ligation of portal vein branch induces dna polymerases α, δ, and ϵ in nonligated lobes

JOURNAL OF SURGICAL RESEARCH 65, 15–24 (1996)ARTICLE NO. 0337

Ligation of Portal Vein Branch Induces DNA Polymerasesa, d, and e in Nonligated Lobes1

EIJI TAKEUCHI,* YUJI NIMURA,* SHIN-ICHI MIZUNO,* MASATO NAGINO,* MAMI SHOJI-KAWAGUCHI,†SHUNJI IZUTA,† AND SHONEN YOSHIDA†,2

*First Department of Surgery and †Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control,Nagoya University School of Medicine, Nagoya 466, Japan

Submitted for publication March 4, 1996

trophy in the future remnant liver before partial hepa-Ligation of a portal vein branch supplying 70% of tectomy. Currently we perform percutaneous transhe-

the rat liver causes compensatory hypertrophy of the patic portal vein embolization before extensive livernonligated hepatic lobes with concomitant atrophy of resections for biliary tract cancer in order to preventthe ligated lobes. To elucidate the mechanism of this hepatic failure [4–9]. Using rat models, we have shownresponse, the induction of the replication enzymes that activity of DNA polymerase a in crude liver extractDNA polymerases a, d, e, as well as proliferating cell is a good marker for the regenerating capacity of hepa-nuclear antigen (PCNA), were investigated in nonli- tocytes [10, 11]. Using this marker, it was seen thatgated lobes after portal branch ligation. The induction portal branch ligation strongly induced hepatocyte pro-patterns were compared with the well studied liver liferation in nonligated lobes, with the activitiy of DNAregeneration after 70% partial hepatectomy. DNA poly-

polymerase a reaching maximum levels at 48 hr [12],merases a, d, and e in the liver were extracted with 5in the case of partial hepatectomy, maximum activitymM KCl (low-salt extract), then with 600 mM KCl (high-was attained by 26 hr [11]. We have also shown thatsalt extract). DNA polymerases a, d, and e in low-saltexternal biliary drainage [13] suppressed the regener-extract were partially separated on a hydroxyapatiteating capacity in cholestatic rat liver after partial hepa-column and quantified. All enzyme activities in thetectomy [14, 15]. In contrast, the regeneration inducednonligated lobes started to increase within 24 hr andby portal branch ligation was relatively unsuppressedreached maximum levels by 48 hr after portal branchby external biliary drainage [12]. These results suggestligation. These patterns were quite similar to thosethat portal branch ligation might induce hepatocyteobtained with the remnant liver after partial hepatec-

tomy. In low-salt extract, DNA polymerase d and ewere proliferation in the nonligated lobes by a mechanismprominent, while, in high-salt extract, largely DNA different from proliferation following partial hepatec-polymerases a and some activity of e were recovered. tomy. A number of pathways might potentially inducePCNA was also induced after both portal branch liga- cell growth in the liver, depending on various prolifera-tion and partial hepatectomy, reaching maximum lev- tion stimuli. These signal transduction pathways con-els at 48 hr. From the similar changes in DNA polymer- verge at DNA replication via protein kinase cascadesases and PCNA, our data indicate that portal branch that induce the transcription of DNA replication en-ligation induces hepatocyte proliferation in the nonli- zymes and factors.gated lobes in a way similar to partial hepatectomy. In the present study, we focused on the induction ofq 1996 Academic Press, Inc. DNA replication enzymes, especially DNA polymerases

a, d, and e. In general, eukaryotes have five species ofDNA polymerases, a, b, g, d, and e [16], and among

Ligation of a portal vein branch of the liver causes these polymerases a, d, and e are implicated in nuclearcompensatory hypertrophy of the nonligated hepatic DNA replication. DNA polymerase a, complexed withlobes, with concomitant atrophy of the ligated lobes [1]. DNA primase, is implicated in the initiation of bothKinoshita et al. [2] and Makuuchi et al. [3] embolized Okazaki fragment synthesis on the lagging strand andthe human portal branch by percutaneous transhepatic leading strand synthesis [17]. DNA polymerase d, origi-portal catheterization to induce compensatory hyper- nally distinguished from DNA polymerase a by pos-

sessing 3 *–5* exonuclease activity [18–23], is believed1 This work was supported in part by a Grant-in-Aid for General to replicate the leading strand in association with

Scientific Research (Grant No. 04404050) from the Ministry of Edu- PCNA, an auxiliary factor for this enzyme [24–26].cation, Science and Culture, Japan, and the grants from the Uehara DNA polymerase e is also thought to be involved inMemorial Foundation.

leading strand synthesis [27–30]. The exact roles of2 To whom correspondence should be addressed. Fax: 052-744-2457. these DNA polymerases are still under debate. In order

15 0022-4804/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID JSR 4902 / 6n14$$$$21 09-17-96 00:19:36 srga AP: Surg Res

