Increased spermidine or spermine level is essential for hepatocyte growth factor-induced DNA synthesis in cultured rat hepatocytes

GASTROENTEROLOGY 1994;106:1024-1031

Increased Spermidine or Spermine Level is Essential for Hepatocyte Growth Factor-Induced DNA Synthesis in Cultured Rat Hepatocytes IKKO HIGAKI,* ISA0 and SHUZO OTANI*

MATSUI-YUASA,’

MASANOBU

TERAKURA,*

HIROAKI

KINOSHITA,§

*Second Department of Biochemistry, Osaka City University Medical School; ‘Department of Food and Nutrition, Faculty of Science of Living, Osaka City University; and “Second Department of Surgery, Osaka City University Medical School, Osaka, Japan

Background/Aims: Hepatocyte growth factor is a potent mitogen for mature hepatocytes and seems to act as a trigger for liver regeneration. Hepatocyte growth factor was first purified from human and rabbit plasma and rat platelets. Additionally, putrescine, spermidine, and spermine are widely distributed in many different cells; intracellular concentrations of these polyamines are closely related to cell proliferation. The present study examined whether polyamine metabolism is involved in hepatocyte growth factor-induced DNA synthesis in primary cultured rat hepatocytes. Methods: Hepatocy-tes were isolated from rats by the collagenase perfusion method. Ornithine decarboxylase and Sadenosylmethionine decarboxylase activities were measured as the release of 14C02 from L-[1-14C]ornithine and Sadenosyl-L-[carboxyI-14C]methionine, respectively. Results: a-Difluoromethylornithine inhibited hepatocyte growth factor-induced DNA synthesis by only 21%. On the other hand, methylglyoxal bis(guanylhydrazone) completely inhibited hepatocyte growth factorinduced DNA synthesis to nontreated control level. The inhibitory effect of methylglyoxal bis(guanylhydrazone) on hepatocyte growth factor-induced DNA synthesis was reversed by exogenously added spermidine or spermine. Conclusions: Spermidine or spermine is essential for hepatocyte growth factor-induced DNA synthesis in primary cultured rat hepatocytes.

many

different

polyamines

H

for mature

factor

hepatocytes

(HGF)

and seems

for liver regeneration

after various

purified

and rabbit

based

from

on its ability

tocytes.“2 with

human

HGF

hepatic

animals

with

kinetics

of this

round

of DNA

gesting

that

levels

experimentally response synthesis HGF

plays

stimuli.

regeneration

forms

of liver injury,

ischemia,”

nine

HGF

in a time-

of rat hepa-

in the plasma

in the

plasma3

induced is rapid during a key

and

liver damage. precedes

the

in initiating

of The first sugthis

process.3*4 Putrescine,

spermidine,

and

spermine

the

after

activities

of

S-adenosylmethiokey

enzymes

in mammalian

many

liver

and polyamines The

and

pathway

many

different

of the cells, are

growth-pro-

reported

and

that HGF

dose-dependent

rat hepatocytes.13

ined

the

role

of ODC,

of polyamine and

is essential cultured

inhibitor

in primary study,

cul-

we exam-

by

using

a-

inhibi-

bis(guanylhydrazone) of SAMDC,

and

level of spermidine

for HGF-induced

activity

an irreversible

methylglyoxal

cellular

ODC

metabolism

(DFMO),

a reversible

that an increased

fashion

In the present

difluoromethylornithine (MGBG),

induced

DNA

found

or spermine

synthesis

in primary

rat hepatocytes.

Materials and Methods Materials Male Wistar rats weighing 150-200 g were used for preparation of hepatocytes. The protocol of the experiment was by the Animal

Research

care of the animals of this institution

Committee

of Osaka City

was in accordance

Osaka City University). Insulin, dexamethasone, spermidine, spermine, and S-adenosyl-L-methionine products

of Sigma Chemical

with

the

(Guide for AnimaL Experimentation, putrescine, were the

Co. (St. Louis, MO). MGBG

was

of patients or serum*

liver regeneration, role

increased

tured

standards

was first

regeneration. (ODC)

from

hepatectomy,‘.”

