J Neurosurg 76:502-506, 1992

Development of multidrug resistance in a primitive neuroectodermal tumor cell line DAVID M. TlSltLER, M.D., AND COREr RAFFEI, M.D., PH.D.

Division ( f Neuros urgery, Childrens ttospital of Los Angeles, and Department of Neurosurgery, University of Southern California School of Medicine, Los Angeles, California ~" Drug resistance remains a formidable obstacle to the successful treatment of pediatric primitive neuroectodermal tumors. Resistance to chemotherapeutic agents may be related, in part, to expression of the multidrug resistance gene I (MDR1). The protein product of this gene, P-glycoprotein, confers resistance to multiple unrelated antineoplastic drugs. The cell line DAOY, derived from a primitive neuroectodermal tumor, was used as an in vitro model to examine the development of drug resistance. Cell lines resistant to actinomycin D were developed by the growth of DAOY in increasing concentrations of the drug. The ICso(concentration of drug needed to induce a 50% reduction in cell growth) of the resultant lines to actinomycin D was more than l0 times that of the parental line. The resistant lines were cross-resistant to VP-16 (etoposide), despite lack of previous exposure to this drug. The resistance to actinomycin D was attenuated in the presence of verapamil, a known inhibitor of P-glycoprotein. The MDR 1 gene was not expressed by the parental DAOY line at the messenger ribonucleic acid (RNA) and protein level. Expression of the MDRI gene was documented in the resistant lines by RNA blot and immunoblot techniques. These results suggest that exposure to chemotherapeutic drugs can induce classical multidrug resistance in primitive neuroectodermal tumors. KEY WORDS multidrug resistance gene glycoprotein 9 actinomycin D

EVERAL mechanisms have been described 22 by which tumor cells cultured in vitro may express drug resistance. Multidrug resistance, in which a tumor cell becomes resistant to a number of drugs, has often been associated with the expression of the multidrug resistance gene (MDR1).23"26 This gene encodes a 170-kD membrane-associated glycoprotein that serves as an energy-dependent drug effiux p u m p . 1k3~ The presence of the protein in the cell membrane leads to lower sublethal intracellular drug concentrations. Few stable cell lines have been derived from primitive neuroectodermal tumors of the central nervous system. 7'g'12 The cell line DAOY was derived from a 4year-old boy with a posterior fossa desmoplastic primitive neuroectodermal tumor] 2 To examine whether MDRI may have a role in the resistance to chemotherapy seen in primitive neuroectodermal tumor, we chose to determine if expression of the gene could be induced in DAOY. In this paper, we describe the development of DAOY cell lines that are resistant to actinomycin D. The resistant lines, but not the parental line, express the MDRI gene product, P-glycoprotein.



primitive neuroeetodermal tumor


Materials and Methods

Cell Culture The DAOY cell line was supplied by Dr. Henry Friedman. The cells were maintained in high-glucose Dulbeeco's modified Eagle's medium supplemented with 10% fetal bovine serum, L-glutamine (0.3 mg/ml), penicillin (100 U/ml), and streptomycin (100 U/ml). Actinomycin D-resistant lines were developed by exposing cells to sublethal concentrations of the drug for six passages, then increasing the drug concentration. Cells were initially exposed continuously to 10-6 mM actinomycin D, which was then increased in steps to a final concentration of 2.5 • 10-5 raM. Resistant lines were established at 10-5 and 2.5 x t0 -5 raM, and named DAOYAR1 and DAOYAR2, respectively.

