Vol. 187, No. 2, 1992 September

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1992

16,

Activation

BIOCHEMICAL

of

Pages

Human

Multidrug

Resistance-l

Heat Miki

Miyazaki,

Kimitoshi

Ken-ichi Departments

of

Received

July

17,

Kohno,

Matsuo.

Biochemistry

Shock

Nasu*

in

Response to

Stress

Takeshi

Masaru

and

Gene Promoter

677-684

Uchiumi, and

Medicine*,

Oita

Oita 879-55,

Japan

Hideyuki

Michihiko

Tanimura,

Kuwano

Medical

University,

Hasama-machi,

1992

Summarv : The multidrug resistance (MDRl) gene encodes a P-glycoprotein, which catalyzes the energy-dependent efflux of anticancer agents. Various environmental stresses including heat shock can induce the expression of endogenous MDRl genes. In order to study the regulatory mechanisms of MDRl gene expression, we have established human cancer KB cell lines which could stably integrate bacterial chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDRl promoter. Kst-6 has an integrated plasmid, pMDRCAT1, containing the human MDRl promoter of -2 kilobases. The MDRl gene promoter contains a typical heat shock element (HSE) motif located -152 bp to -178 bp from the initiation site. Heat shock at 45 * C for 90 min significantly induced CAT activity in Kst-6 cells. Northern blot analysis showed a 4-5 fold increase in CAT mRNA levels in Kst-6 cells. Deletion analysis of the MDRl promoter demonstrated that the induction of CAT activity was observed in Kxh-14 cells containing a HSE-deleted MDRl promoter construct, pMDRCAT7. However, further deletion analysis showed that heat shock could not induce CAT activity in Khp-1 cells containing -76 +121 base sequence of the promoter, suggesting that a new heat shock responsible element was located at between -136 and -76. Gel shift assay showed that the heat shock factor (HSF) could bind to the HSE motif located at -152 bp to -178 bp in the MDRl promoter. We also found that one distinct DNA-protein complex formed specifically within the MDRl promoter region -99 to -66 was not significantly increased, but relatively more stabilized under mild denaturing condition in the nuclear extract of heat-shocked cells. In our present assay system, activation of the MDRl promoter in response to heat shock appears to be mediated through both a new heat shock % 1992 Academic mess, 1nc responsive element and MDRl specific transcription factor.

A variety cells

respond

to heat

genes expression temperature (HSF)

WE)

(5).

inducible

contrast,

shock

and The

is mediated

induce

heat shock genes

chemical induction by the

resulting

stresses, of mammalian binding

in many

of

in

transcripts

by

Expression in response

mRNA

levels

various

DNA

damaging

of a gene encoding to various

DNA

damaging

of a representative

but not by the DNA

damaging

switches

a transcription

stresses

for DNA

in

heat

shock

activating

in

cultured

Chinese

8, a DNA

agents, but not by protein,

heat

to a shock

as the heat shock element RNA and other heat shock

polymerase

heat shock

Most

organisms.

heat shock genes in response

to a short highly conserved DNA sequence known Fomace ad (6) have isolated heat shock protein

ovary cells. is activated

stresses

(l-4).

upshift

factor

shock,

of cellular

hsp70,

repair

hamster enzyme,

heat shock (7). is elevated

by

By heat

agents (7).

677

All

Copyright 0 1992 rights of reproduction

0006-29 I X/92 S4.00 by Academic Pre.s.s. Inc. irr an\ form reserved.

Vol.

187, No. 2, 1992 The

multidrug MDRI

efflux gene

gene pump

arsenite various

The

7-8 fold shock

multidrug

MDRl

shock

enhance

stresses

that

agents anticancer

the human

MDRl

promoter

chloramphenicol

established

human

cancer

CAT

fused gene (18).

(19)

and

some

human

activate

MATERIALS

the MDRl

the

MDR

promoter

cell

lines

agents

cell lines. gene

promoter

(20,

kidney levels

agents

which 21)

such

stably

express

reported study,

in human

cancer

in

as vinka

line

human

cell

cells.

