©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Antisense to DNA Methyltransferase mRNA Induces DNA Demethylation and Inhibits Tumorigenesis (*)

(Received for publication, October 10, 1994; and in revised form, January 24, 1995)

A. Robert MacLeod Moshe Szyf (§)

From the Department of Pharmacology and Therapeutics, McGill University, Montreal H3G 1Y6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Many tumor cell lines overexpress DNA methyltransferase (MeTase) activity; however it is still unclear whether this increase in DNA MeTase activity plays a causal role in naturally occurring tumors and cell lines, whether it is critical for the maintenance of transformed phenotypes, and whether inhibition of the DNA MeTase in tumor cells can reverse transformation. To address these basic questions, we transfected a murine adrenocortical tumor cell line Y1 with a chimeric construct expressing 600 base pairs from the 5` of the DNA MeTase cDNA in the antisense orientation. The antisense transfectants show DNA demethylation, distinct morphological alterations, are inhibited in their ability to grow in an anchorage- independent manner, and exhibit decreased tumorigenicity in syngeneic mice. Ex vivo, cells expressing the antisense construct show increased serum requirements, decreased rate of growth, and induction of an apoptotic death program upon serum deprivation. 5-Azadeoxycytidine-treated cells exhibit a similar dose-dependent reversal of the transformed phenotype. These results support the hypothesis that the DNA MeTase is actively involved in oncogenic transformation.


INTRODUCTION

Vertebrate DNA is methylated at the 5-position of the cytosine residues in the dinucleotide sequence CpG(1, 2) . Twenty percent of the CpG sites are nonmethylated, and these sites are distributed in a nonrandom manner to generate a pattern of methylation that is site-, tissue-, and gene-specific(1, 2, 3) . Methylation patterns are formed during development: establishment and maintenance of the appropriate pattern of methylation is critical for development (4) and for defining the differentiation state of a cell(5, 6, 7) . The pattern of methylation is maintained by the DNA MeTase (^1)at the time of replication(8) , and the level of DNA MeTase activity and gene expression is regulated with the growth state of different primary (8) and immortal cell lines(9) . This regulated expression of DNA MeTase has been suggested to be critical for preserving the pattern of methylation(8, 9, 10) .

An activity that has a widespread impact on the genome such as DNA MeTase is a good candidate to play a critical role in cellular transformation. This hypothesis is supported by many lines of evidence that have demonstrated aberrations in the pattern of methylation in transformed cells. While many reports show hypomethylation of total genomic DNA (11) as well as individual genes in cancer cells(12) , other reports have indicated that hypermethylation is an important characteristic of cancer cells(13) . First, large regions of the genome such as CpG-rich islands (14) or regions in chromosomes 17p and 3p that are reduced to homozygosity in lung and colon cancer, respectively, are consistently hypermethylated(15, 16) . Second, the 5` region of the retinoblastoma (Rb) and Wilms Tumor (WT) genes are methylated in a subset of tumors, and it has been suggested that inactivation of these genes in the respective tumors resulted from methylation rather than a mutation(17) . Third, the short arm of chromosome 11 is regionally hypermethylated in certain neoplastic cells(15) . Several tumor suppressor genes are thought to be clustered in that area(18) . If the level of DNA MeTase activity is critical for maintaining the pattern of methylation as has been suggested before(8, 9, 10) , one possible explanation for this observed hypermethylation is the fact that DNA MeTase is dramatically induced in many tumor cells well beyond the change in the rate of DNA synthesis(13, 19) . The observation that the DNA MeTase promoter bears AP-1 sites (20) and is activated by the Ras-AP-1 signaling pathway (21) is consistent with the hypothesis that elevation of DNA MeTase activity is an effect of activation of the Ras-Jun signaling pathway(22) .