Colony-Forming Efficiency Assay For all experiments, 60-mm Petri dishes were prepared 24 hours in advance by seeding heavily irradiated (40 Gy) DAOY feeder cells into 4 ml growth medium. The optimum number (5 • 104) of feeder cells was

J. Neurosurg. / Volume 76 /March, 1992

Drug resistance of neuroectodermal tumors TABLE 1 Concentrations oJ'drug needed to induce a 50% decrease in cell growth (ICso) of DAOY, DAOYAR1. and DAOE4R2 cell line.; Cell Line

Actinomycin D


Actinomycin D + 30 mM Verapamil


9 . 0 x t0-SmM


9.1 x 10-SmM


3.5 x 10 3 mM

4,25 ug/ml

5.5 x l0 m mM


4.7 • lO -3 mM

5,3 ug/ml

9.5 x 10-4 mM

determined in preliminary experiments. Cells were treated with actinomycin D or VP-16 (etoposide) at various concentrations for I hour. After the treated cells were plated, the dishes were incubated for 2 weeks at 37~ in a humidified atmosphere containing 5% CO2 and 95% air, after which they were fixed and stained with 0.125% crystal violet in methanol and the colonies were counted. Plating efficiencies were determined by dividing the number of colonies on the plates by the number of cells seeded. Surviving fractions were calculated by dividing the plating efficiency of treated cells by the plating efficiency of untreated cells. DNA Blotting Cellular deoxyribonucleic acid (DNA) from logarithmically growing cells was isolated by a salting-oat technique. ~9Ten #g of DNA was digested with EcoRI (a restriction enzyme derived from Escherichia colt RY13). The resulting digests were blotted as previously described. 24 The probe used, pMDR5a, HGM locus MDR 1, was supplied by Dr. Michael Gottesman.3~ The intensity of the autoradiographic bands was determined by densitometry* and was used to estimate the copy number for amplified genes. The densitometry value for the resistant lines was compared to that of the parental line. An intensity ratio of greater than 5 was taken to represent gene amplification.

with an equal volume of sample buffer (62.5 mM TrisHC1, pH 6.8, 10% vol/vol glycerol, 2% sodium dodecyl sulfate (SDS), 5% vol/vol 2-mercaptoethanol, and 0.001% bromophenol blue) and was sized electrophoretically on a 7.5 % SDS-polyacrylamidegel with running buffer (25 mM Tris base, 62.5 mM glycine, and 0.1% SDS). Following electrophoresis, the gel was electroblotted to nitrocellulose~ in transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol). The nitrocellulose filter was stained for P-glycoprotein using an anti-P-glycoprotein monoclonal antibody strel~avidin alkaline phosphatase conjugate.{}2~ Results Resistance to Actinomycin D The colony-forming efficiency assay was used to determine the ICso (concentration of drug needed to induce a 50% decrease in cell growth) of the parental DAOY cell line and the actinomycin D-resistant derivative cell lines (Table 1). The ICso of both resistant lines was more than 10 times that of the parental line. If the resistance to the drug in the actinomycin D-resistant lines is conferred by the expression of the MDR 1, crossresistance to other drugs should be present in these lines. The ICso of the cell lines to VP-16, another drug recognized by P-glycoprotein, is also shown in Table 1. Although they had not been exposed to VP-16, both actinomycin D-resistant lines exhibited an ICso to VP16 about five times that of the parental line. Verapamil is a known inhibitor of the P-glycoprotein pump. As shown in Table I, the ICs0 of actinomycin D for the resistant cell lines is reduced in the presence of verapamil. Verapamil does not alter the ICso of the parental DAOY cell line.

Immunoblotting Total protein was isolated using a previously described method? 5Protein (60 #g) in 20 #l was combined

Expression of the M D R 1 Gene To test for the presence of MDR 1 messenger RNA (mRNA), total cellular RNA from each cell line was analyzed with a probe specific for MDR1 (pMDR5a). As seen in Fig. 1, MDRI transcripts are detectable in both resistant cell lines, but not in the parental cell line, indicating that expression of MDR1 mRNA has been induced by exposure of the cells to actinomycin D. To verify that no MDR1 transcripts were present in the parental cell line, polymerase chain reaction, a technique that greatly increases the sensitivity of mRNA detection, was performed. In this technique, a single mRNA molecule from a specific gene can be amplified 230 times. Primers were devised that spanned an intron (the noncoding part of a gene that does not become a part of the mRNA), so that a 226-base-pair product would be expected from mRNA and a 750-base-pair product would be obtained from genomic DNA. In

* Densitometer, Model 620, manufactured by Bio-Rad, Richmond, California. t Nylon membrane manufactured by Biotrace RP, Gelman Sciences, Ann Arbor, Michigan.

