multiple mechanisms of resistance to af ...cancerres.aacrjournals.org/content/47/8/2056.full.pdf ·...

[CANCER RESEARCH 47, 2056-2061, April 15, 1987]

Multiple Mechanisms of Resistance to af-Diamminedichloroplatinum(II) inMurine Leukemia LI 210 Cells1

Victoria M. Richon, Nancy Schulte, and Alan Eastman2

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68 IOS

ABSTRACT

As an experimental model for resistance to ciVdiamminedichloro-platinum(II) (</\-l)!)!'). murine leukemia LI210 cells have been exposedto a stepwise increase in c/s-DDP concentration to produce a variety ofresistant cell lines. Intraspecies hybrids of the sensitive and resistantcells were made to determine whether cis-DDP resistance is a dominantor recessive trait. Hybrid cells displayed a partial degree of resistance ascompared to the parental cells. To determine whether this was due to asingle codominant trait or contribution from a variety of resistancemechanisms, the cells and hybrids were investigated for alterations inthe accumulation of drug, as well as alterations in glutathione levelswhich might inactivate the drug. The cÃ¯s-DDP-resistant cells demonstrated both a 50% reduction in accumulation of drug and a 1.7-foldincrease in intracellular glutathione. Reducing the glutathione levels inthese cells with buthionine sulfoximine did not sensitize them to cis-DDP. The hybrid cells had the same accumulation and the same levelsof glutathione as the cw-DDP-sensitive cells. Parallel studies were performed with cells resistant to l,2-diaminocyclohexaneplatinum(II) analogues. These cells also demonstrated reduced drug accumulation but noincrease in glutathione. Therefore, both a decrease in accumulation andincrease in glutathione may mediate resistance. Both mechanisms represent recessive traits as demonstrated in the cell hybrids. These mechanisms can only account for a small part of the resistance in these cells.A major, dominant mechanism occurs after the DNA has been platinated,but it remains to be determined whether this involves DNA repair,postreplication repair, or some other as yet unidentified process.

INTRODUCTION

Resistance to cancer chemotherapeutic drugs is a major limitation to their clinical use. In order to examine the developmentof resistance to the platinum antitumor drugs, cell strains havebeen developed with varying levels of resistance to c/s-DDP.3The mechanism of the antitumor action of c/s-DDP is notcompletely understood, however, a multitude of evidence suggests that the critical intracellular target is DNA (1). Theplatinum drugs passively diffuse into the cell, and form anaquated, charged intermediate that interacts with nucleophilicsites within the cell. C/s-DDP reacts bifunctionally to formDNA-intrastrand, DNA-interstrand, and DNA-protein crosslinks (2-4). Initially, it was believed that the DNA-interstrandcross-link was the most important cytotoxic lesion (5); however,other results have implicated intrastrand cross-links (6, 7).

Resistance to c/s-DDP has been investigated in many celllines. The drug used as the selecting agent for the developmentof resistance can determine which mechanism of resistancedevelops within the cell (8). For instance, when human epithelial cells (9) or human ovarian carcinoma cells (10) were exposed

Received 7/21/86; revised 12/15/86; accepted 1/18/87.The costs of publication of this article were defrayed in pan 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.

' This work was supported by National Cancer Institute Research Grants CA36010, CA 36039, and CA 00906 and Cancer Center Support Grant CA 36727.

2To whom requests for reprints should be addressed, at University of NebraskaMedical Center, Eppley Institute for Research in Cancer and Allied Diseases,42nd and Dewey Avenue, Omaha, NE 68105-1065.

3The abbreviations used are: ci'i-DDP, cu-diamminedichloroplatinum(II); cis-

DEP, c/i-dichloro(ethylenediamine)platinum(II); DACH-Pt, 1,2-diaminocyclo-hexaneplatinum(II); HAT, hypoxanthine (0.1 HIMI. aminopterin (0.4 /.M). andthymidine(I6 n\i).

to increasing concentrations of the heavy metal cadmium, thecells became resistant to cadmium and cross-resistant withplatinum compounds. The resistant cells have an increasedintracellular concentration of metallothionein which may inactivate the drug before it can produce the toxic lesion withinthe cell. Secondly, when murine leukemia LI210 cells wereselected for resistance to the alkylating agent melphalan, theywere found to be cross-resistant with c/s-DDP (5). Althoughinitial studies suggested that a defective uptake mechanism mayunderly the resistance (11), more recent evidence suggests thatthis resistance is due to an increased level of glutathione whichbinds to and thus inactivates the drugs before they reach thecritical DNA target (12).