stimuli.5~6”2

We recently

tor

for recovery

(SAMDC),

biosynthetic

of these

proliferation.5.6

liver injury,‘“*”

for liver

decarboxylase

moting

cell

such as partial

decarboxylase

dramatically

to

is important

are responsible

polyamine

concentrations

related

and chemical

ornithine

University;

and rat platelets

mitogenesis

increase and

mitogen

to act as a trigger

plasma

to stimulate

failure’

is a potent

closely

Liver

approved growth

epatocyte

cells; intracellular are

are found

in

Abbreviations used in this paper DFMO, adifluoromethylornithine; HGF, hepatocyte growth factor; MGBG, methylglyoxal bis(guanylhydrazone); ODC, ornithine decarboxylase; PCNA, proliferating cell nuclear antigen; PVDF, polyvinyldifluoride; rhHGF, recombinant human hepatocyte growth factor: SAMDC, Sadenosylmethionine decarboxylase; SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline containing 0.05% Tween 20. 0 1994 by the American Gastroenterological Association 0016-5065/94/$3.00

April 1994

POLYAMINE

purchased

from Aldrich

L-[l-‘“C]otnithine methionine

from Nippon

cell nuclear antigen

(Arkansas)

was from

human Ltd.

growth

(Tokushima,

Research

Japan)

Institute,

Hepatocyte Male cytes judged

from

Viability by ttypan

purity

immu-

> 99%) (Mettell

1 X lo-’

L dexamethasone. in a humidified changing

in the absence hours.

Then

thHGF

were

was

and harvested

Protein

CO,

concentrations

of radioactivity

as

as described counts

in 35-

DNA

from

The

a tubber

policeman

containing ylene

in 300

diaminetettaacetic

air. After

for additional

fresh

medium

thiothteitol. minutes

and were

at 4°C.

Then

16

0.25

and 80 nmol of L-otnithine stopper

syringe

needle.

350 absorbed transferred

(5 pL). The

on the paper disk, Daiichi

was measured 5801; mined

Beckman,

Tokyo,

by the release

‘“Clmethionine.‘”

The

was trapped

incubated

was sealed syringe

nee-

(Sigma

to

protein

Chemical

(Packon the

sodium

(SDS),

0.15

scintillation

SAMDC

were

The

as

amount

of Button.“.‘”

were washed

1% Nonidet

deoxycholate, mol/L NaCl,

of

Calf thy-

for 3 minutes

to PVDF

membrane

was blocked

albumin).

the PVDF

Tween (1:500

lin G conjugated After

developed

of SDS Protein

protein

Twenty

once

with

assay kit

micrograms gels and casein.

Ttis-buffeted

saline

TBST

with a chemiluminescent

and incu-

and 0.2%

with the primary three

times

with

bovine

antibody,

with

with anti-mouse

washing

transfer,

0.5%

(5 minutes)

in 0.05%

was washed

with

TBST

(5

immunoglobu-

with horse radish petoxidase

extensive

gel electto-

of Laemmli.”

at 4°C

20 (TBST)

each) and incubated

Cells

in the presence

After electtophotetic

After incubation

membrane

at 4°C.

SDS-polyactylamide

overnight

was washed

Co.), sulfate

and 2 mmol/L

by a Bio-Rad

membrane.

bated in anti-PCNA serum

PAGE

as a standard.

was run on 12.5%

0.05%

Chemical dodecyl

minutes

to the method

transferred

phos-

at 100,OOOg for 30 minutes

buffet.

of protein

containing

(Sigma EDTA,

for 30

with

in SDS-polyactylamide

sample

the paper

times

10 mmol/L Ttis-HCl

sodium

1 mmol/L

fluoride

were measured

Then

P-40

by centtifugation

(PAGE)

three

with

0.1%

concentrations

(1:2500

TBST,

detection

in 0.5%

the blots

were

system.

Measurement of Polyamine Concentrations

(LS

was detet-

washed

was measured

was expressed

of DNA.

saline and disrupted

according

The incubated

S-adenosyl-L-[catboxyl-

cells

incorporation

fluid

counter

activity

The incorporation

Co.) was used as a standard.

was performed

Radioactivity

scintillation

from

photesis

TBST).

by the Soluene-

Japan).

