169

Biochimica et Biophysica Acta, 1139 (1992) 169-183 © 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

Review

BBADIS 61169

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines Dorte Nielsen and Torben Skovsgaard Department of Oncology, Universityof Copenhagen, Herlev Hospital, Herlev (Denmark) (Received 10 December 1991)

Key words: P-glycoprotein; Cell line; Multidrug resistance

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169

lI. Correlation between resistance, accumulation, and p-glycoprotein expression? . . . . . . . . . . . . . . . A. Cell lines selected for resistance to daunorubicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cell lines selected for resistance to doxorubicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cell lines selected for resistance to vinca alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cell lines selected for resistance to colchicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cell lines selected for resistance to actinomycin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

170 171 171 174 175 176

III. Summary of results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overexpression of P-glycoprotein and decreased accumulation/increased efflux . . . . . . . . . . . B. Overexpression of P-glycoprotein without increased effiux . . . . . . . . . . . . . . . . . . . . . . . . . . . C. No expression of P-glycoprotein, no accumulation defects . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Decreased accumulation/increased efflux without P-glycoprotein overexpression . . . . . . . . . . E. Quantification of P-glycoprotein and transport properties in series of cell lines . . . . . . . . . . . .

176 176 177 177 177 178

IV. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179

Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

180

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

180

I. Introduction T u m o r cell resistance to cytotoxic drugs is consid e r e d o n e o f the m a j o r obstacles to successful c h e m o therapy. M u l t i d r u g r e s i s t a n c e ( M D R ) d e s c r i b e s t h e

Correspondence to: D. Nielsen, Department of Oncology, University of Copenhagen, Herlev Hospital, Herlev Ringvej, DK-2730 Herlev, Denmark. Abbreviations: MDR, multidrug resistance; P-gp, P-glycoprotein; mRNA, messenger RNA; DNR, daunorubicin; DOX, doxorubicin; VCR, vincristine; VBL, vinblastine; COL, colchicine; ACTD, actinomycin D; MIT, mitoxantrone; VER, verapamil;; SDS-PAGE, sodiumdodecyl sulfate polyacrylamide gel electrophoresis.

s i m u l t a n e o u s expression o f cellular resistance to a wide r a n g e o f u n r e l a t e d drugs primarily o f n at ur a l origin. T h e p h e n o t y p e is f r e q u e n t l y o b s e r v e d in r o d e n t and h u m a n cell lines s e l e c t e d for resistance to a single a g e n t . T h e drugs m o s t o f t e n involved in M D R are antibiotics or alkaloids o f fungal or p l an t origin, including a n t h r a c y c l i n e s , A C T D , v i n ca a l k a l o i d s a nd colchicine ( C O L ) . A l t h o u g h studies have shown the o c c u r r e n c e of a l t e r e d t o p o i s o m e r a s e II or e n h a n c e d g l u t a t h i o n e t r a n s f e r a s e in cell lines expressing M D R , i n c r e a s e d expression o f a 170 k D a p l a s m a m e m b r a n e g l y co p r o t ei n , P-gp, is the most c o n s i s t e n t c h a n g e in M D R cell lines. F u r t h e r m o r e , t r a n s f e c t i o n e x p e r i m e n t s with the P-gp c o m p l e m e n t a r y D N A s e q u e n c e have

17{} revealed that increased expression of P-gp is sufficient lk}r development of an MDR phenotype [1]. Other experiments have demonstrated that MDR cells are able to maintain decreased drug accumulation. Since Dan~a in 1973 [2] proposed the existence of an active drug extrusion in tumor cells resistant to DNR, increased activity of an energy-dependent drug efflux pump in M D R cells has been the working model for most investigators. The primary sequence of P-gp has been determined from sequence data obtained from complementary DNA. The protein consists of 1276-1280 amino acids with a tandemly duplicated structure. Each half of the molecule contains a nucleotide-binding site and reveals six hydrophobic regions that fit all criteria for transmembrane domains. A remarkable degree of sequence homology has been detected between P-gp and bacterial transport proteins [3]. Furthermore, several experiments have shown that a number of radio-labelled drugs and photoactivated drug analogs bind to the protein [4,5]. A model for the function of P-pg has been proposed in which drugs bind directly to P-gp which then acts as a carrier protein that actively extrudes drugs through a pore or channel in the membrane formed by transmembrane domains of the molecules using energy derived from ATP hydrolysis [6]. The genetic and biochemical aspects of M D R have recently been reviewed [7-9]. In this article we focus on re-examining cell lines selected for resistance to a single agent in order to point out what we perceive to be controversies and contradictions in regarding P-gp as a multidrug transporter. Consequently, possible changes in topoisomerase II or glutathione transferase isozymes are not discussed. Furthermore, although reversal of resistance by addition of calcium channel blockers is regarded as one of the characteristics of cell lines overexpressing P-gp, this subject is not reviewed, as the mechanism of these compounds in respect to circumvention of resistance is still not clarified.

II. Correlation between resistance, accumulation, and P-glycoprotein expression? Assuming that P-gp acts as a drug transporter mediating drug effiux, one would expect a close connection between P-gp expression, reduction in drug accumulation, and, if no other resistance mechanisms are operating, drug resistance. Furthermore, all M D R cells which overexpress P-gp ought to have increased drug effiux and probably also vice versa. The situation may be symbolized as shown in Fig. 1A. In order to review whether the above-mentioned relationship exists in cell lines expressing MDR, we have collected papers describing cell lines selected for resistance to drugs which, according to usual practice,

S e n s i t i v e cells

M D R cells

A S e n s i t i v e cells

Fig. 1. The correlation between P-glycoprotcm and reduced drug accumulation in cell lines selected for resistance to M D R compounds. The figure only symbolizes the qualitative relationship and is not mathematically correct. • Total number of cell lines with Pglycoprotein expression; o, Total number of cell lines with reduced drug accumulation. A.: The 'ideal" situation regarding P-gp as an effiux pump, i.e., in all cells overexpressing P-gp increased resistance and decreased accumulation are demonstrated. B.: The "actual" situation, cell lines overexpressing P-gp without both increase in resistance and reduction in drug accumulation have been reported.

are associated with the M D R phenotype (the classical anthracyclines, the vinca alkaloids, COL and ACTD). Thus, cell lines selected for resistance to cytostatic agents commonly associated with the at-MDR phenotype (i.e., epipodophyllotoxins and amsacrine) are excluded, although P-gp expression is described in some of the cell lines. Only cell lines which have been systematically investigated for both P-gp expression and transport properties are included. We have endeavoured to collect as many cell lines as possible, although obviously we may have missed some. However, we believe that the number of collected cell lines enables us to draw conclusions about the relationship between P-gp expression and changes in drug accumulation. Experiments for determination of drug accumulation are generally performed by incubating cells with radiolabelled drugs for a period of several minutes or hours and therefore reflect net accumulation (influx and effiux). Only in a limited number of cell lines has the effiux been determined. In this review, cell lines with increased expression of P-gp and decreased drug accumulation will be considered as supporting the hypothesis of P-gp as multidrug transporter unless separate effiux experiments demonstrate the opposite. However, it is important to remember that this assumption is an absolute minimum. If P-gp is to be considered as a 'genuine drug-pump,' a significant reduction in steady-state accumulation, and not merely a decreased influx, must be demonstrated (Fig. 2). Furthermore, the effiux must be blocked by addition of

171

Cellular drug CGIIC

..........# i

Time ~

T'la'm¢ r

Fig. 2. - - , wild-type tumor cells; - - - - - , resistant tumor cells. A.: Time course uptake showing significant decrease in steady-state accumulation. B.: Time-course uptake showing reduced accumulation, which could be explained by decreased influx.

