Báo cáo khoa học: Role of the plasma membrane leaflets in drug uptake and multidrug resistance ppt

Thus, the sec-ond phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip-flop of dye across the pla

multidrug resistance

Hagar Katzir*, Daniella Yeheskely-Hayon*, Ronit Regev and Gera D Eytan

Department of Biology, The Technion – Israel Institute of Technology, Haifa, Israel

Introduction

P-glycoprotein [Pgp; multidrug resistance protein

(MDR) 1] (ABCB1) [1] and the multidrug

resistance-associated protein (MRP) 1 (ABCC1) [2] were

recog-nized as serious impediments to cancer chemotherapy

through their ability to eliminate drugs from cells

Both proteins are members of the ABC transporters

superfamily [3] Pgp efficiently exports amphipathic

somewhat basic drugs, such as paclitaxel (taxol),

anth-racyclines and Vinca alkaloids The hydrophobic parts

of these drugs allow their rapid insertion in the mem-brane The hydrophilic residues prevent rapid flipping

of the drug from the extracellular leaflet to the cyto-plasmic leaflet of the membrane, slowing down entry into the cell; indeed, for an anthracycline such as doxorubicin, this takes approximately 1 min, giving the Pgp pump ample opportunity to deal with the influx [4–6] This rate of spontaneous flip-flop is rele-vant because estimates of the turnover number of Pgp

Keywords

MDR1; MRP1; multidrug resistance;

P-glycoprotein; plasma membrane

Correspondence

G D Eytan, Department of Biology,

The Technion – Israel Institute of

Technology, Haifa, Israel

Fax: +972 4 822 5153

Tel: +972 4 829 3406

E-mail: eytan@tx.technion.ac.il

Website: http://biology.technion.ac.il

*These authors contributed equally to this

work

(Received 5 November 2009, revised 13

December 2009, accepted 18 December

2009)

doi:10.1111/j.1742-4658.2009.07555.x

The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion

by P-glycoprotein and multidrug resistance-associated protein 1 Drug accumulation was monitored by fluorescence resonance energy transfer from trimethylammonium-diphenyl-hexatriene (TMA-DPH) located at the outer leaflet to a rhodamine analog Uptake of dye into cells whose mito-chondria had been inactivated was displayed as two phases of TMA-DPH fluorescence quenching The initial phase comprised a rapid drop in fluo-rescence that was neither affected by cooling the cells on ice, nor by activ-ity of mitochondria or ABC transporters This phase reflects the association of dye with the outer leaflet of the plasma membrane The sub-sequent phase of TMA-DPH fluorescence quenching occurred in drug-sensitive cell lines with a half-life in the range 20–40 s The second phase of fluorescence quenching was abolished by incubation of the cells on ice and was transiently inhibited in cells with active mitochondria Thus, the sec-ond phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip-flop of dye across the plasma membrane and slow diffusion from the inner leaflet into the cells Whereas activity of P-glycoprotein prevented the sec-ond phase of fluorescence quenching, the activity of multidrug resistance-associated protein 1 had no effect on this phase Thus, P-glycoprotein appears to pump rhodamines from the cytoplasmic leaflet either to the outer leaflet or to the outer medium

Abbreviations

CCCP, carbonyl cyanide m-chlorophenylhydrazone; FRET, fluorescence resonance energy transfer; MDR, multidrug resistance;

MRP1, multidrug resistance-associated protein; Pgp, P-glycoprotein; TMA-DPH, trimethylammonium-diphenyl-hexatriene;

TMRM, tetramethylrhodamine methyl ester.

substrates are in the range 1–10 s)1, which is fast

com-pared to the flip-flop rates of drugs, such as

doxorubi-cin [6,7]