Fraction

fraction

by the method

hepatocytes

0.5%

minutes

and the paper disk was then

liquid

Japan).

di-

(5 FL)

IL) was impaled

Tokyo,

of 14C02

7.5)

was added to

test tube

The

using bovine serum albumin

at 30,OOOg for 20

5 mL toluene

Chemicals,

in a Beckman

(pH

50 PL Soluene-350

Grove,

The CO* released

with

mmol/L

with a disposable

to a vial containing

(Omnifluot;

Ttis

PCi of L-El-‘*C]otnithine

Co., Downers

washed

by three cycles of freezing

dle. A paper disk (8 mm) containing ard Instrument

were

and 2.5

centrifuged

equipped

The

was added to the cell cul-

pet microgram

(pH 7.5) containing

and boiled

0.1 mmol/L eth-

90 PL of the supetnatant

a glass tube containing with a tubber

cells

phosphate,

(EDTA),

then

PL). similar

of [3H]Thymidine Into Acid-Insoluble

previously.”

Rat

with

saline and harvested

The cells were disrupted

and thawing

(5

of 14C02 pto-

by a Bio-Rad

into the acid-insoluble

phate-buffeted

by the release of ‘“CO2

/.LLof 50 mmol/L

acid

tube

Western Blot Analysis

cells were cultured

incubated

200 pmol/L pytidoxal

at 4°C.

to a glass

VA) using bovine serum albu-

(1 PCiidish)

was determined

mus DNA

times.

was determined

L-[1-‘4C]ornithine.‘5

were measured

Richmond,

pet minute

moli

of Enzyme Activities

three times with phosphate-buffeted

and thawing

by estimation

ture 4 hours before the cells were harvested.

were pelleted

activity

was added

The

used in assay of ODC activity.

phenylmethylsulfonyl

ODC

of freezing

S-adenosyl-L-methionine

{‘H]Thymidine

the

>95%

and 95%

in the

at the indicated

Measurement

2.8

phosphate.

from S-adenosyl-L-[catboxyl-‘4C]methionine

at 37°C for 24 hours

and hormones

cultured

pytidoxal

cycles

phosphate

dithiothteitol,

at 30,OOOg for 20 minutes

was measured

min as a standard.

perfusion

and 1 X lo-’

with fresh medium,

of the serum cells

nmol

Dow

Hepato-

collagenase

The cells were cultured

the medium

20

activity

assay kit (Bio-Rad,

before

The cells were plated

of 5%

by three

Measurement Incorporation

water freely.

mol/L insulin,

atmosphere

and

duced

mmol/L

1025

PCi S-adenosyl-L-[catboxyl-‘4C]methionine

Co.

mm collagen-coated dishes at a density of 5 X lo4 cells.0.2 mL-’ . cm-’ m Williams’ medium E supplemented with 5% fetal calf serum,

PL)

the method

and

1.1

and 200 PmoliL

0.125

SAMDC

te-

Pharmaceutical

hepatocytes

blue exclusion.

(5

was from

for 18 hours

by the

of isolated

containing

PL of the supetnatant

containing

and Culture

to drink

tats

in 300 PL of 55 mmol/L sodium

7.5)

were disrupted 90

PROLIFERATION

respectively.

tats were fasted

isolated

[mouse])

IN HEPATOCYTE

and were then centrifuged Then

Recombinant

P. McCann

OH),

but were allowed

were

method.”

and Dr.

Preparation

Wistat

experiments

MA).

by Otsuka

Cincinnati,

was

proliferating

anti-mouse

factor (thHGF;

supplied

(pH

cells

petoxidase

(Boston,

policeman

buffet

(Buckinghammembrane

blot chemiluminescence

NEN

tubber

mmol/L puttescine,

anti-PCNA

Denmark);

and Western

were kindly

and

(31 Ci/mmol)

Japan);

with horseradish

DuPont

hepatocyte

DFMO

(PVDF)

(monoclonal

(Glosttup,

G conjugated

Pel-Fteet

International

Co. Ltd. (Tokyo,

(PCNA)

was from Dakopatts

agent

and [6-3H]thymidine

Polyvinyldifluotide

Genetics

WI),

S-adenosyl-L-[catboxyl-‘“C]-

from Ametsham

England).