It has recently been demonstrated that forced expression of exogenous DNA MeTase cDNA causes transformation of NIH 3T3 cells supporting the hypothesis that overexpression of DNA MeTase can cause cellular transformation(23) . The critical question that remains to be answered is whether indeed the level of expression of the endogenous DNA MeTase plays a causal role in tumors that are induced by naturally occurring oncogenic signal transduction pathways. To address this question, we have chosen the adrenocortical carcinoma cell line Y1 as a model system. Y1 is a cell line that is derived from a naturally occurring adrenocortical tumor in LAF1 mice(24) . Y1 cells bear a 30-40-fold amplification of the ras proto-oncogene(25) . If the level of expression of DNA MeTase activity is critical for the oncogenic state, then the transformed state of a cell should be reversed by partial inhibition of DNA methylation. We have previously demonstrated that forced expression of an ``antisense'' mRNA to the most 5` 600 bp of the DNA MeTase message (pZalphaM) can induce limited DNA demethylation in 10T1/2 cells(7) . To directly test the hypothesis that the tumorigenicity of Y1 cells is controlled by the DNA MeTase, we transfected either pZalphaM or a pZEM control into Y1 cells. We demonstrate that inhibition of DNA MeTase activity causes demethylation of Y1 DNA and results in reversal of the tumorigenic phenotype suggesting that DNA MeTase plays a critical role in tumorigenesis.


MATERIALS AND METHODS

Cell Culture and DNA-mediated Gene Transfer

Y1 cells were maintained as monolayers in F-10 medium which was supplemented with 7.25% heat-inactivated horse serum and 2.5% heat-inactivated fetal calf serum (Immunocorp, Montreal)(23) . 5-azaCdR was from Sigma, all other media and reagents for cell culture were obtained from Life Technologies, Inc. Y1 cells (1 times 10^6) were plated on a 150-mm dish (Nunc) 15 h before transfection. The pZalphaM expression vector (7) encoding the 5` of the murine DNA methyltransferase cDNA (10 µg) was co-introduced into Y1 cells with 1 µg of pUCSVneo as a selectable marker by DNA-mediated gene transfer using the calcium phosphate protocol, and G418-resistant cells were cloned in selective medium (0.25 mg/ml G418)(26) . Anchorage-independent growth in soft agar (1 times 10^3 cells) was as described previously(27) .

DNA and RNA Analyses

Genomic DNA was prepared from pelleted nuclei, and total cellular RNA was prepared from cytosolic fractions according to standard protocols(26) . MspI or HpaII restriction enzymes (Boehringer Mannheim) were added to DNA at a concentration of 2.5 units/µg for 8 h at 37 °C. At least three different DNA preparations isolated from three independent passages were assayed per transfectant. To quantify the relative abundance of DNA MeTase mRNA, total RNA (3 µg) was blotted onto Hybond N using the Bio-Rad slot-blot apparatus. The filter-bound RNA was hybridized to a P-labeled 1.31-kb cDNA probe encoding the putative catalytic domain of the mouse DNA MeTase(3170-4480) (28) and exposed to XAR film (Kodak). The relative amount of total RNA was determined by measuring the signal obtained after hybridization to an 18 S RNA-specific P-labeled oligonucleotide(29) . The autoradiograms were scanned with a Scanalytics scanner (one-dimensional analysis), and the signal at each band was determined and normalized to the amount of total RNA at the same point. Three determinations were performed per RNA sample.

Nearest Neighbor Analysis

Two µg of DNA were incubated at 37 °C for 15 min with 0.1 unit of DNase, 2.5 µl of [alpha-P]dGTP (3000 Ci/mmol from Amersham), 2 Kornberg units of DNA polymerase (Boehringer) were then added, and the reaction was incubated for an additional 25 min at 30 °C. Fifty µl of water were then added to the reaction mixture, and the nonincorporated nucleotides were removed by spinning through a Microspin S-300 HR column (Pharmacia). The labeled DNA (20 µl) was digested with 70 µg of micrococcal nuclease (Pharmacia) in the manufacturer's recommended buffer for 10 h at 37 °C. Equal amounts of radioactivity were loaded on TLC phosphocellulose plates (Merck), and the 3` mononucleotides were separated by chromatography in one dimension (isobutyric acid:H(2)O:NH(4)OH in the ratio 66:33:1). The chromatograms were exposed to XAR film (Eastman-Kodak), and the autoradiograms were scanned by scanning laser densitometry (Scanalytics one-dimensional analysis). Spots corresponding to cytosine and 5-methylcytosine were quantified.

Assay of DNA MeTase Activity

To determine nuclear DNA MeTase levels, cells were maintained at a nonconfluent state and fed with fresh medium every 24 h for at least 3 days prior to harvesting, and DNA MeTase activity was assayed as described previously(9) .

