Mechanisms of Ageing and Development
101 (1998) 153 – 166

Epigenetic control of programmed cell death:
inhibition by 5-azacytidine of
1,25-dihydroxyvitamin D3-induced programmed
cell death in C6.9 glioma cells
C. Canova *, G. Chevalier, S. Remy, P. Brachet, D. Wion
INSERM U 298, CHU Angers, F-49033 Angers Cedex, France
Received 29 May 1997; accepted 19 November 1997

Abstract
In mammalian DNA cytosine methylation occurs speciﬁcally at CpG dinucleotide. Although the full array of function of DNA methylation is yet to be elucidated, it is well
established that DNA methylation is an important mechanism involved in gene expression,
DNA replication and cancer. Rat glioma C6.9 cells undergo programmed cell death (PCD)
after treatment with 1,25-dihydroxyvitamin D3 (1,25-D3). Hence, these cells were used to
study whether DNA methylation was involved in the control of PCD. We found that
1,25-D3-mediated PCD of C6.9 cells was suppressed by exposure of the cells to the DNA
demethylating agents 5-azacytidine (5-AzaC) and 5-aza-2%-deoxycytidine. This effect remains
detectable several cell divisions following removal of 5-AzaC and, therefore, involves DNA
methylation as an epigenetic regulatory mechanism of PCD. Accordingly, internucleosomal
fragmentation, a feature of apoptosis that is detected in 1,25-D3-treated cells, is no longer
observable after treatment of these cells with 5-AzaC. However, 5-AzaC does not totally
suppress the responsiveness of C6.9 cells to 1,25-D3 since the induction of the c-myc gene
remains unaffected. These results suggest that a change in DNA methylation pattern could
suppress 1,25-D3-mediated PCD through the expression of previously hypermethylated genes
such as proto-oncogenes with death-repressor activity, endogenous virus sequences or even
genes inducing change in the differentiated state of these cells. © 1998 Elsevier Science
Ireland Ltd.
Keywords: Apoptosis; Glioma; Methylation; Programmed cell death; Vitamin D
* Corresponding author. Tel.: +33 241 731070; fax: + 33 241 731630.
0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 1 7 2 - 3

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1. Introduction
Programmed cell death (PCD) is now widely recognized as a mechanism for
selective elimination of cells during ageing and development. This naturally occurring form of cell death is involved in many fundamental biological mechanisms,
including metamorphosis, morphogenesis, hormone-induced tissue remodelling and
tissue homeostasis (Kerr et al., 1972; Tenniswood et al., 1992; Wyllie, 1992).
Accumulating data have demonstrated that PCD is a gene-directed process. Depending on the experimental systems used, the involvement of several genes such as
c-myc (Evan et al., 1992), interleukin 1i converting enzyme (Miura et al., 1993),
nur77 (Liu et al., 1994) or p53 (Clarke et al., 1993; Lowe et al., 1993) in PCD have
been reported. PCD is also involved in the elimination of transformed or virus-infected cells (Williams, 1994; Vaux et al., 1994; Shen and Shenk, 1995). This process
has then been interpretated as a protective mechanism of multicellular organisms to
cellular transformation and viral infection. However, many cancer cells and viruses
encode proteins with death-repressor activity that counteract this host apoptotic
response. For instance, these include the cellular bcl-2 proto-oncogene (Vaux et al.,
1988; Hockenbery et al., 1990) and adenovirus protein E1B-19K (Rao et al., 1992),
Epstein – Barr virus protein BHFR1 (Henderson et al., 1993), baculovirus gene
product p35 (Hershberger et al., 1994), cowpox protein CrmA (Gagliardini et al.,
1994), and herpesvirus 8 protein KSbcl-2 (Cheng et al., 1997).
In mammalian DNA 2 – 7% of cytosine residues are present as 5-methylcytosine.
Patterns of methylation, which frequently occur in the dinucleotide 5%-CG-3%, are
both cell-type speciﬁc and highly conserved. This epigenetic modiﬁcation of eukaryotic DNA has been proposed to be involved in repair of DNA, maintenance of
chromosome structure, mutagenesis, genomic imprinting and cellular ageing (Holliday and Pugh, 1975; Sager and Kitchen, 1975; Jones and Taylor, 1980; Razin and
Riggs, 1980; Ehrlich and Wang, 1981; Catania and Fairweather, 1991; Naveilhan et
al., 1994a). In addition, considerable evidence has accumulated over the past 20
years suggesting a causal relationship between hypermethylation and gene inactivity. Thus, expression of housekeeping genes can be correlated to their hypomethylated state, while hypermethylation has been shown to restrict the transcription of
several genes in every tissue other than those where they are normally expressed
(Holliday and Pugh, 1975; Sager and Kitchen, 1975; Jones and Taylor, 1980; Razin
and Riggs, 1980; Ehrlich and Wang, 1981). Similarly, several viral DNA sequences
were found to be extensively methylated in latently infected cells, whereas corresponding virus-producing cells contain unmethylated viral DNA (Desrosiers et al.,
1979; Sutter and Doerﬂer, 1980; Groudine et al., 1981; Harbers et al., 1981;
Jaenisch et al., 1985; Bednarik et al., 1990). Inaccurancy in the regulation of DNA
methylation has also been suggested to be involved in tumour promotion by
facilitating aberrant gene expression (Counts and Goodman, 1995a,b; Huang et al.,
1997).
C6.9 cells are rat glioma cells which have been reported to induce a cell death
programme following exposure to 1,25-dihydroxyvitamin D3 (1,25-D3) (Baudet et
al., 1996a). These cells, after treatment for 24 h with 1,25-D3, keep an apparently

