Download Multidrug Resistance in a Human Small Cell Lung Cancer Cell Line

(CANCER RESEARCH 47, 2594-2598, May 15, 1987]
Multidrug Resistance in a Human Small Cell Lung Cancer Cell Line Selected
in Adriamycin1
Shelagh E. L. Mirski, James H. Gerlach, and Susan P. C. Cole2
Ontario Cancer Treatment and Research Foundation, Kingston Regional Cancer Centre, Kingston, Ontario, Canada K7L 2V7 fS. P. C. C.J; Departments of Oncology
fS. E. L. M., S. P. C. C.J and Microbiology and Immunology {S. P. C. C.J, Queen's University, Kingston, Ontario, Canada K7L 3N6; and Ontario Cancer Institute,
Princess Margaret Hospital, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M4X1K9 [J. H. G.J
ABSTRACT
A multidrug resistant variant (H69AR) of the human small cell lung
cancer cell line NCI-H69 was obtained by culturing these cells in grad
ually increasing doses of Adriamycin up to 0.8 *tMafter a total of 14
months. H69AR expresses the multidrug resistant phenotype because it
is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide,
gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine. H69AR is also similar to other multidrug resistant cell lines in
that it displays little or no cross-resistance to bleomycin, S-fluorouracil,
and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine. H69AR has increased cell-cell adhesiveness com
pared to H69, but a similar growth rate in vitro and tumorigenicity in
nude mice. When cultured in the absence of Adriamycin, there is a 40%
decrease in resistance by 35 days of culture, compared to cells in
continuous culture in drug, but no further decrease in resistance up to
181 days. Monoclonal antibodies to P-glycoprotein have no detectable
reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques. Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced
levels of P-glycoprotein. H69AR will provide a useful model for the study
of multidrug resistance in human small cell lung cancer.
INTRODUCTION
Lung cancer is the leading cause of cancer death in American
men aged 35 or older and is increasing in incidence in women
in whom it is predicted that it will surpass breast cancer as the
leading cause of cancer death during the 1980s (1). Twenty-five
% of autopsied lung cancer patients have the histológica! type
designated "small cell." The histológica! distinction between
S( ï<" and nonsmall cell lung cancer is of major clinical
importance because of the different responses to therapy of
these two tumor types. Non-SCLC is treated with surgery and/
or radiotherapy. SCLC does not respond well to surgery or
radiotherapy alone but regimens of combination chemotherapy
or chemotherapy and radiotherapy have resulted in increases in
median survival to 1 year compared to 7 weeks with supportive
care only. However subsequent relapse is common with a 2year survival of only 10% (2). The high rate of relapse and
failure of chemotherapy is believed to be due to a large degree
to drug resistant cells either existing prior to or arising during
treatment.
A MDR phenotype has been observed in a variety of mam
malian cell lines which provides a model for the study of this
clinical problem (3). Even though selected by a single agent,
Received 9/29/86; revised 12/18/86; accepted 2/19/87.
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 in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by grants to S. P. C. C. from the Medical Research Council of
Canada (MA 9355) and the Clare Nelson Bequest of Kingston General Hospital.
2To whom requests for reprints should be addressed.
3The abbreviations used are: SCLC, small cell lung cancer; MAb, monoclonal
antibody; PBS, phosphate-buffered saline; ADM, Adriamycin; MDR, multidrug
resistant(ce); FBS, fetal bovine serum; CHO, Chinese hamster ovary; MTT, 3|4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide; ELISA, enzymelinked immunosorbent assay; ID»,mean drug dose that inhibits growth by 50%.
these cell lines are resistant to a wide range of chemically and
functionally unrelated drugs. In most cases, this cross-resistance
has been closely associated with the increased expression of Pglycoprotein, a M, 170,000 plasma membrane glycoprotein (3,
4). Because of the clinical importance of MDR in SCLC, we
have derived a MDR variant (designated H69AR) of the human
SCLC cell line NCI-H69 using doxorubicin (ADM) as the
selecting drug. In this paper we describe the preliminary char
acterization of this cell line and show that, although it exhibits
the MDR phenotype, enhanced expression of P-glycoprotein is
not detectable.
