Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine

Vol.

162,

August

No.

3, 1989

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

15, 1989

1402-1408

Pages

PHOTOAFFINITY LABELING OF P-GLYCOPROTEIN IN MULTIDRUG RESISTANT CELLS WITH PHOTOACTIVE ANALOGS OF COLCHICINE AHHAD R. SAFA*,

July

D. MEHTA, and MICHAEL AGRESTI

JOINT SECTION OF HEMATOLOGY/ONCOLOGY OF CHICAGO AND MICHAEL REESE MEDICAL CENTERS CHICAGO, IL 60616

UNIVERSITY Received

NITIN

3, 1989

gf colchicine, Jii(p-azido[3,5(\2tQNABCI) and l-?&azido-[3IINASC) were synthesized and used to identify colchicine-specific acceptor(s) in membrane vesicles from hamster lung cells. prominent 150-180 kDa vesicles from DC-3F/VCRd-5L cells. The photolabeled polypeptide was immunoprecipitated by monoclonal antibody C219 specific for the MDR-related P-glycoprotein (P-gp) indicat'n the identity of this protein with P-gp. Colchicine at 1000 UM reduced [ j3H NABC photolabeling of P-gp by vincristine, vinblastine, doxorubicin 72%. Furthermore, 100 uM of cIgj5c hicine, and actinomycin D inhibited I: IINASC photolabeling by 45, 88.8, 911& 61.5, methotrexate did not affect the [ IlnAsc and 51% respectively. However, indicating the multidrug specificity of the P-gp photolabeling of P-gp, 0 1989 colchicine acceptor for drugs to which these cells are resistant. Academic Press, Inc.

Multidrug a

wide

resistance

variety

derivatives dependent

P-gp

(3).

and

that

transport

proteins (7), displays

sites (4-6).

an energy-dependent certain

The

efflux

compounds

*To whom correspondence University of Chicago Chicago, IL 60637.

reversing

should Medical

0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

and

overexpression

of

P-glycoprotein

cells

cDNA for

ATPase the

to

hamster

cytotoxic

MDR phenotype

in

sites

Hence, hydrohobic

in

and mouse domains

in

photoactive

(12).

role

P-gp results

and ATP

be addressed at Division Center, 5841 S. Maryland

1402

membrane

a crucial

human,

(8-lo), activity

pump for

integral

an

ATP binding

binds

blockers

energy-

plays the

to

synthetic

an increased

has 12 transmembrane to

protein

their

with cDNA encoding

protein

homologous

calcium channel Mg*+ -dependent

to cross-resistance

drugs display

of this

refers

MDR phenotype

(P-gp). Analysis

binding

vinblastine pure form likely

the

cells

cytotoxic

of sensitive

demonstrated

nucleotide

and for

with

mechanism

MDR phenotype

in cancer

product

P-glycoprotein transfection

encoding two

Cells

efflux

MDR since

(MDR)

natural

(1,2).

glycoprotein, the

of

with

bacterial analogs

(ll), P-gp drugs

of

and its is in

(13-15). of Hematology-Oncology, Ave., Box 420,

most MDR

Vol. 162, No. 3, 1989

Recently,

we

photoactive was

demonstrated

analogs

inhibited

by

actinomycin

D, but

P-gp, colchicine

since

above

studies

binding

site

as

not

vinblastine

identify the

and our

natural

analogs as

the

on

other

of

findings

In

colchicine

which

synthesized

for

colchicine.

(8,9)

MDR cells

imply

that

are P-gp

by to

(3). a

The separate

tritiated

were

site

and

resistance of

study,

binding

(8,9)

transported agents

existence

accepter

with

doxorubicin

display

this

colchicine to

by

cytotoxic

P-gp

blockers

presumably

gene

possible

P-gp.

a specific drugs

is

P-gp product

suggest

of a specific

previous

the

of

channel

inhibited

Colchicine

other

colchicine

labeling

and calcium

partially

as to

P-gp with

and with

presence

cross-reactive

(16,17)

by colchicine.

photoactive

indicated

photoaffinity

transfected

collectively for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

vinblastine

well

directly

drug-acceptor

of

cells

radioiodinated

results

BIOCHEMICAL

and used

on P-gp resistant.

may have

and to

Our data which is These

overlapping

sites.

