-adenosylmethionine synthetase is overexpressed in murine ...min at 94°c, annealing for 1 min at...

[CANCER RESEARCH 59, 1852–1856, April 15, 1999]

Advances in Brief

S-Adenosylmethionine Synthetase Is Overexpressed in Murine Neuroblastoma CellsResistant to Nucleoside Analogue Inhibitors ofS-AdenosylhomocysteineHydrolase: A Novel Mechanism of Drug Resistance1

Rama S. Dwivedi,2 Ling-Jia Wang, and Bernard L. MirkinChildren’s Memorial Institute for Education and Research, Children’s Memorial Medical Center, Departments of Pediatrics [R. S. D., L-J. W., B. L. M.] and MolecularPharmacology and Biological Chemistry [B. L. M.], Northwestern University Medical School, Chicago, Illinois 60614

Abstract

S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), which cat-alyzes the synthesis of AdoMet from methionine and ATP, is the majormethyl donor for transmethylation reactions and propylamino donorfor the biosynthesis of polyamines in biological systems. We havereported previously that wild-type C-1300 murine neuroblastoma(wMNB) cells, made resistant to the nucleoside analogue (Z)-5*-fluoro-4*,5*-didehydro-5*-deoxyadenosine (MDL 28,842), an irreversible in-hibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1),express increased AdoMet synthetase activity (M. R. Hamreet al.,Oncol. Res., 7: 487– 492, 1995). In the present study, immunoblotanalyses of AdoMet Synthetase with isoform-specific (MATII) antibod-ies demonstrated an elevation in the AdoMet synthetase immunopro-tein in nucleoside analogue-resistant MNB cells (rMNB-MDL) whencompared to wild-type, nonresistant MNB cells. An increase of 2.1-foldwas observed in thea2/a2* catalytic subunit, which differed signifi-cantly from the much smaller increment in the noncatalytic b-subunitof AdoMet synthetase. Densitometric analyses revealed that an in-creased expression of AdoMet synthetase in rMNB-MDL cells was dueto overexpression of thea2 (M r 53,000; 2.6-fold) anda2* (M r 51,000;1.8-fold) subunits. AdoMet synthetase mRNA expression in rMNB-MDL cells was remarkably greater than wMNB cells, as determined byquantitative competitive reverse transcription-PCR (QC-PCR) analy-sis. DNA (cytosine) methyl transferase expression, measured by reversetranscription-PCR analysis, was also elevated significantly in rMNB-MDL cells. In contrast, Western blot analyses demonstrated down-regulation (1.6-fold) of AdoMet synthetase in doxorubicin-resistanthuman leukemia cells (HL-60-R) expressing multidrug resistance pro-tein when compared with wild-type, nonresistant HL-60 cells. Theresistance of rMNB-MDL cells to nucleoside analogue inhibitors ofS-adenosylhomocysteine hydrolase correlates directly with overexpres-sion of the a2/a2* subunits of AdoMet synthetase. Cellular adaptationallows sufficient AdoMet to be synthesized, so that viability of the MNBcells can be maintained even in the presence of high AdoHcy concen-trations. This novel mechanism of drug resistance does not appear torequire multidrug resistance protein (P-glycoprotein) overexpression.

Introduction

AdoMet3 synthetase (ATP:L-methionine S-adenosyltransferase;EC 2.5.1.6), the enzyme responsible for synthesis of AdoMet fromL-methionine and ATP, plays a major role in the development ofacquired drug resistance to nucleoside analogue inhibitors of AdoHcyhydrolase (EC 3.3.1.1) by murine neuroblastoma tumor cells (1).AdoMet is the major donor of methyl groups for transmethylationreactions in eukaryotic systems (2) and is essential for normal cellularmetabolism. Inhibition of this enzyme alters metabolic function, ulti-mately leading to cell death. Three different isoforms of mammalianAdoMet synthetase,a (MAT-I), b (MAT-III), and g (MAT- II) havebeen identified (3). MAT-I and MAT-III are products of the samegene,MAT1A,and predominantly expressed in adult liver. MAT-II isa product of theMAT2A gene and mainly present in kidney, brain,lymphocytes, testis, and lens and fetal, newborn, and early regener-ating liver (4). The tetramer MAT-I (Mr 200,000) and dimer MAT-III(Mr 100,000) are oligomeric forms of thea1 subunit, whereas MAT-II(Mr 185,000) is a heterooligomeric complex comprised ofa2/a29 andb subunits. Thea29 subunit of MAT-II is considered to represent aposttranslational modification of thea2 subunit during biologicalmaturation. MAT-II, predominantly expressed in fetal liver, is grad-ually replaced by MAT-I and MAT-III (5). In carcinogen-inducedhepatocarcinoma, the expression of MAT-I and III decreases whileMAT-II increases, depending on the stage of carcinogenesis (6).

