Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. C Cordon-Cardo, J P O'Brien, J Boccia, D Casals, J R Bertino and M R Melamed J Histochem Cytochem 1990 38: 1277 DOI: 10.1177/38.9.1974900 The online version of this article can be found at: http://jhc.sagepub.com/content/38/9/1277

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0022-1554/90/$3.30 The Journal of Histochemistry Copyright

©

1990

and

by The

Cytochemistry

Histochemical

Vol.

Society.

Original

Expression of the Multidrug (P-.Glycoprotein) in Human C.

J.

CORDON-CARDO,2

and

M.

R.

Memorial Received

P.

J.

O’BRIEN,

BOCCIA,

D.

non to

whereby a single

resistance

J.

CASALS,

Cancer

publication

Cenier

November

13.

New l98)

Thrk.

and

in

New

York

10021.

revised

form

February

21.

1990:

accepted

tumor cytotoxic

to a variety

R.

April

1990 USA.

BERTINO,

resistance” cells

in culture,

agent,

(1). The importance our understanding

sistance

to multiple

drugs

describes

when

simultaneously

ofstructurally

pounds advance

(MDR)

and

selected develop

functionally

the

phenome-

for resistance broad unrelated

ofthis laboratory model is that of the complex clinical problem which

is characteristic

of many

crosscorn-

it may of rehuman

WORDS:

KEY

Multidrug

histochemistry;

resistance;

(9Al835).

MDR1;

Differentiation;

P-glycoprotein;

Human

cells

170,000-180,000

tumors;

lmmuno-

Cancer

(Pgp) rived

consistently

membrane

(2-5).

Amplified

chemo-

overexpress

glycoprotein

DNA

multidrug-resistant

known

sequences

cell

lines

from

have

been

an

Mr

as P-glycoprotein

experimentally shown

de-

to encode

Pgp

(6-10). The amino acid sequence of Pgp has been deduced from full-length or partial eDNA sequences of human, mouse, or Chinese tein

hamster of

Pgp

1280

amino

This

molecule

putative AlP tural homology Supported in part by NCI Grants CA-47538. CA-47179, CA-l4134, and by The Brookdale Foundation.JPO’B is a Special Fellow ofThe Leukemia Society of America. 2 Correspondence to: Carlos Cordon-Cardo, MD, PhD, Memorial Sloan-Kettering Cancer Center, Department of Pathology, 1275 York Ayenue, New York, NY 10021.

1990

therapy.

halves.

tumors.

12.

renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern ofstaining varied among different cases ofa given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety ofnormal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion ofxenobiotics. Pgp expression in capillanes ofthe brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanetuanes for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding. (J Hiswchem Cytochem 38:1277-1287, 1990)

Multidrug-resistant

“multidrug

1277-1287, Pr,nted:n

Article

Introduction term

9. pp.

Resistance Gene Product Normal and Tumor Tissues’

We have characterized the normal human tissue distribunon and tumor expression of the human multidrug resistance gene (MDRJ) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells ofcapillary blood vessels at blood-tissue barncr sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon,

The

No.

MELAMED

Sloan-Kettering kr

38.

Inc.

coding is believed

effort

which

human

contains

MDRI

consists

active

to better

efflux

transport for pump

understand role

in mediating

gene

oftwo

gene

MDR of

the normal one

bacterial systems

by functioning

broad

specificity

function form

and

has revealed

and

export

a pro-

homologous

domains

analysis and

encodes

highly

12 transmembrane

to be responsible drug

its potential

The

acids

binding sites. Sequence between the MDRI

periplasmic

dependent

genes.

of drug

two

struc-

genes

en-

(11-13).

Pgp

as an energy(14,15).

of this protein resistance

In

an

and in hu-

1277

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

CORDON-CARDO,

1278

man

tumors,

we have

expression

in tumors,

Materials

and

examined

its normal

using

immunohistochemical

tissue

distribution

and

Methods

25 germ

cell tumors,

20 melanomas,

14 sarcomas,

five

pheochromocytomas. seven neuroblastomas, and five astrocytomas also were examined. Tissues were histologically well preserved. Fresh tissues were immersed in isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds (Miles Laboratories; Naperville, IL). and stored at - 70’C until needed. Alternatively, tissues were fixed in buffered 10% formaldehyde, dehydrated in graded alcohols and xylol. and embedded in paraffin. Reagents. 612 were

Mouse

supplied

immunoglobulins

of the

preparations rum albumin

antibodies (San

IgGi

(MAb)

Diego,

CA).

subclass.

HYB-24l

These

They

were

and

antibodies utilized

HYB-

was drained PBS

in

PBS),

100

tion

Ig preparation

control,

tigen

anthranylate

tions

(bcr-25;

at 20 jig/mI

purified

mouse

directed

Science.

New

York,

Malvern,

against

of E. colt was used

synthase Oncogene

MAb

(Centocor;

(see below),

washed

horse

(blocking

the

same

(20 ag/ml).

were:

(a) goat

affinity-purified

antibodies,

(Tago; Burlingame. +

light

anti-mouse

chain

IgG

peroxidase

specific)

affinity-purified

Immunocytocbemical fresh

mm.

Both

cells

antibodies

indirect

immunoperoxidase

were used, according ary antibodies pI ofO.3%

incubated with

with

distilled

used

previously

as indicated

(5 mg ofDAB hydrogen the

H20,

cold

mm.

counterstained

DAB

After

with

complex (16,17).

methods The

second-

(DAB)

solution

was filtered

treatment,

cells

hematoxylin,

and

10

were mounted

was

To ensure

that

tissue

sections

Cryostat-cut

tissue

0.1% hydrogen washed three

sections

(4-8

1% formalin

peroxidase

.tm thick) in PBS (pH

activity

peroxide in distilled times in PBS for 5 mm

H2O each

were

used,

7.4),

was quenched

cold

would

or fixed

acetone,

or 95%

for 10 mm. Tissue and then incubated

and

the

normal

was removed

overnight

at 4’C.

were

as indicated

used

The

immunoreactions

the sections and

devel-

were

mounted

washed

with

with

permount

4.

