(CANCER RESEARCH 48, 4477^*483, August 15, 1988]

Properties of Verapamil-hypersensitive Multidrug-resistant Chinese HamsterOvary Cells1

J. Roger VVarr,2Michael Anderson, and Jill Fergusson

Department of Biology, University of York, Heslington, York, YO1 5DD, United Kingdom

ABSTRACT

Two vincristine-resistant Chinese hamster ovary cell lines have beenshown previously to be hypersensitive to the calcium channel blocker,verapamil. They are now shown to be hypersensitive to the membrane-active agent quinidine sulfate and to the calcium channel blockers dilti-azem and nicardipine. Hypersensitivity to quinidine sulfate implies thatcalcium channels are not the primary target for these drug effects onthese cell lines and is consistent with our previous observation that theircalcium accumulation is normal in the presence and absence of verapamil.The two cell lines have elevated levels of membrane P-glycoprotein andof two cytosolic proteins, M, 27,000 and pi 6.0 and 6.4. Revertants havenormal levels of these cytosolic proteins, suggesting that these proteinsmay play a role in conferring resistance. [3H)Verapamil accumulation by

the two cell lines is lower than in controls. One of the cell lines has beenhybridized to normal cells and the vincristine resistance and verapamilsensitivity of three hybrid clones has been determined. Vincristine resistance is semidominant but verapamil hypersensitivity is completely re-

INTRODUCTION

Multidrug resistance involves the simultaneous developmentof resistance to Vinca alkaloids, Adriamycin, actinomycin D,and certain other functionally unrelated drugs. The phenomenon has been studied extensively in human and rodent cell lines(1-3). It is usually associated with increased rates of drug effluxand with the presence of elevated levels of an M, 170,000-180,000 membrane glycoprotein, called the P-glycoprotein (2,4). Although MDR3 cell lines share common basic features,

differences do exist between them. Commonly, resistance isgreatest to that drug which was present during the selection ofresistance and the relative resistance to different drugs mayvary between cell lines (2). Some resistant cell lines havechanges in low molecular weight cytosolic proteins which maypossibly play a secondary role in modulating the probablecentral role of the P-glycoprotein in multidrug resistance (5-8).

In many cases, multidrug resistance is partially reversible bythe presence of verapamil or other calcium channel blockers (9,10). Acquired and innate multidrug resistance can both bereversed by calcium channel blockers (11). This phenotypicreversal is due to the abolition of the elevated drug efflux ratesof MDR cells by the presence of these agents. The molecularmechanisms involved are not yet fully understood, but it is ofinterest that verapamil inhibits binding of photoaffinity-labeledVinca alkaloids to the P-glycoprotein (12) and the P-glycoprotein binds various calcium channel blockers (13).

During a study (14) of two CHO MDR cell lines, VCR/Aand VCR/T, we observed a different form of interaction between the cells and verapamil. The two cell lines were 100-fold

Received 12/14/87; revised 4/4/88; accepted 5/2/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from the Yorkshire Cancer Research

Campaign.2To whom requests for reprints should be addressed.3The abbreviations used are: MDR, multidrug resistant; CHO, Chinese ham

ster ovary.

more sensitive to verapamil alone than the cells from whichthey were derived. Reversion of vincristine resistance duringgrowth in vincristine-free medium was correlated with reversionof this verapamil hypersensitivity, indicating that the two aspects of the cell phenotype have a common underlying cause.Cano-Gauci and Riordan (15) have also recently observed thatMDR CHO cells are unusually sensitive to verapamil, andTwentyman et al. (16) have observed verapamil hypersensitivityin a MDR small cell lung cancer cell line.

