Cancer Research Targets Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 1852-1856, April 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dwivedi, R. S.
Right arrow Articles by Mirkin, B. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dwivedi, R. S.
Right arrow Articles by Mirkin, B. L.
[Cancer Research 59, 1852-1856, April 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

S-Adenosylmethionine Synthetase Is Overexpressed in Murine Neuroblastoma Cells Resistant to Nucleoside Analogue Inhibitors of S-Adenosylhomocysteine Hydrolase

A Novel Mechanism of Drug Resistance1

Rama S. Dwivedi2, Ling-Jia Wang and Bernard L. Mirkin

Children’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 Molecular Pharmacology and Biological Chemistry [B. L. M.], Northwestern University Medical School, Chicago, Illinois 60614


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), which catalyzes the synthesis of AdoMet from methionine and ATP, is the major methyl donor for transmethylation reactions and propylamino donor for the biosynthesis of polyamines in biological systems. We have reported 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 inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), express increased AdoMet synthetase activity (M. R. Hamre et al., Oncol. Res., 7: 487–492, 1995). In the present study, immunoblot analyses of AdoMet Synthetase with isoform-specific (MATII) antibodies demonstrated an elevation in the AdoMet synthetase immunoprotein in nucleoside analogue-resistant MNB cells (rMNB-MDL) when compared to wild-type, nonresistant MNB cells. An increase of 2.1-fold was observed in the {alpha}2/{alpha}2' catalytic subunit, which differed significantly from the much smaller increment in the noncatalytic ß-subunit of AdoMet synthetase. Densitometric analyses revealed that an increased expression of AdoMet synthetase in rMNB-MDL cells was due to overexpression of the {alpha}2 (Mr 53,000; 2.6-fold) and {alpha}2' (Mr 51,000; 1.8-fold) subunits. AdoMet synthetase mRNA expression in rMNB-MDL cells was remarkably greater than wMNB cells, as determined by quantitative competitive reverse transcription-PCR (QC-PCR) analysis. DNA (cytosine) methyl transferase expression, measured by reverse transcription-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-resistant human leukemia cells (HL-60-R) expressing multidrug resistance protein when compared with wild-type, nonresistant HL-60 cells. The resistance of rMNB-MDL cells to nucleoside analogue inhibitors of S-adenosylhomocysteine hydrolase correlates directly with overexpression of the {alpha}2/{alpha}2' subunits of AdoMet synthetase. Cellular adaptation allows sufficient AdoMet to be synthesized, so that viability of the MNB cells can be maintained even in the presence of high AdoHcy concentrations. This novel mechanism of drug resistance does not appear to require multidrug resistance protein (P-glycoprotein) overexpression.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
AdoMet3 synthetase (ATP: L-methionine S-adenosyltransferase; EC 2.5.1.6), the enzyme responsible for synthesis of AdoMet from L-methionine and ATP, plays a major role in the development of acquired drug resistance to nucleoside analogue inhibitors of AdoHcy hydrolase (EC 3.3.1.1) by murine neuroblastoma tumor cells (1) . AdoMet is the major donor of methyl groups for transmethylation reactions in eukaryotic systems (2) and is essential for normal cellular metabolism. Inhibition of this enzyme alters metabolic function, ultimately leading to cell death. Three different isoforms of mammalian AdoMet synthetase, {alpha} (MAT-I), ß (MAT-III), and {gamma} (MAT- II) have been identified (3) . MAT-I and MAT-III are products of the same gene, MAT1A, and predominantly expressed in adult liver. MAT-II is a product of the MAT2A gene and mainly present in kidney, brain, lymphocytes, testis, and lens and fetal, newborn, and early regenerating liver (4) . The tetramer MAT-I (Mr 200,000) and dimer MAT-III (Mr 100,000) are oligomeric forms of the {alpha}1 subunit, whereas MAT-II (Mr 185,000) is a heterooligomeric complex comprised of {alpha}2/{alpha}2' and ß subunits. The {alpha}2' subunit of MAT-II is considered to represent a posttranslational modification of the {alpha}2 subunit during biological maturation. MAT-II, predominantly expressed in fetal liver, is gradually replaced by MAT-I and MAT-III (5) . In carcinogen-induced hepatocarcinoma, the expression of MAT-I and III decreases while MAT-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 methyltransferases and can influence AdoMet-associated cellular methylation processes (2) . The concentration of AdoMet and magnitude of AdoMet synthetase activity modulate the transmethylation reactions that regulate functional gene expression in biological systems.

