Amplified, overexpressed and rearranged epidermal growth factor receptor gene in a human astrocytoma cell line

Vol.

131,

August

No. 30,

1, 1985

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207-215

Pages

AMPLIFIED, 0vERJzxPREssED AND REARRANGEB EPIDERMAL GROWTH PACMR RECEPTOR GENE IN A HlJMAN ASTROCYTCMA CELL LINE Jorge J. Gregory

Filmusl,

Michael

Cairncross'

N. Pollaklr*

and Ronald

N. Buick'

lOntario Cancer Institute and Department of Medical Biophysics, of Toronto, 500 Sherbourne Street, Toronto, Ontario, Canada,

University

2 The London Received

June

28,

Regional

Cancer

Centre,

London,

Ontario,

lK9

M4X

Canada

1985

We report here that SK-MG-3, a human astrocytoma cell line with a high tEGF) receptors, has an amplified and number of epidermal growth factor show any overexpressed EGF receptor gene. Northern blot analysis did not EGF receptor gene-related mRNA species. No amplification or abnormal was noted in 21 other astrocytoma cell lines. In contrast to rearrangement we have not other cell lines that have EGF receptor gene amplifications, detected inhibition of in vitro proliferation of the SK-MG-3 line by EGF. 0 1985

Academic

Press,

The finding the

erb-B

growth

that

factor

It

homologous

receptor

gene

of this

gene

has been clearly and

concentrations human

of avian

to the kinase

(EGFRG) (1,2), be

may

cerebrospinal

graded exhibits

4 of

qlioblastoma

human

erythroblastosis

portion

suggests

involved

in

can

stimulate

(6). of

primary

that

of

virus,

the

epidermal

amplifications

the oncoqenic

or

process

in

EGF receptor GM examined EGF receptor One cell

in

growth (3,4,5)

qlioblastomas

(GM) (astrocytoma

of mouse and

EGF

morphologically grade

III

or

IV)

level, and subsequently they demonstrated have an amplified EGFRG gene (8). number line,

in

22 cell

SK-MG-3,

*Present address: Department of Medicine, McGill of Oncology, Sir Mortimer Davis Jewish General Catherine Road, Montreal, Quebec, Canada, H3T lE2. Abbreviations: receptor gene; kilobase.

the

culture

human plasma have been reported in Liberman et al. (7) reported that human

multiforme

10 primary

or IV astrocytomas.

cells

in Recently

an elevated

We have estimated

EGF qlial

those

to

fluid

proportion

as

shown that

normal comparable

a significant

III

is

gene

neoplasms.

astrocytes

that

the transforming

oncoqene,

rearrangements certain

Inc.

EGF, epidermal growth factor; GM, qlioblastoma multiforme;

lines

derived

exhibited

University Hospital,

from

an unusually

grade high

and Division 3755 Cote Ste.

EGFRG, epidermal growth factor FCS, fetal calf serum; kb,

0006-291X/85 207

$1.50

Cowright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

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RESEARCH COMMUNICATIONS

number of specific

EGFbinding sites as estimated by [125~1~~~ binding studies. All cell lines were subsequently screened for EGFRGamplification, and SK-MG-3 was the only line with detectable amplification and rearrangement of the EGFRG. Despite the rearrangement, no abnormal EGFRG-related mRNAspecies were detected. Since other cell lines which have an amplified EGFRGare growth-inhibited by EGF (9,10), we examined the effect of this growth factor on the proliferation

of SK-MG-3 cells.

