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Department of 1 Pediatrics and 2 Pathology and 3 The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3-fold changes in expression of 44 genes that play roles in angiogenesis, apoptosis, cell adhesion, transcriptional regulation, and signal transduction. The gene encoding heat shock protein 47 (Hsp47), a collagen-specific molecular chaperon, was up-regulated >80-fold after 5-Aza-dC treatment. Expression studies confirmed that Hsp47 is silenced in a subset of NB cell lines and tumors. We also show that silencing of Hsp47 in NB cells is associated with aberrant methylation of promoter CpG islands and that Hsp47 expression can be restored after treatment with 5-Aza-dC. A strong correlation between Hsp47 and collagen type I and IV expression was seen in NB cells. Interestingly, tumorigenicity was inversely correlated with the level of collagen expression in NB cell lines, and higher levels of collagen were detected in mature NB tumors that are associated with favorable outcome compared with undifferentiated, advanced-stage NBs. Our studies support a role for Hsp47 in the regulation of collagen type I and IV production in NB cells and suggest that the level of collagen expression may influence NB tumor phenotype. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epigenetic changes can commonly be reversed in vitro after treatment with agents that modulate DNA methylation (8, 9, 10) . In our studies, restoration of TSP-1 expression was detected in the MYCN-amplified NB cell line NBL-W-S within 24 h of 5'-Aza-2'-deoxycytidine (5-Aza-dC) treatment (8) . Similarly, other NB cell lines with methylated TSP-1 responded to treatment, but longer exposure to 5-Aza-dC was required to induce expression. We also found that 5-Aza-dC treatment led to significant inhibition of NB growth in vivo and that in a subset of the NB xenografts, TSP-1 was up-regulated and angiogenesis was impaired (8) . However, because 5-Aza-dC is known to globally modulate gene expression, we hypothesized that treatment with this agent affected many other genes that play a role in the regulation of NB tumor growth.
In an effort to identify the genes and biological pathways that are affected by 5-Aza-dC treatment in NB cells, we performed genome-wide gene expression analysis of control and 5-Aza-dC-treated NBL-W-S NB cells. Forty-four genes displayed differential levels of expression (3-fold) in the arrays performed with treated versus control NB cells. One gene, SERPINH1 (hereafter referred to as Hsp47), the gene encoding Hsp47, was up-regulated >80-fold after 5-Aza-dC treatment. Hsp47, a collagen-specific molecular chaperone, is believed to play an important role in the synthesis, processing, and secretion of collagen (13, 14, 15, 16, 17)
. Our studies demonstrate that Hsp47 is epigenetically silenced in a subset of NB tumors and cell lines. We also found a strong correlation between Hsp47 and collagen type I and type IV expression. Interestingly, nontumorigenic NB cell subclones expressed high levels of Hsp47 and collagen, whereas all of the cell lines with low to undetectable levels of Hsp47 protein and collagen are highly tumorigenic in nude mice. High levels of collagen type I expression were also detected in primary tumors with morphological evidence of differentiation, whereas undifferentiated tumors lacked collagen expression, suggesting that collagen may influence NB phenotype.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microarray Analysis.
Total RNA was isolated from the 5-Aza-dC-treated and untreated NBL-W-S cell line using TRIzol reagent (Invitrogen) and was cleaned using RNeasy mini columns (Qiagen, Valencia, CA) according to the manufacturer’s protocol. cDNA synthesis was performed using the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen). The manufacturer’s protocol was modified by the use of an HPLC- purified T7-(dT)24 primer (GenSet Oligos; Proligo, Boulder, CO). The double-stranded cDNA product was purified by phenol-chloroform extraction using Phase Lock Gels (Eppendorf Scientific, Westbury, NY) followed by ethanol precipitation. cRNA was synthesized using a BioArray High Yield RNA Transcript Labeling kit (Enzo, Farmingdale, NY) according to the manufacturer’s protocol.
