INCREASED DNA METHYLTRANSFERASE EXPRESSIONIN RHABDOMYOSARCOMASBin CHEN1*, Xiuli L IU1, Van H. SAVELL 1, Bradley R. DILDAY 1, Michael W. JOHNSON1, Jesse J. JENKINS2 AND DAVID M. PARHAM1

1Department of Pathology, University of Arkansas for Medical Sciences and Arkansas Children’s Hospital, Little Rock, AR, USA2Department of Pathology and Laboratory Medicine, St. Jude Children’s Research Hospital, Memphis, TN, USA

In normal somatic cells, the methylation pattern of DNA isstably maintained by DNA (cytosine-5-)-methyltransferase(DNA methyltransferase). Increased expression of DNAmethyltransferase has been detected in many types of humancancer and has been thought to play an important role intumorigenesis. In our study, we developed a standardizedreverse transcription-polymerase chain reaction (RT-PCR)assay to determine the mRNA levels of DNA methyltransfer-ase in rhabdomyosarcoma, the most common soft tissuecancer in children. Using this assay, expression of DNAmethyltransferase was analyzed for 32 rhabdomyosarcomasand 12 normal skeletal muscle samples. All tumor samples, ofwhich 18 were embryonal and 14 were alveolar subtype,showed increased expression of DNA methyltransferase afternormalization to b-actin. Compared to normal skeletalmuscle, the average increase of DNA methyltransferaseexpression was 6.7-fold (6.7 6 0.96) in the embryonal tumorsand 3.7-fold (3.7 6 0.46) in the alveolar rhabdomyosarcomas.The difference in the average increase of the DNA methyl-transferase expression was statistically significant in the 2rhabdomyosarcoma subtypes, which have distinct etiologiesand clinical behaviors. Our results are consistent with previ-ous reports that an increase in DNA methyltransferaseactivity is associated with neoplastic transformation; how-ever, the role of increased DNA methyltransferase expres-sion in the development and progression of rhabdomyosar-coma needs to be investigated in future studies. Int. J. Cancer83:10–14, 1999.r 1999 Wiley-Liss, Inc.

Methylation at the 5 position of cytosines in the CpG dinucleo-tides is the major modification of DNA in higher eukaryotes and isinvolved in a number of biological processes including X inactiva-tion, genomic imprinting, aging and mutagenesis (reviewed byBaylin et al., 1998). The methylation pattern of DNA is stablymaintained by DNA (cytosine-5-)-methyltransferase (DNA methyl-transferase) that recognizes and methylates hemi-methylated DNAafter replication (Baylinet al.,1998). Growing evidence indicatesthat the normal pattern of DNA methylation is altered in cancercells, with an overall decrease in the genomic content of the58-methylcytosines (Flatauet al., 1983; Gama-Sosaet al., 1983),hypermethylation and inactivation of tumor suppressor genes(Baylin et al.,1998), and relatively infrequent hypomethylation inindividual genes or chromosomal regions (de Smetet al.,1996; Jiet al.,1997; Vachtenheimet al.,1994). Although the mechanismsunderlying the alterations of DNA methylation are unclear, in-creased levels of DNA methyltransferase may be associated withcancer development. Increased DNA methyltransferase activitywas found in colon cancer cells and paralleled tumor progression(el-Deiry et al., 1991). Similarly, increased expression of DNAmethyltransferase was detected in leukemia (Melkiet al., 1998)and hepatocarcinoma (Sunet al.,1997). Forced expression of DNAmethyltransferase in murine NIH3T3 cells led to genomic hyper-methylation and neoplastic transformation (Wuet al., 1993),whereas decrease of the enzyme activity was found to slow downtumor progression (Lairdet al.,1995). Increased DNA methyltrans-ferase activity was found to be an early event in the development ofcarcinogen-induced lung tumors from a susceptible mouse strain,suggesting that elevated DNA methyltransferase expression alsomay be involved in the initiation of tumorigenesis (Belinskyet al.,1996).

