The direct activation of human multidrug resistance gene (MDR1) by anticancer agents

Vol. 165, No. 3, 1989 December

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages

29, 1989

THE

DIRECT ACTIVATION

Kimitoshi

OF HUMAN MULTIDRUG RESISTANCE BY ANTICANCER AGENTS

KOHNO, Shin-ichi

SATO, Hiroshi

and Michihiko Department

Received

November

TAKANO, Ken-i&i

Oita

Medical

Oita

879-56,

Japan

School,

The development in cancer

increased to

encoded is

Decreased

expression

multidrug

resistant

precedes The

MDRl

shown

(11,12). blastoma

is

cell that

tumor

the

express

levels

Hazama-cho

expressed

In these

to be regulated in several

at the

loss

time

by Bourhis

specimens,

In these

cell

does not

lines,

the

correlate

line,

with

in which

of amplified

over-

(8).

in revertants DNA (9).

of Further,

of MDRl gene

drug-resistant

cell

at transcriptional

lines, level.

including

following

al. (13) indicates -et -~ one of 15 (6%) tumors from

initial that, patients

neurochemotherapy among 41 neurowho have 0006-291x/89

1415

P-

vincristine(VCR)-

KB cell

observed

(Z-4).

of a membrane

gene amplification

human cancers

of relapse

agents

levels

established

for

cross-resistant

unrelated

of transcription

(10).

appears

study

without

clinical

selected

found

increased

human cancer

activation

a major

been

(5,6).

without

gene (MDRl) is observed of MDRl gene expression in regulation of MDRl gene presence of anticancer activated directly on the daunomycin, adriamycin of MDRl mRNA expression drugs that select the

lines

of expression

previously

from

line the

and leukemia Recent

repeatedly

of MDR 1 gene was also

gene amplification gene

lines

gene was observed

MDRl gene expression blastoma

but (VJ-300)

line

of MDRl

has been

have

We have

expression

it

Unexpectedly,

cell

by the MDRl gene

(7). cell

remains

and functionally

cell

amplified

copy number

resistance

of structurally

resistant

glycoprotein MDRl gene

(1).

to one drug

range

Many multidrug

resistant

of multidrug therapy

resistance

a broad

MATSUO

1989

of a multidrug-resistance SUMMARY : Enhanced expression in some cancer patient, but any regulatory mechanism this phenomenon is not yet known. In this study, the by transient expression assays in the was analysed We found that MDRl promoter could be agents. addition of anticancer agents including vincristine, and colchicine. The results suggest that the level including is associated with previous chemotherapy, multidrug resistance phenotype. 0 1989 Academic Pre5.s. Inc.

problem

GENE (MDRl)

KUWANO

of Biochemistry,

20,

1415-1421

not $1.50

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 165, No. 3, 1989

received (42%)

BIOCHEMICAL

chemotherapy treated

samples,

MDRl gene

therapeutic

agents

at transcriptional To test

is

not

levels

levels

themselves

of MDRl transcripts

These

might

while

of MDRl expression.

amplified.

possibility,

5'-flanking

enhancer

element

determine

the

directly

often

function

chloramphenicol

we have region

which

promoter

of the

act

of these region

in

11 of 26

In these

evidences

suggest

cause

activation

Using

agent

themselves

transient

the

clinical

that

chemoof MDRl gene

gene

we linked are

(14).

To

sequences

useful

we examined

and

DNA sequences

coding

which

the MDRl gene

promoter

manner

to the

system,

cis-regulatory

termed

specific

(CAT)

activate

identified

MDRl gene,

DNA elements,

of MDRl

expression

could

recently

tissue

acethyl-transferase

(15).

MATERIALS

high

show high

level. this

DNA elements,

containing

exhibits

tumors

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reporter

whether

of function

anticancer

or not.

