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[Cancer Research 64, 3465-3473, May 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Microarray Analysis of Epigenetic Silencing of Gene Expression in the KAS-6/1 Multiple Myeloma Cell Line

Celine Pompeia1, David R. Hodge1, Christoph Plass3, Yue-Zhong Wu3, Victor E. Marquez2, James A. Kelley2 and William L. Farrar1

1 Laboratory of Molecular Immunoregulation, and 2 Laboratory of Medicinal Chemistry, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, and 3 Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The control and cure of cancer is an important public health issue because cancer is a significant contributor to patient mortality. Because a majority of patients suffer serious side effects or develop resistance to traditional forms of cancer treatment, the development of drugs as an adjunct to, or replacement for, current chemotherapy and radiotherapy protocols is of paramount importance. One of the great difficulties in such a quest is the vast variability inherent to malignant cells, thus requiring drugs that are targeted to the common features found in neoplastic disease. Recently, attention has focused on some types of neoplastic growth that are thought to evolve from chronic inflammatory states (1, 2, 3) . In these cases, cancer cells are able to proliferate by using inflammatory signals as growth signals and to evade cell death by inactivating apoptotic pathways triggered in some cases by immune surveillance and are themselves capable of promoting and inducing angiogenesis.

Many tumor cells respond to, or become, autocrine producers of the inflammatory cytokine interleukin 6 (IL-6), using it for growth and survival. This phenomenon is interesting because, in normal cells, IL-6 functions mainly to mediate and augment inflammatory responses. IL-6 has been associated with the etiology of, and as a promoter of, several forms of cancer, including hepatic and prostate cancers and multiple myeloma (4, 5, 6, 7) . DNA methylation is associated with several changes in chromatin structure, including the regulation of histone methylation and acetylation and the recruitment of proteins to the methylated sites. This usually leads to the obstruction of the promoter, which hinders gene transcription, and to subsequent gene silencing (8) . Our laboratory has reported that IL-6 exerted a regulatory influence on DNA methylation via the transcriptional activation of FLI-1, a transcription factor that up-regulated the expression of DNA methyltransferase-1 (DNMT-1; Refs. 9 , 10 ). We have also observed that IL-6 contributes to the epigenetic silencing of important cell cycle and tumor suppressor genes.4 Therefore, it is conceivable that some malignant tumors may develop from chronic inflammatory activity based on the influence IL-6 exerts on the epigenetic control of genes, such as tumor suppressors and modulators of differentiation and apoptosis.

Many of the traits exhibited by cancer cells can be attributed to epigenetic changes in gene expression induced by methylation of DNA. Numerous tumor suppressor genes and proapoptotic genes, for example, are down-regulated or silenced in cancer cells because of promoter hypermethylation (11 , 12) . For this reason, some chemotherapy protocols have included the use of drugs that inhibit DNA methylation. Among such drugs are several analogs of deoxycytidine, the targeted nucleoside for methylation, such as 5-azacytidine and 5-aza-2'-deoxycytidine. Additionally, 5-aza-2'-deoxycitidine has been used extensively to study the reactivation of silenced genes in numerous cell types. Although these drugs have shown some efficient antitumor activity (reviewed in Ref. 13 ), they are highly unstable in vivo and toxic to normal cells. Recently, a new drug, originally developed as a cytidine deaminase inhibitor, zebularine, has been shown to inhibit DNA methylation and is a promising candidate for cancer therapy because of its lower toxicity and increased stability under physiological conditions (14 , 15) .

To assess the importance of epigenetic regulation of gene expression in cancer cells and to explore the role of IL-6 as an inducer of gene methylation and silencing, we used zebularine to demethylate DNA in the IL-6-responsive multiple myeloma cell line KAS-6/1. After treatment with zebularine, cells were washed and allowed to grow in medium supplemented with 10 ng/ml IL-6 to assess the consequences of gene remethylation. The overall effect of zebularine on KAS-6/1 cell DNA methylation, and the silencing effect of IL-6, were evaluated by cDNA microarrays and Restriction Landmark Genomic Scanning (RLGS) analysis. Our data indicate that vital genes were subject to epigenetic regulation via DNA methylation and suggest that this particular tumor type may have developed, in part, as a result of the epigenetic silencing of differentiation modulators, tumor suppressors, and apoptotic mediators. Furthermore, because IL-6 was needed to restore the original DNA methylation levels present before drug treatment, the results obtained confirmed the important role of IL-6 in methylation and subsequent down-regulation or silencing of gene expression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
KAS-6/1 cells (16) were kindly provided by Dr. Dianne Jelinek, of the Mayo Clinic, Rochester, MN. Zebularine was synthesized in Dr. Victor E. Marquez’s Laboratory of Medicinal Chemistry (National Cancer Institute, NIH, Frederick, MD; Ref. 14 ). Affymetrix DNA microarrays were obtained from Affymetrix (Santa Clara, CA). Fetal bovine serum was from Gemini Bio-Products (Woodland, CA). RPMI 1640, ampicillin/streptomycin solution, and L-glutamine were from Cellgro (Mediatech, Herndon, VA), IL-6 was obtained from PeproTech (Rock Hill, NJ). Agarose, Taq polymerase, and dNTPs were from Invitrogen (Carlsbad, CA). DNA molecular weight standard (100 bp) was from New England BioLabs (Beverly, MA), and other reagents were obtained from Sigma (Saint Louis, MO), unless otherwise stated in the methods.