Tis-

to fixation

paraffin-embedded

treated

by washing

with

pH

tissues

0.0025%

pronase

for 4-6

7.6,

in PBS

were

containing

in distilled

Trypsin:

sections

NY)

treated

with

for 30 mm.

2.25.

washing

of the were

pre-

mm. 0.2%

(Calbiochem;

The

reaction

glycine

was

(Sigma;

St

0.1%

The

pepsin

reaction

(Sigma)

in 0.01

was stopped

N

by repeated

H20. were

in Tris

treated

buffer,

by incubation

with

pH

with

0.1% for

7.6,

soybean

trypsin 5-20

trypsin

(Gibco;

mm.

Grand

The

Is-

reaction

inhibitor(Sigma)

was diluted

PBS.

Saponin:

tilled

deparaffinized

enzymes

sections

were

Frozen

subtype

primary

(bcr-25;

that

were

with

They

were

not

0.05% then

appropriate

with

were

in dis-

times

in PBS.

several

Negative

used

controls

antibody

by a similar

antibody

see above),

or substitution

ofthe

for

normal

titration

included

of the same primary

pro-

(Sigma)

cell lines and/or

antigen(s)

controls.

pre-digested

saponin

washed

and paraffin-embedded

the

as well as positive

of the

treated

H20 for 30 mm.

expressing

reagents. tion

of Deparaffinized owing

tis-

of the substitu-

species

antibody

and by PBS

alone.

Results

with

unfixed

Analysis

of Monoclonal

Detecting

P Glycoproteins

Mouse

HYB-241

MAb

lar epitope of Pgp terminal intracellular by the MDR3

shared

with

antibodies

sections were with block-

cell line,

by incubation

antibody

10%

serum

and washed

adhere, slides were cleaned in 95% alcohol and subbed in 0.3% gelatin solution containing 0.05% chromium potassium sulfate in distilled water. Frozen and deparaffinized tissue sections were used in this study. for 10 mm with either ethanol. Endogenous

with

blocking

H2O.

or detergents

MO).

Specificity Technique.

The

deparaffinized,

in distilled

with

permount. lmmunohistochemical

were

sections HCI buffer, pH

sues

for

in 100 ml ofPBS

tetrahydrochloride

for 6-12

acetone

Diaminobenzidine

The

sections

teolytic

was performed

with

described

above.

peroxide).

cells

fixed

solu-

After

protocols:

CA) in Tris buffer,

Controls.

and avidin-biotin

to methods

were

used as chromogen 100

and

were enzymes

complexes

formalin-fixed,

following

2. Pepsin:

(heavy in PBS)

mm.

washed

LaJolla,

in

in PBS),

several

primary

Pronase:

stopped

dilution

DAB

mm.

counterstained

peroxide

with

avidin-biotin

extensively

6-12

H20,

sections

hydrogen

20

with

and

study, the

land,

dilution

Immunocytochemistry

on microslides

under

Louis,

3.

(1:100

incubated

terminated

an-

(l100

distilled

Digestion and Detergent Treatment To assess the possibility of masking

with

1.

in PBS)

lgG

for

PBS)

permount.

treated

serum)

in

The

for

several times in PBS, and incubated

were

Enzymatic sue Sections.

specific)

dilution

anti-mouse

complexes CA).

Technique.

grown

( I :50

horse

(1100

tetrahydrochloride

sections

tissue 1%

(llOO

(16,17).

dilu-

chain

in

were

antibodies Sections

As a positive

light

+

conjugated

CA); and (b) biotinylated

followed by avidin-biotin-peroxidase (Vector Laboratories; Burlingame,

using

(gamma

sections

above.

with

with

oped by using DAB (see above). Finally. dH2O. counterstained with hematoxylin.

As a

working

15 mm

sections

secondary

washed

bio-

dilution

peroxide).

tissue

paraffin-embedded for

Deparaffinized serum

hydrogen

appro-

washed

anti-mouse

optimal

(5 mg ofDAB

the

1 hr.

were

complexes

with

mounted

for

horse

for

All inBlocking

Sections

secondary

titrated

sI ofO.3%

were

and

quenched

and

100

incubated

sections

Formalin-fixed,

Ig

the cell surface

at the

NY)

PA).

with

for 20 mm.

incubated

avidin-biotin-peroxidase

and

the

treated

as purified

of PBS

hematoxylin,

control, purified MAb against cytokeratins and other intermediate filaments (e.g.. neurofilaments) were also used at the same concentrations (20 ag/ml; Cambridge Research Laboratories, Cambridge. MA). Secondary antibodies used

ml

treatment,

then

with

MELAMED

in wet chambers.

established.

for 45 mm also previously

by

PBS-BSA)

antibody

previously

DAB was used as chromogen

was filtered

with

in 2%

BERTINO,

temperature

the primary been

incubated

followed

CASALS,

serum

at room

offand

antibodies,

(see above). in

horse

having

and

antigens

are mouse

normal

dilution

tinylated

BOCCIA,

performed

priate

at approximately 20 ig/ml in PBS containing 2% bovine se(PBS/BSA). Mouse MAb C2l9 was used for the present study

as a purified negative

monodonal

by Hybritech

(10% were

serum with

Tissues. Histologically normal adult human tissues and tumors were obtained from surgical pathology specimens within 1-2 hr ofresection and autopsy specimens within 10 hr of death. Several normal specimens from each organ site of different individuals, ranging from two to 12 samples, were used for the present study. A wide variety of human tumors, includ85 carcinomas,

serum

ing

cubations

techniques.