noglobulin

Co. (Milwaukee,

Ciimol),

(57 Ci/mol),

were purchased shire,

Chemical

(53

METABOLISM

with

a

liceman

in 300

cells were harvested

PL of ice-cold

with a tubber

5% ttichlotoacetic

po-

acid centti-

1026

HIGAKI

GASTROENTEROLOGY

ET AL.

fuged for removal

of the protein.

amine

in the acid extract

liquid

chromatography

Japan)

equipped

of polyamine

(Shimadzu

with fluorescence

was performed

150 mm; particle

The concentration

was analyzed

detector.*’ Shimadzu

ric acid as solvent A and solvent A-methanol was eluted

with solvent

solvent

B at 4°C for 3 minutes

solvent

gradient

The gradient 22 minutes concentrations

using

The separation (4.6 X

Techno-Research of 10 mmol/L

l-

changed

(1:3) as solvent

A at 96°C and with

and then with a programmed

the linear

gradient

curve number

from 4°C to 55°C with

at a flow rate of 0.7 mlimin. were expressed

as picomoles

Various

Kyoto,

acid sodium salt- 100 mmol/L sodium perchlo-

B. The sample

Results

of poly-

LC-6A; Shimadzu,

Inc., Kyoto, Japan) with a solvent composed hexanesulfonic

No. 4

by high-performance

on a STR ODS-II column

size, 5 pmol/L;

Vol. 106,

solvent

cells, and the activities

of ODC (Figure

B in of

DNA.

1A) and SAMDC

6 hours after the addition (Figure 1B) were determined in primary cultured rat hepatocytes. Both of the enzyme activities

began

to increase

reached a maximum

with

with

1 ng/mL

5 ng/mL

(Figure activity

increased

and

HGF. 4 hours

of HGF and peaked at 8 hours; thereaf-

2A). To determine is required

we used DFMO.

HGF

(60 pmol/L)

Figure 2A shows that ODC activity after the addition

ter it decreased and returned 0.

The polyamine per micrograms

of HGF were added to the

concentrations

to O-hour level by 16 hours whether

an increase

for HGF-induced

Five millimolars

DNA

of DFMO

inhibited

the increase

in ODC activity

inhibited

HGF-induced

C3H]thymidine

in ODC synthesis,

completely

(Figure

2A) but

incorporation

by

Statistical Analysis The results are expressed cance of differences t test. Differences

as means t SD. The signifi-

in assay values was evaluated

by Student’s

with P values < 0.05 were considered

sig-

nificant.

“0

Figure 1. Effect of HGF on (A) ODC and (6) SAMDC activity in primary cultured rat hepatocytes. The cells were cultured as described in Materials and Methods. The culture medium was changed with the addition of HGF at the various concentrations, and cells were cultured for 6 hours. Values are expressed as means + SD of five dishes. *P < 0.01 compared with 0 ng/mL HGF. **P < 0.05 compared with 0 ng/mL HGF.

4

(I

1ime

12

16

:

P.

after HGF (hr)

figure 2. (A) Time course of ODC activity in primary cultured rat hepatocytes after the addition of HGF and DFMO. The culture medium was changed with the addition of HGF (10 ng/mL) in the absence (0) or presence (0) of 5 mmol/L DFMO. Values are expressed as means 2 SD of five dishes. *P < 0.01 compared with the presence of DFMO. **P i 0.05 compared with the presence of DFMO. (6) Effect of exogenously added putrescine, spermidine, or spermine on [3H]thymidine incorporation in primary cultured rat hepatocytes treated with DFMO. The culture medium was changed with the addition of HGF (10 ng/mL) in the absence or presence of 5 mmol/L DFMO. At 8 hours, 0.1 mmol/L putrescine (Put), 0.1 mmol/L spermidine (Spd), or 0.1 mmol/L spermine (Spm) was added. A 4-hour pulse of [3H)thymidine (1 pCi/dish) was begun 20 hours after the addition of HGF. Incorporation is expressed as means 2 SD of five dishes. “P < 0.01 compared with control; “P < 0.01 compared with the addition of HGF: ‘P < 0.01 compared with the addition of HGF in the presence of DFMO.

