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Biochimica et Biophysica Ac
Blockade of murine erythroleukemia cell differentiation by
hypomethylating agents causes accumulation of discrete small poly(A)�
RNAs hybridized to 3V-end flanking sequences of hmajor globin gene
Ioannis S. Vizirianakis*, Asterios S. Tsiftsoglou
Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki,
Thessaloniki GR-54124, Greece
Received 15 July 2004; received in revised form 2 September 2004; accepted 3 September 2004
Available online 21 September 2004
Abstract
Induction of murine erythroleukemia (MEL) cell differentiation is accompanied by transcriptional activation of globin genes and
biosynthesis of hemoglobin. In this study, we observed cytoplasmic accumulation of relatively small RNAs of different size (150–600
nt) hybridized to a1 and hmajor globin DNA probes in MEL cells blocked to differentiate by hypomethylating agents (neplanocin A,
3-deazaneplanocin A and cycloleucine). These RNAs lack poly(A) tail and appear to be quite stable. Search within the 3V-endflanking sequences of hmajor globin gene revealed the presence of a B1 repeat element, several ATG initiation codons, a GATA-1
consensus sequence and sequences recognized by AP-1/NF-E2 and erythroid Krqppel-like factor (EKLF) transcription factors. These
data taken together indicate that exposure of MEL cells to hypomethylating agents promotes accumulation of relatively small discrete
RNA transcripts lacking poly(A) tail regardless of the presence or absence of inducer dimethylsulfoxide (DMSO). However, the
relative steady-state level of small RNAs was comparatively higher in cells co-exposed to inducer and each one of the
hypomethylating agents. Although the orientation of these RNAs has not been established as yet, the possibility these small poly(A)�
RNAs which are induced by hypomethylating agents may be involved in the blockade of MEL cell differentiation program is
discussed.
D 2004 Elsevier B.V. All rights reserved.
Keywords: MEL; Cell; Differentiation; Hypomethylating agent; Inducer; Globin gene; Transcription; Neplanocin A; 3-Deazaneplanocin A; Cycloleucine;
Silent DNA; DNA methylation; RNA methylation
1. Introduction
The precise cellular and molecular mechanisms involved
in initiation of commitment of murine erythroleukemia (MEL
or Friend) cells to terminal erythroid maturation are still not
fully elucidated, regardless of the remarkable progress
achieved in this field [1–3]. Earlier studies with metabolic
0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2004.09.003
* Corresponding author. Tel.: +30 2310 997658; fax: +30 2310
997645.
E-mail address: [email protected] (I.S. Vizirianakis).
inhibitors (e.g. cordycepin, cycloheximide) revealed that
initiation of commitment to terminal maturation depends on
the synthesis of new RNA transcripts and proteins [1,4,5]. In
addition, erythroid differentiation of MEL cells was shown to
be associated with DNA hypomethylation [6], as well as
transmethylation of RNA [7,8] and other biochemical events
reviewed elsewhere [1–3].
We have shown earlier that induction of erythroid
differentiation of MEL cells is inhibited by N6-methyl-
adenosine (N6mAdo), neplanocin A, 3-deazaneplanocin A
and cycloleucine [7,9], all of which inhibit active
methylation cycle within the cells [10–13]. These agents
ta 1743 (2005) 101–114
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114102
are claimed to have selective inhibitory action on intra-
cellular methylation cycle reactions as reported elsewhere
[7,10–13]. In particular, neplanocin A, 3-deazaneplanocin
A and N6mAdo are inhibitors of S-adenosylhomocysteine
(AdoHcy) hydrolase (E.C. 3.3.1.1) (Ki values for the
isolated AdoHcy hydrolase 2�10�9, 5�10�11 and 2�10�4
M, respectively), whereas cycloleucine is an inhibitor of S-
adenosylmethionine (AdoMet) synthetase (E.C. 2.5.1.6)
(Ki value for the isolated AdoMet synthetase 4.8�10�5 M)
[12,14,15]. The hypomethylating agents block initiation of
commitment and prevent accumulation of hemoglobin in
inducer-treated MEL cells presumably by interrupting a
central process required for commitment to terminal
erythroid maturation [7–9].
Interestingly enough, during the course of our work, we
observed that exposure of MEL cells to each of these
agents, (N6mAdo, neplanocin A, 3-deazaneplanocin A and
cycloleucine), led to cytoplasmic accumulation of discrete
RNAs detected at first by using a mouse genomic DNA
fragment containing the hmajor globin gene as well as its
flanking sequences [7,9]. In the present study, we extended
these observations in order to: (a) analyze the expression
pattern of these RNAs in MEL cells induced to differ-
entiate as well as in cells blocked to commit by the
hypomethylating agents; (b) assess the specificity of such
RNAs with respect to genes other than hmajor globin; (c)
estimate the size of RNA transcripts; and (d) demonstrate
their origin by extensive Northern blot analysis using small
genomic DNA sequences fragments as probes.
Here, we wish to report that: (a) the relatively small
discrete RNA transcripts accumulate in the cytoplasm of cells
exposed to hypomethylating agents in the absence or
presence of the inducer. These RNAs were entirely absent,
however, in MEL cells exposed to neither agent nor
dimethylsulfoxide (DMSO) only; (b) such new RNA tran-
scripts were not detected in the cytoplasmic RNA hybridized
with 32P-labeled probes derived from genes other than globin
including those encoding for transferrin receptor (TfR),
GAPDH, h-actin and ribosomal protein S5 (rpS5); (c)
Northern blot hybridization analysis with probes generated
from different parts of the hmajor globin gene flanking regions
revealed that the detected new RNA transcripts lack poly(A)
tail and it is likely to be encoded by sequences located at the
3V-end flanking sequences of the mouse hmajor globin gene.
This region is enriched in ATG initiation codons, consensus
sequences for transcription factors GATA-1, AP-1/NF-E2
and erythroid Krqppel-like factor (EKLF) as well as it has aB1 repeat element. These data indicate for the first time that
treatment of MEL cells with three hypomethylating agents
that block MEL cell differentiation promotes cytoplasmic
accumulation of relatively small poly(A)� RNAs that are
likely to originate from the 3V-end flanking sequences of
hmajor globin gene. We are in the process of characterizing
these RNAs and investigating whether these molecules
contribute to the blockade of differentiation of MEL cells
exposed to hypomethylating agents.
2. Materials and methods
2.1. Chemicals and biochemicals
DMSO was purchased from Mallinckrodt, Inc. (St.
Louis, MO, USA). Neplanocin A and 3-deazaneplanocin
A were kindly donated by Dr. V.E. Marquez (National
Cancer Institute, USA), whereas cycloleucine was obtained
from Sigma Chemical Co. (St. Louis, MO, USA). All
restriction enzymes were obtained from New England
Biolabs, Inc. (Hertfordshire, England).