MATERIALS AND METHODS
Drugs. Gramicidin D, 1-dehydrotestosterone, lidocaine, 5/3-pregnan3a-ol-20-one, 5j3-pregnan-3j3-ol-20-one, deoxycortisone, dexamethasone, dibucaine, tetracaine, and procaine were obtained from Sigma
Chemical Co. Acivicin was the generous gift of Dr. R. A. Whitney,
Department of Chemistry, Queen's University. The remaining drugs
were obtained from the pharmacy at the Kingston Regional Cancer
Centre.
Cell Culture. The human SCLC cell line NCI-H69 (H69) was kindly
provided by J. Minna (National Cancer Institute, Bethesda, MD). It
was routinely cultured in RPMI 1640 medium (GIBCO) supplemented
with 5 or 10% heat-inactivated FBS, 4 HIM L-glutamine, 50 nM 2mercaptoethanol, and 1 HIMsodium pyruvate. CHO cell lines AuxBl
and CHRCS (S) were cultured in a-minimal essential medium supple
mented with 10% FBS, 4 HIML-glutamine, 50 MM2-mercaptoethanol,
and 1 mM sodium pyruvate. Cultures were checked monthly for Mycoplasma contamination using the 4',6-diamidino-2-pheny!indole
DNA-binding assay (6) and found to be negative.
A multidrug resistant subline of H69 was obtained by culturing the
cells in gradually increasing doses of ADM. After 8 months, cells which
grew in 0.4 ¡MI
ADM were obtained. After a further 6 months, cells
which grew in 0.8 ^M ADM were obtained. This cell line has been
designated H69AR and has been maintained by alternate feedings with
drug-free medium or medium containing 0.8 «¿M
ADM.
The stability of the resistant phenotype was determined by culturing
continuously in medium with either 0.8 ¿>M
ADM or no drug and
assessing relative resistance after various periods of time up to 5
months.
Attempts to derive multidrug resistant variants of other SCLC lines
including MAR (a generous gift of Prof. A. Neville, Ludwig Institute
for Cancer Research, London, United Kingdom), NCI-H209, NCIHI 28 (generous gifts of J. Minna, National Cancer Institute), and QU
AD (7) in liquid culture or in soft agar have been unsuccessful to date.
Growth Curve. The growth curves of H69AR and H69 were deter
mined by seeding 1 x 10s cells/ml in triplicate wells in a 24-well plate
(Costar). Cell counts were done on days 3, 4, and 7 using a hemocytometer with trypan blue exclusion as an indicator of viability.
Tumorigenicity in Athymic Mice. The tumorigenicity of H69 and
H69AR was determined by s.c. injection of 1.3 x IO7 viable cells of
each type in a volume of 0.2 ml PBS into the left flank of 5- to 6-weekold male BALB/c nu/nu mice. Tumor size was measured in two
dimensions, and the area was estimated. Tumors that developed after
injection of H69AR were excised, placed into culture, and tested for
sensitivity to ADM after 4 weeks. The experiment was performed three
times with 3-4 mice in each experimental group.
Chemosensitivity Testing. The resistance of H69AR to other antineo-
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MULTIDRUG
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plastic agents and its collateral sensitivity to a number of anesthetics
and steroids were tested using the MTT assay (8). In brief, H69 and
H69AR cells were harvested by centrifugation 48-72 h after feeding
and plated at 2.5 x 10*cells/well in 96-well plates. Preliminary exper
iments showed that cultures initiated at this cell density continue to
grow exponentially for at least 7 days. After incubation at 37°Cfor 24 h, drugs were added and the plate incubated at 37°Cfor 7 days. Three
100
h before the end of drug exposure time, 25 n\ of MTT (Sigma; 2 mg/
ml in PBS) were added and the plate incubated at 37°Cfor an additional
3 h. Isopropanol:! N HC1 (25:1) was added to solubilize the forma/an
crystals, and then the absorbance at 570 nm was determined using a
Dynatech MR600 microtitre plate reader. Within each experiment,
determinations were done in quadruplicate, and each drug was tested
in at least two separate experiments in most cases. Controls included
wells with cells but no drugs (base line) and wells with medium and the
highest drug concentration but no cells. The percentage of viability was
expressed as a percentage of the base-line absorbance at 570 nm. The
relative resistance of H69AR compared to H69 is expressed as the ratio
of drug concentrations which decrease the base-line absorbance by 50%.
The H69AR cell line was cultured in drug-free medium at least 48 h
before testing.