AdoHcy, generated by the transfer of a methyl group from AdoMet,is converted to adenosine and homocysteine by AdoHcy hydrolase.AdoHcy is a competitive inhibitor of protein carboxylases and meth-yltransferases and can influence AdoMet-associated cellular methyl-ation processes (2). The concentration of AdoMet and magnitude ofAdoMet synthetase activity modulate the transmethylation reactionsthat regulate functional gene expression in biological systems.

Inhibition of AdoMet synthetase gene expression and enzymicactivity results in cell death (7). AdoMet synthetase-mediated meth-ylation of DNA at CpG sites catalyzed by DNA methyl transferase, inparticular, is associated with the functional and transcriptional inac-tivation of a number of genes during the process of cellular differen-tiation. Impaired regulation of protein expression or mRNA synthesisby gene methylation (8) constitutes an important mechanism of drugresistance (9, 10). This investigation has examined the role of AdoMetsynthetase (MAT-II) gene overexpression in the development of nu-

Received 9/25/98; accepted 3/2/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This investigation was partially supported by grants from the Pharma Foundation (toR. S. D.), Illinois Department of Public Aid and United States Department of Health andHuman Services (to R. S. D.), Anderson Foundation (to B. L. M.), the Medical ResearchInstitute Council (to B. L. M.), and the Children’s Memorial Institute for Education andResearch (CMIER) Program in Cancer Biology and Therapeutics (to B. L. M.).

2 To whom requests for reprints should be addressed, at Children’s Memorial Institutefor Education and Research, Northwestern University Medical School, 2300 Children’sPlaza, Mail Box 204, Chicago, IL 60614. Phone: (773) 880-8222; Fax: (773) 880-3282;E-mail: ramaa@nwu.edu.

3 The abbreviations used are: AdoMet,S-adenosylmethionine; MAT, methionine ad-enosyltransferase; AdoHcy,S-adenosylhomocysteine; RT-PCR, reverse transcription-PCR; QC-PCR, quantitative competitive RT-PCR; MTase, DNA (cytosine) methyl trans-ferase; MDL, (Z)-59-fluoro-49,59-didehydro-59deoxyadenosine; wMNB, C-1300 murineneuroblastoma; rMNB-MDL, nucleoside analogue resistant C-1300 murine neuroblas-toma; MDR, multidrug resistance protein; MRP, multidrug resistance associated protein;GAPDH, glyceraldehyde-39-phosphate dehydrogenase; AZT, 29,39-dideoxy-39-azidothy-midine.

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cleoside analogue-induced resistance in murine neuroblastoma(rMNB-MDL) cells.

Materials and Methods

Chemicals and Drugs

All chemicals used in these studies were of tissue culture and molecularbiology grades and purchased from Sigma Chemical Co. (St. Louis, MO). TheECL chemiluminescence reagents were obtained from the Amersham LifeSciences Co., and PCR primers were from Macromolecular Resources (Ft.Collins, CO).

Cell Lines

The C-1300 wild-type murine neuroblastoma (wMNB) cell line was origi-nally obtained from the EG & G Mason Research Institute (Worchester, MA)and has been maintained in this laboratory without any apparent change intumorigenicity since 1980. Human leukemia cells (HL-60) were acquired fromthe American Type Culture Collection (Rockville, MD), and clones resistant todoxorubicin were isolated after long-term incubation with this drug at concen-trations ranging from 1029 to 5 3 1026 M. The resistant subclone, HL-60-R,was grown in DMEM supplemented with 1 mM glutamine and 10% fetalbovine serum. HL60-R cells served as the positive control for expression of theMDR phenotype.

The nucleoside analogue resistant murine neuroblastoma cell line (rMNB-MDL) was established by continuous incubation of wMNB in MDL 28,842 ata concentration varying from 13 1029 M to 1 3 1027 M (1). The cloned cellline rMNB-MDL has been sustained for over 4 years in DMEM supplementedwith 10% FCS, 10,000 units/ml penicillin, and 100mg/ml streptomycin with-out any apparent loss of drug resistance.

