Expression of the MRP and MDR1 Multidrug Resistance Genes ...clincancerres.aacrjournals.org/content/clincanres/3/1/115.full.pdf · Expression of the MRP and MDR1 Multidrug Resistance

Vol. 3, 115-122, January 1997 Clinical Cancer Research 115

Expression of the MRP and MDR1 Multidrug Resistance Genes in

Small Cell Lung Cancer’

Barbara G. Campling,2 Leah C. Young,

Kathy A. Baer, Yuk-Miu Lam, Roger G. Deeley,

Susan P. C. Cole, and James H. Gerlach

Cancer Research Laboratories [B. G. C., L. C. Y., K. A. B., R. G. D.,S. P. C. C., J. H. G.] and Departments of Oncology [B. G. C.,R. G. D., S. P. C. C., J. H. G.l, Pathology [B. G. C., L. C. Y., R. G. D.,S. P. C. C.], Community Health and Epidemiology [Y-M. L.J, andBiochemistry [R. G. D., J. H. G.], Queen’s University, Kingston,

Ontario, K7L 3N6 Canada

ABSTRACT

Acquired multidrug resistance is a major obstacle to acure for small cell lung cancer (SCLC). Overexpression ofthe MDR1 gene occurs infrequently in multidrug-resistant

SCLC cell lines. The multidrug resistance protein (MRP)can confer multidrug resistance, but its role in clinicallyacquired drug resistance is unknown. The purpose of thisstudy was to measure expression ofMRP and MDR1 mRNA

in cell lines and clinical samples from SCLC patients and tocorrelate the results with drug sensitivity profiles. Twenty-

three SCLC cell lines and iO tumor samples from SCLC

patients were examined. Samples expressing MRP andMDR1 were identified by reverse transcription-PCR, andlevels of MRP mRNA in the cell lines were measured byquantitative reverse transcription-PCR. One of 23 cell lines(4%) expressed MDR1 mRNA, whereas MRP expression was

detected in i9 of 23 cell lines (83%). There was a significant

correlation between doxorubicin resistance and MRP ex-pression levels (r = 0.422; P = 0.045). Of the 10 clinicalsamples, 3 expressed only MRP, 2 expressed only MDR1, and4 expressed both drug resistance genes. In summary, MRP is

frequently expressed in clinical samples and cell lines fromSCLC patients, and the levels correlate with doxorubicinresistance in unselected SCLC cell lines. Expression ofMDR1 can be detected in clinical samples of SCLC but israrely found in cell lines from drug-resistant patients. Thesemultidrug resistance proteins may contribute to the multi-

Received 5/24/96; revised 10/9/96; accepted 10/23/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I Supported by the Medical Research Council of Canada. B. G. C. is aClinician Scientist, and S. P. C. C. and J. H. G. are Career Scientists of

the Ontario Cancer Treatment and Research Foundation. L. C. Y. issupported by the Ontario Cancer Treatment and Research Foundation.R. G. D. is the Stauffer Professor of Cancer Research at Queen’s Uni-

versity.

2 To whom requests for reprints should be addressed, at The Ontario CancerTreatment and Research Foundation, Kingston Regional Cancer Centre, 25

King St. West, Kingston, Ontario, K7L 5P9 Canada. Phone: (613) 545-6357; Fax: (613) 544-9708; E-mail: [email protected].

factorial problem of clinically acquired drug resistance inSCLC.

INTRODUCTION

Chemotherapy is the primary treatment modality for

SCLC,3 but despite high initial response rates, most patients

eventually die with drug-resistant tumors (1). In many in vitro

systems and in some clinical settings, resistance to multiple

chemotherapeutic agents is caused by overexpression of P-gp, a

membrane transport protein encoded by the MDR1 gene, which

actively effluxes many natural product-type drugs from cells (2,

3). However, data from large panels of cell lines indicate that

P-gp overexpression occurs infrequently in multidrug-resistant

SCLC cell lines (4-7). Despite these findings, P-gp overexpres-

sion cannot been ruled out as a factor contributing to multidrug

resistance in SCLC. Because most studies have used established

cell lines rather than tumor samples, it is possible that P-gp

expression may have been present in the original tumor but was

lost during culture in vitro. Alternatively, other mechanisms of

resistance may be more important in SCLC tumors. A novel

transport protein, termed MRP, that is overexpressed in a doxo-

rubicin-selected SCLC cell line has now been cloned (8). Like

P-gp, MRP is a member of the ATP-binding cassette superfam-

ily of transport proteins, and cDNA transfection studies dem-

onstrate that MRP can confer multidrug resistance (9, 10). It is

not known whether this protein is involved in clinically acquired

drug resistance in SCLC.