Cell ELISA. The expression of P-glycoprotein as detected by the
MAb C219 (9) was assessed in a cell ELISA ( 10). H69, H69AR, AuxB 1,
and CHRC5 were washed with PBS and fixed in 70% methanol at
-20°C for 5 min. The cell concentration was adjusted such that 5 x
10" cells in 50 ^1 PBS were layered per well in a 96-well plate (Falcon
3912). The plates were dried overnight in a 37'C warm room. The cells
-00
overnight with shaking. The blot was incubated with approximately 5
x 10* cpm/ml of I25l-labeled MAb for 16 h at 4'C with shaking. The
blot was washed extensively in PBS, dried, and exposed with intensi
fying screens on Kodak X-AR5 film at -70°C.
-4
LOG DRUG CONC (;jM)
Fig. 1. Resistance to Adriamycin of H69, H69AR, and H69AR which had
been passed as a solid tumor in BALB/c nu/nu mice and subsequently cultured
I'Mvitro (H69AR passed in mouse). D, 116'»;
O, H69AR passed in mouse; •,
H69AR. Points, mean of quadruplicate determinations. SEs were less than 10%
of the mean values and have been omitted for clarity. CONC, concentration.
Table 1 Stability ofH69AR resistance to ADM
Time (days)35
were rehydrated in PBS and nonspecific binding was blocked with 1%
bovine serum albumin/5% normal goat serum. MAb C219, an irrelevant
MAb 1-7-1 (11), or medium were added to the wells and binding of
antibody detected using horseradish peroxidase conjugated goat antimouse IgG plus IgA plus IgM affinity purified F(ab'): fragments
(Cappel No. 23470) with o-phenylenediamine and hydrogen peroxide
used as substrates. Color development was measured by scanning at
490 nm on a Dynatek MR600 microtitre plate reader.
P-GIycoprotein Detection by Western Blotting. Radioiodinated MAb
C219 and C494 were used to detect P-glycoprotein in crude membrane
preparations of H69 and H69AR as described previously (9). Membrane
preparations of AuxBl and CHRC5 were included as controls in all
steps of the procedure. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was performed using a modification (12) of the proce
dure of Fairbanks et al. (13) and the gel was replica blotted onto
nitrocellulose paper essentially by the method of Towbin et al. (14).
Nonspecific binding of antibody was blocked with 10% bovine serum
albumin in PBS and 15 HIMsodium azide for 2 h at 37°Cor at 4'C
-4.7
off
ADM*0.49
on
ADM'0.78
49
1.89
2.61
IOS
1.36
2.51
140
0.89
3.98
171
0.91
1.47
181ID«,'H69AR
1.20(MM)H69AR
2.00
" Assessed in quadruplicate using the MTT assay.
* Cells were cultured in the absence of ADM from day 0.
c Cells were cultured in the presence of 0.8 ^M from day 0.
experiments, using a total of 11 mice given injections of H69
and 12 mice given injections of H69AR, there was no significant
difference in the rate of tumor growth.
Stability of the Resistant Phenotype. The drug resistance
phenotype of H69AR was stable when these cells were passaged
in BALB/c nu/nu mice as solid tumors and returned to culture
for 4 weeks in the absence of ADM before testing for drug
resistance (Fig. 1).
The resistance of H69AR which had been grown in the
absence of drug was compared to H69AR grown continuously
in 0.8 MMADM using the MTT assay (Table 1). By 35 days of
culture without'ADM, the ID50 of H69AR cells had decreased
A multidrug resistant variant of H69 was obtained by culturing these cells in gradually increasing doses of ADM up to 0.8
UM after a total of 14 months.
Relative Resistance to ADM. The relative resistance to ADM
of H69AR compared to H69 was determined using the MTT
assay (Fig. 1). In this experiment the ratio of the ID50 for each
cell line indicates a 32-fold relative resistance to ADM of
H69AR compared to H69. The results from additional experi
ments are presented in Tables 2 and 3.
Growth of H69 and H69AR in Vitro. When grown in RPMI/
10% FBS in the absence of ADM and at a starting cell density
of 1 x 10* cells/ml, H69 and H69AR have the same rate of
to about 60% of that of H69AR cultured continuously in the
presence of 0.8 /¿M
ADM. Although there was some variation,
the resistance of cells cultured without ADM remained at about
60% of those grown continuously in ADM up to 181 days in
culture.