Pgp has been proposed to function as a

‘hydropho-bic vacuum cleaner’, extracting its substrates directly

from the lipid core of the membrane rather than from

the aqueous phase [8] This idea is supported by data

showing that the apparent affinity of a drug for

bind-ing to purified Pgp is highly correlated with its lipid–

water partition coefficient [9] Subsequently, this model

has been refined and Pgp has been suggested to act as

a plasma membrane flippase, moving drug molecules

from the cytoplasmic leaflet to the extracellular leaflet

[10] Romsicki and Sharom [11] have shown that Pgp

reconstituted into proteoliposomes transports lipid

analogs from the cytoplasmic leaflet to the

extracellu-lar leaflet In contrast, it has been demonstrated that

reconstituted Pgp and the bacterial multidrug

trans-porter, LmrP, expel drugs from the cytoplasmic leaflet

of the membrane to the aqueous medium rather than

to the extracellular leaflet [12,13] The question

remains as to whether the release of drugs from the

cytoplasmic leaflet of the plasma membrane into the

cytoplasm is fast, resulting in a practical equilibrium

between the drug concentrations in the cytoplasmic

leaflet and the cytoplasm, or whether the release is

slow, and drugs taken up into cells accumulate in the

cytoplasmic leaflet prior to being released into the

cytoplasm

In the latter case, Pgp functioning as a flippase will

have the added advantage of capturing incoming drugs

before they reach the cytoplasm and at a transient high

local concentration Moreover, in the latter case, Pgp

is expected to handle incoming drugs more efficiently

compared to drugs already present in the cell interior

By contrast, in the case where drug concentrations in

the cytoplasm and the cytoplasmic leaflet are in

equi-librium, Pgp is expected to treat incoming drugs and

drugs already present within the cell in a similar

manner

By contrast to Pgp, MRP1 functions as a

glutathi-one–X conjugate pump It not only transports a

vari-ety of drugs conjugated to glutathione, sulfate or

glucuronate, as well as anionic drugs and dyes, but

also neutral⁄ basic amphipathic drugs and even

nions Previously, it has been assumed that the

oxya-nions arsenite and antimonite and the neutral⁄ basic

drugs are cotransported by MRP1 with glutathione

[14] However, recent data indicate that the

mechanis-tic interaction between the transported neutral⁄ basic

drugs and the glutathione is more complicated [15]

The hydrophilic nature of some MRP1 substrates

makes it unlikely that MRP1 functions as a flippase

and extracts these substrates from the inner leaflet of the plasma membrane Rather, MRP1 pumps these substrates directly from the cytoplasm

The experiments conducted in the present study were designed to dissect the cellular uptake of MDR-type drugs into its constituent steps: uptake into the extra-cellular leaflet, flip-flop across the lipid core of the membrane and movement to the cytoplasmic leaflet of the plasma membrane First, an awareness of such data should help to resolve an outstanding question: is there a kinetic barrier between the cytoplasmic leaflet

of the plasma membrane and the cytoplasm? Such a putative barrier would result in the cytoplasmic leaflet constituting a kinetic compartment separate from the extracellular leaflet and from the interior of the cell In the case where the cytoplasmic leaflet does constitute a separate compartment, the accumulation of drug within this would be accomplished prior to saturation

of the total cellular content of the drug By contrast,

in the case where there is no kinetic barrier, drug accu-mulation within the cytoplasmic leaflet would proceed

in parallel with total drug accumulation within the cells Second, measurement of drug accumulation in the cytoplasmic leaflet should help determine whether Pgp removes its substrates from the cytoplasmic leaflet,

as predicted by the vacuum cleaner model, whereas MRP1 extracts its substrates from the cytoplasm Tetramethylrhodamine methyl ester (TMRM) served

as a highly fluorescent probe representing the MDR-type drugs [16] TMRM accumulation in the plasma membrane leaflets was assayed in cells over-expressing either Pgp or MRP1 and their sensitive parental lines TMRM accumulation was monitored as fluorescence resonance energy transfer (FRET) from trimethyl-ammonium-diphenyl-hexatriene (TMA-DPH) to the TMRM present in the membrane or very close to it Because of its polar nature, TMA-DPH, unlike its ana-log diphenyl hexatriene, has a high specificity for the plasma membrane in intact cells [17,18]; TMA-DPH is located within the lipid bilayer close to the outer sur-face The probe has been reported to be useful for measurements of plasma membrane fluidity and for studies on cellular exocytosis [19] Kessel [20] found similar values for TMA-DPH accumulation in drug-resistant P388 cells and wild-type cells; no differences were observed in the fluorescence anisotropy and life-time of TMA-DPH between these cell lines, which would indicate that there are no MDR-related differ-ences in the binding of TMA-DPH to different cellular components On the basis of the overlap between the fluorescence-emission spectrum of TMA-DPH and the excitation spectrum of TMRM, FRET can occur, pro-vided that the probe and the drug are sufficiently close

Thus, the degree of TMA-DPH fluorescence quenching

by TMRM may provide information on the amount of

TMRM associated with the plasma membrane, as

described previously for synthetic and natural

mem-brane vesicles [21,22]