To investigate the role of MRP and MDRI in SCLC, we

examined 23 SCLC cell lines and 10 cryopreserved tumor

samples obtained from SCLC patients at various stages of

treatment. Six of these cryopreserved samples were the original

specimens from which cell lines in this collection were derived.

Expression of MRP and MDRI mRNA was examined using

RT-PCR, and levels of MRP mRNA were quantitated by Q-PCR. Results were correlated with the drug sensitivity profiles

and the treatment histories of the patients from whom the cell

lines were derived.

MATERIALS AND METHODS

Cell Lines. A collection of 23 unselected SCLC cell lines

was established from patients at various stages of treatment.

Table I summarizes the features of these cell lines, including the

source of the tumor tissue from which the lines were derived, the

treatment received by the patients at the time the line was

established, as well as subsequent treatment and response. The

conditions for establishing and culturing the cell lines have been

3 The abbreviations used are: SCLC. small cell lung cancer; P-gp.P-glycoprotein; MRP, multidrug resistance protein; RT-PCR, reverse

transcription-PCR; Q-PCR, quantitative RT-PCR; VP-l6, etoposide;

TFRR, transferrin receptor; AUC, area under the curve.

on May 9, 2018. © 1997 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

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Cell line Source” Prior treatment” Subsequent treatment’ Response” MDR1 MRP

NCI-H69 Pleural effusion CMC-VAP Unknown - - -

NCI-H209 Bone marrow None Unknown - - +

SHP-77 Primary tumor None None - + +

AD-A Subcutaneous lesion� CA, VP/CP, RT Mitox PD - +

BK-T Primary tumor None CAV NA - -

LG-T Lymph node None CAV, VP/CP CR - -

HG-E Pleural effusion None None - - +

JO-E Pleural effusion CAV, VP/CP, RT None - - +

WL-E Pleural effusion VP/CP, CAV None - - +

JN-M’ Bone marrow None CAV, VP/Carb PD - +

SH-A Lymph node� CAV. VP/CP CAy, VP/CP PR - -

MM-I Pleural effusion CAV, VP/CP Unknown - - +

LD-T Primary tumor None CAV, VP/CP NA - +

MO-A Lymph node� CAy, VP/CP CAV, VP/CP CR - +

OS-A Subcutaneous lesion” CAV, VP/CP None - - -

SV-E Pleural effusion CAV VP/CP PD - +

LV-E Pleural effusion Oral VP None - - +

JS-E Pericardial effusion CAV, VP/CP None - - +

GL-E Pericardial effusion CAy, VP/CP, RT Oral VP PD - +

SM-E Pleural effusion CAV VP/CP, CAV PD - +

TY-E Pleural effusion CAV, VP/CP None - - +

YR-A Subcutaneous lesion’ None Oral VP PD - +

HA-E Pleural effusion CAV, VP/CP. RT, Oral VP None - - +

“ Tumor cells from which the cell line was derived.

“ Therapy that the patient had received at the time that the cell line was established. CMC-VAP, cyclophosphamide, methotrexate, CCNU,vincristine, Adriamycin, procarbazine: CAV, cyclophosphamide, Adriamycin. and vincristine: VP/CP. VP-16 and cisplatin; RT, radiotherapy; Carb,

carboplatin; Mitox, mitoxantrone.

‘ Therapy that the patient received after the cell line was established.

“ The response of the patient to further chemotherapy (if given) after the cell line was established. CR, complete response: PR, partial response:

PD, progressive disease; NA, not assessable; -, patient was not treated or treatment status is unknown.‘. Needle aspirate.

116 Multidrug Resistance in Small Cell Lung Cancer

Table I SCLC cell lines

described previously (1 1 ). Nineteen of the cell lines were de-

rived from patients treated at this center, and the origin of those

lines obtained from other investigators has been noted previ-

ously ( 12). The doxorubicin-selected multidrug-resistant my-

eloma cell line, 82261Dox40, obtained from Dr. W. S. Dalton,

was used as a positive control for MDRI expression (13), and

the doxorubicin-selected multidrug-resistant SCLC cell line,

H69AR, was used as a positive control for MRP expression (14).

The cell lines were harvested in exponential growth phase

(approximately 2-5 X l06/ml).

Cryopreserved Tumor Samples. Tumor specimens

were obtained for diagnostic or therapeutic indications from

SCLC patients treated at the Kingston Regional Cancer Centre

from 1986 to 1994. With the patient’s informed consent, a

sample was sent for research purposes as part of a study ap-

proved by the Research Ethics Board of Queen’s University.