(13).

gene

(17).

the MDRl gene whether

We

alkaloid

assays with

the MDRl

we examined

of the cell

carcinogens

rodent

that serum starvation

can activate

In this

levels

fused

in other

a

MDRl mRNA levels in (16) have reported that

expression

(CAT)

carcinoma

(11,12),

15) enhance Chin fi &

in transient

acetyltransferase KB

mRNA

mRNA

P-glycoprotein,

has HSE and expression

such as hepatectomy

chemotherapeutic

gene

termed

in a human

MDRJ

We have recently

anticancer

cancer

increase

RESEARCH COMMUNICATIONS

transporter

gene promoter

by heat

cannot

environmental

anticancer

reported

activate

stable

the

AND BIOPHYSICAL

(lo), retinoic acid and sodium butylate (14, cell lines in vitro as well as tissues in vivo.

anthracycline have

heat

Other

(10).

encodes

(8, 9).

is increased

However,

(10). lines

MDRl

BIOCHEMICAL

could

the We

gene

MDRl have

promoter-

or hydroxyurea promoter heat shock

in the could

cells.

AND METHODS

Materials : Acetyl- CoA was obtained from PL- Pharmacia. Silica gel plates (60F254) were purchased from Merk (Germany). [14C]chloramphenicol [y-32P]-ATP and [a-32P]-dCTP were from New England Nuclear. Hybrisol 1 was purchased from Oncor (Gaithersburg, MD). The plasmids used in these studies included pMDRCAT containing various lengths of the MDRl promoter directing expression of CAT (17, 19) (see Fig. 1). : KB cells were grown in Eagle MEM (minimal essential Cell culture and transfection medium) supplemented with 10% newborn bovine serum and antibiotics (22, 23). To establish stable transfectants, mixtures of reporter plasmid (30 pg) and pRSV-neo (1 pg) Growing colonies in were added to KB cell (0.5 - 1 x lo7 cells) by electroporation. selection medium containing 800 pg/ml G418 (GIBCO. NY) were cloned, expanded and tested for CAT activities. For transient CAT assay, only reporter plasmids (4 Jrg/ml) were added to KB cells by using Lipofectin TM reagent (BRL, Bethesda, MD). CAT assays were performed as previously described before (17, 19). Northern analvsis : Total RNA was isolated using guanidine isothiocyanate (19, 24). RNA samples (10 ~.tg/ Jane) were separated on a 1 % formaldehydeagarose gel, stained and photographed. RNAs were transferred to Nytran with 10 x ssc. The filters were prehybridized at 42-C for 4 hr and hybridized for 18 hr with Hybrisol 1. The CAT probe (EcoRlBamHl fragment) and hsp70 cDNA probe (American Type Culture Collection, Rockville, MD) were labeled by the random priming method (25). Filters were washed as previously described (19) and exposed to Kodak XAR- 5 film at -70 * C. Nuclear extract nreparation and Pel shift assay : Nuclear extracts were prepared from KB cells treated with heat (45’ C) for various times as described previously (26). Oligonucleotide (D2, D3, D4 and HSE) were synthesized on an Applied Biosystems 391 DNA synthesizer. Dl (Xbal-Xbol fragment) was gel-purified and radioactively labeled with [y-32P]-ATP and T4 polynucleotide kinase at the 5’ termini. D2, D3 and D4 were also labeled, annealed and purified on a 15% polyacrylamide gel as described previously (26) Dl - D4 were used as probes (see Fig. 5). Binding reactions were performed as previously described (26), with the exception that they contained lug of each poly (dJdC), poly (dA- dT) and E. coli DNA as non specific competitors. 678

Vol.

187,

No.

BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

gene

promoter

RESEARCH

COMMUNICATIONS

RESULTS To

understand

environmental

stimuli,

Kac-7,

Kxb-10,

lengths

of MDRl

containing could

and followed

Kst-6

cells

at time

mRNA

and hsp70

mRNA

for indicated

cells.

Kst-6

than

from

the initiation

bp is inverted induction

site of MDRl

CAT

activity

was significantly

increased

transfected

with

(Fig.

3A).

induced

These

were

HSE transient

was

in

KB

and

MDRl with

A;

Xb

mRNA

in

at 24 hr over

the

indicated

Khp-6

was not sequence

promoter various

between (see

pMDRCAT5

I

.:;

.:

:.,

-197LQ@

YP

... :

..,:.