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normal morphology and still divide during the following 3 days. However, they
suddenly start to round up and die around day 5 (Naveilhan et al., 1994b). This
process is accompanied by the induction of c-myc gene and by DNA fragmentation
(Baudet et al., 1996a,b), two features that occur during the course of PCD. Thus,
in addition to its potential for the treatment of brain glial tumors and in the
understanding of the mechanisms by which 1,25-D3 can mediate its antitumoral
effects (DeLuca and Ostrem, 1986; Reichel et al., 1989; Pols et al., 1990), these
results suggest that C6.9 cells provide a useful in vitro experimental model to study
hormone-induced PCD. To further elucidate the molecular mechanisms involved in
this process we investigated the relationship between DNA hypomethylation and
PCD.

2. Material and methods

2.1. Cell culture
The cell line used in this study, the clone C6.9 (Baudet et al., 1996b), was isolated
from the rat glioma C6 cell line (Benda et al., 1968). These cells were isolated by the
method of limiting dilution in microtitre plates. All experiments described were
performed between passages 5 and 20. Cells were grown in Ham’s F12 medium
containing 10% fetal calf serum. For the experiments, cells were plated at a density
of 2 – 4×103 cells/cm2 and incubated for 24 h in a serum-free medium consisting of
F12 medium supplemented with insulin (2.5 vg/ml), transferrin (2.5 vg/ml) and
sodium selenite (2.5 ng/ml). All media were supplemented with penicillin (100
U/ml), streptomycin (100 vg/ml) and amphotericin B (0.25 vg/ml). 1,25-D3 (10 − 7
M) or vehicle (ethanol) were added to cell cultures for 24 h. At the end of this
incubation time, cells were cultured in serum-free medium devoid of 1,25-D3 during
24 h. For experiments with 5-azacytidine (5-AzaC), the drug was introduced into
the medium at day 2, and after 48 h (day 4) the medium was removed, the cells
were washed twice and replaced in serum-free medium. Alternatively, C6.9 cells
were treated during 5 days with 5-AzaC (3 mM) in a F12 medium supplemented
with 10% fetal calf serum. Subsequently, they were washed twice and cultured for
2 days in F12 medium with fetal calf serum. After incubation in serum-free medium
for 24 h, these cells were treated with 1,25-D3 for 24 h. Thereafter, the medium was
removed and cells were incubated in serum-free medium which was changed after
3 and 5 days.

2.2. MTT cell 6iability assay
The MTT assay is based on the capacity of mitochondrial dehydrogenases of
viable cells to convert the water-soluble tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to water-insoluble purple formazan
product. MTT stock solution (5 mg/ml) was added to the cultures and incubated
for 4 h. Then, the formazan product was solubilized with acidic isopropanol, and

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absorbance was measured at a wavelength of 570 nm according to the manufacturer’s instructions (Sigma, USA).

2.3. DNA isolation and analysis
Cells treated at day 0 for 24 h with 10 − 7 M 1,25-D3 were cultured for 6 days in
a serum-free medium with or without a 48 h treatment with 3 vM 5-AzaC at day
2. At day 7, adherent and ﬂoating cells were pooled and pelleted. Pellets were lysed
and then incubated for 1 h in 50 mM Tris–HCl (pH 8.0), 50 mM EDTA, 0.5%
(w/v) sodium lauryl sarcosinate and 0.5 mg/ml proteinase K. After a 1 h treatment
with DNAse-free RNAse at 50°C, the samples were mixed with 1% low-gelling-temperature agarose at 62°C, loaded into the dry wells of a 1.5% agarose gel and size
fractionated by electrophoresis (Baudet et al., 1996a).