atically. Increased expression of P-gp was demonstrated in 43 (Table VI). Among these, a reduction of intracellular drug level was found in 42. Furthermore, increased effiux a n d / o r a significant decrease in steady-state accumulation was demonstrated in 27 cell lines (A1847 [19], A2780 [21], CEM [23], DLD [26], H69 [29], K562 [38], K562 [39], KB [41], LoVo [36], LoVo [42], LoVo [44], LoVo [26], MCF-7 [47], MCF-7 [50], MES-SA [51], MOLT4 [34], SW620 [54], 8226 [58], EMT6 [63], H1 [65], HCT [66], MEL PC4 [71], MEL C7D [71], P388 [72], S180 [79], UV2237 [82], V79 [83]. Lankelma et al. [22] have recently provided experimental evidence for effiux against a concentration gradient. However, Toffoli et al. [26] in five sub]ines of the human colon carcinoma cell line SW948 which were 3-5-fold resistant to DOX and 2-fold cross resistant to VCR, respectively, found enhanced mdrl m R N A and increased expression of P-gp, but were unable to detect either reduction in the intracellular drug accumulation or increase in effiux. Furthermore, despite considerable decreases in drug accumulation, the effiux was not significantly changed in three cases [67,74,78]. Ganapa t h i e t al. [67] r e p o r t e d t h r e e s u b l i n e s o f L 1 2 1 0 w h i c h

inhibitors of energy production (both oxidative phosphorylation and glycolysis) and by analogs which act as competitive inhibitors. II-A. Cell lines selected for resistance to DNR Cell lines selected for resistance to DNR are given in Table I. Seven cell lines have been studied systematically. In all cases, there was agreement between the occurrence of enhanced P-gp expression and decreased cellular accumulation (Table VI), but a significant decrease in steady-state accumulation a n d / o r increased effiux have been reported in only four cell lines (K562 [10], DC-3F [12], EHR2 [14], P388 [17]). II-B. Cell lines selected for resistance to D O X Cell lines selected for resistance to DOX are given in Table II. 61 cell lines have been investigated system-

overexpressed P-gp [68] and were 2-, 10- and 40-fold resistant to DOX, respectively. The cellular accumulation was 15-50% lower in the resistant cell lines compared to the sensitive. However, in effiux experiments, the cell lines retained approximately the same amount of drug. In a 10-fold resistant subline of P388 with increased expression of P-gp and increased m R N A Goidenberg et al. [74] demonstrated a 1.6-fold decrease in drug accumulation, but Deffie et al. [75] were unable to detect any significant increase in effiux of DOX. Furthermore, the accumulation was not enhanced by addition of sodium azide (1 mM) or dinitrophenol (1 mM). Ramu et al. [78] measured the fluorescence of accumulated DOX, which was rapidly quenched by DNA in the cell nuclei of a resistant subline of P388. The authors were unable to detect an active efflux mechanism, but found that the drug pas-

TABLE I Cross resistance, P-glycoprotein expression, and drug accumulation in cell lines selected for resistance to daunorubicin Cell lines are derived in humans and rodents and given in alphabetic order (see Appendix). Numbers in parentheses refer to fold of resistance compared to the parent line. Abbreviations: NR, not reported; R, resistant. Parent line

Primary resistance

Cross resistance

P-glycoprotein

Decreased accumulation/ increased effiux

References

K562 CHO DC-3F EHR2 L1210 P388 S180

DNR(15) DNR(37) DNR(883) DNR(60) DNR(30) DNR(12) DNR(73)

DOX(24), VCR(14) VBL(39), COL(21) VCR(285), ACTD(263) VCR( >> 15), VP16(50) DOX(51), ACTD(20) NR DOX(275), ACTD(50)

+ + + + + + +

+/ + +/NR +/NR + /+ +/NR +/NR +/NR

[10] [11] [12,13] [14,15] [16] [17] [181

172 sively d i f f u s e d i n t o cells a n d t h a t t h e d i f f u s i o n in r e s i s t a n t cells was c o n s i d e r a b l y l o w e r t h a n in t h e sensitive cells. In o t h e r cases, a m i s s i n g c o r r e l a t i o n b e t w e e n P - g p e x p r e s s i o n a n d t r a n s p o r t p r o p e r t i e s was o b s e r v e d . T h u s , S i e g f r i e d et al. [79] d e s c r i b e d f o u r s u b l i n e s o f

m u r i n e s a r c o m a 180. T h e s u b l i n e s w e r e 7 - 11-, 40- a n d 122-fold r e s i s t a n t c o m p a r e d to the p a r e n t line. T h e level o f P - g p e x p r e s s i o n c o r r e l a t e d w i t h t h e d e g r e e of r e s i s t a n c e [80]. H o w e v e r , a 4 0 % d e c r e a s e in t h e intrac e l l u l a r s t e a d y - s t a t e c o n c e n t r a t i o n of D N R was obs e r v e d in all t h e sublines. K a t o et al. [38] r e p o r t e d

TABLE II Cross-resistance, P-glycoprotein expression, and drug accumulation in cell lines selected for resistance to doxorubicin

Cell lines are derived in humans and rodents and given in alphabetic order (see Appendix). Numbers in parentheses refer to fold of resistance compared to the parent line. Abbreviations: NR, not reported; R, resistant; S, sensitive. Parent line

Primary resistance

Cross-resistance

P-glycoprotein

Decreased accumulation/ increased effiux

References

A 1847 A2780 A2780 GEM

DOX(6) DOX(150) DOX(5) DOX(24)

NR NR VBL(1) DNR(18), VCR(1), COL(l), VP16(15) DNR(90), VCR(100), COL(20), VP16(70) VCR(34), COL(10) ACTD(3-5), VCR(4-8)

+ + -

+/ + +/ + ( - )/+ -/NR

[19,20] [21 ] [22] [23]

+

+/NR

+

+/ + +/ +

[24,25] [26]

VCR(6), COL(0.5), VP16(38) VCR(750), COL(80) DNR(85), VCR(112), COL(16), VP16(12) DNR(50), VCR(8), ACTD(6) DNR(75), VCR(25), COL(10), ACTD(15) VCR(25), ACTD(17), VP16(837) VCR(35-221), VP16(4-9) DNR(80), VCR(630), ACTD(108), VP16(44) VBL(43), COL(19) VCR(200), ACTD(9), VP16(63) DNR(29), VCR(27), VP16(21) VCR(R), VP16(R) VCR(R), VP16(R) ACTD(6-9), VCR(10-15)

-

+/NR

[27,28]

+ -

+/NR -/NR

[29,30] [31 ]

-

+/ +

[32,33]

-

+/( + )

[34,35]

-

+/ +

[36,37]

+

+/ +

[38]

+

+/ +

[39]

+ +

+/ + +/ +

[40,41] [36]

+

+/ +

[42,43]

+ +

+/ +/ + +/ +

[44]

+ +

+/NR +/NR

[45] [46,47]

-

-/NR

[48]

+ +

-/NR +/ + +/ +

[49] [50] [51,52]

+ -

+/NR +/ +

[34] [53]

+

+/NR

[54]

DOX(83) COR-L DLD1 GLC 4

DOX(23) DOX(9-11) (5 sublines) DOX(44)

H69 H69

DOX(85) DOX(100)

HE60

DOX(111)

HL60

DOX(80)

HT1080

DOX(222)

K562 K562

DOX(13-155) (3 sublines) DOX(134)

KB LoVo

DOX(97) DOX(129)

LoVo

DOX(29)

LoVo

LS180 MCF-7

DOX(4) DOX(90) DOX(19-23) (5 sublines) DOX(R) DOX(192)

MCF-7

DOX(900)

MCF-7 MCF-7 MES-SA

DOX(10) DOX(75) DOX(25,100) (2 sublines)

MOLT4 SK-MEL 170 SW620

DOX(15) DOX(178)

NR VBL(274), ACTD(357), VP16(175) DNR(326), VBL(5) VM26(26) VBL(3) VCR(190) DNR(25,160), VCR(100,240), VP16(15,30) DNR(R) NR

DOX(R, 112) (2 sublines)

DOX(102): VBL(40), COL(35)

LoVo

[26]

173 three sublines of K562 which were 13, 104 and 155-fold resistant to DOX, respectively. The cell lines showed decreased drug accumulation (48, 35 and 16% of the parent line) and increased efflux of DOX proportional with the degree of resistance. However, the cell lines, which accumulated 35 and 16% compared with the

parent line, had the same degree of P-gp as detected by Western blotting. In 18 pairs of cell lines overexpression of P-gp could not be detected (Table VI). Among these the transport properties of the resistant cell lines were unchanged in seven (CEM [23], H69 [31], MCF-7 [48], MCF-7 [49],

TABLE II (continued) Parent line

Primary resistance

Cross-resistance

P-glycoprotein

Decreased accumulation/ increased efflux

References

SW948

DOX(3-5) (5 sublines) DOX(5-10) (3 sublines) DOX(R)

ACTD(4-5), VCR(2)

+

- / -

[26]

DNR(4-5), VCR(2-16)

+/ -

+/NR

[55-57]

DNR(4), VCR(17), VP16(45) DNR(20-70), VCR(69-540), VP16(53-750) VCR(197,270), VP16(5,95) vinca alkaloid(S), VP16(17) VCR(1149), VP16(l18) DNR(27), VCR(4), COL(4) NR

-

+/NR

+

+/NR

+

+/NR

[58]

-

-/NR

[59]

+ +

+/NR +/NR

[60] [61]

+

+/NR

[62]

VCR(43), COL(50), VP16(54) DNR(35), VCR(19), VP16(17) NR NR NR

+

+/NR

[63]

+

+/NR

[64]

+ + +

+/NR +/NR +/ -

[65] [66] [67,68]

DNR(24), ACTD(12) VCR(R), VP16(40) (R) VCR(1-2), VP16(8-62)

+ + + -

+/NR +/NR +/NR -/NR

[16] [69] [70] [71]

VCR(16), VP16(74) VCR(16,21), VP16(86,133) VCR(1,2), VP16(11)