16 JOURNAL OF SURGICAL RESEARCH: VOL. 65, NO. 1, SEPTEMBER 1996

collected (high-salt extract). The solution was diluted fivefold withto measure the activities of DNA polymerases a, d, andbuffer C. Then, DNA polymerase a activity in high-salt crude extracte, we performed a two-step extraction and further par-was determined.tially separated these enzymes in a low-salt extraction The low-salt extract was filtered through cheesecloth and applied

on a hydroxylapatite column, seeking to avoid the to a DEAE-cellulose (3.51 20 cm, Whatman DE-52) previously equil-cross-contamination among similar activities. DNA ibrated with buffer A. The column was eluted with buffer B con-

taining 300 mM KCl. The fractions containing DNA polymerasespolymerase a was largely recovered in the high-saltwere pooled and diluted twofold with buffer A (except KCl was omit-extract. The main aim of the present study is to deter-ted). The diluted fraction was applied to a hydroxyapatite columnmine whether or not the induction patterns of these (1.3 1 50 cm, Gigapite, Toa-Gosei, Nagoya, Japan) previously equili-

replication enzymes after portal branch ligation differ brated with buffer D. The column was washed with buffer D andfrom those prevailing in the case of partial hepatec- then eluted with a gradient (250 ml) from 20 to 800 mM potassium

phosphate at pH 7.2. DNA polymerases d, a, and e were eluted intomy, reflecting possible differences in proliferationthis order as triple peaks between 20 and 190 mM potassium phos-stimuli.phate. The activities of DNA polymerases a, d, and e were calculatedas the total sum of the activities in the each fraction from the hy-

MATERIALS AND METHODS droxyapatite column, respectively. The fractions containing DNApolymerases a, d, and e were pooled and dialyzed against buffer E,

Animals. Six-week-old adult male rats of the Donryu strain, and stored at 0207C. The separated DNA polymerase d was appliedweighing 240–260 g were purchased from Chubu Shiryo Co. (Na- to a HiTrap-SP column (1 cm, Pharmacia LKB Biotechnology) equili-goya, Japan). Rats received food and water freely in an air-condi- brated with buffer F. The column was washed with buffer F andtioned room. After maintenance for 1 week, they were divided into then eluted with a gradient (50 ml) from 0 to 1 M KCl to purify DNAthree groups: a 70% partial hepatectomy group, a 70% portal branch polymerase d.ligation group, and a sham-operated group. Laparotomy was done by High-salt extract was diluted fivefold with buffer A (except KCla midline incision under ether anesthesia. In the partial hepatectomy was omitted) and then centrifuged at 20,000g for 30 min at 47C andgroup, the median and the left lateral lobes (70% of total liver vol- supernatant was collected and filtered through cheesecloth. In thisume) were resected according to the procedure of Higgins and Ander- case, DEAE-cellulose column chromatography was omitted to avoidson [31]. In the portal branch ligation group, the portal branch sup- inactivation of DNA polymerases by an unknown reason. So, theplying the same lobes was ligated according to the procedure of extract was applied to a hydroxyapatite column (1.3 1 50 cm, Giga-Steiner and Martinez [32]. Rats were sacrificed under ether anesthe- pite, Toa-Gosei, Nagoya, Japan) previously equilibrated with buffersia at 12, 26, 48, 72, and 96 hr and at 1 week (for blood analysis) D. The column was washed with buffer D and then eluted with aafter the operation. The remnant livers after partial hepatectomy, gradient (250 ml) from 20 to 800 mM potassium phosphate, pH 7.2.and the ligated and nonligated lobes after portal branch ligation, The activities of DNA polymerases a, d, and e were calculated as thewere obtained. The sham-operated rats were sacrificed immedately total sum of each response activity in the each fraction from theafter laparotomy, and the livers were removed. The right lateral and hydroxyapatite column. The fractions containing DNA polymerasescaudate lobes represented 70 and 30% of total liver volume. The a, d, and e were pooled and dialyzed against buffer E and stored atlivers were weighed and stored at 0807C until their use. All animals 0207C.received humane care in compliance with the Guidelines for Animal Mouse PCNA was expressed in Escherichia coli transfected withExperiments established at Nagoya University. a recombinant plasmid carrying PCNA-cDNA [Ref. 34; a kind gift

Reagents. Unlabeled deoxyribonucleoside triphosphates (dNTPs) from Dr. Akio Matsukage, Aichi Cancer Center] and was purified bywere purchased from Yamasa Shoyu Co. Ltd. (Chiba, Japan). Radio- columns using HiLoad 16/10 Q Sepharose HP and an anti-PCNAactive compounds were obtained from ICN Pharmaceuticals (CA). antiboby-conjugated column as described [35].Activated calf thymus DNA was prepared as described previously Assay conditions for DNA polymerases. DNA polymerase a was[33]. Poly(dA)-oligo(dT)12–18 (A/T Å 20) and poly(dA-dT) were from assayed in the reaction mixture (25 ml) containing 80 mM potassiumPharmacia LKB Biotechnology (Uppsala, Sweden). Anti-mouse phosphate (pH 7.2), 8 mM 2-mercaptoethanol, 8 mM MgCl2, 80 mMPCNA monoclonal antibody was purchased from Oncogene Science, dATP, 80 mM dGTP, 80 mM dCTP, 40 mM [3H]dTTP (500 cpm/pmol),Inc. (New York, NY). All other reagents were from commercial and 200 mg/ml activated calf thymus DNA [36]. DNA polymerase dsources. was assayed in the reaction mixture (25 ml) containing 40 mM