2.4. Northern blot analysis
Total cellular RNA was prepared according to the LiCl/urea method (Auffrey
and Rougeon, 1980), size-fractioned in 1.5% agarose gel, transferred to a Hybond
N membrane (Amersham, UK) and hybridized as previously described (Wion et al.,
1991). For control purposes, blots were probed with a glyceraldehyde-3-phosphate
dehydrogenase probe (GAPDH) (Fort et al., 1985).
3. Results

3.1. Treatment of C6.9 cells with 5 -AzaC inhibits the PCD induced by 1,25 -D3
Treatment of cultured cells with 5-AzaC is known to induce DNA hypomethylation and therefore transcriptional activation of previously unexpressed genes (Jones
and Taylor, 1980). To investigate whether modiﬁcations in the pattern of methylation of C6.9 cell DNA can alter the cell death programme induced by 1,25-D3,
these cells were cultured for 5 days in the presence of 3 vM of 5-AzaC. After this
treatment, they were cultured for 3 days in a medium devoid of 5-AzaC and then
treated for 24 h with 1,25-D3 as previously described (Naveilhan et al., 1994b;
Baudet et al., 1996a,b). Results presented Fig. 1D show that 5-AzaC pretreatment
renders the cells resistant to the PCD induced by a subsequent treatment with
1,25-D3. An important feature of the PCD induced by 1,25-D3 in C6.9 cells is the
time course of the process which triggers cell death occurring 5 days after the
addition of the hormone (Naveilhan et al., 1994b; Baudet et al., 1996a,b ). To
investigate the ability of 5-AzaC to inhibit the lethal effect of 1,25-D3 in cells
already committed to die, naive C6.9 cells that had been treated for 24 h with
1,25-D3 (day 0) were then exposed to 5-AzaC (3 vM) from day 2 to day 4. Results
presented in Fig. 1F and Fig. 2 demonstrate that 5-AzaC is able to inhibit the cell
death programme even though it had already been triggered by 1,25-D3 pretreatment. Similar results were obtained with 5-aza-2%-deoxycytidine, another potent
inhibitor of DNA methylation (Jones and Taylor, 1980) (data not shown).

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Fig. 1. 5-AzaC treatment suppresses the 1,25-D3-induced PCD in C6.9 cells. (A) Cells were treated for
24 h with vehicle alone and then cultured in serum-free medium for 4 days. (B) Cells were treated with
10 − 7 M 1,25-D3 for 24 h, then rinsed and cultured in a serum-free medium without 1,25-D3. (C, D)
Cells pretreated with 5-AzaC (see Section 2) were treated for 24 h with vehicle alone (C) or 10 − 7 M
1,25-D3 (D) and then cultured in a serum-free medium without 1,25-D3. (E, F) Naive C6.9 cells were
treated with vehicle alone (E) or 10 − 7 M 1,25-D3 (F) during 24 h in serum-free medium. Then they were
cultured in serum-free medium with 5-AzaC (3 mM) from day 2 to day 4. Photographs were taken on
day 5.

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3.2. Effect of 5 -AzaC treatment on DNA fragmentation, a feature of apoptosis
encountered in C6.9 cells treated with 1,25 -D3
A feature currently observed during apoptosis is the internucleosomal cleavage of
nuclear DNA which is considered to arise from the activation of cellular endonuclease(s) (Wyllie, 1980). Internucleosomal degradation of nuclear DNA was therefore
assessed qualitatively by agarose gel electrophoresis. Results presented in Fig. 3
show that electrophoretic patterns of DNA degradation were observed depending
of the treatment with 5-AzaC. Hence, the internucleosomal fragmentation normally
observed in 1,25-D3-treated cells was no longer detected when DNA was extracted
from cells treated with both 1,25-D3 and 5-AzaC (Fig. 3).

Fig. 2. Concentration-dependent protective effect elicited by 5-AzaC in 1,25-D3-treated C6.9 cells. C6.9
cells cultured in serum-free medium were exposed for 24 h to 1,25-D3 and then treated with the indicated
concentrations of 5-AzaC. MTT was performed at day 7.

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Fig. 3. Inhibition of internucleosomal DNA fragmentation by 5-AzaC in C6.9 cells treated with 10 − 7 M
1,25-D3. DNA was extracted at day 7 from C6.9 cells and the formation of oligonucleosomal fragments
was determined by agarose gel electrophoresis. Lane 1, molecular weight markers; lane 2, DNA from
control cells; lane 3, DNA from cells treated for 24 h with 10 − 7 M 1,25-D3 and then cultured for 6 days
in serum-free medium; lane 4, DNA from cells treated from day 2 to day 4 with 3 vM 5-AzaC; lane 5,
DNA from cells treated for 24 h with 10 − 7 M 1,25-D3 and then exposed to 3 vM 5-AzaC from day 2
to day 4.