In vitro transcription was carried out at 37°C for 5 h, and the biotin-labeled cRNA obtained was purified using the RNeasy Mini kit (Qiagen) and was then fragmented for 35 min at 94°C in 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate. The labeled, fragmented cRNA was added to a 300 µl volume of hybridization mixture, which included final concentrations of 0.1 mg/ml herring sperm DNA (Promega, Madison, WI), 0.5 mg/ml acetylated BSA (Promega), and 2x 4-morpholinepropanesulfonic acid Hybridization Buffer (Sigma, St. Louis, MO). This mixture also included the following hybridization controls: 50 pM of oligonucleotide B2 and 1.5, 5, 25, and 100 pM of cRNA BioB, BioC, BioD, and Cre, respectively (Affymetrix, Santa Clara, CA). We hybridized 250 µl of the mixture to an Affymetrix HG-U133A microarray containing 
22,000 probe sets for 16 h at 45°C in a hybridization oven with constant rotation. The microarrays were washed with nonstringent (6x SSPE, 0.01% Tween 20, and 0.005% Antifoam) and stringent (100 mM 4-morpholinepropanesulfonic acid, 0.1 M NaCl, and 0.01% Tween 20) buffers. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR) and the signal amplified using antibody solution. The streptavidin-phycoerythrin stain contained 2x Stain Buffer (final concentration: 100 mM 4-morpholinepropanesulfonic acid, 1 M NaCl, 0.05% Tween 20, 0.005% Antifoam, 2 µg/µl acetylated BSA, and 10 µg/ml streptavidin-phycoerythrin; Molecular Probes). The antibody amplification solution contained 2x Stain Buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml normal goat IgG (Sigma), and 3 µg/ml biotinylated antistreptavidin antibody (Vector Labs, Burlingame, CA).
The arrays were scanned using a fluorometric scanner (HP GeneArray Scanner; Hewlett Packard, Palo Alto, CA) at the excitation wavelength of 488 nm. The scanned images were analyzed using quality control measures established by Affymetrix. Probe signal intensity values were extracted from the array images using Affymetrix Microarray Suite 5.0 software. Data where the presence/absence call was "absent" across all of the samples were excluded. Affymetrix Data Mining Tool 3.0 software was used to calculate fold changes using average signal intensities between groups. Fold change between two comparison groups of 3 was used as the cutoff point for comparison.
Expression of Hsp47 in NB Cell Lines.
Hsp47 mRNA expression in NB cell lines was measured using semiquantitative reverse transcription-PCR. Total RNA (2.0 µg) was reverse-transcribed in a final volume of 20 µl, and 1 µl of the diluted reaction mixture was subsequently amplified by PCR. Hsp47 sense and antisense primer sequences are shown in Table 1
, and ß2-microglobulin was used as a loading control with template diluted 1:500. Each cycle consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. The PCR products were subjected to 1.0% agarose gel electrophoresis. The level of Hsp47 and Hsp70 protein was examined by Western blot analysis with mouse monoclonal anti-Hsp47 and anti-Hsp70 antibodies (1:1000 dilution; Stressgen Biotechnologies Inc., San Diego, CA) using methods described previously (8)
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. Q-solution (Qiagen) was added to the reaction mix. Each cycle consisted of denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s. The PCR products were separated by electrophoresis on a 2.5% agarose gel and visualized under UV illumination using ethidium bromide staining. Universal Methylated DNA (Intergen), which is enzymatically methylated human genomic DNA, was used as a positive control for MSP. For bisulfite DNA sequencing, 190-bp PCR products were gel purified and cloned into the pCR2.1-TOPO vector (Invitrogen) according to the manufacturer’s protocol. Plasmid DNA was purified with the QIAprep Spin Miniprep kit (Qiagen). Individual plasmids were then sequenced using the ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA).
Immunofluorescence.