2.2. Cell cultures
Cells employed throughout this study were MEL-745PC-
4A a clone of MEL-745 cells obtained after subcloning and
subsequent testing of clones derived for high degree of
inducibility. All cultures were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% (v/v)
fetal calf serum (FCS, Gibco, Long Island, NY) and
antibiotics (penicillin and streptomycin 100 Ag/ml). Cells
were incubated at 37 8C in a humidified atmosphere
containing 5% CO2 and maintained at densities that
permitted logarithmic growth (1�105–1�106 cells/ml). Cell
viability was assessed by trypan blue exclusion as reported
elsewhere [7,8].
2.3. Induction and assessment of differentiation
Cells were incubated with no drug and the inducing agent
in the absence or presence of an inhibitor as indicated in the
text. At certain timed-intervals during incubation, the
proportion of differentiated (hemoglobin-producing cells)
was assessed cytochemically with benzidine–H2O2 solution
as described elsewhere [7,8].
2.4. Assessment of the steady-state level of RNA transcripts
by Northern blot hybridization analysis
Cytoplasmic RNA prepared from control and MEL
cells treated with agents at various timed-intervals, as
indicated in the text, was assessed for the steady-state level
of RNA transcripts coded by hmajor globin, a1-globin,
mouse TfR, h-actin, GAPDH and rpS5 genes, with the use
of [32P]-labeled probes, as previously described [7,8]. The
former three genes are regulated and expressed devel-
opmentally in differentiating cells, while the latter three
are considered housekeeping genes. The probes used were
a 7.3 kb genomic DNA fragment bearing the hmajor globin
gene [7], a ~3 kb SacI/SacI genomic DNA fragment
encoding for the a1-globin mRNA kindly donated by Dr.
N. Anagnou (University of Athens, Greece), a 2.28 kb
DNA fragment encoding for mouse TfR mRNA kindly
donated by Dr. K. Pantopoulos (EMBL, Heidelberg,
Germany), a 426 bp encoding for rat GAPDH [31], a
715 bp cDNA fragment encoding for the full-length of
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 103
rpS5 gene previously isolated in our laboratory [16], and a
350 bp PstI/HindIII cDNA fragment encoding for mouse
h-actin mRNA [7].
2.5. Preparation of DNA hybridization probes from the
different regions of mouse 7.3 kb EcoRI/EcoRI genomic
DNA fragment
Basically, two sets of DNA probes were prepared and
used as hybridization probes in Northern blot analysis of
total cytoplasmic RNA in order to map the area of origin
of new RNA transcripts. Probes designated as C, D, E and
F were generated by the use of polymerase chain reaction
(PCR) and primers as follows: Probe C (length 132 bp):
sense primer 5V-GGCCAATCTGCTCACACAGGA-3V, andantisense primer 5V-GATGTCTGTTTCTGGGGTTGT-3V;Probe D (length 91 bp): sense primer 5V-TGCACCT-GACTGATGCTGAGAAG-3V and antisense primer 5V-CCTGCCCAGGGCCTCACCACCAA-3V; Probe E (length
879 bp): sense primer 5V-CTGCTGGTTGTCTACCCT-TGG-3V and antisense primer 5V-CTGTGGGAATAT-
GGAAGAACC-3V; Probe F (length 224 bp): sense
primer 5V-CTGCTGGTTGTCTACCCTTGG-3V and anti-
sense primer 5V-CCTGAAGTTCTCAGGATCCAC-3V.The probes A, B, G, H, I and J were generated by diges-
tion of 7.3 kb DNA fragment with restriction enzymes as
follows: Probe A is a 191 bp EcoRI/PstI DNA genomic
fragment; Probe B is a PstI/HindIII DNA genomic DNA
fragment; Probe G is a 1028 bp HindIII/HindIII DNA
genomic fragment; Probe H is a 903 bp PstI/XbaI DNA
genomic fragment; Probe I is a 3920 bp XbaI/EcoRI
DNA genomic fragment; and Probe J is a 1429 bp
EcoRV/EcoRI DNA genomic fragment. All probes are
illustrated in Fig. 5.
2.6. DNA methylation analysis at CCGG sites within the 7.3
kb DNA fragment
MEL cells were incubated with each hypomethylating
agent in the absence or the presence of DMSO and at
various incubation times cells were collected by cen-
trifugation at 600�g for 15 min. The genomic DNA was
then isolated by the PUREGENEk DNA isolation kit
(Gentra Systems, Minneapolis, USA). Digestion of DNA
(4–8 Ag/sample) with EcoRI (20 units) was followed by
digestion separately with 20 units of endonucleases
HpaII and MspI (New England Biolabs) carried out in
the appropriate buffers recommended by the manufac-
turer. After incubation at 37 8C overnight to ensure
maximum digestion, DNA samples were subjected to gel
electrophoresis through 1.2% agarose in a Tris acetate/
EDTA buffer. After denaturation, DNA was transferred to
Nylon filters (S&S NYTRAN-N, Schleicher and Schuell,
Dassel, Germany) in 1.5 M NaCl/0.15 M sodium citrate
(pH 7.4) and cross-linked to filters under UV. Hybrid-
ization was performed overnight at 65 8C, by using as
probe the 7.3 kb genomic DNA fragment labeled with
a-[32P]-dCTP (sp. act. 3000 Ci/mmol, IZOTOP, Buda-
pest, Hungary) by random priming (Invitrogen Life
Technologies). The filters were then washed and exposed
to autoradiography films (Kodak, Rochester, NY, USA)
at 70 8C for 1–4 days.
3. Results
3.1. Dose-dependent blockade of cell growth and eryth-
roid differentiation of MEL cells by hypomethylating
agents
It was quite important from the beginning of these
experiments to establish appropriate culture conditions in
order to study the effects of each hypomethylating agent
(neplanocin A, 3-deazaneplanocin A, cycloleucine), on
cell growth and differentiation without killing the cells.
Therefore, exponentially growing cells (MEL-745A-PC-4)
were exposed separately to varying concentrations of each
one of these hypomethylating agents in the absence (Fig.
1A) and/or in the presence of inducer DMSO (Fig. 1B,C).
About 48 h later, the effect of each agent on cell growth
was assessed, whereas the proportion of hemoglobin-
producing cells (benzidine-positive cells) was determined
96 h following treatment. As shown in all three cases, the
agents decreased cell growth either in the absence (Fig.
