Journal of General Virology (1991), 72, 1105-1111. Printed in Great Britain
1105
The inhibitory effects of oligonucleotides, complementary to Marek's
disease virus mRNA transcribed from the BamHI-H region, on the
proliferation of transformed lymphoblastoid cells, MDCC-MSB1
Mikiko Kawamura, 1 Masanobu Hayashi, 1. Tatsuya Furuichi, ~ Meihan Nonoyama, 2
Emiko lsogai 3 and Shigeo N a m i o k a I
1Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan,
2Department of Virology, Tampa Bay Research Institute, 10900 Roosevelt Boulevard North, St Petersburg,
Florida 33716, U.S.A. and 3Department of Preventive Dentistry, School of Dentistry, Higashi Nippon Gakuen University,
Tobetsu 069, Japan
An oligonucleotide complementary to the splice donor
sequence of the 1-8 kb gene family produced from the
B a m H I - H region of Marek's disease virus (MDV)
DNA inhibited the proliferation of the MDV-derived
lymphoblastoid cell line, MDCC-MSB 1 (MSB-1), but
not that of the avian lymphoid leukosis-derived
lymphoblastoid cell line, LSCC-BK3. Colony forma-
tion in soft agar was also inhibited by treatment of
MSB-1 cells with the antisense oligonucleotide. It is
hypothesized that expression of the 1.8 kb gene family
produced from the B a m H I - H region is directly associated with the maintenance of the tumorigenic state of
transformed Marek's disease-derived lymphoblastoid
cells.
Introduction
A variety of transformed lymphoblastoid cell lines
which contain multiple copies of the MDV genome have
been established from MDV-induced lymphomas or
transplantable lymphomas in chickens (Akiyama et al.,
1974; Hahn et al., 1977). There was no amplification of
the 132 bp repeat sequence within the BamHI-D and -H
regions of latent viral DNA in the transformed lymphoblastoid cells (Hayashi et al., 1988) and the 1-8 kb
transcript was produced from the BamHI-D and -H
regions of MDV DNA in the transformed cells (Bradley
et al., 1989a). These reports suggest that maintenance
of the tumorigenic state of MDV-derived lymphoblastoid cell lines might be associated also with the
expression of the 1.8 kb gene family from the BamHI-D
and-H regions. However, direct proof of the function of
the 1-8 kb mRNA has been lacking.
It has been shown that antisense oligonucleotides and
their analogues can be used as tools for inhibiting viral
replication, for blocking splicing and translation of
mRNA, and for regulating specific gene expression
(Zamecnik & Stephenson, 1978; Smith et al., 1986;
Lemaitre et al., 1987; Agrawal et al., 1988; MarcusSekura, 1988; Mercola et al., 1988; Gewirtz et al., 1989).
To obtain insight into the role of the 1.8 kb transcripts
from the BamHI-D and -H regions of MDV DNA in the
maintenance of the tumorigenic state, we investigated
the effects on transformed lymphoblastoid cell proliferation of treatment with oligonucleotides complementary
to the splice donor sequence of the 1.8 kb transcript.
Marek's disease virus (MDV) is an avian herpesvirus
which induces lymphoproliferative disease and demyelination of peripheral nerves in chickens. The viral D N A
is a linear double-stranded molecule of 180 kbp (Lee et
al., 1971), of which the BamHI, Sinai and BglI restriction
maps have been established (Fukuchi et al., 1985a).
It has been demonstrated that serial passage of
virulent MDV in primary chicken embryo fibroblasts in
vitro results in a loss of virion tumorigenicity (Churchill
et al., 1969; Fukuchi et al., 1985b). This loss of
tumorigenicity strongly correlates with an expansion in
two particular regions, a 1-5 kbp BglI-PstI subfragment
of the BamHI-D and -H regions in the TRL and IRL,
respectively, of the viral genome. This heterogeneity in
size (expansion) in the BamHI-D and -H regions was
found only in viral DNA from the non-pathogenic
strains of MDV (Fukuchi et al., 1985b). Maotani et al.