Immunoblot Analysis

AdoMet Synthetase Expression.wMNB and rMNB-MDL cells were har-vested in log phase using a lysis buffer containing 10 mM Tris-HCl (pH 7.4),1 mM DTT, 1 mM EDTA, and the protease inhibitors phenylmethylsulfonylfluoride (1 mmol/L), leupeptin (2mg/ml), pepstatin (1mg/mL), and aprotinin(2 mg/ml). Approximately 83 108 cells/ml were lysed by three 15-s burstswith a 50 Sonic Dismembranator (Fisher Scientific, Pittsburgh, PA) set at 50%intensity. The lysed cellular suspension was centrifuged at 10,0003 g for 15min at 4°C to obtain the soluble fraction. The protein concentration of thesupernatant was determined using a Micro BCA Protein Assay reagent kit(Pierce Chemical Co., Rockford, IL). The supernatant fractions (20mg ofprotein/lane) were electrophoresed on 10% SDS-polyacrylamide slab gels andthen transferred to an Immobilon-P transfer membrane (Millipore, Bedford,MA) by electroblotting. The transfer membrane was incubated for 1 h in PBScontaining 1% (w/v) BSA, 5% (w/v) nonfat dry milk, and 0.1% (w/v) Tween20 to prevent nonspecific binding of antibodies. Each membrane was incubatedfor 1 h at room temperature with polyclonal AdoMet synthetase antibodyspecific for thea and b forms of AdoMet synthetase (11). Immunoreactivebands were visualized by use of the ECL Western blotting detection kit(Amersham, Arlington Heights, IL). Molecular weight markers consisting ofb-galactosidase (Mr 116,000), phosphorylase-A (Mr 94,000), BSA (Mr

67,000), ovalbumin (Mr 43,000), lactate dehydrogenase (Mr 35,000), andsoybean trypsin inhibitor (Mr 21,500) were used to identify specific proteinbands.

RT- PCR Analysis of AdoMet Synthetase mRNA (MAT-II). Total RNAwas extracted from MNB cell lines using the Tri Reagent-RNA/DNA/ProteinIsolation reagent (Molecular Research Center, Inc., Cincinnati, OH). RNA (1mg) was incubated for 15 min at 42°C in a reaction mixture of 20ml containing50 mM Tris-HCl (pH 8.3), 75 mM KCL, 2 mM MgCl2, 10 mM DTT, 1 mM ofdeoxynucleotide triphosphate, 1unit/ml of RNase inhibitor from human pla-centa, 2.5mM of random hexamer primers, and 20 units of Avian myeloblas-tosis virus reverse transcriptase to synthesize the cDNA. The cDNA productwas subjected to PCR using a GeneAmp DNA Amplification kit (Perkin-Elmer, Branchburg, New Jersey) and thermocycler (M. J. Research, Inc.,Watertown, MA).

The AdoMet synthetase (MAT-II) sense primer 59-TGC CTT GGT TAC

GCC CTG ATT CTA-39 (nucleotides 583 to 606), antisense primer 59-CCATAG GCT GCA GTC CTC TGA TAA -39 (nucleotides 1175 to 1198) (12),GAPDH sense primer 59-ATT CTA CCC ACG GCA AGT TCA ATG G-3(nucleotides 173 to 197), antisense primer 59-AGG GGC GGA GAT GATGAC CC -39 (nucleotides 376 to 396; Ref. 13), DNA methyltransferase senseprimer, 59-GTGCGAGACACGATGTC-39(nucleotides 4337 to 4352), andantisense primer 59-CTGTCCAGGATGTTGCCG-39(nucleotides from 4934to 4953; Ref. 14) were designed for the PCR reactions. All PCR primers wereprocured from Macromolecular Resources (Ft. Collins, CO). The PCR reac-tions were carried out to 26, 35, and 25 cycles for AdoMet synthetase, DNAcytosine-MTase, and GAPDH, respectively. Denaturation was performed for 1min at 94°C, annealing for 1 min at 54°C, and polymerization for 2 min at72°C. The last cycles were carried out for 7 min at 72°C, followed by gradualcooling to ambient temperature.