1A) or in the presence of DMSO (Fig. 1B) in a dose-
dependent fashion but to a different extent. Interestingly,
cycloleucine was 4-log less toxic to the cells as compared
to neplanocin A and 3-deazaneplanocin A. This agent
caused inhibition of cell growth at relatively high
concentration (N10�3 M). Comparatively, all three agents
caused less inhibition of cell growth in the presence of
DMSO rather in the absence of the inducer (Fig. 1B
versus Fig. 1A). Co-treatment of MEL cells with each
hypomethylating agent and DMSO prevented erythroid
differentiation and markedly reduced production of
hemoglobin (Fig. 1C). Neplanocin A was the most potent
inhibitor followed by 3-deazaneplanocin A. Cycloleucine
was much less potent inhibitor of differentiation since it
modulated erythroid differentiation at concentration higher
than 10�4 M. At concentration higher than 10�4 M, 20–
25% of cells continued to differentiate even in the
presence of cycloleucine (see Fig. 1C). These data
enabled us to select the optimum inhibitory concentration
of each agent for our studies (1�10�6 M for neplanocin
A, 1�10�5 M for 3-deazaneplanocin A and 4�10�2 M
for cycloleucine). At these concentrations, all agents
increase the SAH/SAM ratio and inhibit RNA methyl-
ation in MEL cells as we have previously shown [7].
Cells exposed to each one of the three agents under
conditions described remained quite viable in proportion
as shown by the trypan blue exclusion test illustrated in
Table 1 [7].
Table 1
Effect of neplanocin A, 3-deazaneplanocin A and cycloleucine on cell
viability in control and DMSO-treated MEL cells
Treatment Concentration (M) Viability (%)
None – 93
DMSO 0.210 93
Neplanocin A 1�10�6 87
3-Deazaneplanocin A 1�10�5 82.1
Cycloleucine 4�10�2 87.4
DMSO+neplanocin A 0.210+1�10�6 82.8
DMSO+3-deazaneplanocin A 0.210+1�10�5 76.6
DMSO+cycloleucine 0.210+4�10�2 74.9
Exponentially growing MEL-745PC-4A cells were incubated with and
without DMSO, as well as treated separately with neplanocin A, 3-
deazaneplanocin A and/or cycloleucine in the absence or the presence of
DMSO at the concentrations indicated. Cell viability was determined after
72 h incubation by trypan blue exclusion as previously shown [7]. Each
value indicates the mean of two separate experiments.
Fig. 1. Concentration-dependent effects of neplanocin A, 3-deazanepla-
nocin A and cycloleucine on cell growth and differentiation of MEL
cells. MEL-745PC-4A cells were incubated with varying concentrations
of neplanocin A (–.–), 3-deazaneplanocin A (–o–) and/or cycloleucine
(–E–) in the absence (panel A) and presence (panels B and C) of DMSO
(1.5% v/v). Cell growth (panels A and B) was determined after 48 h by
counting the cell number with the use of a hemocytometer under a light
microscope, whereas the proportion of differentiated (hemoglobin-pro-
ducing) cells (panel C) was assessed after 96 h as previously described
[7,8]. Each value represents the mean of two separate experiments.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114104
3.2. Cytoplasmic accumulation of relatively short RNAs in
MEL cells exposed to hypomethylating agents in the
presence or absence of DMSO
Cytoplasmic RNA isolated from MEL cells exposed to
each one of the three hypomethylating agents in the presence
of DMSO and hybridized with multi-primed 32P-labeled 7.3
kb mouse genomic DNA fragment bearing the hmajor globin
gene and flanking sequences, revealed accumulation of
nascent hmajor globin mRNA as expected as well as of
discrete RNA transcripts of relatively short molecular weight
(Fig. 2A–D). The steady-state level of these RNAs was
higher in cells exposed to each hypomethylating agent in the
presence of DMSO from the beginning as compared to that
seen in cells exposed to the same hypomethylating agent in
the absence of DMSO. These data are in agreement with our
previous observation showing that N 6mAdo promoted
similar pattern of cytoplasmic accumulation of RNA tran-
scripts of hmajor globin gene [9]. Notably, cells exposed to
each hypomethylating agent in the absence of DMSO
produced negligible quantities of nascent hmajor globin
mRNA. These data (Fig. 2A–D) indicate that: (a) all three
hypomethylating agents promote accumulation of discrete
RNA transcripts, an event not seen in cells exposed to
DMSO alone; (b) the level of these short RNAs increased
after 60–72 h exposure to hypomethylating agents only (Fig.
2B,C,D). DMSO enhanced the steady-state level of such
RNAs in each occasion suggesting that these RNAs are
accumulated at higher levels in cells exposed to inducer
regardless of whether erythroid maturation is blocked.
Moreover, these data suggest that DMSO treatment modu-
lates the onset of appearance of these RNAs in cells treated
with hypomethylating agents.
3.3. The detection of short-end RNA transcripts was specific
to bmajor and a1-globin gene DNA probes
To demonstrate whether the appearance of new RNA
transcripts is specific for hmajor globin mRNA, we screened
Fig. 2. Assessment of the effect of neplanocin A, 3-deazaneplanocin A and cycloleucine on the steady-state accumulation of hmajor globin RNA transcripts in
control and differentiating MEL cells. MEL-745PC-4A cells were incubated in DMEM supplemented with FCS (10% v/v) and DMSO (1.5% v/v) (panels A,
E), or exposed separately to neplanocin A (1�10�6 M) (panels B, F), 3-deazaneplanocin A (1�10�5 M) (panels C, G) and/or cycloleucine (4�10�2 M) (panels
D, H) in the absence or presence of DMSO (1.5% v/v). At times indicated (numbers above the panels), cells were removed from culture and total cytoplasmic
RNA was isolated. Ten micrograms of cytoplasmic RNA were electrophoretically separated on 1% agarose gel, transferred and immobilized onto a nylon
membrane (Hybond-N, Amersham) and hybridized at 65 8C with [32P]-labeled genomic DNA fragment coding for hmajor globin gene (7.3 kb), as described
earlier [7]. The filter was then washed at 65 8C and autoradiographed using Kodak XAR-5 film. The autoradiograms obtained are shown above (panels A–D).
Panels E–H show the corresponding ethidium bromide staining patterns of electrophoresed RNA samples (the positions of 28S and 18S rRNAs are indicated).
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 105
Northern blots with DNA probes originated from mouse a1-
globin gene as well as from genes encoding TfR, h-actin,rpS5 and GAPDH. In addition to hmajor probe, hybridization
with a 32P-labeled a1-globin DNA probe also led to the
detection of short-end RNA transcripts whose level was
once again enhanced by DMSO (Fig. 3A–D). In contrast,
the use of DNA probes encoded either by the developmen-
tally regulated TfR gene (Fig. 4), or by the housekeeping
genes h-actin, rpS5 and GAPDH (data not shown) failed to
detect short-end RNA transcripts in MEL cells exposed to
each hypomethylating agent in the absence and/or presence
of DMSO. These findings indicate that the phenomenon of
appearance of small RNA transcripts appears to be unique
for globin genes in MEL cells exposed to hypomethylating
agents.