Multidrug Resistance of H69AR. To determine whether
H69AR exhibited the MDR phenotype, the relative resistance
of H69AR compared to H69 was assessed with a panel of drugs
(Table 2). These results show that H69AR expresses the MDR
phenotype as it is cross-resistant to anthracycline analogues
including daunomycin, epirubicin, menogaril, mitoxantrone, as
well as to acivicin, etoposide, gramicidin D, colchicine, and the
Vinca alkaloids, vincristine and vinblastine.
Although H69AR was resistant to colchicine and vincristine,
the dose/response curves to these drugs were unusual. H69 cells
were very sensitive to these drugs (ID50 < 10~5 n\i). With
growth with a doubling time of about 24 h (Fig. 2).
Tumorigenicity. The tumorigenicity of H69 and H69AR was
assessed by measuring tumor size in BALB/c nu/nu mice after
s.c. inoculation of 1.3 x IO7 cells (Fig. 3). In three separate
H69AR cells, the viability decreased to a certain level (usually
less than 50%) and then did not decrease any further with an
increase in drug dose. For example, in one experiment, viability
remained at 35% of control values from 0.001-100 UMcolchi-
RESULTS
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MULTIDRUG
RESISTANT SMALL CELL LUNG CANCER
Table 2 Relative resistance ofH69AR compared to H69
(MM)DrugAdriamycinDaunomycinEpirubicinMenogari!MitoxanlroncColchicineVincristineH69AR3.16
ID»"
resistance*100.041.3
0.825
1.993.98
0.348
0.3160.619
0.020
0.0250.047
<0.001
0.0005<0.001
79.684.7
>348.0
632.0>619.0
LU
OD
0.468
0.6310.448
0.422
0.39810.0
0.0150.006
0.0003
6.310-'0.050
x
0.008
0.0040.631
«0.01<0.001
1,560.0
10,000.09.052.8
99.515.8
»1.5>6.0
02468
>1.00
0.00075.6
<0.000110-'5x
x IO-2
4.5 x IO-4
Etoposide
6.3
13.2
5-Fluorouracil
24.0
Bleomycin (milli1.62.010.0
units/ml)CarboplatinAcivicinGramicidin
TIME
2.82 x IO"5
0.39
0.22
13.5
1.62.00.47
(DAYS)
Fig. 2. Growth of H69 and H69AR in vitro in the absence of ADM. Cultures
were set up in triplicate wells at 1 x 10*cells/ml on day 0. Results are from one
experiment and similar results were obtained in a second experiment. O, H69AR,
mean ±SE (hur\): •.H69, mean ±SE.
IO-4
7.9
3.2
<1 x 10-'Relative
5.5 x IO-1H690.0316
Vinblastine
>104
7,000112.0
2.5
>55.0
15.8
500
16.2
59.0
400
1.8
1.01.021.3
300
Lu
8.0
28.20.010
0.71.78«0.003
11.4
15.9»3.322,430.0
D1.6
1.I2X 10-'2.5
0.25.63
°Assessed in quadruplicate by the MTT assay. Each line is the result from one
experiment.
* Ratio of ID»H69AR/ID„H69.
M
200
CC
O
100
10
Table 3 H69AR tested for collateral sensitivity to various drugs
TIME
20
(DAYS)
30
40
Fig. 3. Tumorigenicity of H69 and H69AR cells in nude mice. Each mouse
resistance'100.00.711.01.01.01.010.00.631.01.063.01.0
received a s.c. innoculation of 1.3 x IO7cells in 0.2 ml PBS on day 0. •,H69; O,
H69AR. Curves, tumor growth in an individual mouse. Results are from one
-Dehydrotestosterone50-Pregnan-3a-ol-20-one5/3-Pregnan-3/3-ol-20-oneDeoxycortisoneDexamethasoneAdriamycinLidocaineDibucaineTetracaineAdriamycinProcaineID»H69
experiment. Similar results were obtained in two additional experiments.
Experiment"123DrugAdriamycin1
MTT assay because it was also observed in experiments where
viable cells were counted using a hemocytometer and trypan
blue as an indicator of viability.
Collateral Sensitivity. To determine whether H69AR exhib
ited collateral sensitivity, the effect of a panel of steroids and
anesthetics on cell viability was assessed (Table 3). H69AR
showed a slightly enhanced sensitivity to 1-dehydrotestosterone
and to lidocaine compared to H69 (relative resistance 0.71 and
" Adriamycin is included in each experiment as a positive control, demonstrat
0.63, respectively). H69 and H69AR were equally sensitive to
ing the relative resistance of H69AR to this drug.