QC-PCR Analysis of AdoMet Synthetase mRNA (MAT-II). AdoMetsynthetase was quantitatively determined by the procedure described in thePCR MIMIC Construction kit (Clontech, Palo Alto, CA). A heterologous DNAfragment was used as internal standard (PCR MIMIC) using the compositePCR sense primer 59-TGC CTT GGT TAC GCC CTG ATT CTA ATT CTACCC ACG GCA AGT TCA ATG G-39 and the antisense PCR primer 59-CCATAG GCT GCA GTC CTC TGA TAA AGG GGC GGA GAT GAT GACCC-39containing human AdoMet sequences ligated to rat GAPDH. A 266-bpamplified product of the internal standard (PCR MIMIC) was excised andpurified with the QIAquick gel extraction kit (Qiagen, Germany) and quanti-tatively analyzed by measuring its absorbance at 260 nm. Purified internalstandard (266 bp) was readily distinguished from AdoMet synthetase PCRproduct (616 bp) on an agarose gel. A standard curve for the internal standardwas prepared using 10-fold serial dilutions (107 to 1022 attomoles) of thereverse-transcribed cDNA products. PCR conditions were optimized to assurethat the reaction had linear amplification. The PCR-amplified products ofAdoMet synthetase, DNA cytosine-MTase, and GAPDH were detected byelectrophoresis of the amplified products on a 1.5% agarose gel using SYBRgreen dye (Molecular Probes, Eugene, OR). Gels were scanned, integrated, andanalyzed by densitometric analysis using a Phospho Imager (DU 70; Beckman,Schaumberg, IL).

Statistical Analysis

Densitometric analyses of Western blot and RT-PCR data were collatedfrom three individual experiments. The Western blot and PCR data presentedin this study are representative of one of the three experiments performed. Dataare presented as means6 SE. Statistical analyses were carried out using theone- or two-way ANOVA as appropriate, followed by the Newman-Keules testfor multiple comparison.P , 0.05 was deemed significant.

Results

AdoMet Synthetase Expression in Wild-Type (wMNB) and Nu-cleoside Analogue-resistant (rMNB-MDL) Murine Neuroblas-toma Tumor Cells. A significant increase in the expression of thecatalytic subunit (a2/a29) of AdoMet synthetase was observed inrMNB-MDL cells when compared with wMNB cells. Western blotanalyses using AdoMet synthetase (MATII)-specific antibodies dem-onstrated a 2.6- and 2.1-fold increase in thea2 (Mr 53,000) anda29(Mr 51,000) subunits of MAT-II AdoMet synthetase, respectively, inrMNB-MDL cells (Fig. 1A). Expression of the regulatoryb subunit(Mr 38,000) of AdoMet synthetase in wMNB and rMNB-MDL cellsdid not differ (Fig. 1B). In contrast, immunogenic expression of thea2and a29 subunit (Fig. 2A) andb subunit (Fig. 2B) of AdoMet syn-thetase in doxorubicin-resistant HL-60-R cells was decreased.40%(P , 0.05) when compared with the wild-type HL-60 cells.

QC-PCR Analysis of AdoMet Synthetase mRNA Expression inWild-Type (wMNB) and Nucleoside Analogue-resistant (rMNB-MDL) Murine Neuroblastoma Tumor Cells. The expression ofAdoMet synthetase mRNA was determined by RT-PCR analysis. An;3-fold increase was observed in rMNB-MDL cells when comparedwith wMNB cells (data not shown). This elevated expression of

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AdoMet synthetase mRNA correlated positively with the increment inimmunogenic protein as well as enzymatic activity of AdoMet syn-thetase described previously in rMNB-MDL cells (1).

AdoMet synthetase mRNA expression was quantitatively deter-mined by QC-PCR analysis. Target (AdoMet synthetase) and MIMIC(AdoMet synthetase and GADPH, a heterologous internal standard)PCR primers were designed to produce PCR products of 616 bp(AdoMet synthetase) and 266 bp (GAPDH), respectively. An equalamount of cDNA was amplified in a 10-fold dilution of the PCRMIMIC. Amplified PCR products were subjected to SYBR gel elec-trophoresis and densitometric analysis for integration. The QC-PCRanalyses (Fig. 3) demonstrated a 7.6-fold increase (n 5 8, P , 0.01)in the expression of AdoMet synthetase mRNA in rMNB-MDL cellswhen compared with wMNB cells. A significant increment in DNA(cytosine) methyltransferase mRNA expression was also observed inrMNB-MDL cells by RT-PCR analysis, which exceeded to that ofwMNB cells by ;3 fold (Fig. 4). GAPDH expression did not differbetween wMNB and rMNB-MDL cells (data not shown).

