J Neurosurg 72:96-101, 1990

Amplification and expression of a multidrug resistance gene in human glioma cell lines TSUYOSHI MATSUMOTO, M.D., EIICHI TANI, M.D., KEIZO KABA, M.D., NOBUO KOCHI, M.D., HIDEKI SHINDO, M.D., YOSHIHIRO YAMAMOTO, HIROMI SAKAMOTO~ M.D., AND JUNICHI FURUYAMA, PH.D.

Departments of Neurosurgery and Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan u," Two human glioma cell lines were examined for multidrug resistance (MDR). A vincristine (VCR)-resistant glioma cell line showed a cross resistance to Adriamycin (doxorubicin, ADR) and etoposide (VP- 16) to varying extents, suggesting the presence of MDR; the resistance to VCR was considerably decreased by calcium entry blockers. On the other hand, another VCR-sensitive glioma cell line exhibited no cross resistance to ADR or VP-16. Double minute chromosomes and homogeneously staining regions as well as clonal aberrations of chromosome 7 were not observed in cytogenetic studies of multidrug-resistant and multidrug-sensitive glioma celt lines. In Northern and Southern blot analyses, MDR gene 1 (MDR 1) messenger ribonucleic acid (mRNA) was shown to be overexpressed without any amplification of the MDRI gene in multidrug-resistant glioma cell lines as compared to multidrug-sensitive glioma cell lines. It would be reasonable to suggest that amplification of the MDR 1 gene may not be a sine qua non for acquisition of MDR and that the MDR 1 mRNA level may be well correlated with the extent of MDR. KEY WORDS brain neoplasm c a l c i u m entry blocker *

T

HE development of multidrug resistance (MDR) is a c o m m o n clinical problem in the treatment of malignant tumor. Characteristics of multidrug-resistant cell lines include the following: l) cross resistance to m a n y naturally occurring lipophilic cytotoxic drugs, such as plant alkaloids and antitumor antibiotics; 2 2) decreased accumulation of these drugs resulting from an increase in drug efflux~ 14'21'47'55 3) phenotypic reversal of M D R by several agents including verapamil; 21'49'55 4) increased amounts of a 170-kD surface glycoprotein (P-glycoprotein); 23'4~ and 5) amplification and overexpression o f the M D R gene 1 (MDRI) gene. 16'18,34,36-38,4t,53 In a previous study, 22 a vincristine (VCR)-resistant h u m a n glioma cell line showed a decreased intracellular accumulation of VCR mediated by an increase in effiux of the drug and a reversal of V C R resistance by calcium entry blockers (CEB's) and calmodulin inhibitors. In this communication, we examined a cross resistance of VCR-resistant h u m a n glioma cell line to Adriamycin (doxorubicin, A D R ) and etoposide (VP-16), and the cytogenetic characteristics and the copy n u m b e r o f the M D R 1 gene, as well as the level of M D R 1 messenger ribonucleic acid ( m R N A ) in h u m a n glioma cell lines. 96

9 glioma

9 multidrug resistance gene

9

Materials and M e t h o d s

Glioma Cell Lines Two h u m a n glioma cell lines were used in the present study: a h u m a n glioblastoma-derived cell line (GB-l) established in our laboratory and the U-373 M G h u m a n glioma cell line obtained from a h u m a n t u m o r cell bank.* Both glioma cell lines were maintained in tissue culture with Dulbecco's modified Eagle's m i n i m u m essential m e d i u m supplemented with 10% fetal calf serum and antibiotics (penicillin G, 100 U/ml, and streptomycin, 100 t~g/ml). The m e a n (_+ standard deviation) cell doubling time for both glioma cell lines was 42 _+ 2 hours. Evaluation o f Antitumor Activity In Vitro The GB-1 and U-373 M G cells were plated onto 60m m diameter Falcon plastic dishes at a density of 2 x 105 cells/dish and incubated at 37~ in a humidified atmosphere o f 5% CO2 and 95% air for 2 days. The * U-373 MG cell line obtained from American Type Culture Collection, Rockvitle, Maryland.