Strand-specific Reverse-transcribed PCR

Total RNA (1 µg) prepared from each transfectant was reverse-transcribed with either a sense primer corresponding to bases 1-30 in the published mouse DNA MeTase cDNA sequence(28) , 5` GCAAACAGAAATAAAAAGCCAGTTGTGTGA 3` to detect antisense RNA or an antisense primer corresponding to bases 475-451, or 5` CCACAGCAGCTGCAGCACCACTCT 3` to detect sense DNA MeTase RNA using the conditions described above. RNA incubated with reverse transcriptase in the absence of primers was used as a control. The reaction was terminated by heating to 95 °C for 10 min. The reverse-transcribed cDNA was subjected to amplification in the presence of both primers using the Hot Tub amplification protocol conditions described above. The DNA was amplified for 40 cycles of 2 min at 95 °C, 2 min at 60 °C, and 0.5 min at 72 °C. The reaction products were separated on an agarose gel, Southern blotted onto Hybond N filter, and hybridized with a P-labeled internal oligonucleotide corresponding to bases 190-211: 5` AAATGGCAGACTCAAATAGAT 3`. The conditions used (40 cycles of amplification) do not provide a quantitative assessment of the level of mRNA, but were used to exclude the possibility that small levels of antisense mRNA is present in the control transfectants.

Tumorigenicity Assays

LAF-1 mice (Bar Harbor) (6-8-week-old males) were injected subcutaneously (in the flank area) with 10^6 cells. Mice were monitored for the presence of tumors by daily palpation. Mice bearing tumors of greater than 1 cm in diameter were sacrificed, while tumor-free mice were kept for 90 days.

Electron Microscopy

Cells were fixed in glutaraldehyde (2.5%) in cacodylate buffer (0.1 M) for 1 h and further fixed in 1% osmium tetroxide. The samples were dehydrated in ascending alcohol concentrations and propylene oxide followed by embedding in Epon. Semithin sections (1 µm) were cut from blocks with an ultramicrotome and counterstained with uranyl acetate and lead citrate. Samples were analyzed using a Philips 410 electron microscope(30) .


RESULTS

Expression of Antisense to the DNA Methyltransferase mRNA in Y1 Cells Results in Limited Inhibition of DNA Methylation

To directly inhibit DNA methylation in Y1 cells, we introduced either the DNA MeTase antisense expression construct pZalphaM (encoding 600 bp from the 5` of the DNA MeTase cDNA in the antisense orientation) or a pZEM control vector (7) into Y1 cells by DNA-mediated gene transfer as described under ``Materials and Methods.'' Six G418-resistant colonies were isolated and propagated for both constructs. All antisense transfectants (determined by a preliminary Southern blot analysis) exhibited distinct morphological differences from the pZEM transfectants or nontransfected Y1 cells. Based on Northern blot analysis of the antisense mRNA expression, three independent pZalphaM transfectants (4, 7, 9) were selected for further characterization.

To verify that the antisense mRNA strand is transcribed in the pZalphaM transfectants, we employed reverse-transcribed PCR analysis using strand-specific primers as described under ``Materials and Methods.'' The experiment presented in Fig. 1demonstrates that when the sense oligonucleotide is used for reverse transcription (transcribing the antisense mRNA strand), the expected 0.475- kb amplification product is observed only in pZalphaM transfectants. As expected, the endogenous DNA MeTase sense mRNA is amplified in both pZEM and pZalphaM transfectants when the antisense oligonucleotide is used for reverse transcription (sense). Interestingly, a smaller amplification product (0.375 kb) is also seen in the pZalphaM lines suggesting that another splice variant of DNA MeTase mRNA is transcribed in these transfectants. The biological significance of the induction of the smaller variant in the pZalphaM transfectants is unclear.


Figure 1: Expression of pZalphaM in Y1 adrenocortical cells. To determine the strand specificity of the RNA transcribed by the pZalphaM vector, we employed a reverse transcriptase-PCR analysis using a sense-specific primer (lanes labeled SENSE), an antisense-specific primer (lanes labeled alpha-SENSE), or no primers (lanes labeled NO RT). Total RNA (1 µg) was reverse-transcribed with either a sense-specific primer, an alpha-sense-specific primer, or no primers. Resulting cDNA was then subjected to PCR with the complementary oligonucleotide (sense or alpha-sense) as described under ``Materials and Methods,'' one-tenth of the PCR reaction was Southern-blotted and hybridized with a P-labeled oligonucleotide encoding a sequence included in the amplified mRNA region (see ``Materials and Methods'' for description of the sequences of the primers). Sense DNA MeTase (SENSE) is observed in both pZalphaM and control pZEM transfectants as expected (475-bp product). An antisense transcript is seen only in pZalphaM transfectants. An unexpected additional sense amplification product of 375 bp is seen in all the pZalphaM transfectants.