3.4. The short-end RNAs may originate from the 3V-flankingsequences of bmajor globin gene locus
The appearance of new RNA transcripts in the cytoplasm
of MEL cells exposed to hypomethylating agents prompted
us to investigate their origin. We generated several DNA
probes from the 7.3 kb EcoRI/EcoRI genomic DNA
fragment, as indicated in Fig. 5, and used them accordingly
Fig. 3. Assessment of the effect of neplanocin A, 3-deazaneplanocin A and cycloleucine on the steady-state accumulation of a1-globin RNA transcripts in
control and differentiating MEL cells. MEL-745PC-4A cells were incubated separately in culture with each methylation inhibitor in the absence and presence
of DMSO (1.5% v/v) as indicated under Fig. 2 and the steady-state level of a1-globin mRNA cytoplasmic accumulation was assessed by Northern blot
hybridization analysis using a genomic SacI/SacI DNA fragment (~3 kb) described under Materials and methods. The autoradiograms showing the steady-state
level of a1-globin mRNA and other RNAs hybridized by the same probe are indicated above (panels A–D). Panels E–H show the corresponding ethidium
bromide staining patterns of electrophoresed RNA samples (the positions of 28S and 18S rRNAs are indicated).
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114106
as hybridization probes to demonstrate the origin of these
RNAs. Since the only known gene within the 7.3 kb
genomic DNA fragment was that of hmajor globin we
searched the flanking regions. Extensive Northern blot
hybridization analysis done with the use of several 32P-
labeled DNA fragments revealed that none of the eight
DNA probes generated and used (probes A–H in Fig. 5)
enabled us to detect the small RNA transcripts in MEL cells
exposed to neplanocin A, 3-deazaneplanocin A, or cyclo-
leucine in the absence or presence of DMSO (see Fig. 6 for
probe F; Fig. 7 for probe H; Fig. 8A for probe C; Fig. 8B for
probe D; Fig. 8C for probe E; Fig. 8D for probe G; Fig. 9A
for probe A; Fig. 10A for probe B). Interestingly, however,
we observed that the only two DNA probes used to detect
the new RNA transcripts were those generated from the 3V-
downstream flanking sequences of hmajor globin gene (DNA
probes I and J in Fig. 5) (see Figs. 8E and 10B for probe I;
and Figs. 9B and 10C for probe J). The estimated size of the
detected RNA transcripts ranged from 150 to 600 nt as
shown in Fig. 10. These data indicate that exposure of MEL
cells to hypomethylating agents promotes accumulation of
relatively short RNA transcripts hybridized to DNA
sequences originated from the 3V-downstream region of
hmajor globin gene locus.
3.5. The detected RNA transcripts lack poly(A) tail
To further characterize the detected RNAs, we first asked
if they bear poly(A) tail as the majority of mRNAs by
separating cytoplasmic RNA into poly(A)� and poly(A)+
Fig. 4. Assessment of the effect of neplanocin A, 3-deazaneplanocin A and
cycloleucine on the steady-state accumulation of TfR RNA transcripts in
control and differentiating MEL cells. Membranes bearing the same RNA
samples shown in Fig. 3 were stripped and re-probed with a DNA genomic
fragment encoding for mouse TfR gene in order to assess the cytoplasmic
accumulation of TfR RNA transcripts. The results obtained are shown
above (panels A–D).
Fig. 5. Map of the different DNA probes originated from the 7.3 kb EcoRI/EcoRI genomic DNA fragment and used for Northern blot hybridization studies.
The mouse genomic 7.3 kb EcoRI/EcoRI DNA fragment is illustrated along with the different segments generated and used as probes to map the area of origin
of new RNA transcripts detected upon incubation of MEL cells with methylation inhibitors. The hmajor globin gene with the three exons and two introns is
shown at the 5V-end of this genomic fragment. The probes designated C, D, E and F shown at the bottom were generated by PCR, whereas these named as A, B,
G, H, I and J were generated by digestion of 7.3 kb DNA fragment with the restriction enzymes indicated at the top.
Fig. 6. Northern blot hybridization analysis of cytoplasmic RNA by using
the exon-2 DNA sequences of hmajor globin gene as probe. The same
membranes shown in Fig. 4 were stripped and re-hybridized with the
radiolabeled probe F shown in Fig. 5 which contains the full-length of
exon-2 of hmajor globin gene. The results shown in panels A–D indicate no
detection of discrete small weight RNA transcripts.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 107
Fig. 7. Northern blot hybridization analysis of cytoplasmic RNA with
probes encoding the exon-3 and the 3V-UTR DNA sequences of hmajor
globin gene. The same membranes shown in Fig. 6 were stripped and re-
hybridized with the radiolabeled probe H shown in Fig. 5 which contains
the full-length of exon-3 and 3V-UTR sequences of hmajor globin gene. The
results shown in panels A–D indicate no detection of discrete small weight
RNA transcripts.
Fig. 8. Northern blot hybridization analysis of cytoplasmic RNA by using
each one of the probes C, D, E, G and I generated from the 7.3 kb genomic
DNA fragment. MEL-745PC-4A cells were incubated in DMEM supple-
mented with 10% v/v FCS with DMSO, or exposed separately to
neplanocin A, 3-deazaneplanocin A and/or cycloleucine in the absence or
the presence of DMSO as indicated in Fig. 2. At times indicated (numbers
above the panels), cells were removed from culture and total cytoplasmic
RNAwas isolated. Ten micrograms of cytoplasmic RNAwere then assessed
by Northern blot hybridization analysis by using the following probes that
have previously been indicated in Fig. 5: in Panel A the probe C
corresponds to the 5V-UTR region of hmajor globin gene; in Panel B the
probe D encodes the exon-1 of hmajor globin gene; in Panel C the probe E
encodes the exon-2 and the intron-2 of hmajor globin gene; in Panel D the
probe G corresponds to the 5V-UTR, the exon-1, the intron-1, the exon-2
and part of the intron-2 region of hmajor globin gene; in Panel E the probe I
spans throughout the 3V-flanking sequences of hmajor globin gene. Panel F
shows the corresponding ethidium bromide staining patterns of electro-
phoresed RNA samples. The positions of 28S and 18S rRNAs are also
indicated by the arrows. Note that the detection of the discrete small weight
RNA transcripts upon exposure of control and/or DMSO-treated MEL cells
to each one of the methylation inhibitors is achieved only with the use of
probe I shown in panel E.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114108
fractions by oligo(dT)-cellulose chromatography. While
three known nascent mRNAs encoding for hmajor globin,
TfR and GAPDH were detected in the poly(A)+ RNA
fraction of cytoplasmic RNA (Fig. 11B) as expected, to our
surprise the small RNAs were found exclusively in the
poly(A)� RNA fraction (Fig. 11A). Therefore, we reason
that these poly(A)� RNA transcripts must represent RNA
species accumulated in the cytoplasm of MEL cells exposed
to hypomethylating agents.
3.6. DNA sequences located downstream of bmajor globin
gene locus contain unique cis-elements and consensus
binding sites for GATA-1, NF-E2 and EKLF transcription
factors
The data presented thus far revealed that the short
RNAs are hybridized with the probes I and J that originate
from the 3V-end flanking sequences of hmajor globin gene.