J. Neurosurg. / Volume 72/January, 1990

Multidrug resistance gene in glioma cell lines Definitions of Abbreviations

ADR = Adriamycin AHC-52 = methyl 2-(N-benzyl-N-methylamino) ethyl 2,6-dimethyl-4(2-isopropylpyrazolo [ 1,5-a]pyridine-3-yl) - 1,4-dihydropyridine3,5-dicarboxylate c D N A = complementary DNA CEB = calcium entry blocker DM = double minute chromosome DNA = deoxyribonucleic acid dpm = disintegrations per minute EcoRI = a restriction endonuclease EDTA = ethylenediaminetetra-acetic acid HindIII = a restriction endonuclease HSR = homogeneously staining region IC~o = concentration of drug inducing a 10% decrease in cell growth IC5o = concentration of drug inducing a 50% decrease in cell growth kb = kilobase pairs MDR = multidrug resistance MDR1 = human multidrug resistance gene 1 mRNA = messenger RNA P-glycoprotein = a 170-kD surface glycoprotein pMDR5A = a complementary DNA of multidrug resistance gene 1 RNA = ribonucleic acid SSC = standard saline citrate TE = 10 mM Tris-HC1, pH 7.5/1 mM ethylenediaminetetra-acetic acid U-373 MG = human malignant glioma-derived cell line VCR = vincristine VP- 16 = etoposide

cells grown in logarithm phase were then treated with graded concentrations of CEB alone (10 - 7 to 1 0 - 4 M) or of an antitumor agent (10 - ~ to 10 -8 M of VCR, 10 -'~ to 10 -8 M of ADR, or 10 -'2 to 1 0 -9 M of VP-16) in the absence or presence of CEB, and followed by incubation for two cell-doubling times of each glioma cell line. The cytotoxic activities were measured by determining the concentration of CEB alone inducing a 10% decrease in cell growth compared to control (IClo) and the concentration of VCR, ADR, or VP-16 in the absence or presence of CEB inducing a 50% decrease in cell growth as compared to control (IC50). The initial cell n u m b e r was subtracted in the calculation. The levels o f relative resistance to VCR, ADR, and VP-16 in glioma cells were determined by dividing the IC50 of GB-1 cells by that of U-373 M G cells. All experiments were carried out three times, each in triplicate. The CEB's used were: verapamil; methyl 2-(N-benzyl-N-methylamino)ethyl 2,6-dimethyl-4-(2-isopropylpyrazolo [ 1,5-a]pyridine-3-yl)- 1,4-dihydropyridine-3,5-

J. Neurosurg. / Volume 72/January, 1990

dicarboxylate (AHC-52);t brovincamine (a selective slow CEB); and flunarizine (a nonselective CEB). 54 The CEB's used were dissolved in phosphate-buffered saline (PBS). The final concentrations of CEB's used together with VCR, A D R , or VP-16 were nontoxic or slightly toxic (IC,o).