Inhibition of AdoMet synthetase gene expression and enzymic activity results in cell death (7) . AdoMet synthetase-mediated methylation of DNA at CpG sites catalyzed by DNA methyl transferase, in particular, is associated with the functional and transcriptional inactivation of a number of genes during the process of cellular differentiation. Impaired regulation of protein expression or mRNA synthesis by gene methylation (8) constitutes an important mechanism of drug resistance (9 , 10) . This investigation has examined the role of AdoMet synthetase (MAT-II) gene overexpression in the development of nucleoside analogue-induced resistance in murine neuroblastoma (rMNB-MDL) cells.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Chemicals and Drugs
All chemicals used in these studies were of tissue culture and molecular biology grades and purchased from Sigma Chemical Co. (St. Louis, MO). The ECL chemiluminescence reagents were obtained from the Amersham Life Sciences Co., and PCR primers were from Macromolecular Resources (Ft. Collins, CO).

Cell Lines
The C-1300 wild-type murine neuroblastoma (wMNB) cell line was originally obtained from the EG & G Mason Research Institute (Worchester, MA) and has been maintained in this laboratory without any apparent change in tumorigenicity since 1980. Human leukemia cells (HL-60) were acquired from the American Type Culture Collection (Rockville, MD), and clones resistant to doxorubicin were isolated after long-term incubation with this drug at concentrations ranging from 10-9 to 5 x 10-6M. The resistant subclone, HL-60-R, was grown in DMEM supplemented with 1 mM glutamine and 10% fetal bovine serum. HL60-R cells served as the positive control for expression of the MDR phenotype.

The nucleoside analogue resistant murine neuroblastoma cell line (rMNB-MDL) was established by continuous incubation of wMNB in MDL 28,842 at a concentration varying from 1 x 10-9M to 1 x 10-7M(1) . The cloned cell line rMNB-MDL has been sustained for over 4 years in DMEM supplemented with 10% FCS, 10,000 units/ml penicillin, and 100 µg/ml streptomycin without any apparent loss of drug resistance.

Immunoblot Analysis
AdoMet Synthetase Expression.
wMNB and rMNB-MDL cells were harvested 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 phenylmethylsulfonyl fluoride (1 mmol/L), leupeptin (2 µg/ml), pepstatin (1 µg/mL), and aprotinin (2 µg/ml). Approximately 8 x 108 cells/ml were lysed by three 15-s bursts with a 50 Sonic Dismembranator (Fisher Scientific, Pittsburgh, PA) set at 50% intensity. The lysed cellular suspension was centrifuged at 10,000 x g for 15 min at 4°C to obtain the soluble fraction. The protein concentration of the supernatant was determined using a Micro BCA Protein Assay reagent kit (Pierce Chemical Co., Rockford, IL). The supernatant fractions (20 µg of protein/lane) were electrophoresed on 10% SDS-polyacrylamide slab gels and then transferred to an Immobilon-P transfer membrane (Millipore, Bedford, MA) by electroblotting. The transfer membrane was incubated for 1 h in PBS containing 1% (w/v) BSA, 5% (w/v) nonfat dry milk, and 0.1% (w/v) Tween 20 to prevent nonspecific binding of antibodies. Each membrane was incubated for 1 h at room temperature with polyclonal AdoMet synthetase antibody specific for the {alpha} and ß forms of AdoMet synthetase (11) . Immunoreactive bands were visualized by use of the ECL Western blotting detection kit (Amersham, Arlington Heights, IL). Molecular weight markers consisting of ß-galactosidase (Mr 116,000), phosphorylase-A (Mr 94,000), BSA (Mr 67,000), ovalbumin (Mr 43,000), lactate dehydrogenase (Mr 35,000), and soybean trypsin inhibitor (Mr 21,500) were used to identify specific protein bands.