The source of the tumor cells and the treatment history of the

patients is indicated in Table 2. Effusion samples were centri-

fuged and washed in RPM! 1640 medium, and viable tumor

cells were separated from RBCs and non-viable cells on a

Ficoll-Hypaque density gradient. The proportion of tumor cells

present was determined by examination of cytospin prepara-

tions. Only samples containing more than 90% tumor cells were

cryopreserved. The cells were frozen in a viable state in 10%

DMSO in RPM! 1640 medium with 20% fetal bovine serum and

stored in liquid nitrogen. Cell lines were subsequently derived

from six of these tumor samples, as indicated in Table 2. In

addition, the cell line JN-M was established from a marrow

sample taken prior to treatment from the same patient as tumor

sample no. 4, which was a pleural effusion obtained at recur-

rence after chemotherapy treatment.

Drug Sensitivity Testing. The sensitivity of each cell

line was determined using a modified 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide assay (15). Four drugs, i.e.,

doxorubicin, vincristine, VP-I6, and cisplatin were tested, usu-

ally within 3 weeks of mRNA isolation. The dose-response

curves for the four drugs were summarized by calculating the

area under the curve, using the trapezoidal method as described

(15).

mRNA Isolation, cDNA Synthesis, and Oligonucleotide

Synthesis. Polyadenylated mRNA was isolated from approx-

imately i0� cells using the Micro-FastTrack mRNA isolation kit

(Invitrogen, San Diego, CA). The cDNA synthesis reaction

mixture consisted of 20 mr�i of each dinucleotide triphosphate

(Pharmacia, Biotech, Inc., Baie d’Urf#{233},Quebec, Canada), 10

mM Dli’, S ng/�i.1 random hexanucleotide primers (Pharmacia

Biotech, Inc.), I .35 units/p.l RNAguard (Pharmacia, Biotech,

Inc.), 1 unit/pA avian myeloblastosis virus reverse transcriptase

(Life Sciences, Inc., St. Petersburg, FL), and reverse tran-

scriptase buffer (40 mM MgCl2, 250 mrvi KC1, and 250 msi

Tris-HC1, pH 8.3, at 42#{176}C).mRNA (0.3 p.g) was added to each

20 �jJ cDNA synthesis reaction. To minimize variation between

samples, all cDNA synthesis reactions were carried out simul-

taneously under the same conditions. The cDNA synthesis re-

action was incubated at 37#{176}Cfor I h followed by enzyme



Clinical Cancer Research 117

Table 2 Clinical samples

The name of the corresponding cell line established from the same sample as in Table I is indicated. Cell line JN-M was established from a

marrow sample taken prior to treatment from the same patient as tumor sample no. 4. The results of RT-PCR screening for MRP and MDR I expression

are shown.

Sample no. Source” Prior treatment”

Subsequent

treatment’ Response”

Corresponding

cell line MDRI MRP

I Pleural effusion None None - HG-E - +

2 Pleural effusion None RT. CAV PR - - +

3 Pleural effusion VP/CP, CAV None - WL-E - +

4 Pleural effusion CAy, VP/CP None - JN�Me + +

5 Pleural effusion CAV VP/CP PD SV-E + +

6 Pleural effusion CAV None - - + -

7 Pleural effusion CAV, VP/CP None - - + +

8 Pericardial effusion CAV, VP/CP None - JS-E + -

9 Pleural effusion Oral VP None - LV-E + +

10 Pericardial effusion CAV. VP/CP. RT Oral VP PD GL-E - -

“ Tumor cells from which the cell line was derived.

h Therapy that the patient had received at the time that the cell line was established. CMC-VAP, cyclophosphamide. methotrexate. CCNU,

vincristine, Adriamycin, procarbazine: CAV. cyclophosphamide, Adriamycin. and vincristine; VP/CP. VP-l6 and cisplatin: RT. radiotherapy; Carb.

carboplatin; Mitox. mitoxantrone.

C Therapy that the patient received after the cell line was established.

�1 The response of the patient to further chemotherapy (if given) after the cell line was established. CR. complete response; PR. partial response:

PD, progressive disease; NA. not assessable; -, patient was not treated or treatment status is unknown.‘. The cell line JN-M was established from a bone marrow aspirate taken prior to therapy. A clinical sample from the same patient (no. 4) was

obtained at relapse following partial response to chemotherapy.

inactivation at 95#{176}Cfor 3 mm. The resulting cDNA was diluted

10-fold in sterile water.

The 25-mer primers specific for MRP, MDRI, and the

human TFRR were synthesized on a Biosearch 8750 DNA

synthesizer and purified by thin layer chromatography (Queen’s

University DNA Synthesis Laboratories, Kingston). The down-

stream (antisense) primers were biotinylated using biotin phos-

phoramidite (Prime Synthesis, Ashton, PA). The internal stand-

ard, TFRR, was used to control for variations in mRNA

extraction and cDNA synthesis. TFRR was selected as an inter-

nal standard because it is expressed ubiquitously at relatively

low levels (similar to that anticipated for MRP and MDRI), and

expression levels are not cell cycle dependent.