Cytogenetic Studies After subculture, the cells were grown for 48 to 72 hours, at which time metaphase arrest was initiated by the addition of colchicine (0.1 ug/ml), and the incubation continued for 2 89hours. C h r o m o s o m e preparations were made according to the air-drying technique of Tjio and Whang. 45 Conventional Giemsa staining and trypsin Giemsa banding 39 were performed. Isolation o f Nucleic Acids High-molecular-weight genomic deoxyribonucleic acid (DNA) was isolated from exponentially grown glioma cells by the m e t h o d of Maniatis, et al. 29 Briefly, the genomic D N A was prepared by digestion with proteinase K / s o d i u m dodecyl sulfate (SDS), followed by gentle extraction with buffer-saturated phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). The R N A was removed with ribonuclease A (200 ~g/ml) at 37~ for 45 minutes, and the protein was digested with proteinase K (200 #g/ml) for 60 minutes at 37~ After being purified several times with phenol/chloroform, D N A was precipitated with sodium acetate a n d ethanol and suspended with 10 m M Tris-HC1, p H 7.5/1 m M ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer). Total cellular R N A was isolated from exponentially grown glioma cells by the methods of Chirgwin, et al.,8 and Maniatis, et al? 9 T h e cells were washed three times with PBS and lysed with guanidine thiocyanate buffer, followed by homogenization in the same buffer. Each homogenate was layered over a CsC1 cushion and centrifuged for 18 hours at 30,000 rpm.z~ The R N A pellets were suspended with TE buffer, extracted with phenol/ chloroform, precipitated twice with ethanol, and dissolved in TE buffer. Southern and Northern Blot Hybridizations After digestion from E c o R I (a restriction endonuclease derived f r o m Escherichia coli RY13) u n d e r conditions r e c o m m e n d e d by the supplier, the D N A samples (15 ug) were electrophoresed on 0.8% agarose in 1 • Tris-borate-EDTA buffer, and transferred f r o m the gel to a nitrocellulose filter by the m e t h o d o f Southern. 43 Total R N A samples (15 #g) were analyzed by electrophoresis on 1% agarose containing 0.66 M formaldehyde and were transferred directly to a nitrocellulose filter after two rinses of the gel in 10 • t AHC-52 was kindly provided by Dr. M. Inaba, Japanese Foundation for Cancer Research, Tokyo, Japan. Centrifuge SW40 rotor manufactured by Beckman Instruments, Inc., Palo Alto, California.

97

T. Matsumoto, et al. TABLE 1

TABLE 3

Drug resistance in human glioma cell lines*

Cytotoxicity of vincristine in human glioma cell lines*

Drug

IC~o(nM) Against:

GB- 1 U-373 MG VCR 73.5 _+ 3.6 (66) 1.12 _ 0.14 (1) ADR 34.2 _+ 3.1 (5) 7.50 __+0.30 (1) VP-16 1.24 + 0.14 (7) 0.17 _+0.03 (1) * Values are the mean _+standard deviation. Numbers in parentheses indicate levels of relative resistance which are determined by dividing the ICs0 of GB-1 cells by that of U-373 MG cells. See Definitions of Abbreviations table.

TABLE 2

CEB's Added

ICso (nM) of VCR Against:

GB-1 U-373 MG none 73.5 + 3.6 1.12 _+0.14 verapamil 2.04 _+0.31 (36) 0.54 + 0.14 (2) AHC-52 2.10 + 0.25 (35) 0.63 -- 0.12 (2) brovineamine 5.95 - 0.45 (12) 0.95 + 0.25 (1) flunarizine 8.90 + 0.90 (8) 1.06 _+0.14 (1) * The cytotoxicity of vincristine t o GB-1 cells is enhanced by the addition of calcium entry blockers (CEB's). Values are mean -+ standard deviation. Numbers in parentheses indicate the increase in cytotoxicity, which is calculated by dividing the ICs0 obtained with vincristine alone by that with vincristine and each of the CEB's. See Definitions of Abbreviations table.

Cytotoxicity of calcium entry blockers in human glioma cell lines* Calcium Entry Blocker

IC~0 (#M) Against:

GB-1 U-373 MG verapamil 6.0 + 1.4 (5) 8.8 _ 2.1 (5) AHC-52 5.8 --- 0.1 (4) 4.0 ___0.3 (4) brovincamine 6.4 _+0.9 (5) 8.5 - 0.5 (5) flunarizine 4.4 + 0.6 (2) 5.0 +_0.5 (2) * Values are the mean 4- standard deviation. Numbers in parentheses indicate concentrations of calcium entry blockers used with vincristine in the present study. See Definitions of Abbreviations table.