RT- PCR Analysis of AdoMet Synthetase mRNA (MAT-II).
Total RNA was extracted from MNB cell lines using the Tri Reagent-RNA/DNA/Protein Isolation reagent (Molecular Research Center, Inc., Cincinnati, OH). RNA (1 µg) was incubated for 15 min at 42°C in a reaction mixture of 20 µl containing 50 mM Tris-HCl (pH 8.3), 75 mM KCL, 2 mM MgCl2, 10 mM DTT, 1 mM of deoxynucleotide triphosphate, 1unit/µl of RNase inhibitor from human placenta, 2.5 µM of random hexamer primers, and 20 units of Avian myeloblastosis virus reverse transcriptase to synthesize the cDNA. The cDNA product was 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 5'-TGC CTT GGT TAC GCC CTG ATT CTA-3' (nucleotides 583 to 606), antisense primer 5'-CCA TAG GCT GCA GTC CTC TGA TAA -3' (nucleotides 1175 to 1198) (12) , GAPDH sense primer 5'-ATT CTA CCC ACG GCA AGT TCA ATG G-3 (nucleotides 173 to 197), antisense primer 5'-AGG GGC GGA GAT GAT GAC CC -3' (nucleotides 376 to 396; Ref. 13 ), DNA methyltransferase sense primer, 5'-GTGCGAGACACGATGTC-3' (nucleotides 4337 to 4352), and antisense primer 5'-CTGTCCAGGATGTTGCCG-3' (nucleotides from 4934 to 4953; Ref. 14 ) were designed for the PCR reactions. All PCR primers were procured from Macromolecular Resources (Ft. Collins, CO). The PCR reactions were carried out to 26, 35, and 25 cycles for AdoMet synthetase, DNA cytosine-MTase, and GAPDH, respectively. Denaturation was performed for 1 min at 94°C, annealing for 1 min at 54°C, and polymerization for 2 min at 72°C. The last cycles were carried out for 7 min at 72°C, followed by gradual cooling to ambient temperature.

QC-PCR Analysis of AdoMet Synthetase mRNA (MAT-II).
AdoMet synthetase was quantitatively determined by the procedure described in the PCR MIMIC Construction kit (Clontech, Palo Alto, CA). A heterologous DNA fragment was used as internal standard (PCR MIMIC) using the composite PCR sense primer 5'-TGC CTT GGT TAC GCC CTG ATT CTA ATT CTA CCC ACG GCA AGT TCA ATG G-3' and the antisense PCR primer 5'-CCA TAG GCT GCA GTC CTC TGA TAA AGG GGC GGA GAT GAT GAC CC-3' containing human AdoMet sequences ligated to rat GAPDH. A 266-bp amplified product of the internal standard (PCR MIMIC) was excised and purified with the QIAquick gel extraction kit (Qiagen, Germany) and quantitatively analyzed by measuring its absorbance at 260 nm. Purified internal standard (266 bp) was readily distinguished from AdoMet synthetase PCR product (616 bp) on an agarose gel. A standard curve for the internal standard was prepared using 10-fold serial dilutions (107 to 10-2 attomoles) of the reverse-transcribed cDNA products. PCR conditions were optimized to assure that the reaction had linear amplification. The PCR-amplified products of AdoMet synthetase, DNA cytosine-MTase, and GAPDH were detected by electrophoresis of the amplified products on a 1.5% agarose gel using SYBR green dye (Molecular Probes, Eugene, OR). Gels were scanned, integrated, and analyzed by densitometric analysis using a Phospho Imager (DU 70; Beckman, Schaumberg, IL).

Statistical Analysis
Densitometric analyses of Western blot and RT-PCR data were collated from three individual experiments. The Western blot and PCR data presented in this study are representative of one of the three experiments performed. Data are presented as means ± SE. Statistical analyses were carried out using the one- or two-way ANOVA as appropriate, followed by the Newman-Keules test for multiple comparison. P < 0.05 was deemed significant.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
AdoMet Synthetase Expression in Wild-Type (wMNB) and Nucleoside Analogue-resistant (rMNB-MDL) Murine Neuroblastoma Tumor Cells.
A significant increase in the expression of the catalytic subunit ({alpha}2/{alpha}2') of AdoMet synthetase was observed in rMNB-MDL cells when compared with wMNB cells. Western blot analyses using AdoMet synthetase (MATII)-specific antibodies demonstrated a 2.6- and 2.1-fold increase in the {alpha}2 (Mr 53,000) and {alpha}2' (Mr 51,000) subunits of MAT-II AdoMet synthetase, respectively, in rMNB-MDL cells (Fig. 1A)Citation . Expression of the regulatory ß subunit (Mr 38,000) of AdoMet synthetase in wMNB and rMNB-MDL cells did not differ (Fig. 1B)Citation . In contrast, immunogenic expression of the {alpha}2 and {alpha}2' subunit (Fig. 2A)Citation and ß subunit (Fig. 2B)Citation of AdoMet synthetase in doxorubicin-resistant HL-60-R cells was decreased >40% (P < 0.05) when compared with the wild-type HL-60 cells.



