0022-1554/91/$3.30

The Journal of Histochemistry and Cytochemistry Copyright C) 1991 by The Histochemical Society,

Vol. 39, No. 1, pp. 1-6, 1991 Inc.

Printed

Article

Original

Ultrastructural Localization EGF Receptor in Human by In Situ Hybridization DOMINIQUE

LE GUELLEC,1

Laboratoire

d’Histologie

Laboratoire

de Biologic

Received

for publication

&p#{233}rimentale,

UPR

M#{233}dicale (L1PYD),

March

of mRNA Breast Cell

LUCIEN CNRS Centre

28, 1990 and

FRAPPART, 412

(DLG),

Regional

in revised

The EGF receptor (EGF-R), a 170 KD transmembrane glycoprotein, is found at a highievelin the BT2O human mammary carcinoma cell line (1 ± 0.4 x 1O sites per cell). In this study, we examined the expression of the EGF-R gene in BT2O cell line by in situ hybridization at the light and electron microscopiclevelusing a human eDNA, corresponding to EGF-R transmembrane and protein kinase domains, labeled with E3H1-, [35S]-, or [32P]-d-ATP. Two treatments were tested to embed cells in Lowiayl resin: the first used fixation and dehydration by progressivelowering of temperature, the second quick freezing and cryosubstitution. The best ultrastructural preservation was obtained with the second procedure without modification of the hybridization

Introduction Epidermal growth factor (EGF), a polypeptide with a molecular weight of 6045, induces a variety of biochemical events that can culminate in stimulation of DNA replication and cell division in many normal and malignant cells, including those ofhuman breast epithelium (Imai et at., 1982; Carpenter and Cohen, 1979). The EGF receptor (EGF-R) is a 170 lD transmembrane glycoprotein found on many cell types (Carpenter, 1983; Adamson and Rees, 1981), which can be divided into an extracellular domain that serves to bind the ligand, EGF or transforming growth factor a (IDFa), a putative short transmembrane domain, and an intracytoplasmic domain carrying tyrosine kinase activity (Downward et al., 1984). Overproduction of the EGF-R has been detected in several types ofcancers (Pfeiffer et a!., 1989; Gullick et al., 1986; Yamamoto et al.,

1986;

Libermann

et al.,

1985),

including

human

breast

carci-

noma (Ro et al., 1988). Kageyarna et a!. (1988) have shown that the EGF-R gene, when overexpressed, can function as an oncogene. EGF-R content is usually expressed at the highest level in the less differentiated breast cancer cell lines, such as MDA-468 (Filmus et

and

Encoding for the Cancer Line BT2O

PIERRE

Universit#{233} Claude

de Lutte

form July

contre

30, 1990; accepted

1

Correspondence

to: D. Le Guellec,

Laboratoire

1918, 69622

d’Histologie Villeurbanne

ExCedex,

DESPREZ

YVES

Bernard,

Ic Cancer

69622

Leon

Villeurbanne,

BCrard,

August

69373

and

Lyon,

France.

4, 1990 (0A1939).

signal. EGF-R mRNA was observed principally at the cytoplasmic level, on organelles involved in the protein synthesis process. Labeling was also located on the miaovilli which extend into the intercellular space, suggesting that some mRNA would be located in sites where EGF-R is utilized. Some mRNA was observed in the nucleus. This study demonstrates that post-embedding in situ hybridization, after quick freezing and cryosubstitution, is a powerful EM in situ hybridization procedure to study the expression of the EGF-R gene. (J Hiscochem Cytochem 39:1-6, 1991) KEY

In situ hybridization; microscopy.

EGF receptor;

WORDs:

Electron

BT2O cell line;

al., 1985) and BT2O (Lebeau and Goubin, 1987). It has been in our laboratory that in the BT2O cell line the EGF-R gene

plified results

and overexpressed, and that treatment with in an increased expression of EGF-R mRNA

data). To study cisely the hybridization microscopic described,

the expression corresponding procedure levels.

of the EGF-R mRNA, at the

Some

and according

EM

gene

1,25(OH)2D3 (unpublished

and to localize

we have developed an light (LM) and electron

hybridization

to the authors

methods

hybridization

shown is am-

have

prein situ (EM) been

is performed

before (Guitteny and Bloch, 1989; Wolber et a!., 1989; Brangeon et al., 1988; Trembleau et al., 1988; Harris and Croy, 1986), after (Escaig-Haye et al., 1989; Webster et al., 1987; Binder et al., 1986), or without

(Morel

et al.,

1986,

1989a,b)

the

embedding

of cells

The aim of this work was to localize at the LM and EM levels the mRNA coding for the EGF receptor in BT2O human mamor tissue.

mary carcinoma probes.