A different pattern of cross-resistance is found in L1210 cellsdeveloped for resistance to c/s-DDP by continual exposure toincreasing concentrations of this drug (13, 14). The resultingresistance is specific for c/s-DDP and other closely relatedstructural analogues. These cells remain sensitive to DACH-Ptanalogues, melphalan, and cadmium. This pattern of resistancehas also been reported for a human squamous cell carcinoma(15). Similarly, if resistance is developed by continual exposureto DACH-Pt-carboxyphthalate, the cells display a lack of cross-resistance to c/s-DDP, melphalan, and cadmium (16). Themechanism of resistance in this case is unknown, but couldinclude decreased accumulation, intracellular inactivation, repair of the damaged DNA, or tolerance of unrepaired damage.

In an attempt to more fully understand the development ofresistance that is specific for platinum antitumor complexes,intraspecies hybrids have been made between c/s-DDP-sensitiveand -resistant LI210 cells. These hybrids have been examinedfor their degree of drug resistance. The results of such a studyindicate whether the resistance is a dominant or recessivegenetic trait. The hybrids also provide a model system to beginto investigate the possible mechanisms of resistance and therebyto more fully understand the mechanism of action.

MATERIALS AND METHODS

Chemicals. The following drugs were obtained from the indicatedsources: cw-DDP (Platino!) and DACH-Pt-carboxyphthalate, BristolLaboratories, Syracuse, NY; melphalan, Drug Development Branch,National Cancer Institute, Bethesda, MD; DACH-Pt-SO4 was synthesized and donated by Dr. Abdul Khokhar, M. D. Anderson Hospitaland Tumor Institute, Houston, TX; [3H]DACH-Pt-Cl2 was donated byDr. Stephen Chancy, University of North Carolina; DL-buthionine-S,A-sulfoximine, Chemical Dynamics Corporation, South Plainfield, NJ.[3H]m-DEP was synthesized as previously described (4). Cis-DDP wasdissolved according to the manufacturer's recommendations. Mel

phalan was dissolved in 0.1 N HC1. DACH-Pt-SO4 was dissolved inwater and used within 1 h.

Cell and Culture Conditions. Sensitive murine leukemia 1.1210 cells,LI210/0, and the cw-DDP- and DACH-Pt-resistant sublines have beenpreviously described (16, 17). The cw-DDP-resistant cell lines L1210/DDP5 and L1210/DDPi0 were developed by growth in incrementalincreases in drug concentration over several years and are now maintained in 5 and 10 Mg/ml e/i-DDP, respectively. The DACH-Pt-resistantsubline was developed in a similar manner using DACH-Pt-carboxyphthalate for initial selection and is now maintained in 4 ng/ml of

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RESISTANCE TO cis-DDP IN L1210 CELLS

DACH-Pt-SO4. Suspension cultures of the cell lines are maintained inMcCoy's 5a (modified) medium supplemented with penicillin (300

U/ml), streptomycin (300 U/ml), fungizone (l Â¿ig/ml),and 15% calfserum (17).

An LI210/0 double mutant resistant to ouabain and 6-thioguaninewas developed for hybrid selection. LI210/0 cells were mutagenizedwith 200 /ig/ml ethylmethanesulfonate for 20 h at 37'C. After a 6-day

expression time, the mutagenized cells were selected for resistance to 1mM ouabain. The ouabain-resistant cells were again mutagenized withethylmethanesulfonate followed by a 6-day expression time, and amutant clone was selected by addition of 10 Â«jg/mlof 6-thioguanine inthe culture medium. The double mutant was designated as I 121(1TOO. These cells were unable to grow in HAT-containing medium.