This observation prompted us to examine the sequence
characteristics of this region illustrated in Fig. 5. The entire
Fig. 9. Northern blot hybridization analysis with the probes A and J
encoding sequences that are located in distant 5V- and 3V-flanking sequencesof hmajor globin gene locus. MEL-745PC-4A cells were incubated in culture
with either DMSO alone, or with DMSO in the presence of either
neplanocin A, 3-deazaneplanocin A and/or cycloleucine for 60 h as
indicated in Fig. 2. Untreated cells were used as control experiment.
Constant amount of cytoplasmic RNA (10 Ag) derived from each sample
was assessed by Northern blot hybridization analysis using the following
probes illustrated in Fig. 5. Panel A: probe A was derived from the 5V-flanking sequences of hmajor globin gene. Panel B: probe J spans 3V-flanking sequences far away of hmajor globin gene. Both probe A and J
detected nascent hmajor globin mRNA, but only probe J detected small
molecular weight RNAs. Corresponding ethidium bromide staining pattern
of electrophoresed RNA samples is shown in panel C. Note that the
detection of the discrete small weight RNA transcripts upon exposure of
DMSO-treated MEL cells to each one of the three methylation inhibitors is
achieved only with the use of probe J shown in panel B.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 109
nucleotide sequence of the BALB/c mouse h-globincomplex spans almost 55.9 kb as had been previously
reported by Shehee et al. [17], (accession number
X14061). The 7.3 kb genomic DNA fragment used as
hybridization probe to detect the new RNA transcripts in
MEL cells exposed to hypomethylating agents corresponds
to sequences spanning from 36,884 bp to 44,192 bp (the
numbers refer to the sequence data deposited at Gen/EMBL
with accession number X14061). This region contains the
hmajor globin gene (38,207–39,683 bp) and a B1 repeat
element (42,382–42,545 bp) along with two short direct
repeats (SDR) located at the 5V-end (5V SDR: 42,367–42,381bp) and 3V-end (3V SDR: 42,546–42,560 bp) of B1 element
[17] (see Fig. 12). This B1 repeat element is being
transcribed by RNA polymerase III and it has been
previously identified by hybridization to human Alu repeat
elements [18]. More interestingly, however, detailed struc-
tural analysis enable us to reveal in the region upstream of
the B1 repeat element the presence of consensus binding
sites for the transcription factors GATA-1 (binding site
AGATAAGG at position 42,283– 42,290 bp) as well as for
AP-1/NF-E2 (binding site TGCTGACTCA at position
42,463–42,272 bp) (see Fig. 12). In addition to these two
consensus sequences for GATA-1 and AP-1/NF-E2 tran-
scription factors, another binding site for EKLF has been
revealed internally; the B1 element at the 42,528–42,537 bp
region (binding site CCCCCCCCCA) (Fig. 12). The
identified GATA-1 binding site is identical to the one
previously found within the promoter of hmajor globin gene
(at position 38,076–38,083 bp; promoter site �216 to �209
bp) (Fig. 12), which it is known to play a crucial role in the
transcriptional activation of hmajor globin gene [19].
Furthermore, we identified another identical GATA-1 bind-
ing site located within the bh3 gene (at position 31,153–
31,160 bp). In addition, several ATG initiation codons
localized around the GATA-1 and AP-1/NF-E2 binding sites
were identified in the 3V-downstream sequences of hmajor
globin gene, the most of which found to be at their 3V-endsequences near the B1 repeat element (Fig. 12). The
existence of so many structural elements within the 3V-endflanking sequences of hmajor globin gene tend to suggest that
this DNA region is probably involved in the appearance of
small RNAs detected upon treatment of MEL cells with
hypomethylating agents.
3.7. DNA methylation analysis within the region of 7.3 kb
genomic DNA fragment in MEL cells exposed to hypome-
thylating agents
Since we employed hypomethylating agents, it was
reasonable to investigate whether exposure of MEL cells
to these agents alters the methylation status of CpG sites
located within the region of interest. The 7.3 kb genomic
DNA fragment contains a CCGG consensus methylation
sequence at position 40,523 bp that is recognized by the
methylation-sensitive restriction isoschizomer enzymes
HpaII and MspI. HpaII does not cut when the internal
cytosine is methylated, whereas MspI does so regardless
of whether the cytosine is methylated or not. Thus, we
applied restriction enzyme analysis complemented by
molecular hybridization as previously shown [16]. If
sequence CCGG was being methylated, then MspI
produces two fragments of 3641 and 3663 bp, in contrast
to HpaII which leaves the fragment with intact size (7.3
kb). DNA extracted from control, DMSO- and/or hypo-
methylating agent-treated cells was at first digested with
EcoRI and then with MspI and/or HpaII and was
analyzed by molecular hybridization, as shown in Fig.
13. The results have shown that the CCGG site remained
methylated and it is not affected by either neplanocin A
or 3-deazaneplanocin A (Fig. 13). Similar results were
obtained upon treatment of MEL cells with cycloleucine
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114110
(data not shown). These data indicate that methylation at
the specific CCGG site examined is not altered. There-
fore, the appearance of small RNAs induced by the
hypomethylating agents in MEL cells is not related to
changes in the level of DNA methylation of the region
tested. It is likely, however, that changes to other DNA
methylation sequences may be related to the blockade of
differentiation program of MEL cells by hypomethylating
agents with the possible involvement of transcription
factors or with specific changes in chromatin superfine
structure.
4. Discussion
Despite the plethora of data accumulated during the last
years concerning the in vitro MEL cell differentiation
program, the precise molecular mechanism(s) which govern
this process are as yet not fully delineated. Furthermore, the
detailed mechanism by which RNA and DNA metabolic
inhibitors like cordycepin [4,5], 3-deazaadenosine [20], S-
5V-isobutylthioadenosine and 5V-methylthioadenosine [21]
block differentiation are still not clear. The data presented in
this study indicate for the first time that three agents
(neplanocin A, 3-deazaneplanocin A and cycloleucine),
known to inhibit DNA and RNA methylation by blocking
the active methylation cycle induced cytoplasmic accumu-
lation of discrete RNAs of relatively small molecular
weight. The appearance of four to five different size RNAs
(150–600 nt) lacking the poly(A) tail in MEL cells exposed
to three different hypomethylating agents suggest that their
accumulation is likely to result from unique, although
unknown, effects of these agents. The observations, how-
ever, that the steady-state level of these RNAs was higher in
cells co-exposed to hypomethylating agents and DMSO
indicate that the inducer of differentiation favors the
accumulation of such RNAs. Although it is not clear at
the moment whether DMSO/hypomethylating agent-treated
cells become committed to terminal maturation without
hemoglobin synthesis or both commitment to terminal
maturation and hemoglobin production are blocked, it is
essential to ask the following questions: (a) How specific
the accumulation of these RNAs is and where do these
poly(A)� RNA species originate from? (b) Are these RNAs
authentic products resulting from transcription of DNA
sequences located within the h-globin gene family complex,
or are abnormal products coming from mis-processed
Fig. 10. Detection of discrete small RNA transcripts in cytoplasmic RNA
prepared from methylation inhibitor-treated MEL cells with probes
corresponding to 3V-flanking sequences of hmajor globin gene. MEL-
745PC-4A cells were incubated in culture with DMSO or exposed
separately to neplanocin A, 3-deazaneplanocin A and/or cycloleucine in
the absence or presence of DMSO for 60 h as indicated in Fig. 2. Untreated
cells were also used as control experiment. A constant amount of
cytoplasmic RNA (10 Ag) derived from each sample was assessed by
Northern blot hybridization analysis using probe B originated from 5V-flanking sequences, and probe I and J originated from the 3V-flankingsequences of hmajor globin gene shown in Fig. 5. The results obtained are
shown in Panel A for probe B, in Panel B for probe I and in Panel C for
probe J. Panel D shows the corresponding ethidium bromide staining
pattern of electrophoresed RNA samples. The arrows show the molecular
weight of different size RNA ladder markers for comparative study. The
position of 28S and 18S rRNAs is also indicated. Note that the detection of
the discrete small weight RNA transcripts upon exposure of DMSO-treated
MEL cells to each one of the three methylation inhibitors is achieved only
with the use of probes I and J as illustrated in panels B and C.