The mechanism responsible for inhibition of gene expression by antisense is still unclear; however, some models suggest that degradation of the hybrid RNA by RNase H might be involved. To determine whether expression of an antisense to the DNA MeTase can lead to a reduction in the steady state level of endogenous DNA MeTase mRNA, we isolated total RNA from the antisense transfectants and the pZEM controls. DNA MeTase activity is regulated with the state of growth of cells(8, 9) ; therefore, all cultures were maintained at the logarithmic phase of growth and fed with fresh medium every 24 h for 3 days prior to harvesting. Total RNA isolated from the transfectants was subjected to a Northern blot analysis and sequentially hybridized with a probe to the putative catalytic domain of the mouse DNA MeTase mRNA (MET 3`) and an 18 S rRNA-specific P-labeled oligonucleotide probe as described under ``Materials and Methods.'' A result of such an analysis is presented in Fig. 2. Scanning of the autoradiogram indicates that the relative abundance of the 5-kb DNA MeTase mRNA (Fig. 2, top panel) relative to 18 S rRNA (Fig. 2, bottom panel) is reduced 2-fold in the three antisense transfectants. To quantify expression of DNA MeTase, the different RNA samples were subjected to a slot-blot analysis and sequential hybridization with the DNA MeTase and 18 S rRNA probes. The relative level of DNA MeTase mRNA in the different samples was determined by scanning densitometry. The results of such an analysis show that the pZalphaM transfectants exhibit an average decrease of 45% and a maximal decrease of 58% in the abundance of DNA MeTase mRNA relative to the pZEM controls (p < 0.001). The mean value for the control group was 0.480, S.D. = 0.104, the mean for the antisense group was 0.280, S.D. = 0.066.


Figure 2: DNA MeTase expression, activity, and genomic methylation levels of pZalphaM transfectants. Total cellular RNA (10 µg) prepared from pZalphaM lines (4, 6, 7, and 9), pZEM transfectants (1 and 7) and from Y1 controls was subjected to Northern blot analysis and hybridization with a 1.3-kb DNA MeTase 3`-cDNA probe (encoding bases 3170-4480 from the cloned mouse cDNA(27) ). The filter was stripped and rehybridized with an 18 S rRNA probe. Relative MeTase expression was determined by densitometric analysis (see text).



We next compared the DNA MeTase enzymatic activity present in nuclear extracts prepared from antisense transfectants relative to control pZEM transfectants using S-[methyl-^3H]adenosyl-L-methionine as the methyl donor and a hemimethylated double-stranded oligonucleotide as a substrate. The results of two experiments with triplicate determinations each indicate that the three pZalphaM transfectants express a lower level of DNA MeTase activity than the control transfectants with an average inhibition of DNA MeTase activity of 42% and a maximum of 48% relative to control (p < 0.05).

Whereas our experiments demonstrate that the DNA MeTase antisense transfectants bear a lower level of DNA MeTase activity than the control transfectants, it is important to note that we measured only steady state levels in the transfectants. It is hard to assess the actual level of inhibition of DNA MeTase activity at the time of transfection, when a higher copy number of DNA MeTase antisense RNA might have been present in the cell. The steady state level of DNA MeTase mRNA might reflect an equilibrium of different cellular regulatory controls over the level of DNA MeTase activity in the cell. To directly demonstrate that expression of the DNA MeTase antisense leads to inhibition of DNA methylation activity in the cell, we determined whether it leads to a general reduction in the level of methylation of the genome. We performed a ``nearest neighbor'' analysis using [alpha-P]dGTP as described previously(6) . This assay enables one to determine the percentage of methylated and nonmethylated cytosines residing in the dinucleotide sequence CpG(6) . The results of three such experiments show that the mean value for the pZEM controls as a group was 9.7% nonmethylated cytosines, S.D. = 2.13, the mean value for the antisense lines as a group was 23.83% cytosine, S.D. = 5.88. p < 0.001.

In summary, our experiments demonstrate that expression of an antisense to the DNA MeTase mRNA leads to partial inhibition of DNA MeTase mRNA and DNA MeTase enzymatic activities and a significant reduction in the level of genomic cytosine methylation.

