24

40

28s ias

Culture

Time

(h)

Biq. 1. Time COUrSe of change in expression of c-w mRBAduring primary culture of adult rat hepatocytes. Isolated hepatocytes were cultured at 5 x 104 cells/cm* in serum-free medium. RNA was extrtimes, acted at the indicated separated by electrophoresis and probe. (A) Radioautograph of the blots. hybridized with c-myc 28s and 18s indicate ribosomal IWAs. (B) Quantification of the blots by densitometry. Values are means for triplicate experiments. 482

the

20 h or 40 h increased

in the

are consistent

20 h.

in transcription, in nuclei

As shown in Fig.

in cell

to a very

expression

liver(Fig.

of

of to

a plateau

mRNA levels

and normal

culture

density-dependency

increased

The increased

expression

These results

Culture

A

after

mRNA level

was due to increase

the

esse-

at a density

reaching

in

initially

to the maximum level

40 h.

density.

decrease

cells/cm2.

showing

least

whether

mRNA by cells

markedly

again,

and plated

culture

decreased

was similar

of

by cell

during c-myc

rats

mRNA was

c-myc

Then it

hepatocytes

we examined

was regulated c-myc

their

culture

as shown by comparison 40-h

but

level.

which

level,

2 h, persisted

from

of

10 h and increased

plateau

c-myc

expression

for

lo-fold

low level

their

from adult

Vol.

178,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Fiq. 2. Stimulation of transcription of the c-w gene in adult hepatocytes during culture. (A) Transcription after culture for h. Culture conditions were as for Fig. 1. After 40 h, the nuclei were isolated and transcription of the c-w gene was measured. Transcription in nuclei from normal liver. pSP65 is a control vector.

As we

in the ced the density. ml)

not

expression

of

We found

that

controlled

rig/ml),

pg/ml)

shown).

density-dependent

of mitogens,

presence

IL-lf3(2

gon(0.5

observed

previously

c-myc

whether

mitogens

cultured

hepatocytes

at

insulin(O.1

5% calf

serum,

had no influence

Therefore, entirely

PM),

expression by cell

expression

of the

density.

c-myc

There

are

only influen-

low

EGF(10 rig/ml),

dexamethasone(1

on the

(B)

DNA synthesis

we examined by

rat 40

cell

TGF-(3(1

).IM) and glucaof

gene

c-myc(data seems

several

to

be

recent

20 h 40 h

O312.5 Cell

Density

(X10GL91cm2)

Fiq. 3. Effect of cell density on expression of c-myc mRNA in Cells were cultured at the ifiaicated cell cultured hepatocytes. densities for 20 h and 40 h, then total RNA was extracted and c-Myc mRNA was measured by Northern separated by electrophoresis. blot hybridization analysis as described in Materials and Methods. 28s and 18s indicate ribosomal RNAs. 2.5 Kb (A) Radioautograms. indicates c-z mRNA. (B) Quantitative analysis of results of (A). Values are means for triplicate l , 40 h culture. 0, 20 h culture; experiments. 483

ng/

Vol.

178,

No.

reports

of

but

regulation

the

BIOCHEMICAL

2, 1991

c-myc

expression of

expression is

previously.

suppress

expression

of

suppress

stimulated

DNA synthesis

suggest late

is that

these

of c-this

ing

from

their

attachment

to

finding (data

that not

liver

in --

and cultured they

mechanism(s) involve

growth

phase

from

also

present

of

changes

this of

the

was not

cell

that

on cells

culture

in of

is

vivo is

the

expressunknown.

hepatocytes,

affected

result-

and their

supported

by the

by cell

present

that

move from

the

growth

shift

membrane

shown to

findings

phase

--in

are

not

density

at

low levels

hepatocytes

isolated

culture[l7-201.

to various cycle

been

these

explanation

iS likely

density

cell

the

contact

during

results,it

sensitive

of

did

As c-myc

act

2 h of

stress

This

increase

have

transient

after

proteins

low cell

become

first

phase

Several

they

factors

been

and IL-l$

Gl phase[41,

tight

dishes.

transient

vivo

the

has not

hepatocytes[l61.

early

was due to

plastic

at

for

TGF-8

although in

a maximum

release

this

the

density

that

mRNA,

COMMUNICATIONS

hepatocytes[l4,151,

by cell

suppressive

The reason

shown).

From the

where

in

mRNA with

Possibly

in

completed

Gl phase.

ion

c-myc

RESEARCH

cultured

interesting

reported

expression

BIOPHYSICAL

in primary

its It

AND

is

still

Go to

the

Gl phase,

conditions. unknown,

The but

may

and cytoskeleton.

REFERENCES 1. Guillouzo, A. & Guguen-Guillouzo, C. eds (1986) Isolated and Cultured Hepatocytes, John Libbey Eurotext, London/Paris 2. Grunnet, N. & Quistorff, B. eds.(1991) Regulation of Hepatic Function, Munksgaard, Copenhagen G.K. (1990) FASEB J. 4, 176-189 3. Michalopoulos, Lu, X.P., Brenner, D.A., Fey, G.H., Martinez-Conde, 4. Koch, K.S., A. & Leffert, H.L. (1990) In Vitro Cell Dev. Biol. 26, loll1023 T., Tomita, Y. & Ichihara, A. (1983) J. Biochem. 94, 5. Nakamura, 1029-1035 6. Nakamura, T., Yoshimoto, K., Nakayama, Y., Tomita, Y. & Ichihara, A. (1983) Proc. Natl. Acad. Sci. USA, 80, 7229-7233 7. Nakamura, T., Nakayama, Y. & Ichihara, A. (1984) J. Biol. Chem. 259, 8056-8058 8. Noda, C., Ohguri, M. & Ichihara, A. (1990) Biochem, Biophys. Res. Commun. 168, 335-342 9. Makino, R., Hayashi, K. & Sugimura, T. (1984) Nature 310., 697698 lO.Thompson, N.L., Mead, J-E., Braun, L., Goyette, M., Shank, P.R. & Fausto, N. (1986) Cancer Res. 46, 3111-3117 484

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ll.Tanaka, K., Sato, M., Tomita, Y. & Ichihara, A. (1978) J. Biothem. 84, 937-946 12.Nawa, K., Nakamura, T., Kumatori, A., Noda, C. & Ichihara, A. (1986) J. Biol. Chem. 261, 16883-16888 13.Feinberg, A.P. & Vogelstein, B.A. (1984) Anal. Biochem. 137, 266-267 14.He)hne, M., Becker-Rabbenstein, V., Kahl, G.F. & Taniguchi, H. (1990) FEBS Lett. 273, 219-222 15,Sawada, N. (1989) Exp. Cell Res. 181, 584-588 16.Nakamura, T., Arakaki, R. & Ichihara, A. (1988) Exp. Cell Res. 179, 488-497 17.Kato, S., Aoyama, K., Nakamura, T. & Ichihara, A. (1979) J. Biochem. 86, 1419-1425 18.Nakamura, T., Tomomura, A., Noda, C., Shimoji, M. & Ichihara, A. (1983) J. Biol. Chem. 258, 9283-9289 lg.Schetz, E.G., Li, D., Omiecinski, C.J., Muller-Eberhard, U., Kleinman, H.K., Elswick, B. & Guzelian, P.S. (1988) J. Cell. 134, 309-323 Physiol. 20.Emi, Y., Chijiwa, C. & Omura, T. (1990) Proc. Natl. Acad. Sci. USA, 87, 9746-9750

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