The phenomenon of verapamil hypersensitivity of MDR linesis of considerable interest inasmuch as the study of an unusualsubset of MDR lines may provide insight into the range ofpossible variations in cellular physiology which may be significant for multidrug resistance in general. We have thereforestudied our MDR-verapamil-hypersensitive cell lines further.Initially, we examined whether these cell lines are hypersensitive to calcium channel blockers other than verapamil. We havepreviously found that calcium accumulation in the presence andabsence of verapamil is normal in these cell lines, suggestingthat calcium channels may not be involved in hypersensitivity(14). With this in mind, we also determined the sensitivity ofour cells to quinidine sulfate, a membrane-active agent whichdoes not have direct effects on calcium channels (17). We haveexamined the rate of accumulation of verapamil by our celllines because this is an obvious factor which could be involvedin verapamil hypersensitivity. In order to explore the biochemical basis of the mutant phenotype we have compared thecytosolic and membrane proteins of resistant cells to those ofcontrols. We have also hybridized the vincristine-resistant-verapamil-hypersensitive cells to normal cells. The study of thephenotype of such hybrids should reveal the consequences ofdiluting the abnormal gene products with their normal counterparts. Such information can then be used as one considerationin speculation on molecular mechanisms involved in the drug-resistant verapamil-hypersensitive phenotype.

MATERIALS AND METHODS

Cell Culture and Cell Lines. Culture conditions and media and theorigin of the two CHO cell lines VCR/A and VCR/T and theirrevenants has been described previously (14). AuxBi, E29, and themultidrug-resistant cell line CHRC5 (18) were kindly provided by Dr.V. Ling of the Ontario Cancer Institute.

VCR/A, VCR/T, and E29 (the cell line from which they werederived) have a nutritional requirement for adenosine. AuxBi andCHRC5 have a triple requirement for adenosine, glycine, and thymi-dine. Medium was therefore supplemented with 10 n%/m\ of each ofthese where necessary.

Viability curves in drugs were determined by plating around 200cells/well in quadruplicate wells of 24 multiwell dishes (Nunc). Themull ¡wellswere stained after 8 days and colonies of over SOcells werecounted. The />,nvalues quoted are the drug concentration at which thecolony-forming efficiency is 10% ofthat observed in drug free medium.

Cell Hybridization. Cells of each of the two cell lines (10'') being

hybridized were plated together in 60-mm plates and incubated for 24h in nonselective medium. Each plate was then treated with 50% (w/v)polyethylene glycol 1000 for 1.5 min before being rapidly washed threetimes with 2.5 ml of medium lacking fetal calf serum and then incubated

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for 24 h in 5 ml of nonsclective medium. Hybrid cells were then selectedby plating 5 x IO4 cells/ 100-mm plate into selective medium lacking

adcnosine, glycine, and thymidine and in which fetal calf serum hadbeen replaced by dialyzed fetal calf serum. Slight background growthof AuxBi was observed in this medium; therefore 0.37 ftM vincristinewas also added during VCR/T x \u\H, hybridizations to select againstAuxBi. Hybrid clones were isolated from separated colonies after 10 to14 days using cloning rings.

Detection of P-Glycoprotein. Cells of each of the cell lines (3-4 x10") were grown as a monolayer using 22- x 22-cm plates, rinsed twicewith Dulbecco's phosphate-buffered saline, harvested with a plastic

scraper, resuspended in the saline, and disrupted in a Polytron PT IO/35 cell disruptor (Kinematica. Lucerne, Switzerland) on setting 6 for 2min and 30 s. Preparation of the "16/31" membrane fraction from a

discontinuing sucrose gradient was then performed according to themethod of Riordan and Ling (19) and 100 ^g of protein of eachpreparation were run on a 7% acrylamide gel.

Proteins were blotted for 16 h at 60 V onto nitrocellulose and theblots were blocked with 3% bovine serum albumin and 1% rabbit serumin phosphate-buffered saline containing 0.3% Tween. The P-glycopro-tcin was visualized in the Western blots using the mouse monoclonalantibody C219, which is directed against the cytoplasmic domain ofthe P-glycoprotein (20) which was kindly provided by Drs. Ling andEvernden-Porelle of the Ontario Cancer Institute. Antibody (2.5 ng/ml) was added to the blot for 2 h. After conjugation with anti-IgGhorseradish peroxidase conjugate for 90 min, the bands were visualizedusing 100 ng/m\ diaminobenzidine and 0.0075% hydrogen peroxide.