5/8-pregnan-3/3-ol-20-one, 5/î-pregnan-3a-ol-20-one, deoxycor* Assessed in quadruplicate by MTT assay.
' Ratio of ID5oH69AR/ID»H69.
tisone, dexamethasone, dibucaine, tetracaine, and procaine.
P-Glycoprotein. P-Glycoprotein is recognized in Chinese and
cine. Similarly, in another experiment, H69AR cultures re
Syrian hamster, mouse, and human MDR cell lines by M Ah
mained 30% viable over the range of 0.0001-60 /¿M
vincristine.
C219, and in Chinese and Syrian hamster and human cell lines
The ID50 for both H69 and H69AR exposed to vinblastine was by MAb C494 (9). To determine whether H69AR expressed Plower than the lowest drug concentration tested (10~3-10~6 ¿¿M
glycoprotein, H69AR and H69 were tested in a cell ELISA
using MAb C219 (Fig. 4). AuxBl was negative and CHRC5 was
in three experiments) but only with H69AR did the viability
plateau. This phenomenon was not due to an artifact of the positive for P-glycoprotein as expected. Expression of P-gly2596
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MULTIDRUG
RESISTANT
SMALL CELL LUNG CANCER
H69AR is similar to other human MDR lines in that it
displays little or no cross-resistance to bleomycin, 5-fluorouracil, and platinum-containing drugs (3, 4, 16, 21, 22) (Table 2).
In the CHO derived MDR cell lines, a collateral sensitivity has
been observed to some local anesthetics and hormones (28). In
apparent contrast to the CHO cells, H69AR has only a slight
collateral sensitivity to l-de hydro testosterone and to lidocaine
and is equally as sensitive as H69 to the remaining steroids and
anesthetics tested (Table 3).
Multidrug resistant Chinese hamster lung cells described by
Biedler et al. (29) displayed an altered cell morphology and
patterns of growth including increased cell-substrate and cellcell adhesiveness and weak tumorigenicity /'// vivo compared to
0.3
0.2
N
O)
0.1
m o
o
01
<D
X
No 1st Ab
1-7-1
ei
IO
X
C 219
Fig. 4. Immimologie;!I detection of P-glycoprotein with MAb C219 in a cell
ELISA. Bars, mean absórbante of tests performed in duplicate. Negative controls
included wells with an irrelevant MAb (1-7-1) or no first antibody.
coprotein was not detectable above background levels in either
H69 or H69AR.
H69 and H69AR were also tested for P-glycoprotein by
immunoblotting.
Crude membrane fractions of AuxBl,
CHRC5, H69, and H69AR were made, separated on electrophoretic gels, and replica blotted on to nitrocellulose paper.
The blot was incubated with '"I-labeled C219 or C494. Autoradiographs showed a band corresponding to P-glycoprotein in
extracts of CHRC5 as expected, but not in AuxBl, H69, or
H69AR (results not shown).
DISCUSSION
By continuous culture of the human SCLC cell line H69 in
gradually increasing doses of ADM we have obtained over a
period of 14 months a cell line, H69AR, which will grow in 0.8
iiM ADM. When compared to its parent line, H69AR is 10- to
100-fold more resistant to ADM. H69AR is also resistant to a
panel of anthracycline analogues, Vinca alkaloids, colchicine,
acivicin, and etoposide (Table 2) and thus possesses the MDR
phenotype which has been described in numerous rodent and
human cell lines (3, 4, 15-26).
Variation in the level of resistance of H69AR (but not the
pattern of resistance) was observed with all drugs tested. Such
variation has been noted by others (22). We have no explanation
for this phenomenon but note that it is composed of variations
in the fix,, for both the parent (H69) and resistant line
(H69AR). The unusual dose response curves with the Vinca
alkaloids in which total cell kill is not achieved even at high
drug concentrations have been observed by others. For example,
Hill and Bellamy (27) have described a HN-1 human squamous
cell carcinoma line and its VP 16-213 resistant variant VPR,
which exhibited a plateau survival fraction after exposure to
vincristine. The VPR cells had a decreased slope in the expo
nential region and a higher plateau level of survival, indicating
cross-resistance to vincristine when cell survival was assessed
by colony formation in soft agar.
sensitive cells. H69AR also displayed increased cell-cell adhe
siveness, growing in spheroids rather than loose aggregates as
did the parent H69. However, in contrast to the results of
Biedler et al. (29), there was no significant difference between
H69 and H69AR growth rates in vitro or tumorigenicity in vivo.