Buffers. The following buffers were used: buffer A, 5 mM Tris– Hepes–KOH (pH 6.5), 1 mM MgCl2, 10 mM KCl, 2 mM DTT, 0.03%HCl at pH 7.5, 0.25 M sucrose, 1 mM dithiothreitol (DTT), 1 mM Triton X-100, 2% glycerol, 80 mg/ml bovine serum albumin, 50 mMphenylmethylsulfonylfluoride (PMSF), 10 mM sodium bisulfite, 2 dATP, 50 mM [3H]dTTP (500 cpm/pmol), and 45 mM poly(dA-dT) [21].mM benzamidine, 0.1 mM (ethylenedinitrilo) tetraacetic acid DNA polymerase e was assayed in the reaction mixture (25 ml) con-(EDTA), and 5 mM KCl; buffer B, 50 mM Tris–HCl at pH 7.5, 0.25 taining 50 mM bisTris–HCl (pH 6.7), 5 mM MgCl2, 1 mM dithiothrei-M sucrose, 1 mM DTT, 1 mM PMSF, 10 mM sodium bisulfite, 2 mM tol, 50 mM [3H]dTTP (500 cpm/pmol), 100 mg/ml bovine serum albu-benzamidine, 0.1 mM EDTA, 600 mM KCl, and 0.5% Nonidet P-40; min, and 25 mg/ml poly(dA)-oligo(dT)12–18 [37].buffer C, 25 mM Tris–HCl at pH 7.5, 1 mg/ml bovine serum albumin After incubation for 30 min at 377C, acid-insoluble radioactivity(BSA), 1 mM DTT, and 10% glycerol; buffer D, 20 mM potassium was measured [33]. One unit of DNA polymerase catalyzed the incor-phosphate (K2HPO4 and KH2PO4) at pH 7.2, 10% glycerol, and 0.5 poration of 1 nmol of dNTPs into DNA at 377C in 60 min under themM DTT; buffer E, 20 mM KPO4 at pH 7.2, 55% glycerol, 1 mM conditions above.DTT, 0.5% Triton X-100 (Sigma Chemical Co.), and 0.2% Polybuffer

Measurement of bilirubin and enzymes in blood. Blood was with-74 (Pharmacia Fine Chemicals); and buffer F, 50 mM Tris–HCl atdrawn from the abdominal aorta at the time of sacrifice, and serumpH 7.5, 5% glycerol, and 0.5 mM DTT.was obtained by centrifugation (3,000 rpm, 20 min). Total bilirubin

Enzyme extraction. Two livers resected at the same time (total 8 concentration (T-Bil) and activities of glutamic oxaloacetic transami-to 20 g) were homogenized with a Polytron (Kinematica, Switzerland) nase (GOT), glutamic pyruvic transaminase (GPT), and alkalineand sonicated for 30 sec using a Micro Ultrasonic Cell Disrupter

phosphatase (ALP) were then measured as described [38–41].(Kontes) in 9 vol of buffer A. One milliliter of the homogenate (crudeMeasurement of DNA content. DNA content was measured usingextract) was used for measurement of DNA content and immunoblot-

a fluorescent reagent (Hoechst 33258; Sigma Chemical Co., St. Louis,ting. The remainder was centrifuged at 20,000g for 20 min twice atMO) according to the method of Labarca and Paigen [42].47C and supernatant was collected (low-salt extract). Then, DNA

Other methods. Sodium dodecyl sulfate–polyacrylamide gel elec-polymerase a activity in low-salt crude extract was determined. Thetrophoresis and immunoblotting were performed according to Laem-pellet was homogenized with a Polytron in 5 vol of buffer B and

then centrifuged at 20,000g for 20 min at 47C and supernatant was mli [43] and Towbin et al. [44], respectively.


17TAKEUCHI ET AL.: LIGATION OF PORTAL BRANCH INDUCES DNA POLYMERASES a, d, AND e

ligation were significantly higher than those after par-tial hepatectomy (Table 1).

Activities of DNA polymerases a, d, and e. Two-stepextraction was employed in this study, because the sep-aration of DNA polymerases d and e from a can beachieved with the low-salt extract but is difficult withthe extract obtained by the one-step extraction withhigh-salt used in previous studies [10, 11, 12, 14, 15].The activity with activated calf thymus DNA templatewas measured using low-salt extract (Table 2). Thisactivity, largely due to DNA polymerase a [35], startedto increase before 20 hr and reached maximum at 48hr in the nonligated lobes after portal branch ligation.The same pattern was observed with remnant liversafter partial hepatectomy (Table 2). Then, this low-saltextract was subjected to DEAE-cellulose and hydroxy-apatite column chromatography (Fig. 3). DNA polymer-ases were eluted in the order d, a, and e from a potas-sium phosphate gradient. Both partial hepatectomy

FIG. 1. Changes in wet weight of the liver. Each point representsand portal branch ligation induced high activities ofthe mean { SD for 5–10 animals. *P õ 0.05, statistically significantthese polymerases after 72 hr, compared with those indifference between partial hepatectomy group and portal branch liga-

tion group according to Student’s t test. s, remnant liver after partial sham-operated liver. The data are summarized in Fig.hepatectomy; j, nonligated lobes after portal branch ligation; n, 4. After portal branch ligation, activities of DNA poly-ligated lobes after portal branch ligation. merases a, d, and e started to increase within 24 hr,

reaching their maximum levels at 48 hr in the nonli-gated lobes (Fig. 4A). Similarly, after partial hepatec-

Statistics and image analyses. The data were expressed as the tomy, activities of these three enzymes increased andmean { standard deviation. Statistical analyses were performed us-reached their maximum levels at 48 hr in the remnanting Student’s t test. Differences with a P value of less than 0.05 were

considered statistically significant. The gel blottings were analyzed liver (Fig. 4B).by MASTERSCAN (CSPI). The activity with activated calf thymus DNA in 600

mM KCl crude extract, largely representing DNA poly-merase a, also increased with time, reaching maximumRESULTSat 48 hr in both the nonligated lobes after portal branchligation and remnant liver after partial hepatectomy

Wet weight of the liver. After partial hepatectomy,the wet weights of the remnant liver increased withtime to 2.4-fold at 96 hr. Similarly after portal branchligation, the nonligated lobes enlarged 2.0-fold at 96hr, while the ligated lobes shrunk to 16% of originalweights at 96 hr after portal branch ligation. The in-crease in weights of the remnant liver at 72 and 96hr after partial hepatectomy were slightly higher thanthose of the nonligated lobes after portal branch liga-tion (Fig. 1).