Results

Drug Sensitivities o f G l i o m a Cell Lines The sensitivities o f G B - 1 a n d U-373 M G cell lines to VCR, A D R , a n d V P - 16 were expressed as the IC50 value o f each drug a n d also as relative resistance with respect to the U-373 M G cell ( T a b l e 1). T h e GB-1 cell was 66fold m o r e resistant to V C R t h a n was the U-373 M G cell, a n d fivefold a n d s e v e n f o l d m o r e resistant to A D R a n d VP-16, respectively, t h a n was the U-373 M G cell.

Effects o f Calcium E n t r y Blockers s t a n d a r d saline c i t r a t e (SSC: 0.15 M N a C I / 0 . 0 1 5 M s o d i u m citrate, p H 7.0) buffer. Filters were b a k e d in a v a c u u m oven for 2 h o u r s at 80"C. T h e p M D R 5 A p r o b e w u s e d was 1.35 kilobase pairs (kb) E c o R I - E c o R I D N A f r a g m e n t e n c o d i n g a b o u t onethird o f the c o d i n g r e g i o n o f a full-length M D R 1 c o m p l e m e n t a r y D N A ( c D N A ) , 5~ a n d labeled to specific activities o f 1 • l09 d p m / / z g b y r a n d o m p r i m i n g ~3 with 32p-deoxycytidine t r i p h o s p h a t e (3000 Ci/mmol).ll B a k e d filters were i n c u b a t e d for 16 hours at 42"C in 50% f o r m a m i d e / 5 • SSC/5 x D e n h a r d t ' s s o l u t i o n / 0.5% S D S / 5 0 #g o f d e n a t u r e d s a l m o n s p e r m D N A p e r m l / 1 0 % d e x t r a n sulfate c o n t a i n i n g 32p-labeled probe. Filters were w a s h e d twice for 15 m i n u t e s each at r o o m t e m p e r a t u r e in 2 • S S C / 0 . 1% SDS, o n c e for 30 m i n u t e s at r o o m t e m p e r a t u r e in 0.5 x S S C / 0 . 1 % SDS, a n d once for 30 m i n u t e s at 42"C in 0.2 x SSC/0.1% SDS, dried, a n d a u t o r a d i o g r a p h e d at - 8 0 " C for 2 to 4 days with intensifying screens. T h e M D R I gene c o p y n u m b e r a n d the level o f M D R I m R N A were d e t e r m i n e d b y densit o m e t r y o f the a u t o r a d i o g r a m s .

wpMDR5A probe kindly provided by Dr. I. Pastan, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and by Dr. K. Ueda, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan. II 32p-deoxycytidine triphosphate supplied by Amersham International, Buckinghamshire, England. 98

T h e CEB's used d e m o n s t r a t e d s o m e cytotoxicity to GB-1 a n d U - 3 7 3 M G cell lines, a n d their cytotoxicities were expressed as IC~o v a l u e o f each CEB (Table 2). Consequently, the n o n t o x i c o r slightly toxic concentrations (IC~0) o f C E B ' s were u s e d to e x a m i n e their effect on the c y t o t o x i c i t y o f V C R , A D R , a n d VP-16; these c o n c e n t r a t i o n s were 5 izM for verapamil, 4 u M for AHC-52, 5 u M for b r o v i n c a m i n e , a n d 2 ~ M for flunarizine. T h e IC50 value o f V C R was m a r k e d l y decreased in VCR-resistant GB-1 cells b y the a d d i t i o n o f C E B ' s (Table 3), suggesting t h a t t h e resistance to V C R was considerably d e c r e a s e d b y CEB's, p a r t i c u l a r l y b y verap a m i l a n d A H C - 5 2 (these p r o d u c e d a 36-fold a n d 35fold decrease o f V C R resistance, respectively). O n t h e o t h e r h a n d , t h e ICso v a l u e o f V C R in U-373 M G cell was n o t so c h a n g e d b y t h e a d d i t i o n o f CEB's (Table 3), suggesting t h a t U-373 M G cell is rather VCR-sensitive.

