Identification of a characteristic cytosolic polypeptide of rat preneoplastic hepatocyte nodules as placental glutathione S-transferase

Vol. 143, No. 1, 1987 February 27, 1987

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 98-103

IDENTIFICATION OF A CHARACTERISTIC CYTOSOLIC POLYPEPTIDE RAT PRENEOPLASTIC HEPATOCYTE NODULES AS PLACENTAL GLUTATHIONE S-TRANSFERASE T.H.

Rushmore, *K.

R.N.S. Sato,

Sharma, M.W. R.K. Murray,

Roomi, L. Harris, and E. Farber

*K.

OF

Satoh,

Departments of Pathology and of Biochemistry, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada MSS lA8 *Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, Japan Received

December

31.

1986

Evidence is presented that a distinctive type of glutathione-S-transferase and a cytosolic polypeptide of M (GSHTase-P), 52,000 (P-52), each appearing in greatly increased amounts in hepatocyqe nodules during liver carcinogenesis in the rat, are so far indistinguishable. The probable identity of the two polypeptides was established with the use of SDS polyacrylamide gel electrophoresis and Western blot techniques with purified GSHTase-P and P-52 and their respective antibodies and by comparison of the sequence of the first 26 N-terminal amino acids. Since the enzyme and the polypeptide are each considered to be the best available early markers for hepatocyte nodules, as putative precancerous lesions, their probable identity makes them attractive cellular components for in depth studies on their transcriptional and translational regulation and their use in new approaches to the sequential analysis of liver carcinogenesis. 0 1987 Academic press, hc.

Cytosol

from

different

models

SDS-PAGE marked

from increase

tentatively 21,000

preneoplastic of that

of

in

the

considered (1).

liver

Recently,

hepatocyte carcinogenesis, normal

liver

amount previously Sato

and

of

nodules

(HN),

showed

a series

(1). a to

co-workers

a

of

difference Mr

molecular

have

in

six

of differences

A prominent

polypeptide have

generated

isolated

was

26,000 weight and

on a

(P-26), of

about

purified

a

Abbreviations: GSHTase-P, glutathione-S-transferase (EC2.5.1.18); HN, hepatocyte (“hyperplastic”, “neoplastic”) nodules; GluTase, y-glutamyltransferase (EC2.3.2.2); RH, resistant hepatocyte; PMSF, phenylmethylsulfonylfluoride; CM-sephadex , carboxymethyl cellulose-sephadex; GSH, glutathione; BSA, bovine serum albumin; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TBS, 20 mM Tris, 500 mM NaCI, pH 7.5; TTBS, TBS containing 0.05% Tween-20; PBS, 10 mM Na2HP04, 150 mM NaCI, pH 7.2. 0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

98

BIOCHEMICAL

Vol. 143, No. 1, 1987

neutral to

form

be

of GSHTase

present

in

normal

control

enzyme

was

positive

foci

Since in

HN

blot

identity

acids

high

be

if

the

in

and HN,

increased

P-52

similar

and

have

in

anti-GSHTase-P

markedly

biologic not

P-52

of

the

by the

have

conditions,

identical

and

their

techniques,

are

GSHTase-P,

concentrations

Using

and

similar

and

supported amino

to

placenta,

RESEARCH COMMUNICATIONS

shown

it

comparison antibody,

localized

in

to this

GluTase-

in HN (5).

related

Western

rat

(2-5).

GSHTase-P

GSHTase-P

not

shown

under

closely

relatively

liver

and

from

AND BIOPHYSICAL

observation

liver

we

weights

postulated

that

and

appear

they

(4,6).

Through

the

respective

antibodies

and

of SDS-PAGE

the

close

evidence two

molecular

is

presented

nodule that

for

polypeptides.

the

sequences

use of

This of the

first

were

purified and

similarity conclusion

if is

26 N-terminal

identical.