Materials

and Methods

MATERIALS. &Hydroxysuccinimidy1-4-azido[~&-3H]benzoate (47.7 CiJmmol) was purchased from New England Nuclear; sodium I: Iliodide (2200 Ci/mmol) was purchased from Amersham; N-hydroxysuccinimidyl-4-azidobenzoate and Nhydroxysuccinimidyl-4-azidosalrcylte were obtained from Pierce; actinomycin l?, doxorubicin, colchicine and methotrexate were purchased from Sigma. Vinblastine and Vincristine were obtained from Eli Lilly & Company (Indianapolis, IN). Aminohexanoyldeacetylcolchicine trifuaroacetate was custom synthesized by Molecular Probes, Inc. (Eugene, OR). Monoclonal antibody C219 specific for P-gp was purchased from Centacor (Malvern, PA). All other chemicals were obtained commercially and were of reagent grade. The photoactive colchicine analog N-(p-azido[3,5-3H]benzoyl)SYNTHESIS. aminohexanoyldeacetylcolchichine (NABC) was synthesizTd by azidobenzoylation aminohexanoyldeacetylcolchine trifluoroacetate (AHDC) with Nof hydroxysuccinimidyl-4-azidobenzoate (18,19) . the presence Tf triethylamine. The photoactive analog was purifiiid as previously described The product gave a single UV-absorbing spot on a silica gel TLC plate (9). [Solvent, with fluorescence indicator (Analtek; Newark, DE) chloroform:methanol:H20 of 80:20:3 (v/v), Rf=0.8; solvent, benzene:methanol The synthesis of the radioactive photoaciive analog, N3:l (v/v) (&vazido-~3~{~g*53a H]benzoyl)aminohexanoyldeacetylcolchicine was cc HINABC) similar to that described for the nonradioactive compound using J-hYdroxYsuccinimidyl-4-azido[3,5-3H]benzoate (47.7 Ci/mmol) (8,18,19). The photoaffinity analog l-(p-azidosalicyl)aminohexanoyldeacetylcolchicine (NASC) was synthesized from ADHC and &hydroxysuccinimidyl-4The corresponding azidosalicylate and purified asI&scrib.ed for NABC. I]salicyl)aminohexanoyldea@ylcolchicine active analog N-(e-azidoC3raQE Ilsalicylate ([ I]NASC) was prepared with F&hydroxysuccinimidyl-4-azido[3(7,9) and purified similarly to NABC. exhibited a UV-visible absorption spectrum The photoactive analogs consisting of a composite of the absorption spectra of colchicine and either the azidobenzoate or azidosalicylate chromophore. Chemical structure of NABC and NASC were analyzed by low and high-resolution fast atom bombardment (FAB) Analytical and Consulting Laboratories Inc. mass spectroscopy by Shrader The accurate mass measured for the molecular ion (MH)+ of NABC (Detroit, MI). was 616.27 corresponding to an elemental composition of C33H37N507+H. 1403

BIOCHEMICALAND

Vol. 162, No. 3, 1989

BIOPHYSICAL RESEARCH COMMUNICATIONS

the accurate mass measured corresponding to an of colchicine, C3H1NABC and [:

Similarly, 632.27 structure

for

the

molecular

ion

of

NASC was The

Cell lines were cultured as described previously CULTURE. Vincristine-resistant DC-3F/VCRd-5L cells (2750-fold resistant to and cross-resistant to doxorubicin (220-fold), actinomycin D and colchicine (lOOO-fold) were selected from Chinese hamster lung as described previously (20,21).

CELL (9,20,21). vincristine) (lOOO-fold) DC-3F cells

MEMBRANE PREPARATIONS. cavitation and the differential Protein concentrations (22). (Bio-Rad; Richmond, CA).

Cell

membrane vesicles were prepared by nitrogen centrifugation procedure described by Lever were determined with the Bio-Rad protein assay

Photolabeling of membrane vesicles (25 ug of PHOTOAFFINITY LABELING. protein) was performed in 10 mM Tris-HCl (pH 7.4), 250 mM sucrose, 10 mEJ NaCl, 1.5 mM MgCl and 3 mM ATP ontaining 4% Me2S0 and 0.5 uM of either [ H]NABC (47.7 Ci/mmi!) or 40nM [12' I]NASC (220 Ci/nmol) in a final volume of 0.050 After preincubation in the presence or absence of inhibitors irradiation ml. was accomplished using a UV lamp (302 or 366 nm) for 20 min at 25*C. Sodium dodecyl sylfate poly~~r#lamide gel electrophoresis (SDS-PAGE). Samples labeled with [ H]NABC or [ I]NASC were resolved on a 5-15% SOS-PAGE gradient gel containing 4.5 M urea. Fluorography, autoradiography and quantitation of radiolabeling were carried out as previously described (783). IMMUNOPRECIPITATION. Immunoprecipitations of deoxycholate-solubilized C125]NASC photolabeled membrane vesicles (50 ug of protein) were performed using monoclonal antibody C219 specific for P-gp as previusly described (7-9); normal mouse serum was the control.