Demethylation of Specific Genes in Y1 and pZalphaM Transfectants

To further verify that expression of pZalphaM results in demethylation and to determine whether specific genes were demethylated, we resorted to a HpaII/MspI restriction enzyme analysis followed by Southern blotting and hybridization with specific gene probes. HpaII cleaves the sequence CCGG, a subset of the CpG dinucleotide sequences, only when the site is unmethylated, while MspI will cleave the same sequence irrespective of its state of methylation. By comparing the pattern of HpaII cleavage of specific genes in cells expressing pZalphaM with that of the parental Y1 or cells harboring only the vector, we determined whether the genes are demethylated in the antisense transfectants. We first analyzed the state of methylation of the steroid 21-hydroxylase gene (C21)(29, 31) . This gene is specifically expressed and hypomethylated in the adrenal cortex, but is inactivated and hypermethylated in Y1 cells(29, 31) . We have previously suggested that hypermethylation of C21 in the Y1 cell is part of the transformation program that includes the shutdown of certain differentiated functions(29) . DNA prepared from Y1, pZalphaM(4, 7, 9) , and pZEM (1 and 7) transfectants was subjected to either MspI or HpaII digestion, Southern blot analysis, and hybridization with a 3.8-kb BamHI fragment containing the body of the C21 gene and 3` sequences (Fig. 3, bottom panel, for physical map). Full demethylation of this region should yield a doublet at 1 kb, an 0.8-kb fragment, and a 0.4-kb fragment, as well as a number of low molecular weight fragments at 0.1-0.4 kb. As observed in Fig. 3, the C21 locus is heavily methylated in Y1 cells, as well as the control transfectant, as indicated by the high molecular weight fragments. Only a relatively weak digestion product is seen at 1.9 kb (Fig. 3). This pattern of hypermethylation of C21 which is observed in Y1 cells and different control transfectants, that were analyzed in our laboratory in the last 5 years, is markedly stable. On the other hand, the antisense transfectant's DNA is significantly hypomethylated at this locus as indicated by the relative diminution of the high molecular weight fragments and relative intensification of the partial fragment at 1.9 kb. The appearance of new partial fragments in the lower molecular weight range between 1 and 0.4 kb indicates partial hypomethylation at a large number of HpaII sites contained in the 3` region of the C21 gene (see physical map)(29, 31) . The pattern of demethylation, indicated by the large number of partial HpaII fragments, is compatible with a general partial hypomethylation rather than a specific loss of methylation in a distinct region of the C21 gene.


Figure 3: The pattern of methylation of C21 hydroxylase in pZalphaM transfectants and pZEM controls. Genomic DNA (10 µg) was extracted from the transfected lines and subjected to digestion with either MspI (M) or HpaII (H), Southern blot transfer, and hybridization with a P-labeled DNA probe (3.8-kb genomic fragment of the C21 gene, see bottom panel for physical map). The open arrows indicate HpaII fragments resulting from demethylation of the different sites in the C21 gene in pZalphaM transfectants. Complete digestion of the region will yield 0.36- and 0.16-kb fragments.



To determine whether demethylation is limited to genes that are potentially expressible in Y1 cells such as the adrenal cortex-specific C21 gene (29) or if the demethylation is widely spread in the genome, we tested the methylation state of the MyoD (32) and p53 5` locus. Specific demethylation of MyoD and the p53 fragment was seen in the pZalphaM transfectants (data not shown).

Morphological Transformation Loss of Anchorage-independent Growth and Inhibition of Tumorigenicity of Y1 Cells Expressing Antisense to the DNA MeTase

As the level of DNA MeTase activity is regulated with the state of growth and is induced in transformed cells and in tumors in vivo(8, 9, 13, 19) , we determined whether expression of the DNA MeTase antisense construct results in a change in the tumorigenic potential of Y1 cells. A comparison of pZalphaM transfectants and controls showed a small but statistically significant reduction in the growth rate of antisense lines relative to the Y1 controls especially at higher densities (which is statistically significant, p < 0.001). This may reflect contact-inhibited growth and increased serum requirements of the antisense lines (data not shown). The morphological properties of the pZalphaM transfectants further support this conclusion (Fig. 4). While control Y1 and pZEM cells exhibit limited contact inhibition and form multilayer foci, pZalphaM transfectants exhibit a more rounded and distinct morphology and grow exclusively in monolayers, and, in many cases, pZalphaM cells form distinct cellular processes (Fig. 4).