Results

FRET from TMA-DPH to TMRM in cells sensitive

to anticancer drugs

The association of the dye, TMRM, with the surface

of cells was monitored as FRET from TMA-DPH

located at the outer leaflet of the plasma membrane to

this dye [23,24] The background fluorescence of

TMA-DPH in the aqueous medium appeared to be

negligible Immediately upon the addition of cells to a

medium containing TMA-DPH, the fluorescence of the

latter increased by at least a factor of 100 as a result

of adsorption of dye on the outer leaflet of their

plasma membrane [25] Steady-state fluorescence was

reached after < 5 min The fluorescence of

TMA-DPH was observed immediately at the periphery of the

cells (data not shown) After prolonged incubation,

additional fluorescence was observed within the cells

However, this fluorescence, presumably located in the

mitochondria and endosomes [23], was faint compared

to the fluorescence at the periphery of the cells The

quenching pattern of TMA-DPH fluorescence by

TMRM was unaffected by the preincubation period of

TMA-DPH with the cells (Fig 1) Upon the addition

of TMRM to cells preincubated with TMA-DPH,

quenching of the fluorescence of the TMA-DPH

occurred in two steps: an initial fast drop in

fluores-cence followed by a slow further fluoresfluores-cence

quench-ing The simultaneous addition of the two dyes to cells

resulted in a slow quenching similar to the second step

that was observed when TMRM was added after

TMA-DPH Presumably, when the two dyes are added

together, the initial quenching of fluorescence occurred

faster than the adsorption of TMA-DPH to the cells

and fluorescence quenching as a result of the added

TMRM prevented the rapid initial drop in fluorescence

observed when TMRM was added to cells

preincubat-ed with TMA-DPH Thus, the measurpreincubat-ed FRET

occurred from the TMA-DPH located at the surface of

the plasma membrane and not from TMA-DPH

located within the cells

The rapid initial quenching of the fluorescence was

essentially complete within 1 s after the addition of

TMRM The extent of the initial quenching was linear

with the outer concentration of TMRM up to a

con-centration of 25 lm Because the initial rapid drop in

TMA-DPH was not modulated by low temperatures (Fig 1), it reflects the absorption of TMRM to the outer leaflet of the plasma membrane After the initial rapid quenching phase, a slower quenching phase was observed at ambient temperatures, although not on ice Thus, the slower fluorescence quenching reflected TMRM crossing a lipid barrier located in the plasma membrane

The main intracellular accumulation site of rhodam-ines inside cells is the mitochondria This accumulation could be eliminated by poisoning the mitochondria either with the uncoupler, carbonyl cyanide m-chloro-phenylhydrazone (CCCP), or the respiration inhibitor, sodium azide Poisoning the mitochondria had no effect on the initial rapid phase of TMA-DPH fluores-cence quenching by TMRM By contrast, poisoning the mitochondria accelerated the second phase of TMA-DPH fluorescence quenching by TMRM The resulting curve could be fitted to a first-order reaction with half-lives in K562, GLC4 and 2008 cells of

36 ± 5, 19 ± 4 and 21 ± 6 s, respectively (Fig 2)

To determine whether the second phase of TMA-DPH fluorescence quenching by TMRM in the

B A

TMA-DPH fluorescence 3 min

C D

Fig 1 TMA-DPH fluorescence quenching by TMRM K562 cells were incubated in the presence of glucose and sodium azide (10 m M ) at 37 C and their fluorescence was monitored continu-ously using the excitation and emission wavelengths of TMA-DPH fluorescence Trace A: 2 l M TMA-DPH was added at the time point marked by the thin arrow and 25 l M TMRM was added at the time point marked by the thick arrow Trace B: 2 l M TMA-DPH was added at the time point marked by the thin arrow and, 5 s later,

25 l M TMRM was added Trace C: 2 l M TMA-DPH and 25 l M

TMRM were added together at the time point marked by the arrows Trace D: Cells were incubated for 10 min at 37 C with

2 l M TMA-DPH Subsequently, the cells were cooled by incubation

on ice for 5 min and, at the time point marked by the arrow, 25 l M

TMRM was added The extent of fluorescence drop presented in trace D was equivalent to 0.26 ± 0.05 of the fluorescence observed before the addition of the TMRM.

presence of CCCP reflects the total cellular uptake of

TMRM, the time course of the quenching was

com-pared with the time course of TMRM uptake into the

cells The time course of TMRM uptake into cells

con-sists of two stages: a first rapid stage that reflects

bind-ing of TMRM to the outer leaflet of the plasma

membrane and a subsequent uptake of TMRM into

the cells [16] The uptake of TMRM into K562 and

GLC4 cells occurred with half-lives of 3.7 ± 0.4 and

1.3 ± 0.2 min, respectively (as calculated based on

data presented in Fig 3) Thus, the FRET was

four-to six-fold faster compared four-to the four-total uptake of TMRM into the cells and the kinetics of the two movements are separate