Two Dimensional Gel Electrophoresis. Cells (3.5 x 105/well) wereplated in 96-well plates and preincubated for 16 h prior to a 6-hincubation in 50 ¿ilof methionine-free medium supplemented with 10¿iCi("Sjmethionine per well. The cells from each well were lysed in 50/il O'Farrell lysis buffer (21) with 20% sodium dodecyl sulfate. Gel

electrophoresis was according to the method of Laemmli (22) andO'Farrell (21) using ampholine mixtures of either 3 parts pH 5-7, 1

part pH 4-6, and 1 part pH 3.5-10 (Fig. 7) or 3 parts pH 5-7, 1 partpH 6-8, and 1 part pH 3.5-10 (Fig. 8). For each sample 7 x 10" cpm

were loaded into tubes for isoelectric focusing for 15 h at 400 V,followed by focusing for l h at 800 V. Following electrophoresis in thesecond dimension in 12% acrylamide, the gels were fluorographed withPPO before being in contact with Kodak X-Omat S 1596 film for 15

days.Verapamil Accumulation. Cells were plated in 24-well multiwells

(Nunc) 3 days prior to the addition of verapamil. Plating was at 4 xIO4cells/well for E29 and at 8 x IO4cells/well for VCR/A and VCR/

T to allow for the slower growth rate of the resistant cells. At timezero, 1 ¿iMverapamil (specific activity, 300 ^Ci/Mmol) was added inprcgassed medium and was aspirated off at stated time points. Rapidwashing of individual wells with three changes of 1 ml of Tris-bufferedsaline was rapidly followed by submerging each 24-multiwell in threechanges of 1.5 liters of Tris-buffered saline. Cells were extracted fromeach well with 0.5 ml of 0.3 N NaOH for 1 h, neutralized with 0.5 mlof 0.3 N HC1, and transferred to scintillation vials for counting alongwith two washes of 0.5 ml of Tris-buffered saline.

RESULTS

Sensitivity to Membrane-active Agents. We have shown previously that the vincristine-resistant CHO cell lines VCR/Aand VCR/T are unusually sensitive to verapamil (14). In orderto assess whether this was a specific response to verapamil oran example of a less specific response, we examined the resistance levels of these cell lines to the calcium channel blockersdiltiazem and nicardipine and to the membrane-active agentquinidine sulfate. The results are shown in Figs. 1-3 andsummarized in Table 1.

Our data indicate that the vincristine-resistant cell lines arehypersensitive to all of these agents. In the cases of diltiazemand nicardipine, the cell lines show considerable hypersensitiv-ity, although a small percentage of cells of both cell lines are

100-

30-

£ 10-

S 3Hs

0310 20

Diltiazem30 40

Cone (/uM)SO

Fig. 1. Colony-forming ability of E29, VCR/A, and VCR/T in diltiazem.Standard errors (him) are shown where they exceed 10% of the plotted values.

2 4Nicardipine Cone (/uM>

Fig. 2. Colony-forming ability of E29, VCR/A, and VCR/T in nicardipine.Standard errors (ears) are shown where they exceed 10% of the plotted value.

too .-^E29

Fig. 3. Colony-forming ability of E29, VCR/A, and VCR/T in quinidinesulfate. Standard errors (him) are shown where they exceed 10% of the plottedvalue.

able to form colonies in intermediate concentrations of thesedrugs (Figs. 1 and 2). These survival curves resemble the bi-phasic curves previously observed when these cell lines aregrown in verapamil (14).

The two cell lines are also hypersensitive to quinidine sulfatebut in this case there is an exponential kill rather than a biphasicsurvival curve (Fig. 3) and the overall reduction in resistancelevels of the two cell lines in quinidine sulfate is much smallerthan to the other drugs. The Dto values for VCR/A and VCR/T in quinidine sulfate were 84 and 92 ¿¿M,respectively, compared to a DIOvalue of 133 pM for control cells. In verapamil

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Table 1 Drug sensitivity of VCR/A and VCR/TDrug sensitivity is expressed as /;,„values which are defined as described in

"Materials and Methods." Numbers in parentheses are the ratio of the DIOvalue

for VCR/A or VCR/T compared to the corresponding value for the control cellline, E29. Data on colchicine and Adriamycin cross-resistance (14) are includedfor comparison.

