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(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- 2594 Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1987 American Association for Cancer Research. MULTIDRUG RESISTANT SMALL CELL LUNG CANCER 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 2595 Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1987 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1987 American Association for Cancer Research. 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 2597 Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1987 American Association for Cancer Research. MULTIDRUG RESISTANT 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. 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A., Chang, T.-d., Melera, P. W., and Biedler, J. L. Gene amplification-associated cytogenetic aberrations and protein changes in vincristine-resistant Chinese hamster, mouse, and human cells. J. Cell Biol., 700: 588-597, 1985. 2598 Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1987 American Association for Cancer Research. 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/47/10/2594 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 14, 2017. © 1987 American Association for Cancer Research.