Similar in vitro growth characteristics for paired sensitive and
resistant human leukemia cell lines have been observed previ
ously (17, 19). In contrast, the human sarcoma MDR cell line,
MES-SA, described by Harker and Sikic (21), and the SCLC
MDR cell line, H69/LX4, described by Twentyman et al. (22)
grew at about 70% of the rate of the parent line in vitro. Whether
the altered growth characteristics observed in these cells reflect
cell membrane changes which may be involved in the mecha
nism of MDR is unknown and remains to be determined.
The experiments aimed at determining the stability of multidrug resistance suggest that the drug resistant phenotype of
H69AR may be complex and possibly has two components.
One component is lost within 35 days of culture in the absence
of ADM, resulting in a 40% decrease in resistance compared
to cells in continuous culture in drug. The second component
is relatively stable, so that cells cultured in drug free medium
for up to 181 days are still 60% as resistant as those cultured
with ADM. Similarly, Twentyman et al. (22) found a partial
loss of resistance in H69/LX4 within 3 weeks, but no further
loss up to 9 weeks in drug-free medium.
The MDR phenotype has been associated with changes in
cellular protein composition and gene amplification (30, 31).
In particular, the expression of P-glycoprotein, a M, 170,000
plasma membrane-associated glycoprotein, is elevated in MDR
cell lines (4,16, 21, 25, 26, 32-34). In addition, P-glycoprotein
overexpression has been detected in tumor samples from pa
tients with ovarian carcinoma who were resistant to multidrug
therapy (35). Although the exact function of P-glycoprotein is
unknown, it is the molecular alteration found to be most
consistently associated with the MDR phenotype (4). The level
of P-glycoprotein expression has been correlated with the de
gree of resistance (32), and the transfer of genomic DNA from
an MDR line to a sensitive line resulted in the acquisition of
both the MDR phenotype and P-glycoprotein (12). Recently, a
complementary DNA has been isolated which confers multidrug resistance in a drug-sensitive cell, clearly demonstrating a
functional role for P-glycoprotein in the expression of the MDR
phenotype (36). To our knowledge, H69AR is the first MDR
cell line in which P-glycoprotein is not detectable using imnni
nological detection methods. The possibility exists that H69AR
expresses a form of P-glycoprotein that is not detected by MAbs
C219 and C494. However, Southern and RNA blot analysis
with the pCHPl probe (31) for P-glycoprotein indicates that
the gene for P-glycoprotein is not amplified, rearranged, or
overexpressed in H69AR cells (within the boundaries recog
nized by this probe) (37). Taken together, these results appear
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MULTIDRUG
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SMALL CELL LUNG CANCER
to suggest that cellular changes other than enhanced expression
of P-glycoprotein are responsible for the M DR phenotype
observed in H69AR cells.
The development of drug resistance has been associated with
specific chromosomal alterations: homogeneously staining re
gions or double minute chromosomes (38, 39). Karyotypic
analysis of H69 and H69AR showed a marked increase in the
number of double minute chromosomes per cell in the drug
resistant cells compared to the parental cells, with the number
of double minute chromosomes per cell returning to near pa
rental levels in cells cultured in the absence of drug (37). The
relationship of this observation to the multidrug resistance of
H69AR is unknown at the present time.
We have recently produced a panel of murine Mabs specific
for H69AR cells. These MAbs will be useful in the isolation
and characterization of membrane components involved in the
M DR phenotype in H69AR SCLC cells. Furthermore, these
MAbs may prove to be valuable aids in the detection of drugresistant cells in SCLC patients.
ACKNOWLEDGMENTS
We wish to thank Norbert Kartner for his generous provision of
MAbs C219 and C494 and Dr. Victor Ling and Dr. Jeff Trent for
helpful discussions. The excellent technical assistance of Ingrid Lou wman, Elisabeth Vreeken, and Deanna Evernden-Porelle is gratefully
acknowledged. This paper is dedicated to the memory of Ingrid Louwman.
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Multidrug Resistance in a Human Small Cell Lung Cancer Cell
Line Selected in Adriamycin
Shelagh E. L. Mirski, James H. Gerlach and Susan P. C. Cole
Cancer Res 1987;47:2594-2598.
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