Rhabdomyosarcoma is the most common soft tissue malignancyin childhood (reviewed by Barr, 1997). These tumors originatefrom undifferentiated mesenchymal cells and resemble developingstriated muscle. Within the class of pediatric rhabdomyosarcomas,there are 2 major subtypes, embryonal and alveolar, which presentwith biologically and clinically distinct behaviors. Alveolar rhabdo-myosarcomas usually have a more aggressive clinical behavior andare associated with a worse outcome than all embryonal variants(Barr, 1997). An early study described variable levels of genomic5-methylcytosines in rhabdomyosarcomas, although no tumorsubtypes were noted (Flatauet al., 1983). Results from ourlaboratory have revealed sequence-specific alterations in DNAmethylation in rhabdomyosarcomas (Chenet al.,1998). We foundthat the upstream region of the humanMyoD1gene is hypomethyl-ated in rhabdomyosarcomas compared with normal differentiatedmuscle, and that embryonal and alveolar rhabdomyosarcomas havedistinct patterns of altered CpG methylation in this region (Chenetal., 1998). To determine whether the observed methylation alter-ations are correlated with changes in DNA methyltransferaseexpression, we developed a standardized reverse transcription-polymerase chain reaction (RT-PCR) assay to measure the mRNAlevels of DNA methyltransferase transcripts in the 2 subtypes ofrhabdomyosarcomas. Our results indicate that expression of DNAmethyltransferase is increased in all rhabdomyosarcoma samples,and that the mean levels of increased gene expression are statisti-cally different in the 2 subtypes of rhabdomyosarcoma.

MATERIAL AND METHODS

Tumors and normal muscle samples

Frozen rhabdomyosarcoma specimens were obtained from St.Jude Children’s Research Hospital (Memphis, TN) and from a bankof tumor tissues derived from a previous Intergroup Rhabdomyosar-coma Study (Parhamet al., 1991). All tumor samples weresectioned for histological analysis, and only blocks that containedgreater than 80% tumor cells were used in this study. Normalskeletal muscle specimens from the same age group as therhabdomyosarcoma patients (5–16 years of age) were obtainedfrom Departments of Pathology at Arkansas Children’s Hospitaland the University of Arkansas for Medical Sciences (Little Rock,AR). All tissue samples were snap-frozen in liquid nitrogen-cooledisopentane and stored at280°C until RNA extraction. All proce-dures for handling the specimens were in agreement with properethical standards and were approved by the Human ResearchAdvisory Committee of the University of Arkansas for MedicalSciences.

Grant sponsor: The Dean’s CUMG Development Fund, University ofArkansas for Medical Sciences; Grant sponsor: Arkansas Science andTechnology Authority; Grant number: 98-B-34; Grant sponsor: AmericanCancer Society Institutional Research Grant (Arkansas Cancer ResearchCenter); Grant number: IRG-91–012–06; Grant sponsor: National CancerInstitute; Grant numbers: P30 CA217657 and 5 U10 CA24507–09.

*Correspondence to: Slot 820, Arkansas Children’s Hospital, 800Marshall Street, Little Rock, AR 72202, USA. Fax: (501) 320-3912.E-mail: chenbin@exchange.uams.edu

Received 20 January 1999; Revised 13 March 1999

Int. J. Cancer:83,10–14 (1999)

r 1999 Wiley-Liss, Inc.

Publication of the International Union Against CancerPublication de l’Union Internationale Contre le Cancer

Diagnosis and subclassification of rhabdomyosarcomaDiagnosis was obtained from previous IRS review or published

retrospective analyses, and was verified based on standard histo-logic criteria. To confirm the subclassification of alveolar rhabdo-myosarcoma, all tumor samples were analyzed for thePAX3-FKHRandPAX7-FKHRfusion transcripts as described previously (Chenet al.,1998).

RNA isolation and reverse transcription (RT)Total RNA was extracted with the Purescript RNA Extraction Kit

according to the manufacturer’s instructions (Gentra Systems,Minneapolis, MN). The concentration of the extracted RNA wasdetermined by spectrophotometry. The first strand cDNA wassynthesized as previously described (Chenet al., 1998) with thefollowing modifications. A 20-µl mixture, consisting of 1 µg RNA,40 U AMV reverse transcriptase (Promega, Madison, WI), 100pmol random hexamers (Amersham, Arlington Heights, IL), 40 URNasin (Promega) and 1 mmol/L deoxyribonucleotide triphos-phates (dATP, dCTP, dGTP, and TTP) in a buffer containing 50mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2 and10 mmol/L DTT, was incubated at 37°C for 30 min and then at42°C for 30 min. The first strand of cDNA specific for DNAmethyltransferase orb-actin, used for standardization of the duplexamplification assay, was generated from RNA isolated from analveolar rhabdomyosarcoma cell line RH30 (ATCC, Monassas,VA) by replacing the random hexamers with 20 pmol of primerMTR (58 TTC TCA TCC TGG TCT TTG T38) or ACTR (58 CTCCTT CTG CAT CCT GTC 38), respectively, followed by incubatingat 45°C for 60 min. The reaction was subsequently heated to 90°Cto denature the reverse transcriptase.