Cell Treatment.
The human multiple myeloma cell line KAS-6/1 was grown in RPMI 1640 supplemented with 10% fetal bovine serum, 10 ng/ml IL-6, 50 IU/ml ampicillin, 50 µg/ml streptomycin, 2 mM L-glutamine in a humidified incubator, at an atmosphere of 5% CO2, at 37°C. Control cells received no treatment; zebularine-treated cells received 200 µM drug hereafter referred to as zebularine for a period of 48 h; "remethylated" cells were treated with zebularine as described, washed 3 times with fresh medium, diluted 10-fold in fresh medium containing 10 ng/ml IL-6, and allowed to grow for 1 week.

RLGS.
RLGS was performed as described in Hatada et al. (17) . Cell genomic DNA was extracted, and sheared ends were blocked with nucleotide analogs ({alpha}-S-dGTP, {alpha}-S-dCTP, dideoxyadenosine triphosphate, and dideoxythymidine 5'-triphosphate) in the presence of DNA polymerase I. Fragments were then cut with NotI, which is specific for nonmethylated strands, and cut ends were radiolabeled with [{alpha}-32P]dGTP. This method ensured that highly methylated regions of DNA were not radiolabeled. The resulting fragments were further cut with EcoRV and subjected to electrophoresis on a 0.8% agarose tube gel. The DNAs were cut in situ with HinfI and the tube gel was placed on the top of a 5% polyacrylamide gel for a second dimension electrophoresis of the DNA. The gels were dried and exposed to an X-ray film. Differences observed in the intensity of spots indicate changes in the degree of DNA methylation between samples. RLGS fragments were cloned from the two-dimensional gel using the strategy described by Smiraglia et al. (18) . Sequences were aligned using BLAST to DNA databases such as GenBank and BLAT, for alignment to the University of California-Southern California human genome to map their positions and identify putative gene promoter regions.

RNA Extraction.
RNA was extracted from cells using the TRIzol reagent (Invitrogen), as recommended by the manufacturer. Briefly, cells were lysed in a solution of phenol and guanidine isothiocyanate, and on the addition of chloroform, the aqueous phase, containing the RNA, was isolated from the organic phase containing solvents, denatured proteins, and DNA. The RNA was then precipitated with isopropanol, washed with 75% ethanol, dried, and dissolved in water. RNA quantity and quality were assessed by agarose gel electrophoresis (1% gel), spectrophotometry (wavelength ratio 260 and 280 nm). The RNAs used for microarray analysis were also evaluated by high-pressure liquid chromatography by Expression Analysis (Durham, NC).

Affymetrix Microarray Analysis.
Samples of RNA from control (untreated), zebularine-treated, and remethylated cells were processed by Expression Analysis (Durham, NC). The steps involved in Affymetrix microarray assays were as follows: generation of a double-stranded cDNA using the RNA as a template; in vitro transcription for the synthesis of an antisense complementary biotin-labeled cRNA; hybridization to the microarray chip; stringency washing of the chip; and scanning of the bound fluorescence tags on the chip. The data obtained was then processed by a series of programs to normalize the data, statistically determine whether a given transcript was present or not, and whether there were significant differences in expression between the different duplicates (experiments 1 and 2) or groups (control, zebularine-treated, and remethylated). The Affymetrix statistical algorithms were run using the Affymetrix Microarray Suite version 5.0, a proprietary group of programs. Standard statistical techniques were used and later validated using an experimental design called the "Latin square," which is developed using naturally absent transcripts spiked at known concentrations. This test allows the evaluation of the significance of values given the complex background of the microarrays.

A detection P was calculated for each probe set by the signal difference from perfect-match probes and mismatch probes, rendering a discrimination score that was used in a one-sided Wilcoxon’s signed-rank test. The signal from each probe set was a quantitative value that represented the relative expression level and was calculated using the one-step Tukey’s biweight estimate. The comparison between chips was done by two algorithms, one that generated a "change P" (using Wilcoxon’s signed-rank test) and the other for the quantitative estimate of the gene expression, which was associated with a signal log ratio (calculated with Tukey’s biweight method). Before chips were compared, scaling and normalization were also carried out using several methods (not detailed by Affymetrix).

The microarray assay was carried out with two sets of chips, U133A and U133B, with a total of 45,000 probe sets representing more than 39,000 transcripts, derived from ~33,000 well-substantiated human genes (based on the genome Build of April 2001). The set design uses sequences selected from GenBank, dbEST, and RefSeq. The U133A set was generated from cDNA data sequences previously represented on the Human Genome U95Av2 Array, whereas the U133B was generated primarily from EST clusters. Each array has ~22,000 "probe sets," each containing 11 DNA oligonucleotides of 25 bases corresponding to different regions of the 3' end of a given transcript. In some cases, several probe sets exist for the same gene, so that different splice forms or transcripts with different polyadenylation sites can be assessed. Three sets of chips were analyzed for each group of cells: two with probes generated from the same RNA (experiments 1 and 2) and one with probes generated from RNA obtained in a different experiment (experiment 3). To ascertain the accuracy of our first result (experiments 1 and 2), we repeated the microarray analysis de novo with total RNAs from the KAS 6/1 cells treated as described previously. Results obtained from this confirmatory work is presented in experiment 3.