View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Expression of AdoMet synthetase subunits in wild-type (wMNB) and nucleotide analogue-resistant murine neuroblastoma (rMNB-MDL) cells. Western blot (upper panel) and histogram of integrated densitometric units (lower panel) for each electrophoretic lane are shown. A, {alpha}2 subunit (Mr 53,000) and {alpha}2'subunit (Mr 51,000). B, ß subunit (Mr 38,000). Protein samples (20 µg/lane) were electrophoresed in a 10% SDS-PAGE as described in "Materials and Methods." Bars, SE.

 


View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Expression of AdoMet synthetase subunits in wild-type (HL60) and doxorubicin-resistant human leukemia (HL60-R) cells. Western blot (upper panel) and histogram of integrated densitometric units (lower panel) for {alpha} subunits (A) and ß subunit (B) of AdoMet synthetase are shown. Protein samples (20 µg/lane) were electrophoresed in a 10% SDS-PAGE as described in "Materials and Methods." Bars, SE.

 
QC-PCR Analysis of AdoMet Synthetase mRNA Expression in Wild-Type (wMNB) and Nucleoside Analogue-resistant (rMNB-MDL) Murine Neuroblastoma Tumor Cells.
The expression of AdoMet synthetase mRNA was determined by RT-PCR analysis. An ~3-fold increase was observed in rMNB-MDL cells when compared with wMNB cells (data not shown). This elevated expression of AdoMet synthetase mRNA correlated positively with the increment in immunogenic protein as well as enzymatic activity of AdoMet synthetase described previously in rMNB-MDL cells (1) .

AdoMet synthetase mRNA expression was quantitatively determined 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 equal amount of cDNA was amplified in a 10-fold dilution of the PCR MIMIC. Amplified PCR products were subjected to SYBR gel electrophoresis and densitometric analysis for integration. The QC-PCR analyses (Fig. 3)Citation demonstrated a 7.6-fold increase (n = 8, P < 0.01) in the expression of AdoMet synthetase mRNA in rMNB-MDL cells when compared with wMNB cells. A significant increment in DNA (cytosine) methyltransferase mRNA expression was also observed in rMNB-MDL cells by RT-PCR analysis, which exceeded to that of wMNB cells by ~3 fold (Fig. 4)Citation . GAPDH expression did not differ between wMNB and rMNB-MDL cells (data not shown).



View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		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-PCR was performed using specific primers designed for target (AdoMet synthetase) and mimic (internal standard) samples as described in "Materials and Methods." Electrophoretic lanes represent 10-fold dilutions of MIMIC PCR products. GAPDH expression was not altered in wMNB and rMNB-MDL cells (data not shown). Lower panel, integrated densitometric units 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 of molecules of mRNA/µg total RNA). These data are representative of three independent experiments.

 


View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		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 described in "Materials and Methods." Agarose gel (top panel) of the RT-PCR-amplified products and histogram of integrated densitometric analyses (lower panel) for each respective electrophoretic lane are shown. GAPDH amplification was used as a positive control and did not differ in wMNB and rMNB-MDL cells. These data are representative of three independent experiments.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
A major problem encountered in the chemotherapy of neuroblastoma, similar to that with other neoplasms, has been their intrinsic or acquired drug resistance (15) . This phenomenon represents a major obstacle to successful treatment. Factors contributing toward the development of drug resistance in neuroblastoma include overexpression of the multidrug resistance gene (MDR1), which encodes for a Mr 170,000 plasma membrane P-glycoprotein (16) , and the multidrug-resistance-associated protein gene (MRP; Ref. 17 ), encoding a Mr 190,000 membrane bound non-P-glycoprotein (18) . Expression of DNA topoisomerase II and glutathione S-transferases (8) , p53, BCL2, and the BAX gene families (19) have also been found to play an important role in the development of drug resistance in a variety of tumors.