(1986) reported that the heterogeneity was due to the
amplification of a 132 bp repeat sequence found within
the BamHI-D and -H fragments; a 1.8 kb gene family
was produced from the BamHI-D and -H regions only by
pathogenic strains of MDV (Bradley et al., 1989a). It has
been reported recently that the disappearance of the
m R N A associated with attenuation and the loss of
tumorigenicity was due to truncation of the 1.8 kb
transcript (Bradley et al., 1989b).
0000-9914 © 1991 SGM
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1106
M. Kawamura and others
Methods
Oligonueleotides. The oligonucleotides used in this study were
synthesized using the phosphoamidite method on a Beckman DNA
synthesizer (model System plus 1) and purified by HPLC. Oligonucleotide A1 was complementary to the splice donor sequence of exon I,
from nucleotides 243 to 260, 3' to the Sinai site; oligonucleotide A2 was
complementary to the splice donor sequence from nucleotides 363 to
380, 3' to the Sinai site (Fig. 1). Oligonucleotides S1 and $2 were the
complementary strands of oligonucleotides A1 and A2, respectively.
Two primer oligonucleotides for the polymerase chain reaction (PCR)
( T G A C A C G G C T C T G G G T G G G A and TATAGCCTGATGATACATCATCTC) were complementary to the sequences from nucleotides - 9 7 to - 7 6 at a site 5' to the PstI site in exon II of the 1.8 kb
mRNA, and from nucleotides 123 to 142 at a site 3' to the PstI site.
Cell growth experiment. MDV-induced, transformed lymphoblastoid
cell lines MDCC-MSB1 (MSB-1) and MDCC-JP1 (JP-1) were
obtained from Dr S. Kato, Osaka, Japan. A transformed lymphoblastoid cell line, LSCC-BK3 (BK-3), was derived from a bursal lymphoma
of a chicken with avian lymphoid leukosis (ALL) (Hihara et al., 1980).
These cell lines were cultivated in RPMI 1640 medium supplemented
with 10% foetal calf serum (FCS), 2 mM glutamine and 100 units (12)
penicillin G/ml, in a humidified atmosphere containing 5% CO2.
Logarithmically growing cells were pelleted at 1000g, washed with
phosphate-buffered saline (PBS) and resuspended at a density of
3 x 106/ml with FCS-free RPMI 1640 medium. After incubation of the
cells with oligonucleotides at concentrations from 1 to 5 laM for 1 h at
37 °C, the cells were diluted to a density of 5 x 105/ml by addition of
medium with FCS and again incubated in the presence of oligonucleotides at concentrations from 1 to 5 ~tM.After staining with trypan blue,
the number of viable cells was counted.
Growth in agarose. MSB-1 cells (1 x 104) were plated in 60 mm Petri
dishes in 2 ml RPMI 1640 medium with 10% FCS and 0.35% agar
[Special Agar (Noble), Difco], with or without oligonucleotides, over a
2ml 0.9% agar layer in the same medium. After cells had been
incubated at 37 °C for 14 days, colonies were stained with Giemsa; only
colonies of more than 30 cells were scored.
Incorporation o f oligonucleotides into cells. An oligonucleotide was
dephosphorylated by bacterial alkaline phosphatase (Takara Shuzo)
and then radiolabelled with [~,-3~P]ATP (4500 Ci/mmol; ICN
Radiochemical) using T4 polynucleotide kinase (Takara Shuzo)
(Maniatis et al., 1982). After incubation of MSB-I cells (5 x 10S/ml)
with radiolabelled oligonucleotide at 5 ~tM (specific activity 3.3 x 107
c.p.m./~tmol) for 24, 48 and 72 h, incorporation of oligonucleotides into
cells was determined according to the method of Miller et al. (1977).
After the addition of 10 ml ACS-II (Amersham) to an aliquot of cell
lysate, the radioactivity was counted with an Aloka LSC-750 liquid
scintillation counter. Radioactive compounds in the medium and the
cell lysate were analysed by 4% agarose gel electrophoresis (Nusieve
GTG agarose; FMC Bioproducts). The gel was dried and subjected to
autoradiography.