AND METHODS

Cell line and culture : We have mainly used CV-1 cell derived from African green monkey kidney which was obtained from Japanese Cancer Research Resource Bank(JCRB). CV-1 cells were grown as monolayer culture at 37°C in 95% air and 5% CO2 in minimal essential medium (MEM) supplemented with 10% fetal calf serum, penicillin G (100 U/ml) and kanamycin (60 pg/ml). Properties of KB and its multidrug-resistant VJ-300 are described elsewhere (8). Drugs and chemicals : Vincristine were obtained from Sigma Chemical from Nihon Kayaku Co., Tokyo. Silica gel plate 60F254 purchased chloramphemicol from New England

(VCR), adriamycin, daunomycin and colchicine Co. St.Louis MO. Etoposide was obtained Acetyl-CoA was purchased from DL-Pharmacia ; from Merk (Federal Republic Germany) ; 14CNuclear.

DNA transfection and CAT assay : For all constructions, plasmid DNA was by isolated using standard procedures including two cycles of purification centrifugation in CsCl gradients. Transient transfection protocol for CAT CV-1 cells were seeded into assay utilized the DEAEgdextran method (15). loo-mm dishes at 3-5 x 10 cells and grown overnight. Concentration of Concentration of DEAE-dextran is plasmid used for transfection was 10 pg/ml. cells were subjected to 200 pg/ml for CV-1 cell. After 8 hr transfection, DMSO shock for 2 min, followed incubation for 48 hr. Indicated drugs were and CAT assay was carried then added to culture medium for transfected cells, cells were out 24 hr later. For DNA transfection into KB and VJ-300 cells, Transfection procedures were transfected by using Lipofectin T"Reagent (BRL). used The assay for according to instructions provided by the manufacturer. CAT enzymatic activity in transfected cells was followed after the procedure reported by Gorman --et al. (16). RESULTS AND DISCUSSION Restriction containing structure Fig.lB. monkey

map of two genomic

parts

of pMDRCAT reporter After kidney

clones

of the human MDRl gene the

plasmids

EMBL-MDR-1

is

shown

used

in

this

transfection of these plasmids the effects of a transient

CV-1 cells,

1416

and EMBL-MDR-2 The shematic

in Fig.lA. study

is also

shown

into African green exposure to anticancer

in

BIOCHEMICAL

Vol. 165, No. 3, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

5

EMBL-MDR-2 S I

EMBL-MDR-1

SS,t?tEEE

Fragment

I

Fragment

D

St I

S! S!

E

c

I

H

H

Sp AC

Sm AC * ’

-

St I

pMDRCAT

2

pMDRCAT

3

-3

I

s

lK2

-

mRNA

B pMDRCAT

5

s-

t

CAT

POSY A

I-,CATI I

-+-aq 1-m

pMDRCATL

b-+-CR-J-

pMDRCAT5 pMDRCATI-E

Y

pMDRCATI-I

1-4

CAT

HFragmentib

I_)

CAT

HFragmenlDi-

pMDRCAT

1-D

1 CAT

HENHANCER~

Fi;;lge~~)Restriction map of genomic clones containing parts of the human . S, SalI; E. EcoRl; St, Styl. The 2 kb Styl-Sty1 fragment is the segment of EMBL-MDR-2 containing the promoter region and the first axon of the human MDRI gene, which is indicated by solid line. The open circle and arrow indicate the initiation site of transcription. Both fragment 1 and fragment D are the segment of EMBL-MDR-1 containing the enhancer region of the human MDRl gene. (B)Schematic representation of the recombinant plasmids used in this study. Characteristic feature of the MDRI promoter is on the top with relevant restriction enzyme sites. H, HindIII; Sm, SmaI; AC, AccI; Sp, SphI. pMDRCATl-1 and 1-D are MDRl enhancer containing constructs as described elsewhere (13). pMDRCATl-E indicates the construct containing SV40 enhancer.

agents

were

remarkably

the

up

enhancer

or the

pMDRCATl-E

in

enhancer

5-fold

It

containing

in Fig.2, within

was not

CAT activities 24

fail

the presence

whereas

of the

CAT activities

be probably

to be further

constructs

in

of pMDRCAT1 were

VCR addition,

after

Decreased

of VCR might

remains

hr

observed

of MDRl gene.

the presence

of VCR.

enhancer

to

enhancement

VCR-induced

effect

As shown

examined. increased

due to the

SV40

of cytotoxic

studied

why CAT

activities

of

to be activated

in cells

by the

drug

treatment. To determine the

activation

introduced. Only

the

location

pMDRCAT5

450 bp upstream

failed is

experiment,

CAT activity

pMDRCATl-E,

was

region

of the

differential

We then

of functionally

significant

of MDRl gene by VCR treatment, enough decreased is

rates

of

asked

whether

manner,

and also

gene.