A comparison of TMA-DPH fluorescence quenching

by TMRM observed in normally respiring cells and in cells whose mitochondria had been poisoned reveals that the active uptake of TMRM into the mitochon-dria interferes with the second phase of TMA-DPH quenching (Fig 2) Initially, there is significant inhibi-tion of the quenching in the respiring cells, which is subsequently relieved, presumably as a result of satura-tion of the mitochondria with TMRM As shown in

Table 1, poisoning of the mitochondria with either CCCP or sodium azide resulted in little change in their ATP content These cells relied mainly on glycolysis for their ATP supply and only poisoning the mito-chondria and glucose deprivation lead to a reduction

in cellular ATP content Thus, the effect of the mito-chondrial poisons on the secondary fluorescence drop

is not the result of an indirect effect mediated by ATP depletion

FRET from TMA-DPH to TMRM in multidrug resistant cells

Over-expression of Pgp by K562 cells had no effect on the rapid initial drop in TMA-DPH fluorescence induced by TMRM By contrast, it eliminated the sec-ond phase of drop in TMA-DPH fluorescence induced

by TMRM (Fig 4A) The activity of Pgp completely cancelled the slow phase of fluorescence drop, both in cells with active mitochondria and in cells whose mito-chondria had been poisoned This effect of the over-expressed Pgp was partially reversed as a result of the

K562

A

B

5 min C

GLC4

A

3 min

B

2008

A

2 min

B

A

B

C

Fig 2 Effect of poisoning the mitochondria on TMA-DPH fluores-cence quenching by TMRM (A) K562, (B) GLC4 or (C) 2008 cells were incubated at 37 C either in the absence (trace A) or presence

of either 1 l M CCCP (trace B) or 10 m M sodium azide (trace C).

2 l M TMA-DPH was added at the time points marked by the thin arrows and 25 l M TMRM was added at the time points marked by the thick arrows TMA-DPH fluorescence was monitored continu-ously The curves represent at least four separate experiments The curves describing the second, slow, phase of TMA-DPH fluo-rescence quenching by TMRM in the presence of either CCCP or sodium azide were fitted to the first-order reaction y = a · exp(–k · t) + c, where t is the time period elapsed from the addition of the dye and k is the reaction constant; a and c represent the extent of the secondary fluorescence drop and the fluorescence remaining after both phases of fluorescence quenching, respectively The k values obtained served to calculate the half-life of the fluorescence quenching All fluorescence values are expressed as fractions of the TMA-DPH fluorescence exhibited by the cells just before the addition of the TMRM dye The r 2 values obtained were > 0.95.

modulation of Pgp activity by the chemosensitizers,

cyclosporine A, verapamil and reserpine, or by

deple-tion of cellular ATP These treatments had no

signifi-cant effect on either the initial fast phase of

fluorescence in the Pgp-over-expressing cells or

fluores-cence quenching in wild-type cells (Fig 5) Inhibition

of Pgp with cyclosporine A caused a parallel increase

in the amount of TMRM taken up by the cells as well

as the extent of the second phase of TMA-DPH

fluo-rescence quenching by TMRM (Figs 6 and 7) By

con-trast to over-expression of Pgp, over-expression of

MRP1 had no apparent effect on fluorescence

quench-ing of TMA-DPH by TMRM (Fig 4B, C) As

expected, MRP1 activity had no apparent effect on the

rapid initial quenching of TMA-DPH fluorescence

Moreover, MRP1 over-expression did not affect the

subsequent slow fluorescence quenching of TMA-DPH

fluorescence, either in respiring cells or in cells whose

mitochondria had been poisoned

Discussion

Cellular uptake of the rhodamine, TMRM, was analy-sed using FRET from the dye TMA-DPH located at the surface of the cell plasma membrane to the incom-ing rhodamine dye The quenchincom-ing of TMA-DPH occurred in two distinct phases: an initial rapid phase followed by a slower phase with a measurable kinetics Because the initial quenching phase was very rapid and was unaffected by low temperatures, it represents the adsorption of dye to the cell surface The subse-quent phase was eliminated at low temperatures and thus involves transport across or into the lipid core of the plasma membrane This temperature-dependent flu-orescence quenching phase exhibited the following characteristics (a) In the presence of mitochondrial poisons, it occurred as a single first-order reaction (b) Active uptake of the TMRM by respiring mitochon-dria transiently inhibited the temperature-dependent fluorescence quenching This inhibition could be pre-vented by mitochondrial poisons such as the uncou-pler, CCCP, and the respiration inhibitor, sodium azide Treatment of cells with these poisons did not deplete the ATP content of the cells Thus, the fluores-cence quenching observed in the presence of these poisons, especially the hydrophilic azide ion, does not reflect a direct effect on the plasma membrane (c) The temperature-dependent fluorescence quenching was prevented by the activity of over-expressed Pgp Fluorescence quenching could be restored by modu-lation of Pgp activity, either by its specific inhibitors