Standardization of duplex polymerase chain reaction (PCR)The first strand cDNA specific for DNA methyltransferase was

diluted serially and mixed with a constant volume of theb-actin-specific cDNA. The ratios of the DNA methyltransferase to theb-actin cDNA were 0, 1:20, 1:10, 1:5, 1:2, 1:1, 2:1 and 4:1,respectively. Then the mixtures were used as templates for duplexamplification of DNA methyltransferase andb-actin. The 15-µlPCR reaction consisted of 100 ng of cDNA, 10 pmol each of theDNA methyltransferase primers (MTF: 58 AAG TGA AGC CCGTAG AGT G 38 and MTR), 3 pmol each of theb-actin primers(ACTF: 58 ACT CTT CCA GCC TTC CTT38 and ACTR), 3 µCia-32P-dCTP, 2.5 nmol of each dNTP and 2 units of Taq DNApolymerase (Perkin Elmer, Foster City, CA) in a buffer containing1.5 mmol/L MgCl2 and 50 mmol/L KCl. Amplification wasperformed for all samples using 18, 20, 22 or 25 cycles of 94°C for30 sec, 55°C for 30 sec and 72°C for 30 sec. The PCR productswere electrophoresed through an 8% native polyacrylamide gel for3 hr at 200 V. Then the gel was transferred onto blotting paper,dried, followed by autoradiography. The intensity of the DNAmethyltransferase andb-actin bands was quantified with a Cyclonephosphor-imager (Packard, Meriden, CT). All primers were se-lected based on the published coding sequences of the DNAmethyltransferase gene (Yoderet al., 1996) and theb-actin gene(Ponteet al.,1984).

To test whether the starting amount of cDNA could affect theratio of DNA methyltransferase relative tob-actin, the first strandcDNA from the RH30 cell line was reverse-transcribed withrandom hexamers, and 1/20, 1/10, 1/5, 1/2, 1, 2, 4 or 8 µl of the RTreaction was used in the duplex amplification. The PCR reactionwas performed as described in the above paragraph. After quantifi-cation with a Packard Cyclone phosphor-imager, the intensity ofDNA methyltransferase relative tob-actin was plotted and thelinearity analyzed using Microsoft Excel software (Microsoft,Redmond, WA).

Determination of DNA methyltransferase expressionin rhabdomyosarcomas

The mRNA levels of DNA methyltransferase expression inrhabdomyosarcomas and normal muscle specimens were deter-

mined using optimized PCR conditions. Simultaneous amplifica-tion of DNA methyltransferase andb-actin was performed for 22cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. Thereaction mixture was then electrophoresed through an 8% polyacryl-amide gel for 3 hr at 200 V. The gel was dried and analyzed with aPackard Cyclone phosphor-imager. The intensity of the 267-bpDNA methyltransferase and the 169-bpb-actin amplicon wasquantified and their ratio determined for each reaction. The meanDNA methyltransferase/b-actin ratio (arbitrarily set as 1.0) and thestandard error of the mean (SEM) in the normal muscle sampleswere used to assess levels of DNA methyltransferase expression inthe rhabdomyosarcomas. Normalized values over 3 SEM wereconsidered to be increased expression. All samples were analyzedat least 3 times.

Immunohistochemistry and determination of proliferative indexin rhabdomyosarcomas

Proliferative index of the rhabdomyosarcomas was determinedby immunohistochemistry using Ki-67, a monoclonal antibody(MAb) commonly used to assess the growth fraction of neoplastictissues (Brown and Gatter, 1990). Paraffin blocks of formalin-fixedtumor tissue that were available for Ki-67 immunostaining in-cluded 9 alveolar rhabdomyosarcomas and 12 embryonal rhabdo-myosarcomas. Ki-67 immunohistochemistry was performed on5-µm sections from these blocks, using pre-diluted MAb cloneMIB1 (DAKO, Carpenteria, CA). Prior to primary antibodyincubation, antigen retrieval was performed by submersion of thesections in an antigen retrieval solution (DAKO) and placing thesolution in a pressure cooker for 75 min, as suggested by themanufacturer. Following equilibration to room temperature, immu-nostaining was accomplished with an automated DAKO immunos-tainer using the labeled streptavidin-biotin-complex procedure.After development of the peroxidase reaction using 3,38-diaminobenzidine-HCl, the sections were counterstained withhematoxylin. A section of tonsil with active germinal centers wasused as a positive control, and pre-diluted normal mouse serum(DAKO) was substituted for the primary antiserum in each case asnegative controls. Proliferative indices were derived by countingthe number of MIB1-positive cells in a total of 1,000 consecutivetumor cells in randomly chosen 4003 fields. The results wereexpressed as a percentage of positive cells over total cells counted.Statistical comparison of the proliferative index results betweenalveolar and embryonal rhabdomyosarcomas was performed usingthe Student’st-test.