Fig.4

whether

clearly

for

constructs activation

response.

containing

by the

clearly

deletion drug-induced

VCR-induced

of enhancer

again

promoter

to show the

to VCR.

the

agents

for were (Fig.3).

In this pMDRCATl-D and

constructs,

exposure

involved,

sequences

Since

probably

specific act

by

transcription.

drug other

showed

activation chemical

that

was observed agents

VCR-induced 1417

could activation

in

activate

dose-dependent the MDRl

was increased

as a

Vol.

165,

No.

3, 1989

BIOCHEMICAL

AND

RELATIVE

AcCM c;”

BIOPHYSICAL

RESEARCH

CAT

COMMUNICATIONS

ACTIVITY

I

1 *

1)

163 1

DMSO

5 122 1

.;

182

*a

pMDRCATlE

*.+

+

v

5

*

5

VCR

108 1

a

Fig..?. Activation of MDRl promoter by VCR. Expression of CAT activity in CV-1 cells transfected with indicated recombinant plasmids and exposed to rig/ml of VCR or DMSO for 24 hr. Relative enhancement of the CAT expression was expressed as the fold increse of CAT activity of pMDRCAT1 obtained in absence of drug.

10 the

FOLD IDUCTION

VCR

pMORCATl

pMORCAT2

x

2.5

x

2.0

x

0.6

x

0.5

x

3.5

x

2.5

x

2.0

x

0.7

[+ pMORCATI-0 [

+ -

pSV2CAT

-

+ pMURCAT

2

+

pMDFKAT3 +

pMORCAT4 [+ pMDRCAT5 [+

Fig.3. constructs. increase

of CAT activity in CV-1 cells transfected Expression CAT activity of VCR treated cell was expressed in CAT activity obtained in the absence of drug.

1418

as

with the

deletion fold of

Vol.

165,

No.

BIOCHEMICAL

3, 1989

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

FOLD !NDUCTION

AcCM

I

1.0

Vincristine

I. 4 2.0 4.3 2.6 Adriamycin

1.4

Colchicine

3.2

Daunomycin

2.0

Etoposide

1.0

(nglml)

Fig.4. Effects of VCR and other anticancer agents on the activation gene. Expression of CAT activity in pMDRCATl-transfected cells various concentration of VCR and indicated concentration of other agents. Concentration of other agents except VCR was 4 times than that of LD of KB cells and was equivalent to 10 rig/ml of LDgO is the dosz'required to block the initial survival by 90%.

function 40 rig/ml could

of VCR.

activation

Among several

effectively

activate

activated

weakly

multidrug

resistant

colchicine

(3,17).

(Fig.4).

was observed

anticancer

agents

and an DNA topoisomerase cell

II

lines

which

Colchicine

also

gene.

The levels

MDRl gene It

by drugs

is

cancer

is

into

overexpressed

was transfected CAT activity

to examine

observed

the presence

P-glycoprotein

as shown

in

by the

could

often

not.

observed

after

appear

exposure

agents

in

to

to activate

the MDRI

varied

between

activation

2-

of the

cells.