Semiquantitative RT-PCR.
Total RNA was used as a template for the synthesis of cDNA using the "1st Strand cDNA Synthesis kit for RT-PCR (AMV)," according to the manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). The RNA (1 µg/20 µl) was added to a mix containing 10 mM Tris (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 mM dGTP, 80 ng/ml oligo-p(dT)15 primer, 2.5 units/µl RNase inhibitor, and 1 unit/µl AMV reverse transcriptase. After primer annealing at room temperature, the cDNA synthesis proceeded at 42°C, and the final reaction was stopped by enzyme inactivation at 99°C for 5 min. The final product was further diluted with 30 µl water.

The PCR was carried out with 20 mM Tris (pH 8.4), 50 mM KCl, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.2 mM dGTP, 1.5 mM MgCl2 (unless otherwise stated), 0.2 µM each primer, 1 unit AmpliTaqDNA polymerase (Invitrogen), and 40 µl/ml cDNA template. After an initial step for enzyme activation (2 min, 96°C), cycles consisting of 30 s denaturing (96°C), 30 s annealing, and 30 s extension (72°C) were performed. Primers, annealing temperature, and cycle number are indicated in Table 1Citation . The PCR conditions were optimized to obtain data from the exponential phase of the reaction, when relative levels of expression could be assessed. The housekeeping gene glyceraldehyde 3-phosphate (GAPDH) was used to normalize data. After PCR, the DNA products were run on a 2% Tris-acetate-EDTA (0.5x TAE), ethidium bromide-stained agarose gel. The molecular weight of the PCR products was estimated by comparison with the 100-bp molecular weight standard. The fluorescence of each band was quantified using LabWorks Analysis Software, Version 3.0.02.00, UVP (Upland, CA).


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  		Table 1 PCR primers and conditions for each of the analyzed genes

 
RNA Protection Assay.
RNase protection assay was carried out with the "Multi-Probe RNase Protection Assay System," human apoptosis-related set (hAPO-2c; BD Biosciences, Franklin Lakes, NJ), according to the manufacturer’s instructions. The kit contains DNA templates for [{alpha}-33P]-labeled RNA antisense synthesis. The probe mixture was added to the sample RNA and allowed to hybridize. Nonannealing RNA was then digested with RNases and protected RNA fragments were resolved in a 6% polyacrylamide-urea gel. The gel was dried and exposed to X-ray film. Internal standards [L32 and glyceraldehyde 3-phosphate (GAPDH)] were used to normalize for sample loading.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RLGS.
The overall effect of zebularine on KAS-6/1 genomic DNA methylation was evaluated using RLGS (Fig. 1Citation ; Table 2Citation ). This technique uses methylation-sensitive restriction enzymes with a high CG and CpG island content to cleave genomic DNA at the CpG islands. Cleaved sites are radioactively labeled and, after further digestion with restriction enzymes, the fragments are sequentially resolved in agarose and polyacrylamide gels, yielding a two-dimensional level of resolution. As shown in Fig. 1Citation , changes in the intensity of the fragments indicate methylation control. Darker spots correspond to unmethylated DNA and are found more frequently in zebularine-treated-derived DNA, as compared with control DNA (untreated) and DNAs obtained after wash-out of zebularine from the cells, associated with the culture of the cells for several days in the presence of IL-6 ("remethylated"). The patterns of the DNA fragments obtained from control and remethylated cells were very similar, indicating that the removal of zebularine and addition of IL-6 restores control DNA methylation patterns.



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  		Fig. 1. Restriction Landmark Genomic Scanning (RLGS) two-dimensional gel film X-rays. The genomic DNA from each sample was digested with NotI, radioactively labeled, digested with EcoRI, run on an agarose tube gel; digested in situ with HinfI and run on a 5% polyacrylamide gel. Regions with good resolution were selected to pick spots with various intensity. Each spot received identification. Arrows, DNA fragments (spots) that appear in zebularine-treated cells and are absent, or found at a lower intensity, in DNA fragments from control and remethylated cells. Alphanumerical codes to the right correspond to the names assigned to each of these regulated spots.

 

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  		Table 2 Overall results of Restriction Landmark Genomic Scanninga

After exposure of the two-dimensional gel to an X-ray film, the spot intensities and patterns among the control, zebularine-treated, and remethylated samples were compared. In all cases, the intensity of the control and the remethylated spots was the same.

 
Of a total of 1360 spots, 23 DNA fragments were darker in DNA derived from cells treated with zebularine. These 23 well-resolved spots were identified, and 7 were sequenced. The results are shown in Table 2Citation .