DNA content. The total amount of DNA in the non-ligated lobes increased 2.0-fold at 96 hr after portalbranch ligation, the same degree of increase as for wetweight. The total DNA contents of the remnant liverincreased after partial hepatectomy and enlarged 2.3-fold at 96 hr (Fig. 2).

Enzyme activities and total bilirubin concentration.Serum total bilirubin and alkaline phosphatase levelsrapidly increased and reached their maximums at 12hr after partial hepatectomy. After portal branch liga-tion, they increased more slowly, reaching lower maxi-mums at 72 hr. By 1 week postoperatively, both serum

FIG. 2. Changes in total DNA contents of the liver. Each pointconcentrations were normal in both groups. GOT andrepresents the mean { SD for 5–10 animals. *Põ 0.05, statisticallyGPT levels reached their maximums at 12 hr after ei- significant difference between partial hepatectomy group and portal

ther operation and then decreased to control level by branch ligation group. s, remnant liver after partial hepatectomy;j, nonligated lobes after portal branch ligation.96 hr. The levels of GOT and GPT after portal branch



TA

BL

E1

Ch

ange

sin

Lev

els

ofS

eru

mT

otal

Bil

iru

bin

,AL

P,G

OT

,an

dG

PT

Hou

rsaf

ter

oper

atio

n

Gro

up

012

2648

7296

168

T-B

il(m

g/dl

)P

arti

alh

epat

ecto

my

0.02{

0.04

0.50{

0.16

*0.

35{

0.15

*0.

39{

0.16

*0.

34{

0.19

0.24{

0.15

0.10{

0P

orta

lbr

anch

liga

tion

0.02{

0.04

0.07{

0.05

*0.

10{

0*0.

26{

0.08

*0.

45{

0.10

0.20{

0.11

0.10{

0A

LP

(IU

/L)

Par

tial

hep

atec

tom

y35

6{

8910

64{

234*

1070{

253*

1108{

292*

1034{

215

642{

79*

565{

38*

Por

tal

bran

chli

gati

on35

6{

8946

1{

54*

490{

60*

543{

80*

764{

315

537{

40*

402{

44*

GO

T(I

U/L

)P

arti

alh

epat

ecto

my

153{

7815

96{

266*

1005{

272*

505{

159*

396{

66*

274{

7419

5{

24P

orta

lbr

anch

liga

tion

153{

7827

57{

473*

2246{

718*

1771{

668*

604{

171*

320{

120

160{

22G

PT

(IU

/L)

Par

tial

hep

atec

tom

y40{

854

8{

174

275{

85*

104{

23*

96{

1565{

1840{

8P

orta

lbr

anch

liga

tion

40{

865

5{

135

444{

146*

414{

158*

152{

7075{

2437{

3

Not

e.T

he

data

repr

esen

ted

the

mea

n{

SD

for

5–

10an

imal

s.*

Põ

0.05

,st

atis

tica

lly

sign

ifica

nt

diff

eren

cebe

twee

npa

rtia

lh

epat

ecto

my

grou

pan

dpo

rtal

bran

chli

gati

ongr

oup.

TA

BL

E2

Ch

ange

sin

Act

ivit

ies

ofD

NA

Pol

ymer

asesa

,d,a

nde

DN

Apo

lym

eras

ea

(hr

afte

rop

erat

ion

)d

(hr)

e(h

r)

Gro

up

Ext

ract

ion

012

2648

7296

048

048

Par

tial

hep

atec

tom

yL

owsa

lt1.

52.

96.

98.

67.

94.

42.

88.

50.

55

Hig

hsa

lt26

22.7

46.7

78.8

72.7

35.8

26

3.1

10.6

Por

tal

bran

chli

gati

onL

owsa

lt1.

51.

84.

89.

29.

27.

62.

89

0.5

6.8

Hig

hsa

lt26

19.5

37.8

83.2

7965

.82

4.4

3.1

7

Not

e.T

he

data

wer

eex

pres

sed

asu

nit

spe

rm

illi

gram

DN

Aof

orig

inal

tiss

ues

.A

ctiv

itie

sof

DN

Apo

lym

eras

ea

wer

em

easu

red

inlo

wan

dh

igh

salt

extr

acts

,an

dth

ose

ofd

ande

ofbo

thlo

wan

dh

igh

salt

extr

acts

at0

and

48h

raf

ter

the

oper

atio

nw

ere

mea

sure

daf

ter

chro

mat

ogra

phic

sepa

rati

onw

ith

ah

ydro

xyla

pati

teco

lum

n.

AID JSR 4902 / 6n14$$4902 09-17-96 00:19:36 srga AP: Surg Res


FIG. 3. Elution profiles of DNA polymerases a, d, and e from hydroxylapatite column chromatography in low salt extract. (A) Sham-operated liver. (B) Nonligated lobes at 72 hr after portal branch ligation. (C) Remnant liver at 72 hr after partial hepatectomy. n, DNApolymerase a; j, DNA polymerase d; 1, DNA polymerase e.