or by depletion of cellular ATP

The temperature-dependent fluorescence quenching reflects the transfer of TMRM from its location at the surface of the cells toward an inner location Because the cationic rhodamine dye is amphipathic, it is practi-cally insoluble in the lipid core and is expected to be

GLC4 K562

0

Time (min)

–6 cells)

Fig 3 TMRM uptake into K562 (left) and GLC4 (right) cells K562 or GLC4 cells were incubated with 25 l M TMRM in the presence (circles)

or absence (squares) of 1 l M CCCP Samples were withdrawn at various time points and the amount of TMRM associated with the cells was determined by the quantitative procedure described in the Experimental procedures The data describing the dye uptake into the cells whose mitochondria were poisoned with CCCP were fitted to a first-order reaction with r 2 > 0.95, as described in Fig 2.

Table 1 Effect of mitochondrial poisons on the cellular ATP

con-tent of K562 cells K562 cells or their Pgp over-expressing sub-line,

K562 ⁄ ADR, were incubated for 30 min at 37 C in the absence or

presence of 10 m M glucose, 10 m M deoxyglucose, 1 l M CCCP or

1 m M azide Cell samples were withdrawn and their ATP content

was determined ATP content is expressed as a percentage of the

ATP content of the control K562 cells and their Pgp

over-express-ing cells (4.6 ± 0.6 and 5.3 ± 0.7 nmolÆ10)6cells, respectively).

K562 wild-type

Pgp over-expressing cells

localized at the surfaces of the plasma membrane.

Theoretically, this fluorescence quenching could be the

result of TMRM transfer from the cell surface further

into the outer leaflet of the membrane or flip-flop across the membrane and residence in the inner leaflet

of the membrane The observation that active uptake

of the TMRM by the mitochondria delays the temper-ature-dependent fluorescence quenching is inconsistent with the possibility that quenching occurs as a result

of dye moving within the outer leaflet of the plasma membrane The lipid core of the plasma membrane constitutes the main barrier to TMRM transport across the membrane and the mitochondria cannot affect the TMRM concentration bound at the outer leaflet Thus, the temperature-dependent fluorescence quenching reflects the flip-flop of TMRM from the outer leaflet of the plasma membrane to the inner

K562/ADR

A

5 min B

B

GLC4/ADR

A

2 min B

2008/MRP1

A

2 min

B

A

B

C

Fig 4 TMA-DPH fluorescence quenching by TMRM in Pgp or

MRP1 over-expressing cells Pgp over-expressing cells, (A)

K562 ⁄ ADR, or MRP1 over-expressing cells, (B) GLC4 ⁄ MRP1 and

(C) 2008 ⁄ MRP1, were incubated at 37 C either in the absence

(trace A) or presence (trace B) of 1 l M CCCP 2 l M TMA-DPH was

added and the cells were incubated for a further 10 min 25 l M

TMRM was added at the time points marked by the arrows

TMA-DPH fluorescence was monitored continuously The curves

repre-sent at least four separate experiments The curves describing the

second phase of TMA-DPH fluorescence quenching by TMRM in

presence of CCCP were fitted as a first-order reaction with

r 2 > 0.95, as described in Fig 2.

Sensitive K562 cells

A

B

A

B C D

3 min

D E

Resistant K562/ADR cells

B A B

C D

3 min

D E

Fig 5 Effect of Pgp modulation on TMA-DPH fluorescence quenching by TMRM (A) K562 cells or (B) their Pgp over-express-ing sub-line, K562 ⁄ ADR, were incubated at 37 C in the presence

of glucose and 1 m M azide and either in the absence (trace A) or presence of 10 l M cyclosporine (trace B), 100 l M verapamil (trace C) or 30 l M reserpine (trace D) Cells presented in trace E were depleted of ATP by incubation for 30 min at 37 C in the presence

of deoxyglucose instead of glucose and 1 m M azide At the time points marked by the arrows, 2 l M TMA-DPH was added and, after

a further 5 min of incubation, 25 l M TMRM was added TMA-DPH fluorescence was monitored continuously The curves represent at least four separate experiments.