Fig. 11. The discrete RNA transcripts detected in methylation inhibitor-
treated MEL cells lack poly(A) tail. MEL-745PC-4A cells were incubated
in culture with either DMSO alone, or with DMSO in the presence of
neplanocin A for 72 h as indicated in Fig. 2. Untreated cells served as a
control experiment. Total cytoplasmic RNA derived from such treated cells
was then separated into the poly(A)� and the poly(A)+ RNA fractions using
oligo(dT)-cellulose column as previously shown [7]. Northern blot hybrid-
ization analysis then carried out and the results obtained are shown above.
In panel A, hybridization was performed at 65 8C with [32P]-labeled 7.3 kb
EcoRI/EcoRI genomic DNA fragment. Panel B: after the stripping of the
membrane shown in panel A, hybridization was repeated simultaneously
with three different [32P]-multi-primed labeled probes encoding the exon-2
of hmajor globin (probe F in Fig. 5), the TfR and the GAPDH genes in order
to assess the relative purity of the poly(A)+ RNA fraction. Note, as shown
in panel A, that the detection of the discrete small weight RNAs upon
exposure of DMSO-treated MEL cells to neplanocin Awas possible only in
the samples of total cytoplasmic RNA (lane 3) and exclusively in poly(A)�
RNA fraction (lane 4), suggesting that small RNAs lack poly(A) tail.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 111
globin-like RNA precursors which are unable to be
polyadenylated in the presence of hypomethylating agents?
(c) What is their orientation (sense or antisense), and why do
they lack poly(A) tail? (d) To what extent do these RNAs
share structural homology? Finally, it would be very
interesting to investigate whether these RNA species may
be responsible for blockade of MEL cell differentiation seen
following treatment with hypomethylating agents. Unfortu-
nately, the answers to some of these questions are not
presented in this paper at this stage, since additional work is
needed to further characterize these RNAs and then
investigate their role in differentiation. The fact that these
RNA species were detected only by using DNA hybrid-
ization probes originated from a- and h-globin genes
indicates that these RNAs are exclusively related to the
globin gene family and not to other non-globin genes tested
(TfR, h-actin, GAPDH, rpS5). We cannot rule out, however,
the possibility that these short RNA transcripts may
originate from other regions of the mouse genome that
contain sequences structurally homologous to those being
present within the 3V-end flanking sequences of hmajor
globin gene.
Previous work has shown the presence of antisense RNA
molecules of hmajor globin gene in the cytoplasm of MEL
cells as well as in erythroid spleen cells and reticulocytes
from anemic mice [22,23]. These antisense globin RNA
transcripts are detected in full size or as truncated molecules.
They are complementary to the spliced hmajor globin mRNA
synthesized by the mouse erythroid cells, and they are
implicated in a mechanism that gives rise to new sense-
strand hmajor globin mRNA by the involvement of an RNA-
dependent RNA polymerase [23]. The fact that the detected
poly(A)� RNA species presented in this paper do not
hybridize with DNA probes derived exclusively from the
hmajor globin coding sequences, it tends to suggest that they
represent a different group of RNA molecules, although we
do not know yet their orientation (sense or antisense). On
the contrary, we know that these poly(A)� RNAs are
detected in Northern blots only with DNA probes derived
from downstream DNA sequences of hmajor globin gene,
and for this reason, they do not seem to be generated from
premature globin-like RNA molecules attacked by specific
endoribonucleases as those recently characterized and being
involved in the cleavage of h- and a-globin mRNAs
intracellularly [24,25].
The Northern blot hybridization experiments of cyto-
plasmic RNAs isolated from MEL cells exposed to
hypomethylating agents and presented in this paper have
revealed that besides the transcriptional activation of hmajor
gene, DNA sequences located at 3V-flanking region may be
also selectively activated. A careful look at this DNA locus
indicated the presence of two consensus sequences for
GATA-1 and AP-1/NF-E2 transcription factors. These
sequences are located upstream of a previously identified
B1 repeat element, the mouse equivalent of the Alu element
seen in human DNA [17,26]. The fact that preliminary
results from electrophoretic mobility binding assays by
using DNA fragments from this region have shown binding
of several transcription factors (data not shown), tends to
suggest that the identified sequences may have some
functional role in transcription, a result that needs further
verification. Alternatively, the presence in the 3V-down-stream sequences of hmajor globin gene of the consensus
Fig. 12. Diagrammatic presentation of unique DNA sequences located within the 3V-flanking sequences of mouse hmajor globin gene. Detailed examination of
sequences located at the 3V-flanking region, (position 42,201–42,759 bp at the sequence deposited at Gen/EMBL with accession numberX14061), of hmajor
globin gene revealed the presence of several consensus sequences for the binding of the NF-E2, GATA-1 and EKLF transcription factors shown in boxes at the
lower right side of the panel. In addition, this area has several ATG initiation codons (shown in bold and italics) and a B1 element whose sequence is
underlined. The sequences of SDR that are located at the 5V- and 3V-end of B1 element is shown in lowercase letters. Finally, the GATA-1 consensus sequence
known to exist within the promoter region of hmajor globin gene (position �220 to �208 bp), which is identical to that identified at the 3V-flanking sequences isalso illustrated in a large box at lower left panel.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114112
binding sequences for GATA-1 and AP-1/NF-E2 tran-
scription factors in the vicinity of B1 repeat element may
suggest some specific role in the expression pattern of hmajor
gene during development. This is an attractive direction,
since it is well known that these two transcription factors
have crucial roles in the regulation of globin gene
expression through binding in consensus DNA sequences
located within the locus control region (LCR) and promoter
site of the genes [27–29]. Although no data exists to support
such an assumption, this hypothesis is further strengthened
by recent findings that implicate the function of B1 repeat
family in complex regulatory elements involved in post-
transcriptional control of gene expression in the mouse
genome [30]. In particular, the insertion of a B1 repeat
element in the 3V untranslated region of the rabbit h-globingene conferred growth- and transformation-dependent tran-
scriptional regulation of this gene after its transfection into
the cells. This effect was attributed to the function of B1
repeat element that cooperated with other regulatory
sequences in a way that somehow modulated the RNA
polymerase II transcription machinery [30]. In the light of
these structural elements being involved in the 3V-down-stream region of hmajor globin gene, the data presented
suggest, but by no means prove, that the detected small size
RNAs stem from locally transcriptionally activated DNA
sequences. Unfortunately, no conventional transcriptional
units have been detected in these 3V-flanking sequences
regardless of the existence of the B1 element, of several
ATG initiation codons and consensus sequences for the
binding of the transcription factors AP-1/NF-E2, GATA-1
and EKLF. Therefore, we cannot rule out the possibility that
transcriptional units might be located somewhere else within
the mouse genome which is activated by hypomethylating
agents and produce such RNAs which share structural
complementarities with the 3V-downstream sequences of
hmajor globin gene used as probes for detection.