Figure 4: Morphological transformation of Y1 cells transfected with pZalphaM. Phase contrast microscopy at times 200 magnification of living cultures of Y1 clonal transfectants with pZalphaM and pZEM controls. Equal numbers of cells were plated (1 times 10^5 cells per well in a six-well dish), and pictures were taken 72 h after seeding.



The ability of cells to grow in an anchorage-independent fashion is considered to be an indicator of tumorigenicity(27) . A soft agar assay performed in triplicate showed that the pZalphaM transfectants demonstrate a significant decrease in their ability to form colonies in soft agar: pZEM 1 and 7 form an average of 38 and 37 colonies, respectively, while pZalphaM transfectants 4, 7, and 9 formed an average of 12, 15, and 18 colonies, respectively. Moreover, the colonies that do form are significantly smaller and contain fewer cells.

Another indicator of the state of transformation of a cell is its serum dependence. Tumor cells exhibit limited dependence on serum and are usually capable of serum-independent growth(33) . Factors present in the serum are essential for the survival of many nontumorigenic cells. As observation of the pZalphaM transfectants indicated that they expressed enhanced dependence on serum and limited survivability under serum-deprived conditions, we determined whether this limited survivability involved an enhancement or induction of an apoptotic program. While the control cells exhibited almost 100% viability up to 72 h after transfer into serum-deprived medium, all pZalphaM transfectants showed up to 75% loss of viability at 48 h.

To test whether the serum-deprived pZalphaM cells were dying as a result of an activated apoptotic death program, cells were plated in starvation medium and harvested at 24-h intervals, and total cellular DNA was isolated from the cells and analyzed by agarose gel electrophoresis. After 48 h in serum-starved conditions, pZalphaM transfectants exhibit the characteristic 180-bp internucleosomal DNA ladder while the control pZEM transfectants show no apoptosis at this time point (Fig. 5A).


Figure 5: Survival and apoptosis of pZEM transfectants in serum-deprived medium. A, the indicated transfectants were plated in 1% serum-containing medium and harvested after 1 and 2 days. Total cellular DNA was isolated, separated by agarose gel electrophoresis, transferred to nitrocellulose membrane, and probed with P-labeled Y1 genomic DNA. A 180-bp internucleosomal ladder characteristic to cells dying via apoptosis can be seen in the pZalphaM transfectants only. B, Y1 transfectants were grown in 1% serum medium for 24 h, fixed, and analyzed by electron microscopy for early signs of apoptotic death; I-III are various sections (the magnification is indicated) of Y1 pZalphaM transfectants and pZEM control lines.



To determine whether cells expressing antisense to the DNA MeTase exhibit early morphological markers of apoptosis, cells were serum-starved for 24 h, harvested, and analyzed by electron microscopy. Fig. 5B shows representative electron micrographs of several blocks of control pZEM and pZalphaM transfectants at various magnifications (I-III). The control cells have a fine uniform nuclear membrane whereas the pZalphaM cells exhibit the cardinal markers of apoptosis(34) : condensation of chromatin and its margination at the nuclear periphery (panels I and II), chromatin condensation (panel II), nuclear fragmentation (panel III), formation of apoptotic bodies, and cellular fragmentation. Whereas it is still unclear whether apoptosis upon serum deprivation is directly enhanced by demethylation or is an indirect effect of the change in the transformed state of the transfectants, the serum deprivation-induced cell death is another indicator of the reversal of cellular transformation by DNA MeTase antisense.

To determine whether demethylation can result in inhibition of tumorigenesis in vivo, we injected 1 times 10^6 cells for each of the Y1, pZEM, and pZalphaM (4, 7, and 9) transfectants subcutaneously into the syngeneic mouse strain LAF-1. The presence of tumors was determined by palpation. While all the animals injected with Y1 or pZEM cells formed tumors, animals injected with the pZalphaM transfectants had very few tumors arise (Fig. 6A; p > 0.005).