MATERIALS

AND

METHODS

Resistant hepatocyte (RH) nodules were produced by the method of Prior to harvesting the Solt and Farber (7) in male Fischer 344 rats. nodules, the livers were perfused with ice cold 0.25 M sucrose containing 1 mM PMSF (Sigma) as protease inhibitor and 1 mM EDTA. The nodules, grayish-white and up to 1 cm in diameter, were dissected free from the surrounding pale brownish red liver. Cytosol fractions from nodules and from control normal livers were prepared by homogenizing tissue (1 gm per Homo10 ml of medium) in 0.25 M sucrose containing PMSF and EDTA. genates were centrifuged at 105,000 x g for 1 hr at 4OC and the clear estimaton was by the supernatants were frozen at -70° until used. Protein bovine serum albumin as standard. method of Lowry et al. (8), using The GSHTase-P was purified from the cytosol of rat placenta at the 15th day of gestation (2), by sequential chromatography on CM-Sephadex C-50, hydroxylapatite and GSH affinity columns and finally on immunoaffinity columns coupled with antibodies against GSHTase AC subunits and GSHTase BL subunits to remove very small amounts of contaminating AC and BL GSHTases. Highly purified preparations of P-52 were prepared from the cytosol of HN by sequential chromatography on S-hexylglutathione, CM-Sephadex and DEAE-Sephacel columns. Antibodies to purified GSHTase-P and to purified P-52 were prepared in rabbits by intracutaneous injections with complete Freund’s adjuvant. Polypeptides SDS-PAGE was performed as described previously (I). electrophoretically transferred to from unstained SDS-PAGE gels were nitrocellulose paper with a Trans-blot apparatus (Bio-Rad) at 85 volts for After 4 hours at 4OC using the buffer system of Towbin et al. (9). the P-26 or GSHTase-P subunit was visualized by immunoblot transfer, analysis using either antibody to P-52 or GSHTase-P (10,ll).

99

Vol. 143, No. 1, 1987

BIOCHEMICAL

RESULTS Fig. from

1A shows

control

GSHTase-P. in

the

HN

band

rat

liver

The

band

cytosol

and

blot)

antibody

to

properties

of

antibodies

2 is

antibody,

control

liver

reaction

of

(1)

is

this

the

prepared Also,

HN,

the

are

corn munication)

.

A nodules

similar

been

designate

GSHTase-P

very

level

lung.

In

in

their

study,

has

been

like

GSHTase

7-7

unlike

towards

ethacrynic

also

high

has

activity

in

and acid

(14).

toward

rat

the

The

similarity

in

the

their

reaction

with

other

ethacrynic

purified acid.

100

cytosol

from

(B).

The

clearly

evident.

in SDS-PAGE

and

livers

and

livers

two

different

antibody

Rushmore,

personal

GSHTase-P,

which

HN

is in

was

not

(13).

P-52,

like

at a

kidney

and

used.

and This

has

and

authors

present

levels

GSHTase-P

GSHTases,

P-52

These

(12).

high

hepatomas

of

control

al.

resembling

Highly

is

use

both

to

from

the

with

a GSHTase

P-52

several

With in the

and

et

of antibodies

react

the

relatively

primary

use

preparation

of

respect

7-7, at

the

preparation

Jensson

a homodimer from

with

positions

Cameron

purified

purified

similar

using

by

but

reacting

GSHTase-P

sections

(5,

liver

this

after

is present

purified

conclusion

rat

of

immunoblot

with

immunoblot

in

staining

as GSHTase

region

position

GSHTase-P.

with

are

suggested

and

The

to

data,

that

the

histological

glutathione-S-transferase, 7-7,

in

identical

the

1B.

against

each

of

tentative

has

low

in

P-26 weight

evident.

respect

of

of P-52

P-52

patterns

preparations

set

than

against

purified

molecular

and

Fig. in

preparation and

of

of cytosols

apparent.

visible

antibody

preparations.

in

amount

now

GSHTase-P

antibodies

shown

rather

small

are

SDS-PAGE

a similar

P-52

and

between

to the

GSHTase-P

on SDS-PAGE

26,000

CSHTase-P

is readily

shown

the

at about

and

and

HN

correspondence

is

P-52

against

with

P-52

CSIiTase-P

Fig.

prepared

Thus,

the

pattern

liver

appearing

to CSHTase-P

In

rat

RESEARCH COMMUNICATIONS

DISCUSSION

polypeptide

corresponding

of

AND

and

and

those

(Western

this

a typical

AND BIOPHYSICAL

A

GSHTase GSHTase,

high GSHTase

activity 7-7,

Vol.

143,

No.