Cytogenetic Studies T h e GB-1 a n d U - 3 7 3 M G cells exhibited several n u m e r i c a l a n d s t r u c t u r a l c h r o m o s o m e aberrations. T h e stemline c h r o m o s o m e n u m b e r o f G B - 1 a n d U-373 M G cells was h y p o t r i p o i d with a m o d a l c h r o m o s o m e n u m ber o f 66 (range 63 to 70) in G B - I cells a n d o f 67 (range 60 to 69) in U - 3 7 3 M G cells. T h e r e was no evidence o f d o u b l e m i n u t e c h r o m o s o m e s (DM's), h o m o g e n e o u s l y staining regions ( H S R ' s ) , o r c l o n a l a b e r r a t i o n s o f chrom o s o m e 7 in GB-1 o r U - 3 7 3 M G cells.

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Multidrug resistance gene in glioma cell lines TABLE 4 Characterization of human glioma cell lines* Cell Line

Relative Resistance to: MDR1 mRNA MDRI Gene VCR ADR VP- 16 Expression Copy No.

GB-1 66 5 7 26 1 U-373 MG 1 1 1 1 1 * Relative resistance is determined by dividing the IC50 of GB-1 cells by that of U-373 MG cells. Levels of MDR1 mRNA expression and MDR 1 gene copy number are determined by densitometryof the autoradiograms. Results are relative to the MDR1 mRNA expression and MDRI gene copy number in U-373 MG cells, respectively.See Definitions of Abbreviationstable.

Analysis o f M D R 1 Gene Amplification The DNA extracted from GB-1 and U-373 M G cell lines was digested with E c o R I and hybridized with the 32p-labeled p M D R 5 A probe. Southern blot analysis revealed four EcoRI D N A fragments (two prominent (1.0 kb and 1.8 kb), one moderate (3.0 kb), and one weak (6.0 kb)) equally in both cells (Fig. 1). The M D R 1 gene copy number was determined by densitometry of the autoradiograms, showing no clear difference between the GB-1 and U-373 M G cells (Table 4). Consequently, the resistance to a n t i t u m o r agents used in the GB- 1 and U-373 M G cells might not correlate with the MDR1 gene copy number. In addition, the M D R 1 gene in GB1 and U-373 M G cells could not be rearranged, mainly because the four D N A fragments from both cells were equal in size with each other. Expression o f M D R 1 m R N A Northern blot analysis was performed using 15 izg of RNA from the GB-1 and U-373 M G cell lines. The 32p_ labeled p M D R 5 A probe hybridized with a 4.5-kb RNA species from both cells, which was expressed highly in GB-1 cells and slightly in U-373 M G cells (Fig. 1). The M D R 1 m R N A levels were measured by densitometry of the autoradiograms, and expressed 26-fold more in GB-1 cells than in U-373 M G cells (Table 4), showing a good correlation of the M D R 1 m R N A levels with the extent of resistance to a n t i t u m o r agents used, particularly VCR. Discussion The resistance of V C R in glioma cell lines was directly related, at least in part, to the decreased intracellular level of V C R caused by an active effiux of intracellular VCR (as reported in the previous study 22) and was overcome by treatment with CEB's. In addition, the VCR-resistant GB- 1 glioma cell line was cross resistant to A D R and VP-16, although the cellular levels of cross resistance were rather low, suggesting that the VCR-resistant GB-1 cell line showed MDR. Shen, et al., 4~ and Ueda, et al., 5~ reported that the relative resistance to colchicine, vinblastine, and A D R was highly variable in multidrug-resistant h u m a n KB car-

J. Neurosurg. / Volume 72/January, 1990

FIG. 1. Southern and Northern blot hybridizations. DNA (left) and RNA (right) isolated from human glioma cells hybridize with pMDR5A probe labeled with [32p]-deoxycitidine triphosphate by random priming. HindlII-digested X phage DNA is used as a size standard. Numbers indicate kilobase pairs. See Definitions of Abbreviations table.