The sequences of the primers used for PCR are as

follows: MRP, upstream primer (5 ‘-AGTGACCTCTGGTC-

CTTAAACAAGG-3’) and downstream primer (5’-GAGG-

TAGAGAGCAAGGATGACTTGC-3 ‘); MDR 1 , upstream

primer (5 ‘-ACACCCGACTTACAGATGATGTCTC-3 ‘) and

downstream primer (5 ‘-CGAGATGGGTAACTGAAGT-

GAACAT-3’); TFRR, upstream primer (5’-GGATA-

I and downstream primer

(5 ‘-TGGAAGTAGCACGGAAGAAGTCTCC-3’).

RT-PCR and Q-PCR. Each PCR reaction contained

dinucleotide triphosphates ( 1 20 p.M; Pharmacia), Vent

NEBuffer (New England Biolabs, Mississauga, Ontario, Cana-

da), upstream (sense) primer (0.2 pM), downstream (antisense)

primer (0.2 pM), 0.005 unit/pA Vent DNA polymerase (New

England Biolabs), and cDNA (10 pi per 50 p.1 PCR reaction).

The reaction mixture was overlaid with light mineral oil. RT-

PCR screening of the cell lines and clinical samples was per-

formed independently with the primers specific for MRP,

MDRI, and TFRR.

The PCR was performed on a PHC-l Dri-Block thermo-

cycler (Techne, Cambridge, United Kingdom). Reactions were

allowed to proceed through one cDNA second strand synthesis

cycle of 95#{176}Cfor 1 .5 mm, 58#{176}Cfor I mm, and 72#{176}Cfor 3 mm,followed by 27 PCR cycles of 95#{176}Cfor 45 s, 58#{176}Cfor I mm,

and 72#{176}Cfor 1 mm, with a final PCR cycle of 95#{176}Cfor 45 s,

58#{176}Cfor I mm, and 72#{176}Cfor S mm. For reactions with the

MRP-specific primers, the annealing temperature was 52#{176}C.The

expression of MRP and TFRR was quantified by Q-PCR using

“mimic” standards in those cells lines which were shown to

express MRP by RT-PCR. The “gene-mimic” is a competitive

standard comprised of a heterologous Ba,nHIlEcoRI DNA frag-

ment of the human v-erbB gene (Clontech MIMIC Construction

kit, Mississauga, Ontario, Canada) that is flanked on either side

by the DNA sequence complementary to the gene-specific

primer of interest. This allows both the target cDNA and the

gene-mimic to be coamplified by the same primers. When the

target DNA and the gene-specific mimic DNA are present in

equal concentrations in the PCR reaction mixture, they compete

equally for the primers and result in equal quantities of ampli-

fication product.

For each cDNA sample, five PCR reactions were per-

formed using a 5-fold dilution series of the cDNA template. The

reaction components for each sample were assembled in a

master mixture, and a known amount of the gene-mimic stand-

ard was added, resulting in a series ofcompetitive reactions with

a constant concentration of the gene-mimic standard and van-

able concentrations of the target cDNA. For each sample. the

cDNA concentration at which the PCR product of the target

cDNA and the gene-mimic were present in equal amounts was

determined by interpolation. The level of MRP mRNA was

normalized to the level of TFRR expression in the same cell line

and then expressed as a percentage of the MRP level in H69AR

cells. The conditions for the Q-PCR were the same as for the

RT-PCR except that 36 cycles of amplification were carried out.



C-)

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VP-16

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C.)

‘4:

7c

Cisplatin

1o0�

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25

��HHnnnnnHHHHiffi�wwu�u�u�

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Cell Line

Fig. I Chemosensitivity was measured using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The dose-response

curves were summarized by calculating the AUC. The cell lines were ranked in order of AUC. Results for doxorubicin, vincristine, VP- 16, and

cisplatin are shown.


50

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Doxorubicin Vincristine

�

�thIc/)>.IO�,(coth>OL�O�Jc1: ,

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. C,)Cell Line

Chemiluminescent Detection of PCR Products. Thedownstream primers contained a biotin group to allow for