Cell Fusion. Cell fusion was carried out as described by Ramshaw etal. (18). L1210/TGO cells (2 x IO6)were mixed with the cw-DDP- orDACH-Pt-resistant cells (2 x IO6).The cells were centrifuged together

at 900 x g for 5 min, the supernatant removed, the cells suspended inthe remaining medium and 1 ml of fusion solution was added dropwisewith constant agitation over a 90-s period. The fusion solution consistedof prepurified polyethylene glycol 1000 (19) at 48% in McCoy's 5a

(modified) medium. The fusion was stopped by adding 20 ml of phosphate buffered saline over 3 min. The cells were allowed to rest for 5min, centrifuged, and resuspended carefully in medium so as not tobreak up all of the small clumps. The cells were diluted with 50 ml ofMcCoy's 5a medium containing 1 mM ouabain and HAT, then 0.2 ml

was plated into each well of a 96-well cluster dish. Wells whichcontained single colonies were detected after 10-15 days. Ten singlecolonies from each fusion were transferred to 15-ml culture tubes. Thecolonies were initially grown in ouabain-HAT medium and then sub-cultured in the absence of selection conditions. At least four clones andmass culture hybrids from each fusion were tested for their drugsensitivity.

Karyotype Analysis. Cells growing exponentially in suspension wereincubated with 0.25 Mg/ml colcemid for l h at 37"C. The cells were

washed with hypotonie sodium citrate and fixed in methanol-aceticacid. Slides were prepared, stained with Wright's stain, and 20 repre

sentative chromosome spreads were photographed and counted.Determination of Drug Sensitivity. Sensitivity to cw-DDP, DACH-

Pt, and melphalan was determined by measuring the inhibition of cellgrowth. Approximately, 5 x IO4 cells/ml were incubated in triplicatewith varying concentrations of drug over a 3-day period. The resultantcell number was determined using a Coulter Counter Model /., (CoulterElectronics, Hialeah, FL).

Accumulaton Studies. L1210 cells I Id'') were incubated with 0.1 Mg/ml of [3H]cw-DEP (5 /Â¿Ci)or 0.31 mg/ml [3H]DACH-Pt-CI2 (5 nCi) at37Â°Cin a total volume of 10 ml of medium. The incubation was stopped

at 0, 2, 4, and 6 h. The cells were washed two times with phosphatebuffered saline. The cell pellet was dissolved in 0.5 ml of NCS tissuesolubilizer (Amersham Corp., Arlington Heights, IL) for 2 h, neutralized with glacial acetic acid, and the radioactivity was determined usinga Beckman LS2800 liquid scintillation counter.

Due to the differences in the size of the various cells and hybrids,the accumulation experiments were normalized to the surface area ofthe cells. The cell surface area was determined using the CoulterCounter Model Zf (Coulter Operator's Reference Manual).

In dose response experiments, 10" I 121(1cells were incubated in a

total volume of 10 ml with 0-10 Â¿ig/mlc/s-DEP and 0.1 Â¿"g/mlof[3H]c/s-DEP. After a 6-h incubation at 37'C, the cells were processed

in the same manner as the above accumulation experiments.Glutathione Assay. The fluorometric method for the determination

of reduced glutathione in tissue (20, 21) was modified to determine theglutathione levels in murine leukemia 11210 cells. Cell samples wereresuspended at IO7 cells/ml PO4-EDTA (100 mM Na2HPO4, 5 mMEDTA, pH 8.0), sonicated and precipitated in 5% 11,!'<),. The resultingsupernatant was diluted 1:10 in PO4-EDTA buffer. The final assaymixture (2 ml) contained 100 /<!of the diluted cell supernatant 1.8 mlof PO4-EDTA buffer, and 100 M>of o-phthaldialdehyde (1 mg/mlmethanol). After thorough mixing and incubation at room temperaturefor 15 min, the fluorescence emission at 420 nm was determinedfollowing excitation at 350 nm on a Turner model 430 spectrofluo-

rometer. The protein concentration of an aliquot of the cell sample wasdetermined by the method of Lowry el al. (22).