Fig. 13. Analysis of methylation of CCGG sites located within the 7.3 kb genomic DNA fragment in MEL cells exposed to neplanocin A and 3-
deazaneplanocin A. MEL-745PC-4A cells were incubated in culture with neplanocin A and 3-deazaneplanocin A in the absence or presence of DMSO as
described under Fig. 2. At times indicated (numbers above the panels), cells were removed from culture and nuclear DNA was isolated. Constant amount of
DNA (4–8 Ag) was digested first with EcoRI and then by either MspI or HpaII. The DNA digestion products were separated in 1.2% agarose gel, transferred
onto Hybond-N membrane and hybridized with [32P]-multi-primed labeled 7.3 kb genomic DNA fragment as indicated under Materials and methods. Panel A:
digestion of DNA with both EcoRI and HpaII followed by hybridization. Note that the EcoRI/EcoRI 7.3 kb genomic DNA fragment remained intact upon
HpaII digestion as shown by the arrowhead. Panel C: digestion of DNAwith both EcoRI andMspI and hybridization. In this caseMspI digestion produced two
fragments from the EcoRI/EcoRI 7.3 kb genomic DNA fragment at the expected size (3641 and 3663 bp) as shown by the arrowhead. The first lane in each
panel (A, C) represents the results obtained with the DNA isolated from control untreated MEL cells and digested with only EcoRI. The corresponding
ethidium bromide staining patterns of digested DNAs are illustrated in panel B (EcoRI and HpaII digestion) and panel D (EcoRI and MspI digestion). Size of
molecular weight DNA markers are indicated in the left of each panel.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 113
Experiments in our laboratory are underway to clone and
characterize these RNA transcripts and then demonstrate if
the identified consensus sequences for the GATA-1 and AP-
1/NF-E2 are indeed functional. Moreover, it would be of
great interest to delineate how DNA regions, which are silent
in untreated MEL cells, are somehow activated, and how this
process leads to the blockade of erythroid differentiation
program. Since the appearance of these short poly(A)�
RNAs is seen exclusively in cells exposed to hypomethylat-
ing agents, but not in control cells, we tend to conclude that
their production may be due to the direct action of
hypomethylating agents on chromatin transcriptional state,
or alternatively, that these agents may exert their action
indirectly on chromatin by modulating the active methyl-
ation cycle. The latter focuses on data that strongly support
the notion of a close relationship between DNA methylation
state and chromatin conformation status in a way that affects
gene transcription and represents a research area that has
received a lot of attention in recent years [31,32]. Although
at present, we have no clue as to what these small RNAs do,
we may ask whether these RNAs act at the transcriptional or
even posttranscriptional level to abrogate erythroid differ-
entiation. This may be a novel mechanism for regulating
gene expression posttranscriptionally. Alternatively, the
detected poly(A)� RNAs might be encoded by genes that
belong to the family of non-coding RNAs (ncRNAs) which
are well known to have important roles in processes like
transcriptional regulation, RNA maturation, stability and
translation as well as protein degradation and translocation
[33,34]. In any case, however, these processes impinge on
the crucial molecular mechanisms that govern the initiation
of MEL differentiation program and justify our attempt to
isolate, clone and characterize the detected poly(A)� RNAs
presented in this paper.
Acknowledgements
We would like to thank Mr. Sotirios Tezias for technical
help and Dr. Aristidis Kritis for sequence alignment at the
initial phase of this work. This work was supported in part
by the EKVAN Project from the Greek Secretariat Science
and Technology awarded to Dr. A.S. Tsiftsoglou.
References
[1] P.A. Marks, M. Sheffery, R.A. Rifkind, Induction of transformed cells
to terminal differentiation and the modulation of gene expression,
Cancer Res. 47 (1987) 659–666.
I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114114
[2] A.S. Tsiftsoglou, I.S. Pappas, I.S. Vizirianakis, Mechanisms involved
in the induced differentiation of leukaemia cells, Pharmacol. Ther. 100
(2003) 257–290.
[3] A.S. Tsiftsoglou, I.S. Pappas, I.S. Vizirianakis, The developmental
program of murine erythroleukemia cells, Oncol. Res. 13 (2003)
339–346.
[4] N.M. Kredich, Inhibition of nucleic acid methylation by cordycepin,
J. Biol. Chem. 255 (1980) 7380–7385.
[5] R. Levenson, J. Kernen, D. Housman, Synchronization of MEL cell
commitment with cordycepin, Cell 18 (1979) 1073–1078.
[6] J.K. Christman, N. Weich, B. Schoenbrun, N. Schneiderman, G. Acs,
Hypomethylation of DNA during differentiation of Friend erythro-
leukemia cells, J. Cell Biol. 86 (1980) 366–370.
[7] I.S. Vizirianakis, A.S. Tsiftsoglou, Induction of murine erythroleuke-
mia cell differentiation is associated with methylation and differential
stability of poly(A)+ RNA transcripts, Biochim. Biophys. Acta 1312
(1996) 8–20.
[8] I.S. Vizirianakis, A.S. Tsiftsoglou, N6-methyladenosine inhibits
murine erythroleukemia cell maturation by blocking methylation of
RNA and memory via conversion to (N6-methyl)-adenosylhomocys-
teine, Biochem. Pharmacol. 50 (1995) 1807–1814.
[9] I.S. Vizirianakis, W. Wong, A.S. Tsiftsoglou, Analysis of inhibition of
commitment of murine erythroleukemia (MEL) cells to terminal
maturation by N6-methyladenosine, Biochem. Pharmacol. 44 (1992)
927–936.