(Table 2). However, the maximum activities of DNA low-salt extract (Fig. 3). The activities of each enzymespecies are summarized in Table 2. Similarly, the activ-polymerase a in 600 mM KCl crude extract were 9ities of DNA polymerases d and e also increased 3- totimes higher than those in 5 mM KCl crude extract.5-fold at 48 hr after the two operations compared toAlthough it seems that the high-salt extract still con-those in sham-operated liver.tains DNA polymerases d and e besides a, they are

very unstable on DEAE-cellulose column for unknown Identification of each enzyme species. The activitiesreasons (data not shown). Therefore, we applied the of DNA polymerases a, d, and e measured here werehigh-salt extract directly on the hydroxyapatite column not due to contaminating DNA polymerases b or g,

since the potent inhibitor of DNA polymerases b and(Fig. 5) but the separation was less clear then with the



mouse PCNA antibody (Fig. 7A). Densitometry of thegel staining was performed as shown in Fig. 7B. Theamount of PCNA increased after either portal branchligation or partial hepatectomy, and reached maximumat 48 hr, similarly to the induction of DNA polymer-ases.

DISCUSSION

We have shown that the compensatory hypertrophyof the nonligated hepatic lobes after portal branch liga-tion results from a strong induction of hepatocyte pro-liferation [12, this study]. Previously we have shownthat DNA polymerase a is present in proliferating cellsthroughout G1, S, M, and G2 phases [45]. By measuringthis enzyme activity in crude liver extract, we havedemonstrated that the activity reached maximum at48 hr after portal branch ligation, while after partialhepatectomy, it reached maximum at 26 hr [12]. Fur-thermore, these two induction systems differ from eachother in the sensitivity to external biliary drainage [12,14, 15]. These results prompted us to compare the in-duction of DNA polymerases more precisely. Here wedemonstrated that the activities of DNA polymerasesa, d, and e after portal branch ligation reached maxi-mum at 48 hr by measuring partially purified enzymesafter two successive chromatographic separations.Contrary to our expectation, the induction pattern ofall three enzyme activities after partial hepatectomywas similar to that after portal branch ligation. Theinductions of PCNA after portal branch ligation andafter partial hepatectomy both reached maximum at

FIG. 4. Changes in activities of DNA polymerases a, d, and e in 48 hr. The reason for the apparent discrepancy betweenlow salt extract calculated from hydroxylapatite column chromatog- results using crude extracts and partially purified frac-raphy. Activities are expressed as units per mg DNA of original tions is not clear, but it is conceivable that the crudetissues. (A) Nonligated lobes after portal branch ligation. (B) Rem-

extracts contain an intrinsic inhibitor of the DNA poly-nant liver after partial hepatectomy. n, DNA polymerase a; j, DNAmerase reaction, which have been removed by two col-polymerase d; 1, DNA polymerase e.umn separations. It is concluded that responses tothese two liver regeneration stimuli are similar with

g, 2 *,3 *-dideoxyTTP did not affect these activities. Fur- respect to the DNA replication machinery. The incorpo-ther, potassium phosphate, an inhibitor of DNA poly- ration of [3H]thymidine pulse-labeled at various timesmerases b and a stimulator of g, did not affect those after portal branch ligation reached maximum at 24 hractivities (data not shown). DNA polymerase d can be [46], the same interval as for partial hepatectomy. Thedistinguished from e by its PCNA-dependency. The hy- prolonged induction of DNA polymerases may indicatedroxyapatite fractions in low salt extract, correspond- that, after the first burst of DNA synthesis, the regen-ing to DNA polymerase d, did not respond to PCNA. eration capacity of liver may still increase until 48However, after further purification of this fraction on hours after either portal branch ligation or partial hep-HiTrap-SP column, the activity became highly PCNA atectomy.dependent (Fig. 6). The maximum stimulation was In regenerating liver after both partial hepatectomy28.5-fold with poly(dA-dT) template and 1.7-fold with and portal branch ligation, all replicative DNA poly-poly(dA)-oligo(dT)12–18 template with the addition of merases, a, d, and e, increased simultaneously. Yang260 ng mouse PCNA. The fractions of DNA polymerase et al. [47] have obtained similar results by using crudee were not affected by the addition of mouse PCNA. extracts and by immunochemistry. We measured theThese results strongly suggest that the DNA polymer- activities of these enzymes more precisely. In low saltase d fraction from hydroxyapatite column was satu- extract, the activity of DNA polymerase d was higherrated with PCNA but was dissociated from this cofactor than those of a and e, while in high salt extract, theon the HiTrap SP column. activities of DNA polymerases a and e were higher.

The maximum activities of DNA polymerase a in 600Induction of PCNA. Immunoblotting was per-formed with crude extracts (4 mg protein) at several mM KCl crude extract were 9 times higher than those

in 5 mM KCl crude extract. The rates of increase at 48time points after portal branch ligation using an anti-



FIG. 5. Elution profiles of DNA polymerases a, d, and e from hydroxylapatite column chromatography in high salt extract. (A) Sham-operated liver. (B) Nonligated lobes at 48 hr after portal branch ligation. (C) Remnant liver at 48 hr after partial hepatectomy. n, DNApolymerase a; j, DNA polymerase d; 1, DNA polymerase e.

hr after partial hepatectomy were 3.0-, 4.5-, and 3.8- ary drainage suppressed regeneration in cholestatic ratliver after partial hepatectomy, while it failed to suppressfold for DNA polymerases a, d, and e, respectively.