chemiluminescent detection of the PCR products. PCR-ampli-

fled samples were separated on a 2% agarose gel, stained with

ethidium bromide, and photographed. The PCR products were

then transferred to a Zetaprobe membrane (Bio-Rad, Missis-

sauga, Ontario, Canada) by downward alkaline transfer (0.4 M

NaOH, 0.6 M NaCI); the membrane was UV-irradiated and then

agitated briefly in neutralizing buffer (1.5 M NaCl, 0.5 M Tris,

pH 8.0). The membrane was blocked in SDS buffer (5% w/v

SDS, 17 mM Na2HPO4, and 8 mr�i NaH2PO4) for 30 mm,

agitated in blocking buffer containing 4 units/iOO ml streptavi-

din-alkaline phosphatase conjugate (Boehringer Mannheim) for

10 mm, washed for 15 mm in a 1:10 dilution of blocking buffer,

and finally agitated in a wash buffer (1 m�i MgC12, 10 mM NaC1,

and 10 mM Tris-HC1, pH 9.5) for 30 mm. Lumi-Phos 530

(Boehringer Mannheim, Laval, Quebec, Canada) was applied,

and the membrane was placed within plastic page protectors and

allowed to incubate at 37#{176}Cfor 1.5-2.5 h. X-OMAT film

(Kodak) was exposed to the membrane for 30-60 s at room

temperature. The intensity of the bands on the film was deter-

mined on a laser scanning densitometer (Molecular Dynamics,

Sunnyvale, CA) using Image Quant 3.3 software.

Correlation Analysis. Statistical analyses of drug sensi-

tivity, levels of MRP expression, and chemotherapy treatment

histories were performed using the Systat software package

(version 5.0 for DOS). The cell lines were classified as treated

or untreated according to whether the patients from whom the

cell lines were derived had received chemotherapy at the time

the cell line was established (Table 1). The drug sensitivity data

were normally distributed. In contrast, the MRP mRNA levels

were highly skewed toward low values. Consequently, a loga-

rithmic transformation of the MRP values was performed to

approximate more closely a normal distribution. The cell line

H69AR was not included in the correlation analysis, because

unlike the other cell lines, it had undergone in vitro selection in




Table 3 Pearson correlation analysis

Pearson correlation matrix is shown. The correlation coefficientsare indicated, and the P values indicating the statistical significance of

the correlations are shown in parentheses below the correlation coeffi-

cients.

Dox Vinc VP-l6 CisPt

Dox” 1.000

(0.000)Vinc” 0.739c

(<0.001)

1.000(0.000)

VP�l6a 0.360

(0.091)0.347

(0.104)1.000

(0.000)CisPt” 0.523k

(0.01 1)

0.624’

(0.001)0.539’

(0.008)1.000

(0.000)MRP” 0.422c

(0.045)0.336

(0.1 16)

0.205

(0.347)

0.080

(0.7 16)

a Correlation between sensitivity to doxorubicin (Dox), vincristine

(Vinc), VP-l6, and cisplatin (CisPt).b Correlation between drug sensitivity and MRP levels.

C Statistically significant correlations.

doxorubicin. Furthermore, this cell line is highly drug resistant

and has exceptionally high levels of MRP mRNA, resulting in

an undue influence on the correlation analysis. Pearson corre-

lation coefficients were calculated for all pairwise data combi-

nations, including sensitivity to doxorubicin, vincristine, VP-16,

and cisplatin, MRP mRNA levels, and treatment history.

RESULTS

Drug Sensitivity. The sensitivities of the 23 cell lines to

doxorubicin, vincristine, VP-16, and cisplatin are shown in Fig.

I . The dose-response curves were summarized by calculating

the AUC as described previously (15). The cell lines displayed

a spectrum of drug responsiveness. One of the cell lines, SV-E,

was highly resistant to the natural product drugs, doxorubicin,

vincristine, and VP-16, but had an intermediate level of sensi-

tivity to cisplatin. The cell line NCI-H69 (from which H69AR

was derived) was among the most sensitive to doxorubicin,

vincristine, and cisplatin but not to VP-l6. The results for

H69AR, which was the most resistant to doxorubicin and yin-

cristine, are shown for comparative purposes only.

The correlation between response to each of the four drugs

tested and response to the other three drugs is shown in Table 3.

Doxorubicin, vincristine, and VP-16 are natural products, and

resistance to these agents may be conferred by either MRP or

P-gp. On the other hand, neither MRP nor P-gp would be

expected to confer resistance to cisplatin. There was a highly

significant correlation between responsiveness to doxorubicin

and vincristine (r = 0.739; P = 0.0001). However, no signifi-

cant correlation was observed between VP-I 6 and either doxo-

rubicin (r = 0.360; P 0.091) or vincristine (r = 0.347; P =

0. 104). There was an unexpected correlation between respon-

siveness to cisplatin and the other drugs, including doxorubicin

(r 0.523; P = 0.010) and VP-16 (r 0.539; P = 0.008). The

association between cisplatin and vincristine was particularly

striking (r = 0.624; P 0.001).

Screening for MRP and MDR1 Expression in Cell Lines

and Clinical Samples. MRP expression was detected in 20 of

the 23 SCLC cell lines, whereas expression of MDRI was

detected in only 1 of 23 cell lines, i.e., SHP-77 (Table 1).

Because of the low mRNA yield from the clinical specimens, 33

cycles of amplification were required to obtain levels of the

control gene, TFRR, that were comparable to those obtained for

the cell lines. The results of screening the 10 cryopreserved

SCLC tumors for MRP and MDRI expression are shown in Fig.