POLYAMINE METABOLISM

April 1994

only 21% (Figure

2B). The inhibitory

on HGF-induced

13H]thymidine

versed by exogenously

added 0.1 mmol/L

was re-

putrescine,

mmol/L spermidine, or 0.1 mmol/L spermine after HGF and DFMO addition (Figure 2B). As shown in Figure

3A, SAMDC

hours after the addition remained

at a high

L spermine

effect of DFMO

incorporation

activity

0.1

8 hours

increased

3

of HGF, peaked by 6 hours, and

level until

12 hours.

Thereafter

it

addition To

IN HEPATOCY-TE PROLIFERATION

was added exogenously

8 hours after MGBG

3B).

(Figure determine

HGF-induced

which

DNA

polyamine

synthesis,

addition

of DFMO

addition

and MGBG

of 5 mmol/L

DFMO

completely

inhibited

corporation

to nontreated

is

(Figure

control

level;

spermine

crease in SAMDC

DFMO

and MGBG

caused by HGF MGBG

HGF-induced

{3H]thymidine

treated

level; these inhibitory

control

were reversed when 0.1 mmol/L

(Figure

completely incorporation

3A).

inhibited to

non-

effects of MGBG

spermidine

or 0.1 mmoli

addition.

when 0.1 mmol/L

levels of PCNA (Figure showed

DNA

HGF-induced

spermidine hepatocyte

leled that seen in Figure Dose responses

proliferation.

inhibition

at 20 pmol/L.

amine concentrations tine concentration

Figure 3. (A) Time course of SAMDC activity in primary cultured rat hepatocytes after the addition of HGF and MGBG. The culture medium was changed with the addition of HGF (10 ng/mL) in the absence (0) or presence (0) of 20 umol/L MGBG. Values are expressed as means 2 SD of five dishes. *P < 0.01 compared with the presence of MGBG. (8) Effect of exogenously added spermidine or spermine on [3H]thymidine incorporation in primary cultured rat hepatocytes treated with MGBG. The culture medium was changed with the addition of HGF (10 ng/mL) in the absence or presence of 20 pmol/L MGBG (MGBGzO) or 30 pmol/L MGBG (MGBG,O). At 8 hours, 0.1 mmol/L spermidine or 0.1 mmol/L spermine was added. A 4-hour pulse of [3Hjthymidine (1 @/dish) was begun 20 hours after the addition of HGF. Values are expressed as means 2 SD of five dishes. aP < 0.01 compared with control; bP i 0.01 compared with the addition of HGF; ‘P < 0.01 compared with the addition of HGF in the presence of MGBG (20 pmol/L): dP < 0.01 compared with the addition of HGF in the presence of MGBG (30 umol/L).

also for

This result paral-

effects on SAMDC

activity

are shown in Figure 6A displaying

7.5 pmol/L

MGBG

The effects of HGF and polyamine Time after HGF (hr)

of PCNA is necessary

4.

incorporation

at roughly

protein

of cell proliferation

blot analysis

and B. Both curves are similar, inhibition

reversed

is important

we measured

or spermine

of MGBG

and [iHfthymidine

or spermine marker

5A and B). Western that

the inhibitory

was added exogenously.

synthesis,

as another

or 0.1

8 hours after

was not completely

that spermidine

for HGF-induced

in-

spermidine

In contrast,

putrescine

MGBG

the inhibitory

was added exogenously

effect of DFMO and MGBG To confirm

4). Combined

[‘H)thymidine

mmol/L

activity

for

combined

and 20 pmol/L

HGF-induced

effect was reversed when 0.1 mmol/L

20 and 30 pmol/L

essential

we examined

decreased and returned to O-hour level by 18 hours. Twenty-micromolar MGBG completely inhibited the inBoth

1027

half-maximal and maximal

inhibitors

on poly-

are shown in Figure 7. The putrespeaked

by 8 hours and decreased

by

16 hours after the addition

of HGF.

The addition

mmol/L

inhibited

the increase in the

DFMO

completely

of 5

Figure 4. Effect of exogenously added putrescine, spermidine, or spermine on [3H]thymidine incorporation in primary cultured rat hepatocytes treated with DFMO and MGBG. The culture medium was changed with the addition of HGF (10 ng/mL) in the absence or presence of DFMO (5 mmol/L) and MGBG (20 pmol/L). At 8 hours, 0.1 mmol/L putrescine, 0.1 mmol/L spermidine, or 0.1 mmol/L spermine was added. A 4-hour pulse of [3H]thymidine (1 uCi/dish) was begun 20 hours after the addition of HGF. Values are expressed as means -t SD of five dishes. aP < 0.01 compared with control; bP < 0.01 compared with the addition of HGF; ‘P < 0.01 compared with the addition of HGF in the presence of DFMO and MGBG.