cinoma cells. Scotto, et a/., 38 demonstrated that the development of resistance to a single drug and M D R in Chinese hamster lung cells was a closely related but uncoupled event: the degree o f resistance was better correlated with the selected agent than it was with the extent of MDR. The m e c h a n i s m o f transport for each antitumor agent is not necessarily exactly the same but possesses similar characteristics. Multidrug resistance is connected with elevated levels of P-glycoprotein, which is considered as a possible transporter that p u m p s out a n t i t u m o r agents. 3,5,1~176 P-glycoprotein possesses an adenosine triphosphatase activity, 2~a binding site for V C R and ADR,11 a binding site for CEB's,l~ and a phosphorylation site which might be involved in the regulation of its function. 19 Some CEB's can bind to P-glycoprotein, and the binding of some antitumor agents can be inhibited competitively by CEB's and other potentiating agents. 10.11Thus, CEB's could inhibit the effiux of a n t i t u m o r agents, thereby overcoming MDR. 22'47'48 The dihydropyridine derivative AHC-52 shows little activity as a slow CEB (M Inaba: personal communication), but it overcomes M D R as m u c h as verapamil does. On the other hand, 99

T. Matsumoto, et al. brovincamine acts as a CEB similarly to verapamil, 24 but overcomes M D R much less than verapamil does. There was no clear evidence o f correlation between the inhibition of calcium entry and the overcoming ability of M D R in C E B ' s , 10'33'47thus, the molecular mechanism of action o f CEB's and of P-glycoprotein remains to be identified. The development o f M D R in cultured cells correlated with the increased expression and amplification of a gene or family o f genes encoding P-glycoprotein. 23"34'38'53 The extent of M D R in the same cell lines was also shown to correlate with increased expression and amplification o f an independently cloned D N A sequence for the MDR1 gene. 18'36'37'41 The cross hybridization of M D R 1 c D N A segments with c D N A ' s for Pglycoprotein confirmed that the M D R 1 gene coded for P-glycoprotein. 52 Multidrug-resistant glioma cells showed an overexpression o f M D R 1 m R N A without M D R 1 gene amplification on Northern and Southern blot analyses. In addition, karyotypic analysis showed that DM's and HSR's, as well as clonal aberrations of chromosome 7 that are indicative o f gene amplification, 1,4.6.16.25,26.28,30. 31,34.35,42,44,46,49 were not present in multidrug-resistant GB-1 cells. In h u m a n leukemia and ovarian carcinoma cell lines, selected for high levels of MDR, amplification of the MDR1 gene was c o m m o n l y observed. 41 However, the same cell lines with rather lower levels of M D R showed an increased MDR1 m R N A expression without MDR1 gene amplification, 15m suggesting an increase in the rate of transcription, m R N A stability, or posttranscriptional events necessary for maturation and delivery of R N A to the cytoplasm. 7'9'~2'32A similar observation, a dissociation of MDR1 m R N A expression and MDR1 gene amplification, was reported in several multidrug-resistant h u m a n t u m o r cells. 17'27'38"4j These results indicate that increased expression of MDR1 m R N A could precede M D R I gene amplification and that MDR1 m R N A expression correlates well with extent of MDR. The levels of MDR1 m R N A detected in m a n y h u m a n tumors are comparable to those in the GB-1 glioma cell line, suggesting that, in clinical situations, gene activation might be a more c o m m o n means of developing resistance than gene amplification. ~5 The ability to identify a group of tumors that show M D R by measuring the levels of M D R 1 m R N A could have m a n y important consequences. First, determination of the level of MDR1 m R N A could allow the identification o f tumors likely to fail to respond to a given drug. Second, the level of MDR1 m R N A could be used as a guide for the selection of drugs. Third, the development of resistance to a given agent during the initial therapy could be assessed in recurrent tumors. Acknowledgment

The authors thank Dr. K. Ueda, Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan, for his helpful advice. 1O0

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Manuscript received April 6, 1989. Address reprint requests to: Eiichi Tani, M.D., Department of Neurosurgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan. 101