Materials p#{233}rimentale,43 blvd du 11 Novembre France.

in USA.

cell

line

using

3H-,

35S-,

or 32P-labeled

cDNA

and Methods

Cell Culture. The BT-20 human mammary carcinoma cell line was supplied by Professor W. L McGuire (University ofTexas, San Antonio). This cell line was devoid ofestrogen receptor and progesterone receptor, as shown

1

Downloaded from jhc.sagepub.com by guest on June 4, 2016

2

LE

by Horwitz cell

et al. (1978).

line:

75 fmol

patrick

et al. 1984).

level

of estrogen

and

a very

MCF-7

low level

37C

(Fitzpatrick

(2 mM) were

Molecular domains, Medicine,

cDNA

were

labeled

FRG)

with

UK) (for Ci/mmol)

provided

Texas).

cpm/pg

“random

for “5, through

and

Blot

and

procedures

Southern

and

performed

according

cultivated -80C

on coverslips buffer,

before for

15 mm

in PBS.

They

were

dehydrated For

the

In the first

in 0.2

M phosphate

in phosphate

stored

ded in Lowicryl viscosity

In the nitrogen

K11M.

Thin

sections

nm)

collodion

and

incubated

(this

step

now

been

x

For the

EM study, x

1

PBS,

incubation x

SSC

tions

were

diluted 32P),

and

40% 2-5 stained

0.5

coated

x

thin

for

by Northern

a major

one

at

a ratio be

In the

detected was

and

10 KB

of4:1.

(Figure

prolonged.

shown

that

level

obtained

there

probe

another MCF-7

blot

and

la),

even

A Southern

is an amplification

in the

MCF-7 selective

A before

probe.

when blot

hybridization

We

at 5.6 EGF-R

the

human have

de-

of the EGF-R, (Figure

KB

transcripts

la) could

autoradiographic

analysis

(Figure

corresponding

cell

ofBT-20

gave reproducible

line,

in poorly

by hybridization

domain

expressed cell

gene

differentiated

to

cxib)

10 times

has the

line.

cells with a human labeling were

(Figure clearly

EGF-R cDNA

2a). Slides

treated

negative

(Figure

in1

hr

MCF7

BT2OMCF7kb

1 hr, rinsed

dehydrated

conembedfrozen

1o

by freezeand

embed-

it has a lower

is that

ofthe

sam-

5.6

(-60C).

grids

et al.

immersed

28S

previously

coated

solution

for 1 hr

DNA

1

rinsed x

at 42C.

L4 nuclear

emulsion

of Caro

or 1-6 months X (Eastman

acetate

and

in 50%

of denatured were

Kodak;

examined

Ficol

from

herring

probe.

After

in 50% Grids

St Priest, After

5-15

3H) of incubation, aJeol

18S

and

sperm, 4 hr of

0.5

containing

Rochester,

4

400,

formamide,

15 mm,

(Ilford;

with

out

formamide,

BSA,

SSC for

et al. (1962). (for

was carried

1988).

,

(0.02%

for 30 mm

method

355),

uranyl

well

rinsed

quickly

temperature

500 tg/ml

xg/ml sections

Ilford

the

(for

MCF-7

of the EGF-R

in the

+ ) mRNA with the EGF-R cDNA two main messages for the cytoplasmic

with RNAse

PBS,

and

hybridization

solution

SSC for 15 mm,

in Microdol with

were

dextran, 0.25

SSC

x

with

using

weeks

were developed were

the 2

for

were

resin

(Le Guellec

sections

with

at 37C,

for 30 mm,

report

10%

for 15 mm,

line

and

paraformaldehyde

1% Denhardt’s

For the LM study, the

tRNA

1 mg/mi

cell

cells

in

x

the infiltration

on nickel

in 4 x SSC,

1% Denhardt’s

polyvinylpyrolidone), and

captured

1 hr 30

dehydrated

KF 80 (Reichert-Jung)

were

at lower

for and

Ia-

in Lowicryl

Reichert-Jung)

facilitates

A (Boehringer)

and control,

stored

in increasing

cells

Cells

probe

For a second

pre-hybridized,

Bi-

France).