RESULTS

Properties of Parental and Hybrid Cells. The L1210/TGOcell line is resistant to 10 Mg/ml 6-thioguanine and 1 HIMouabain, however, it is sensitive to growth in HAT-containingmedium. These cells retain the same sensitivity to cis-DDP asthe parent sensitive cell line (Ref. 16 and Table 1). The drug-resistant cell lines are unable to grow in 1 mM ouabain but theirgrowth in HAT-containing medium is uninhibited. The c/s-DDP-resistant cell lines were developed by exposing the L121O/0 cells to stepwise increments in drug concentration over severalyears (Fig. 1). L1210/DDP5 cells are 48-fold resistant to cis-DDP and 6.6-fold resistant to DACH-Pt-SO4. As the resistanceto c/s-DDP was doubled to 110-fold in the L1210/DDPi0 cells,the resistance to DACH-Pt-SO4 rose to 18-fold. The cross-

resistance has therefore become quite significant. L1210/DACH cells are 33-fold resistant to DACH-Pt-SO4 and 6-foldcross-resistant to cw-DDP. The resistance in all of the cells isa very stable trait. The cells have been grown in the absence ofdrug for up to 2 years with no decrease in the level of resistance.

The sensitivity of the isolated cell hybrids to growth in cw-DDP and DACH-Pt-SO4 was assessed to determine whetherc/s-DDP and DACH-Pt resistance in the LI210 cells is adominant or recessive trait. The cell hybrids made between theL1210/TGO and L1210/DDP, cell lines (TGO:DDP5) displayed a partial degree of resistance to c/s-DDP (Table 1). TheTGO:DDP5 hybrid cells maintained 25% of the resistance ofthe L1210/DDP5 cells to cis-DDP, and 50% of the cross-resistance to DACH-Pt-SO4. The TGO:DACH hybrids retained13% resistance to DACH-Pt but practically all of the cross-resistance to c/s-DDP. At least four individual cloned hybrids,as well as uncloned populations displayed this partial degree ofresistance toward c/s-DDP and DACH-Pt-SO4. These resultsdemonstrate that c/s-DDP and DACH-Pt resistance in LI210cells is a codominant trait. All parent cell lines displayedpseudodiploid metaphase spreads with an average of 38 chromosomes (Table 1). The hybrid cells maintained approximately70 chromosomes for over 3 months.

Melphalan Cross-Resistance of Parental and Hybrid Cells.The L1210-sensitive, -resistant, and -hybrid cell lines wereinvestigated for their resistance to the alkylating agent melphalan (Table 1). Melphalan was chosen for investigation dueto the previous observation that LI210 cells, which were selected for resistance to melphalan, were found to be cross-resistant to c/s-DDP (5). All resistant and hybrid cells exhibiteda low degree of melphalan resistance as compared to the L1210/TGO cell line. The degree of melphalan resistance, therefore,was found not to parallel the degree of c/s-DDP- or DACH-Pt-resistance in the parental or hybrid cells.

Accumulation of c/s-DEP. One possible mechanism for resistance is decreased accumulation of drug in the cell. In orderto investigate this, accumulation experiments were performedwith the radiolabeled analogue of c/s-DDP, ['HJc/s-DEP. This

analogue is also an effective antitumor agent that demonstratesthe same profile of sensitivity and resistance as that observedfor c/s-DDP in murine leukemia LI210 cells (13, 17) andproduces adducts at identical sites in DNA (3).

The accumulation of [3H]c/s-DEP was linear over a 6-h

incubation time (Fig. 2A) and doses from 0.1 to 10 Mg/ml (datanot shown) in both sensitive, resistant, and hybrid cells. Accumulation in the L1210/0 cells (data not shown) was the same

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RESISTANCE TO ciÃ-DDP IN LI 210 CELLS

Table 1 Properties of parental and hybrid cells

CelllineParent

cellsL1210/TGOL1210/DDP,L1210/DDP.OL1210/DACHHybrid

cellsTGO:DDP,TGO:DDP,oTGO:DACHChromosome

modalvalue(range)40

(38-42)38(37-40)38(34-44)39(37-40)74(67-81)70

(63-80)74(67-84)cts-DDP0.125.80

(48)'13.20(110)0.70

(5.8)1.55(12)1.90(15)0.60

(5.0)IDÂ«,

(Mg/ml)"DACH-Pt-SOÂ«0.221.40(6.4)3.92

(18)7.20(33)0.73

(3.3)1.32(6.0)0.98

(4.4)Melphalan0.642.66

(4.8)3.09(5.6)1.50(2.3)2.16

(3.9)1.40(2.6)1.32(2.0)HAT

sensitivitySRRRRRROuabainsensitivityRSSSRRRSurface

area(Mm2)460

Â±49*439

Â±52469Â±54418Â±42632

Â±24614Â±33620

Â±62* IDÂ»is the concentration effective in inhibiting 50% of the cell growth measured after 3 days of continuous exposure to the drug."Mean Â±SD of four determinations.' Values in parenthesis, fold resistance.