Cell Lines. The human melanoma cell line BRO and a variant transfected with a human MDRI cDNA under control of a CMV promotor and HBV polyadenylation signals (BRO/T cell line) were supplied by Dr. Piet Borst (Netherlands Cancer Institute). Parental neuroepithelioma MC-IXC and the drug-resistant variant MC-IXC/VCR. were provided by Dr. June L. Biedler (Memorial Sloan-Kettering Cancer Center).

ing

O’BRIEN,

there

and

HYB-612

each

recognize

(18-21). Mouse MAb epitope containing gene

product

for Pgp is demonstrated transfected

Antibodies

with

is no detectable

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

(22,23).

The specificity

by their

and expressing

binding

an extracellu-

C219 binds to a carboxy an amino acid sequence binding

the MDR1

to the non-transfected

of these

to the BRO/T gene,

whereas

parental

BRO

P-GLYCOPROTEIN

Table

1.

IN HUMAN

Distribution

of

Pgp

NORMAL

AND

TUMOR

human

normal

tissues

in

1279

TISSUES

Table

by

Continued

1.

immunohistochemistry” HYB-241.

Tissue

FIYB-6)2

C-219

Tissue

FIYB-24l,

0

0

Hepatocytes

0

0

Biliary

Melanocvtes

0

0

Acrosvringeum

A

A

vu

A

,

Acinar

0

0

0

0

Keratinocytes Langerhans

cells

Sweat

glands

Sebaceous

glands

Endothelial

C-2l9

Central

nervous

--

Purkinje

Glomeruli

Distal

Pt-rineurium Endoneurium

Urothclium

‘

Ureter

0

0

Bladder

0

0

Colieting

0

0

0

ducts

Prostate

Endorine

system

Adrenal

gland

(;ortical

cells

T-cells

0

0

Chromaffin

B-cells

0

0

#{149}lhyroid

0

0

0

0

pulp pulp

cells

Pancreas

(endocrine)

Reproductive

system

Thymocvtes

.

.

0

0

0

0

0

0

Breast

Cervix

Thvmus 0

0

Uterus

(endometrium)

Epithelial

cells

0

0

Ovary

(germinal

cells)

0

0

Hassall’s

bodies

0

0

Testes

(germinal

cells)

0

0

Respiratory

Placenta

system A

Bronchial

A

cells 0

0

Esophagus

0

0

Stomach

0

0

Gastrointestinal

Small

Smooth

cell line (24). to increase

In addition, intracellular

sensitivity

HYB-241 drug

in MDR

and

HYB-6l2

accumulation

cells

and

have

been

to partially

Skeletal

We utilized

MAb

on

Human

0

0

0

0

Adipocvtes

0

0

0

0

shown

tory

restore

placental

HYB-241,

HYB-612,

and

Tissues C219

Pgp

of

sections Significant cells,

did levels

including

not

improve

of Pgp but

not

Pgp

are

found

limited

digestion

of deparaffinized

a wide

to those

observations

on specific

presented

tissue

with

variety

of epithe-

secretory/excre-

in

and

C219

fibroblasts

medium

size

and

larger

endothelial which

241

than

and

HYB-612

ducts No

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

blood

with were

with

C219

these

MAb

HYB-241,

human

epidermal

immunoreactive;

the

and connective was observed on me-

staining

the epidermis. vessels

Endothelial

were

non-reactive;

dermis

prominent (see

on

cells.

glands

cells in the papillary was more

within

het.

below.

with

Sebaceous

unreactive.

immunoreactivity,

and cell types

staining

.

is found

endothelial

discussed

no reactivity

cells within

staining;

expression

1 and

and

luminal

ofcapillary

tissues

Table

glands

were

.

prominent

was luminal.

or dendritic

staining:

on a subset

showed

Sweat

of staining

lanocytes

levels

Immunohistochemical

ever, capillary

detection. on

and

keratinocytes.

and

and enzymatic

addition,

are

pattern

embedding,

In

trophoblasts

HYB-612,

to localize

#{149} . homogeneous undetectable

0.

functions.

expression in histological sections from normal adult human tissues. lmmunostaining on frozen tissue sections was intense, reproducible, and reliable. Immunoreactivity was lost after tissue fixation paraffin

matrix

Immunorcactivity: staining;

Detailed

Normal

0

0

muscle

Skin.

of Pgp

0

0

Fibroblasts

tissues

Expression

0

Chondrocytes

erogeneous

(20).

0

vessels

Interstitial

( continued)

tissue

Organs

A

intestine

connective

muscle

Blood

system

intestine

Large

and

Heart (myocardium)

-.

Pneumocvtes

.

(trophoblasts)

Vascular

Lung

tissue

0

0

Spleen

drug

0

0

system

White

0

0

nodes

Red

.

0

0

0

tubules

0

.

0

cells

Lymph

0

tubules

0

nerves

Heinatopoietic

‘

cells

system

Proximal

cells

Endothelial

tat

Urinary

system

cells

Peripheral

e

(exocrine)

Kidney

Neurons

Glial

0 ducts

Pancreas

cells

Fibroblasts

hal

HYB-612

Liver

Skin

below).

with

cells how-

showed

patchy

MAb

HYB-

CORDON-CARDO,

1280

O’BRIEN,

BOCCIA,

CASALS,

w,.,-..

BERTINO,

:‘

MELAMEE)

.

‘I

‘s

.

.

...

.

b.*

;.

).

..

..

.s..-’..

-8-. “I...

‘ .

,

.