[10] M. Caboche, J.-P. Bachellerie, RNA methylation and control of
eukaryotic RNA biosynthesis: effects of cycloleucine, a specific
inhibitor of methylation, on ribosomal RNA maturation, Eur. J.
Biochem. 74 (1977) 19–29.
[11] R.I. Glazer, M.C. Knode, Neplanocin A: a cyclopentenyl analog of
adenosine with specificity for inhibiting RNA methylation, J. Biol.
Chem. 259 (1984) 12964–12969.
[12] R.I. Glazer, K.D. Hartman, M.C. Knode, M.M. Richard, P.K. Chiang,
C.K.H. Tseng, V.E. Marquez, 3-Deazaneplanocin: a new and potent
inhibitor of S-adenosylhomocysteine hydrolase and its effects on
human promyelocytic leukemic cell line HL-60, Biochem. Biophys.
Res. Commun. 135 (1986) 688–694.
[13] C.K.H. Tseng, V.E. Marquez, R.W. Fuller, B.M. Goldstein, D.R.
Haines, H. McPherson, J.L. Parsons, W.M. Shannon, G. Arnett, M.
Hollingshead, J.S. Driscoll, Synthesis of 3-deazaneplanocin A, a
powerful inhibitor of S-adenosylhomo-cysteine hydrolase with potent
and selective in vitro and in vivo antiviral activities, J. Med. Chem. 32
(1989) 1442–1446.
[14] P.K. Chiang, G.A. Miura, S-Adenosylhomocysteine hydrolase, in:
R.T. Borchardt, C.R. Creveling, P.M. Ueland (Eds.), Biological
Methylation and Drug Design, Humana Press, Clifton, NJ, 1986,
pp. 239–251.
[15] J.R. Sufrin, J.B. Lombardini, D.L. Kramer, V. Alks, R.J.
Bernacki, C.W. Porter, Methionine analog inhibitors of S-
adenosylmethionine biosynthesis as potential antitumor agents,
in: R.T. Borchardt, C.R. Creveling, P.M. Ueland (Eds.), Biological
Methylation and Drug Design, Humana Press, Clifton, NJ, 1986,
pp. 373–384.
[16] I.S. Vizirianakis, I.S. Pappas, D. Gougoumas, A.S. Tsiftsoglou,
Expression of ribosomal protein S5 cloned gene during differentiation
and apoptosis in murine erythroleukemia (MEL) cells, Oncol. Res. 11
(1999) 409–419.
[17] W.R. Shehee, D.D. Loeb, N.B. Adey, F.H. Burton, N.C. Casavant, P.
Cole, C.J. Davies, R.A. McGraw, S.A. Schichman, D.M. Severynse,
C.F. Voliva, F.W. Weyter, G.B. Wisely, M.H. Edgell, C.A. Hutchison
III, Nucleotide sequence of the BALB/c mouse beta-globin complex,
J. Mol. Biol. 205 (1989) 41–62.
[18] L.W. Coggins, J.K. Vass, M.A. Stinson, W.G. Lanyon, J. Paul, A B1
repetitive sequences near the mouse beta-major globin gene, Gene 17
(1982) 113–116.
[19] K. Macleod, M. Plumb, Derepression of mouse hmajor globin gene
transcription during erythroid differentiation, Mol. Cell. Biol. 11
(1991) 4324–4332.
[20] M.L. Sherman, T.D. Shafman, D.R. Spriggs, D.W. Kufe, Inhibition of
murine erythroleukemia cell differentiation by 3-deazaadenosine,
Cancer Res. 45 (1985) 5830–5834.
[21] P.P.D. Fiore, M. Grieco, A. Pinto, V. Attadia, M. Porcelli, G.
Cacciapuoti, M. Carteni-Farina, Inhibition of dimethylsulfoxide-
induced differentiation of Friend erythroleukemic cells by 5V-methylthioadenosine, Cancer Res. 44 (1984) 4096–4103.
[22] V. Volloch, Cytoplasmic synthesis of globin RNA in differentiated
murine erythroleukemia cells: possible involvement of RNA-depend-
ent RNA polymerase, Proc. Natl. Acad. Sci. U. S. A. 83 (1986)
1208–1212.
[23] V. Volloch, B. Schweitzer, S. Rits, Antisense globin RNA in mouse
erythroid tissues: structure, origin, and possible function, Proc. Natl.
Acad. Sci. U. S. A. 93 (1996) 2476–2481.
[24] N.D. Rodgers, Z. Wang, M. Kiledjian, Characterization and purifica-
tion of a mammalian endoribonuclease specific for the a-globin
mRNA, J. Biol. Chem. 277 (2002) 2597–2604.
[25] A. Stevens, Y. Wang, K. Bremer, J. Zhang, R. Hoepfner, M. Antoniou,
D. Schoenberg, L.E. Maquat, h-Globin mRNA decay in erythroid
cells: UG site-preferred endonucleolytic cleavage that is augmented
by a premature termination codon, Proc. Natl. Acad. Sci. U. S. A. 99
(2002) 12741–12746.
[26] D. Labuda, D. Sinnett, C. Richer, J.M. Deragon, G. Striker, Evolution
of mouse B1 repeats: 7SL RNA folding pattern conserved, J. Mol.
Evol. 32 (1991) 405–414.
[27] A.B. Cantor, S.H. Orkin, Transcriptional regulation of erythropoi-
esis: an affair involving multiple partners, Oncogene 21 (2002)
3368–3376.
[28] K.S. Choe, F. Radparvar, I. Matushansky, N. Rekhtman, X. Han,
A.I. Skoultchi, Reversal of tumorigenicity and the block to differen-
tiation in erythroleukemia cells by GATA-1, Cancer Res. 63 (2003)
6363–6369.
[29] T. Sawado, K. Igarashi, M. Groudine, Activation of beta-major globin
gene transcription is associated with recruitment of NF-E2 to the beta-
globin LCR and gene promoter, Proc. Natl. Acad. Sci. U. S. A. 98
(2001) 10226–10231.
[30] F. Vidal, E. Mougneau, N. Glaichenhaus, P. Vaigot, M. Darmon, F.
Cuzin, Coordinated posttranscriptional control of gene expression by
modular elements including Alu-like repetitive sequences, Proc. Natl.
Acad. Sci. U. S. A. 90 (1993) 208–212.
[31] K.D. Robertson, DNA methylation and chromatin-unraveling the
tangled web, Oncogene 21 (2002) 5361–5379.
[32] M.S. Turker, Gene silencing in mammalian cells and the spread of
DNA methylation, Oncogene 21 (2002) 5388–5393.
[33] S.R. Eddy, Non-coding RNA genes and the modern RNAworld, Nat.
Rev., Genet. 2 (2001) 919–929.
[34] G. Storz, An expanding universe of noncoding RNAs, Science 296
(2002) 1260–1263.
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