BIOCHEMICAL

1, 1987

1234

14-

AND

BIOPHYSICAL

56

1234567

Analysis nodules.

COMMUNICATIONS

oA

Fig. 1. hepatocyte

RESEARCH

of

B cytosolic

polypeptides

of

control

livers

and

of

A. SDS-PAGE (12.5% polyacrylamide) using 20 ).KJ protein/lane except for lane 6 for which about 5 ug protein were used. The gel was stained with Coomassie Blue B. The markers for estimation of M phosphorylase B (94,000), bovine serum albumin (67,000), o&lb$cz (47,000). carbonic anhydrase (30,000) and lysozyme (14,000). control liver; P-52;

1. control purified

liver; 2, 4, affinity 6, purified

HN; 3, purified GSHTase-P.

affinity cytosol

purified (step

1)

cytosol from

(step 1) from HN; 5, highly

B. lmmunoblot (Western blot) analysis of a gel identical to that shown in A using GSHTase-P antibody. After transfer, the nitrocellulose paper was treated with Blotto (5% non-fat dry milk in PBS) for 1 hour at room temperature and then incubated in Dlotto containing antibody to GSHTase-P (diluted 1 :200) for 4 hours at room t,%perature (18). The paper was then washed in Blotto and treated with I Protein A for 2.5 hours at room temperature (0.5 uCi/lO ml). The blot was washed in PBS and the polypeptides reacting with the primary antibody were detected by autoradiography. Lanes 1,2,3,4,5 and 7 correspond to lanes l-6 in Fig. In lane 6, 5 )q of purified P-52 was used. The antibody used was 1A. against GSHTase-P.

Preliminary the

first

(15)

and

the

almost

protein

26

N-terminal

in

GSHTase

with

on

7-7 that

comparable HN

resistance

the

sequence

amino

certainty

Since unusual

data

acids (14).

a of

this

is

P-52,

acids to

several

CSHTase-P

and

in

that lines

P-52

found of

CSHTase

indicate in

GSHTase-P

evidence 7-7

that

indicate

are

the

same

activities.

characteristic new

amino

identical

Thus,

enzyme

have

of

liver

biochemical cell

101

population

phenotype to

many

that xenobiotics

confers and

Vol.

143,

No.

1, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

94 67 47 -

A Fig. 2. hepatocyte

B

Analysis nodules.

of

cytosolic

polypeptides

of

control

livers

and

of

A. SDS-PAGE (15% polyacrylamide) using 6 w protein/lane. The gels were stained with silver nitrate as described by Wray et al. (19). The markers for estimation of M were the same as in Fig. 1A. 1, control liver; 2, HN; 3, P-52, 4, CSHTa&-P. B. Immunoblot (Western blot) analysis of a gel identical to that shown in Fig. 2A. After transfer, the nitrocellulose paper was treated with 3% gelatine in TBS for 1 hour at room temperature, then incubated in TTBS (TBS containing 0.05% Tween-20) containing antibody to P-52 (diluted 1:300) for 4 hours at room temperature. The paper was washed in TTBS and incubated in TTBS containing 1% gelatin ml goat anti-rabbit diluted 1:3000 for 1 hour at room HRP-conjugated IgC ( Bio-Rad) temperature. The polypeptides reacting with the primary antibody were visualized by immersion in development solution (100 ml TBS containing 60 1.11 30% H202 and 60 mg 4-chloro-1-Napthol) for 10 min at room temperature. The lanes are the same as in Fig. 2A.

to

exposure

consider

toxic

environmental

GSHTase-P

Since the

to

this

polypeptide

putative

prove

to

another shows

preneoplastic be

carcinogenesis promotion

as

a

and of

in

the

(16,171,

component a

large

and

focus

useful

components

for

of

increase

this in

precancerous studies

of

development

of

hepatocarcinogenesis.

102

is

attractive

resistance

activity

and

in it

gene

expression

novel

assay

for

to

spectrum.

hepatocytes, altered a

it

amount might

initiation

in well

during and

BIOCHEMICAL

Vol. 143, No. 1, 1987

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS This the

work

NC1 (NIH).

was THR

supported

by grants

is in receipt

from

of an NIH

NC1 and

MRC

of Canada

and

Fellowship.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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