After alignment with public databases, all of the sequenced fragments were found to correspond to CpG islands. Such alignment, particularly to the human DNA genome, enabled us to map the sequenced fragments and, in some cases, associate them with putative gene promoters. Five sequences aligned with known genes, one with a predicted open reading frame and one with an expressed sequence tag. The first known gene is the poly(rC)-binding protein 1 (PCBP1), a multifunctional protein that binds RNA and is involved in translation and RNA stability. PCBP1 is known to participate in the increased stability of mRNAs containing internal ribosome entry segments. Next, the microtubule-associated protein 1 light chain 3 ß (MAP1LC3B) protein, which functions in both neurogenesis and autophagy. Third, the estrogen receptor 1 (ESR1), that was found to be down-regulated by demethylation from its basal level in untreated cells. Fourth, the SET binding protein 1 (SETBPI), binds to SET, the translocation breakpoint-encoded protein found in acute undifferentiated leukemia, which participates in protein transport and transcriptional regulation. Fifth, the N-deacetylase/N-sulfotransferase (heparin glucosaminyl) 3 (NDST3) enzyme, responsible for heparin and protein deacetylation and sulfation. This finding is interesting because of the role heparin is believed to play in cell proliferation, metastasis, angiogenesis, and cell adhesion (reviewed in Ref. 19 ).

Microarray.
On verification that zebularine reduced DNA methylation levels in KAS-6/1 cells, we decided to perform a systematic study on genes epigenetically regulated by zebularine using total cell RNA as a probe in the expression cDNA microarray technique. This approach has already been used to study methylation-regulated gene transcription, e.g., 5-aza-deoxycytidine-treated human fibroblast and bladder cancer-derived RNA revealed many genes differentially regulated by this demethylating agent in normal versus cancer cells using the microarray technique (20) . Several criteria were used to select probe-sets of interest: (a) results derived from drug-treated cells had to be at least 2-fold higher than those derived from control or remethylated cells; (b) such differences between control, drug-treated, and remethylated signals had to be statistically significant (P <= 0.05); and (c) for positive regulation with the drug, gene expression had to be considered present in the probe set in all experiments (specialized software was used to determine whether a given gene set differed significantly from the background hybridization signal).

Fig. 2Citation is an example of the genes that are up-regulated by drug treatment in relation to control and remethylated-derived RNA. The X and Y axes correspond to the fraction between the signal from the treated probe sets and that of the control and remethylated probe sets, respectively. High values in the abscissa indicate that demethylation resulted in an increase in gene expression. Probe sets corresponding to high Y-axis values indicate that the removal of zebularine and the recovery of the cells in IL-6-containing medium down-regulates gene expression. The black diamonds ({diamondsuit}) correspond to the total probe sets and, overlaying them in white squares ({square}, are the probe sets that correspond to transcripts considered significantly different between both demethylated and control, and demethylated and remethylated data (P <= 0.05). Although statistical programs consider the differences significant, from a biological perspective differences of at least 2-fold are most likely relevant. Therefore, only the probe sets corresponding to white boxes in the upper right quadrant of the plot were used for further analyses, because they indicate genes up-regulated by zebularine and down-regulated in the absence of the drug and in the presence of IL-6.



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  		Fig. 2. Gene regulation by zebularine (Array U188A, average of experiments 1 and 2). Full diamond-shaped symbols, the relative intensity of zebularine/remethylated samples (Y axis) and zebularine/control samples (X axis). The intensity of each sample corresponds to an average from experiments 1 and 2 of the results of each probe set present in Array U188A. The empty boxes, data considered significant between groups by the Affymetrix statistical suite (P <= 0.05). The gray lines, the cutoff value of 2 in each axis. Only probe sets corresponding to white squares with a value within the upper right quadrant were used for further analysis. These correspond to genes that were up-regulated at least 2-fold by zebularine and then down-regulated at least 2-fold after remethylation.

 
The overall results obtained from the microarray studies are summarized in Table 3Citation . The number of probe sets up-regulated by demethylation represents a small fraction of the total probe sets: 0.19% and 0.58% of genes up-regulated by drug in experiments 1 plus 2 and 3, respectively (U133A chip). These values corresponded to 0.11 and 0.39% for chip U133B. The results were always higher for experiment 3 because experiments 1 and 2 were analyzed together, which led to the exclusion of duplicates that were not considered compatible with each other. The overlap of up-regulated probe sets by zebularine between all of the experiments was of 9 (0.04%) and 2 (0.0088%) for chips U133A and U133B, respectively (Table 4ACitation and Table 4BCitation ).


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  		Table 3 Genes up-regulated by zebularine and identified by microarray analysis

 

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  		Table 4 Gene expression in probe sets up-regulated by zebularine

The microarray expression data are shown for the probe sets up-regulated by zebularine according to three criteria: presence of signal in zebularine-derived samples, increase in signal of at least 2-fold, and significant difference between the zebularine signal and that of control and remethylated samples. Furthermore, only genes that fitted all of the criteria in all of the experiments are shown. The columns show, respectively, the probe set identification number (id); the average value obtained, respectively, with control, zebularine-treated (Zebul), and remethylated (Remeth) samples from experiments 1 and 2 [(1+2)]. The fraction between the value obtained with zebularine and that of the control or remethylated samples are in the two following columns, respectively. Control, zebularine-treated, and remethylated values, as well as the fraction values obtained in experiment 3 [(3)] follow in columns 7–11, respectively. Column 12 contains a brief description of the gene associated with the probe set, and the last column indicates whether there is a CpG island in the promoter of the gene (see Table 3Citation ). The value 1 is assigned when at least one significant CpG island is found in the promoter region of the gene associated with the probe set; otherwise, the value 0 is assigned.