,Nitrocellulose supplied by Micron Separations, Inc., Westboro, Massachusetts. wStrepavidin alkaline phosphatase conjugate supplied by Tago, Inc., Burlingame, California.

RNA Blotting Total cellular ribonucleic acid (RNA) was isolated with an acid guanidinium isothiocyanate-phenol-chloroform method? The RNA was sized electrophoretically on a 1% agarose formaldehyde gel, transferred to a nylon membrane,t cross-linked with ultraviolet light, and probed with pMDR5a. 2q Polymerase Chain Reaction Polymerase chain reaction for MDR 1 messenger RNA and ~-actin messenger RNA was performed. 29

J. Neurosurg. / Volume 76 / March, 1992


D. M. Tishler and C. Raffel

FIG. 1. To detect the presence of MDR 1 messengerRNA in three cell lines, an RNA blot was performed using a probe specificfor MDRI. Total cellular RNA was separated by size on a geI and transferred to a nylon membrane as described in the text: the probe used was pMDR5a. Lanes 1, 2, and 3 contain total cellular RNA from the DAOY, DAOYARI, and DAOYAR2 cell lines, respectively.Note the 4.5-kilobase(kb) message in the Iwo resistant lines, but not in the parental line.

FiG. 2. With synthetic oligonucleofideprimers specificfor MDRI, polymerase chain reaction was employed to amplify MDRI messenger RNA 23~ times. The resultant amplified reaction products were analyzed as described in the text. The 226-base-pair product (lap) expected from MDRI messenger RNA was detected in both of the resistant lines (DAOYARI (Lane 2) and DAOYAR2 (Lane 3)), but not in the parental line (DAOY (Lane I)).

addition, the primers were specific for MDR1 and did not give a 226-base-pair product when analyzed on an MDR2 complementary DNA template (data not shown). As seen in Fig. 2, no polymerase chain reaction product could be detected in the parental cell line; in both resistant cell lines, the 226-base-pair product was easily identified. To demonstrate the integrity of the RNA preparations, polymerase chain reaction was performed using ~-actin primers. In all three cases, the expected product was easily identified (data not shown), indicating that the absence of MDRI transcripts in the parental cell line was not due to degradation of the parental cell line RNA. Immunoblot analysis of protein from the three cell lines also demonstrated that the MDR1 gene was being expressed in the resistant cell lines, as P-glycoprotein was detected in both resistant cell lir/es but not in the parental cell line (Fig. 3). This result confirms those presented above for mRNA at the protein level.

Gene A mplification Increased MDRI gene expression is sometimes accomplished in vitro by gene amplification. A blot analysis of DNA from the DAOYAR2 cell line was compared to that of the parental cell line to see whether gene amplification had occurred. No amplification was 504

FIG. 3. Immunoblot for P-glycoprotein. Total protein from the DAOY (Lane t) and DAOYARI (Lane 2) cell lines was separated by size on a gel and transferred to a nitrocellulose membrane as described in the text. The membrane was then probed with an antibody specificfor P-glycoprotein.The expected 170-kD protein is detected in the resistant cell line, but not the parental cell line. Lane 3 is a P-glycolarotein standard.

identified. To determine if a small population of the cells in the DAOYAR2 cell line had an amplified MDRI gene, eight subclones of the line were isolated and analyzed for gene amplification. No amplification was detected in any clone.