eliminated).

in a previous

SSC,

cx-

out

and

of temperature,

of this

which

were

In Situ Hybridization. as described

use.

to embed

with

has

1

cells

dehydrated

the cryobloc

advantage

K4M,

possible (100

temperature

(CS Auto;

water,

as control

cDNA.

of RNAse

distilled

the expression

BT-20

In situ hybridization

and

solution

in 2 or 4%

procedure,

before

The

than it

MgCl2,

fixed

with

was used as EGF-R

poly(A

not

blot

carried

for 30 mm

at room

second

fragment

activity

a la

Appliquee Villeurbanne,

above.

differentiated

posure

paraformaldehyde

to embed

lowering

University,

100 .&g/ml

with

We have compared

with

modifications.

1% Denhardt’s

15 mm,

a cryoapparatus

at - 35C

pies and makes

for

propane

in liquid using

7.4,

by progressive

nitrogen-cooled

substitution

64

DNA

K (Boehringer),

treatments

were

pH

two times

with

rinsed

kbBT2O

two

cells

buffer,

K4M.

and

some

in 5 mM

in 4 x SSC,

19

specific

Electronique

Bernard

Results

tected

were

or Southern

were

with

in 2 or 4%

hydrated

Rsa I Puc

treated

as described

et al. (1982).

temperature

of proteinase

we used

buffer

fixed

at 37C,

ethanol.

procedure,

in Lowicryl

and

incubated

of ethanol

in liquid

were

in 100%

resin.

ded

Cells

in 5 j.tg/ml

EM study,

centrations

were

(AS 32P 2

probes

RNA blot

1988)

,

were

mm

de Microscopic (Claude

the same

cells

of

Poole,

precipitation.

pre-treatments

et al.

pH 7.4, at room

use.

cubated and

(Le Guellec

labeled

by ethanol

of Maniatis

For the LM study,

previously

0.2 M phosphate

method

Centre

An

with

mammary

Mannheim,

or [3H]-d-ATP cpm/pg for

The

the

probes

Amersham,

by Northern

of

with The

(Boehringer;

Analysis.

analysis to the

Cell Pre-treatment. as described

Blot

their

homology

i0

kinase

College

et al. (1984).

Ci/mmol;

1.3 KB

protein

complete

followed

and

was a human

for 3H. The

columns,

at

in air. Serum

and

Ci/mmol) of4 x

Controls. beled

grown

antibiotics,

(Baylor

3000

cpm/pg

108

were

in the

et I Ia Geologic

DESPREZ

France).

study

method

ologie

supplemented

M. Hughes

has

(AS

G-50

CO2

by Ullrich

primed”

3 x

Sephadex

Northern traction

at

probe

published

of[a32P]-d-CTP

Cells

transmembrane

LM) or [35S]-d-ATP (AS 1000 (for EM) to a specific activity

purified

Cells

in this

a high

microscope

et al. 1978)

salts,

(Flobio,

by Dr.

The

first

by the

used

it possesses

without 5%

Gibco

to EGF-R

sequence 50 liCi

calfserum

containing

(Fitz-

1984).

Earle’s

in this

of protein (Horwitz

et al.,

fetal

probe

was kindly

Houston,

EGF-R

were

The

as control;

with

from

corresponding

and

5%

obtained

Probe.

cDNA

and

is observed

receptor

medium

atmosphere

medium

EcoRl

was used

of EGF-R Eagle’s

receptor

specifically/mg

progesterone

in a humidified

culture

of EGF

bound

and

modified

L-glutamine

level

cell line

receptor

in Dulbecco’s with

A high

[ ‘251]-EGF

of

FRAPPART,

GUELLEC,

2 x

SSC

the

5cc-

France) days the

(for grids

NY).

Sections

1200EX

electron

1a (a) Northern

LIb

blot analysis of 5 tg of poly(K) mANA from BT-20 cells and MCF-7cells hybridized with (32P]-EGF-R cDNA. Two messages are detected at 10 KB and 5.6 KB. In McF.7 cell line, no EGF-R transcripts could be detected. (b) Southern blot analysis of 10 tg of DNA from BT-20 cells and MCF-7 cells. A band ofthe EGF-R gene is detected at 4.8 KD in BT-20 cells, with an intensity 10 times higher than the level obtained in MCF-7 cells. Figure

1.