3.3. Inhibition of PCD by 5 -AzaC does not suppress the induction of c-myc
obser6ed in 1,25 -D3 -treated cells
The c-myc gene has been reported to induce apoptosis in ﬁbroblasts cultured in
serum-free medium (Evan et al., 1992). This ﬁnding has led to the demonstration
that the expression of this proto-oncogene was also increased in C6.9 cells undergoing 1,25-D3-induced PCD (Baudet et al., 1996a,b). Therefore we investigated the
fate of c-myc induction in cells treated with 5-AzaC. For this purpose, Northern
blot analyses were performed with RNA extracted either from control cells, or from
cells treated at day 0 for 24 h with 1,25-D3 and then exposed or not to 5-AzaC
from day 2 to day 4, or from cells treated with 5-AzaC alone from day 2 to day 4.
Results presented in Fig. 4 show that the inhibition of PCD induced by 5-AzaC
treatment is not accompanied by a concomitant inhibition of the expression of the

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c-myc gene. It is also noticeable that the up-regulation of the vitamin D receptor
gene (VDR) already observed in 1,25-D3-treated cells (Naveilhan et al., 1994b;
Baudet et al., 1996b) is not affected by 5-AzaC treatment.
In ﬁbroblasts, Myc-induced apoptosis can be inhibited by speciﬁc cytokines such
as IGF-1 (insulin-like growth factor) and PDGF (platelet-derived growth factor)
(Harrington et al., 1994). It is therefore possible that a consequence of the
treatment of C6.9 cells with 5-AzaC could be de novo expression of genes encoding
for cytokines able to suppress PCD. We therefore determined whether an inhibitory
soluble factor of PCD was produced by 5-AzaC-treated cells. Fig. 5 shows that the
effect was not due to a soluble factor since the cytotoxic effect of 1,25-D3 was still
observed on naive cells when 1,25-D3 was added to cocultures of 5-AzaC treated
and naive cells physically separated by a ﬁlter membrane (0.2 mm pore size). This
result suggests that a soluble factor produced by the 5-AzaC-treated cells was not
involved in the effect observed.

Fig. 4. Expression of c-myc gene in C6.9 cells treated for 24 h with 10 − 7 M 1,25-D3 and then exposed
to 3 vM 5-AzaC from day 2 to day 4. Total RNA was extracted from cells 3 days (lanes 1 – 4) after a
pretreatment for 24 h with vehicle alone (lane 1), or with 10 − 7 M 1,25-D3 (lane 2), or after treatment
with 3 vM 5-AzaC (lane 3), or after a pretreatment for 24 h with 10 − 7 M 1,25D3 and then treatment
with 3 vM 5-AzaC (lane 4). Blots were serially hybridized with radiolabelled c-myc, VDR and GAPDH
probes.

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Fig. 5. Effects of a coculture of 5-AzaC-treated and naive C6.9 cells on 1,25-D3-induced cell death.
1,25-D3 was added at day 0 for 24 h. Then, serum-free culture medium was replaced every 2 days. MTT
assay was performed at day 7.

3.4. 5 -AzaC treatment does not suppress ceramide-induced PCD in C6 glioma cells
N-Acetylsphingosine (C2-ceramide) has been reported to induce PCD in C6
glioma cells (Yoshimura et al., 1997). The protective effect of 5-AzaC against this
PCD was therefore examined. For this purpose C6.9 cells pretreated with 5-AzaC
for 24 h were then exposed to C2-ceramide. Data presented in Fig. 6 show that
5-AzaC treatment was unable to prevent C2-ceramide-induced cell death. This
suggests that C2-ceramide was acting through mechanisms at least partially distinct
from those relevant to the action of 1,25-D3 and that demethylation of DNA by
5-AzaC did not provide a general protection against PCD.