RESULTS

Standardized RT-PCR assay

Detecting differences in mRNA levels by RT-PCR can bedifficult because the amplification may not be linear, especially athigh cycle numbers. To ensure an accurate reflection of changes inDNA methyltransferase expression, we first tested the linearity ofthe duplex amplification using different initial levels of DNAmethyltransferase transcripts and different numbers of PCR cycles.An example of the duplex amplification usingb-actin as an internalcontrol is shown in Figure 1a. The intensity of the DNAmethyltrans-ferase amplicon relative tob-actin was determined by phosphor-imaging and was plotted against the initial amount of the DNAmethyltransferase cDNA (Fig. 1b). Linear amplification relative tob-actin was observed for all levels of DNA methyltransferase using22 PCR cycles (Fig. 1).

To test whether the initial amount of cDNA template could affectthe amplification efficiency, varying amounts of total cDNA fromthe RH30 cell line were used as templates for duplex amplificationof DNA methyltransferase andb-actin. As shown in Figure 2, theintensity of DNA methyltransferase amplicon relative tob-actinremained essentially the same (, 5% difference) for all templateamounts, indicating that the duplex amplification is able to reflectthe original level of DNA methyltransferase mRNA using amountsof cDNA that vary over 2 orders of magnitude.

11DNA METHYLTRANSFERASE IN RHABDOMYOSARCOMA

Detection of PAX3-FKHR and PAX7-FKHR fusion mRNATo confirm the histologic diagnosis of the alveolar rhabdomyo-

sarcomas, RT-PCR analysis was performed for each tumor to detectthe PAX3-FKHR or PAX7-FKHR mRNA transcripts. Of the 14alveolar rhabdomyosarcomas,PAX3-FKHRfusion transcripts wereobserved in 10 tumors andPAX7-FKHRtranscripts in 4 tumors. Allembryonal rhabdomyosarcomas were negative forPAX3-FKHRandPAX7-FKHRfusion transcripts (Table I).

Expression of DNA methyltransferase in rhabdomyosarcomasExpression of DNA methyltransferase was determined for 32

rhabdomyosarcomas and 12 normal muscle samples by the standard-ized duplex PCR assay usingb-actin as the internal control.Compared with the mean level of the DNA methyltransferasemRNA in the normal controls, all rhabdomyosarcomas showedincreased expression of the gene (Table I). The highest increasewas detected in the embryonal tumor E6, in which the DNAmethyltransferase mRNA level was 16.5-fold higher than the meanexpression in normal muscle. The lowest level of the DNAmethyltransferase expression was observed in tumor E3, whichshowed a 1.7-fold increase after normalization tob-actin (Fig. 3).An example of DNA methyltransferase expression in the rhabdo-myosarcomas is shown in Figure 3.

Although expression of DNA methyltransferase was found to beincreased in all rhabdomyosarcoma samples, the average increaseof the gene expression was different in the 2 tumor subtypes.Compared with the mean expression level in normal muscle, theaverage increase in the DNA methyltransferase expression was6.7-fold (6.76 0.96,p , 0.001 by the Student’st-test) in the 18embryonal rhabdomyosarcomas, but was 3.7-fold (3.76 0.46,p ,0.001 by the Student’st-test) in the 14 alveolar rhabdomyosarco-mas. The difference in the mean levels of the DNA methyltransfer-

(a)

(b)

FIGURE 1 – Example of the standardized duplex reverse transcription-polymerase chain reaction (RT-PCR) assay for detecting relativechanges of DNA methyltransferase. (a) A polyacrylamide gel contain-ing RT-PCR reactions with constantb-actin cDNA and serially dilutedDNA methyltransferase cDNA. The first strand cDNA specific forDNA methyltransferase was mixed with theb-actin-specific cDNA andused as templates in 22 cycles of PCR amplification. The ratios of theDNA methyltransferase to theb-actin cDNA are shown above the gel.(b) The intensity of the DNA methyltransferase bands relative to that ofb-actin was quantified by phosphor-imaging and plotted against theamount of initial DNA methyltransferase template. Regression analysiswas performed using Microsoft Excel.