a b c

DrugVincristine

ColchicineAdriamycinVerapamilDiltiazemNicardipineQuinidine sulfateE290.03

0.170.15

469516.6

133O,o

(fiM)VCR/A1.65(62)

4.02 (24)0.69 (4.6)0.37 (0.008)4.8 (0.05)0.79 (0.05)

84 (0.63)VCR/T1.75(66)

6.50 (38)2.93(19.5)0.44 (0.009)

17.0 (0.18)1.52(0.09)

92 (0.69)

5 15-

¡10-

05-

E29

10(minutes)

20

Fig. 4. [3H]Verapamil accumulation by E29, VCR/A, and VCR/T. Standard

errors (bars) are shown where they exceed 10% of the plotted value.

the Dio value for control cells is around 100-fold that for VCR/A and VCR/T.

Verapamil Accumulation. In order to study the mechanism ofverapamil hypersensitivity, we measured the rate of accumulation of [3H]verapamil in the resistant cell lines and in control

cells. The concentration of verapamil which was used for theseexperiments was 1 pM which is within the range of concentrations over which VCR/A and VCR/T are sensitive to verapamilbut control cells are not (14). In both VCR/A and VCR/T therewas a greatly reduced level of verapamil accumulation (Fig. 4).In the case of VCR/A, accumulation was around 20% of controlvalues; in the case of VCR/T, it was around 40%.

Membrane and Cytosolic Proteins. Many multidrug-resistantcell lines express high levels of an Mr 170,000 or 180,000membrane glycoprotein. This protein is usually referred to asthe P-glycoprotein. We examined whether the P-glycoproteinis expressed at high levels in VCR/A and VCR/T cells. Membrane protein preparations were immunostained with a monoclonal antibody which is specific for P-glycoprotein (20). Strongstaining around MT 180,000 was obtained with membrane proteins of VCR/A and VCR/T (Fig. 5) and of a control cell lineCHRC5, which is a colchicine-resistant mutant known to express P-glycoprotein in its membrane (4). Antibody-positiveprotein is not seen at the same loading of membrane proteinfrom E29, the cell line from which VCR/A and VCR/T wereselected. Heavy staining of gels showed up smaller amounts ofsome antibody-positive material at other molecular weights inVCR/A and VCR/T, as has been reported previously forCHRC5 (23).

We compared the cytosolic proteins of VCR/A and VCR/Twith those of the parental cell line E29 by means of twodimensional gel electrophoresis and observed certain reproduc-

-200K

- 1 16K

-97K

-66K

.... to nFig. 5. Immunochemical detection of P-glycoprotein in Western blots of

membrane preparations of VCR/A and VCR/T using antibody C219. CHRC5 isa positive control, and £29is a negative control.

Lane a, E29; Lane ft, VCR/A; Lane c, VCR/T; Lane d. CHRC5. The molecularweight markers were from the Bio-Rad high molecular weight marker kit andwere (in thousands): 200K, myosin; 1I6K, galactosidase; 97K, rabbit musclephosphorylase b; 66K, bovine serum albumin. Arrowhead, heavily stained P-glycoprotein band.

ible differences (Fig. 6). Both VCR/A and VCR/T showedmore intense staining of two M, 27,000 protein spots in comparison with control cells. The spots had pi values of 6.4 and6.0 and were designated Sr and 82, respectively. As they have asimilar molecular weight it is possible that they are chargevariants of a single protein species. The extent of their elevationappeared to differ slightly in the two cell lines. In order toinvestigate whether these elevated proteins are associated withthe resistance phenotype, we examined their levels in revertantstrains which had been derived from VCR/A and VCR/T byprolonged growth in drug-free medium (14). In these revertantcell lines, vincristine resistance has fallen from around 100-folddown to around 5- to 10-fold. They are no longer hypersensitive

to verapamil (14). It was found that Si and 82 were no longerelevated in the revertants (Fig. 6) indicating a correlation between their presence and a high level of resistance.