The present study has demonstrated the presence of a novel drug resistance mechanism in murine neuroblastoma cells resistant to the irreversible inhibitor of S-adenosylhomocysteine hydrolase, MDL 28,842. These data reveal a significant and selective increase in AdoMet synthetase expression (MAT-II) in rMNB-MDL cells as determined by Western blot analysis using AdoMet synthetase (MAT-II)-specific antibody. The elevation in immunogenic AdoMet synthetase protein correlates with our earlier finding of a 2.0-fold increase in the enzymatic activity of AdoMet synthetase in rMNB-MDL cells when compared with wMNB cells (1) . Overexpression of AdoMet synthetase mRNA as demonstrated by QC-PCR analysis should favor hypermethylation of CpG dinucleotides (2) predominantly found in the promoter regions of the growth regulatory and tumor suppressor genes of rMNB-MDL cells. Although global and specific DNA methylation 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 length and constitute about 1% of the vertebrate genome. These are located upstream in the promoter regions of many regulatory genes. In the presence of DNA (cytosine) methyltransferase, they are targets for DNA methylation and produce 5-methylcytosine, which undergoes deamination to form thymidine (20) . Methylation of cytosine at a CpG dinucleotide increases the probability of a cytosine to thymine or corresponding guanine to adenine transition mutation. The high frequency of mutagenesis observed at CpG sites is due to differences in the repair efficiencies of premutagenic lesions generated by methylation. The G:C mismatch resulting from deamination of 5-methylcytosine is believed to be more difficult for repair by uracil-DNA glycosylase than G:U mismatches, which are generated from the spontaneous deamination of cytosine. Presumably this occurs because thymine, a normal component of DNA, unlike uracil (21) , is not recognized by the DNA repair mechanism(s). It has been shown that in human colonic tumors, excision of uracil is ~6000 fold more efficient than thymine 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 dependent, may also cause inactivation or silencing by methylation of functional 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 increased DNA methylation (2) .

Drug-induced DNA hypermethylation of cytosine within CpG islands can alter gene function by transcriptional inactivation of growth regulatory and tumor suppressor genes (24) and thereby induce drug resistance in tumor cells (9) . It has been shown that AZT, an inducer of DNA hypermethylation, produces AZT resistance by transcriptional inactivation of thymidine kinase (TK-) genes. Subsequent exposure of AZT-resistant cells to the demethylating agent 5-aza-2'-deoxycytidine is associated with the transcriptional inactivation of TK[minus] genes (9) . Epigenetic changes due to hypermethylation induced transcriptional inactivation of the deoxycytidine kinase gene, and resistance to ara-C has been demonstrated previously (25) . Hypermethylation-induced conformational changes in DNA strands by cisplatin has also been documented to play an important role in cisplatin-induced drug resistance (26) .

Hypermethylation of CpG dinucleotides within the CpG island of the p53 tumor-suppressing gene and its functional inactivation have been suggested to occur in various human cancers. It has been shown recently that methylated CpG sites in the p53 gene exhibit preferential binding for a variety of carcinogens, including benzo(a)pyrene and aflatoxin B. Increased hypermethylation of CpG islands in other tumor suppressor genes such as retinoblastoma (Rb; Ref. 23 ), von Happel-Lindau (VHL; Ref. 27 ), and H19 gene (28) and their functional inactivation has also been demonstrated previously. A reduced transcriptional activity of mouse metallothionine (mMT1) promoter in transient transfection experiments has been correlated due to the methylation of CpGs located at the preinitiation domain of the promoter (29) . Thus, the epigenetic effects of DNA methylation due to an increased expression of AdoMet synthetase gene in resistant MNB tumor cells provide an important potential mechanism of drug resistance (30) in rMNB-MDL cells, whereas drug resistance in doxorubicin-resistant HL-60 cells is primarily due to the expression of MDR genes.

Although the existence of other drug resistance mechanism(s) in rMNB-MDL cells, in addition to overexpression of AdoMet synthetase, cannot be ruled out, the modulation of AdoMet synthetase gene expression in neuroblastoma cells as a means of preventing or reversing acquired resistance to nucleoside analogues represents an innovative possibility worthy of further investigation.


   ACKNOWLEDGMENTS
 
We are thankful to Dr. Caiping Wang and Ms. Janet Devine for helpful suggestions and critical reading of the manuscript. Dr. H. L. LeGros, the University of Tennessee, Memphis, is thankfully acknowledged for providing AdoMet (MAT-II)-specific antibodies.