Figure 6: In vivo tumorigenicity of pZalphaM transfectants. A, parental Y1 cells, a pZEM control line, and three pZalphaM transfectants (4, 7, and 9) were tested for their ability to form tumors in syngeneic LAF-1 mice. Tumor formation was assessed by palpation for 2 months after injection. The number of mice forming tumors is tabulated. The statistical significance of the difference between the control and antisense transfectants was determined using a test; p > 0.001. * indicates that these tumors were negative for pZalphaM expression. B, loss of antisense DNA MeTase expression in tumors derived from antisense transfectants. RNA (10 µg) isolated from the indicated tumors was subjected to Northern blot analysis and hybridization with the 0.6-kb MET cDNA probe. Expression of the 1.3-kb antisense message is seen only in the original cell lines pZalphaM (4, 7, and 9) and is undetectable in tumors arising from pZalphaM transfectants or Y1 cell lines even after long exposure. The filter was stripped of radioactivity and rehybridized with a P-labeled oligonucleotide corresponding to 18 S rRNA(28) .



One possible explanation for the fact that a small number of tumors did form in animals injected with the pZalphaM transfectants is that they are derived from revertants that lost expression of the antisense to the DNA MeTase under the selective pressure in vivo. RNA was isolated from tumors arising from the pZalphaM transfectants, and the level of expression of the 0.6-kb antisense message was compared with the transfectant lines in vitro (Fig. 6B). The expression of the antisense message is virtually nonexistent in the tumors derived from pZalphaM transfectants even after long exposure of the Northern blots, supporting the hypothesis that expression of an antisense message to the DNA MeTase is incompatible with tumor growth in vivo.

DNA Demethylation Induced by 5-AzaCdR Results in Reversal of Cellular Transformation ex Vivo

To further verify that inhibition of DNA methylation results in reversal of cellular transformation and to exclude the possibility that the effects observed are nonspecific results of antisense expression we used an inhibitor of DNA methylation 5-azadeoxycytidine (5-azaCdR) that acts at a different site than antisense RNA(35) . 5-azaCdR is a deoxycytidine analogue that inhibits DNA methylation once it is incorporated into DNA. It has been suggested that an irreversible complex is formed between the DNA MeTase enzyme and the C-6 position of the cytosine moiety(36) . We treated Y1 cells with concentrations of 5-azaCdR ranging from 0 to 10 µM every 12 h for 72 h. 5-azaCdR increases the proportion of cytosine to methylcytosine in the DNA by 1.6-fold in a dose-dependent manner (0-5.0 µM) as determined by a nearest neighbor analysis. Over the same concentration range, cell viability in low serum is reduced from 80% to 40%, and the ability of cells to form colonies in soft agar is reduced by 50-fold. No differences were seen in the ability of cells to form colonies on regular plastic dishes. The 5-azaCdR-treated cells exhibited dose-dependent morphological changes similar to those observed in the pZalphaM transfectants (Fig. 7). This experiment suggests that 5-azaCdR treatment reversed the transformed phenotype of Y1 cells but did not affect their viability.


Figure 7: Morphological change in Y1 cells treated with 5-azaCdR. Y1 cells were treated with concentrations of 5-azaCdR ranging from 0-10 µM every 12 h for 72 h. Phase contrast microscopy at times 200 magnification of living cultures of the treated cells is presented.




DISCUSSION

This paper tests the hypothesis that overexpression of the DNA MeTase plays a causal role in cellular transformation by expressing an antisense message to the DNA MeTase in an adrenocortical carcinoma cell line. Expression of an antisense DNA MeTase (Fig. 1) leads to: (i) a limited reduction in DNA MeTase steady state mRNA and protein levels (Fig. 2), (ii) a general but limited reduction in the methylation content of the genome (Fig. 2), (iii) demethylation of regions aberrantly methylated in this cell line such as the adrenal specific 21-hydroxylase gene (Fig. 3), (iv) morphological changes indicative of inhibition of the transformed phenotype, (v) inhibition of anchorage-independent growth as determined by soft agar assays, (vi) inhibition of serum-independent survivability and induction of apoptosis under serum-deprived conditions, as well as (vii) inhibition of tumorigenesis in syngeneic mice (Fig. 6) and (viii) inhibition of DNA methylation by 5-azaCdR, which acts at a site completely different from antisense to the DNA MeTase, also results in reversal of transformation indicators ex vivo. The fact that a 2-fold inhibition in DNA MeTase expression is sufficient to induce such profound changes in the state of transformation of Y1 cells is in accordance with previously published data showing that a 2-3-fold elevation in DNA MeTase activity by forced expression of an exogenous DNA MeTase in NIH 3T3 can induce cellular transformation of these cells (23) . Whereas antisense expression is considered one of the most direct means to inhibit gene expression, no experimental method is devoid of potential complications. 5-azaCdR, which is the most commonly used DNA methylation inhibitor, has side effects(36, 37) . However, the fact that both inhibitors had similar effects strongly validates our conclusions. The fact that 5-azaCdR inhibited transformation indicators but not the survival of the cells and their ability to form colonies, and the fact that the reversal of transformed phenotype was expressed weeks after the inhibitor had been removed is consistent with the model that 5-azaCdR triggered a change in the cellular program rather than a cytotoxic or cytostatic effect. It stands to reason that this change in program was triggered by the initial demethylation event caused by the drug.