We also consistently observed the appearance of two cytosolicprotein spots of about M, 53,000 and 54,000 on the VCR/Agels, compared to control cells (Fig. 7). There appeared to be asimultaneous reduction in the intensity of the a-tubulin spot,

indicating that the new spots could possibly be tubulin variants.The appearances of the new spots or the reduction in the a-tubulin spot were not observed in VCR/T gels. This proteindifference did not revert during the reversion of resistance from100-fold, and therefore, if it is associated with vincristine re

sistance, it can be associated only with the residual, stable lowlevel resistance found in the revertant.

Dominance Relationships of Vincristine Resistance and Verapamil Hypersensitivity. VCR/T cells were fused with a vincris-tine-sensitive CHO cell line (AuxBi) following polyethyleneglycol treatment. VCR/T x AuxBi hybrids were selected asdescribed in "Materials and Methods" and recloned. Their

hybrid nature was confirmed by means of chromosome analysis.Thirty metaphase spreads of each hybrid were examined following Leishman staining and in every case between 40 and 44chromosomes were observed (E29, AuxBi, and VCR/T have20-22 chromosomes). The dominance relationships of vincris

tine resistance and verapamil hypersensitivity were then examined by plating three independently isolated hybrids in vincristine and in verapamil. For comparison, E29 (the sensitive

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-55K

-43K

A AS1 S 2

-26K

T . -55K

-43K

r «-26K

bS1 S 2 •_ S\ S 2

-55K

-43K

-26K

-55K

-43K

AS1 -26K

AS1 S2

-26K

Fig. 6. Elevation of M, 27,000 proteins in VCR/A and VCR/T and their subsequent reduction in revenant cell lines VCR/A-REV and VCR/T-REV. a, E29; *,VCR/A; c, VCR/T; d, VCR/A-REV; e, VCR/T-REV. Arrowheads, positions of the M, 27,000 spots S, and S2. A, actin spot; T, tubulin region. The molecular weightmarkers were l4C-labeled standards from BRL and were (in thousands): 43K, ovalbumin; 26K, a-chymotrypsin.

parent of VCR/T), VCR/T, and a hybrid of E29 x AuxB, wereplated concurrently with the three VCR/T x AuxBi hybrids.

The data are shown in Figs. 8 and 9. It can be seen that allthree VCR/T x AuxB, hybrids behaved similarly. The vincris-tine resistance of VCR/T was dominant, but the dominancewas not complete as the hybrid resistance levels were slightlyless than those of VCR/T alone. In contrast, the hybrids allhad levels of verapamil resistance at least as great as £29andas the E29 x AuxBi hybrid showing that the verapamil hyper-sensitivity of VCR/T is completely recessive. The two aspectsof the mutant phenotype, vincristine resistance and verapamilhypersensitivity, therefore exhibit different dominance relationships in hybrids with sensitive cells.

DISCUSSION

The two cell lines studied during this work exhibit somefeatures which group them with other M DR cell lines and somewhich make them distinctive. They have previously been foundto be cross-resistant to Adriamycin and to have reduced vincristine accumulation (14) and now have been found to haveelevated levels of the P-glycoprotein, all of which are typical ofMDR cell lines. Our interest in these cell lines stems from theirdistinctive feature, verapamil hypersensitivity. Our objectivesare to discover what other distinctive cellular changes haveoccurred in verapamil-hypersensitive cell lines, to establishwhether such changes play a role in verapamil hypersensitivity

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-55K -55K

T -43K -43K

Fig. 7. Appearance of M, 53,000 and 54,000 protein spots in VCR/A as compared to control E29 cells. A, actin spots, a, E29; ft, VCR/A.

100

a> 10-

0 0.05 0.1Vtncristine Cone </jM)

Fig. 8. Colony-forming ability of three VCR/T x AuxB, hybrids in vincristinein comparison to AuxB,, VCR/T, and a hybrid of E29 X AuxB,. •,E29; A,VCR/T; x, E29 x AuxB,; O, A, D, three VCR/T x AuxB, hybrids.