Downloaded from jhc.sagepub.com by guest on June 4, 2016

EGF-R

UITRASTRUCTURAL

mRNA

HYBRiDIZATION

3

I

: #{149}: .

..

-.

.

..

S

. ..

.

.

#{149}s 5. .

#{149}1,

_... . I-

‘I

.1

#{149}:;--

‘4;4’:.,.

-‘

1I’

2a

1,

#{149}

-_

Figure 2. In situ hybridization using a 32P-Iabeled cytoplasm of the cell. (b) No labeling is observed

2b).

The

label

2a).

The

BT-20

ganelles,

and

On the

other

appeared cells

granular

many cell

hybridization

with

alive

followed

relatively

good

was better

when

with

Lowicryl

liquid

polysomes

(Figure

by Lowicryl

resin

without propane

(Figure rich

in or-

present. label

com-

was observed

resin

was added, ofultrastructure fixation, (Figure

with

3).

Many

(Figure microvilli

were

present

3) where labeling was observed located on the nucleus (Figure

at the periphery

tritiated probe, but the resolution time was much shorter.

This

fix-

embedding

gave

The

structure

a

similar.

35S probe

study

shows

in situ hybridization cell cultures. This

direct

embedding

freezing

in

bleau

envelope of the

(Figure

4c).

cell (Figure

(Figure 4d). Some silver grains were 4c). The results obtained with 3H

the

expression

the EGF receptor in the at the LM and EM level.

but mRNAs were was obtained

4b).

4a) and also on the nuclear

were

ground than the exposure

gave

over the

a slightly

higher

back-

and

was acceptable

Discussion

after

Cytoplasm was the most heavily labeled compartment (Figures 4a-4c). Labeling seemed to be located on granular endoplasmic reticulum

or 35S probe

is observed

5 tm.

Hybridization on various

A 2% paraformaldehyde K4M

on BT-20 human mammary carcinoma cell line. (a) Dense labeling RNAse A. Original magnifications: a x 4400; b x 2600. Bars =

was rare (Figure

labeling

No

cDNA with

not

of the cell architecture.

glutaraldehyde The best preservation K11M

were

probe.

0.1%

nitrogen-cooled

were

12b

-

the cytoplasm EM

a selective

4).

the control

preservation

less detectable.

over reticulum

level showed

of the

I

human EGF-R after treatment

in classical

endoplasmic

hand,

solution

concentrated observed

at the ultrastructural partments

:

#{149}.

is that

just after

it is possible

and it is not

cell fixation. to treat

detergent

selective

and

extraction

Downloaded from jhc.sagepub.com by guest on June 4, 2016

necessary

to perform

sections

encoding

for

of nucleotide

because

sequence

hybridiza-

can be stored,

different

Moreover,

is not useful

in situ

material with

methods.

protease

gene

breast cell line BT-20 that a post-embedding

can be employed at the EM level on has some advantages over the pre-

Embedded

serial

ply immunocytochemical

EGF-R

as described by Wolber et al. (1989), TremGuitteny and Bloch (1989). The primary

hybridization

advantage

tion

method method

et al. (1988),

of the

malignant human We demonstrate

probes

the they

and

or to ap-

application can bring

of about

and a loss of structural

4

LE GUELLEC,

dehydration

-

r

steps The

the

temperatures

higher

substitution K11M resin

only

to be the

critical

is offered

the

by rapid

35’C, respectively), at low temperature.

low viscosity

of this

a modification

after quick-freezing, embedding.

freeze-substitution,

dehydration steps

freezing

and

and

thesis process. Labeling seems to be located though this label appears specific, because

in controls,

a quantitative

the accuracy

The Lowicryl than K4M that it has a conditions,

signal

low-temperature encoding for the the protein syn-

on the microvilli. it is repeated

oflabeling

should

Al-

and

or 3H-labeled

between

probes,

resolution

otinylated sensitivity

3S probe

seems

and exposure

time.

EGF-R cDNA, (unpublished

This already

not

be precise

by

study.

specificity of the labeling is shown by the control blot, and LM hybridization results. Compared

The Northern

at

freeze-

of the hybridization

The ultrastructural localization of the mRNAs receptor on cytoplasm is in agreement with

observed

DESPREZ

dehydration

owing to the fact In our experimental

we have not observed

EGF

part

way to circumvent

followed by low-temperature embedding. allows one to embed at a lower temperature

(-60#{176}Cand

p.

seem

procedure.