1028

HIGAKI

putrescine

GASTROENTEROLOGY

ET AL.

concentration

caused by HGF. In contrast,

by 8 hours and peaked by 16 hours after the addition HGF.

The addition

MGBG dine

completely

concentration

spermine

of 5 mmol/L inhibited caused

concentration

DFMO

by HGF

increased

of HGF. inhibited

spermine

concentration

mmol/L

DFMO

Changes

caused

did not inhibit

in polyamine

(Figure

by HGF. it (Figure

concentrations

However, after

are shown in Table

nously

added putrescine,

spermidine,

exoge-

1. Exoge-

or spermine

in cells, but each polyamine

5

7C).

added polyamines

converted

of 20

the increase in the

nously

accumulated

7B). The

The addition

spermine

polyamines

are widely

were

added was not

into the other polyamines.

-*

116kD

-

66kD

-

42kD

-

30kD

-

after various

documented 1 ng/mL

that DNA

HGF

HGF

induced

dependent

ODC

manner

In the present mine

metabolism

using

inhibitors

et a1.23 previously

began to increase with

cultured

trescine

and

was reversed

and SAMDC

rat hepatocytes.

activities

in a dose-

similar to that seen in DNA synthesis. study,

we examined

in HGF-induced

DNA DFMO

concentrations

DNA synthesis

synthesis

by

enzymes decreased but

in pu-

inhibited

by only 21%. The inhibi-

on HGF-induced

by exogenously

DNA

synthesis

added putrescine,

spermi-

On the other

caused the accumulation

the role of polya-

biosynthetic

rat hepatocytes.

spermidine

or spermine.

with 5 ng/mL

cultured

of polyamine

primary

dine,

synthesis

ODC ac-

fashion in primary

Nakamura

in primary

tory effect of DFMO . , .;

stimuli.‘-”

HGF and reached its maximum

(60 pmol/L)

is associated

shown that HGF induced

rat hepatocytes.13

cells

and differentia-

synthesis

in a time- and dose-dependent

cultured

and

in many different

that polyamine

We have recently tivity

spermidine,

role in cell growth

with liver regeneration

HGF-induced 200kD

putrescine,

distributed

and play an essential tion.22 It is known

by 8 hours and peaked

I6 hours after the addition completely

or 20 pmol/L

the increase in the spermi-

pmol/L

MGBG

The

of

No. 4

Discussion

the

addition of MGBG increased the putrescine concentration (Figure 7A). The spermidine concentration increased

Vol. 106,

hand,

of putrescine

MGBG,

which

and decreased

in

A

B Figure 5. Western blot analysis of PCNA levels in primary cultured rat hepatocytes. The culture medium was changed with the addition of HGF (10 ng/mL) in the absence or presence of DFMO (5 mmol/L) and MGBG (20 pmol/L). At 8 hours, 0.1 mmol/L putrescine. 0.1 mmol/L spermidine, or 0.1 mmol/L spermine was added. Cells were harvested 24 hours afterthe addition of HGF, and cellular proteins (20 pg) were separated by acrylamide gel electrophoresis. (A) lmmunoblot analysis of PCNA. Lanes: M, marker proteins; 1, control; 2, HGF: 3, HGF + DFMO + MGBG; 4. HGF + DFMO + MGBG + putrescine; 5, HGF + DFMO + MGBG + spermidine; 6, HGF + DFMO + MGBG + spermine. (6) Densitometric analysis of relative amounts of PCNA.

Figure 6. Dose responses of (A) MGBG effects on SAMDC activity and (8) [3H]thymidine incorporation in primary cultured rat hepatocytes. SAMDC activity and [3H]thymidine incorporation were measured at 6 and 24 hours, respectively. A 4hour pulse of [3Hjthymidine (1 uCi/ dish) was begun 20 hours after the addition of HGF. Values are expressed as means 2 SD of five dishes. *P < 0.01 compared with 0 pmol/L MGBG. **P < 0.05 compared with 0 pmol/L MGBG.