+

+/NR +/NR

-

-/NR

VCR(3), VP16(15) VCR(5-21), VP16(22-50) VCR(64), ACTD(223) DNR(11,16), ACTD(18), VP16(4,6) VCR (615), VP16(28) VBL(R) DNR(11-154), VCR(8-12), ACTD(7-10) DNR(60-125), ACTD(32-88) DNR(50), VCR(109) VCR(260), ACTD(27) Vinca alkaloids(R)

+

+/NR +/NR

+ +

+/ + +/ -

[72,73] [74,75]

+ + +

+/NR +/ +/NR

[76,77] [78] [79,80]

+

+/NR

[18]

+ + +

+/ + +/ + +/NR

[81,82] [83] [84]

SW1573

DOX(40-2000) (4 sublines) 8226 8226

DOX(11,138) (2 sublines) DOX(8)

8226 CHO

DOX(1245) DOX(28)

DM15 EMT6

DOX(8-270) (3 sublines) DOX(69)

F4-6

DOX(51)

H1 HCT L1210

DOX(R) DOX(200-400) DOX(10,40) (2 sublines) DOX(45) DOX(40) DOX(R) DOX(5-17) (3 sublines) DOX(44) DOX(98,180) (2 sublines) DOX(3,5) (2 sublines) DOX(10) DOX(12-54) (4 sublines) DOX(69) DOX(5,10) (2 sublines) DOX(703) DOX(R) DOX(7-122) (4 sublines) DOX(98-340) (2 sublines) DOX(140) DOX(3000) DOX(R)

L1210 L1210 M109 MEL PC4

MEL C7D

P388 P388 P388 P388

S180 UV2237 V79 3LL

174 8226 [59], MEL-PC4, MEL-C7D [71]), of which two were selected in V E R in addition to D O X [48,59]. Furthermore, one cell line had minor changes in net accumulation (A2780 [22]). Despite that P-gp was not enhanced, considerable reductions in intracellular drug levels were demonstrated in nine cell lines. In a 44-fold-resistant small cell lung carcinoma cell line (GLC4) without overexpression of m d r l m R N A [28] Zijlstra et al. [27] found a 45% decrement in intracellular level of DOX. In a large cell lung carcinoma cell line (COR-L) without overexpression of P-gp (Western blot), both significantly reduced drug accumulation and increased energy-dependent effiux was demonstrated [24,25]. In a Ill-fold-resistant subline of HL60 with no m d r l m R N A expression (slot blot) and a very low level of P-gp (Western blot) [33], the net accumulation was decreased to 40% due to increased effiux, which could be blocked by incubation with sodium azide and deoxyg]ucose [32]. The influx in this cell line was unchanged. Furthermore, in an 80-fold-resistant subline of HL60 after prolonged incubation (1 h), Marsh et al. [34] demonstrated increased effiux of DOX. In a 222fold-resistant fibrosarcoma cell line in which P-gp was not detectable with Western blot Slovak et al. [36] found increased effiux of DOX. In a 178-fold-resistant subline of SK-MEL-170 Panneerselwam et al. [53] demonstrated increased effiux and decreased drug ac-

cumulation. Using S D S - P A G E without application of monoclonal antibody, the authors revealed the presence of several high molecular weight proteins; however, none of the proteins was identical to P-gp. In addition, Slapak et al. [71] have investigated resistant sublines of the murine erythroleukemia cell lines M E L PC4 and M E L C7D and found that changes in drug accumulation preceded increases in m R N A and overexpression of P-gp in both series of cell lines. Furthermore, utilizing the RNase protection assay, Baas et al. [56] studied a series of sublines of the human squamous lung carcinoma cell line SW1573. In cell lines with a low degree of resistance the authors were unable to detect increased expression of m R N A despite accumulation defects. However, the same cell lines (with one exception) have been studied by Keizer et al. [55], who used an enzyme-linked immunosorbent assay for detection of P-gp and found enhanced expression of P-gp proportional with the degree of efflux in all investigated cell lines.

I I - C Cell lines selected f o r resistance to vinca alkaloids

Cell lines selected for resistance to vinca alkaloids are shown in Table III. Fourteen cell lines have been investigated systematically. All cell lines overexpressed P-gp and had decreased drug accumulation (Table VI). However, increased effiux a n d / o r reduced steady-state

TABLE III Cross-resistance, P-glycoprotein expression and drug accumulation in cell lines selected for resistance to vinca alkaloids

Cell lines are devided in humans and rodents and given in alphabetic order (see Appendix). Numbers in parentheses refer to fold of resistance compared to the parent line. Abbreviations: NR, not reported; R, resistant. Parent line

Primary resistance

Cross-resistance

P-glycoprotein

Decreased accumulation/ increased efflux

References

CEM

VBL(10- > 2 0 0 0 ) (7 sublines) VCR(22600)

VBL(186), DNR(152), VCR(2023), VP16(44) DNR(61), DOX(61), VBL(4480), VM26(35) DNR(5) DOX(8,15)

+

+/ +

[85,86]

+

+/ +

[87]

+ +

+/NR +/NR

[43,88] [89]

DOX(422), COL(171) DNR(18), DOX(10), COL(21), VP16(5) DOX(R), VP16(R) DNR(2), DOX(2), COL(4) DNR(3), COL(3) DOX(5,20), COL(24,39)

+ +

+/NR +/NR

[40,41] [90]

+ +

+/NR +/NR

[91,92] [93]

+ +

+/NR +/NR

[94]

DNR(178), ACTD(267) DOX(35), COL(64) VP16(45) DOX(166,234), COL(268,382) ACTD(13)

+ +

+/ + +/NR

[13,95] [63]

+

+/NR

[96,97]

+

+/NR

[98,99]

CEM GM3639 HL60 KB KB

VCR(59) VCR(25,140) (2 sublines) VBL(213) VCR(394)

MCF-7 7932

VCR(14) VCR(24)

CHO DC-3F EMT6 J774.2 SEWA

VBL(6) VCR(92,98) (2 sublines) VCR(2750) VCR(56) VBL(1017,1083) (2 sublines) VCR(170)

175 TABLE IV Cross-resistance, P-glycoprotein, and drug accumulation in cell lines selected for resistance to colchicine Cell lines are derived in humans and rodents and given in alphabetic order (see Appendix). Numbers in parentheses refer to fold of resistance compared to the parent line. Abbreviations: NR, not reported; R, resistant. Parent line

Primary resistance

Cross-resistance

P-glycoprotein

Decreased accumulation/ increased effiux

References

KB

COL(2-128) (6 sublines) COL( > 33) (3 sublines) COL(R) COL(3-300) (9 sublines) COL(170-1200) (10 sublines) COL(16,181) (2 sublines) COL(11) COL(530,1500) (2 sublines)

DOX(1-39), VCR(1-59)

+

+/+

[100,101]

DOX(24- > 33), VCR( > 20)

-

+/NR

[102]

NR DNR(4- > 25), VBL(2-49)

+ +

+/NR +/-

[ 103] [104,105]

NR

+

+/N R

[62,106]

DOX(17,183), VCR(12,43), VP16(19,69) NR DNR(81,NR), VBL( 17,N R)

+

+/NR

[63]

+

+/NR +/N R

[107]

MCF-7 CC531 CHO DM 15 EMT6 J774.2

accumulation were demonstrated in only nine cell lines (CEM [86], CEM [87], HL60 [89], KB [41], 7932 [93], DC-3F [95], EMT6 [63], J774.2 [97], SEWA [98]). Among series of cell lines, Yang et al. [97] have investigated two individually selected sublines of J774.2 which were 1017 and 1083-fold resistant to VBL, respectively. The cell lines had the same transport properties (net accumulation was approx. 10% of the parent line) but a 2-fold difference in P-gp expression. However, the authors isolated two different precursors of P-gp (120 and 125 kDa). The precursors were encoded by the mdrla and mdrlb gene, respectively, and had different numbers of binding sites for VBL.