HSE

~. .. ...

,,:.,,.:..:.

in the

(Fig.

3A).

at all

of MDRl

promoter

activation

..:...

CAT

However,

enhanced

inducibility.

MDRl

1).

region,

in

promoter

response

in Khp-6 shock

between

-136

determined

to heat

constructs.

shock

Transient

lmes

st

.:.:. :

: .:

tl

t121

CAT

Kst-6

.,

Kac-7

.‘.

Kxb-IO

-258

m.

pMDRCAT6 pMDR

CAT?

pMDRCAT8

: : :::. ..::

198

m..:: .: .:.: ,:.:...:.: ,.::. - 136 r.:.:.:. -76

Kxh - lr, Khp-6

ml.

Schematic representation of plasmids and stable KB cell lines. The pMDR CAT1 The other promoter length of about 2,000 bases (StyI-StyI:- +121). promoter lengths are: pMDRCAT5, AccI-Sty1 (-258 - +121); pMDRCAT6, XbaI-Sty1 (-198 has

MDRl

Solid +121); pMDRCAT7, XhoI-Sty1 (-136 - +121); and pMDRCAT 8, HphI-Sty1 (-76 - +121). KB cell lines which stably integrate lines indicate two strong HSEs and their direction. each plasmid are listed. Relevant restriction site are indicated as follows; St: StyI, AC: Southern blot analysis demonstrated the copy number Accl, Xb: Xbal, Xh: Xhol, Hp: HphI. of the integrated MDRCAT gene in various stable cell lines as listed in Fig.1 : 1-2 (Kst-6); 4-6 (Kac-7); lo-15 (Kxb-10); 8-10 (Kxh-14); 1-2 (Khp-6).

679

on

Although activity

by heat

We further

bp

bp and +26

of heat shock Fig.

Cell

pMDRCAT

already

increase

+13

the effect cells

in its promoter

by heat shock activity

X,h

periods.

min.

Plasmids St

of CAT

was observed

about 4- to 5-fold

the motif

HSE motif

the heat shock

for

period,

activity

bp and -178 bp, and +13 bp and +26

that the DNA

cells

CAT

for the expression

to 45’ C for

in hsp70

We compared

Kxh-14 cells

for

responsible

exposed

while

(10).

the upstream

responsible

assay

-165

8 fold

kinetics

By contrast,

gene promoter

data suggested

increased

‘time

increase

CAT

to 45’ C for

At each indicated

to 45 * C for 60-90

in Kxh-14

were exposed

promoter

at 37’ C.

activity

Kst-6,

pMDRCAT1

gene

significantly

cells

Kxb-10,

with

MDRI

to

by various

periods

between

pMDRCAT8,

-76 bp was

whether

in

does not contain

cells

bp and

exposure

are located

lines

Heat shock

20-fold

in a 3’- to 5’-direction

of

pMDRCAT7

during

motifs

cell

transfected

cells

response

gene driven

whether

Kst-6

in

KB

the CAT

used Kst-6

We examined

when

activated cancer

examined

to heat shock stress.

a more

appeared HSE

expressed

We

2A).

is

human

We initially

after heat shock at 45 * C for 15 min. Two

using

stably

the stress, and CAT

mRNA 2B,

various

promoter.

out (Fig.

0 in Kst-6

As seen in Fig. CAT

gene

hr after

control

1).

incubation

assay was carried

which

(Fig.

in response

12-24

established

and Khp-6

MDRI

be activated

MDRI

have

promoter

CAT

just

the

we

Kxh-14

human

90 min

how

Vol.

187,

No.