Our experiments support a previously proposed hypothesis that overexpression of DNA MeTase is an important component of an oncogenic pathway(s)(22) . Since Y1 is a line derived from a naturally occurring tumor (24) which bears amplified copies of Ras(25) , it is possible that hyperactivation of the DNA MeTase is triggered by the Ras-Jun signaling pathway(21, 22) . The DNA MeTase promoter bears a number of AP-1 sites(20) , and we demonstrated that the activity of the DNA MeTase promoter is dependent on binding of AP-1 (21) and that down-regulation of the the Ras-Jun pathway in Y1 cells results in inhibition of DNA MeTase activity, hypomethylation, and reversal of the transformed phenotype. (^2)Our data might explain previous observations demonstrating an increase in DNA MeTase activity (13, 19) in cancer cells by suggesting that this increase is critical for the transformed state.

What is the possible mechanism by which hypermethylation can cause cellular transformation? The answer to this question is still elusive and could not be resolved by the data presented in this paper; however, several hypotheses have been previously suggested. One plausible explanation that has been previously suggested by Baylin and his colleagues (38) is that methylation may establish abnormalities of chromatin organization which in turn mediate the progressive losses of gene expression associated with tumor development. One interesting class of genes that might be affected are the tumor suppressor genes. There is evidence that ectopic inactivation of tumor suppressor genes by methylation contributes to cancer(39, 40) . The promoter region of the RB-1 gene was found to be methylated in 6 of 77 retinoblastomas (17) , and the 5` region of the WT-1 gene was methylated in 2 out of 29 Wilms tumors(40) , while the gene methylated was otherwise grossly normal. However, there is no evidence that tumor suppressor genes are the critical targets for hypermethylation in cancer cells or that tumor suppressor genes are selectively demethylated in the DNA MeTase antisense transfectants. Also, our unpublished data do not suggest any induction in the level of expression of these genes in the pZalphaM transfectants.

Another interesting mechanism that has been suggested by Jones and his colleagues is that methylated CpGs are hot spots for mutations by deamination of the methylated cytosine into thymidine(41) . This kind of change induced by methylation will not be reversible, the fact that we could reverse transformation by inhibiting DNA MeTase suggests that other mechanisms must be involved. While inhibition of gene expression by methylation is the best analyzed function of DNA methylation, one should bear in mind that any function of the genome might be modified by methylation. Sites that are especially sensitive to changes in methylation might be controlling DNA functions such as repair, replication, and susceptibility to death program-related endonucleases.

One question that remains to be answered is how to explain the contradiction between the fact that DNA MeTase is overexpressed in cancer cells and the observed regional hypomethylation of the genome of many cancer cells(11, 12) . However, there are no data at this stage to resolve this apparent contradiction. While additional experiments will be required to address these questions, this paper demonstrates that inhibition of DNA methylation leads to a reversal of the transformed state and that DNA methylation plays a critical role in cellular transformation.


FOOTNOTES

*
This paper was supported in part by a grant from the Cancer Research Society and the NCI (Canada). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Scientist of the National Cancer Institute (Canada). To whom correspondence and reprint requests should be addressed. Tel.: 514-398-7107; Fax: 514-398-6690; mcms{at}musica.mcgill.ca.

(^1)
The abbreviations used are: MeTase, methyltransferase; 5-azaCdR, 5-azadeoxycytidine; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.

(^2)
A. R. MacLeod, J. Roleau, and M. Szyf, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Gary Tanigawa, Marc Pinard, and Shyam Ramchandani for critical reading of the manuscript and stimulating discussions, as well as Vera Bozovic for excellent technical assistance, Allan Forester for help with the photography, and Marie Ballak for her contribution to the electron microscopy analysis.


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