leaflet The expected distance between TMRM located

at the inner leaflet of the plasma membrane and

TMA-DPH located at the outer leaflet is somewhat

< 3 nm (i.e a distance that could allow FRET

between these dyes)

The results obtained with FRET from TMA-DPH

to TMRM located at the outer leaflet of the plasma membrane suggest that uptake of TMRM occurs in distinct steps: rapid binding to the outer surface of the cells, flip-flop across the plasma membrane, accu-mulation of dye in the cytoplasmic leaflet of the plasma membrane and release into the cell interior The initial binding of dye to the cells, evident as the TMRM-mediated initial drop in TMA-DPH fluores-cence, appears to be instantaneous, even at low temperatures Therefore, it is very rapid, possibly limited by the diffusion of dye toward the cell Local-ization studies of multidrug-type drugs and modula-tors suggest that, upon association of the TMRM with the plasma membrane, it is located between the phosphate of the lipid headgroups and the upper segments of the lipid hydrocarbon chains [26]

The subsequent accumulation of dye in the cytoplas-mic leaflet of the plasma membrane comprises a fast process compared to the total uptake of dye into the cells, indicating that, in kinetic terms, the cytoplasmic leaflet comprises a compartment separate from the cytoplasm The accumulation of dye in the cytoplas-mic leaflet is the outcome of a balance between the rate of flip-flop across the membrane from the outer leaflet to the cytoplasmic leaflet of the plasma mem-brane and the release from the cytoplasmic leaflet into the cell Analysis using a kinetic model of drug uptake into cells similar to the previously reported models [27,28] suggests that a significant accumulation in the cytoplasmic leaflet of the plasma membrane takes place only when the release from the plasma mem-brane into the cytoplasm occurs at a rate similar to that of the flip-flop of dye across the plasma mem-brane In the case where the release into the cytoplasm

is fast compared to the flip-flop, the amount of dye accumulated in the cytoplasmic leaflet will be insignifi-cant By contrast, in the case where diffusion into the cells is slower than the flip-flop across the plasma membrane, it will constitute the limiting step of dye uptake into the cells

The secondary drop in the TMRM-mediated TMA-DPH fluorescence observed in cells whose mitochon-dria were poisoned, reflects the flip-flop rate of dye from the outer leaflet of the plasma membrane to the cytoplasmic leaflet The apparent half-life of the flip-flop was in the range 20–40 s in the various cell lines investigated in the present study This half-life value was similar to the flip-flop value of doxorubicin observed in cell-free systems such as liposomes and iso-lated erythrocyte membranes [4,5] The half-life of the flip-flop observed as the drop in TMA-DPH fluores-cence is a minimum value because the step subsequent

0.0

0.1

0.2

1.0

TMA-DPH fluorescence 5.0

K562

5 min Sensitive cells

Fig 6 Effect of various cyclosporine concentrations on TMA-DPH

fluorescence quenching by TMRM in Pgp over-expressing cells,

K562 ⁄ ADR, or K562 sensitive cells, were incubated in the presence

of 1 l M CCCP, 2 l M TMA-DPH and various concentrations of

cyclo-sporine A (l M concentrations are indicated) for 15 min and then

25 l M TMRM was added at the time points marked by the arrows.

TMA-DPH fluorescence was monitored continuously The curves

represent at least four separate experiments The curves describing

the second phase of TMA-DPH fluorescence quenching by TMRM

in presence of CCCP were fitted as a first-order reaction with

r 2 > 0.95, as described in Fig 2.

1.5

1.0

0.20 0.15

0.10 0.5

0.05

–6 cells)

cells 0.0

Fig 7 Effect of cyclosporine A on FRET from TMA-DPH to TMRM

and TMRM uptake in Pgp over-expressing cells K562 ⁄ ADR cells

were treated as described in Fig 6 The amount of TMRM that

was associated with cells during 30 min of incubation (circles) was

determined quantitavely as described in the Experimental

proce-dures The extent of the second phase of fluorescence quenching

by TMRM (squares) was determined by fitting the relevant curves

from Fig 6 to equations describing a first-order reaction, as

described in Fig 2.