BIOCHEMICAL

2, 1992

A

AND

Kst- 6

BIOPHYSICAL

B[

0 2 z IL 0

RESEARCH

45”C(mtn)

COMMUNICATIONS

0 15 30 60 90

b 0

12

24

40

b

(hr)

u. Induction of CAT and hsp70 by heat shock. (A) Induction of CAT activity by heat shock in Kst-6. Subconfluent cells were heat shocked at 45. C for 90 min. allowed to recover at 37-C and harvested for CAT assay after time indicated : 0, no heat shock; 12, 12 hr recovery after heat shock; 24, 24 hr recovery; 48. 48 hr recovery. (B) Northern blot analysis of CAT and hsp70 mRNA in heat shocked cells. Kst-6 cells were exposed to 45. C for 15, 30, 60, and 90 min and then RNA extracted. Upper panel shows CAT mRNA. Middle panel shows hsp70 mRNA. Lower panel shows ethidium bromide staining of gel, and rRNAs were indicated.

Kxb-

Kac-7

10

A mm

0 12 24

OIIl

48

0

122448

m (A) Induction of CAT by heat shock in KB cell lines carrying MDRI promoter with or without HSE. Kac7: a transfectant with pMDRCAT5; KxblO: a transfectant with pMDRCAT6; Kxhl4: a transfectant with pMDRCAT7; Khp-6: a transfectant with pMDRCAT8 (see Fig. 1). These cell lines were treated as in Fig. 2 (A) legend. (B) Induction of CAT activity by heat shock in transient transfection. Subconflent cells were transfected with various plasmids with Lipofectin and treated at 45’ C for 90 min after 24 hr as indicated by (+). CAT assays followed 48 hr after heat shock stress. 1: pMDRCAT5, 2: pPMDRCAT6.3: pMDRCAT7.4: pMDRCAT8.5: pSVOCAT.

680

Vol.

187, No. 2, 1992

BIOCHEMICAL

01

-198

AND BIOPHYSICAL

-137

02

-149

-115

03

-116

04

B

01 1

2

4

-96 -99

02

3

12

D3 3

RESEARCH COMMUNICATIONS

4

1

-62 D4

2

3

4

1

2

HS C-1

c 3

4

1

HS (+)

23456123456

Fig. 4. (A) Schematic representation of the DNA fragments of MDRI promoter for gel shift assays. Dl: -198 to -137 bp; D2: -147 to -115 bp; D3: -116 to -96 bp; D4: -99 to -62 bp were used as probes. Two solid lines indicate the location and orientation of two strong HSE motifs (10). The sequences of synthetic HSE oligonucleotides are 5’AATTCCCTGGAATATTCCCGATA-3’. (B)Gcl shift assays with DNAfragments of MDRl promoter. Subconfluent cells were exposed 45’ C for 0 (0) or 30 (30) min. and then nuclear proteins were extracted. Each nuclear extract (2pg/assay) was incubated with labeled DNA fragments (Dl)or synthetic oligonucleotides (D2-4) of MDRl promoter and subjected to gel electrophorcsis. (a) and (b) indicate DNA-protein complexes. In nuclear extracts from the heat-treated cells at 45-C for 30 min were competition assays, incubated with the labeled probe of MDRI promoter in the absence or presence of 50 molar excess of the unlabeled competitors. Lane 1; nuclear extracts from non treated cells Lane 2.3 and 4; nuclear extracts from heat treated cells. Lane 3; synthetic HSE was used as a competitor Lane4; unlabeled DNA same as probe was used as a competitor. (C)Detergent stability of the complex (b) formation in the gel shift assays. Nuclear for 30min prior extracts (4pg/assay)wcre incubated with either NP-40 or urea at 37-C to the binding reaction. HS (-) indicates that nuclear extracts from untreated cells was incubated with labeled D4. HS(+) indicates that nuclear extracts from the cells treated with heat shock at 45~ C for 30min was used for binding assays. Lane 1; control, Lane 2; nuclear extraets were incubated at 37’ C for 30min. without dctcrgcnts. Lane 3; 0.5% NP40, Lane 4; 1% NP-40, Lane 5; 0.5M urea, Lane 6; 1M urea.

assay

system

similar

to pMDRCAT1,

increased showed heat

when

with

pMDRCAT7

either

that HSEs

showed

an

apparent

pMDRCAT5

and pMDRCAT6

pMDRCAT8

or pSVOCAT

were

not directly

involved

increase

CAT

However,

(Fig. 3B). were

in

transfected.

in the induction

activity CAT

activity was not

Transient

CAT

at levels

activity

assays again in response

to

shock. In

order

MDRl

promoter,

tested

gel shift

(Fig. 4A).

to

examine

we prepared assays with The complex

whether nuclear various

transacting extracts

from

end-labeled

(a) increased

factors

several 681

KB

DNA fold

play cells

a role

treated

fragments

with

between

after heat shock when

in

activating

heat -198 Dl

the

shock

and

and -62

bp

was used as

Vol.