 
To evaluate the function of the genes up-regulated by demethylation, several databases were searched, particularly those from the Gene Ontology (GO) Consortium. A main function was assigned to each gene shown in Table 5Citation . The regulated probe sets were most associated with metabolism, intracellular signaling, transcriptional regulation, and proliferation (with the exception of one chip) genes. Genes associated with RNA binding/processing, chromatin/DNA, replication/DNA repair, and cell structure, exhibited relatively few changes on demethylation.


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  		Table 5 Gene function of up-regulated genes

Each probe set found to be up-regulated by zebularine was assigned a main function based on Gene Ontology (GO) Consortium annotations (53) , GeneBank and UniGene annotations, publications, and bioinformatics prediction tools, such as SMART. "(1+2)" indicates the combined results from experiments 1 and 2 (using the same RNAs) and "(3)" corresponds to experiment 3 (using RNAs obtained in an independent experiment). The second line, GO annotations, indicates the number and percentage of probe sets with any GO annotation, according to the Affymetrix download of July 2003 (updated every 3 months).

 
Some of the genes regulated by methylation, as assessed by the microarray analysis, matched the genes identified by RLGS. These include RLGS fragment 5E2, which codes for a microtubule-associated protein. The fragment was present exclusively in the drug-treated-derived samples, indicating that the promoter for this gene was demethylated facilitating transcription. There are two probe sets corresponding to this gene, 208785_s_at and 208786_s_at, both from chip U133A. The fractions obtained between demethylated and control or remethylated-derived samples were as follows: 1.32/1.19 (experiments 1 + 2), 1.52/1.46 (experiment 3) for 208765_s_at; and 1.18/0.98 (experiments 1 + 2), 1.4/1.68 (experiment 3) for set 208786_s_at. The restriction landmark genomic sequencing fragment 3F53, corresponding to SETBP1 overlaps the promoter corresponding to probe set 205933_at. The expression fractions were: 1.12/1.16 (experiments 1 + 2) and 1.43/1.56 (experiment 3).

RT-PCR.
To confirm data obtained in the microarrays, semiquantitative reverse-transcription PCRs (RT-PCRs) were carried out for nine probe sets (Fig. 3Citation and Table 6Citation ). We found up-regulation of all chosen genes in cells treated with zebularine, and the relative change correlated well with the microarray results; therefore, microarray analysis alone is reliable, at least for initial studies, to globally assess the affects of zebularine on gene expression. The most pronounced regulation by zebularine was found for aldehyde dehydrogenase 12 (ALDH12), a gene that metabolizes retinoic acid to its active retinoic-acid receptor-binding form. All of the other results from RT-PCR and microarrays indicate an up-regulation of ~2–3-fold on zebularine treatment.



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  		Fig. 3. Semiquantitative reverse transcription-PCR for microarray genes up-regulated by zebularine. RNAs from control, zebularine-treated, and remethylated cells were extracted and used as templates for the synthesis of cDNAs (the RNA was the same as that used in microarray experiments 1 and 2). After normalization of cDNA concentrations, PCR reactions were carried out with primers designed to hybridize with different exons of each gene. The number of cycles and PCR conditions were optimized to obtain semiquantitative data (see "Materials and Methods"). The PCR reaction products for glyceraldehyde 3-phosphate (GAPDH) are shown as control for equal total cDNA template added to each PCR reaction.

 

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  		Table 6 Microarray and reverse transcription-PCR (RT-PCR) comparison

The bands obtained by RT-PCR were quantified by densitometry and corrected for the glyceraldehyde 3-phosphate (GAPDH) levels. The fraction between values obtained with zebularine and control or zebularine and remethylated samples were then calculated. These fractions were then compared with those obtained in the microarray experiments [averages of experiments (exp) 1 and 2]. Genes depicted in bold correspond to those considered significantly altered in microarray experiments 1, 2, and 3.

 
RNase Protection Assay.
We noted some effects of demethylation on the expression levels of several apoptotic related genes in the microarrays and used the RNase protection assay, with probes associated with apoptotic markers, to confirm our microarray data. As shown in Fig. 4Citation , up-regulation of BCL-W, BAD, BAK, and BAX by demethylation was detected, whereas there was no change in BCL-X and MCL-1 expression. This result was consistent with our microarray data and reinforced the validity of the results. BCL-W expression values induced by drug versus control, and drug versus remethylated fractions were 1.15/1.36 (experiments 1 + 2) and 1.29/1.28 (experiment 3), respectively. The BAD gene corresponds to three probe sets, one of which remained unchanged in both experiments, another that was not detected in any of the experiments, and a third that, although not detected in experiments 1 + 2, showed up-regulation in experiment 3; the fractions found were 1.70/1.87. These results suggest BAD might be alternatively spliced or merely that the probes used were not optimum. BAK was also found to be up-regulated in experiment 3, fractions were 1.59/1.40. Isoform {delta} of BAX was found to be up-regulated in experiments 1 + 2, with fractions of 2.95/2.85.