II-D. Cell lines selected for resistance to COL Cell lines selected for resistance to COL are given in table IV. Eight cell lines have been reported. P-gp was increased in six, which all had decreased drug accumulation (table VI). In two instances a significant decrease in steady-state accumulation was demonstrated (EMT6 [63], J774.2 [107]). Furthermore, the effiux of drug was investigated in two series of cell lines. Fojo et al. [101] in the K B / C O L cell lines found a correlation between drug resistance and increased effiux. However, a correlation was also demonstrated for the initial rate of uptake (determined at 1 rain) and addition of VER increased cytotoxicity of COL without

TABLE V Cross-resistance, P-glycoprotein expression and drug accumulation in cell lines selected for resistance to actinomycin D Cell lines are derived in humans and rodents and given in alphabetic order (see Appendix). Numbers in parentheses refer to fold of resistance compared to the parent line. Abbreviations: NR, not reported, R, resistant. Parent line

Primary resistance

Cross-resistance

P-glycoprotein

Decreased accumulation/ increased effiux

References

CHO

ACTD(36)

+

+/NR

[108]

DC-3F

ACTD(37670 000) (4 sublines) ACTD( > 1200) ACTD(R)

DNR(32), DOX(30) VCR(29), VP 16(24) DNR(29-2240), VCR(189-1400), COL(180-410) NR DOX(R), VCR(R), VP16(R) VCR(307), COL(989) VCR(17- f 19)

+

+/NR

[109,110]

+ +

+/NR +/NR

[62] [ 111,112]

+ +

+/NR +/NR

[110] [98,99]

+

+/NR

[113]

DM15 P388 QUA SEWA WEHI

ACTD(9355) ACTD(5 - 115 ) (4 sublines) ACTD(R)

DOX(R), VCR(R)

176 any apparent effect on drug effiux. In C H O / C O L cells, uptake of COL was found to remain linear over the first 2 h [105] and no effiux of drug could be demonstrated. A very tight binding of COL to cytoplasmic tubulin could explain the result. In three cases, a correlation between P-gp and transport properties could not be demonstrated. Lothstein et al. [107] generated three resistant sublines of the mouse macrophage-like cell line J774.2. Two sublines were 530 and 1500-fold resistant to COL, respectively, and one revertant was l 1-fold resistant. The revertant lacked P-gp as determined by SDS-PAGE and staining with silver (this could be due to the detection limit of the assay), whereas the expression of P-gp increased gradually with resistance in the other sublines. The steady-state association of COL, however, was nearly identical in the three sublines (26, 32 and 26% of the parent line, respectively). Furthermore, Twentyman et al. [63] have developed two sublines of a mouse mammary carcinosarcoma cell line (EMT6) which were 16- and 181-fold resistant to COL, respectively. The steady-state accumulation of D N R was reduced in both cell lines to 16% of the parent line. However, the 181-fold resistant cell line expressed a considerably higher degree of P-gp (more than 2-fold as determined by intensity of autoradiography of bands after Western blot). Curt et ai. [102] reported the development of three resistant sublines of MCF-7 in which P-gp was not enhanced (examined with SDSP A G E of [14C]glucosamine labelled cell lysates). The tubulin content and affinity for COL were unchanged in the resistant cells. The resistant sublines were all defective in drug uptake. The behavior of P-gp, however, in gel electrophoresis has been found to be significantly affected by solution procedures and type of electrophoresis; thus, the absence of a 170 kDa band in gel electrophoresis ought not to be regarded as a proof of absence of P-gp overexpression [80].

TABLE

VI

Relationship between P-glycoprotein expression and drug accumulation NR, not reported, Drug

-

protein

P-glyco-

Daunorubicin Total number

7

Decreased accumulation Increased efflux

+

7

-

0

+

2

-

0

NR

5

I)

-

Doxorubicin Total number

43 *

Decreased accumulation Increased efflux

18 *

+

42

9

-

1

9

+

15

6

-

4

1

24

11

14

0

NR

Vinca a l k a l o i d s Total number Decreased accumulation Increased efflux

+

14

-

0

+

3

-

-

0

-

NR

11

-

6

2

+

6

2

-

0

+

1

-

1

NR

4

7

0

+

7

-

-

0

--

+

-

Colchicine Total number Decreased accumulation Increased efflux

2

Actinomycin D Total number Decreased accumulation Increased efflux

.

.

.

NR All r e v i e w e d cell

II-E. Cell lines selected for resistance to actinomycin D Table V shows cell lines selected for resistance to ACTD. Seven pairs of cell lines have been reported. All overexpressed P-gp and had decreased drug accumulation (Table VI). In two cell lines, a significant reduction in steady-state accumulation has been reported (SEWA [98], W E H I [113]).

+ P-glycoprotein

Decreased MDR

of

compounds

Increased efflux

77 *

20 *

+

76

11

-

1

9

NR

-

+

21

6

-

5

0

51

14

NR * SW-1573,

--

lines

Total number accumulation

.

7

5-10-fold

resistant

to

DOX

[55,561

omitted

due

to

conflicting results.

IlL Summary of results and discussion

IliA. Overexpression of P-gp and decreased accumulation / increased efflux Among 77 cell lines with increased expression of P-gp, 76 cell lines accumulated less drug than their sensitive counterparts. Supporting the hypothesis of P-gp as multidrug transporter, enhanced effiux or significantly reduced steady-state accumulation was

demonstrated in 46 cell lines. Furthermore, if inhibitors of energy production such as cyanide, sodium azide, or dinitrophenol were added to the incubation medium in absence of glucose, the accumulation was increased. In addition, Lankelma et al. [21], in a flow-

177 through system, demonstrated effiux of drug against a concentration gradient.

III-B. Ouerexpression of P-gp without increased efflux In five cases P-gp expression was not followed by increased effiux. In four instances, the authors found a decreased net accumulation, but were unable to demonstrate any drug effiux (L1210/DOX [67], P388/DOX [75], P388/DOX [78], C H O / C O L [1051). Among these, the findings of Carlsen et al. [105] could be explained by tight binding of COL to tubulin. Furthermo/'e, quenching could confound the results of Ramu et al. [78]. In addition, Toffoli et al. [26] were unable to detect any significant differences in either steady-state accumulation or efflux of DOX in five SW948 cell lines 3-5-fold resistant to DOX (Table II). The authors stressed that the results could be due to the detection limit of the experimental procedure being too high to detect variations in transport. However, the results are supported by the findings of Mickley et al. [114] and Bates et al. [115], who investigated the effects of a variety of differentiating agents on human colon carcinoma and neuroblastoma cell lines, respectively. In a HCTll5 cell line, addition of butyrate induced a 5-fold increase in expression of mdrl and P-gp followed by a considerable fall in drug accumulation. In a SW620 cell line addition of butyrate resulted in increased expression of mdrl, mdrl mRNA, and P-gp, but the transport of VBL was not significantly changed and no cross resistance to VBL or DOX was found. However, recently the authors demonstrated increased efflux of COL in the SW620 cell line and found that butyrate in both cell lines modulated P-gp by phosphorylation, which could explain the different specificity of P-gp in the two cell lines [116]. However, addition of retinoic acid to the neuroblastoma cell line SK-N-SH resulted in increased expression of P-gp without any significant changes in drug accumulation. The level of P-gp expression in the above-mentioned cell lines was comparable to levels associated with easily detectable decreases in drug accumulation in cell lines selected in vitro for resistance. In addition, Volm et al. [117] have shown that the P-gp expression in a L1210 cell line increased within a few hours after treatment with DOX. In spite of a high P-gp expression, the authors detected only a slight increase in tumor cell resistance.

III-C. No expression of P-gp, no accumulation defects The resistance of 20 cell lines without P-gp varied from 3-2045 fold, as compared to their parent lines, suggesting that other mechanisms (i.e., o.ttalitative or quantitative changes in topoisomerase II) were responsible for resistance, nine cell lines were not defective in drug accumulation.

III-D. Decreased accumulation/increased efflux without P-gp ouerexpression Decreased accumulation of one or more of the classical MDR drugs was demonstrated in 11 cell lines (GLC4/DOX [27], C O R - L / D O X [24], HL60/DOX [32], H L 6 0 / D O X [34], H T 1 0 8 0 / D O X [36], L o V o / D O X [44], S W 1 5 7 3 / D O X [56], M E L PC4/DOX, MEL C 7 D / D O X [71], MCF-7/COL [102], J774.2/COL [107]). Given the variability in methodology and amounts of different drugs utilized for determination of transmembrane transport, the comparison of data from different laboratories should be performed with caution. However, comparing the steadystate level of intracellular drug in resistant ceils with and without expression of P-gp, it is obvious that cell lines without P-gp expression accumulated more drug. Thus, in resistant cell lines expressing P-gp, the intracellular drug concentration at steady-state was most often reduced to 20% or less compared to the parent lines, whereas most of the cell lines not overexpressing P-gp accumulated approx. 50% compared to their sensitive counterparts. However, in a J774.2 cell line l 1fold resistant to COL, the steady-state association of COL was only 26% of that in the sensitive cell line [107] (Table IV). In six pairs of cell lines without overexpression of P-gp, increased drug effiux has been demonstrated (A2780/DOX [22], C O R - L / D O X [25], HL60/DOX [32], H L 6 0 / D O X [34], HT1080/DOX [36], SKM E L / D O X [53]. Among these, the A2780/DOX subline described by Mazzoni et al. [22] retained 90% of drug compared to the sensitive counterpart. The efflux pattern in the HL60/DOX cell line reported by Marsh et al. [34] did not resemble the efflux of typical MDR cell lines, as drug effiux occurred only after prolonged incubation time (> 60 rain). The absence of P-gp overexpression in the SKMEL/DOX cell line reported by Panneerselwam et al. [53] could be due to the assay procedure (gel electrophoresis without application of monoclonal antibody). Hindenburg et al. [118] found that the decreased accumulation and increased effiux (which could be blocked by incubation in sodium azide and deoxyglucose) in a HL60/DOX could be explained by changes in intracellular drug distribution. Although the mechanisms in the above-mentioned studies could be explained by assay conditions or findings other than membrane changes, they did not exclude that membrane-associated mechanisms distinct from P-gp could contribute to or account for effiux in some cell lines. Furthermore, Coley et al. [25] demonstrated energy-dependent effiux in a lung carcinoma cell line selected for resistance to DOX. Slovak et al. [36], in a HT1080/DOX cell line, found a 2-fold decrease in net accumulation and a 50% decrease in drug retention after 1 h (same retention as a LoVo/DOX subline expressing P-gp). The hypothesis is further