These results suggest that DNA polymerases d and e regeneration after portal branch ligation [12, 15], sug-gesting a difference in the proliferation signaling sys-also have important roles in nuclear DNA replication

as well as DNA polymerase a. Our purified polymerase tems. Nakamura et al. [48] found the hepatocyte growthfactor (HGF). In liver injury, another factor, injurin [49],d was stimulated 28.5-fold by the addition of mouse

PCNA, unlike DNA polymerase e. The induction of is first induced, and stimulates lung tissue to expressHGF [50], which induces hepatic growth. Alternatively,PCNA synchronized well with DNA polymerases.

In previous studies, we have shown that external bili- Michalopoulos et al. [51, 52] have proposed that de-



creased liver capacity for clearance of HGF released fromstorage sites initiates hepatocyte proliferation.

In portal branch ligation, the ligated lobes mayplay some roles. Kerr et al. [53] have demonstratedthat portal branch ligation induces apoptosis in theligated lobes by electron microscopy. This is accompa-nied by loss of liver volume as shown in Fig. 1. How-ever, the ligated lobes may temporally maintain adegree of function, which could explain the lower andslower increase of total serum bilirubin in portalbranch ligation than in partial hepatectomy (Table1). Likewise, the uptake of indocyanine green [54] orbile acids [55] are impaired after partial hepatec-tomy. The remnant liver after partial hepatectomymust bear burdens of both cell proliferation and liverfunctions. These might be why the nonligated lobesare able to regenerate despite loss by external biliarydrainage, differing from the remnant liver after par-tial hepatectomy. Alternatively, the ligated lobesmay initiate strong growth signals including theHGF pathway to overcome the suppressive effects ofexternal biliary drainage. But the exact mechanismmust wait further study.

With respect to clinical application, Uesaka et al.FIG. 7. Immunoblottings for PCNA after portal branch ligation.[56] reported that the functional capacity of nonem-

(A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis andbolized lobes as assessed by biliary indocyanineimmunoblotting were performed as described under Materials andgreen (ICG) excretion was enhanced at 11 days afterMethods: mouse PCNA, 1 mg; crude extract 4 mg at 0, 12, 26, 48, 72,

portal vein embolization in humans. Nagino et al. [5] and 96 hr after portal branch ligation. (B) Densitometry. The amountreported that from computed tomographic volumetry of PCNA was measured using a densitometer as described under

Materials and Methods. j, nonligated lobes after portal branch liga-and the ICG disappearance rate in human, portaltion; s, remnant liver after partial hepatectomy.vein embolization produced a compensatory hyper-

trophy within 11 days without seriously affectinghepatic function. From this, portal vein embolization

ACKNOWLEDGMENTSprior to hepatic resection can be recommendedfor inducing liver regeneration in the future remnant Authors thank Dr. Akio Matsukage of Aichi Cancer Center Re-

search Institute for providing PCNA-recombinant plasmid, Dr. Keikoliver, in order to prevent postoperative hepaticTamiya-Koizumi, Dr. Motoshi Suzuki, and Dr. Hideto Yoshida offailure.the Research Institute for Disease Mechanism and Control, NagoyaUniversity School of Medicine, for helpful discussion. We are gratefulto Mr. Y. Ito, Miss M. Takahashi, and Mrs. T. Tomita for excellenttechnical assistance.

REFERENCES

1. Rous, P., and Larimore, L. D. Relation of the portal blood toliver maintenance. J. Exp. Med. 31: 609, 1920.

2. Kinoshita, H., Sakai, K., Hirohashi, K., Igawa, S., Yamasaki,O., and Kubo, S. Preoperative portal vein embolization for hepa-tocellular carcinoma. World J. Surg. 10: 803, 1986.

3. Makuuchi, M., Thai, B. L., Takayasu, K., Takayama, T., Ko-suge, T., Gunven, P., and Yamazaki, S., et al. Preoperative por-tal embolization to increase safety of major hepatectomy forhilar bile duct carcinoma: A preliminary report. Surgery 107:521, 1990.

4. Nagino, M., Nimura, Y., and Hayakawa N. Percutaneous trans-hepatic portal embolization using newly devised catheters: Pre-liminary report. World J. Surg. 17: 520, 1993.

5. Nagino, M., Nimura, Y., Kamiya, J., Kondo, S., Uesaka, K., Kin,Y., Hayakawa, N., and Yamamoto, H. Changes in hepatic lobeFIG. 6. Effect of mouse PCNA on DNA polymerase d. The activi-volume in biliary tract cancer patients after right portal veinties of DNA polymerase d in the presence or absence of PCNA (260embolization. Hepatology 21: 434, 1995.ng) were measured by using the template of poly(dA-dT) or poly(dA)-

oligo(dT)12–18 . Mouse PCNA was obtained as described under Materi- 6. Nagino, M., Nimura, Y., Kamiya, J., Kondo, S., Uesaka, K., Kin,Y., Kutsuna, Y., Hayakawa, N., and Yamamoto, H. Right or leftals and Methods.



trisegment portal vein embolization before hepatic trisegmen- H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-d.Nature 362: 515, 1987.tectomy for hilar bile duct carcinoma. Surgery 117: 677, 1995.

25. Prelich, G., Tan, C. K., Kostura, M., Mathews, M. B., So,7. Nimura, Y., Hayakawa, N., Kamiya, J., Kondo, S., and Shio-A. G., Downey, K. M., and Stillmann, B. Functional identity ofnoya, S. Hepatic segmentectomy with caudate lobe resection forproliferating nuclear antigen and a DNA polymerase-d auxil-bile duct carcinoma of the hepatic hilus. World J. Surg. 14: 535,iary protein. Nature 326: 517, 1987.1990.