   FOOTNOTES
 
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.

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

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

3 The abbreviations used are: AdoMet, S-adenosylmethionine; MAT, methionine adenosyltransferase; AdoHcy, S-adenosylhomocysteine; RT-PCR, reverse transcription-PCR; QC-PCR, quantitative competitive RT-PCR; MTase, DNA (cytosine) methyl transferase; MDL, (Z)-5'-fluoro-4',5'-didehydro-5'deoxyadenosine; wMNB, C-1300 murine neuroblastoma; rMNB-MDL, nucleoside analogue resistant C-1300 murine neuroblastoma; MDR, multidrug resistance protein; MRP, multidrug resistance associated protein; GAPDH, glyceraldehyde-3'-phosphate dehydrogenase; AZT, 2',3'-dideoxy-3'-azidothymidine. Back

Received 9/25/98. Accepted 3/ 2/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Hamre M. R., Clark S. H., Mirkin B. L. Resistance to inhibitors of S-adenosyl-L-homocysteine hydrolase in C1300 murine neuroblastoma tumor cells is associated with increased methionine adenosyltransferase activity. Oncol Res., 7: 487-492, 1995.[Medline]
  2. Chiang P. K., Gordon R. K., Tal J., Zeng G. C., Doctor B. P., Pardhasardhi K., McCann P. P. S-Adenosylmethionine and methylation. FASEB J., 10: 471-480, 1996.[Abstract]
  3. Kotb M., Mudd S. H., Mato J. M., Geller A. M., Kredich N. M., Chou J. Y., Cantoni G. L. Consensus nomenclature for the mammalian methionine adenosyltransferase gene and gene products. Trends Genet., 13: 51-52, 1997.[Medline]
  4. Kotb M., Geller A. M. Methionine adenosyltransferase: structure and function. Pharmacol. Ther., 59: 125-143, 1993.[Medline]
  5. Gil B., Casado M., Pajares M. A., Bosca’ L., Mato J. M., Martin-Sanz P., Alvarez L. Differential expression pattern of S-adenosylmethionine synthetase isozymes during rat liver development. Hepatology, 24: 876-881, 1996.[Medline]
  6. Tsukada K., Okada G. S-Adenosylmethionine synthetase isozyme patterns from rat hepatoma induced by N-2-fluorenylacetamide. Biochem. Biophys. Res. Commun., 94: 1078-1082, 1980.[Medline]
  7. Cai J., Mao Z., Hwang J-J., Lu S. C. Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells. Cancer Res., 58: 1444-1450, 1998.[Abstract/Free Full Text]
  8. Alton P. A., Harris A. L. Annotation. The role of DNA topoisomerases II in drug resistance. Br. J. Haematol., 85: 241-245, 1993.[Medline]
  9. Nyce J. W. Drug-induced DNA hypermethylation and drug resistance in human tumors. Cancer Res., 49: 5829-5836, 1989.[Abstract/Free Full Text]
  10. Costello J. F., Futscher B. W., Kroes R. A., Pieper R. O. Methylation related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of O-6-methylguanine DNA methyltransferase gene in human glioma cell lines. Mol. Cell Biol., 14: 6515-6521, 1994.[Abstract/Free Full Text]
  11. LeGros H. L., Geller A. M., Kotb M. Differential regulation of methionine adenosyltransferase in superantigen and mitogen stimulated human T lymphocytes. J. Biol. Chem., 272: 16040-16047, 1997.[Abstract/Free Full Text]
  12. Horikawa S., Tsukada K. Molecular cloning and developmental expression of a human kidney S-adenosylmethionine synthetase. FEBS Lett., 312: 37-41, 1992.[Medline]
  13. Tso J. Y., Sun X-H., Kao T-H., Reece K. S., Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAase: genomic complexity and molecular evolution of the gene. Nucleic Acids Res., 13: 2485-2502, 1985.[Abstract/Free Full Text]
  14. Yoder J. A., Yen R. C., Vertino P. M., Bestor T. H., Baylin S. B. New 5' regions of the murine and human genes for DNA (cytosine-5)-methyltransferase. J. Biol. Chem., 271: 31092-31097, 1996.[Abstract/Free Full Text]
  15. Bradley G., Ling V. P-glycoprotein, multidrug resistance and tumor progression. Cancer Metastasis Rev., 13: 223-233, 1994.[Medline]