NB cells were cultured on coverslips overnight and were then fixed in 1% paraformaldehyde in PBS for 15 min at room temperature. After permeabilization with 70% ethanol for 2 h at -20°C, the coverslips were rinsed and then blocked with 5% nonimmune horse and 5% nonimmune rabbit serum in PBS for 1 h at room temperature. The cells were then incubated with either a mixture of the primary antibodies (5 µg/ml mouse anti-Hsp47 monoclonal IgG; Stressgen) and 1:25 diluted goat anti-type I or IV collagen (Chemicon International, Inc., Temecula, CA). All of the antibody incubations were carried out in PBS containing 5% nonimmune horse serum and 5% nonimmune rabbit serum. Horse antimouse IgG coupled to FITC (Vector Laboratories Inc.) and rhodamine-conjugated rabbit antigoat IgG (Vector) were used as the secondary antibodies at a dilution of 1:200. The coverslips were mounted in a drop of slow anti-Fade (Molecular Probes) to reduce photobleaching. Hoechst dye (Sigma) was used to counterstain nuclei.
Histological sections from a human ganglioneuroma (GNR) were also immunostained using mouse anti-type I collagen (the monoclonal antibody developed by Dr. Heinz Furthmayr was obtained from the Development Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA) and S-100 (NeoMarkers, Fremont, CA) monoclonal antibodies. Briefly, tumor tissue was fixed in 10% buffered formalin and embedded in paraffin. Four-µm thick sections were rehydrated in graded alcohols and rinsed in PBS. Antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a boiling steamer for 20 min. Sections were incubated with primary anti-pro-
1(I) NH2-terminal propeptide antibody (1:40) and S-100 (1:100) in a humidity chamber overnight at 4°C, visualized with antimouse fluorescence and Texas Red-labeled horse IgGs (Vector Labs) at room temperature for 30 min. Sections were counterstained and sealed with Vectashield mounting medium with 4',6-diamidino-2-phenylindole (Vector Labs).
Western Blot Analysis of Type I and IV Collagen.
Lysates were prepared from primary tumors as described previously (20)
, and protein was quantified using the Bio-Rad method (Richmond, CA) following the manufacturer’s instructions. Western blot analyses were performed with 10 µg of protein/lane and anti-collagen type I antibody (from the Development Studies Hybridoma Bank; 1:1000 dilution) and anti-collagen type IV antibody [anti-2 (IV) antibody; 1:500 dilution (Chemicon)] using methods described previously (8)
. Western blot studies were similarly performed with 10 µg CM, collected from NB cell lines and Schwann cells and concentrated 20-fold using Centricon-3 concentrators (Millipore) as described (8)
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NB Xenograft Studies.
Female 4–6-week-old homozygous athymic nude mice (Harlan, Madison, WI) were inoculated s.c. into the right flank with 1 x 107 cells from the NBL-W-S cell line. Once tumors were palpable, mice were treated with three doses of 5-Aza-dC (5 mg/kg/dose) in 1 day with 3-h intervals. Mice were sacrificed at day 9 after 5-Aza-dC treatment to analyze Hsp47 re-expression and collagen IV expression at that time point posttreatment. Animals were treated according to NIH guidelines for animal care and use, and protocols were approved by the Animal Care and Use Committee at Northwestern University.
Immunohistochemical Studies.
Tumor tissue was fixed in 10% buffered formalin and embedded in paraffin. Four-µm thick sections were rehydrated in graded alcohols and rinsed in PBS. Antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a boiling steamer for 20 min. Sections were incubated with a 1:3000 and 1:50 dilution of primary anti-Hsp47 antibody (Stressgen) and collagen IV antibody (Chemicon), respectively, in a humidity chamber overnight at 4°C, and developed with peroxidase-labeled dextran polymer followed by diaminobenzidine (DAKO Envision Plus System; DAKO Corporation, Carpinteria, CA). For double labeling, sections were incubated with anti-Hsp47 and anticollagen IV antibodies and then developed with diaminobenzidine and 3-amino-9-ethylcarbazole (DAKO Envision Plus System) for Hsp47 and type IV collagen, respectively. Sections were counterstained with Gill’s hematoxylin. For negative controls, primary antibody was omitted. Hsp47 and collagen IV staining above the background in the tumor cytoplasm or in the extracellular matrix (ECM) was scored as positive. Histological sections from human GNRs (n = 6), ganglioneuroblastomas (n = 2), and NBs (n = 5) were also immunostained with a mouse anti-type I collagen (1:40 dilution; from the Development Studies Hybridoma Bank) using similar techniques.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3-fold) was detected for 44 genes; 27 were up-regulated, whereas 17 were down-regulated (Table 2)
. As expected, TSP-1 was up-regulated (>18-fold) after treatment with 5-Aza-dC. We also found changes in expression of genes that encode proteins that play roles in apoptosis, cell adhesion, transcriptional regulation, and signal transduction. Significant up-regulation of two heat shock response genes was observed, with a >10% increase in the gene coding for heat shock 70 kDa protein 1A, and a >80-fold increase in the level of expression of Hsp47 after 5-Aza-dC treatment.
