(Received for publication, October 10, 1994; and in revised form, January 24, 1995)
From the
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.
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 ()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 (pZM) 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 pZ
M 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.
To verify that the antisense mRNA strand is
transcribed in the pZM 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 pZ
M transfectants. As expected, the endogenous
DNA MeTase sense mRNA is amplified in both pZEM and pZ
M
transfectants when the antisense oligonucleotide is used for reverse
transcription (sense). Interestingly, a smaller amplification product
(0.375 kb) is also seen in the pZ
M 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 pZ
M transfectants is unclear.
Figure 1:
Expression of
pZM in Y1 adrenocortical cells. To determine the strand
specificity of the RNA transcribed by the pZ
M vector, we employed
a reverse transcriptase-PCR analysis using a sense-specific primer (lanes labeled SENSE), an antisense-specific primer (lanes
labeled
-SENSE), or no primers (lanes labeled NO
RT). Total RNA (1 µg) was reverse-transcribed with either a
sense-specific primer, an
-sense-specific primer, or no primers.
Resulting cDNA was then subjected to PCR with the complementary
oligonucleotide (sense or
-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 pZ
M and control pZEM transfectants as expected
(475-bp product). An antisense transcript is seen only in pZ
M
transfectants. An unexpected additional sense amplification product of
375 bp is seen in all the pZ
M
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 pZ
M 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 pZM transfectants. Total cellular
RNA (10 µg) prepared from pZ
M 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-H]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 pZ
M
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 [-
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.
Figure 3:
The pattern of methylation of C21
hydroxylase in pZM 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 pZ
M 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
pZM transfectants (data not shown).
Figure 4:
Morphological transformation of Y1 cells
transfected with pZM. Phase contrast microscopy at
200
magnification of living cultures of Y1 clonal transfectants with
pZ
M and pZEM controls. Equal numbers of cells were plated (1
10
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 pZM
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 pZ
M 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 pZM 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
pZ
M transfectants showed up to 75% loss of viability at 48 h.
To test whether the serum-deprived pZM 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, pZ
M
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 pZ
M 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 pZ
M 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 pZM transfectants at various magnifications (I-III). The control cells have a fine uniform nuclear
membrane whereas the pZ
M 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 10
cells for each of the Y1, pZEM, and pZ
M (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
pZ
M transfectants had very few tumors arise (Fig. 6A; p > 0.005).
Figure 6:
In vivo tumorigenicity of
pZM transfectants. A, parental Y1 cells, a pZEM control
line, and three pZ
M 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 pZ
M
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 pZ
M (4, 7, and 9) and is undetectable in tumors
arising from pZ
M 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 pZM 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 pZ
M 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 pZ
M 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.
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 200 magnification of living cultures of
the treated cells is presented.
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. ()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 pZM 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.