‘

.

a

,

-

‘.

A’ ‘%L

#{163}.

.l’

.

.-

‘r’

ik

.-

. .

..‘.

.,

,w

:-

-,

\i:t\

:

,,.,.

.

tl

:J

‘%_L.

.:

4

.

c..&

-J

.

p #{149}1I

I #{149}_,

‘)

0

4

%

a

..

.

_

.

yf

,*

.

-

-0

-

: -

.,-, ...

...

‘I

:

#{149} !:4

i.:.

.

-.

ii’

1

.

‘

:

-

k

.

=

7

-.b-:-

J

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

-

.

‘:‘

P-GLYCOPROTEIN

Nervous frontal

IN HUMAN

System.

cortex),

cord.

No

Areas

expression

cells

ofsmall

dothelial homogeneous

staining

with

staining

was

low).

No

cells.

Peripheral

nerves,

were

unreactive

for

were

blood

bronchi

C219

on

stained

not

cells

showed

and

En-

tissue

(see

cells,

in thymus and

red

and

lymph

pulp

cells

of spleen,

all

failed

to stain.

macrophages

cells lining

revealing

an

mesenchymal

bronchi

was

patchy

the trachea

apical

were

then

tive.

of

the Endothelial

staining

irrelevant

mouse

Epithelial

neous

Stain-

MAb

epi-

(IgGi

of the

cells

luminal

ing

for

pattern

HYB-241

munoreactivity

sub-

was

as

negative

control.

Gastrointestinal were

Tract.

negative.

stained

In

diffusely

of the

small

the with

intestine

staining

was

observed

lumen

of the

small

staining

was

of

surface

epithelial

pattern.

Cells

and

were

colon

in the

apical

large

stronger

in

biliary

cells

brush

the

in the

however,

border

(Figure and

were

(Figures

612

liver pole

1G and

stained

showed

strongly

all three

for

1H).

C219

Expression

intense lining

The

intensity

HYB-612

than

not

for

the of that

a luminal

cells

pattern

Mesenchymal

and

lining

the

of immunoreac-

cells

and

Kuppfer

a weak

and

was

stronger

cells

Strong lary

geneous diffuse

cells

ofthe

exocrine

pattern

of

reactivity.

in

epithelial

Urinary

cells

Tract.

of Henle’s found

unreactive, bladder Endocrine present

of

convoluta

loops

and

showed

in distal and

tubules

System. in the

no

the

kidney,

pars

recta

minimal and

transitional had

pancreatic

Homogeneous cortex,

no

(Figure

stem,

seen

cells none

Glomeruli the

ureter

were and

un-

Pgp.

whereas

intense

vessels

unreactive.

However,

showed

a heteroge-

strong

and

but

not

homogeneous

lF),

stain-

whereas

weak

all myocardial with

MAb

to

include

of Pgp

was

found

brain

and

im-

fibers

stained

HYB-241

or

HYB-

sites

vascular

Capillaries tubules,

beds.

ofthe

ofcapillanies

dothelial

in the

cells

also

of the

central

were

cells

(Figure

extend cells

in stained

in

with

the

endothelial

as scattered

endometnium,

HYB-24l

the

these

surrounding

as well

cervix,

with

localized

Finally,

dermis, the

brain-

observation

stained

those

2)

system, 1E),

this

immunoreactive.

papillary

(Table

nervous

patchily

mainly

of capil-

organs

cortex

Endothelial

testes, also

of capillaries

bronchi,

other

we now

nerves

were

endothelial

several

cerebral

cord,

ofperipheral

antibodies.

and

of the spinal

Cells on

to capillaries

seminiferous

gus,

the

and

other

Endothelial

in

different

endoneurium

and

en-

esophaHYB-6l2

(see

2).

Expression

cells

of the

ofPgp

Immunopathological tumors,

roblastomas,

and that

3 summarizes

Human

carcinomas,

although

were

Tumors performed

sarcomas,

melanomas.

as previously

seen

on studies

including

cinomas,

were

immunoreactivity

chromaffin

(see

unreactive.

C219.

muscle

In addition

phasized and

Pgp

were

in prox-

whereas

of

were

(Figure for

on

cerebellum,

Table

1A). Epithelial ducts.

cells

and

differences

immunoreactivity, collecting

hetero-

ducts.

was present with

epithelial

detectable

adrenal

major

immunoreactivity

tubules

pars

showed

Immunostaining lining

Intense

convoluted

between

pancreas

detectable

ovary

1J).

expression blood

cells

unreactive. Acinar

HYB-6l2

ofPgp

(24,25). at

HYB-24l

epithelial

no

and

pits

immunoreactivity

but

However,

with

antibodies.

intense

had

sur-

immunoreac-

immunoreactivity.

C219,

II and

in cicells

crypts

ofcells lC).

HYB-241

of

located

unreactive;

bowel for

canalicular

ducts for

tivity

the

(Figures

biliary

was

stomach, a secretory

mucosa

were

cervix

observed

MAb

testis

oocytes

detectable

endothelial

of the endometnium

ofskeletal

with

including

HYB-612

nary

cells of the esophageal

C2l9.

their

was

Epithelial

and

again

Hepatocytes

imal

intensely

ovaries

prostate.

and

was

testes

demonstrated

and

Pgp

was de-

and

spermatocytes

of the

in

of

Pgp.

for Pgp

capillary

uterine

trophoblasts

A subgroup class)

the

types

cells

isNeu-

no detectable

gland

No

However,

of

cell

epithelial

Placental

in alveoli

of bronchial

other

mature

tubules

cells

All

had

mammary

more

seminiferous

was

unneactive.

immunostaining

adult

unreactive.

cells.

were

tract

Focal

and

staining Pancreatic

examined.

of

were

or Sertoli

glandular

reactivity

cells

ovaries

Leydig

rounding

major

unreactive.