178 supported by observations from cell lines selected for resistance to the anthracene derivate MIT. In an MIT resistant human colon carcinoma cell line (WiDr), in which P-gp was not detected by immunoblot, Dalton et al. [119] found decreased accumulation ( W i D r / M I T retained 49% DOX and 81% MIT compared to WiDr) and enhanced effiux. Taylor et al. [50] described a subline of MCF-7 which was 1208-fold resistant to MIT and did not overexpress P-gp, but accumulated significantly less drug than the sensitive counterpart due to increased effiux. Furthermore, Taylor et al. [49] (preliminary results) generated a subline of MCF-7 which was 531-fold resistant to MIT and had decreased drug accumulation without any detectable P-gp (method not reported). Nakagawa et al. [120] described a subline of MCF-7 which was 3000-fold resistant to MIT and had enhanced effiux but did not overexpress P-gp.

III-E. Quantification of P-gp and transport properties in series of cell lines Comparing one resistant cell line with its sensitive counterpart can yield important information about cellular changes accompanying development of resistance, However, only by comparing sublines with different degrees of resistance one can determine whether any property is invariably associated with resistance (and other cellular changes) in a predictive and quantitative way. A correlation between P-gp overexpression, decreased net accumulation and resistance level has been demonstrated in several of the series of cell lines mentioned above. However, in five of the series, the degree of P-gp expression did not correlate with decreases in intracellular drug concentration ( K 5 6 2 / D O X [38], S 1 8 0 / D O X [79], J774.2/VBL [96], E M T 6 / C O L [63], J 7 7 4 . 2 / C O L [107]). Furthermore, Slapak et al. [71], in two series of erythroleukemia cell lines resistant to DOX, found that changes in transport emerged before overexpression of P-gp could be detected. Baas et al. [56], in a series of S W 1 5 7 3 / D O X cell lines, found similar results using the RNase protection assay for d e t e r m i n a t i o n of P-gp. However, the S W 1 5 7 3 / D O X cell lines were also investigated by Keizer et al. [55] who determined P-gp immunohistochemically and found correlation between P-gp expression and transport changes. In conclusion, in several of the cell lines selected for resistance to the classical M D R compounds, overexpression of P-gp is followed by decreased steady-state accumulation of drug/increased effiux. However, the condition given in Fig. 1A is far from fulfilled. Several exceptions can be pointed out, and Fig. 1B illustrates the experimental data. Furthermore, in only nine of 20 cell lines absence of P-gp is accompanied by minimal or insignificant changes in accumulation. In addition, in series of cell lines in which quantification of P-gp

and accumulation defects have been attempted the correlation is often complicated. Several explanations could account for these discrepancies. Most important, the only relevant relationship is between the number of P-gp molecules on the cell surface and the transport properties measured. Most studies measured total P-gp or total P-gp mRNA. Measuring total protein or RNA is not completely satisfactory, as it has been shown that some of the protein could be inside the cell [121] and, thcreforc. was not functional. Furthermore, Choi ct al. [122] have shown that mutations in the mdrl gene affect thc spectrum of drugs transported by P-gp. Recently, Yang et al. [97], in J774.2 cells selected for resistance to VBL, have demonstrated the existence of 2 mdrl genes encoding for different P-gp molecules which possess different affinities for metabolitcs. Furthermore, co-amplified genes adjacent to the mdr 1 gene or cellular constituents present in the wild-type cell may modulate the phenotype. Transcipts from the human mdr3 gene have been shown to undergo differential splicing [123]; although it is not known whether the different transcripts each encode for a functional P-gp molecule, splicing could account for some of the complexity. Furthermore, the function of P-gp could be modulated either by phosphorylation due to protcin kinase C activation [124-126] or by glycosylation [127,128]. In addition, Bates et al. [116] have shown that differentiating agents (sodium butyratc) arc able to modulate P-gp by phosphorylation. Circumstances concerning the methods could also account for some of the discrepancies. Still, several investigators coming from different laboratories have generated cell lines in which a connection between P-gp expression and accumulation defects does not exist. Thus, the proposal of P-gp as multidrug transporter is an attractive hypothesis. However, the suggestion is - although not excluded contradicted by findings in several MDR cell lines. Furthermore, comparing P-gp to bacterial transport proteins the hypothesis has several weaknesses. Bacterial carrier molecules are substrate specific, transport the solutes in an osmotic-, energy- and temperaturedependent, saturable manner, have a characteristic binding constant, and can be blocked by competitive and non-competitive inhibitors. P-gp appears to recognize a wide range of different lipophilic cations. Purified P-gp has been shown to possess ATPase activity. However, Hamada et al. [129] have reported the activity to be quite low (1.2 nmol A T P / m g per rain). whereas Shimabuku et al. [130] found the activity to be 80-400 nmol A T P / m g per rain. Furthermore, results concerning osmotic dependence in MDR membrane vesicles are conflicting. Thus, Horio et al. [131] found osmotic dependence of VBL accumulation in MDR membrane vesicles, whereas Cornwell et al. [132] and Sehested et al. [133] were unable to demonstrate os-

179 motic dependency of vinca alkaloid binding to vesicles of K B / C O L or E H R / D N R ceils, respectively. The best evidence of the function of P-gp has been provided by transfection studies. Thus, Choi et al. [134] have infected NIH 3T3 cells with a recombinant retrovirus carrying the human m d r l gene and selected ceils with increased P-gp expression by immunofluorescence labelling and flow sorting. The results of this study indicated that P-gp density in the plasma membrane could be sufficient to determine the level of MDR. However, in general the transfected cells were selected in medium containing drug, i.e., 10 n M / m l VCR [135] or 0.06/zg/ml COL [1]. Furthermore, although Lincke et al. [135] have generated transfected cells with relatively high resistance (100-fold), resistance levels were in general low (approx. 10-fold to selecting agent) and the relative resistance to different drugs varied in different clones [1]. In addition, the only study concerning transport in transfected cells [136] has failed to demonstrate competetive inhibition by VCR or anthracyclines on VBL-efflux. Furthermore, Belli et al. [137] have fused P-gp isolated from DOX resistant cells with sensitive cells. Incorporation of P-gp in sensitive cells resulted in a significant increase in resistance (90-fold), and the fused cells effluxed DOX more rapidly within the first 5 rain of observation. However, uptake (steady-state accumulation at 60 min in 0.3 /xg/ml DOX) was unchanged and blocking of the efflux by inhibitors of energy production was not attempted. Thus, no transfection study has yet provided data which undeniably indicate that P-gp functions as a drug transporter. However, if active efflux is not the mechanism of MDR, what then is the mechanism? Furthermore, if P-gp does not function as a multidrug transporter, then what is the function of the molecule? Two other models have been proposed for the decreased accumulation in MDR cells. Based on results from transport studies in C H O / C O L cell lines, Ling et al. [7] have proposed a model in which an energy-dependent permeability barrier, operating with greater efficiency in resistant cells, controls drug entry into cells. The finding of decreased influx in seven ( E H R / D N R [14], MOLT4/DOX [34], P388/DOX [72], P388/DOX [78], DC-3F/VCR [95], K B / C O L [101], C H O / C O L [105]) of eight investigated MDR cell lines could support this model. Furthermore, several studies have demonstrated that the influx could be enhanced by addition of inhibitors of energy production [14,72]. In contrast, Yang et al. [44] in a preliminary report, described a LoVo cell line 90-fold resistant to DOX with enhanced efflux, but no significant changes in K m or Vm,Xof influx (method not reported). A 'membrane trafficking' model for resistance has been introduced by Beck [138]. According to this hypothesis, the drugs are expelled via an exocytotic process which includes binding to P-gp. Supporting this

model, several authors have found significant increases of the endosomal volume and plasma membrane traffic, and VER has been shown to inhibit plasma membrane traffic in resistant cells [139,140]. However, generalized alterations could also account for/contribute to resistance. Thus, there is no doubt that P-gp plays a significant role in the development of resistance, and it is likely that insertion of large quantities of P-gp in plasma membranes has profound effects on the packing and structural order of the membrane. Furthermore, various changes in membrane lipids, structural order, and fluidity have been described. However, no consistent pattern has emerged [141]. Knowledge about MDR and the function of P-gp could be provided from studies of the biochemical basis for MDR. Several authors have focused on the regulation of the intracellular pH and membrane potential. The intracellular pH in resistant cells was higher than in sensitive cells (0.1-0.44 units), and in a series of cell lines with different degrees of resistance pH increased with resistance [142-144]. Furthermore, a lower membrane potential has been documented in resistant ceils [145]. Studies of anthracycline resistance have revealed a significant correlation between pH/surface charge and resistance [146,147]. These findings have led Thiebaut et al. [144] to suggest that P-gp is indirectly a proton pump and that cells may contain an endogeneous substrate(s) for the transporter. In contrast, Gosland et al. [148] suggested that the physiological role of P-gp was transport of bilirubin, as this compound enhanced drug accumulation in MDR cells and inhibited ATP-dependent drug binding in MDR plasma membrane vesicles.