187,

No.

BIOCHEMICAL

2, 1992

This

a probe

(Fig.

4B).

synthetic

HSE

and Dl,

find

any specific

DNA

protein

band

the complex

(b)

physical

properties.

this of

next

various

mild

or

untreated

I.OM

the 50 molar

excess

in nuclear

extracts

denaturing urea

the 50 molar

agents

in

(b)

We found

promoter

extracts

either

from

the cells

of the complex

and subjected

or

nuclear

extracts

from

gene promoter

contains

several

HSE,

factor.

However,

treated

with

heat

to examine

the

cells

assay (Fig.

stable

heat-treated

band

synthetic

(b)

shift

more

not

The

heat-stressed

to gel

relatively

of

that one distinct

specific

untreated

remained

excess

We could

to HSF.

of D4 but not with

stability

from

COMMUNICATIONS

D4 was used as a probe.

to MDRl

the detergent

that complex

of

with

used as probes. when

augumented

Nuclear

RESEARCH

competed

specifically

correspond

studied

case, we observed NP-40

with

may

was not

We have with

(b)

specifically

D2 and D3 were

abolished

band

shock. treated

when

BIOPHYSICAL

that the band (a) corresponds

(b) was formed

(b) was specifically that

was

suggesting

complex

suggesting

band

AND

were 4C).

up to either

cells

than

In 1%

those

from

cells.

DISCUSSION The

human

MDRl

(10) have reported MDRl

mRNA

not in other

that the MDR

are specifically cell lines

to HSE core sequence, are involved

in the

of MDRl

Three

in one human region.

S-CNNGAANNTTCNNG-3’ heat

shock

induction

fused

kidney

of the HSE are found

5’ untranslated

promoter

motifs

is a heat shock inducible

elevated

(10).

and the rest are located

deletion

gene

HSE

and Chin Cellular

gene. cell

line

upstream

from

(27-29).

To confirm gene,

we

but site

perfectly

whether

generated

and established

levels of

the initiation

of them are almost

gene

&I&l

by heat shock,

Two

of MDRl

to CAT

(lo),

match

these HSEs

a series

stable

of 5’

transfectants

(Fig.

1). Our

present

study

response

to environmental

observed

in

at -152

bp to -178

carrying

heat DNA

promoter. response

sequence

are

Cellular

levels

shock.

of 3- to 8-fold Our

artificial

present constructs

MDRl promoter and cell lines explanation protein

could

Kxh-14,

the MDRl

(Fig. element

involved

in

However,

CAT

1 and 3).

study

is performed the

MDRl

activity These

at between

the

shock

heat

promoter

of CAT

is located

activity

mRNA

levels

is observed

in Khp-6

cells

strongly and

with

human

promoter,

and

from cancer heat

KB

and

showed

cell can

of

more

that

the

also the

that

MDRI

than 30-fold

a relatively

in

small

that a mechanism

that for hsp70 shock

suggest -76

activation

The data suggest

is different

by heat shock is HSE

-136

of Kst-6

in

deleting

data

induced

is activated

pMDRCAT7

in KB cells are increased

by heat shock treatment. promoter

gene

a construct

of CAT

of hsp70 mRNA

of MDRI of

Induction carrying

no induction

pMDRCAT8 responsive

heat shock induction

that

heat shock stress. transfectant,

shock

to heat

increase

demonstrates

bp, whereas

a construct

another these

a stable

also

for

gene by HSF.

lines

transfected

induce

activation

with of

irrespective of the presence or absence of HSE motifs. Assay systems differ between studies by Chin a & (10) and ours, and thus a simple

is not possible. bind

with

a high

Gel

shift

degree

assay was used to determine of specificity 682

to a MDRl

whether

promoter

any specific

regions.

Gel

Vol.