to the flip-flop, namely the release of dye into the

cyto-plasm, can appear to accelerate the rate at which dye

accumulation in the cytoplasmic leaflet of the plasma

membrane reaches steady-state Fast release of dye will

result in shorter apparent half-life of the flip-flop of

dye across the membrane

Surprisingly, and of interest, the active uptake of

dye into the mitochondria prevented the accumulation

of dye in the cytoplasmic leaflet of the plasma

mem-brane Only after a prolonged period, TMRM was

accumulated in the cytoplasmic leaflet of the plasma

membrane, presumably as a result of saturation of the

mitochondria, leading to diminished uptake of TMRM

into the mitochondria Because there are no reports of

direct contact of mitochondria with the plasma

mem-brane, we have to assume that the mitochondria do

not pump the dye directly from the plasma membrane

but, instead, from the cytoplasm adjacent to the

mem-brane On the basis of this observation, it can be

deduced that the limiting step in the release of

dye from the plasma membrane is not the actual

release from the membrane but, instead, the movement

away from the plasma membrane into the cell The

cytoplasm next to the plasma membrane is unstirred

and dense with proteins Moreover, TMRM and

anti-cancer drugs, such as anthracyclines, are positively

charged and therefore bind to acidic groups in proteins

and membranes Thus, their movement into the cell

can be envisaged as a series of binding and releasing

events rather than simple diffusion However, it should

be stressed that although, in kinetic terms, the

com-partment of the plasma membrane includes the

cyto-plasm layer adjacent to the cyto-plasma membrane, the

drop in TMA-DPH fluorescence reflects almost

exclu-sively the dye present in the cytoplasmic leaflet This is

a result of the partition of the dye into the plasma

membrane in preference to remaining soluble in the

aqueous cytoplasm

The data of the FRET from TMA-DPH to TMRM

suggest that Pgp extracts its substrates directly from

the cytoplasmic leaflet of the plasma membrane This

is consistent with the suggestion made by Higgins and

Gottesman [10] that Pgp acts as a flippase transporting

its substrates from the cytoplasmic leaflet of the lipid

bilayer to the outer leaflet or to the external medium

The data reported in the present study, and obtained

in living cells, confirm the finding obtained in

reconsti-tuted proteoliposomes [11,12] and isolated membranes

[29,30] indicating that Pgp and a bacterial multidrug

ABC-transporter extract their substrates from the

cyto-plasmic leaflet of the membrane

By contrast to Pgp, over-expression of MRP1 does

not affect the presence of TMRM in the cytoplasmic

leaflet, but appears to pump it directly from the cyto-plasm Over-expression of MRP1 did not alter the pat-tern of the drop in TMA-DPH fluorescence observed

in the sensitive parent cell lines MRP1 transports, on the one hand, organic anions, such as glutathione con-jugates, and, on the other hand, basic hydrophobic drugs, such as daunorubicin and vincristine [14] It has been suggested that MRP1 has two binding sites: one with high affinity for hydrophobic ligands and the other with high affinity for glutathione [31,32] The results obtained in the present study suggest that both sites are not located within the plasma membrane, but

at its surface The difference in the transport mecha-nisms between Pgp and MRP1, as revealed with FRET from TMA-DPH to TMRM is not the result of higher resistance levels in the Pgp cells Inhibition of Pgp with various concentrations of cyclosporin A allowed for corresponding levels of TMRM accumulation, although in no case was the pattern of TMA-DPH fluorescence drop similar to that observed in sensitive cells, as is the case in MRP1 over-expressing cells The finding that the cytoplasmic leaflet of the plasma membrane constitutes a kinetic compartment separate from the cell interior emphasizes the relevance

of Pgp as a flippase to multidrug resistance Drugs taken up into cells stay in the cytoplasmic leaflet of the plasma membrane for a few seconds before reaching the cell interior Thus, Pgp that extracts its substrates from the cytoplasmic leaflet of the plasma membrane has the opportunity to remove drugs from the cells before they reach the cell interior Pgp is adapted to prevent drugs from entering cells rather than to remove drugs already present in the cells By contrast, transporters such as MRP1 extract their substrates directly from the cytoplasm and are more adapted to remove drugs already present inside the cells than to prevent the access of drugs into the cells This phe-nomenon is especially relevant to drug transcellular transport and multidrug resistance in cell monolayers such as the blood–brain barrier and the epithelia lining the intestine and the nephrons It has been shown that the tight junctions pose a barrier to the movement of lipids between the outer leaflets of the apical and baso-lateral domains of the plasma membrane [33] By con-trast, they do not interfere with the movement of lipids and presumably drugs between the cytoplasmic leaflets

of these domains [33] Transcellular movement across cell monolayers of certain drugs and dyes, such as TMRM, is expected to occur mainly by rapid incorpo-ration into the outer leaflet of the plasma membrane, flip-flop across the lipid core of the membrane, lateral movement in the cytoplasmic leaflet of the plasma membrane from one membrane domain to the other,

flip-flop again across the lipid core of the membrane

and, finally, release from the plasma membrane into

the aqueous phase Thus, drugs and dyes with a high

partition coefficient (membrane⁄ aqueous phase) are

expected to cross cell monolayers via lateral movement

in the cytoplasmic leaflet of the plasma membrane,

with little access into the cells’ cytoplasm Indeed,

kinetic analysis of drug transport across kidney

conflu-ent cell monolayers suggests that hydrophobic drugs

cross the monolayer by lateral transport in the

cyto-plasmic leaflet of the plasma membrane rather than via

the cytoplasm [34]