IV. Summary MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However,

180 scveral exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a singlc agent have becn reported. In many of these, a correlation between P-gp expression and transport propertics can not be established. Co-amplification of genes adjacent to the m d r l gene, mutations [122], splicing of mdr I RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased effiux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (11 An energydependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efl~lux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.

SW1573 Widr 7932 8226 Rodent cell lines CC531

CHO DC-3F DM15 EHR2 EMT6 F4-6 HCT HT1 J774.2 L1210 M109 MEL (PC4/C7D) P388 QUA SEWA S180 V79 WEHI 164 3LL

squamous lung carcinoma colon carcinoma malignant melanoma myeloma

colon adenocarcinoma, chemically induced Chinese hamster ovary Syrian hamster, lung Djungarian hamster, SV40 transformed fibroblast murine, Ehrlich ascites tumor murine, mammary carcinosarcoma murine, Friend leukaemia murine, hepatocytes, SV4~~ transformed Chinese hamster, lung murine, macrophage-like murine, leukaemia murine, lung carcinoma murine, murine, murine, induced murine, murine, Chinese murine, murine,

erythroleukaemia leukaemia methylcholanthreneSEWA ascites tumor sarcoma hamster, lung fibroblast fibrosarcoma Lewis lung carcinoma

References Appendix Human cell lines" A1847 A2780 CEM COR-L DLD 1 GLC4 GM3639 H69 HL6(I HT1080 K562 KB LoVo LS 180 MCF-7 MES-SA MOLT-4 SK-MEL- 170 SW620 SW948

ovarian carcinoma ovarian carcinoma T-cell leukaemia small cell lung carcinoma colon carcinoma small cell lung carcinoma lymphatic leukaemia lymphatic leukaemia promyelocytic leukaemia fibrosarcoma promyelocytic leukaemia squamous cell carcinoma colon carcinoma colon carcinoma breast carcinoma uterine sarcoma T-cell leukaemia malignant melanoma colon carcinoma colon carcinoma

1 Ueda, K., Cardarelli, C., Gottesman, M.M. and Pastan, 1. (1987) Proc. Natl. Acad. Sci. USA 84, 3004-3008. 2 Dams, K. (1973) Biochim. Biophys. Acta 323, 466-483. 3 Hyde, S.C., Emsley, P., Hartshorn, M.J., Mimmack, M.M., Gileadi, U., Pearce, S.R., Gallagher, M.P., Gill, D.R.. Hubbard, R.E. and Higgins, C.F. (19901 Nature 346, 362-365. 4 Cornwell, M.M., Gottesman, M.M. and Pastan, I.H.J. Biol. Chem. 26l, 7921-7928. 5 Naito, M., Hamada, H. and Tsuruo, T. (19881 J. Biol. (;hem. 263, 11887- 1189 I. 6 Chen, C.-J., Chin, J.E., Ueda, K., Clark, D.P., Pastan, I., Gottesman, M.M. and Roninson, I.B. (1986) Cell 47, 381-389. 7 Bradley, G., Juranka, P.F. and Ling, V. (19881 Biochim. Biophys. Acta 948, 87-128. 8 Endicott, J.A. and Ling, V. (1989) Annu. Rev. Biochem. 58. 137-171. 9 Van der Bliek, A.M. and Borst, P. (19891 Adv. Cancer Res. 52, 165-203. 10 Yanovich, S., Hall, R.E. and Gewirtz, D.A. (19891 Cancel" Res. 49, 4499-4503. 11 Kartner, N., Shales, M., Riordan, J.R. and Ling, V. (1983) Cancer Res. 43, 4413-4419. 12 Riehm, H. and Biedler, J.L. (19711 Cancer Res. 31,409-412. 13 Biedler, J.L., Chang, T.-D., Meyers, M.B., Peterson, R.H.F. and Spengler, B.A. (1983) Cancer Treat. Rep. 67, 859-867. 14 Skovsgaard, T. (19781 Cancer Res. 38. 4722-472"/.

181 15 Sehested, M., Skovsgaard, T., Jensen, P.B., Demant, E.J.F., Friche, E. and Bindslev, N. (1990) Br. J. Cancer 62, 37-41. 16 Volm, M., Bak, M., Jr., Efferth, T. and Mattern, J. (1989) J. Cancer Res. Clin. Oncol. 115, 17-24. 17 Nooter, K., Oostrum, R., Jonker, R., Van Dekken, H., Stokdijk, W. and Van den Engh, G. (1989) Cancer Chemother. Pharmacol. 23, 296-300. 18 Volta, M., Bak, M., Jr., Efferth, T. and Mattern, J. (1988) Anticancer Res. 8, 1169-1178. 19 Rogan, A.M., Hamilton, T.C., Young, R.C., Klecker, R.W., Jr. and Ozols, R.F. (1984) Science 224, 994-996. 20 Van der Bliek, A.M., Baas, F., Van der Velde-Koerts, T., Biedler, J.L., Meyers, M.B., Ozols, R.F., Hamilton, T.C., Joenje, H. and Borst, P. (1988) Cancer Res. 48, 5927-5932. 21 Lankelma, J., Spoelstra, E.C., Dekker, H. and Broxterman, H.J. (1990) Biochim. Biophys. Acta 1055, 217-222. 22 Mazzoni, A., Trave, F., Russo, P., Nicolin, A. and Rustum, Y.M. (1990) Oncology 47, 488-494. 23 McGrath, T., Marquardt, D. and Center, M.S. (1989) Biochem. Pharmacol. 38, 497-501. 24 Reeve, J.G., Rabbitts, P.H. and Twentyman, P.R. (1990) Br. J. Cancer 61,851-855. 25 Coley, H.M., Workman, P. and Twentyman, P.R. (1991) Br. J. Cancer 63, 351-357. 26 Toffoli, G., Viel, A., Tumiotto, L., Biscontin, G., Rossi, C. and Boiocchi, M. (1991) Br. J. Cancer 63, 51-56. 27 Zijlstra, J.G., De Vries, E.G.E. and Mulder, N.H. (1987) Cancer Res. 47, 1780-1784. 28 De Jong, S., Zijlstra, J.G., De Vries, E.G.E. and Mulder, N.H. (1989) Proc. Am. Assoc. Cancer Res. 30, 526. 29 Twentyman, P.R., Fox, N.E., Wright, K.A. and Bleehen, N.M. (1986) Br. J. Cancer 53, 529-537. 30 Reeve, J.G., Rabbitts, P.H. and Twentyman, P.R. (1989) Br. J. Cancer 60, 339-342. 31 Cole, S.P.C., Chanda, E.R., Dicke, F.P., Gerlach, J.H. and Mirski, S.E.L. (1991) Cancer Res. 51, 3345-3352. 32 Bhalla, K., Hindenburg, A., Taub, R.N. and Grant, S. (1985) Cancer Res. 45, 3657-3662. 33 Cass, C.E., Janowska-Wieczorek, A., Lynch, M.A., Sheinin, H., Hindenburg, A.A. and Beck, W.T. (1989) Cancer Res. 49, 57985804. 34 Marsh, W., Sicheril D. and Center, M.S. (1986) Cancer Res. 46, 4053-4057. 35 Marquardt, D., McCrone, S. and Center, M.S. (1990) Cancer Res. 50, 1426-1430. 36 Slovak, M.L., Hoeltge, G.A., Dalton, W.S. and Trent, J.M. (1988) Cancer Res. 48, 2793-2797. 37 Slovak, M.L., Coccia, M., Melzter, P.S. and Trent, J.M. (1991) Anticancer Res. 11,423-428. 38 Kato, S., Ideguchi, H., Muta, K., Nishimura, J. and Nawata, H. (1990) Leukemia Res. 14, 567-573. 39 Tsuruo, T., Iida-Saito, H., Kawabata, H., Oh-Hara, T., Hamada, H. and Utakoji, T. (1986) Jpn. J. Cancer Res. 77, 682-692. 40 Shen, D.-W., Cardarelli, C., Hwang, J., Cornwell, M., Richert, N., Ishii, S., Pastan, I. and Gottesman, M.M. (1986) J. Biol. Chem. 261, 7762-7770, 41 Willingham, M.C., Richert, N.D., Cornwell, M.M., Tsuruo, T., Hamada, H., Gottesman, M.M. and Pastan, I.H. (1987) J. Histochem. Cytochem. 35, 1451-1456. 42 Broggini, M., Grandi, M., Ubezio, P., Geroni, C., Giuliani, F.C. and D'Incalci, M. (1988) Biochem. Pharmacol. 37, 4423-4431. 43 Beck, W.T. and Danks, M.K. (1991) in Molecular and cellular biology of multidrug resistance in tumor cells (Roninson, I.B., ed.), pp. 3-55, Plenum Press, New York.