26. Waga, S., and Stillmann, B. Anatomy of a DNA replication fork8. Nimura, Y., Hayakawa, N., Kamiya, J., Maeda, S., Kondo, S.,revealed by reconstitution of SV40 DNA replication in vitro.Yasui, A., and Shionoya, S. Hepatopancreatodoudenectomy forNature 359: 207, 1994.advaned carcinoma of the biliary tract. Hepatogastroenterology

38: 170, 1991. 27. Kesti, T., and Syvaoja, J. E. Identification and tryptic cleavageof the catalytic core of HeLa and calf thymus DNA polymerase9. Nimura, Y., Hayakawa, N., Kamiya, J., Maeda, S., Kondo, S.,e. J. Biol. Chem. 266: 6336, 1991.Yasui, A., and Shionoya, S. Combined portal vein and liver

resection for carcinoma of the biliary tract. Br. J. Surg. 78: 727, 28. Siegal, G., Turchi, J. J., Jessee, C. B., Mallaber, L. M., Bambra,1991. R. A., and Myers, T. W. Structural relationships between two

forms of DNA polymerase e from calf thymus. J. Biol. Chem.10. Kuriki, H., Tamiya-Koizumi, K., Asano, M., Yoshida, S., Kojima,267: 3991, 1992.K., and Nimura, Y. Existence of phosphoinositide-specific phos-

29. Syvaoja, J., and Linn, S. Characterization of a large form ofpholipase C in rat liver nuclei and its change during liver regen-DNA polymerase d from HeLa cells that is insensitive to prolif-eration. J. Biochem. 111: 283, 1992.erating cell nuclear antigen. J. Biol. Chem. 264: 2489, 1989.11. Terasaki, M., Kuruki, H., Nimura, Y., Shionoya, S., Kojima, K.,

30. Araki, H., Ropp, P. A., Johnson, A. L., Johnston, L. H., Morrison,and Yoshida, S. Induction of DNA replication and cell growthA., and Sugino, A. DNA polymerase II, the probable homolog ofin rat liver by obstructive jaundice. Jpn. J. Cancer Res. 82: 170,mammalian DNA polymerase epsilon, replicates chromosomal1991.DNA in the yeast Saccharomyces cerevisiae. EMBO J. 11: 733,12. Mizuno, S., Nimura, Y., Suzuki, H., and Yoshida, S. Portal1992.branch occlusion induces cell proliferation of cholestatic rat

31. Higgins, G. M., and Anderson, R. M. Experimental pathologyliver. J. Surg. Res. 60: 249, 1996.of the liver. I. Restoration of the liver of the white rat following

13. Denning, D. A., Ellison, E. C., and Carey, L. C. Preoperative partial surgical removal. Arch. Pathol. 12: 186, 1931.percutaneous transhepatic biliary decompression lowers opera-

32. Steiner, P. E., and Martinez, J. B. Effects on the rat liver oftive morbidity in patients with obstructive jaundice. Am. J.bile duct, portal vein and hepatic artery ligations. Am. J. Pathol.Surg. 141: 61, 1981.39: 257, 1961.

14. Iyomasa, S., Terasaki, M., Kuriki, H., Nimura, Y., Shionoya, S.,33. Yoshida, S., Kondo, T., and Ando, T. Multiple molecular speciesKojima, K., and Yoshida, S. Decrease in regeneration capacity of

of cytoplasmic DNA polymerase from calf thymus. Biochim. Bio-rat liver after external biliary drainage. Eur. Surg. Res. 24:phys. Acta 353: 463, 1974.265, 1992.

34. Matsuoka, S., Yamaguchi, M., and Matsukage, A. D-type cyclin-15. Suzuki, H., Iyomasa, S., Nimura, Y., and Yoshida, S. Internalbinding regions of proliferating cell nuclear antigen. J. Biol.biliary drainage, unlike external drainage, does not supress theChem. 269: 11030, 1994.regeneration of cholestatic rat liver after partial hepatectomy.

35. Shoji-Kawaguchi, M., Izuta, S., Tamiya-Koizumi, K., Suzuki,Hepatology 20: 1318, 1994.M., and Yoshida, S. Selective inhibition of DNA polymerase e

16. Burgers, P. M. J., Bambara, R. A., Campbell, J. L., Chang, by phosphatidylinositol. J. Biochem. 117: 1095, 1995.L. M. S., Downey, K. M., Hubscher, U., Lee, M. Y. W. T., Linn,

36. Tamai, K., Kojima, K., Hanaichi, T., Masaki, S., Suzuki, M.,S. M., So, A. G., and Spadari, S. Revised nomenclature for eu-Umekawa, H., and Yoshida, S. Structural study of immunoaf-karyotic DNA polymerases. Eur. J. Biochem. 191: 617, 1990.finity-purified DNA polymerase a–DNA primase complex from

17. Tsurimoto, T., Melendy, T., and Stillman, B. Sequential initia- calf thymus. Biochim. Biophys. Acta 950: 263, 1988.tion of lagging and leading strand synthesis by two different

37. Morrison, A., Araki, H., Clark, A. B., Hamatake, R. K., andpolymerase complexes at the SV40 DNA replication origin. Na-Sugino, A. A third essential DNA polymerase in S. cerevisiae.ture 346: 534, 1990.Cell 62: 1143, 1990.