187,

shift

No.

2, 1992

assays

with

of the complex

nuclear

(a) when

motif

-152

either

exogenous

bp to -178 The

heat-treated

more

stable

cells.

We

similar,

property

the factors

bind

that

shock-induced

specific

factors

shock,

polymerase The

agents

heat

mechanism.

Our

is involved gene

of

heat shock

stress.

responsible

for

the

of

heat-treated

of

the

the

of

than

to DNA

complex

of

by the heat

appears

those in

from

to

be

untreated

gel-shift

shock.

is

with

both untreated

(b)

assay

This

suggests

We thus favor

promoter

are that

the hypothesis

mediated

through

mRNA damaging

results

DNA

induction bound

Further

study

agents

heat genes

gene

MDR

promoter

have

of

but

promoter.

activated

by

that DNA

not

and

by

DNA

through

sequence

to purify MDRl

studied

DNA

to be differentially

However,

by heat shock

in progress

activation

is

reported

shock,

-99

be

but not by heat shock.

expression

between

that a by heat

in MDRl

appear

that the 5’ flanking

to the sequence is now

(7)

agents,

by

hsp70

to

gene

and heat shock.

MDRl

of MDRl

shock-induced

MDRl

a al

enhanced

remains

is observed

damaging

8 and

activate

It

human

Fomace

are

suggest

(27) has demonstrated cerevisiae is activated

gene,

the

19-21).

polymerase

factor heat

(b)

mechanism.

by various

might

present

in

the HSE

D3 and D4 (data from

complex

factor(s).

MDRl

promoter

(17,

hsp70

shock

expression with

it competes

D2,

extracts

the

cells

as seen in the DDRA2

that

DNA

in direct

specific

in nuclear

is modified

because

but not with

stability

HSE-independent

agents

(7).

in response and

a

is activated

levels

or Dl

a stabilizing

enhanced

HSF.

motif,

8 gene

gene promoter

formation

COMMUNICATIONS

showed

study by Kobayashi and McEntee gene, DDRA2, of Saccharomvces

observed

agents

regulated

MDRl

of the factors

damaging

cellular

damaging

the

through

also

DNA

although

than

any common

have

various

-76

rather

possibly

whether We

from

activation

cells

formed

detergent

to D4 may require

Another relevant damage-responsive

DNA

(b) is equally

RESEARCH

HSF appears to interact

(see Fig. 4).

extracts

that

heat-treated

of the MDRl

However,

nuclear

BIOPHYSICAL

oligonucleotides

complex

conclude

binding

HSE

AND

from

was used

bp upstream

cells. in

heat

extracts Dl

synthetic

not shown). and

BIOCHEMICAL

damaging a common

between stress, -62

the DNA

-136

and

and also that is activated binding

by

protein

promoter.

ACKNOWLEDGMENTS We thank T.Akashi and T.Umeda in our laboratory for preparing the manuscript. This study was supported by grant-in-aid from cancer research fund from Ministry of Education, Science and Culture, Japan and also from Ministry of Human Health and Welfare, Japan. REFERENCES 1. 2. 3. 4. 5. 6. 7.

Lindquist, S. (1986) Annu. Rev. Biochem. 55, 1151-1191 Milarski, K. L., and Morimoto, R. I. (1986) Proc. Natl. Acad. Sci. USA. 83,9517-9521 Bienz, M., and Pelham, H. R. B. (1986) Cell 45, 753-760 Burdon, R. H. (1986) Biochem. J. 240, 313-324 Sorger, P. K. (1991) Cell 65, 363-366 Fomace AJ Jr. Jr. Hollander-MC and Lamoreaux-E (1989) Exp. Cell Res. 182, 61-74 Fomace, Jr. A. J. Neber, D. W., Hollander, M. C., Luethy, J. D., Papathnasiou, M. Fargoli, J. and Holbrook, N. J. (1989) Mol. Cell. Biol., 9, 4196-4203 8. Gottesman, M. M., and Pastan, I. (1988) J. Biol. Chem., 263, 12163-12166 9. Bradley, G., Juranka, P. F., and Ling, V. (1988) Biochem. Biophys. Acta., 948, 87-128 683

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AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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