POLYAMINE METABOLISM

April 1994

marker

IN HEPATOCYTE PROLIFERATION

of cell proliferation.

as a nonhistone correlates

directly

and DNA synthesis.24-27 the onset of DNA ing S phase.““s

Elevated levels of PCNA appear

synthesis

and become

Exogenously

protein

paralleled

added putrescine,

those obtained

spermidine,

but each polyamine

converted

into the other polyamines.

putrescine

is a potent

stimulator

or sperm-

added was not

It is known

of SAMDC

increases in the spermidine

and spermine

were not observed

putrescine.

The reason for this is not obvious. of transgenic

the human ODC gene.‘“-‘” elevated

et al. who recently

enhanced

and spermine

ODC activity

between putrescine

attributable

to an inhibition

are compatible

and

levels, the concentraremained virtually

altered in the tissue of the transgenic ent blockage

Similar

mice over-expressing

tissue putrescine

tions of spermidine

added

In spite of the fact that these

animals display a strikingly grossly

con-

after exogenously

findings were reported by Halmekyto a number

that

activity.*’

centrations

generated

dur-

incorporation.

ine was accumulated,

However,

before

maximum

The present study showed that changes

by [‘H}thymidine (hr)

proliferation

late G, phase immediately

in the levels of PCNA after HGF

has been described

whose level of synthesis

with the rate of cellular

in the nucleus during

Tie

PCNA

nuclear protein

1029

mice.”

and spermidine

of SAMDC.‘”

un-

The apparwas not

These results

with the views expressed by Davis et a1.34

in their recent review that one of the major functions the polyamine

homeostatic

is to prevent

an excessive

polyamines tylation,

spermidine the extent

which DNA

of inhibition polyamine synthesis,

inhibited control

HGF-induced DNA

synthesis

added spermidine DFMO

DNA

level. The inhibitory

induced

and MGBG was reversed

added exogenously. DFMO

and MGBG

putrescine

or spermine. completely when

to nontreated on HGF-

by exogenously

Combined inhibited

addition

of

HGF-induced

control level; the inhibitory

spermidine

In contrast,

or spermine

the inhibitory

was not completely

was added exogenously.

midine or spermine synthesis,

synthesis

completely

effect of MGBG

was reversed

DNA synthesis to nontreated effect

concentrations,

is important

was

effect of

reversed when

To confirm that sperfor HGF-induced

DNA

we measured protein levels of PCNA as another

of inhibition

after HGF

These

of SAMDC

or spermine

and MGBG synthesis

activity.

for HGF-induced (0.1 - 100 pmol/

The extent

to that of spermine

of recovery of spermi-

(data not shown).

that an increased

or spermine,

cellular

rather than putrescine,

DNA

are essential

for liver

Luk8 showed that the administration inhibitor,

DFMO,

pletely prevented exogenously

to partially

is essen-

added putrescine.

lation

of putrescine

spermine

without

accumulation

tion of DNA

synthesis

regeneration. rats, com-

this was reversed by

Nishiguchi

of interferon

in par-

of a specific ODC

hepatectomized

liver regeneration;

that administration

level of

synthesis.

Previous studies have reported that polyamines, putrescine,

to the

To determine

were added exogenously

addition.

tial for HGF-induced ticular

ace-

synthesis

was similar

by various concentrations

results suggest

spermidine

of DNA

of MGBG

is more important

dine was similar and spermine

cells

of the higher

by excretion,

various concentrations

L) of spermidine of DNA

the spermidine

and spermine

by various concentrations extent

in mammalian

or both.

Moreover, flgure 7. Time course of polyamine concentrations: (A) putrescine, (8) spermidine, and (C) spermine in primary cultured rat hepatocytes. The culture medium was changed with the addition of HGF (10 ng/ mL) in the absence of inhibitors (0) and in the presence of 5 mmol/ L DFMO (0) or 20 pmol/L MGBG (A). Values are expressed as means ? SD of five dishes. *P < 0.01 compared with no inhibitors. **P < 0.05 compared with no inhibitors.