Discussion

A major problem encountered in the chemotherapy of neuroblas-toma, similar to that with other neoplasms, has been their intrinsic oracquired drug resistance (15). This phenomenon represents a majorobstacle to successful treatment. Factors contributing toward the de-velopment of drug resistance in neuroblastoma include overexpres-sion of the multidrug resistance gene (MDR1), which encodes for aMr

170,000 plasma membrane P-glycoprotein (16), and the multidrug-resistance-associated protein gene (MRP; Ref. 17), encoding aMr

190,000 membrane bound non-P-glycoprotein (18). Expression ofDNA topoisomerase II and glutathioneS-transferases (8), p53, BCL2,and the BAX gene families (19) have also been found to play animportant role in the development of drug resistance in a variety oftumors.

The present study has demonstrated the presence of a novel drugresistance mechanism in murine neuroblastoma cells resistant to theirreversible inhibitor of S-adenosylhomocysteine hydrolase, MDL

Fig. 1. Expression of AdoMet synthetase subunits in wild-type (wMNB) and nucleo-tide analogue-resistant murine neuroblastoma (rMNB-MDL) cells. Western blot (upperpanel) and histogram of integrated densitometric units (lower panel) for each electro-phoretic lane are shown.A, a2 subunit (Mr 53,000) anda29subunit (Mr 51,000).B, bsubunit (Mr 38,000). Protein samples (20mg/lane) were electrophoresed in a 10%SDS-PAGE as described in “Materials and Methods.”Bars,SE.

Fig. 2. Expression of AdoMet synthetase subunits in wild-type (HL60) and doxoru-bicin-resistant human leukemia (HL60-R) cells. Western blot (upper panel) and histogramof integrated densitometric units (lower panel) fora subunits (A) andb subunit (B) ofAdoMet synthetase are shown. Protein samples (20mg/lane) were electrophoresed in a10% SDS-PAGE as described in “Materials and Methods.”Bars,SE.

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28,842. These data reveal a significant and selective increase inAdoMet synthetase expression (MAT-II) in rMNB-MDL cells asdetermined by Western blot analysis using AdoMet synthetase (MAT-II)-specific antibody. The elevation in immunogenic AdoMet synthe-tase protein correlates with our earlier finding of a 2.0-fold increase inthe enzymatic activity of AdoMet synthetase in rMNB-MDL cellswhen compared with wMNB cells (1). Overexpression of AdoMetsynthetase mRNA as demonstrated by QC-PCR analysis should favorhypermethylation of CpG dinucleotides (2) predominantly found inthe promoter regions of the growth regulatory and tumor suppressorgenes of rMNB-MDL cells. Although global and specific DNA meth-ylation was not determined in this investigation, the finding that DNA

(cytosine) methyl transferase mRNA expression is elevated in rMNB-MDL cells supports this interpretation.

CpG dinucleotides, comprising CpG islands, are 0.2–1 kb in lengthand constitute about 1% of the vertebrate genome. These are locatedupstream in the promoter regions of many regulatory genes. In thepresence of DNA (cytosine) methyltransferase, they are targets forDNA methylation and produce 5-methylcytosine, which undergoesdeamination to form thymidine (20). Methylation of cytosine at a CpGdinucleotide increases the probability of a cytosine to thymine or corre-sponding guanine to adenine transition mutation. The high frequencyof mutagenesis observed at CpG sites is due to differences in therepair efficiencies of premutagenic lesions generated by methylation.The G:C mismatch resulting from deamination of 5-methylcytosine isbelieved to be more difficult for repair by uracil-DNA glycosylasethan G:U mismatches, which are generated from the spontaneousdeamination of cytosine. Presumably this occurs because thymine, anormal component of DNA, unlike uracil (21), is not recognized bythe DNA repair mechanism(s). It has been shown that in humancolonic tumors, excision of uracil is;6000 fold more efficient thanthymine in affecting this process (22).

The observed increase in AdoMet synthetase and DNA (cytosine)methyl transferase expression is reflected by an increased AdoMet/AdoHcy ratio in rMNB-MDL cells (1). An elevated level of DNA(cytosine) methyl transferase expression, which is cell cycle depend-ent, may also cause inactivation or silencing by methylation of func-tional tumor suppressor genes (23) in drug-resistant tumor cells (20).The significant elevation in AdoMet synthetase mRNA expression,observed in the present study with rMNB-MDL cells, favors increasedDNA methylation (2).