Pneumocytes

of the

using

and

pattern

cells

or absent.

specificity

confirmed

germ

Focal

thyroid.

thyroid

gastrointestinal

cells

1B).

ofthe

ofthe

was not

System.

and

below).

Epithelial

of the

in epithelial

Immature

ofcells

C-cells

gland

Reproductive

(Figure

edge

and cells

in testes

unreactive

luminal

pituitary

tected

perineurial

were

at the

ofLangerhans

be-

examined.

The

lets

The

submeningeal

and

medulla

roendocnine

strong

HYB-612,

less intense

adrenal observed

spinal

cells.

and

meningeal

White

and

was

or glial

capillaries

endoneurial

Pgp.

and

(i.e.,

and

HYB-241

Lymphocytes

strongly,

stained.

parenchyma

1281

TISSUES

antibodies.

Cartilage

of minor

brain neurons

and

in

three

not

TUMOR

was consistently

including

cells,

AND

cerebellum,

for

observed

Tract.

1E).

thelial

vessels

for

was

Respiratory

were

blood

Tissues.

marrow

ing

detected

non-reactive

circulating

(Figure

include

thalamus),

,

was

the

Hematopoietic

Bone

(i.e.

immunoreactivity

whereas

nodes

studied

brainstem

Pgp

NORMAL

Pgp was frequently

reported

(26-28).

homogeneous

heterogeneity the frequency

on

ofPgp

variety

was

expression

of neu-

identified

However, patterns

of expression

a wide

pheochromocytomas,

in car-

it should

be em-

of immunoreactivity

more

common.

in the tumors

Table studied.

Figure 1. Localization of P-glycoprotein in human normal tissues using MAb HYB-241 and avidin-biotin-peroxidase immunohistochemistry. (A) Epithelial cells of the proximal tubules (p1) of the kidney were immunoreactive. Note the lack of reactivity with the glomerular tuft (g), distal tubules (dt), and an arteriole (a). (B) Intense homogeneous immunostaining was observed in the epithelial cells of the adrenal cortex (c), whereas chromaffin cells of the adrenal medulla (m) were unreactive. Note the projections of cortical tissue into the medulla (arrows). (C) Intense immunoreactivity in the apical brush border of cells lining the lumen of the large bowel (arrows). (0) Epithelial cells lining a major bronchus were intensively positive for HYB-241. (E) Frontal cortical brain tissue showing strong immunoreactivity in capillary endothelial cells. (F) Placental trophoblasts were strongly positive for HYB-241 . Distinctive patterns of immunoreactivity were observed when HYB-241 and C219 were used to examine liver and skeletal muscle. (G) Hepatocytes were unreactive for MAb HYB-241. (H) C219 stained the biliary canalicuar pole of hepatocytes. (I) Examination of skeletal muscle showed no reactivity with HYB-241 . (J) Intense staining for C219 was seen in a subset of skeletal muscle fibers. Original magnifications: A,C-J x 200; B x 40. Bars = 50 pm.

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

1282

CORDON-CARDO,

Table

2.

cells

by

Distribution

of

Pgp

in

human

capillary

HYB.241,

FIYB-612

C.219

system

Frontal

cortex

V

Hippocampus

.

Cerebellum Spinal

cord

Meninges Endoneunium

0 0

Penineurium Reproductive

3.

Expression

BOCCIA,

ofPgp

CASALS,

in

human

BERTINO,

tumors

MELAMED

by

immunohistochemistry

Tissue Nervous

Table

endothelial

immunohistochemistry’

O’BRIEN,

Tumor

type

Renal

carcinoma

HYB.24i,

HYB-6i2

C-219

16/21

16/21

Bladdercarcinoma

4/10

Colon

carcinoma

9/

Gastric

carcinoma

4/10

1 1

9/

1 1

I I 3

1/ 3

6/8

Hepatocarcinoma

0/8

0

Lung

carcinoma

2/22

2/22

0

Breast

carcinoma

0/8

0/8

0

Endometrial

0/3

0/3

system

Testes

carcinoma

Adrenal

carcinoma

5/5

5/5

Adrenal

pheochromocytoma

0/5

0/5

2/15

2/15

carcinoma

2/5

2/5

4/5

4/5

0

Seminoma

0

Embryonal

V

0

Teratocarcinoma

Breast

0

0

Choriocarcinoma

0/3

0/3

Placenta

0

0

Melanoma

0/20

0/20

Sarcoma

2/14

2/14

Neuroblastoma

0/7

0/7

0

Ovary

Cervix Uterus

(endometnum)

Skin

Papillary

dermis

Reticular

dermis

Gastrointestinal

V 0

0

Astrocytoma

Total

system

Esophagus

V

0

Stomach

0

0

Small

intestine

0

0

Large

intestine

0

0

0

0

Liver Respiratory

three

specimens

Bronchi

0

V 0

Alveoli

Two

casional

0

Nine

ofthem

system

out

of

0

0

ing

0

0

cases ofgastric

Pancreas

0

0

approximately

Urinary

gland

system

0

Ureter

0

Bladder Prostate

0

0

0

0

Hematopoietic

-

node

Spleen

0,

undetectable

0

(five

0

0

tomas

, homogeneous

staining;

.

weak

and

heterogeneous

levels.

out

of2l

cases,

the remaining Urinary

and cases

five

Renal with

bladder

of infiltrating

tumors

patchy were

transitional TCC

cell carcinomas

a homogeneous

11 cases with

infiltrating

HYB-612

of five

expressed pattern

divided

into

stained

(19

epidermoid

ure

2E).