Experimental procedures

K562, a human leukemia cell line established from a

patient with chronic myelogeneous leukemia in blast

trans-formation [35], was purchased from ATCC (Rockville,

MD, USA) and maintained in RPMI medium (Biological

Industries, Beit-Haemmek, Israel) The K562

Pgp-over-expressing subline was obtained by sequential exposure of

cells to increasing concentrations of doxorubicin and was

maintained in the presence of 0.5 lm doxorubicin 2008

parental cells and their MRP1 over-expressing subline [36]

were kindly provided by P Borst (Netherlands Cancer

Institute, Amsterdam, The Netherlands) and grown in

RPMI-1640 (Sigma-Aldrich, Rehovot, Israel) The CIR

[37], GLC4 cells and MRP1-over-expressing GLC4⁄ ADR

cells [38] were cultured in RPMI 1640 either in the

absence or presence of 1 lm doxorubicin All media were

supplemented with 10% fetal bovine serum, 100 IUÆmL)1

penicillin and 100 lgÆmL)1 streptomycin (Invitrogen,

Rehovot, Israel) and the cells were grown at 37C under

5% CO2⁄ humidified air TMRM, Silicone oil AR200 and

mineral oil were purchased from Sigma-Aldrich Cellular

ATP content was measured by the luciferin-luciferase

assay [39]

Measurement of FRET from TMA-DPH to TMRM

Cells were labeled with TMA-DPH (2 lm) by incubation at

37C The fluorescence of TMA-DPH was monitored

con-tinuously with the temperature maintained at 37C In a

typical experiment, 2· 106cells were incubated with

stir-ring in 2 mL of medium composed of NaCl (132 mm), KCl

(3.5 mm), CaCl2(1 mm), MgCl2(0.5 mm), glucose (10 mm)

and Hepes-Tris buffer (20 mm, pH 7.4) A concentration of

2 lm TMA-DPH was added, leading to a rapid rise in

TMA-DPH fluorescence After further incubation for 10–

15 min, TMRM (25 lm) was added The TMA-DPH

fluo-rescence was monitored continuously in a Varian Cary

Eclipse fluorescence spectrophotometer (Varian Inc., Palo

Alto, CA, USA) using an excitation wavelength of 366 nm

and an emission wavelength of 426 nm

Quantitative determination of the amount of TMRM associated with cells

For determination of the amount of TMRM associated with cells, cells were incubated with the dye in the medium described above Samples containing 4· 105 cells in 0.4 mL of medium were withdrawn and placed in an Eppendorf-style microfuge above a 0.2 mL cushion consist-ing of 95 parts Silicone oil AR 200 (d20= 1.049) and five parts mineral oil (d20= 0.89) After centrifugation for

4 min at 13 200 g at room temperature, the oil cushion was washed three times with water by suction Subse-quently, all of the upper phase, including part of the oil cushion, was removed, leaving a fraction of the oil above the cell pellets The cell pellets were dissolved by the addi-tion of 0.1 mL of guanidine HCl (5 m) buffered with Hepes-Tris (50 mm, pH 7.4), centrifugation for 5 min and incubation for at least 1 h at room temperature The dissolved samples were mixed thoroughly with 0.5 mL of water and centrifuged for 5 min Samples (0.4 mL) were withdrawn from the pellets dissolved in the aqueous phase The fluorescence of TMRM was determined using an exci-tation wavelength of 563 nm and an emission wavelength

of 583 nm To ensure fidelity of the assay, dye-free cell samples were mixed with known amounts of rhodamines and processed as above The rhodamine yield thus obtained matched the amount expected To determine the volume of incubation medium carried through the oil cush-ion together with the cells, a cell sample was incubated on ice with 10 lm acidic dye (calcein) and processed as above The amount of calcein associated with the cells was equivalent to < 0.05% of the sample volume The time period required to separate cells from the external medium was equivalent to 0.5 min All curves were adjusted accordingly

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