2 and summarized in Table 2. Seven of the 10 samples ex-

pressed MRP, and 6 expressed MDR1. Of the 10 clinical sam-

ples, 6 were the original specimens from which cell lines were

derived. It is interesting to note that three of the clinical samples

with detectable MDRI expression (nos. 5, 8, and 9) cone-

sponded to cell lines that had no MDR1 expression (SV-E. JS-E.

and LV-E, respectively). Thus, it appears that the relative levels

of MDRI mRNA may have declined during in vitro culture.

Sample no. 4 was derived from the same patient as cell line

JN-M. The JN-M cell line (established from a bone marrow

aspirate obtained prior to chemotherapy) was negative for

MDRI but positive for MRP expression, whereas the clinical

sample (no. 4; a pleural effusion obtained from the same patient

at recurrence following partial response to chemotherapy) was

positive for both MRP and MDRI . Two of the clinical samples

(nos. 8 and 10) had no detectable MRP expression, whereas

MRP mRNA was detectable at very low levels in the cone-

sponding cell lines (JS-E and GL-E). Because only 10 clinical

samples were studied and only 3 of these samples were derived

from patients who subsequently received chemotherapy, it was

not possible to make any clinical correlations from this data.

Quantitation of MRP Expression in Cell Lines. Levels

of MRP mRNA expression were quantitated by Q-PCR in those

cell lines that were positive by RT-PCR. Since MDRI mRNA

was detectable in only one cell line (SHP-77), no attempt was

made to quantitate levels of expression of this gene. Because of

the limited amount of material available from the clinical spec-

imens, the levels of MRP and MDRI were not quantitated in

these samples.

An example of the Q-PCR for the cell line H69AR is

shown in Fig. 3. Serial 5-fold dilutions of the cDNA, along with

a constant amount of the “mimic” standard were amplifed using

TFRR- and MRP-specific primers. At high concentrations of

MRP cDNA, the mimic standard is not visible due to competi-

tion for primers, whereas at low concentrations of the MRP

cDNA, the “mimic” is able to compete for the primers and is,

therefore, amplified.

The relative levels of MRP expression in the cell lines are

shown in Fig. 4. The results are expressed as a percentage of the

levels in H69AR cells. Five of the 24 cell lines had no detectable

MRP expression, and in the remainder of the cell lines, the

levels ranged from 0. 1 to I 7.5% of the levels detected in

H69AR.

Correlation of MRP mRNA Expression and Drug Re-

sistance. The results of the Pearson correlation analysis are

shown in Table 3. The correlation between resistance to doxo-

rubicin and MRP expression by Q-PCR (r = 0.422) was signif-

icant (P = 0.045). Although there was a significant correlation

between response to doxorubicin and to vincristine (r = 0.739;

p � 0.001), the correlation between MRP mRNA levels and

vincristine resistance, although positive, was not statistically

significant (r = 0.336; P = 0.116). No correlation was found

between MRP levels and sensitivity to either VP-I6 or cisplatin.



123456 7 8 9 10 Sample#

�. *‘� 1r[10* 1* � 4pj� 4� 1� #{149}esa

MRP

MDR1

TFRR

cDNA dilution

.)n

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00

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EO


Fig. 2 Screening for MDRI

and MRP expression in cryo-

preserved SCLC samples. Tencryopreserved tumor samples

were screened for the expres-

sion of MDR1 and MRP usingRT-PCR as described in “Mate-

rials and Methods.” TFRR was

used as an internal control.

‘b ‘�‘.

(vcY�#{234}#{234}-_ 0. O� 0. 0.

TFRR

TFRR-MIMIC

MRP

MAP-MIMIC

Fig. 3 Quantitation of MRP expression using mimic standards. Serial5-fold dilutions of cDNA derived from cell line H69AR along with aconstant amount of the gene-mimic standard were amplified with prim-ers specific for TFRR and MRP.

There was no correlation between the chemotherapy treatment

histories of the patients from whom the cell lines were derived

and either MRP levels or response to doxorubicin, vincristine,

VP-l6, or cisplatin.

DISCUSSION

Ever since combination chemotherapy became the standard

treatment for SCLC nearly two decades ago, there have been no

major improvements in results of therapy of this disease. The

acquisition of resistance to multiple chemotherapeutic agents

continues to be the major impediment to cure (16). Despite the

efforts of many groups, the molecular basis of clinically ac-

quired resistance is not well understood. To identify mecha-

nisms of resistance, we and others have studied cell lines that

have been selected in vitro for drug resistance. Although a

variety of alterations associated with resistance have been iden-

tified, it remains to be determined whether such alterations are

present in patients with drug-resistant tumors and whether they

are responsible for the drug resistance phenotype. In this study,

we examined cell lines and clinical samples obtained directly

from patients with a spectrum of clinically drug-sensitive and

drug-resistant tumors (Tables 1 and 2). Such investigations may

give a better indication of mechanisms that are involved in

clinical drug resistance.