MATERIALSANDMETHODS Cell Lines: The following cell lines originated from astrocytomas grade III or IV were studied: SK-MG-1,-2,-3,-4,-6,-7,-8,-10,-11,-12,-13-,-14,-15, SK-MSand SK-AO2, derived at the Memorial Sloan-Kettering Cancer Center (11,12); U-87MG, U-138MG, U-178MG, U-251MG, U-343MGand U-373MG, derived at the University of Uppsalla (13,14) and T-98 derived by Dr. L. Hayflick (15). A-431, a human epidermoid carcinoma cell line, MDA-468 and MCF-7, human breast cancer cell lines, and 427-N, normal human fibroblasts, kindly provided by Dr. R. Phillips, were used as controls. MDA-468 cells were routinely cultured in L-15 medium, supplemented with 10% fetal calf serum (FCS). All the other cell lines were cultured in alpha mediumalso supplemented with 10%FCS. [1251]EGF-Binding Studies: Binding studies were carried out in triplicate on subconfluent cells grown on 35 mmdishes. Cells were washed twice with binding buffer (serum-free alpha medium with 1 mg/ml bovine serum albumin and 5 mMHEPES,pH 6.8), and then incubated for two hours at 37OC in binding buffer containing 5 x 10mgM EGF (Collaborative Research), a concentration previously noted to saturate available receptors of A-431 cells under these conditions, and about 2 x 105 cpm of [125~]~~~ (150 uCi/mg, New England Nuclear). Cells were then washed 6 times with binding buffer, solubilized in 0.5N NaOH, and radioactivity determined. Specific binding was estimated by subtracting counts bound in the presence of excess unlabelled EGF (10-7M) from total counts bound. The mean number of cells on two companion dishes as counted by hemocyometer following trypslnization was used to estimate the number of binding sites per cell. For certain cell lines, displacement curves for [125~]~~~ by unlabelled EGFwere obtained by incubating cells in varying concentrations of EGF between 5 x 10-12M and 10e7M, and Scatchard plots were derived. Isolation of DNAand Southern Blotting: High molecular weight genomic DNA organic extraction was isolated bv using NaDcdSO4/oroteinase-K lvsis, 716). DNAwas digested with Hind111 or and NaCl/ethanol precipitation to a Zetabind EcoRI, electrophoresed in 0.8% agarose and transferred membrane (AMF CUNO). Hybridizations and washings were performed using high background was reduced as stringent conditions. Probe-related nonspecific XDNAdigested with Hind111 was used as size marker. described (17). by guanidine Isolation of RNAand RNA Blotting: Total RNA was isolated solubilization and centrifugation over a CsCl cushion (18). isothiocyanate was 3 rrg of poly(A)+ RNA, purified by passage over oligo(dT)-cellulose, and electrophoresis was denatured with glyoxal and dimethylsulfoxide performed in a 1.1% agarose gel. The RNAwas then transferred to a Zetabind filter (19) and hybridized using high stringent conditions. 208

Vol. 131, No. 1, 1985

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Probes: The 2.4 kilobase (kb) c-DNA probe et al. of the (20). It encodes a portion a portion of the V-erb-B oncogene. The Hind111 - EcoRI fragmentisolated from the its homologyto the V-erb-B gene.

pE7 has been isolated by Merlin0 EGFRG and is highly homologous to human c-erb-B probe is a 2.5 kb XheB cloK(21) on the basis of

Effect cells

of EGF Concentration on the Proliferation of Cell Lines: 2 x 104 were plated in triplicate in 35 mm multiwell plates containing alpha medium supplemented with 10% FCS and allowed to attach overnight. Cells were then washed twice with serum-free alpha medium and then incubated in alpha media supplemented with 0.1% FCS and varying concentrations of EGF at 37OC in 5% CO2 for one week, with media changes every 48 hours, after which cells were trypsinized and counted with a coulter counter.

RESULTS Estimation

of EGF Binding

astrocytoma binding cells,

cell

assay. while

The SK-MG-3

(data

(SK-MG-S),

curves

lines.

Although

Scatchard

cell

bound

of

equilibrium

plots

receptors

to document

at

5 x lo-'M

Blot

Analysis

screened

for

amplification

blot

from

Cell

Using

line

SKMG-3 SKMG-5 468 A-431 427N

astrocytoma

line,

and A-431), 1.

and receptor where

labelled

glioma

to

those

1

and SK-MG-5

due to receptor

both

normal

Figure

conditions

shows glioma

EGF cell

number

from

there

ligands

human

may (24),

internalization,

lines

tested,

obtained

a cDNA

probe

by

(pE7)

and rearrangements each cell

analysis

Table

typical

be and

Kd was

and estimates measuring

of

specific

EGF.

Southern

DNA extracted

similar

were

EGF/105 similar

constants and/or

[125~]~~~

50 fmols

the SK-MG-3

(22,23) for

a

a

EGF under

(MDA-468

under

using

22

less

shown in Table

of binding

to be 0.7 x lo-'M numbers

from

for

We screened

approximately

lines

are

hazardous

is difficult

estimated

cell

and SK-MG-3

Lines

sites

substantially

Results

extrapolation

heterogeneity

line bound

shown).

is

Cell

EGF binding

and Scatchard

plots

Astrocytoma

specific

lines

not

(427-N)

binding

Southern

in

two EGFRG amplified

fibroblasts

binding

for

the other

conditions

receptor

lines

Sites

line

was digested

was performed

1.

all

of the with

as described

Estimates of EGF Binding Sites for various Cell Lines

Origin

fmol EGF bound/lo5 cells

GM GM Breast tumor Epidermoid tumor Normal fibroblast 209

47 16 163

419 8

the cell EGFRG. EcoRI

lines

were

or Hind111

and

in Methods.

Only

per Cell Estimated EGF receptors number per cell 2.8 x LO5 9.3 x 104 9.7 x 105 2.5 x lo6 5.1 x 104

SK-MG-3

Vol. 131, No. 1, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

B

0.3:m\

. 0.2

-

0.1

-

\

. \ . .

IO

Ii

20

L 40

30

\

.

