5

Vol. 135, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 26, 1986

Pages 262-268

ONCOGENE EXPRESSION Yoshiharu

Motoo,

Wadley

IN HUMAN HEPATOMA CELLS PLC/PRF/S Massoud Mahmoudi, Arthur P. Bollon"

Institutes 9000 Harry Dallas,

Kurt

Osther

and

of Molecular Medicine Hines Boulevard Texas 75235

Received January 7, 1986

of 7 cellular oncogenes in a human SUMMARY: The expression hepatoma cell line PLC/PRF/5 was studied using Northe,rn blot analyses. Among the oncogenes tested, c-&l, c-Q@, c-fms?, cru, c-Ha-and c-u were expressed. The oncogene c-Ki-m was not expreSSed. The length of the mRNAs expressed was almost consistent with published data. Compared to the oncogene expression in Daudi lymphoma cells, the same kind of oncogenes were expressed in PLC/PRF/5 cells, but the intensity of the signal in each oncogene expression was stronger in Daudi cells than in PLC/PRF/5 cells. Considering the cellular localization and the function of each oncogene, the oncogene survey in hepatoma cells broadens the knowledge of hepatocarcinogenesis and the character of human hepatoma cells. 0 1986

Academic

Press,

Inc.

The activation associated

with

oncogenes 6).

u

hepatomas

and ras

However, hepatoma

oncogenes

other

the

hepatoma

cell

investigated tissues

and such oncogenes

could

play

For this there

expression line

reason,

is no report cell

lines.

of 7 different

concerning In this oncogenes

should

be addressed.

$1.50 0 I986 by Academic Press. 0s reproduction m an-v form

Inc. reserved.

262

rat

(12). in

important

oncogene

PLC/PRF/5.

3, 4, 5,

using

be expressed

*TO whom correspondence 0006-291X/86 Copyriphr All rights

liver),

might

in human hepatoma

examined

of the

oncogenes

some

Moreover,

expression

been

of cellular (2,

carcinoma

have

to be

of tumors

11) and human hepatoma

in hepatocarcinogenesis. needed.

reported

The expression

in many types

(or primary

8, 9, 10,

cells,

(1).

studied

hepatoma

(7,

has been

carcinogenesis

has been

As for

mainly

of oncogenes

roles are

surveys

oncogene study,

we

in a human

Vol.

135,

No.

BIOCHEMICAL

1, 1986

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

A human hepatoma cell line PLC/PRF/S was established by Cells: The cells were grown as monolayers in Alexander et al. (13). Eagle's minimal essential medium (MEM) supplemented with 10% Daudi lymphoblastoid cells were fetal bovine serum (FBS). grown in suspension cultures in RPM1 1640 medium with 10% FBS. Both cell lines were obtained from the American Type Culture Collection (Rockville, MD). Human peripheral blood leukocytes were obtained from healthy volunteers. Preoaration of total BhzB and Northern blotting Total RNA from the cells was isolated by extraction with guanfdine thiocyanate followed by centrifugation through a cesium chloride cushion as previously described (14, 15). 40 pg of total RNA from each cell line was denatured with glyoxal and electrophoresed on a horizontal 1.5% agarose gel at 4oC overnight. Transfer, baking and hybridization were carried out as previously described (16, The oncogene DNA probes were nick-translated with 17). The hybridization was done under a stringent a -[32P]dCTP. After washing the condition (in 50% formamide at 420C). filters four times for 5 min each at room temperature in 2X SSC, 0.1% SDS followed by two washes of 15 min each at 500C in 0.1X SSC, 0.1% SDS, they were autoradiographed using intensifying screens at -800C. The oncogene probes used in this study are shown in Probes: Table 1. As Hunter (25) described , oncogenes can be classified into 4 or 5 classes as in Table 1 according to the cellular

Table Cellular

oncogene

Restriction fragment BarnHI-Hind111 BamHI ECoRI-Hind111

1.

Oncogene DNA probes

Size

Cellular location

Function

0.65 kb

Plasma membrane

Tyrosine specific

0.8 kb

Cytoplasm

kinase

0.9 kb

Plasma

and

BamHI

1.0 kb

BamHI

6.6 kb

EcoRI-Bind111

2.0 kb

EcoRI-Hind111

9.0 kb according by Bunter

study

Class Class

Reference 1

(18)

protein

(19)

Cytoplasmic

Class l-

(20)

Secreted

Platelet-derived growth factor like

Class

2

(21)

Plasma membrane

GTP binding

Class

3

(22)

cytoplasmic membranes

Oncogenes were classified eachoncogene as described

used in this

domain of a growth-factor receptor

related

(23) Nucleus to thecellular (25).

263

DNA binding location

Class

4

and the function

(24) of

Vol. 135, No. 1, 1986

localization c-Ha-Collection Oncogene

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and the function. were obtained from the (Rockville, MD). The Science, Inc. (Mineola,

The probes of c-u and American Type Culture rest were purchased from NY).

the

RESULTS Total well

RNA isolated

as Daudi

leukocytes

cells

hybridized

were denatured

oncogene

PLC/PRF/S

and normal

glyoxal,

paper

probes

as

human

with

to nitrocellulose

7 different

To assess

the

methylene

blue. with

The results of the

of

no apparent

and

as described

RNA, the

in

tested

of each

were found

in leukocytes,

transcript

was detected.

the

cells

signal

in Daudi

analyses

, with

gel

was stained

18s and 28.9 rRNA bands

degradation blot

in PLC/PRF/5

that

total

The characteristic

oncogenes

The intensity than

quality

of Northern

were expressed

lower

control)

cells

and Methods.

were observed

All

control)

transferred with

Materials

human hepatoma

(a positive

(a negative

electrophoresed,

with

from

(Fig. are

11. shown in Fig.

exception

as well

observed

cells.