M9/ml<N T (C <Â£>Ã³ Ã³ Ã³ *- *>

15Time (months)

10 15

Fig. 1. Development of resistance to cis-DDP (left) and DACH-Pt-carboxy-phthalate (right) in L1210 cells. Cells were grown in increasing concentrations ofdrug as indicated above the charts. They were subcultured twice weekly at adilution of 1:5. The fold resistance was calculated from the drug concentrationthat caused 50% inhibition of cell growth over a 3-day period.

as in the L1210/TGO. The cw-DDP-resistant cells L1210/DDP5 and L1210/DDPJO demonstrated a 50% decrease inaccumulation of [3H]cw-DEP. The accumulation of [3H]cis-

DEP was also determined in L1210/DDP cells that had beenmaintained frozen and were only 20-fold resistant to c/s-DDP.These cells demonstrated a similar 50% decrease (data notshown). The L1210/DACH cells also exhibited a significantdecrease in accumulation of [3HJcis-DEP (30%; Fig. 2A).

The hybrid cells were also investigated to determine whetherthe decreased accumulation in the L1210/DDPS, 1.12IO/DDP,o, and L1210/DACH cells was a recessive or dominanttrait. The hybrid cells demonstrated a greater accumulation of[3H]cis-DEP (12-40%) than even the LI210/0 cells when measured on a per cell basis. The hybrid cells maintained 70-76chromosomes stably and when viewed microscopically appearedlarger than either the sensitive or resistant cells. Because dif

fusion across a membrane is dependent upon surface area, themedian surface area of the cells was determined using theCoulter Counter. The hybrid cells had approximately a 40%larger surface area than either the sensitive or resistant parentalcells (Table 1). The cell-associated radioactivity was normalizedto cell surface area for that particular cell (Fig. IB). The hybridsTGO:DDP5 and TGO:DACH then exhibited the same level ofaccumulation of [3H]c/s-DEP as the parent-sensitive cell line.

Therefore, it appears that the reduction in accumulation foundin the L1210/DDP5 and L1210/DACH cells is a recessive trait.

An alternate correction for the values could be made usingcell volume. The hybrid cells have approximately a 60% increase in volume compared to the parental lines. The correctedvalues for the hybrids would then be slightly higher relative tothe parent lines. This would not alter the conclusion thatreduced accumulation is a recessive trait.

The TGO:DDPio hybrids demonstrated a 14% decrease inaccumulation as compared to the accumulation measured inthe cw-DDP-sensitive LI210 cells (Fig. IB). At the time ofmeasurement, the TGO:DDP10 cells had increased their resistance from 15-fold to approximately 30-fold solely as a result ofcontinuous growth in culture. The decrease in accumulationmay reflect this increase in resistance. This level of accumulation was still considerably greater than in the I.I2K)/I)OI',,,

parent cell line.Accumulation of DACH-Pt-Cl2. Accumulation of [3H]DACH-

Pt-Cl2 was determined in all the cells (Fig. 3). The L1210/DDP5 and L1210/DDPi0 cells both demonstrated a 36% reduction in accumulation but 6- and 18-fold cross-resistance toDACH-Pt-SO4, respectively. Therefore, the reduced accumulation did not become more prevalent as the degree of resistancewas increased. The L1210/DACH cells demonstrated the greatest effect, a 75% decrease in accumulation of [3H]DACH-Pt-

C12.

Fig. 2. Time-dependent accumulation ofJ'H]cÂ«-DEPin LI210 cells and their hybrids.Cells (1 x 10*) were incubated with 0.1 pg/mlof [3H)c/s-DEP. At 0, 2, 4, and 6 h cell-associated radioactivity was determined in the celllines and hybrids as indicated. The results areexpressed as (A) accumulation per 10" cells

and (111normalized to cell surface area.