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, Hsp47 mRNA was not detected in the untreated NB cells, whereas significantly increased levels of transcript were seen after treatment with 5-Aza-dC for 24 h. To investigate whether Hsp47 was silenced in other NB cell lines, we examined Hsp47 protein expression in a panel of 9 NB cell lines by Western blot analyses. We found that in addition to NBL-W-S, 3 other cell lines had undetectable levels of Hsp47 (SMS-KCNR, NMB, and NBL-W-N), whereas 2 cell lines expressed very low levels of Hsp47 (LA1–55n and SH-SY5Y; Fig. 1B
). Interestingly, high levels of Hsp47 were present in the nontumorigenic S-type NB subclones LA1–5s and SH-EP, whereas all of the cell lines with low to undetectable levels of Hsp47 protein are highly tumorigeneic in nude mice (8
, 21
, 22)
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illustrates the location of the primers used in these assays and the corresponding CpG islands in the Hsp47 promoter. Genomic DNA was isolated from 3 Hsp47-negative NB cell lines (NMB, NBL-W-S, and NBL-W-N), 2 NB cell lines that express low levels of Hsp47 (LA1–55n and SH-SY5Y), and 3 NB cell lines that express high levels of Hsp47 (LA1–5s, SH-EP, and NBL-S). The MSP studies demonstrated that the Hsp47 CpG sites in the transcriptional start site region downstream of the TATA box between bp –24 and +85 were methylated in all 3 of the Hsp47-negative cell lines, whereas these sites were completely unmethylated in the LA1–5s and SH-EP cell lines that express high levels of Hsp47 (Fig. 2B)
. Both methylated and unmethylated alleles were detected in LA1–55n and SH-SY5Y cells, although the intensity of the unmethylated band was much stronger than that of the methylated band. A similar pattern of methylation was observed in these cell lines when the MSP analysis was performed using primers corresponding to the 5' flanking region between bp –351 and –218 (data not shown). To investigate whether aberrant methylation also occurred in vivo, the methylation status of the Hsp47 promoter was analyzed in NB tumor samples. As shown in Fig. 2C
, methylation in the region of the transcriptional start site of the Hsp47 promoter was detected in a subset of primary NB tumors.
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. Cysteines at non-CpG sites were converted to thymine, excluding the possibility that successful amplification could be attributable to incomplete bisulfite conversion (data not shown).
Coexpression of Hsp47, Collagen I, and Collagen IV in NB Cells.
Hsp47 functions as a molecular chaperone for collagen, and its expression has been shown to be closely correlated with collagen production in a number of cell types. As a first step toward determining whether Hsp47 is capable of regulating collagen expression in NB, we double stained 4 NB cell lines with anti-Hsp47 and anticollagen I or IV antibodies, and used FITC- and rhodamine-conjugated secondary antibodies. In the Hsp47-positive cell lines SH-EP and LA1–5s, collagen type I and type IV were present in the cytoplasm. In contrast, neither collagen was detected in the Hsp47-negative cell lines (NBL-W-N and NMB; Fig. 3, A and B
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, Hsp47 mRNA expression was induced in all 3 of the NB cell lines. Moreover, Hsp47 protein was also detected in these cells lines after treatment with 5-Aza-dC for 3 and 5 days, although the level of Hsp47 expression in the NBL-W-N cell line remained low (Fig. 4B)
. In the NMB and NBL-W-S cells, Hsp47 was up-regulated within 3 days of treatment with the demethylating agent (Fig. 4B)
. No change in the level of Hsp70 protein, another heat shock protein, was seen in these cell lines after treatment with 5-Aza-dC (Fig. 4B)
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. Moreover, up-regulation of collagen IV was also detected in the ECM of NBL-W-S xenografts. Double labeling studies indicated that Hsp47 and collagen IV were colocalized as visualized by the dark brown color. However no difference in the level of expression of either Hsp47 or collagen IV was detected in NBL-W-N and NMB xenografts after treatment with 5-Aza-dC (data not shown).