Results The

photoaffinity

analog

azidobenzolation

of

a

'Hlbenzoate.

Similarly, colchicine-specific

retained

the

To

with

and

in

sensitive

(DC-3F)

Fluorography membrane 3) lane

that 1).

and MDR variant of

showed

These

from

was not

detectable

Photolabeling

DC-3F/VCRd-5L in

are since

of

biochemical from

selected labeled

a prominently

vesicles

by amino-

ideal

for

they

have

colchicine

(e.g.,

data).

cells

colchicine,

analogs

properties

vesicles

photoaffinity

colchicine,

interactions

the

membrane

synthesized

from s-hydroxysuccinimidyl-4-

AHDC.

pharmacological

characterize

were analog

and

unpublished

MDR cells,

(DC-3F/VCRd-5L) photoactive

and

of

was prepared

1)

was

l-hydroxysuccinimidyl-4-azido-[3,5-

intracellular

tubulin,

identify

colchicine

C3H]NABC,

derivative

with

[1251]NASC

biological

interact

amine

(Fig.

probing they

colchicine,

(AHDC),

12511salicylate

azido[3-

of

free

hexanoyldeacetylcolchicine

and Discussion

for with

C3H]NABC, photolabeled multidrug

drug-sensitive

specificity

of

resistance 0.5

and

uM

determined

kDa cells by

lung

to drug-

to vincristine of

examined

150-180 resistant

resistance

hamster

DC-3F parental was

1404

basis Chinese

the by

tritiated SDS-PAGE.

polypeptide (Fig. cells performing

in

3, lane (Fig.

2, the

Vol. 162, No. 3, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

DC-3

F

DC-3F/ VCRd-5

L

-II

Colchicine

bCH,

N-(p-azido

[3,fr3H]

benzoyl)

aminohexanoyldeacetylcolchicine

0

0 ”

II NH-C-KHz),-NH-C

OCH,

01

N-(p-azido

[3-“s

Fig.

1.

Fig.

kDa

2.

in In

of colchicine and its photoaffinity 1)aminohexanoyldeacetylcolchicine I]salicyl)aminohexanoyldeacetylcolchicine

the

presence

these

polypeptide

2,

identity

resistant

cells

was

lane of

DC-3F/VCRd-5L for

P-gp

immunoprecipitated

radioactivity

yalogs, N-( -azidoHINABCi2San % N-CL([ IINASC).

the

colchicine

detected

seen

(Fig.

photolabeling in

the

photolabeled of

cell (Fig. by

was

colchicine of

of presence

2, the

lanes 150-180

of

1000

uM

4).

immunoprecipitation

solubilized

was

([

nonradioactive inhibition

by

specific

excess 72%

determined

C219

of

experiments in

(Fig.

The

12345

SDS-PAGE fluorography of [3H]NABC photoaffinity-labeled membrane vesicles (25 ug of protein) of drug sensitive Chinese hamster lung DC-3F cells (lanes 1 and Z), drug-resistant DC-eF/VCRd=5L cells (lanes 3 and 4) and of material immunoprecipitated with P-gp monoclonal antibody C2$3 (lane 5). Photoaffinity labeling was carried out with 0.5uM [ H]NABC (47.7 Ci/mmol) in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 1000 UM colchicine. For immunoprecipitation, 200 Ug C3H]NABC photolabeled deoxycholatesolubilized membrane vesicles were incubated with 10 pg of the P-gp monoclonal antibody C219 and after subsequent incubation ith Protein A-Sepharose, samples were processed for SDS-PAGE and autoradiography The positions of molecular weight standards in kDa are (lane 5). present at the left.

4).

colchicine

aminohexanoyldeacetylcolchicine

Structure

experiments 2 and

I] salicyl)

this

lane

vesicles

the

The

5).

antibody in

L3~]NABC

membrane 2,

150-180

used. 1405

protein

photolabeled, with

the

radiolabeled

indicating

precipitate

kDa

its when

was

detergent-

monoclonal 150-180

antibody kDa

protein

identity

with

P-gp.

nonimmune

mouse

serum

No was

Vol. 162, No. 3, 1989 Similar vesicles

BIOCHEMiCAL

results from

(as

shown

drug-sensitive

photoaffinity

labeled

with

membrane

vesicles

radiolabeled

of the

of

respectively,

and

Furthermore, vincristine, SP. reduced photolabeling of actinomycin the

D, drugs

[1251]NASC

Methotrexate, no

photolabeling result

of

P-gp.

under.

photoactivated

identical

C3H]NABC

or

91.1,

no

200

-

115

-

61.5 the of

activation

the

P-gp

in the

and

85.5% for

resistant

effect

on

photolabeling

light

by these

in

[1251]NASC

regardless

respectively.

line

P-gp

absence

of

demonstrates

the

[1251]NASC was

agents, protein,

of

and inhibited

51%, cell

the

not since

presence

IVGRd-SL

92

45

-

1

Fig.