Drug-induced DNA hypermethylation of cytosine within CpG is-lands can alter gene function by transcriptional inactivation of growthregulatory and tumor suppressor genes (24) and thereby induce drugresistance in tumor cells (9). It has been shown that AZT, an inducer

Fig. 3. QC-PCR analysis of AdoMet synthetase mRNA in wild-type (wMNB) and nucleoside analogue-resistant murine neuroblastoma (rMNB-MDL) cells.Upper panel,QC-PCRwas performed using specific primers designed for target (AdoMet synthetase) and mimic (internal standard) samples as described in “Materials and Methods.” Electrophoretic lanesrepresent 10-fold dilutions of MIMIC PCR products. GAPDH expression was not altered in wMNB and rMNB-MDL cells (data not shown).Lower panel,integrated densitometricunits of the electrophoretic bands corresponding to target and MIMIC PCR products were determined and graphed against the log of ratio of copy numbers of target to MIMIC samples(AdoMet synthetase: MIMIC). Cross-over point (intersect) of target and mimic samples represent the point of equivalence and was used to calculate the copy number (number ofmolecules of mRNA/mg total RNA). These data are representative of three independent experiments.

Fig. 4. RT-PCR analysis of DNA (cytosine) methyl transferase (MTase) in wild-type(wMNB) and nucleoside analogue-resistant murine neuroblastoma (rMNB-MDL) cells.RT-PCR analysis for MTase was performed by using MTase-specific primers as describedin “Materials and Methods.” Agarose gel (top panel) of the RT-PCR-amplified productsand histogram of integrated densitometric analyses (lower panel) for each respectiveelectrophoretic lane are shown. GAPDH amplification was used as a positive control anddid not differ in wMNB and rMNB-MDL cells. These data are representative of threeindependent experiments.

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of DNA hypermethylation, produces AZT resistance by transcrip-tional inactivation of thymidine kinase (TK2) genes. Subsequentexposure of AZT-resistant cells to the demethylating agent 5-aza-29-deoxycytidine is associated with the transcriptional inactivation ofTK[minus] genes (9). Epigenetic changes due to hypermethylationinduced transcriptional inactivation of the deoxycytidine kinase gene,and resistance to ara-C has been demonstrated previously (25). Hy-permethylation-induced conformational changes in DNA strands bycisplatin has also been documented to play an important role incisplatin-induced drug resistance (26).

Hypermethylation of CpG dinucleotides within the CpG island ofthe p53 tumor-suppressing gene and its functional inactivation havebeen suggested to occur in various human cancers. It has been shownrecently that methylated CpG sites in thep53gene exhibit preferentialbinding for a variety of carcinogens, including benzo(a)pyrene andaflatoxin B. Increased hypermethylation of CpG islands in othertumor suppressor genes such as retinoblastoma (Rb; Ref. 23), vonHappel-Lindau (VHL; Ref. 27), andH19 gene (28) and their func-tional inactivation has also been demonstrated previously. A reducedtranscriptional activity of mouse metallothionine (mMT1) promoter intransient transfection experiments has been correlated due to themethylation of CpGs located at the preinitiation domain of the pro-moter (29). Thus, the epigenetic effects of DNA methylation due to anincreased expression of AdoMet synthetase gene in resistant MNBtumor cells provide an important potential mechanism of drug resist-ance (30) in rMNB-MDL cells, whereas drug resistance in doxorubi-cin-resistant HL-60 cells is primarily due to the expression ofMDRgenes.

Although the existence of other drug resistance mechanism(s) inrMNB-MDL cells, in addition to overexpression of AdoMet synthe-tase, cannot be ruled out, the modulation of AdoMet synthetase geneexpression in neuroblastoma cells as a means of preventing or revers-ing acquired resistance to nucleoside analogues represents an innova-tive possibility worthy of further investigation.

Acknowledgments

We are thankful to Dr. Caiping Wang and Ms. Janet Devine for helpfulsuggestions and critical reading of the manuscript. Dr. H. L. LeGros, theUniversity of Tennessee, Memphis, is thankfully acknowledged for providingAdoMet (MAT-II)-specific antibodies.

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1999;59:1852-1856. Cancer Res   Rama S. Dwivedi, Ling-Jia Wang and Bernard L. Mirkin  Mechanism of Drug Resistance

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