18. Byrnes, J. J., Downey, K. M., Black, V. L., and So, A. G. A new 38. Michaelsson, M., Nosslin, B., and Sjolin, S. Plasma bilirubinmammalian DNA polymerase with 3 * to 5* exonuclease activity: determination in the newborn infant: A methodological studyDNA polymerase d. Biochemistry 15: 2817, 1976. with special reference to the influence of hemolysis. Pediatrics19. Weiser, T., Gassmann, M., Thommes, P., Ferrari, E., Hafke- 35: 925, 1965.

meyer, P., and Hubscher, U. Biochemical and functional com- 39. Bergmeyer, H. U., Kreutz, F. H., Pilz, W., Schmidt, F. W., Butt-parison of DNA polymerases a, d, and e from calf thymus. J. ner, H., Lang, H., Rick, W., et al. Recommendations of the Ger-Biol. Chem. 266: 10420, 1991. man Society for Clinical Chemistry. Z. Klin. Chem. Klin. Bio-

20. Goulian, M., Herrmann, S. M., Sackett, J. W., and Grimm, chem. 10: 182, 1972.S. L. Two forms of DNA polymerase d from mouse cells. J. Biol. 40. Wroblewski, F., and LaDue, J. S. Serum glutamic pyruvic trans-Chem. 265: 16402, 1990. aminase in cardiac and hepatic disease. Proc. Soc. Exp. Biol.

21. Syvaoja, J., Suomensaari, S., Nishida, C., Goldsmith, J. S., Med. 91: 569, 1956.Chui, G. S. J., Jain, S., and Linn, S. DNA polymerases a, d, and 41. Hausamen, T. U., Helger, R., Rick, W., and Gross, W. Optimale: Three distinct enzymes from HeLa cells. Proc. Natl. Acad. conditions for the determination of serum alkaline phosphataseSci. USA 87: 6664, 1990. by a new kinetic method. Clin. Chim. Acta 15: 241, 1967.

22. Lee, M. Y. W. T., Jiang, Y., Zhang, S. J., and Toomey, N. L. 42. Labarca, C., and Paigen, K. A simple, rapid, and sensitive DNACharacterization of human DNA polymerase d and its immuno- assay procedure. Anal. Biochem. 102: 344, 1980.chemical relationships with DNA polymerase a and e. J. Biol. 43. Laemmli, U. K. Cleavage of structural proteins during the as-Chem. 266: 2423, 1991. sembly of the head of bacteriophage T4. Nature 237: 680, 1970.

23. So, A. G., and Downey, K. M. Mammalian DNA polymerase a 44. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic trans-and d: Current status in DNA replication. Biochemistry 27: fer of proteins from polyacrylamide gels to nitrocellulose sheets:4591, 1988. Procedure and some applications. Proc. Natl. Acad. Sci. USA

76: 4350, 1979.24. Bravo, R., Frank, R., Blundell, P. A., and Macdonald-Bravo,



45. Nakamura, H., Morita, T., Masaki, S., and Yoshida, S. Intracel- 51. Lindroos, P. M., Zarnegar, R., and Michalopoulos, G. K. Hepato-cyte growth factor (Hepatopoietin A) rapidly increases inlular localization and metabolism of DNA polymerase a in hu-

man cells visualized with monoclonal antibody. Exp. Cell Res. plasma before DNA synthesis and liver regeneration stimulatedby partial hepatectomy and carbon tetrachloride administra-151: 123, 1984.tion. Hepatology 13: 743, 1991.46. Lee, K. C., Kinoshita, H., Hirohashi, K., Kubo, S., and Iwasa,

R. Extension of surgical indication for hepatocellular carcinoma 52. Zarnegar, R., DeFrances, M. C., Kost, D. P., Lindroos, P., andMichalopoulos, G. K. Expression of hepatocyte growth factorby portal vein embolization. World J. Surg. 17: 109, 1993.mRNA in regenerating rat liver after partial hepatectomy. Bio-47. Yang, C. L., Zhang, S. J., Toomey, N. L., Palmer, T. N., andchem. Biophys. Res. Commun. 177: 559, 1991.Lee, M. Y. W. T. Induction of DNA polymerase activities in the

regenerating rat liver. Biochemistry 30: 7534, 1991. 53. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. Apoptosis: Abasic biological phenomenon with wideranging implications in48. Nakamura, T., Nawa, K., and Ichihara, A. Partial purificationtissue kinetics. Br. J. Cancer 26: 239, 1972.and characterization of hepatocyte growth factor from serum

of hepatectomized rats. Biochem. Biophys. Res. Commun. 122: 54. Rikkers, L. F., and Moody, F. G. Estimation of functional he-patic mass in resected and regenerating rat liver. Gastroenterol-1450, 1984.ogy 67: 691, 1974.49. Matsumoto, K., Tajima, H., Hamanoue, M., Kohno, S., Kino-

shita, T., and Nakamura, T. Identification and characterization 55. Nakamura, K., Ichimiya, H., and Nakayama, F. Alteration ofbile acid metabolism in two-thirds hepatectomized rat. J. Gas-of ‘‘injurin’’, an inducer of expression of the gene for hepatocyte

growth factor. Proc. Natl. Acad. Sci. USA 89: 3800, 1992. troenterol. Hepatol. 7: 121, 1992.56. Uesaka, K., Nimura, Y., and Nagino, M. Changes in hepatic50. Yanagita, K., Nagaike, M., Ishibashi, H., Niho, Y., Matsumoto,

K., and Nakamura, T. Lung may have an endocrine function lobar function after right portal vein embolization—An ap-praisal by biliary indocyanine green excretion. Ann. Surg. 223:producing hepatocyte growth factor in response to injury of dis-

tal organs. Biochem. Biophys. Res. Commun. 182: 802, 1992. 77, 1996.


ligation of portal vein branch induces dna polymerases α, δ, and ϵ in nonligated lobes

Documents