(a)

(b)

FIGURE 2 – Effect of varying initial amounts of cDNA template onDNA methyltransferase amplification. (a) Total cDNA from the RH30cell line was diluted serially and used as templates in 22 cycles ofduplex amplification for DNA methyltransferase andb-actin. Theamounts of cDNA added to the PCR reaction are shown above theautoradiogram. (b) The intensity of DNA methyltransferase ampliconrelative to b-actin was quantified by phosphor imaging and plottedagainst the amount of the initial cDNA template. The variation of therelative DNA methyltransferase intensity was less than 5%.

TABLE I – SUMMARY OF DNA METHYLTRANSFERASE EXPRESSION,PROLIFERATIVE INDEX AND FUSION-GENE DETECTION

IN RHABDOMYOSARCOMA SAMPLES

Tumornumber1

Normalized DNAmethyltransferase

expression(Controls: 1.06 0.14)2

Proliferative index(Ki-67 positive cells %)3

PAX3/7-FKHRRT-PCR

E02 7.1 NP —E03 1.7 5.5 —E04 5.1 0.5 —E05 5.7 6.7 —E06 16.5 9.0 —E07 4.1 NP —E12 2.8 3.7 —E13 10.1 0.2 —E14 13.8 1.8 —E15 3.8 NP —E16 3.8 2.8 —E17 4.5 2.0 —E18 5.4 3.5 —E20 6.5 12.5 —E21 3.8 NP —E22 12.8 NP —E28 7.2 1.0 —E31 6.6 NP —A01 2.1 2.0 PAX3-FKHRA02 5.1 20.0 PAX3-FKHRA04 2.4 NP PAX3-FKHRA05 4.8 1.0 PAX3-FKHRA07 3.8 1.0 PAX3-FKHRA09 2.1 14.0 PAX3-FKHRA12 4.8 2.5 PAX7-FKHRA13 3.6 2.6 PAX7-FKHRA16 3.8 NP PAX3-FKHRA17 1.9 NP PAX7-FKHRA18 2.4 NP PAX7-FKHRA19 7.3 1.0 PAX3-FKHRA21 6.0 10.5 PAX3-FKHRA22 1.9 NP PAX3-FKHR

1E02–E31: embryonal rhabdomyosarcomas;A01–A22: alveolar rhab-domyosarcomas–2DNA methyltransferase mRNA in normal skeletalmuscle after normalization tob-actin (mean6 SEM). All tumorsshowed elevated DNA methyltransferase expression as their normal-ized values were over 3 SEM of the controls.–3Percentage of Ki-67-positive cells in a total of 1000 consecutive tumor cells. NP, notperformed. No correlation was found between the Ki-67-positivepercentages from the 12 embryonal and 9 alveolar tumors and theirlevels of DNA methyltransferase expression using Microsoft Excel.

12 CHENET AL.

ase expression were statistically significant (p , 0.05 by theStudent’st-test) in the 2 rhabdomyosarcoma subtypes (Table II).

Proliferative indices in rhabdomyosarcomasOf the rhabdomyosarcomas studied for DNA methyltransferase

expression, formalin-fixed paraffin blocks were available for 12 ofthe 18 embryonal rhabdomyosarcomas and 9 of the 14 alveolarrhabdomyosarcomas. Proliferative index was measured by determin-ing the percentage of Ki-67-positive cells in a total of 1,000consecutive tumor cells. In the 12 embryonal rhabdomyosarcomas,tumor cells that were positive for the Ki-67 antigen rangedfrom 0.2% to 12.5% (Table I), with a mean value of 4.1% (TableII). The Ki-67 positivity ranged from 1% to 20% in the 9 alveolarrhabdomyosarcoma specimens (Table I), with a mean value of6.1% (Table II). Statistical analysis did not indicate a signifi-cant difference in proliferative indices between the 2 subtypesof rhabdomyosarcoma (Student’st-test,p . 0.05). In addition, theKi-67-positive percentages from the 12 embryonal and 9 alveolartumors were plotted against their DNA methyltransferase expres-sion levels using the Microsoft Excel software, and no correlationwas found between the proliferative index and the increase in DNAmethyltransferase expression (not shown).