(TCC)

(four

the

2A). cases)

(six cases). with

16

None

Two

anti-Pgp

2F

(Figure

cell

20% No

cases

sections of

detectable

out

of three

reactive,

with

positive. immunostained

showed

for HYB-

strong

antibodies

used

homoge-

in this

study

of

the

five

pheochromocy-

levels

of

Pgp

(Figure

cancers

(NSCLC)

three

2C).

were

studied

adenocarcinomas)

showed

of the Pgp

five

of stain-

2G).

None

and

of breast

apparently

none

One

tumors

22 NSCLC

Pgp,

(Fig-

reactivity,

tumor

cells

expression

which

staining.

was

detected

was Both

on

two

with

the

carcinoids.

of eight tissue

for

pattern

2D).

heterogeneously

lung

adenocarcinoma.

Similarly,

The

were

detectable

approximately

of

variably

ofthese

three

carcinomas

pulmonary

stained

(Figure

oc-

unreactive.

staining.

studied

all

at surgi-

showed

were

being

and

2B).

two of the

However,

showed

for

showed

MAb. the

none

non-small-cell

Only

were

cells

carcinomas

cases)

with

primary

and

(Figure

superficial

homogeneously

in

ofstaining

immunoreactivity

cell carcinOmas

Pgp

while

(Figures

analyzed

cases

Tumors.

C219,

cases

was heterogeneously

hepatocarcinomas

immunoreactivity

0

site

obtained

superficial,

the

carcinomas

luminal

tumors chemotherapy.

ofthem

of

of tumor

All adrenocortical

patchy

Epithelial

30%

Twenty-two

S

Immunoreactivity:

staining;

and

rest

adenocarcinoma

for MAb

neous

system

Lymph

241

trophoblastic

homogeneous

at the

0/5

53/182

combination

one

The

11 colon

enhanced

reactive

after

tumors,

a strong

Six ofeight

Kidney

a

was

other

staining.

with

gestational

disease

cell

Thyroid

Adrenal

were

of residual

antibodies.

system

Endocrine

All

a

cal resection

0/5

47/182

studied the

levels

cancer

normal showed

three

studied

reacted

parenchymal staining

endometnial

glands

in some

in occasional carcinomas

cells. analyzed

of Pgp.

Figure 2. Localization of P-glycoprotein in human tumors using MAb HYB-241 and avidin-biotin-peroxidase immunohistochemistry. (A) Intense homogeneous pattern of staining in a renal cell carcinoma. (B) Adrenocortical carcinoma showed strong immunoreactivity for HYB-241. (C) Examination of a pheochromocytoma (tm) showed undetectable levels of Pgp when stained with HYB-241. Note the strong immunoreactivity of cortical cells (c) present in the sample. (D) Strong immunoreactivity at the luminal site of a colon carcinoma (arrows). (E) No Pgp expression was detected in epidermoid cell carcinomas of the lung. Differential pattern of immunoreactivity tivity

was observed

HYB-241

and

C219

in hepatocarcinoma

in hepatocellular

cells

carcinomas.

for MAb

C219.

Original

(F) HYB-241

magnification

did not react

with

x 200. Bars

hepatocarcinoma =

50 pm.

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

cells.

(G) Intense

heterogeneous

immunoreac-

P-GLYCOPROTEIN

IN HUMAN

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4

CORDON-CARDO,

1284

Germ

Cell Tumors.

Two out of 15 cases

munoreactive

for

weak

of staining.

pattern

the

antibodies

studied,

These

widespread disease. Two out showed patchy immunoreactivity of five

teratocarcinomas

mainly

at

the

the

three

these

two cases

offive

positive

of cystic

choniocarcinomas

were

from

patients

and

staining

of

adenomatous

studied

tumor

tions from the same cell lines and organs. Satisfactory immunoreactivity was not obtained with either

showed

None

for

Neuroectodermal

Tumors

examined

ofseven

showed

and

roblastomas)

or five

ously

stained

for

of brain

three

of the

of Pgp.

included

had

sarcomas

none

adrenal

neu-

detectable

were

1ev-

reports

the

human

using

microanatomic

and

two different false-positive

epitopes results

topes

shared

by other

a survey

from

failure

ofantibody

localization

of Pgp

of its expression

Three

MAb

in human

recognizing

at

ofPgp were used in this study to minidue to antibody binding ofsimilar epi-

proteins,

and

to minimize

binding

due

false-negative

to tissue-specific

results processing

have

been

directed

developed

tenization parison

with

C219,

MDRJ

have

of HYB-241

has

that although gene, different

and

different

recently

can be identified.

Whereas

human characand

and

com-

has demon-

recognize products of the distinct forms ofthis pro-

MAb

a narrow

to identify

HYB-612,

reported,

all three antibodies electrophoretically

immunoprecipitate

used

of Pgp

in some biochemical

and

been

regions

been

immunohistochemically (24-31). A detailed

of the specificity

strated

against

(15,19,22),

this molecule tumor tissues

HYB-241

well-defined

and band

HYB-612 of

M1

will

180,000,

C219 recognizes both a broad Mr 170,000180,000 narrow M1 180,000 band (19). The availability

species and of MAb capable

distinguishing

the

specific

heterogeneity uct.

The

vation, though

exists existence

for the

it

forms tion

Mr

data

exist

reported

180,000

distinct

in the of Pgp cannot

tissues

proteins

these

of Pgp

addressed

the

obser-

(32). Alcan be

known

whether

any

us to suggest the

Whether present

that

by HYB-241 on

basis

or not

or quantitative by

prod-

a new

presently

the of

masked

processing.

Therefore,

and of

the

or

of

using

epithelial intestine

cells.