system

accumulation

of

et a1.35 showed

suppressed the accumu-

affecting

spermidine

and also suppressed

and

the stimula-

in the liver induced

by partial

1030 HIGAKI ET AL.

GASTROENTEROLOGY Vol. 106, No. 4

Table1. Changes in Polyamine

Concentrations

After Exogenously

Added Polyamines

in Primary Cultured

Rat Hepatocytes

Polyamine concentrations (pmo//pg DNA) Addition

Putrescine

Spermidine

Spermine

Control

2.7 -t 0.4

196.8 2 12.9

105.7

HGF HGF + DFMO HGF + DFMO + Put HGF + DFMO + Spd

2.8 + 0.6 2.0 k 0.2

369.7 253.6

183.7 t 8.1” 171.4 5 15.1

HGF HGF HGF HGF HGF

+ + + + +

260.5 5 24.1

71.8 2 10.7”

404.3

2.1 5 0.2 2.1 67.5 62.8 74.2 63.6

DFMO + Spm MGBG MGBG + Spd MGBG + Spm MGBG + Spd + Spm

2 + 2 ? 2

+ 13.4” k 27/Ib 2 17.3’

249.0 2 23.2 249.7 2 6.2’ 423.8 2 10.0’ 231.1 5 21.1 386.6 -c 13.2’

0.2 5.6b 12.1 8.3 7.6

+ 18.3

161.3 t 5.7

165.3 k 5.4 263.9 96.1 79.8 248.9 269.2

+ 2 % ? 2

12.3” 13.6b 14.3 13.2’ 41.0’

NOTE. After a change to fresh medium, cells were cultured in HGF (10 ng/mL) with or without 5 mmol/L DFMO or 20 pmol/L MGBG. At 8 hours, 0.1 mmol/L putrescine (Put), 0.1 mmol/L spermidine (Spd), or 0.1 mmol/L spermine (Spm) was added. Polyamine concentrations were measured 16 hours after the addition of HGF. Values are expressed as means t SD of five dishes. “P < 0.01 compared with control. *P < 0.01 compared with HGF. ‘P < 0.01 compared with HGF + DFMO. dP < 0.01 compared with HGF + MGBG.

also, the suppression

hepatectomy; completely

nous putrescine regeneration

portant

Furthermore,

exoge-

used to stimulate

liver

1. Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama 0,

and

in animal models of acute liver failure”

alcohol-induced putrescine,

was successfully

References

of DNA synthesis was

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liver injury.36 These results suggest that

rather than spermidine

for liver regeneration.

The

our results and previous

studies

explanation

between

is difference

or spermine,

is im-

difference

2.

between

is not clear; a possible in vitro and in vivo

3.

conditions. It has been documented dine are essential

that putrescine

for proliferating

and spermi-

rat hepatoma

tissue

culture cells.37,38 On the other hand, it has been reported that spermidine

or spermine is required for DNA synthethese results are similar

sis in lymphocytes39-41;

results with cultured rat hepatocytes. stood which polyamine although

is critical

to our

It is not well under-

for proliferating

it may depend on the type of cultured

cells, cells or

tissues. Recently, transient

it was shown that HGF

generation

biphasic production

of inositol

induced

of 1 ,2-diacylglycerol.42

in primary cultured

is important

for ODC

rat hepatoma

tissue culture

duction

in lymphocytes.”

induction

Moreover,

ODC and SAMDC

though

between

caused by HGF

not yet clear.

8.

it 9.

and

it is possible

Ca*+ concentration

volved in HGF-induced changes

7.

by

and that Ca*+

in lymphocytes45

Therefore,

6.

for HGF.*3,44 Ca2+

cells,46 and for SAMDC

an increase in the intracellular the relationship

and a

were induced

rat hepatocytes

acted as one of the second messengers

5.

an early

1,4,5-triphosphate

has been shown that Ca2+ oscillations HGF

4.

inthat

is in-

activity,

al11.

very early biochemical

and polyamine

10.

metabolism

is

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Received June 14, 1993. Accepted November 16, 1993. Address requests for reprints to: lkko Higaki, M.D., Second Depattment of Biochemistry, Osaka City University Medical School, 1454 Asahimachi, Abenoku, Osaka 545, Japan. Fax: (81) 66452030. The authors thank Dr. Tomoyoshi Nishino (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) for providing recombinant human hepatocyte growth factor and Hisako Fujimoto, Toshiko Yoshimata, and Kumle Nagahama for their excellent technical assistance.