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  		Fig. 4. RNase protection assay for apoptosis-related genes. Probes for different apoptosis-related genes consisting of radioactively labeled cRNAs were hybridized with total RNA from control and from zebularine-treated and remethylated cells. On addition of RNases specific for single-stranded RNAs, only double-stranded RNAs remained integral. These were then run on a 6% polyacrylamide gel. After gel drying, the probes were detected on X-ray films. Nonhybridized probes were run in parallel (Lane 4) to localize the bands of interest. These nonhybridized probes are larger than the probes mixed with the RNAs and submitted to RNase digestion because cRNAs don’t hybridize completely to probes, leaving single-stranded probe regions that are shortened by RNases. Probes for L32 and glyceraldehyde 3-phosphate (GAPDH) were added to indicate even loading of samples on the gel.

 
BCL-X showed no significant change in microarray expression data in experiments 1 + 2 and 3 for all three probe-sets representing this gene. As for MCL-1, corresponding to six probe-sets, there was no regulation in five sets and no detection in the last.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Epigenetic silencing of important cell differentiation, tumor suppressor, and apoptotic genes represents a significant impediment to cancer treatment. Here we show that a number of genes the silencing of which could potentially lead to the development of a neoplastic phenotype were reactivated by the use of a single dose of the methylation inhibitor zebularine. Reactivation of these genes using methylase inhibitor drugs may play an important role in cancer treatment (14 , 15) . Zebularine, a compound found recently to inhibit DNA methylation, effectively reversed the methylation status of epigenetically silenced genes in our cell model, suggesting that many tumors probably select for methylation-induced silenced genes as they develop. To evaluate the methylation status of cellular genes, we assessed overall changes in DNA methylation by RLGS, and we systematically searched for specifically targeted genes using the expression microarray technique. Genes of interest were then further analyzed by RT-PCR and RNase protection assay.

The RLGS assay indicated that zebularine caused genome-wide changes in DNA methylation. The analysis of 1360 DNA fragments revealed that 1.7% were demethylated. The patterns of methylation in control cells and in cells subjected to remethylation were unchanged, indicating that the changes induced by zebularine were transient. After drug treatment and rescue (removal of drug), KAS-6/1 cells survived and proliferated only in the presence of IL-6 (16) , suggesting that the cytokine plays an important role in the DNA remethylation process. Because cells rescued from drug treatment in the presence of IL-6 exhibited DNA methylation patterns similar to the untreated control cells, IL-6 apparently plays some role in the epigenetic gene silencing process. This role, particularly as noted by the IL-6-associated remethylation pattern, is consistent with our previous observations that IL-6 up-regulates the transcription of human DNMT-1, an activity essential for DNA methylation (10) .

The CpG island-associated DNA fragments isolated from the RLGS assay showed that promoter methylation was inversely correlated with gene expression, i.e., the higher the methylation, the lower the gene expression. We decided to search for other genes regulated by methylation using the expression microarray technique. Although the RLGS is appropriate to show the relationship between the action of zebularine and DNA methylation, as well as to assess DNA methylation nonspecifically, i.e., irrespective of the genes involved or whether the DNA is associated with coding or noncoding regions, the microarray assay allows a quicker scanning of thousands of transcripts, albeit regulation cannot be directly attributed to methylation, because it may result from an indirect effect of the drug on, for instance, a transcription factor. Nevertheless, some sequences identified by RLGS overlapped promoters of regulated transcripts found by using the microarray approach, thus showing that these techniques are correlated and complementary.

Methylation usually occurs in CpG islands, defined as regions greater than 200 bp with a CG content greater than 50% and an observed to predicted ratio of CG greater than or equal to 0.6 (21) . Gene up-regulation by methylation inhibitors generally occurs by demethylation of the promoters, allowing gene transcription, or by some other unidentified, indirect effect on the gene. Indeed, although the expected ratio of CpG islands in the promoter regions and first exons is around 50% (22) , in our microarray results, we found that 63–71% of the genes regulated by zebularine contained a CpG island within the first 1500 bases upstream from the transcription initiation site, indicating that zebularine preferentially targeted CpG islands. The regulation of many genes devoid of CpG islands could be attributed to indirect regulation by transcription factors that do contain CpG islands and that are regulated by methylation. Additionally, there are reports that methylation may occur on cytosines adjacent to nucleotides other than Gly, particularly Ala (23) , but it is not clear whether they must be in CpG islands. Furthermore, CpG islands may also be found in exons and introns, interfering with gene transcription, and in mRNA splicing. Because we did not search for exon and intron CpG islands, the 29–47% of zebularine-regulated genes, i.e., those without CpG islands in their promoters, may have been directly affected by CpG islands downstream of their promoter regions. Careful examination of our microarray data reveals many unknown open reading frames, representing potential genes, that contain CpG islands in their promoters. These unknown open reading frames have not yet been assigned functional roles and are under investigation.