DISCUSSION

Expression of DNA methyltransferase has been analyzed previ-ously in malignancies from the colon, lung, liver, and bone marrow,using protein-based (Issaet al., 1993; Laird et al., 1995) orRNA-based (Belinskyet al.,1996; el-Deiryet al.,1991; Leeet al.,1996; Melkiet al.,1998) methods. The levels of increase in DNAmethyltransferase expression vary in these studies, depending onthe type of malignancy, the detection assay and the normalizationstandard used in the investigation. For example, DNA methyltrans-ferase expression in colon cancer compared with normal mucosawas reported to be 200-fold increased in an early study using anon-quantitative RT-PCR assay (el-Deiryet al., 1991), 18-foldincreased in a later study using a semi-quantitative RT-PCR assay(Issaet al., 1993) and 3.7-fold (Schmutteet al., 1996) increased

using quantitative studies. In the present study, a standardizedRT-PCR assay usingb-actin as an internal standard was used todetermine the mRNA levels of DNA methyltransferase in embryo-nal and alveolar rhabdomyosarcomas. Increased DNA methyltrans-ferase expression was detected in all rhabdomyosarcoma samplesexamined, with an average increase of 6.7-fold in the embryonalsubtype and 3.7-fold in the alveolar subtype compared with themean level of DNA methyltransferase expression in the normalmuscle controls. These results are consistent with the previousreports that increased expression of DNA methyltransferase isassociated with tumorigenesis.

The significance of increased DNA methyltransferase expressionin colon cancer has been debated, primarily because normal cellsshow increased DNA methyltransferase activity during DNAsynthesis and the observed increase in DNA methyltransferaseexpression could be a consequence of increased cell proliferation(Leeet al.,1996). In our study, to determine whether expression ofDNA methyltransferase in rhabdomyosarcomas was related to cellproliferation, a subset of the tumors were analyzed by immunohis-tochemistry for Ki-67, a nuclear proliferation marker. No correla-tion between the proliferative indices and the levels of DNAmethyltransferase expression was observed, either within theembryonal rhabdomyosarcomas or within the alveolar tumors(Table I). Proliferative activities among different subtypes ofrhabdomyosarcomas were compared by measuring S-phase frac-tion or median S-phase in previous studies, which showed thatalveolar rhabdomyosarcomas usually had higher proliferative activ-ity compared to the embryonal subtype (de Zenet al.,1997; Niggliet al.,1994). In this study, the mean Ki-67 positivity in the alveolartumors (6.1%) was slightly higher than that in the embryonaltumors examined (4.1%); however, this difference was not statisti-cally significant (Table II), possibly due to the relatively smallnumber of tumor samples available for our study. It is unlikely thatthe elevated levels of the DNA methyltransferase mRNA inembryonal and alveolar rhabdomyosarcomas are attributable solelyto increased proliferation rates. Therefore, the increases of DNAmethyltransferase mRNA levels observed in this study shouldrepresent truly increased gene expression in rhabdomyosarcomasindependent of cell proliferation.

Alterations in DNA methylation have been observed consistentlyin many types of human neoplasia (Baylinet al.,1998). Although alarge body of evidence supports the hypothesis that increased DNAmethyltransferase activity is involved in tumor development, therelationship between changes in DNA methylation and the levels ofDNA methyltransferase activity is poorly understood, with theapparently contradictory co-existence of hypomethylation andincreased expression of DNA methyltransferase in cancer cells.Rhabdomyosarcoma, as indicated by the present study and anotherreport from our laboratory, represents an example of this dilemma.Our previous results showed that hypomethylation of theMyoD1upstream region was associated with the characteristicMyoD1expression in rhabdomyosarcomas (Chenet al.,1998), whereas inthe present study, increased DNA methyltransferase expressionwas detected in the same set of rhabdomyosarcomas. The intrigu-ing co-existence of increased expression of DNA methyltransferaseand sequence-specific hypomethylation in rhabdomyosarcoma cellssuggests that, although DNA methyltransferase may have profoundeffects on DNA, its involvement in methylation alterations duringtumorigenesis may be mediated by additional, yet unknownmechanisms, such as factors that determine the enzyme activity,subcellular localization and target specificity of DNA methyltrans-ferase.

In this study, the embryonal subtype of rhabdomyosarcomashowed a greater increase (6.7-fold) in the average level of DNAmethyltransferase expression compared with the alveolar subtype(3.7-fold). The difference of the gene expression between the twosubtypes was statistically significant (p , 0.05) and may haveimportant implications. Embryonal and alveolar rhabdomyosar-coma are genetically and clinically distinct tumors (Barr, 1997).