Smears

but

no specific

of c-Ki-m, \ as in Daudi cells.

in PLC/PRF/f or very

cells faint

band for

was signals

any oncogene

123

Fig.

1

Total (lane 8 pg 1.5%

RIUAfrom PLC/PRF/5 cells (lane 11, Daudi cells 2) and normal human leukocytes (lane 3). of each RNA sample was glyoxalated and run on a agarose gel and stained with 0.2% methylene blue.

264

2.

Vol.

135,

No.

BIOCHEMICAL

1, 1986

AND

c-abl 123

BIOPHYSICAL

RESEARCH

c-f es

c-fms

123

123

COMMUNICATIONS

.

c-myc

c-Ha-ras

123

12

C-Sk 123

3

4.2

Fig.

In larger

2

Northern blot analyses of total RNA from PLC/PRF/5 cells compared to Daudi cells (a positive control) and leukocytes (a negative control) using nick-translated oncogene probes. 40 pg of each RNA sample was denatured with glyoxal, electrophoresed, transferred to nitrocellulose paper and hybridized with the oncoqene probes. A DNA digested with EcoRI and Hind111 ;as used as a marker.Lane 1; PLC/PRF/S, Lane 2; Daudi and Lane 3; leukocyte RNA.

addition bands

to

(>23 kb) were

but

not

in leukocytes.

5.8

kb)

of transcripts

cells. was

The size 4.7,

5.2,

a 2.3

found

There

(kb)

were

2 different in PLC/PRF/5

mRNA of c-u,

and 4.2

kb,

transcript

in PLC/PRF/S

of c-Ra-a

of the

5.0

kilobase

c-m,

of c-a,

and Daudi sizes

2

cells

(1.4

and

and Daudi c-m

and c-g&

respectively.

DISCUSSION An oncogene PLC/PRF/S

using

survey 7 different

was done

in

oncogene 265

human hepatoma probes.

cells

Our data

show

Vol. 135, No. 1, 1986

that

c-&l,

expressed

BIOCHEMICAL

c-m,

c-fma,

c-w,

in the

hepatoma

cells.

expressed.

There

specifically

in

as a positive cells

(12)

control,

are

tissues,

required

for together

there

might

was not

was expressed

The Daudi

cell

line,

signals

than

PLC/PRF/5

that

c-m,

not

(8,

9, 10,

c-&J.

reduced

expression that

maintenance

with

oncogene

hepatomas

used

the

of the

reports

c-Ha--

and Su et al.

11).

of c-IQYE in human

c-s

expression

might

not

above,

in oncogene

be Our

hepatocarcinogenesis.

mentioned

be some differences

suggest

expression

that between

and human hepatomas.

previously 5.0

of

the

reported

et

addition

in

al.

(4).

to the

larger

bands

normal

leukocytes.

mRNAs observed

values.

kb is different

by Slamon

2.3

(>23 kb)

PLC/PRF/5 (281, cellular

it

with in the

might

bands

Our data

tested

in PLC/PRF/S

with

hepatitis

transcript

of

3.7

might

transcript

kb

reported

difference

is unclear.

we found cells

be detected

homology

between B virus,

of the

the

but

not

in

due to the

to c-w

or a

the

show that cells.

there Thus

HBV,

carry

expression disrupt

human hepatoma (26).

human hepatoma

HBV does not

or might

(29).

c-m

of the

association

activate

oncogene

the

and Daudi

genome

Since

(27).

with

mRNA.

is a strong

integrated

consistent

in PLC/PRF/5

of the

c-w

almost

of c-u,

These

infection

the

is

kb transcript

precursor

chronic

from

The nature

of an RNA species

There

However,

size

existence

is

which

cells.

suggesting

The length

In

but

the

the

data,

The c-Ki-Lga

were

expression.

in rat

expressed

hepatoma

and c-u

showed stronger

oncogene

has reported

rat

hepatoma

is reported

c-Ki-~~6i

c-Ha--

was no oncogene the

in every It

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

its

HBV DNA line

own oncogenes

of a neighboring

a cellular

regulator

was no overexpressed far,

cell

and

there

gene oncogenes

is no evidence

for

Vol.

135,

No.

1, 1986

integration

BIOCHEMICAL

of HBV DNA adjacent

oncogenes

cellular

oncogene old

B6C3Fl

contribute In c-a,

delineate

(30)

to any of the

have

in liver

COMMUNICATIONS

known cellular

recently

tumors

reported

arising

an active

spontaneously

Such a new transforming

mice.

conclusion,

cells

c-Ha--

PLC/PRF/5,

but

compared

lymphoma the

among the

c-u,

hepatoma-specific, in Daudi

RESEARCH

in

24

gene might

to hepatocarcinogenesis.

c-frea,

hepatoma

BIOPHYSICAL

(29).

Fox and Watanabe

month

AND

role

cells.

known cellular

oncogenes,

and c-u

expressed

none

of oncogenes

in human

of them were

to the Further

were

c-u,

expression studies

of the are needed

oncogenes to

in hepatocarcinogenesis.

ACKNOWLEDGEMENTS We wish this

to thank

manuscript.

Foundation

This

Ms. Mickie work

and an NIH grant

Cox for

was supported

the

preparation

by the

of

Meadows

to APB.

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