  16. Bates S. E., Mickley L. A., Chen Y-N., Richert N., Rudick J., Biedler J. L., Fojo A. T. Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation. Mol. Cell. Biol., 9: 4337-4344, 1989.[Abstract/Free Full Text]
  17. Cole S. P. C., Bhardwaj G., Gerlach J. H., Mackie J. E., Grant C. E., Almquist K. C., Stewart A. J., Kurtz E. U., Duncan A. M. V., Deeley R. G. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science (Washington DC), 258: 1650-1654, 1992.[Abstract/Free Full Text]
  18. Norris M. D., Bordow S. B., Marshall G. M., Haber P. S., Cohn S. L., Haber M. Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma. N. Engl. J. Med., 334: 231-238, 1996.[Abstract/Free Full Text]
  19. Ling V. P-glycoprotein: its role in drug resistance. Am. J. Med., 99 (Suppl. 6A): 31S-34S, 1995.[Medline]
  20. Gonzalgo M. L., Jones P. A. Mutagenic and epigenetic effects of DNA methylation. Mutat. Res., 386: 107-118, 1997.[Medline]
  21. Mol C. D., Arvai A. S., Slupphaug G., Kavli B., Alseth I., Krokan H. E., Tainer J. A. Crystal structure and mutational analysis of human uracil:DNA glycosylase: structural basis for specificity and catalysis. Cell, 80: 869-878, 1995.[Medline]
  22. Schmutte C., Yang A. S., Beart R. W., Jones P. A. Base excision repair of U:G mismatches at a mutational hotspot in the p53 gene is more efficient than base excision repair of T:G mismatches in extracts of human colon tumors. Cancer Res., 55: 3742-3746, 1995.[Abstract/Free Full Text]
  23. Sakai T., Toguchida J., Ohtani N., Yandell D. W., Rapaport J. M., Dryja T. P. Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am. J. Hum. Genet., 48: 880-888, 1991.[Medline]
  24. Cedar H. DNA methylation and gene activity. Cell, 53: 3-4, 1988.[Medline]
  25. Nyce J. W. Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2'deoxycytidine 5'-triphosphate. Somatic Cell Mol. Genet., 17: 543-550, 1991.[Medline]
  26. Bellon S. F., Coleman J. H., Lippard S. J. DNA unwinding produced by site-specific intrastrand cross-links of the anticancer drug cis-diamminedichloroplatinum(II). Biochemistry, 30: 8026-8035, 1991.[Medline]
  27. Gnara J. R., Tory K., Weng Y. Mutations of the VHL tumor suppressor gene in renal carcinoma. Nat. Genet., 7: 85-90, 1994.[Medline]
  28. Steenman M. J. C., Rainier S., Dobry C. J., Grundy P., Horon I. L., Feinberg A. P. Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilm’s tumor. Nat. Genet., 7: 433-439, 1994.[Medline]
  29. Levine A., Cantoni G. L., Razin A. Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism. Proc. Natl. Acad. Sci. USA, 89: 10119-10123, 1992.[Abstract/Free Full Text]
  30. Nyce J., Leonard S., Canupp D., Schulz S., Wong S. Epigenetic mechanisms of drug resistance: drug-induced DNA hypermethylation and drug resistance. Proc. Natl. Acad. Sci. USA, 90: 2960-2964, 1993.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
S. J. James, S. Melnyk, M. Pogribna, I. P. Pogribny, and M. A. Caudill
Elevation in S-Adenosylhomocysteine and DNA Hypomethylation: Potential Epigenetic Mechanism for Homocysteine-Related Pathology
J. Nutr., August 1, 2002; 132(8): 2361S - 2366.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Kihara, T. Tsunoda, T. Tanaka, H. Yamana, Y. Furukawa, K. Ono, O. Kitahara, H. Zembutsu, R. Yanagawa, K. Hirata, et al.
Prediction of Sensitivity of Esophageal Tumors to Adjuvant Chemotherapy by cDNA Microarray Analysis of Gene-Expression Profiles
Cancer Res., September 1, 2001; 61(17): 6474 - 6479.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dwivedi, R. S.
Right arrow Articles by Mirkin, B. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dwivedi, R. S.
Right arrow Articles by Mirkin, B. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online