100

50

Fig. 9. Colony-forming ability of three VCR/T x AuxB, hybrids in verapamilin comparison to AuxB,, VCR/T, and a hybrid of E29 x AuxB,. Symbols as inlegend to Fig. 8.

and whether any changes in these cell lines relate to the morecommon situation in which vincristine resistance is partially or

completely reversible by verapamil, but the cells are not hypersensitive to it.

We have found that the two cell lines studied here haveelevated levels of two M, 27,000 cytosolic proteins, pi 6.0 and6.4, which is a second distinctive feature of these lines. We havenot detected elevated levels of these proteins in several MDRcells studied in our laboratory which are not verapamil hypersensitive. Other workers have observed changes in low molecular weight proteins in some mult ¡drug-resistant cell lines whichare not verapamil hypersensitive but they appear to be distinctfrom the protein changes studied here. A M, 19,000-22,000protein has been reported in several hamster, mouse, and human MDR lines (5-7). This protein (sorcin/V19) has calciumbinding properties (24). A M, 23,000 protein, pi 4.8, whichappears to be an anionic form of glutathione transferase iselevated in a MDR-resistant human breast cancer cell line (25).As with these other proteins it will be important to establishwhether the presence of the M, 27,000 proteins elevated inVCR/A and VCR/T are merely correlated with the cellularphenotype by chance or whether they play a causal role. Indirectevidence for the latter comes from the fact that the same twospots were elevated in two independently selected cell lines andthat the spots revert to control levels along with the reversionof vincristine resistance and verapamil hypersensitivity. However, both of these observations could be due to the chanceassociation of the genes for Si and Sz with other genes whichcause vincristine resistance and verapamil hypersensitivity on asingle length of DNA which undergoes amplification. Thechange in the protein spot (S3) observed in VCR/A did notrevert along with reversion of most of the vincristine resistance.Our revenant maintained around 5% of the original increase inresistance (14) and we speculate that the overall VCR/A resistance phenotype may be attributed to two components: a majorrevertable component which confers multidrug resistance andverapamil hypersensitivity; and a minor stable componentwhich confers a low level of vincristine resistance, possibly viaa tubulin change. A similar change in an M, 53,000 protein wasnot observed in VCR/T and such changes are not usuallyobserved in other MDR cell lines which are not verapamilhypersensitive. Stable changes in a-tubulins in CHO cell lines

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PROPERTIES OF VERAPAMIL-HYPERSENSITIVE CELLS

which are resistant to antimicrotubular drugs have been reported by others (26, 27); such changes do not lead to multidrugresistance.

We have shown in this work that VCR/A and VCR/T arehypersensitive not only to verapamil but also to diltiazem,nicardipine, and quinidine sulfate. The first two of these areknown to act as calcium channel blockers in muscle cells in amanner broadly similar to that of verapamil (28). However,evidence supporting their action on calcium channels in CHOcells is lacking, and verapamil does not affect calcium accumulation by these cells (14, 15) suggesting some other site ofaction. Quinidine is thought to have rather generalized effectson membrane structure, interacting with phospholipids to reduce the temperature of the liquid-crystalline phase transition(17). The finding that these cells are hypersensitive to quinidineis therefore supporting evidence for our earlier proposal (14)that the cellular abnormality which confers hypersensitivitydoes not involve calcium channels. Cornwell et al. (29) havedrawn a similar conclusion concerning reversal of multidrugresistance in human KB carcinoma cells. They found thatverapamil, diltiazem, and quinidine all reverse multidrug resistance by increasing cellular drug accumulation and that theseagents, but not the more potent calcium channel blocker nitren-dipine, bind specifically to membrane vesicles from MDR KBcells and inhibit vinblastine photoaffinity labeling of the M,170,000 P-glycoprotein.

Hypersensitivity to quinidine does differ in certain respectsfrom hypersensitivity to verapamil, diltiazem, and nicardipine.Firstly, we consistently observed a small percentage of VCR/Aand VCR/T cells growing in intermediate concentrations ofdiltiazem and nicardipine (Figs. 1 and 2) as we previously didin verapamil (Figs. 3 and 4 in Ref. 14). This phenomenon isnot observed when VCR/A or VCR/T cells are plated in quinidine. Secondly, the hypersensitivity to quinidine is less markedthan to the other three drugs. VCR/A and VCR/T are resistantto around one-half the quinidine concentration which is toxicto control platings, whereas most VCR/A or VCR/T cells arekilled by concentrations of verapamil, diltiazem, or nicardipine50 or 100 times less than control cells. There may be aninteresting parallel here with the finding of Tsuruo et al. (30)that although quinidine partially reverses vincristine resistancein tumor cells, its effects are considerably less than those of thecalcium channel blockers.