FIGURE 3 – Representative results of DNA methyltransferase expres-sion in embryonal and alveolar rhabdomyosarcomas, assayed by thestandardized duplex reverse transcription-polymerase chain reaction(RT-PCR) amplification. Levels of DNA methyltransferase mRNA inembryonal (E2, E6, E14, E18, E20, E22 and E31) and alveolar (A4, A5,A16 and A18) rhabdomyosarcomas were quantified by phosphor-imaging and normalized to the co-amplifiedb-actin. Two normalmuscle samples (NM-2 and NM-6) were included in the same run ofthe RT-PCR assay.

TABLE II – COMPARISON OF DNA METHYLTRANSFERASE EXPRESSIONAND PROLIFERATIVE INDEX IN EMBRYONAL AND ALVEOLAR

RHABDOMYOSARCOMAS

Normalized DNAmethyltransferase

expression(mean6 SEM)

Proliferative index(mean Ki-67

positivity 6 SEM)

Embryonal rhabdomyosarcoma 6.76 0.96 4.16 1.1Alveolar rhabdomyosarcoma 3.76 0.46 6.16 2.3Student’st-test (two-tail)1 p 5 0.013 p . 0.05

1Embryonalvs.alveolar rhabdomyosarcoma.

13DNA METHYLTRANSFERASE IN RHABDOMYOSARCOMA

Specific chromosomal translocations, t(2;13) and t(1;13), whichgenerate fusion genesPAX3-FKHRandPAX7-FKHR, are thoughtto be causal for alveolar rhabdomyosarcoma, whereas loss ofheterozygosity for chromosome 11p has been associated with theembryonal subtype (for review, Barr, 1997). With regard toaberrant DNA methylation, the 2 tumor subtypes showed distinctpatterns of methylation alteration in the upstream region of theMyoD1 gene (Chenet al., 1998). Partially demethylatedMyoD1upstream region was found in embryonal rhabdomyosarcomas,while tumors of the alveolar subtype were more extensivelydemethylated in this region (Chenet al., 1998). These previousfindings, combined with the present results, point to the possibilitythat changes in DNA methyltransferase expression could be

orchestrated by different mechanisms in the 2 subtypes of rhabdo-myosarcoma. In summary, our results suggest that increased DNAmethyltransferase expression may be associated with the develop-ment of rhabdomyosarcoma, but the exact role of increased DNAmethyltransferase activity in producing altered DNA methylationin cancer cells awaits further elucidation.

ACKNOWLEDGEMENTS

The authors thank Dr. W. Crist for his review of this manuscript,and Ms. L. Linn, HT, ASCP, for help in immunohistochemistry. Wealso thank the Intergroup Rhabdomyosarcoma Study Group and itsparticipants for their previous contribution of tumor tissue.

REFERENCES

BARR, F.G., Molecular genetics and pathogenesis of rhabdomyosarcoma[Review].J. Pediatr. Hematol. Oncol.,19,483–491 (1997).BAYLIN , S.B., HERMAN, J.G., GRAFF, J.R., VERTINO, P.M. and ISSA, J.P.,Alterations in DNA methylation: a fundamental aspect of neoplasia[Review].Adv. Cancer Res.,72,141–196 (1998).BELINSKY, S.A., NIKULA , K.J., BAYLIN , S.B. and ISSA, J.P., Increasedcytosine DNA-methyltransferase activity is target-cell-specific and an earlyevent in lung cancer.Proc. nat. Acad. Sci. (Wash.),93,4045–4050 (1996).BROWN, D.C. and GATTER, K.C., Monoclonal antibody Ki-67: its use inhistopathology [Review].Histopathology,17,489–503 (1990).CHEN, B., DIAS, P., JENKINS, J.J., SAVELL , V.H. and PARHAM, D.M.,Methylation alterations of the MyoD1 upstream region are predictive ofsubclassification of human rhabdomyosarcomas.Amer. J. Pathol.,152,1071–1079 (1998).DE SMET, C.,DE BACKER, O., FARAONI, I., LURQUIN, C., BRASSEUR, F., BOON,T. and BRASSEUR, F., The activation of human gene MAGE-1 in tumor cellsis correlated with genome-wide demethylation.Proc. nat. Acad. Sci.(Wash.),93,7149–7153 (1996).DE ZEN, L. and 12 OTHERS, Clinical relevance of DNA ploidy andproliferative activity in childhood rhabdomyosarcoma: a retrospectiveanalysis of patients enrolled onto the Italian Cooperative Rhabdomyosar-coma Study RMS88.J. clin. Oncol., 15,1198–1205 (1997).EL-DEIRY, W.S., NELKIN, B.D., CELANO, P., YEN, R.W., FALCO, J.P.,HAMILTON, S.R. and BAYLIN , S.B., High expression of the DNAmethyltrans-ferase gene characterizes human neoplastic cells and progression stages ofcolon cancer.Proc. nat. Acad. Sci. (Wash.),88,3470–3474 (1991).FLATAU , E., BOGENMANN, E. and JONES, P.A., Variable 5-methylcytosinelevels in human tumor cell lines and fresh pediatric tumor explants.CancerRes.,43,4901–4905 (1983).GAMA -SOSA, M.A., SLAGEL, V.A., TREWYN, R.W., OXENHANDLER, R., KUO,K.C., GEHRKE, C.W. and EHRLICH, M., The 5-methylcytosine content ofDNA from human tumors.Nucl. Acids Res.,11,6883–6894 (1983).ISSA, J.P., VERTINO, P.M., WU, J., SAZAWAL , S., CELANO, P., NELKIN, B.D.,HAMILTON , S.R. and BAYLIN , S.B., Increased cytosine DNA-methyltransfer-ase activity during colon cancer progression.J. nat. Cancer Inst.,85,1235–1240 (1993).JI, W., HERNANDEZ, R., ZHANG, X.Y., QU, G.Z., FRADY, A., VARELA, M. andEHRLICH, M., DNA demethylation and pericentromeric rearrangements ofchromosome 1.Mutation Res.,379,33–41 (1997).