4. Discussion
It has been well established for a long time that 5-AzaC and 5-aza-2%-deoxycytidine induce hypomethylation of DNA, and that this process leads to the expression of genes previously hypermethylated. Although DNA methylation is involved
in numerous physiological processes, the possible effect of demethylating agents on
PCD has, to our knowledge, not been extensively studied. In this report we provide
evidence for the ability of these DNA demethylating agents to suppress the cell

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death programme induced by 1,25-D3 in C6.9. Treatment of these cells with
1,25-D3 for 24 h induces PCD 5 days later (Naveilhan et al., 1994b; Baudet et al.,
1996a,b). We demonstrate here that this process can be stopped by a treatment with
demethylating agents from day 2 to day 4. A similar inhibitory effect can be
manifest if 1,25-D3 is added 3 days after the removal of 5-AzaC. According to these
ﬁndings, a classical feature of apoptosis such as DNA fragmentation is no longer
observed in cells primed with 1,25-D3 and then treated with 5-AzaC. Therefore,
these results suggest DNA methylation, an epigenetic modiﬁcation of DNA, as a
regulatory mechanism of programmed cell death. However, 5-AzaC fails to suppress the induction of c-myc observed in 1,25-D3-treated cells. This latter ﬁndings
demonstrates that cells still respond to 1,25-D3 although the cell death programme
is suppressed by 5-AzaC. Induction of c-myc is a known inducer of apoptosis in
serum-deprived ﬁbroblasts (Evan et al., 1992), and this gene has been suggested to

Fig. 6. 5-AzaC pretreatment had no effect on the PCD induced by C2-ceramide. C6.9 cells cultured in
serum-free medium were either treated or not treated for 24 h with 3 vM 5-AzaC and then exposed to
C2-ceramide. MTT was performed at day 3.

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be involved in the PCD studied here (Baudet et al., 1996a,b). Therefore demethylation could have induced member(s) of the bcl-2 family such as bcl-2 and bcl-w
which have been reported to inhibit apoptotic signals (Gonzalez-Garcia et al., 1995;
Gibson et al., 1996), as well as any other gene(s) functioning as dominant
suppressor of PCD. In this regard it should be mentioned that proteins inhibiting
apoptosis have also been associated with many viruses, including avian erythroblasts, hepatitis B, Epstein – Barr, cowpox, herpes simplex and human papilloma
viruses (Gillet and Brun, 1996). Evidence has been obtained that methylation of
endogenous viral genes restricts their expression (Desrosiers et al., 1979; Sutter and
Doerﬂer, 1980; Groudine et al., 1981; Harbers et al., 1981; Jaenisch et al., 1985;
Bednarik et al., 1990). Conversely, treatment with 5-AzaC has been reported to
activate silent viral DNA sequences in different species (Groudine et al., 1981;
Jaenisch et al., 1985; Bednarik et al., 1990). In this context, it is noteworthy that all
rat cells have been reported to contain at least one replication-defective endogenous
retrovirus, as well as several endogenous retrovirus-like sequences (Tsuchida et al.,
1974; Young et al., 1978; Niwa and Sughara, 1981; Hsieh et al., 1987; Hsieh and
Weinstein, 1990). It is therefore possible that treatment of rat C6.9 cells with
demethylating agents activated silent apoptosis-inhibiting endogenous viral sequence(s) responsible for the effect observed. Alternatively, a change in the DNA
methylation pattern could have induced genes initiating a new pathway of differentiation in C6.9 cells and thereby yielding a new cellular phenotype resistant to the
PCD induced by 1,25-D3.
Defective regulation of PCD may play a part in the aetiology of cancer or in
degenerative diseases. With regard to cancer, in addition to glioma cells, apoptotic
or growth inhibitory effects of 1,25-D3 have been demonstrated for breast (James
et al., 1996; Simboli-Campbell et al., 1996), leukaemic (Elnster et al., 1996),
prostatic (Peehl et al., 1994; Hsieh and Wu, 1997), pancreatic (Zugmaier et al.,
1996) and colon (Shabahang et al., 1994) cancer cells. Therefore, it will be of
interest to determine whether hypomethylation conditions also abrogate the effects
of vitamin D in these cell lines. With regard to the involvement of PCD in
degenerative diseases, agents able to modulate this process are potential therapeutic
tools and further characterization of genes involved in the suppression of the PCD
described above is of obvious importance. However, it should be stressed that the
ﬁndings presented are not characteristic of a general anti-apoptotic effect of
demethylating agents. Moreover, we suggest that demethylation will indiscriminantly activate genes some of which can potentially generate both pro-apoptotic
and anti-apoptotic pathways. It can therefore be anticipated that the same
demethylating conditions could have opposite effects and induce apoptosis in other
experimental systems.

Acknowledgements
We thank Dr D. Djakiew for critical reading of this manuscript, Dr. L. Binderup
(Leo Pharmaceutical Products, Denmark) for the gift of 1,25-D3 and Drs H.F.

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DeLuca, M. Yaniv and P. Fort for respectively providing the VDR, c-myc and
GAPDH cDNAs used in this study. This work was supported by l’Association pour
la Recherche sur le Cancer and la Ligue Nationale contre le Cancer (Comite
´
Departemental de Maine-et-Loire).
´
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