The reduced verapamil accumulation in our verapamil-hy-persensitive cell lines was unexpected although is consistentwith a similar phenomenon recently reported by Cano-Gauciand Riordan (15). Our accumulation experiments rule out asimple model of VCR/A and VCR/T verapamil hypersensitivitybeing due to generalized elevation of internal verapamil, butfurther work is now necessary to investigate the possibility ofchanges in verapamil binding to specific cellular components.One speculation could be that VCR/A and VCR/T have a moreextreme change in the state of, or in the membrane environmentof, the P-glycoprotein than is present in other MDR cells.Consequently, interaction with verapamil or other membrane-active agents may induce a more dramatic functional or confor-mational change in the membranes of VCR/A or VCR/Tleading to cell death, rather than the lesser change which occursin other MDR cells and leads only to reversal of drug resistance.

The hybridization data reported here would be consistentwith a model that the vincristine-resistant/verapamil-hypersen-sitive phenotype of VCR/A and VCR/T represents an extremeversion of the vincristine-resistant/verapamil-reversible phenotype frequently seen in MDR cells. When VCR/T cells were

hybridized with control cells, vincristine resistance behavedsemidominantly, but verapamil hypersensitivity was completelyrecessive. One interpretation of this could be that vincristineresistance can gradually increase due to progressive membranechange (such as the presence of increasing quantities of P-glycoprotein) whereas verapamil hypersensitivity is expressedonly above a certain level of membrane abnormality. On thismodel, when VCR/T cells are hybridized with control cells, amembrane is produced which has an intermediate extent of themembrane defect originally present in VCR/T. Although thisleads to an intermediate level of vincristine resistance, it maybe below the threshold level for verapamil hypersensitivity.

ACKNOWLEDGMENTS

We thank Eli Lilly for a gift of vincristine. We are indebted to AnnBamford for technical assistance; C. Hastwell and D. Hunter forassistance in preparation of the figures; Gill Atkinson, Dr. Sue Bougourd, and Lisa Swingler for discussion; and Barbara Alderman fortyping the manuscript.

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2. Gerlach, J. H., Kartner, N., Bell, D. R., and Ling, V. Multidrug resistance.Cancer Surv., 5: 25-45, 1986.

3. Dalton, W. S., Durie, B. G. M., Alberts, D. S., Gerlach, J. H., and Cress, A.E. Characterization of a new drug-resistant human myeloma cell line thatexpresses P-glycoprotein. Cancer Res., 46: 5125-5130, 1986.

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5. Meyers, M. B.. and Biedler, J. L. Increased synthesis of a low molecularweight protein in vincristine resistant cells. Biochem. Biophys. Res. Commun., 99: 228-235, 1981.

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8. Shen, D.-VV.,Cardarelli, C, Hwang, J., Cornwell, M., Richert, N., Isshii, S.,Pastan, L, and Gottesman, M. M. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine,Adriamycin or vinblastine show changes in expression of specific proteins.J. Biol. Chem., 261: 7762-7770. 1986.

9. Tsuruo, T., lida, H., Tsukayoshi, S., and Sukurai, Y. Overcoming of vincris-tine-resistance in P388 leukaemia in vivo and in vitro through enhancedcytotoxicity of vincristine and vinblastine by verapamil. Cancer Res., 41:1967-1972, 1981.

10. Kessel, D. Circumvention of resistance to anthracyclines by calcium antagonists and other membrane-perturbing agents. Cancer Surv., 5:109-127,1986.

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1988;48:4477-4483. Cancer Res   J. Roger Warr, Michael Anderson and Jill Fergusson  Chinese Hamster Ovary CellsProperties of Verapamil-hypersensitive Multidrug-resistant

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