LAIRD, P.W., JACKSON-GRUSBY, L., FAZELI, A., DICKINSON, S.L., JUNG, WE,LI, E., WEINBERG, R.A. and JAENISCH, R., Suppression of intestinalneoplasia by DNA hypomethylation.Cell, 81,197–205 (1995).

LEE, P.J., WASHER, L.L., LAW, D.J., BOLAND, C.R., HORON, I.L. andFEINBERG, A.P., Limited up-regulation of DNA methyltransferase in humancolon cancer reflecting increased cell proliferation.Proc. nat. Acad. Sci.(Wash.),93,10366–10370 (1996).

MELKI, J.R., WARNECKE, P., VINCENT, P.C. and CLARK, S.J., Increased DNAmethyltransferase expression in leukaemia.Leukemia,12,311–316 (1998).

NIGGLI, F.K., POWELL, J.E., PARKES, S.E., WARD, K., RAAFAT, F., MANN,J.R. and STEVENS, M.C., DNA ploidy and proliferative activity (S-phase) inchildhood soft-tissue sarcomas: their value as prognostic indicators.Brit. J.Cancer,69,1106–1110 (1994).

PARHAM, D.M., WEBBER, B., HOLT, H., WILLIAMS , W.K. and MAURER, H.,Immunohistochemical study of childhood rhabdomyosarcomas and relatedneoplasms. Results of an Intergroup Rhabdomyosarcoma study project.Cancer,67,3072–3080 (1991).

PONTE, P., NG, S.Y., ENGEL, J., GUNNING, P. and KEDES, L., Evolutionaryconservation in the untranslated regions of actin mRNAs: DNA sequence ofa human beta-actin cDNA.Nucl. Acids Res.,12,1687–1696 (1984).

SCHMUTTE, C., YANG, A.S., NGUYEN, T.T., BEART, R.W. and JONES, P.A.,Mechanisms for the involvement of DNA methylation in colon carcinogen-esis.Cancer Res.,56,2375–2381 (1996).

SUN, L., HUI, A.M., KANAI , Y., SAKAMOTO, M. and HIROHASHI, S., IncreasedDNA methyltransferase expression is associated with an early stage ofhuman hepatocarcinogenesis.Jpn. J. Cancer Res.,88,1165–1170 (1997).

VACHTENHEIM, J., HORAKOVA, I. and NOVOTNA, H., Hypomethylation ofCCGG sites in the 38 region of H-ras protooncogene is frequent and isassociated with H-rasallele loss in non-small cell lung cancer.Cancer Res.,54,1145–1148 (1994).

WU, J., ISSA, J.P., HERMAN, J., BASSETT, D.E., JR., NELKIN, B.D. and BAYLIN ,S.B., Expression of an exogenous eukaryotic DNA methyltransferase geneinduces transformation of NIH 3T3 cells.Proc. nat. Acad. Sci. (Wash.),90,8891–8895 (1993).

YODER, J.A., YEN, R.W.C., VERTINO, P.M., BESTOR, T.H. and BAYLIN , S.B.,New 58 regions of the murine and human genes for DNA (cytosine-5)-methyltransferase.J. biol. Chem.,271,31092–31097 (1996).

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