44 Yang, L.Y. and Trujillo, J.M. (1989) Proc. Am. Assoc. Cancer Res. 30, 516. 45 Herzog, Z.E., Tsokos, M., Fojo, A.T. and Bates, S.E. (199l) Proc. Am. Assoc. Cancer Res. 32, 368. 46 Fairchild, C.R., Ivy, S.P., Kao-Shan, C.-S., Whang-Peng, J., Rosen, N., Israel, M.A., Melera, P.W., Cowan. K.H. and Goldsmith, M.E. (1987) Cancer Res. 47, 5141-5148. 47 Kramer, R.A., Zakher, J. and Kim, G. (1988) Science 241, 694-697. 48 Chen, Y.-N., Mickley, L.A., Schwartz, A.M., Acton, E.M., Hwang, J. and Fojo, A.T. (1990)J. Biol. Chem. 265, 10073-10080. 49 Taylor, C.W. and Dalton, W.S. (1989) Proc. Am. Assoc. Cancer Res. 30, 530. 50 Taylor, C.W., Dalton, W.S., Parrish, P.R., Gleason, M.C., Bellamy, W.T., Thompson, F.H., Roe, D.J. and Trent, J.M. (1991) Br. J. Cancer 63, 923-929. 51 Harker, W.G. and Sikic, B.I. (1985) Cancer Res. 45, 4091-4096. 52 Sikic, B.I., Scudder, S.A. and Evans, T.L. (1989) in Resistance to antineoplastic drugs (Kessel, D., ed.), pp. 37-47, Boca Raton, Florida. 53 Panneerselwam, M., Bredehorst, R. and Vogel, C.-W. (1987) Cancer Res. 47, 4601-4607. 54 Chao, C.C.-K., Ma, C.M., Cheng, P.-W. and Lin-Chao, S. (1990) Biochem. Biophys. Res. Commun. 172, 842-849. 55 Keizer, H.G., Schuurhuis, G.J., Broxterman, H.J., Lankelma, J., Schoonen, W.G.E.J., Van Rijn, J., Pinedo, H.M. and Joenje, H. (1989) Cancer Res. 49, 2988-2993. 56 Baas, F., Jongsma, A.P.M., Broxterman, H.J., Arceci, R.J., Housman, D., Scheffer, G.L., Riethorst, A., Van Groenigen, M., Nieuwint, A.W.M. and Joenje, H. (1990) Cancer Res. 50, 53925398. 57 Kuiper, C.M., Broxterman, H.J., Baas, F., Schuurhuis, G.J., Haisma, H.J., Scheffer, G.L., Lankelma, J. and Pinedo, H.M. (1990) J. Cell Pharmacol. 1, 35-41. 58 Dalton, W.S., Grogan, T.M., Rybski, J.A., Scheper, R.J., Richter, L., Kailey, J., Broxterman, H.J., Pinedo, H.M. and Salmon, S.E. (1989) Blood 73, 747-752. 59 Bellamy, W., Dalton, W., Gleason, M., Meltzer, P. and Spier, C. (1990) Proc. Am. Assoc. Cancer Res. 31,364. 60 Bellamy, W., Gleason, M., Futscher, B. and Dalton, W. (1991) Proc. Am. Assoc. Cancer Res. 32, 367. 61 Chatterjee, M., Robson, C.N. and Harris, A.L. (1990) Cancer Res. 50, 2818-2822. 62 Gudkov, A.V., Chernova, O.B. and Kopnin, B.P. (1991) in Molecular and cellular biology of multidrug resistance in tumor cells (Roninson, I.B., ed.), pp. 147-168, Plenum Press, New York. 63 Twentyman, P.R., Reeve, J.G., Koch, G. and Wright, K.A. (1990) Br. J. Cancer 62, 89-95. 64 Erttmann, R., Gieseler, F., Wilms, K. and Winkler, K. (1990) Proc. Am. Soc. Clin. Oncol. 9, 56. 65 Garman, D. and Center, M.S. (1982) Biochem. Biophys. Res. Commun. 105, 157-163. 66 Huet, S., Schott, B., Londos-Gagliardi, D., Benchekroun, M.N., Montaudon, D. and Robert, J. (1990) Proc. Am. Assoc. Cancer Res. 31,371. 67 Ganapathi, R. and Grabowski, D. (1988) Biochem. Pharmacol. 37, 185-193. 68 Ganapathi, R., Grabowski, D., Ford, J., Heiss, C., Kerrigan, D. and Pommier, Y. (1989) Proc. Am. Assoc. Cancer Res. 30, 529. 69 Kamath, N., Grabowski, D., Ford, J. and Ganapathi, R. (1991) Proc. Am. Assoc. Cancer Res. 32, 353. 70 Keilhauer, G., Traugott, U., Kluge, M., Jung, S. and Schlick, E. (1990) Proc. Am. Assoc. Cancer Res. 31,367.

182 71 Slapak, C.A., Daniel, J.C. and Levy, S.B. (19901 Cancer Res. 511, 7895-7901. 72 Inaba, M. and Sakurai, Y. (1979) Cancer Lett. 8, 111-115. 73 Shanbaky, N.M., Samy, T.S., Rubin, R. and Krishan, A. (1986) Int. J. Pept. Protein Res. 27, 414-420. 74 Goldenberg, G.J., Wang, H. and Blair, G.W. (1986) Cancer Res. 2978-2983. 75 Deffie, A.M., Alam, T., Seneviratne, C., Beenken, S.W., Batra, J.K., Shea, T.C., Henner, W.D. and Goldenberg, G.J. (1988) Cancer Res. 48, 3595-3602. 76 Capranico, G., Dasdia, T. and Zunino, F. (1986) Int. J. Cancer 37, 227-231. 77 Capranico, G., De Isabella, P., Castelli, C., Supino, R., Parmiani, G. and Zunino, F. (1989) Br. J. Cancer 59, 682-685. 78 Ramu, A., Pollard, H.B. and Rosario, L.M. (1989) Int. J. Cancer 44, 539-547. 79 Siegfried, J.M., Tritton, T.R. and Sartorelli, A.C. (1983) Eur. J. Cancer Clin. Oncol. 19, 1133-1141. 80 Gerlach, J.H., Kartner, N., Bell, D.R. and Ling, V. (1986) Cancer Surveys 5, 25-46. 81 Giavazzi, R., Scholar, E. and Hart, I.R. (1983) Cancer Res. 43, 2216-2222. 82 Giavazzi, R., Kartner, N. and Hart, I.R. (1984) Cancer Chemother. Pharmacol. 13, 145-147. 83 Belli, J.A., Zhang, Y. and Fritz, P. (1990) Cancer Res. 50, 2191-2197. 84 Slate, D.L., Bruno, N., Carver, L.A. and Rabier, M, (1989) Proc. Am. Assoc. Cancer Res. 30, 497. 85 Gerlach, J.H. (1989) in Drug resistance in cancer therapy (Ozols, R.F., ed.), pp. 37-53, Kluwer Academic Publishers, Boston. 86 Beck, W.T. (1983) Cancer Sl'reat. Rep. 67, 875-882. 87 Haber, M., Norris, M.D., Kavallaris, M., Bell, D.R., Davey, R.A., White, L. and Stewart B.W. (1989) Cancer Res. 49, 5281-5287. 88 Slater, L.M., Murray, S,L., Wetzel, M.W., Sweet, P. and Stupecky, M. (1986) Cancer Chemother. Pharmacol. 16, 50-54. 89 McGrath, T. and Center, M.S. (1988) Cancer Res. 48, 3959-3963. 90 Kohno, K., Kikuchi, J., Sato, S.-I., Takano, H., Saburi, Y., Asoh, K.-I. and Kuwano, M. (1988) Jpn. J. Cancer Res. 79, 1238-1246. 91 Hill, B.T. and Whelan, R.D.H. (1982) Cancer Chemother. Pharmacol. 8, 163-169. 92 Hosking, L.K., Whelan, R.D.H. and Hill, B.T. (19911 Proc. Am. Assoc. Cancer Res. 32, 363, 93 Lemontt, J.F., Azzaria, M. and Gros, P. (1988) Cancer Res. 48, 6348-6353. 94 Wart, J.R., Brewer, F., Anderson, M. and Fergusson, J. (19861 Cell Biol. Int. Rep. 10, 389-399. 95 Sirotnak, F.M., Yang, C.-H., Mines, L.S., Oribe, E. and Biedler, J.L. (1986) J. Cell. Physiol. 126, 266-274. 96 Greenberger, L.M., Lothstein, L., Williams, S.S. and Horwitz, S.B. (1988) Proc. Natl. Acad. Sci. USA 85, 3762-3766. 97 Yang, C.-P.H., Cohen, D., Greenberger, L.M., Hsu, S.I.-H. and Horwitz, S.B. (1990) J. Biol. Chem. 265, 10282-10288. 98 Martinsson, T., Dahll6f, B., Wettergren, Y., Leffler, H. and Levan, G. (19851 Exp. Cell Res. 158, 382-394. 99 St~.hl, F,, Martinsson, T., Dahll6f, B. and Levan, G. (19881 Hereditas 108, 251-258. 100 Akiyama, S.-I., Fojo, A., Hanover, J.A., Pastan, I. and Gottesman, M.M. (1985) Somatic Cell Mol. Genet. 11, 117-126. 101 Fojo, A., Akiyama, S.-I., Gottesman, M.M. and Pastan, I. (19851 Cancer Res. 45, 3002-3007. 102 Curt, G.A., Bailey, B.D,, Mujagic, H., Bynum, B.S. and Chabner, B.A. (19841 Proc. Am. Assoc. Cancer Res. 25,337. 103 Van der Vrie, W., Gheuens, E.E.O., Bijma, A., De Bruijn, E.A., Marquet, R.L., Van Oosterrom, A.T. and Eggermont, A.M.M. (1991) Proc. Am. Assoc. Cancer Res. 32, 365. 104 Ling, V., Kartner, N., Sudo, T., Siminovitch, L. and Riordan, J.R. (19831 Cancer Treat. Rep. 67, 869-874.