Discussion The P-glycoprotein molecule is a member of a family of transport proteins the function of which may be to protect organisms from naturally occurring toxins in the diet and environment? 2~3-t~ In the human, many normal tissues express MDRI. Organs with an excretory function, such as the colon, small intestine, kidney, liver, pancreas, and adrenal gland, express MDRI. Protection of the organism may be achieved by secreting toxins into the bile, urine, or gastrointestinal lumen? 2+ Malignancies derived from tissues that normally express MDR 1 are inherently drug-resistant and fail to respond to combination chemotherapy. 5+~+ Normal brain parenchyma does not express MDRI, although P-glycoprotein has been detected on the membrane of normal brain capillary endothelium, suggesting that MDRI may play a role in the formation of the blood-brain barrier. 4.2g In this study, we have demonstrated that MDRI expression can be induced in vitro in the DAOY cell line, derived from a primitive neuroectodermai tumor, by exposure to sublethal concentrations of drug, a J. Neurosurg. / Volume 76/March, 1992

Drug resistance of neuroectodermal tumors technique that has been frequently used to induce in vitro drug resistance, lo,32The parental cell line expressed neither MDRI m R N A (as demonstrated by RNA blotting and polymerase chain reaction) nor P-glycoprotein (as demonstrated by immunoblot). The resistant cell lines derived from the DAOY cell line have all of the features of classic multidrug resistance, including resistance to more than one drug, de novo resistance to a drug not previously seen by the cells, reversal of drug resistance by verapamil, expression of MDR1 mRNA, and expression of P-glycoprotein. Cells that express multidrug resistance in vitro demonstrate a typical pattern of cross-resistance to chemotherapeutic agents? ~ Included in the cross-resistance pattern are the anthracylines, antibiotics (such as actinomycin D), the antimitotics (such as the vinca alkaloids), the epipodophyllotoxins, and colchicine. These drugs differ both in mode of action and chemical structure, are predominantly of plant or fungal origin, and are planar hydrophobic molecules. 2~ The antimitotics and epipodophyllotoxins, which are included in the cross-resistance, are being used more frequently in multidrug regimens directed against primitive neuroectodermal tumors. The extent of MDR1 expression in cell lines in vitro has been correlated with the degree of drug resistance seen in these lines. In some cases, increased expression of MDR1 has been a consequence of gene amplification. 6'27 Gene amplification was not a factor in the resistance of the cell lines presented here, despite exposure of the cells to actinomycin D under conditions lethal to the parental cell line. The expression of the MDR1 gene by central nervous system tumors other than primitive neuroectodermal tumors has been investigated by Matsumoto and coworkers. 17'~8 These investigators examined the drugresistance profiles of five glioma cell lines. Without prior exposure to any drug, one of these lines expressed a pattern of cross-resistance compatible with M D R I expression, and MDR1 expression was demonstrated in this line at the mRNA level. Interestingly, the only cell line to express MDR1 in this work is GB-1, a cell line isolated in the laboratory of Matsumoto, et al. No description or characterization of the cell line was given. It is not clear why this putative glioblastoma cell line should be a constitutive expressor of MDR 1. The work presented here suggests a potential role for P-glycoprotein in acquired drug resistance by human primitive neuroectodermal tumor.


1. Chen C, Chin JE, Ueda K, et at: Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47:381-389, 1986 2. Chin JE, Soffir R, Noonan KE, et al: Structure and expression of the human MDR (P-glycoprotein) gene family. Mol Cell Biol 9:3808-3820, 1989 3. Chomczynski P, Sacchi N: Singie-step method of RNA J. Neurosurg. / Volume 76/March, 1992

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Manuscript received May 19, 1991. Accepted in final form September 16, 1991. Address reprint requests to: Corey Raffel, M.D., Ph.D., 1300 North Vermont Avenue, Suite 906, Los Angeles, California 90027.

J. Neurosurg. / Volume 76/March. 1992

Development of multidrug resistance in a primitive neuroectodermal tumor cell line.

Drug resistance remains a formidable obstacle to the successful treatment of pediatric primitive neuroectodermal tumors. Resistance to chemotherapeuti...
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