TIME (H)

TOO: OACH

TOO: OOPS

TOO: DDP,oL1210/TQO

E

S>_j_iUlo

Â«. L1210/DACH Â°â€¢L1210/OOPS 'g

â€¢L1210/ODP10 %

B. . TOO:DDPs_^Â» TOO: OACH' -* L1210/TQO

, TOO: DDP,o

L1210/DACH

â€¢I 1?10/DDPS

L1210/DDP10

2 4

TIME (H)

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Fig. 3. Time-dependent accumulation ofI'HJDACH-Pt-Ch in LI210 cells and theirhybrids. Cells (1 x 10") were incubated with0.31 mg/ml [3H]DACH-Pt-Cl2. At 0, 2, 4, and6 h cell-associated radioactivity was determined in the cell lines and hybrids as indicated.Results are expressed as (A) accumulation per10" cells and (0) normalized to cell surface

L1210/DDP10

I. 12 IO/DDP5

L1210/OACH

The accumulation of [3H]DACH-Pt-Cl2 was measured in the

hybrid cells, expressed on a per cell basis (Fig. .VI), and normalized to the surface area of the cells (Fig. 3Ã„). TheTGO:DDP5 and TGO:DACH hybrids demonstrated no decrease in accumulation when the data were normalized to cellsurface area. However, they maintained approximately a 3-and 4-fold resistance to DACH-Pt-SO4, respectively. TheTGO:DDPio hybrid line exhibited an 18% decrease in accumulation with 6-fold cross-resistance to DACH-Pt-SO4. Thedecreased accumulation found in the L1210/DDP5 and L1210/DACH cells is a recessive trait as demonstrated in theTGO:DDP5 and TGO:DACH hybrids. Once again, the resultsobtained with TGO:DDPi0 cells are slightly more complex tointerpret and may reflect the attainment of a higher level ofresistance selected for by prolonged growth in culture.

Glutathione Studies. Glutathione levels in all the cells weredetermined (Table 2). The glutathione content was normalizedto protein content of the cells to account for changes in cellsize. The levels of glutathione were significantly higher (approximately 1.7 times) in both the L1210/DDP5 and L1210/DDP,o cells, as compared to the LI210/0 cells. This increasewas not present in the L1210/DACH cells or any of the hybridcells. The increase in glutathione in the L1210/DDP5 andL1210/DDPio cells is therefore a recessive trait as demonstrated in the hybrid cells. LI210/0 and L1210/DDP,0 cellswere incubated with 500 n\i DL-bulhionine-5,/?-sulfoximine for6 h to reduce the glutathione level. Although the glutathione inthe resistant cells was reduced to that of the sensitive cells, thishad no effect on survival of the cells in cw-DDP (Table 3).

DISCUSSION

Although the mechanisms of cellular resistance to platinumcoordination complexes are not understood, several possible

Table 2 Glutathione levels in LÃ¬2ÃŒOcells and hybrids

CelllineParent

cellsL1210/0L1210/DDP,L1210/DDP10L1210/DACHHybrid

cellsTGO:DDP5TGO:DDP,oTGO:DACHGlutathione

(nmol/mgprotein)18.6Â±2.6*(6)c30.0

Â±4.6(5)34.2Â±8.8(5)19.9Â±4.6(4)20.8

Â±3.6(3)23.8Â±0.6(5)16.9Â±1.6(3)P*<0.01<0.01NS*NSNSNS

" Glutathione levels were compared to LI210/0 by the two-tailed rank sum

test.* Mean Â±SD.' Numbers in parentheses, number of determinations on separate cultures.*P> 0.05, and therefore, no significant difference.

Table 3 Effect of glutafnione level on cis-DDP toxicityCells were incubated with or without 500 n\\ BSO* for 6 h prior to a 2-h

incubation with m-DDP. Drug was removed and inhibition of cell growthmeasured after 3 days. Glutathione levels were measured immediately prior toincubation with m-DDP.

Glutathione(nmol/mgprotein)Cell

lineL1210/0

L1210/DDP.O-BSO18.634.2+BSO8.7 17.4cis-DDP

IDÂ»(Mg/ml)-BSO0.48

16.2+BSO0.4516.8* BSO, DL-buthionine-S,/?-sulfoximine.

mechanisms have been proposed (8). These mechanisms include: (a) altered accumulation of the platinum in the cell; (/Â»)a decreased ability of the platinum species once inside the cellto interact with the DNA, which could include inactivation ofthe platinum before interacting with DNA, or alteration in thechromatin to reduce interaction with the drug; (c) an alteredDNA repair mechanism; and (d) the ability to tolerate unrepaired lesions in DNA.