Luminal

but

express

Pgp.

cells of both In

Northern

dot-blot

the

and

planes

separating

this

organ sweat

ofglandular thyroid, in

and

greater

in Figure

ing

C219

as the

ductal

of

expression

cells,

and

posed

extracellular

to be discussed

is seen

the

liver.

biliary

canalicular

ofbiliary

HYB-612.

gene

MDR3

is not

gene

product.

observed

cells.

no hepatocyte

stain-

Epitope

mapping

the

One

possible liver,

studies

an amino

(23). the

epitope

are

limited

of the

gene

MDR3

ofthe

is that

stainexpres-

MDR3

sion is being detected in hepatocytes by C219, whereas MDRI pression is being detected by all three antibodies in the bile Such

the

distinct

a possibility

forms

differentially

is further

in hepatocytes

of the MDRJ expressed

supported

(34).

gene in

by the

Alternatively,

product

hepatocytes

known

the

described vs biliary

pro-

products

reducing the of recognizing

interpretation

therefore,

the

recognized

possibilities

and

acid

Although

Comparison

for MDRJ

in the

of hepa-

and

product

known,

these

epithelial

recognizes

is extracellular.

sequences

pole

ductal

comprising

that

when

As demonstrated

no sequence ofabsolute identity (34,35), that HYB-241 and HYB-612 are capable

ofMDR3

foci

endometnium,

cells

this antibody

sequence

of Pgp

pattern

feature

Pgp

breast,

cell staining

and

or HYB-612

amount

the pole

that

acid

by the

identifies likelihood

stains

by the

amino

by HYB-241

of projections

epithelial

prostate,

to examine

HYB-241

shared

specific

sites

ofendothelial

secretory

biliary

with

sequence

presence

anatomic

of immunoreactivity

are used

demonstrate

sion

data.

pattern

only

is seen

the presence

bronchial

ofthe

subset

the

to false-positive

as a standard

skin,

the

below.

as well

In contrast,

reported

unreported

cells

a particular

1, MAb

tocytes,

cells.

in func-

ofthe

antibodies

ing

different

glands

A distinctive three

and

medulla

Previously

with employing

cortex cannot be reliably disowing to the absence of tissue

structures the

(33).

detail

of its expres-

differences

these into

epithelial

the MDR3

HYB-

RNA

is susceptible

because the Pgp-rich adrenal free of the adrenal medulla, tissue

(23,31),

an analysis

results, sected ofcortical

Such

pars

adrenal

in the cortiThese results

studies

of isolated

expression

what

the an-

analysis

(29).

reports

in which

ofPgp

expression

strongly tubule.

previous

tissues.

Pgp

to epithelial

medulla,

these study

human

detect

proximal

with

adrenal

denatured

we recom-

when

sections

small

three

by HYB-

in the present

antibodies

is restricted

recta

of Pgp

frozen

three

agreement

of the

func-

molecules.

allow form,

above.

qualitative

pars

in general

for C219

that

gene

is not

and

identified

important

described

have be

between

form

Pgp

it is not these

in this study

is a physiologically

hypothesis

for the MDRJ

by Beck and colleagues Mr 180,000 Pgp species

distinguish

differences

tigenically sion

discrete

supports

level

electrophoretically, features

The 612

ofa

of Pgp

protein

has been reported Mr 170,000 and

distinguished structural

tional

forms

at the

irreversibly

glandular

large

expression

recognized

discussed

sec-

any ofthe

are

and

1, all

ofsimple

and

include antibodies

and localize normal and

tein

variety

of

Pgp. Monoclonal

lines

using

and

and

tissue

and reliable untreated or

gland, intense homogeneous expression is identified cal cells but not in the chromaffin cells ofthe medulla.

immunohistochemistry.

least mize

of

detailed

cell

cells ofthe

convoluta

antibodies.

tissues,

fresh

not crypt

are

study

reported

in a wide

sections

MELAMED

frozen

determinants

for analysis

data

in Table

with

the

sections

The

In the kidney,

heterogene-

C219

fixation

tissue

to

exception

in normal

tumors

frozen

As shown

Discussion This

mend

tissue

that

and

formalin

pertain

20 mela-

Similarly,

three

astrocytoma

two of 14 soft tissue the

None

levels

(which

cases

However,

Sarcomas.

detectable

cases ofneuroblastoma

els ofPgp.

HYB-612,

antibodies.

antibodies.

nomas

It is likely

through

compared

deparaffinized

antibodies. 241,

of

immunoreactivity

enzyme-digested

when

BERTINO,

embedded

cells,

areas.

sections

CASALS,

im-

with

tissue

BOCCIA,

and

were

a heterogeneous

cases ofembryonal carcinoma in tumor cells. Similarly, four out

showed

margin

ofseminoma

with

O’BRIEN,

cxduct

expres-

antigenically

above ductal

may

be

cells.

Examination ofskeletal muscle reveals no reactivity with HYBand HYB-612; however, intense immunoreactivity is seen with MAb C219 in a subset ofmuscle fibers, as previously reported (25). 241

Expression

ofPglycoprotein

Excretory/Secretory We

observed

loss

in Epithelial

Cells

with

Functions of Pgp

expression

in formalin-fixed,

paraffin-

Similarly, C219, either

intense

staining

but not in smooth of the antibodies.

Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on October 9, 2012

is seen muscle

in all cardiac or any other

muscle myogenic

fibers cells

with with

P-GLYCOPROTEIN

Expression

IN HUMAN

NORMAL

ofPglycoprotein

Sites

We have

previously

dothelial

cells ofbrain,

reported

the expression

testis,

capillaries,

the cervix, pattern

HYB-241

as in scattered

esophagus

and

and

of Pgp

HYB-612.