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1(I) collagen bands corresponding to the propeptide and N-propeptide forms of the 1(I) chains were observed (17)
, and both low- and high-molecular-weight forms of type IV collagen (13)
were detected in our experiments. In contrast, collagen was not seen in NB cell lines that express low to undetectable levels of Hsp47 (NBL-W-N, NMB, LA1–55n, and SH-SY5Y; Fig. 6A
). Collagen expression was also examined by Western blot in 7 benign GNRs and 5 clinically aggressive advanced-stage NB tumors. Type I collagen was detected in 6 of the 7 GNRs, whereas low to undetectable levels of collagen expression were present in the 5 advanced-stage tumors (Fig. 6B)
. Immunohistochemistry studies similarly demonstrated higher levels of type I collagen expression in GNRs and ganglioneuroblastomas compared with undifferentiated NBs (Fig. 6C)
. In the histologically mature tumors, both ganglion cells and differentiated neuroblasts expressed type I collagen. In contrast, collagen was not detected in tumors composed of undifferentiated neuroblasts. Type I collagen expression was also detected in the Schwann cells in a GNR by immunofluorescence using antibodies directed against collagen type I and S-100 (Fig. 6C)
. In addition, Western blot analyses revealed type I collagen in CM prepared from Schwann cells, further demonstrating that Schwann cells are capable of secreting this protein (Fig. 6D)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3-fold differential levels of expression between control and treated NBL-W-S NB cells. TSP-1 provided an internal control for the microarray studies, and, as expected, this gene was significantly up-regulated in experiments performed with the 5-Aza-dC-treated NB cells. In addition, alterations in the levels of expression of genes that encode cell adhesion molecules and proteins that play a role in transcription regulation, signal transduction, immune response, or apoptosis were observed. The gene encoding Hsp47, a collagen-specific molecular chaperone, displayed the highest level of differential expression, with >80-fold increase in expression after treatment with the demethylating agent. As a first step toward investigating whether the significant induction of Hsp47 after treatment with 5-Aza-dC impacted the biological behavior of NB, its pattern of expression in NB and mechanisms of regulation were additionally evaluated. Expression studies confirmed that Hsp47 was silenced in NBL-W-S cells as well as in a subset of other NB cell lines. A typical CpG island is present at the 5' flanking region of Hsp47 and in the region around the transcriptional start site. MSP and bisulfite sequencing revealed that the promoter region of the Hsp47 gene was methylated in a subset of NB cell lines and primary tumors, and the methylation status was found to be inversely associated with gene expression. Although another member of the heat shock protein family, Hsp70, has been shown to be methylated in primary mouse embryo cells and murine cell lines (23) , to our knowledge this is the first report of aberrant methylation of Hsp47. We also found that treatment with 5-Aza-dC restored Hsp47 expression in Hsp47-negative cell lines in vitro, and up-regulation of Hsp47 was detected in a subset of NB xenografts after administration of 5-Aza-dC in vivo.
In a number of cell types, Hsp47 has been shown to specifically and transiently bind to newly synthesized procollagens, and recent studies indicate that Hsp47 is required to form the rigid triple-helical structure characteristic of mature type I collagen (13, 14, 15 , 24) . Immunofluorescence was used to evaluate collagen expression, and consistent with its role as a collagen molecular chaperone, we found coexpression of Hsp47 with types I and IV collagen in the cytoplasm of NB cells. In contrast, NB cells with low to undetectable levels of Hsp47 did not express collagen. Furthermore, both Hsp47 and collagen IV were up-regulated in NBL-W-S-derived NB xenografts after treatment with 5-Aza-dC, supporting a role for Hsp47 in collagen production in NB.