3.

23456

789

10

11

SDS-PAGE autoradiography of '[Iz51]NASC photolabeled membrane vesicles (25 Pg of protein) of drug sbnsitive Chinese hamster lung DC-3F cells (lanes 1 and 2) and drug-resistant DC-3F/VCRd-5L cells (lanes 3-11) in the absence (lanes 1 and 3) and presence of 100 PM colchicine (COL, lanes 2 and 4), 1000 PM colchicine (lane 5), or 100 PM vincristine (VCR, lane 6) vinblastine (VBL, lane 7), doxorubicin (DOX, lane 8) actinomycin D (ACD, lane g), or methotrexq& (MTX, lane 10). For immunoprecipitation (MCA, lane 11) 200 ng [ I INASC photolabeled deoxycholate-solubilized membrane vesicles and monoclonal antibody C21g were used.

1406

the UV

totally

“0 7

x V

P-

variant,

doxorubicin

and

resistant

conditions,

DC-3F

of

cross-resistance,

significant

DC-3F

h

select

display

inhibition

incidental

to

was

45

photolabeling

Vinblastine,

cells

to which

had The

of

used

P-gp

radiolabeling

by

[1251]NASC

were

[Iz511NASC

that

while

reduced

membrane

cells

of

Radiolabeling

88.8%.

by agent

(23),

of absorption

irradiation

these

when

revealed

cells

was of

drug by

photolabeling an antitumor

cells

sensitive

colchicine

the

to which

cross-resistance

drug

specificity P-gp

obtained DC-3F/VCRd-5L

Autoradiographs

substantially. uM

the

also

DC-3F/VCRd-5L

parental

1000

indicating

were

[1251]NASC.

was increased

100

3)

RESEARCH COMMUNICATIONS

and MDR variant

from

in the

MDR variant

presence

Fig.

DC-3F

photolabeled faintly

in

AND BiOPHYSlCAL

of

Vol. 162, No. 3, 1989

competitors.

BIOCHEMICAL

These the

results

interact

with

acceptor

has an important

Previously, common

binding

verapamil this

study

role

was

shown

(9)

and

can

in

preparation)

site

is

distinct

bind

common

drug-acceptor

lower

to

affinity

for

vinblastine drug thereby

from

may also increasing

and that

this

the

and did

reduce

the

It

displaying

also

Alternatively,

be the

result

binding

of

labeling

may bind

or

that

P-gp

for

the

Vinca

inhibitory

a negative

allosteric

by binding

to

and coupling

of these

to

site(s),

possible

a of

by vinblastine.

photolabeling

affinity

to

binding In contrast,

inhibited

binding

is

the

(16,17).

colchicinc

high

drug

may bind

inhibit

cells is

vinblastine

membrane

verapamail

not

colchicine

that

drugs

MDR phenotype.

DC-3F/VCRd-5L

sites.

[12511NASC the

on P-gp,

photolabeling

colchicine.

on C3H]NABC or

in

suggesting

site

of cytotoxic

vinblastine

also

overlapping

classes

colchicine

colchicine

(manuscript which

that that

to P-gp

verapamil

agents

site

several

in maintaining

it

that

that

binding

site

shows

Furthermore,

indicate

colchicine

or vinblastine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

P-gp

P-gp

at a

that

these

may have

alkaloids effect

caused

effect

a separate

photoactive

of

site

a and by

this

on P-gp

analogs

to P-

9P* Our

data

provide

colchicine

to

mediates

colchicine

P-gp

the and

first

direct

demonstrate

resistance

evidence that

this

of

the

membrane

specific

binding

glycoprotein

of also

in MDR cells.

Acknowledgments for generously supplying the DC-3F and We are grateful to Dr. June L. Biedler DC-3F/VCRd-St cell lines. We thank Mary D. McCauley for her editorial assistance and Cleo Coary for typing the manuscript. This work was supported by Grant CA-47652 (A.R.S.) from the National Cancer Institute and a postdoctoral fellowship award from the Washington Square Health Foundation, Chicago, IL, to Dr. Nitin D. Mehta.

References

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M.M.,

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T.,

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