In the 23 SCLC cell lines examined in this study, there was

a close correlation between response to doxorubicin and to

vincristine. This finding was not unexpected, because doxoru-

bicin and vincristine are both natural product compounds that

1

10

0

Cell Line

Fig. 4 Quantitation of MRP expression SCLC cell lines by Q-PCR.The levels of MRP mRNA are expressed as a percentage of the levels in

H69AR. The cell lines have been ranked in order of MRP levels.

are included in the spectrum of cross-resistance that character-

izes the multidrug resistance phenotype. VP-16 is also included

in the muitidrug resistance phenotype, and thus it is somewhat

surprising that we detected no significant correlation between

response to VP-16 and response to either doxorubicin or yin-

cristine. On the other hand, there was also a close correlation

between response to cisplatin and to doxorubicin, vincristine,

and VP-16. Because cisplatin is not included in the multidrug

resistance phenotype, this finding suggests the presence of re-

sistance mechanisms other than MRP or P-gp in these uns-

elected SCLC cell lines.

Only one of the cell lines, i.e., SHP-77, was positive for

MDR1 expression by RT-PCR. SHP-77 also expressed MRP and

was one of the most resistant cell lines to both doxorubicin and

vincristine but was not highly resistant to VP-16 or cisplatin

(Fig. 1). In contrast to the cell lines, 6 of 10 clinical samples of

SCLC expressed MDR1 (Fig. 2).

P-gp expression does not occur frequently in multidrug-

resistant SCLC cell lines, although it has been detected (17). Lai

et a!. (4) measured expression of MDR1 in lung cancers of all

major histological types as well as corresponding normal lung

tissues and tumor cell lines. In most of these tumors, including

the SCLC samples, the expression of MDR1 mRNA was low or

undetectable. However, it is interesting to note that in three of

the four SCLC samples in which MDR1 levels were measured in

both tumor samples and corresponding cell lines, there was a

decline in MDR1 expression in the derived cell line.

In contrast, two reports suggest that there may be a relation-




ship between clinical drug resistance and MDRI levels in SCLC.

Holzmayer et a!. (18) found that the presence of even very low

levels of MDR1 expression, as detected by PCR, correlated with

lack of response to chemotherapy in seven tumor samples from

SCLC patients. Poupon et aL (19) used Northern blot analysis to

detect MDR1 mRNA in xenografts derived from seven SCLC

patients. Expression of this gene was detected in all but two of the

xenografts, and these two samples corresponded to the two patients

in the series who were long-term survivors. They also noted that the

levels of MDR1 mRNA were higher in tumor samples obtained

directly from patients compared with those which had been pas-

saged in nude mice. Although the results are suggestive, larger

studies are required before firm conclusions can be drawn about the

clinical relevance of MDR1 expression in SCLC.

In our study, MDR1 expression was detected more fre-

quently in clinical samples than in cell lines established from

SCLC patients. Furthermore, in some cases, MDR1 expression

was detectable in the clinical sample, whereas the cell line

established from the same material was negative. In human

tumor cells, increased levels of P-gp are most often due to

increased gene expression rather than gene amplification (16).

This may explain the lack of persistence of P-gp overexpression

when the tumor cells are propagated in vitro. Another possibility

is that the cell types that become established as permanent cell

lines may not reflect the heterogeneity of tumor cells present in

the original sample. Furthermore, small numbers of contaminat-

ing nonmalignant cells in the clinical samples could lead to

false-positive results. The frequent detection of P-gp in clinical

samples of SCLC rather than established cell lines suggests that

this transport protein could play a more significant role in

clinically acquired multidrug resistance in SCLC than previ-

ously thought.

On initial screening, we detected MRP expression in 20 of

our 23 cell lines (83%). It is interesting to note that the cell line

with the highest level of MRP mRNA (SV-E) was also among

the most resistant to doxorubicin, vincristine, and VP-16 but did

not express MDR1 mRNA. However, despite the fact that the

relative resistance of the SV-E cell line to these agents was

comparable to that of H69AR, the level of MRP mRNA expres-

sion in this line was only 17.5% of that of H69AR, suggesting

that MRP expression may not be the only factor accounting for

the resistance of this cell line. In the panel of cell lines, there

was a significant correlation between MRP mRNA levels and

doxorubicin resistance. Although vincristine resistance cone-

lated strongly with doxorubicin resistance, the correlation be-

tween vincristine resistance and MRP mRNA levels, although

positive, was not statistically significant. Furthermore, there was

no apparent correlation between levels of MRP mRNA and

response to VP-16. Although VP-16 is one of the drugs to which

MRP confers resistance, it is possible that other resistance

mechanisms, such as altered topoisomerase II, may obscure

significant correlations with MRP levels. MRP does not confer

resistance to cisplatin (9, 10), and the lack of correlation be-

tween MRP mRNA levels and cisplatin resistance was not

surprising.