11)5 Carlsen, S.A., Till, J.E. and Ling, V. (19761 Biochim. Biophy,,. Acta 455,911(I-912. 106 Gudkov, A.V., Massino, J.S., Chernova, O.B. and Kopnin, B.t'. (1985) Chromosoma 92. 16 24. 107 Lotbstein, L. and Horwitz. S.B. (19861 J. Cell. Physiol. 127. 253-260. 108 Diddens, H., Gekeler, V., Neumann. M. and Niethammer, D. (1987) Int. J. Cancer 40, 635-642. 109 Biedler, J.L. and Riehm, H. (1970) Cancer Res. 311. 1174-1184. 110 Melera, P.W. and Biedler J.L. (1991) in Molecular and cellular biology of multidrug resistance in tumor cells (Roninson, I.B., ed.), pp. 117-145, Plenum Press, New York. 111 Kay, S.B. and Boden, J.A. (1980) Biochem. Pharmacol. 29, 1081-1084. 112 Baguley, B.C., Holdaway, K,M. and Fray, L.M. (19911) J. Natl. Cancer Inst. 82, 398-402. 113 Hofsli, E. and Nissen-Meyer, J. (19891 Int. J. Cancer 44, 149-154. 114 Mickley, L.A., Bates, S.E., Richert, N.D., Currier, S., Tanaka. S., Foss, F., Rosen, N. and Fojo, A.T. (1989) J. Biol. Chem. 264, 18031 18040. 115 Bates, S.E., Mickley, L.A., Chen, Y.-N., Richert, N., Rudick, J.. Biedler, J.L. and Fojo, A.T. (1989) Mol. Cell Biol. 9, 4337-4344. 116 Bates, S.E., Currier, S.J. and Fojo, A.T. (19911Proc. Am. Assoc. Cancer Res. 32, 364. 117 Volta, M., Mattern, J. and Pommerenke E.W. (199l) Antieancer Res. 11,579-586. 118 Hindenburg, A.A., Gervasoni. J.E., Jr., Krishna, S., Stewart, V.J., Rosado, M., Lutzky, J., Bhalla, K., Baker, M.A. and Taub, R.N. (1989) Cancer Res. 49, 4607-4614. 119 Dalton, W.S., Cress, A.E., Alberts, D.S. and Trent, J.M, (19881 Cancer Res. 48, 1882-1888. 120 Nakagawa, M., Dixon, K.H., Gilbert, L., Goldsmith, M.E. and Cowan, K.H. (19911 Proc. Am. Assoc. Cancer Res. 32, 371. 121 Broxterman, H.J., Pinedo, H.M., Kuiper, C.M., Van der ltoeven, J.J.M., De Lange, P., Quak, J.J., Scheper, R.J., KeizeL H.G., Schuurhuis, G.J. and Lankelma, J. (19891 Int. J. Cancer 43, 340-343. 122 Choi, K., Chen, C.-J., Kriegler, M. and Roninson, I.B. (19881 Cell 53, 519-529. 123 Van der Bliek, A.M., Baas, F., Ten Houte de Lange, T., Kooiman, P.M., Van der Velde-Koerst, T. and Borst, P. (19871 EMBO J. 6, 3325-3331. 124 Posada, J., Vichi, P. and Tritton, T.R. (19891 Cancer Res. 49, 6634-6639. 125 Yu, G., Aquino, A., Fairchild, C.R., Cowan, K.H., Ohno, S. and Glazer, R.I. (1990) Proc. Am. Assoc. Cancer Res. 31,366. 126 Gollapudi, S., Patel, K., Jain, V, and Gupta, S. (19911 Proc, Am. Assoc. Cancer Res. 32, 369. 127 Ichikawa, M., Yoshimura, A., Furukawa, T.. Sumizawa, T., Nakazima, Y. and Akiyama, S.-I. (19911 Biochim. Biophys. Acta 1073, 309-315. 128 Greenberger, L.M., Williams, S.S. and Horwitz, S.B. (1987) J. Biol. Chem. 262, 13685-13689. 129 Hamada, H. and Tsuruo, T. (19881 J. Biol. Chem. 263. 14541458. 130 Shimabuku, A.M., Saeki, T., Ueda, K. and Komano. T. (19911 Agric. Biol. Chem. 55, 1075-1080. 131 Horio, M., Gottesman, M.M. and Pastan, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3580-3584. 132 Cornwell, M.M., Gottesman, M.M. and Pastan. I.H. (19861 J. Biol. Chem. 261, 7921-7928. 133 Sehested, M., Bindslev, N., Demant, E.J.F., Skovsgaard, T. and Jensen, P.B. (19891 Biochem. Pharmacol. 38, 3017-3027. 134 Choi, K., Frommel, T.O., Stern, R.K., Perez C.F., Kriegler, M., Tsuruo, T. and Roninson I.B. (1991) Proc. Natl. Acad. Sci. USA 88, 7386-7390. 135 Lincke, C.R., Van der Bliek, A.M., Schuurhuis, G.J. and Van der Velde-Koerts, T. (1990) Cancer Res. 511, 1779-1785.

183 136 Hammond, J.R., Johnstone, R.M. and Gros, P. (1989) Cancer Res. 49, 3867-3871, 137 Belli, J.A., Zhang, Y. and Fritz, P. (1990) Cancer Res. 50, 2191-2197. 138 Beck W.T. (1987) Biochem. Pharmacol. 36, 2879-2887. 139 Sehested, M., Skovsgaard, T., Van Deurs, B. and Winther-Nielsen, H. (1987) J. Natl. Cancer Inst. 78, 171-179. 140 Hindenburg, A.A., Baker, M.A., Gleyzer, E., Stewart, V.J., Case, N. and Taub, R.N. (1987) Cancer Res. 47, 1421-1425. 141 Alon, N., Busche, R., Tfimmler, B. and Riordan, J.R. (1991) in Molecular and cellular biology of multidrug resistance in tumor cells (Roninson, I.B., ed.) pp. 263-276, Plenum Press, New York. 142 Boscoinik, D., Gupta, R.S. and Epand, R.M. (1990) Br. J. Cancer 61,568-572. 143 Keizer, H.G. and Joenje, H. (1989) J. Natl. Cancer Inst. 81, 706-709.

144 Thiebaut, F., Currier, S.J., Whitaker, J., Haugland, R.P., Gottesman, M.M., Pastan, I. and Willingham, M.C. (1990) J. Histochem. Cytochem. 38, 685-690. 145 Hasmann, M., Valet, G.K., Tapiero, H,, Trevorrow, K. and Lampidis, T. (1989) Biochem. Pharmacol. 38, 305-312. 146 Alabaster, O., Woods, T., Ortiz-Sanchez, V. and Jahangeer, S. (1989) Cancer Res. 49, 5638-5643. 147 Ferragut, J.A., Gonzales-Ros, J.M., Ferrer-Montiel, A.V. and Escriba, P.V. (1988) in Membrane in cancer cells (Galeotti, T., Cittadini, A., Neri, G. and Scarpa, A., eds.), pp. 443-445, The New York Academy of Sciences, New York. 148 Gosland, M.P., Brophy, N.A., Duran, G.E., Yahanda, A.M., Adler, K.M., Hardy, R.I., Halsey, J. and Sikic, B.I. (1991) Proc. Am. Assoc. Cancer Res. 32, 426.