Although microarray analysis has the advantage of allowing the screening of a large number of genes, the results must be verified by another method to ensure accuracy. For this reason, several genes were chosen for semiquantitative RT-PCR or RNA protection assay to assess the reliability of the microarray data. The relative values obtained by RT-PCR were very similar to those found using microarrays. Among the criteria to select the genes to be analyzed were the following: consistency between the different microarray assay results; the selection of genes associated with tumorigenesis, such as genes that regulate apoptosis, cell proliferation, DNA repair, and cell differentiation; and the selection of genes associated with IL-6 or inflammation. Several genes found to be silenced after drug removal and IL-6 addition are associated with metabolism, intracellular signaling, transcriptional regulation, and proliferation. The last three functions are closely interconnected and associated with cell transformation and cancer. Genes controlling metabolism are usually not prone to transcriptional regulation because most of them are "housekeeping" genes. However, some cancer cells are known to alter their metabolic pathways to overcome difficulties such as lack of nutrients or hypoxia, and to escape the effects of chemotherapy (24 , 25) .

The gene showing the greatest change in expression levels after drug treatment was ALDH12, important in the metabolism of retinoic acid, a known ligand for the RAR nuclear receptor, which is involved in cell differentiation (26) . There exists an inverse correlation between hematopoietic cell differentiation and carcinogenesis, with highly differentiated, end-stage cells rarely observed as undergoing unregulated proliferation. In contrast, the gene expression profiles of many hematopoietic tumors are more closely compared with those of developing precursor cells, suggesting that many tumors of this type probably arose by the clonal expansion of cells "frozen" in a partially differentiated precursor stage (27) . Given the great changes in the levels of ALDH12 expression, it is probable that the genes regulated by retinoic acid were involved in promoting the differentiation of the KAS-6/1 cells. Furthermore, retinoic acid has been found to induce apoptosis in many cell types, and suppression of its metabolism in this myeloma cell line may promote tumor cell survival (28) . Curiously, ALDH12 was the only gene of the nine chosen for RT-PCR not to contain CpG islands in its promoter (first 1500 bp upstream from transcription initiation site). It may be that methylation occurs despite this, that there are CpG islands upstream or downstream from the region studied, or, most probably, that ALDH12 regulation is indirect, via the regulation of some transcription factor regulated by methylation that binds to the ALDH12 promoter.

Acute myeloid leukemia 1 (AML1), also known as runt-related transcription factor 1 (RUNX1) is one of the transcription factors regulated by zebularine, and is known to be an important factor in developmental hematopoiesis. The AML1/RUNX1 gene has a tumor suppressor activity and is mutated in several types of cancer (29) . AML1 inhibits the transcription of macrophage inflammatory protein 1 (MIP1), which is overexpressed in 70% of the patients with multiple myeloma (30) . Because KAS-6/1 cells are derived from a multiple myeloma, it is possible that they repress AML1 expression to allow overexpression of MIP1, which may protect cells from death and promote their proliferation (31) .

The protein encoded for by the JTV1 (p38) gene (32) , which contains a glutathione S-transferase (GST) domain, was shown to bind damaged DNA, and act as a chaperone. It is possible that JTV1 silencing occurs because it exerts some DNA protection effect. Therefore, silencing of DNA-protective or repair proteins, such as JTV1, may promote cancer. Furthermore, lowered expression of JTV1, with its antioxidant glutathione S-transferase domain, may lead to an accumulation of reactive oxygen species, promoting cell proliferation or mutagenesis.

The ring finger protein 7 (RNF7), also known as Sensitive-to-Apoptosis-Gene (SAG), was also confirmed by RT-PCR assays. RNF7 also has antioxidant activity, being capable of inactivating peroxynitrite, hydrogen, and fatty acid peroxides (33 , 34) . The presence of antioxidants in cancer cells may hinder their proliferation and their mutation rate, depending on the presence of reactive oxygen species (35) , which may explain the fact that RNF7 is down-regulated in control KAS-6/1 cells.

The intercellular adhesion molecule 1 (ICAM1), also known as CD54, the proliferation-associated 2G4 (PA2G4), and the solute carrier family 3 member 2 (SLC3A2) genes are also greatly up-regulated by demethylation. ICAM1 is an adhesion protein found in many cells of the immune system and binds to integrins, which then activate intracellular signaling pathways. This protein is highly expressed in colorectal cancer and breast cancer and is an indicator of inflammation in the latter (36 , 37) . The expression of ICAM1, a p53-responsive gene, may have been induced directly by demethylation or indirectly by p53 transactivation, which was also reactivated in drug-treated KAS-6/1 cells (Ref. 38 and manuscript submitted).4 Furthermore, whereas the expression of ICAM1 mRNA in zebularine-treated cells may seem paradoxical, it could be explained, in part, by the fact that its background expression level in control cells was relatively high, possibly indicating only partial methylation but still sufficient for tumor promotion.

The multifunctional protein, PA2G4, also known as Ebp-1, was first identified as having strong homology with a mitogen-inducible, murine cell cycle gene of the same name (39) . Later work revealed that the coding sequence of PA2G4 was identical to a protein, designated as Ebp-1, that bound to Erb-B3, an inactive tyrosine kinase. PA2G4/Ebp-1 has been shown to participate in the differentiation of human ErbB receptor-positive breast and prostate cancer cells, and is capable of inducing antiproliferative effects by virtue of its interactions with the retinoblastoma (Rb) tumor suppressor protein. The ability of PA2G4/Ebp-1 to complex with Rb enables the resulting heterodimer to recruit histone deacetylases (HDACs), inhibiting the transcription from the cyclin E promoter, an important cell cycle initiator protein (40) . Epigenetic silencing of PA2G4/Ebp-1 could affect cell cycle regulation by impeding this activity.