Although it remains unclear whether Hsp47 and/or type I and type IV collagen directly influence NB growth, high levels of Hsp47 and collagen were detected in nontumorigenic NB subclones. In contrast, this gene was silenced or only detected at low levels in tumorigenic NB cell lines. We also found high levels of secreted types I and IV collagen in the CM collected from the nontumorigenic NB cell lines, whereas collagen was not seen in the CM collected from tumorigenic cell lines. In addition, enhanced levels of Hsp47 and collagen type IV were detected in a subset of the 5-Aza-dC-treated NB xenografts that displayed impaired tumor growth (8) . In primary tumors, type I collagen was expressed in mature GNRs and ganglioneuroblastomas, whereas type I collagen was not detected in more clinically aggressive NB tumors composed of undifferentiated neuroblasts, additionally suggesting that collagen may play a role in influencing NB phenotype. Similarly, previous studies have indicated that collagen may negatively impact the malignant potential of other types of cancer. The level of Hsp47 and type I collagen expression is decreased after the malignant transformation of fibroblasts (14 , 25) . In addition, Hsp47 and type IV collagen are markedly increased during the differentiation of a mouse teratocarcinoma cell line (15) . Furthermore, in head and neck carcinomas, lower levels of collagen XVIII and endostatin, an antiangiogenic molecule derived from collagen XVIII after cleavage, are detected in primary head and neck tumors from patients with metastatic disease compared with nonmetastatic cases (26) .
The ECM, which is largely composed of type I collagen, is known to negatively regulate normal cell proliferation. Although much less is known about the growth regulatory effects of the ECM on malignant cells, several lines of evidence suggest that collagen and/or collagen fragments are capable of influencing tumor growth. Contact with type I collagen has been shown to growth arrest melanoma cells (27) . Similarly, using a three-dimensional ECM that is rich in type I collagen, Hotary et al. (28) demonstrated that the proliferation of tumor cells suspended in three-dimensional gels consisting of protease-resistant type I collagen was fully suppressed. Animal studies also indicate that the composition of the ECM can modulate tumor growth. Secreted protein acidic and rich in cysteine (SPARC)-null mice display alterations in the production and organization of collagen in the ECM, and recent studies show that tumors implanted in these mice exhibit enhanced growth and metastases compared with controls (29) . The increased tumor growth was believed to be due, at least in part, to the more permissive environment of the SPARC-null mice. In addition to intact collagen, proteolytic fragments of noncollagenous domains of collagens IV, XV, and XVIII may also influence tumor growth by inhibiting angiogenesis (30, 31, 32, 33, 34) .
In NB, treatment with the demethylating agent 5-Aza-dC leads to alterations in the level of expression of several genes that are likely to be involved in the regulation of tumor growth. Although the roles that Hsp47 and collagen may play in NB pathogenesis are not known, our results and those reported by others suggest that Hsp47 regulates the production of collagen and that collagen and its cleavage fragments may negatively influence the malignant potential of NB and other types of cancer. Additional functional studies are ongoing in our laboratory to investigate whether NB tumor growth can be directly impacted by contact with type I or IV collagen. Hopefully, these experiments will enhance our understanding of the molecular mechanisms underlying collagen synthesis and function in NB and provide insight into how tumor cell behavior can be influenced by the local microenvironment.
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FOOTNOTES |
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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.
Requests for reprints: Susan L. Cohn, Children’s Memorial Hospital, Division of Hematology/Oncology, 2300 Children’s Plaza, Chicago, IL 60614. Phone: (773) 880-4562; Fax: (773) 880-3053; E-mail: scohn{at}northwestern.edu
Received 3/19/04. Accepted 4/21/04.
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, methylated cytosine; 
, unmethylated cytosine.