Cross-resistance profiles have been helpful in defining theresistance mechanism in a particular cell line. The L1210/DDPcells are very specific in their resistance toward m-DDP andstructurally similar platinum analogues (13). At relatively lowlevels of resistance to cis-DDP, the cells demonstrated less than2-fold cross-resistance to either DACH-Pt analogues or mel-phalan (16, 17). However, at increasing levels of resistance toCK-DDP, cross-resistance to DACH-Pt-SO4 increased markedly, while cross-resistance to melphalan increased minimally(Table 1). This increase in cross-resistance to DACH-Pl-SO4,therefore, does not reflect an increase in general cross-resistancebut probably to a new mechanism which is responsible forresistance to both c/s-DDP and DACH-Pt. This cross-resistancemechanism is probably different than the primary resistance toDACH-Pt-SO4 developed in the L1210/DACH cells becausethese cells are not cross-resistant to m-DDP to a significantextent (16).

Intraspecies hybrids were developed between the sensitiveand resistant L1210 cells. All hybrids exhibited codominanceof the resistant phenotype. Several cases of drug resistance havealso demonstrated an intermediate degree of resistance in intraspecies hybrids (21-26). The best documented case is that ofmethotrexate resistance (24). In one class of mutants, theinhibited enzyme dihydrofolate reducÃaseexhibited a reducedaffinity towards methotrexate, and behaved in a codominantfashion in cell hybrids because both mutant and wild typeenzymes were expressed. A second class involved increasedlevels of dihydrofolate reducÃasethrough a gene amplificationevent which also was expressed codominantly in cell hybrids.An alleniate explana! ion for inlermediate levels of resistance is

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that multiple mechanisms impart both dominant and recessivetraits simultaneously. The stepwise selection procedure required for the development of c/s-DDP resistance may lead tomany mechanisms. It is worth noting that resistance developedvery slowly at first, followed by a rapid increase (Fig. 1).Subsequently, resistance increased again at a very slow rate. Asthe drug concentration increased, perhaps different lesions became critical requiring different mechanisms of resistance. Thiscould particularly be occurring with DNA-damaging agents,such as c/s-DDP and DACH-Pt-SO4, which produce a varietyof different adducts in the DNA, each possibly with varyingtoxic potential.

Two possible mechanisms of resistance were investigated;alterations in accumulation of drug and alterations in glutathi-one levels. The accumulation studies revealed that both c/s-DDP and DACH-Pt-SO4 resistance in the LI210 cells aremediated in part by a decrease in accumulation of the drug.Several c/s-DDP-resistant cell lines with varying levels of resistance to m-DDP demonstrated that the decrease in accumulation is a mechanism of resistance which occurs at lowlevels of resistance and is not significantly increased as the cellsattain higher levels of resistance. The cross-resistance of thec/s-DDP-resistant cells to DACH-Pt may also be mediated inpart by a decrease in accumulation. However, once again thisis a mechanism which occurs at low levels of cross-resistanceand does not correlate with the degree of cross-resistance. Theseresults suggest that cross-resistance to DACH-Pt in the L1210/DDP cells also involves several mechanisms of resistance. Thedecrease in accumulation of drug demonstrated in the c/s-DDP-resistant cells was not apparent in the hybrid cells, and thereforeis a recessive trait.

A decrease in accumulation of [3H]c/s-DEP or [3H]DACH-I'l ( Iâ€¢could indicate an alteration in drug uptake and/or drug

efflux. These studies did not address this question. Kraker etai. (27) have demonstrated a reduction in the intracellularuptake of c/s-DDP in L1210/DDP5 cells. This cell line alsodemonstrated a reduced uptake of DACH-Pt-Cl2. Hromas etal. (28) demonstrated a decrease in m-DDP uptake in the cis-DDP-resistant LI210 leukemia cell line ZCR9, as compared tothe sensitive cell line K25. No change in efflux was detected.Andrews et al. (29) have also demonstrated a decreased uptakeof m-DDP in c/s-DDP-resistant 2008 human ovarian carcinoma cells. All studies, therefore, demonstrate decreases inuptake and suggest that it may be an important mechanism ofm-DDP resistance.