We introduced

gene

cells

the MDRl-CAT

this

of MDRl promoter

cells

pSV2CAT

efficiency.

expression 1419

VJ-300

amplification.

was apparently Initially,

apparatus

of MDRl gene by anti-

and multidrug-resistant

transfection

Fig.5.

transient

only

the MDRl gene well

activation

without

the

of an activation

analysis

whether

KB cells

to normalize

VP-16,

a significant

in human cancer

drug-sensitive

could

selected

chemical

but

of

observed.

of pMDRCAT1 in VJ-300

in KEi cells

Previous

by these

(8).

activate drugs

experiments,

was reproducibly

of interest

agents

constructs which

of activation

in separate

dose

VCR

VCR and daunomycin

adriamycin

inhibitor,

were

could

higher

to

the concentration

of MDRl gene have been

Thus microtubulin-interacting

and 5-folds

at tested,

the MDRl gene whereas

and amplification

Overexpression

that

Maximum

of VCR.

of MDRl exposed anticancer

In this 3-fold result

higher

plasmid

experiment, than

seems to suggest

in VJ-300

of MDRl promoter-CAT

cells. has indicated

Vol. 165, No. 3, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

KB

VJ-300

Fig.5. CAT activity in transfected human drug-sensitive KB cells and multidrugresistant VJ-300 cells. Relative CAT activity was expressed as the percent of activity obtained in pMDRCATl-E after normalization of transfection efficiency by CAT activity of pSVZCAT. The CAT activity of pIDRCATl-E was about 10% of CAT activity of pSV2CAT in both cells.

that

there

was no difference

and its

drug-resistant

results

obtained

in

KB-8-5 with

CAT activity

cell(l8)

VJ-300

cells

was less

than

more resistant in

lo-fold

However, VJ-300 the with

we

could

cells.

drug those

obtained

Drug-induced

pump (6,19). that dioxin (21) the

of this

gene.

transient

the

activation

first

are (see

concentration

mechanism

a cytochrome

regulation

is gene

of the MDRl gene,

--in vivo. report that anticancer

CAT activity

in

contribute

to

Figs.2 the

cellular cytotoxic

to be as a drug

regulation, which

and 5).

protective

it

expression

efflux

has been reported There is

(20). is

cells.

good agreement

below

supposed

of gene

P-450

in

increase

in KB

sequences

results

al.

400-fold

Z-fold

of the

promoter

expression

by Ueda et

was about element

in CV-1 cell

of P-glycoprotein

induced

by carcinogen

(22)

of MDRl gene in assay.

positive

assay drug

As an analogous

is

only

different

of both

observed

enhancement

that

the

used

of MDRl gene may be one of the

function

acute

cell

of MDRl enhancer

further

KB cell

level

VJ-300

We also

These

intracellular

can induce

for

expression acting

the

or xenobiotics This

suggests

to maintain

since

evidence

the

detect

again

activation

mechanism level,

not

that

cellular

KB-8-5

(8).

presence

with

to the

to VCR (lo),

due to the This

activation

KB cells

drug-sensitive

We believe

Although

resistant

to VCR than

the CAT activity

. relate

of MDRl gene and drug-resistance. (18)

between

From our regulatory

factor

a sequence present

agents dependent

study,

may exist 1420

are

clearly

manner

working

constitutively

notion

involved in transient by using is

that

in various

a transtissues

BIOCHEMICAL

Vol. 165, No. 3, 1989

or cancer or by drug

binding

one of a group analogous that

and its

cells,

appearance

dosage

and scheduling

resistant

tumor

be modulated

by the

also

seems possible

that

response

cancer

example,

the resistance

cells That

itself.

of some anticancer for

to stress

In either

in bacteria.

chemotherapy cells,

might in

of drug-resistant

of cancer

24 hr before

activated

SOS system

effect

the

It

proteins.

of genes

to the

activity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

drugs

by attempts

response

take

anticancer

the MDRl gene

on the

case,

agents

these

cell, results

is

somewhat suggest

--in vivo might be a direct consideration may modify the to avoid

the

to intervene

appearance decisively

of drug within

hold.

ACKNOWLEDGMENTS We thank Dr. D. Schlessinger (Washington Univ., St.Louis) for critical reading and comments on this manuscript. We also thank Ms. M. Ando in our laboratory for preparing this manuscript. This study was supported by a grant-in-aid for cancer research from Ministry of Education, Science and Culture of Japan. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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