FRAPPART,

to be a good Recently

which gives optimal results).

study confirms that post-embedding described by Webster et al. (1987),

probe, to

compromise

we have

resolution

used with

bigood

in situ hybridization, Binder et al. (1986),

and Escaig-Haye et al. (1989), is a good tool for EM in situ hybridization studies of mRNA, and shows that this method yields new information on the location of mRNA. A quantitative study is

presently .-)-

under

under #{163}

and

different

The

25-tm

diameter

cisternae,

cells

and

x 5000. Bar

=

some

disadvantage

is the difficulty

the level of the label. This previous observation (Bloch

of Lawrence

and

performance

several observed centrations

Singer

(198 5), who

of short

authors

ofembedding

cell

(Trembleau

fixation

et al.,

observation is in agreement et al. , 1986) and with that have with

1988;

demonstrated

the supe-

formaldehyde.

Binder

However,

et al.,

1986)

have

good detection of mRNAs after fixation with low conof glutaraldehyde (0.1 to 0.5%). We have obtained a

better ultrastructure preservation with quick-freezing and Lowicryl K11M embedding. The two reasons for this are the dehydration and embedding steps. Samples embedded in Lowicryl K4M were dehydrated al. (1985)

by progressive have

shown

of the EGF-R

gene

conditions.

Acknowledgments We wish

1 trn.

cultures in Lowicryl resin. The best fixative for LM or Lowicryl K4M resin proved to be 2% formaldehyde. Addition of glutaraldehyde decreased with our

in the differ-

distribution

the expression

pharmacological

to acknowledge

cal assistance

and

Literature preservation.

the label

to study

,,

Figure a The classical ultrastructure of BT-20 cells shows with many polysomes, few granular endoplasmic reticulum microvilli atthe periphery ofthe cell. Original magnification

nor

way to determine

ent compartments

that

lowering

of temperature.

the high

temperatures

Carlemalm during

et

the early

Ms M.F

Mr A.

Bosch

Lefebvre for

andR.

photographic

Wilemsfortheirtechni. assistance.

Cited

Adamson Biochem

ED, Rees 34:129

Binder

M, Tourmente

hybridization

AR (1981):

at the

5, Roth electron

Epidermal

J, Renaud microscope

growth

factor

receptors.

M, Gehring WJ level: localization

Mol Cell

(1986): In situ of transcripts

on ultrathin sections ofLowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes. J Cell Biol 102:1646 Bloch B, Popovici Bohien P (1986):

gene expression year

experience.

BrangeonJ, small and bridization.

T, Le Guellec

D, Normand

E, Chouham

In situ hybridization histochemistry in the endocrine and central nervous J Neurosci Res 16:183

PrioulJL,

Forchioni

large subunits of rubisco Plant Physiol 86:990

A (1988): using

5, Guitteny

AF.

for the analysis of system tissues: a 3.

Localization ofmRNAs for the electron microscopic in situ hy-

Carlemaim E, Villiger W, HobotJA, AcetarinJD, Kellenberger E (1985): Low temperature embedding with Lowicryl resins: two new formulations and some applications. J Microsc 140:55

Figure 4. In situ hybridization using 35S-Iabeled EGF-R cDNA on BT-20 human mammary carcinoma cell line dehydrated by progressive lowering of temperature and embedded in Lowicryl K4M (a,c,d) or after quick.freezing and Lowicryl K11M embedding (b). Better ultrastructural preservation is obtained with the latter procedure. Labeling is observed principally over the cytoplasm (a,b,c), over the granular endoplasmic reticulum (a, arrows), the nuclear envelope (C, arrow), and over the microvilli (d). Labeling is occasionally observed on the nucleus (C, arrowhead). Original magnifications: a x 18O0O; b x 14,000; c,d x 20,000. Bars - 1 irn.

Downloaded from jhc.sagepub.com by guest on June 4, 2016

ULTRASTRUCTURAL

EGF-R

HYBRIDIZATION

mRNA

5

.-, .‘

a

4c

Downloaded from jhc.sagepub.com by guest on June 4, 2016

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Caro

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Van

RP (1962):

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