It is possible that the relationship between drug response

and MRP expression may be stronger than the correlation anal-

ysis would seem to indicate. As noted, there are other drug

resistance mechanisms that may be involved in SCLC, and these

could obscure significant relationships with drug response. For

example, our analyses showed strong correlations between cis-

platin resistance and resistance to doxorubicin, vincristine, and

VP-16, suggesting the presence of other mechanisms that confer

resistance to all four of these drugs. The correlation coefficients

were similar when only those cell lines derived from untreated

patients were included in the analysis, indicating that these

relationships do not result from prior treatment with these four

drugs. Because cisplatin resistance is not conferred by MRP (9,

10) (and does not correlate with MRP levels in this study), the

correlations of doxorubicin, vincristine, and VP-16 with MRP

mRNA levels could be obscured.

Although increased MRP and MDR1 expression have been

clearly implicated in the resistance of certain tumors, they are

not the only factors that may result in multidrug resistance. For

example, resistance to multiple chemotherapeutic agents has

been associated with increased drug detoxification by glutathi-

one and its associated enzymes (20). Although alterations of

glutathione and related enzymes have been detected in multi-

drug-resistant SCLC cell lines, the functional significance of

these changes remains to be determined (21, 22). In a study

using many of these same unselected SCLC cell lines, we found

no significant correlation between drug response and levels of

GSH and associated enzymes (12).

Increasing evidence has indicated that reduced levels or

function of topoisomerase II are important factors in drug re-

sistance in SCLC as well as other cancers. It is now recognized

that topoisomerase II is the common intracellular target for

several of the natural product drugs that are also part of the

classical “multidrug resistance phenotype.” In this collection of

SCLC cell lines, we have shown that reduced levels of topoi-

somerase IIcz correlate with resistance to a variety of agents,

including some drugs that are not known to exert their cytotox-

icity through this target (23). Other studies using unselected

lung cancer cell lines have also shown an inverse correlation

between topoisomerase II levels and drug resistance (24, 25).

In summary, the data presented here indicate that drug resist-

ance in SCLC is complex and unlikely to be explained by a single

resistance mechanism. Our results further emphasize the impor-

tance of examining clinical samples directly to identify clinically

important resistance mechanisms. Results obtained with estab-

lished cell lines may be misleading, in view of the discordance that

we observed in MDRI expression in tumor samples and cell lines

established from these samples. However, the analysis of clinical

samples is technically demanding and results may be difficult to

interpret. Often the diagnosis of SCLC is made on very small

amounts of tumor tissue, which may be partially necrotic. Further-

more, tumor heterogeneity and infiltration with nonmalignant cells

may pose significant problems.

The ideal method for measuring levels of drug resistance

genes or proteins in clinical samples should be sensitive, repro-

ducible, applicable to small samples, and capable of distinguish-

ing between nonmalignant infiltrating cells and tumor cells. The

quantitative PCR technique used in this study fulfills some of

these criteria but requires a homogeneous population of tumor

cells. Normal bronchial epithelium is known to express MRP,

and this could complicate the analysis of MRP expression in

lung tumor samples. In some cases, measurement of protein

levels may be more informative than gene expression levels.




4 Unpublished observations.

However, the quantitation of MRP protein may be complicated

by its relatively high sensitivity to proteolytic degradation.4 The

use of immunohistochemistry for detecting multidrug resistance

proteins at the cellular level has a number of advantages but may

not necessarily reflect levels of functional protein.

Despite the infrequent expression of P-gp in SCLC cell

lines, this transport protein appears to be more frequently de-

tectable in clinical samples of these tumors. Thus, it is possible

that P-gp may play a significant role in clinical drug resistance

in SCLC. The multidrug resistance protein MRP is detectable in

a majority of cell lines as well as clinical samples of SCLC and

correlates with resistance to doxorubicin in SCLC cell lines. The

difference in expression of these multidrug resistance genes

between cell lines and clinical samples emphasizes the impor-

tance of studying clinical material and correlating results with

drug responsiveness and clinical outcome. An understanding of

clinically significant mechanisms of resistance in SCLC may

lead to effective strategies to overcome this important clinical

problem.

ACKNOWLEDGMENTS

We thank Monika Vasa, Iva Kosatka, and Heather Baker for

excellent technical assistance and Drs. Nidhi Jam and Shelagh Mirski

for helpful suggestions.

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1997;3:115-122. Clin Cancer Res B G Campling, L C Young, K A Baer, et al. small cell lung cancer.Expression of the MRP and MDR1 multidrug resistance genes in

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