I 50

60

B (f mol)

I .I

C c

Od-0

I &yof y &, 11, 0

.\

,,

20 40 100 EGF Molarity x IO“’

B (fmol)

Figure 1. EGF biding to GM cell lines. (A) Specific binding of EGF to SK-MG-3 and SK-MG-5 cells as a function of EGF concentration. !B) and (C) Scatchard plots of binding data for SK-MG-3 and SK-MG-5, respectively.

DNA showed

an

amplification correlating (Table

1).

patterns

amplified

EGFRG.

of the SK-MG-3

cells

with

the

Both

the Hind111

of

the

present

in the

donors

indicating

number

cell

that

restriction

patterns

evidence

of rearrangement

We estimated of

receptor

SK-MG-3 fibroblasts codes

for

gene is Northern

(427-N). a part

of

amplified blot

in is

of in

analysis

species.

Such a situation

rearranged

EGFRG (20).

is

in MDA-468

level

each

cell

kinase

Since

from different The

cell not

lines

of

cells, line

dilutions

intensity

in

normal

EcoRI and Hind111 did not show any

shown).

comparing

successive

the

of

DNA obtained

purpose

we used a genomic

portion

of the

approximately

to investigate

astrocytoma

amplification

For this

we decided

the

in

DNA extracted rearranged.

the EGFRG (data

signal

of the

that

as it found

receptors

and

EGFRG

of the other

the

not as high

EGF

lines

the

the degree with

shows

(Fig.

DNA fragments

cells

is

2

2) and the -EcoRI (not shown) restriction DNA allowed us to detect new bands (arrow) not

SK-MG-3

other

of

Fig.

EGFRG.

signal

intensity

DNA extracted. from normal

Fig.

from human

c-erb-B probe which 3 shows that the

8 times. the EGFRG is

whether

they

has been mRNA was

found

extracted 210

rearranged

express

in A-431 and

in

abnormal cells, Northern

the

SK-MG-3

EGFRG related which blot

also analysis

cells, mRNA have a was

Vol.

131,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

kb - 23.1

-

a

4.3

b

c

d

e

f

kb

-

2.3

03 Figure 2. Southern blot analysis of the EGFRG. 10 ug of DNA were digested with Hind111 and hybridized with a P-labelled pE7 probe. a) SK-MG-3; b) 427-N; c) MDA-468.

Figure 3. Quantification of the EGFRG amplification in SK-MG-3 cells. Serial dilutions of EcoRI-restricted DNA from SK-MG-3 were performed and hybridized with a 32zabelled c-erb-B probe. Small differences in the position of the bands are due to different salt concentrations after the dilution of the DNA. a) 20 ~g; b) 10 ug; c) 5 pg; d) 2.5 ug; e) 1.25 ug; f) 20 ug from normal human fibroblasts (427-N) performed that

as described

the EGFRG is

in Methods

overexpressed

degree of expression = 20-fold amplified express

in

is not EGFRG.

as

Effect and

MDA-468,

two cell

consequence

concentrations EGF

stimulates

concentrations The degree

lines gene

that

stimulate

that of

that

seen in normal

cell

(Table

that

strongly human

can be

(Fig.

as in MDA-468 also shows

are

other

most

of

the

inhibit found

It

have very high

amplification,

growth

stimulation

4

of SKMG-3 Cells

of the

high

cells

It cells, that

the

cells SK-MG-3 growth

in the astrocytoma which

fibroblasts,

1). 211

has been number

observed

4) although which the

mRNA species that are found the most prevalent transcripts.

same

of EGF in the Growth

the pE7 probe.

SK-MG-3

Fig.

EGFRG-related and 5.6 kb species being

10.0

a

the

using

the have

SK-MG-3

in normal

reported

cells,

that

A-431

of EGF receptors

growth-inhibited

a

cells

by

as EGF

19,lO).

Fig.

5 shows

that

cell

line

at

same

of A-431 cells have

the

and MDA-468 (~50%) is 5.1

lo4

cells.

similar

receptors

to per

Vol. 131, No. 1, 1985

BIOCHEMICAL

AND BIOPHYSICAL

160

a

b

RESEARCH COMMUNICATIONS

-

c

60 18s

d

40

d

20

m-8 0

I

I

I

5x1(3*

0

05

04

,I

I

5x10”

I

5xd0

I

5x1eg

5xd

[EGF],M

Figure 4. Northern blot analysis of the EGF receptor. was used as probe a) SK-MG-3; b) MDA-468; c) 427-N.

32F'-labellecl

pE7

Figure 5. Effect of EGF concentration on proliferation of cell lines. Points represent percentage change in cell numbers for cell grown in various media. EGF-supplemented media as compared to cells grown in control D-O, SK-MG-3; O-O, SK-MG-5; 0-0, 427-N; A-A, MDA-468; e-m, A-431.