Lastly, SLC3A2 is involved in cell activation, transformation, inflammation and carcinogenesis. The SLC3A2 protein, also known as CD98, is similar to ICAM1, in that it signals through integrins (41) . CD98 functions also as a L-phenylalanine transporter, and in some myeloma cells, its reduced expression has been correlated with resistance to the drug melphalan (42) . Thus, SLC3A2/CD98 might share similar properties with the major drug resistance protein (MDR-1), also known as P-glycoprotein, which removes chemotherapeutic drugs by active transport.

There are several reports on the repression of apoptosis in cancer cells by DNA methylation (43, 44, 45, 46) . Assuming that dependence on IL-6 during neoplastic development occurs because of its role in mediating gene silencing by methylation, survival in this case means, among other things, evasion of cell death. Therefore, in cells in which proapoptotic genes were epigenetically silenced by methylation, one would expect an up-regulation of antiapoptotic genes by IL-6. We analyzed by RNase protection assay the expression of some apoptosis-associated genes and correlated these results with those obtained by microarray analysis. Genomic demethylation induced the increased expression of BCL-W, an antiapoptotic gene functionally similar to BCL-2. However, as assessed by microarray analysis and RNase protection assay, zebularine induced BCL2-interacting killer (BIK), a member of the BCL2 family that promotes cell death, and several other death-promoting members of the BCL2 family, including the proapoptotic proteins BAD, BAK, and BAX (47) . This increase in proapoptotic BCL-2 family members may reflect a combination of drug-mediated reactivation of other apoptotic inducers such as p53, and the cytotoxic effects of zebularine. Despite the increase in one antiapoptotic protein, the increased expression of four proapoptotic proteins indicates that this multiple myeloma cell line is vulnerable to the removal of methylation-induced epigenetic silencing of apoptotic genes. Because IL-6-mediated resistance to apoptosis is a significant problem in multiple myeloma, the regulation of these apoptotic genes represents an interesting correlation between apoptosis and epigenetic regulation of gene expression.

Several other genes, previously identified as regulated by methylation in cancer are compiled on the website "Genes Affected by Promoter CpG Island Methylation in Aging and/or Cancer."5 This collection contains 66 genes, 35 of which are present in the U133 series of Affymetrix microarrays, corresponding to 212 probe sets. The comparison of the microarray data with the aforementioned collection reveals no gene up-regulated by zebularine by a minimum factor of 2. Only RASSF1 "Ras association (RalGDS/AF-6) domain family 1" was found to be regulated by a minimum factor of 1.5, considering all three experiments. RASSF1 is a gene similar to the RAS genes in that it exerts tumor suppressor effects by interacting with the repair protein XPA (48) and by inhibiting the accumulation of cyclin D1 (49) . The regulation of RASSF1 was confirmed by RT-PCR. The RASSF1 gene has been reported to be epigenetically regulated in many cancers, including multiple myeloma, thus demonstrating that its epigenetic regulation is an important factor in the development and maintenance of neoplasia (50 , 51) .

We have presented an initial exploration into the effects of zebularine on gene expression, using a cellular model in which gene resilencing can be achieved by a pro-inflammatory cytokine, IL-6. The data shown here confirms the effects of epigenetic silencing of important cell-regulatory genes that can be reversed by treatment with DNA methylation inhibitors such as zebularine, as verified by RLGS, microarray, RT-PCR, and RNase protection assay analysis. Also revealed is the requirement for IL-6 in the post-drug recovery phase and its ability to restore epigenetic gene silencing. Because remethylation/resilencing of the genes examined, and subsequent cell survival, correlates with IL-6 treatment, we have demonstrated an important effect of IL-6 on the survival of multiple myeloma cells, and the reestablishment of epigenetic gene silencing.


   ACKNOWLEDGMENTS
 
We thank Dr. Joost Oppenheim for critical review of the manuscript; Dr. Dianne Jelinek, of the Mayo Clinic, Rochester, MN, for kindly providing the KAS-6/1; and Suneetha Betsy Thomas, for technical aid.


   FOOTNOTES
 
Grant support: This work was supported by the Federal funds of the National Cancer Institute, NIH, under contract N01-CO-5600 and the Department of Health and Human Services, by contract with Science Applications International Corporation-Frederick. C. Plass is a Leukemia and Lymphoma Society Scholar and his work is sponsored by NIH Grant CA93548.

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: William L. Farrar, Laboratory of Molecular Immunoregulation, National Cancer Institute-Frederick Cancer Research and Development Center, 1050 Boyles Street, Building 560, Room 31-68, Frederick, MD 21702. Phone: (301) 846-6867; Fax: (301) 846-7042; E-mail: farrar{at}mail.ncifcrf.gov

4 D. R. Hodge, B. Peng, C. Pompeia, S. D. Fox, V. E. Marquez, J. A. Kelley, and W. L. Farrar. IL-6 regulates methylation of tumor suppressor gene promoters, manuscript in preparation. Back

5 Internet address: http://www3.mdanderson.org/leukemia/methylation/cgi.html. Back

Received 12/18/03. Revised 2/ 7/04. Accepted 3/ 2/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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