In those

is weak,

a difference

study, endothelial

cells

Table

in capillary

cases

we extend in endoneu-

bronchi.

examined with the three is consistently seen with

HYB-612

representing bodies.

capillary

observed

the con-

capillary blood yeslocations as phar-

expression

and

en-

skin (24).

with

In the present

as well

of immunoreactivity

ofthe

expression

ofthese anatomic

detection

endometnium,

ofvanious tissues immunostaining 241

the

dermis

ofsuch

(24,36,37).

with

of Pgp on capillary

and papillary

the association

sanctuaries

nial

nived

at Blood-Tissue

tinuous non-fenestrated arrangement sels, as well as the recognition ofthese observation

1285

TISSUES

from

in

2 lists the

endothelial

cells

antibodies. Less intense C219 as compared with

where

no staining

is seen

in binding

affinity

staining with

with

Six of the eight

with

C219

of reactivity

observation

but

Expression

Human Previous

the possibility

using

of MDR

possessing

Pgp,

of Pglycoprotein

observations

clinical

mRNA

such

made

important

material in tumors

as renal

and

Clarification

of

hepatocellular

carcinomas

this

demonstration

nyc

question

in previous

studies

MDR

that

in human

Implications

anti-

Function

have

shown

high

levels

from

cells

normally

carcinomas

and

(26-28).

of Dc-

in a variety of huconfirm some of the

contain

several

tumors most

new and

expressing carcinomas.

analyzed frequently

is summain tumors

diverse

tory

such

cells

in trophoblasts

in the

lining

pancreas. ofparticular tural

the

neuroectodermal

origin,

adrenal

such

as melanomas,

pheochromocytomas,

expression with

had

in the tumors

the

observed

Interestingly,

lack

of Pgp

despite

in placental

the

trophoblasts,

no detectable

intensity

of Pgp

expression

another

for a given

tumor

in normal

the three

had

type,

commonly

The

placenta.

adrenal

The

lack

is con-

chromaffin

expression

gestational

can

vary

of

from

one

sample

to

heterogeneity

tumor. with

of

homology

between

(43-45).

normal

membrane

imity,

and

Pgp

expression

neoplasms.

The

failure

in a variety of clinically unresponsive and epidermoid carcinomas, suggests

that

other

mechanisms

to be operative. in tumors

protein.

Finally,

derived

Examples

cell Several and

from

than

it is also cell

in this

those

clear

types

that

category

involving

that do

Pgp

in certain

can

be

expressed

not

normally

express

this

are some

urinary

bladder

and

normal

tissues

Pgp

here, several in certain

MDR3 in of

including

was

protein

fibrosis

biliary

ducts

barrier

sites

detected

glands

chloride

and

transport,

with

their

co-expression

may

on

be regulated

skin,

cystic

fibrosis

proteins. of Pgp using encourages

clinical

tool.

in a variety

primarily

ofhuman

clinical

to understand tunes of the

Pgp expression tumors.

gene

that

family

antibodies ofthis

expression

and

technique

can now

furtherstudies

as

be de-

are still needed

significance

in the context

tissues. prox-

suggest

ofa

monoclonal

Pgp

tumors,

possible

tissues

evaluation

Although

the

of

gene by ab-

manifestations

as a component

and

organs is of struc-

characterized

a subset

of and

in epithelial

of the

the

is a disease

can tis-

excretory/scene-

and

also

sweat

points normal

of Pgp expression in these ofthe recent demonstration

this

Cystic

to establish

Literature 1.

of this ofother

finding

and

biological

fea-

and

Cited

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Riehm

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de-

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Pgp are likely

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tected

Such a pattern the phenotypic

to detect Pgp expression tumors, such as melanomas resistance

for caution correlations

trophoblastic

considerable

in human

the

need

of kidney

lumens,

identification interest in light

product

a potential

Pgp.

and

observed

cells

origin

intense

expression is commonly seen within a given of Pgp expression in tumor cells is consistent variability

Pgp.

medullary

consistently

examined

tumor

neuroblastomas,

no detectable

ofadrenal

of

or the defini-

recognize

cells at blood-tissue

bronchial

immunohistochemistry

Similarly,

functions, tubules

as well as in endothelial

detectable

C219

not

emphasize the clinicopathological

physiological

of the lung

germ

does

examination

probes,

Pathophysiological

as proximal

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The

the

of Pglycoprotein

in normal or tumor cells with squamous epitheas exemplified by the 18 epidermoid carcinomas

tumors

await

capable of for MDRI.

affecting lung, sweat glands, and pancreas, among other The structural similarities of these proteins, their chromosomal

this protein, such as reConversely, we have found

Pgp expression.

this

carcinomas,

a probe specific

MDRJ-specific

HYB-241

the

no Pgp expression hal differentiation, lacking

pat-

antibodies,

tumors.

for

sues with

cells

findings.

derived from tissues normally nal, colon, and adrenocortical

Pgp

these

stud-

to the

in hepatocellular

will

with

gene product. These findings selecting antibodies to perform

likely

derived

colon

Pgp expression in the 182 human nized in Table 3. Expression is found

cells.

that,

with

cinomas have measured extracted RNA using hybridizing to the MDR3 gene and thus not

these

in

tectable levels of Pgp have also been reported man tumors (38-42). The data reported here

sistent

seen

Similar

C219 is recognizing the MDR3 and not the MDRI gene product. Previous reports describing MDR expression in hepatocellular car-

liver,

studies

ofsuch

liver

carcinomas

HYB-241.

HYB-

Tumors

expression

and

hepatocellular

with

On the basis of the observations reported be made. Pgp expression has been identified

Differential

of

not

in normal

raises

most

C219,

between

them.

stained

tern

We also identified

this

TUMOR

ied

Barrier

macological

AND

teins

from

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