DISCUSSION We report overexpresses Karyotypic double

EGF

minutes

significant

of in

(data for

lines

cells, shown). of primary

receptor abnormalities Our results indicate primary

derived

studies

are (25).

were

cell a

line

derived

consequence

cells but

has

revealed

no homogeneous

GM

(71,

staining

in primary

GMs.

It

from

cell

amplification. 212

regions

line

should

relationship of certain are less

most

were in

a EGF

glioblastomas. common in cell

of the lacking

2 to 3

be useful

between

has been clearly heterogeneous

subpopulations

of

seem to occur

the SK-MG-3 the

human GM,

amplification.

gene

As EGFRG amplifications investigating

a

the presence

comprised of karyotypically is possible, then, that It

derived

from

of

and the neoplastic behavior that EGFRG amplifications

from GM than

GM tumors

subpopulations analyzed here

SK-MG-3

all

further

a

as

not

proportion

a model

SK-MG-3,

receptors

analysis

demonstrated as

that

here

shown that cellular cell the

lines EGFRG

Vol.

131,

No.

The

BIOCHEMICAL

1, 1985

selective

understood. confer

pressures

It

conditions,

by

rendering

(8)

is

in

less

mechanism amplifications

in

monolayer

progression

g

astrocytoma

suggests

that

the

cell

a very

in but

high

extensive

Recently,

Merlin0

lines at

another

carcinoma

squamous

of this gene (20). SK-MG-3 cells paradoxically 20-fold,

are

not

inhibit

have

presence

EGF receptors have

selected

that

and growth

receptors

is

to

growth

that

astrocytoma

there

MDA-468

cell

(10).

is

these

while

cells,

can be proposed cell astrocytoma are growth with

analyzed

is the

but

below EGF

this

by EGF at

receptors

that

(Fig.

growth

grow

the

number

than

inhibition threshold.

concentrations Also,

cells is in the same range as other Kd's known to have normal EGF receptors (33).

213

not similar

the Kd for calculated

than

in the (32)

and cells

present

in

Alternatively, functional

be the case

to

for such

in SK-MG-3

a critical seem

There

underlying

in A-431

EGF

of EGF

concentration

higher

the (31)

of

amount

and

fewer

to EGF.

mechanism

are

in

et al.

of EGF receptors

does not 5).

variant

the number receptors

that

cases,

when a given

lower

(29).

have shown

although

is

the

30

Kawamoto

threshold

here,

of

about to

on exposure

cells,

tumors

amplification

In these

between

absolute

been

been reported.

with

the variants

results

unclear. in SK-MG-3

primary

yet

ability

that

culture

concentrations

(8,30).

and

of EGF-induced

elevated,

EGF

and the molecular

of the fact

line,

stimulated

functional

lines

that

it

an

cells,

The lack

may be a consequence

All

lines

EGF,

and

lines

of their

EGF.

inhibition

a threshold phenomenon remains The number of EGF receptors other

basis

cell

certain

the human tongue

EGFRG.

is a relationship

there

common to all

by

in

an advantage,

amplification

from

cell

of EGFRG

propagated

has a 2 30 fold

of the of

response

exceeded,

inhibition

line,

on the

the parental

receptors

is no evidence

derived

cell

and MDA-468,

concentrations

than

proposed

line

amplification

been

of high

(27)

a four-fold

a

of EGFR has

have not

growth-inhibited

A-431

respectively,

clones

cell

carcinoma

and under

lines

studies

have reported

astrocytomas

the rarity

overexpression

of cell

or EGFRG

represents

represent

least

VlVO

of

primary

selected

might

--in exogenous

to

However,

not

certain

frequency

does not

genetic

et al.

EGFRG in a squamous A-431,

proportion

molecular

levels

the amplification

amplification

lead to counter-selection, may even conditions. In squamous cell carcinomas, significant (28),

under

the

vivo . __

and

reported

cells

that

are

receptor

more aggresive

the possibility

of neoplastic

mitogen

Indeed,

COMMUNICATIONS

propagation

super-sensitive

(26).

differentiated,

with

line

elevated

cells

RESEARCH

cell

to neoplastic

mitogens

in keeping

BIOPHYSICAL

during

that

advantage

autocrine-produced amplification

imposed

is conceivable

a proliferative

AND

since

to other EGF binding for

in

other

cell

this

SK-MG-3 lines

in SK-MG-3 cell

lines

Vol. 131, No. 1, 1985

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS This work was supported by grants from the Medical Research Council Canada and the National Institute of Health (USA) (#CA29526). Michael Pollak is a recipient of a Terry Fox Postdoctoral Fellowship from National Cancer Institute of Canada. We are indebted to Drs. I. Pastan G. Merlin0 for providing the pE1 cDNA probe and to I. Robson for c-erb-B probe. Excellent technical assistance was provided by R. Pullano B.Choo.

of N. the and the and

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