The second mechanism of resistance examined was alterations in glutathione levels. Glutathione has been shown to bean important determinant of the sensitivity of cells to a widevariety of drugs (30). Elevated glutathione has also been shownto be a component of the drug-resistant phenotypes that emergein cells exposed to a number of electrophilic drugs and especially to nitrogen mustards, such as melphalan (12). Melphalan-resistant LI210 cells were shown to be cross-resistant to cis-DDP (5). Although glutathione depletion can sensitize cell linesto melphalan cytotoxicity, the effect on m-DDP cytotoxicitywas minimal (31). Glutathione levels in the cell could mediateplatinum toxicity in at least two different manners. Firstly, oncethe m-DDP or DACH-Pt-SO4 had entered the cell, it coulddirectly bind to the sulfhydryl residues of glutathione forminga thioether. This would inactivate the drug before it could reachthe critical DNA target. Sharma and Edwards (32) demonstrated that 52% of the cytosolic Pt in liver and kidney isassociated with low molecular weight fractions (<M, 1000)which would include glutathione. Normally, Pt is believed to

bind to glutathione in the cytoplasm of the cell. Secondly,glutathione may reduce platinum cytotoxicity by quenchingDNA-Pt monoadducts. c/s-DDP-mediated cross-links betweenglutathione and DNA have been observed (33). This mechanismmay explain the dramatic potentiation of trans-DDP cytotoxicity in glutathione-depleted cells (29). The trans-DDP adductsin DNA are predominantly monofunctional and rearrange tobifunctional adducts much slower than those of cis-DDP (33).Therefore, the high levels of monoadducts may be more easilyquenched by glutathione.

In the present studies a 1.7-fold increase was noted in glutathione in the cells which were 50- and 100-fold resistant to c/s-DDP (Table 2). The DACH-Pt-resistant cells and the hybridcells did not exhibit this increase in glutathione levels, and,therefore, the increase in glutathione levels is also a recessivetrait.

The role, if any, that this increase in glutathione plays in theresistance to c/s-DDP is unknown. Our studies demonstratedthat decreasing the intracellular glutathione levels in the resistant cells to that of the sensitive cells did not sensitize the cellsto c/s-DDP. Andrews et al. (35) have demonstrated that onlyafter a prolonged decrease in the glutathione levels, do the cellsbecome sensitized to c/s-DDP and that this is true for bothsensitive and resistant cells. The 1.7-fold increase in glutathionein the m-DDP-resistant cell lines may play a role in theresistance to c/s-DDP; however, it does not seem to be a simplecorrelation. It is worth noting that the cells with elevatedglutathione are also more resistant to melphalan (Table 1). Inthis case, the glutathione may play a role in drug inactivation.

Both decreased accumulation of c/s-DDP and increased glutathione levels in the cells may be involved in the developmentof resistance to c/s-DDP. Both of these mechanisms can accountfor only a partial degree of resistance and they do not increasein proportion to the level of resistance in the cell lines. Therefore, another mechanism of resistance must also be occurringand this must account for the dominant trait retained in thehybrid cells. This mechanism may involve an increased abilityof the resistant cells to tolerate DNA-Pt lesions. In support ofthis, Strandberg et al. (7) demonstrated that at equitoxic dosesof c/s-DDP, the resistant cells had up to 15-fold higher levelsof DNA-interstrand cross-links. The number of these crosslinks that the resistant cells were able to tolerate increased asthe cells became more resistant to c/s-DDP. The above studyalso demonstrated that at any single dose of c/s-DDP, therewere fewer DNA-interstrand cross-links in the resistant ascompared to sensitive cells. This decrease was the same for allresistant cell lines and can be accounted for by the decrease inaccumulation of the drug reported here.

In summary, this study has demonstrated that c/s-DDP resistance is expressed to an intermediate degree in cell hybrids.This is attributed to multiple mechanisms of resistance including decreased accumulation. Although elevated glutathione hasbeen observed, its role in resistance has not been established. Amajor, dominant mechanism occurs after the DNA has beenplatinated, but it still remains to be determined whether thisinvolves DNA repair, postreplication repair, or some other asyet unidentified process.

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1987;47:2056-2061. Cancer Res Victoria M. Richon, Nancy Schulte and Alan Eastman -Diamminedichloroplatinum(II) in Murine Leukemia L1210 Cells

cisMultiple Mechanisms of Resistance to

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