Southern and Northern blot hybridization. Cellular DNA was
extracted by treatment with proteinase K and SDS followed by phenol
extraction. Cellular DNA was digested with restriction endonuclease
(Nippon Gene) and the DNA fragments were electrophoreticatly
separated on a 0.6~ agarose gel; the separated DNA fragments were
transferred onto nitrocellulose paper by the method of Southern (1975).
Cloned MDV DNA BamHI fragments were 32p-labelled by nick
translation (Rigby et al., 1977). Hybridization was carried out under
conditions described previously (Fukuchi et al., 1985a), i.e. 6 x SSC
(1 × SSC is 0.15 M-NaC1 and 0.015 M-sodium citrate), 5 x Denhardt's
solution (1 x Denhardt's solution is 0-05% bovine serum albumin,
0.05% Ficoll and 0-05~ polyvinylpyrollidone) (Denhardt, 1966) and
50% formamide at 42 °C. Autoradiography using Fuji RX X-ray film
(Fuji Photo Film) was carried out with an intensifying screen at
- 80 °C for 24 h.
Cellular RNA was prepared from MSB-1 cells according to the
method of Silver et al. (1979). Electrophoresis of cellular R N A after
denaturation with 1 M-glyoxat in aqueous 50% DMSO was done by the
method of McMaster & Carmichael (1977). Chicken rRNAs of 28S and
18S were used as size markers.
Synthesis of 32p-labelled RNA probe required cloning of the MDV
BamHI-H fragment into pGem-1 (Promega Biotec). The resulting
plasmid, pGem-H, was linearized by digestion with PvulI or BglI.
Linear pGem-H DNA template (1 Ixg) was transcribed with 10 U T7
RNA polymerase in a 25 ~tl reaction mixture, which contained 40 mMTris-HC1 pH 7.5, 6 mM-MgCI2, 10 mM-NaCI, 10 mM-dithiothreitol,
2 mM-spermidine, 0.5 mM each of ATP, GTP and CTP, 12 ~tM-UTP,
50 ~tCi [~-32p]UTP (3000 Ci/mmol; ICN Radiochemical) and 20 U
RNasin (Promega Biotec), at 37 °C for 60 rain.
PCR o f c D N A , cDNA was synthesized in a 50 ~tl reaction mixture
which contained 5p.g cellular RNA, 4 n g synthetic oligo(dT)ls,
100 mM-Tris-HCl pH 8-3, 4 mM-dithiothreitol, 10 mM-MgC12, 140 mMKCI, 20 llg actinomycin D/ml, 1 mM each of dTTP, dGTP, dATP and
dCTP, and 8 0 U avian myeloblastosis virus reverse transcriptase
(Seikagaku America). The reaction mixture was heated briefly at 90 °C
and then at 42 °C for 2 h. The PCR was performed according to
the protocol provided with the GeneAmp kit (Perkin-Elmer Cetus).
The resulting amplified product was analysed by agarose gel
electrophoresis.
Results
The 1.8 kb m R N A which was transcribed from the
BamHI-H region of MDV D N A consisted of two exons
(Fig. 1). Synthesis of the 1.8 kb m R N A starts from a
locus 260 bp 5' to the SmaI site. Exon I continues to a
locus 260 bp 3' to the SmaI site in a region containing a
donor splice sequence A T G T A T G T G T G G G A G A A A / G T A T G T , or to a locus 380bp 3' to the
Sinai site, containing the donor splice sequence
G C T C G G C G A G T G T T C T / G T A A C T . We selected
the two splice donor sites of exon I as target regions for
the antisense oligonucleotides.
Typical growth curves of transformed lymphoblastoid
MSB-1 cells with or without oligonucleotides are shown
in Fig. 2 and 3. The cells grew exponentially for 72 h to a
density of approximately 2 × 106 to 3 x 106 cells/ml,
after a growth lag of about 12 h either in the absence of
oligonucleotide or with oligonucleotides A2 or $2 in the
medium at 5 ~tM. However, oligonuleotide A1 at concentrations of 3 and 5 ~M inhibited cellular proliferation
(Fig. 3) and the number of viable cells reduced during
incubation up to 96 h. The inhibitory effect was not
observed at a concentration of 1 ~tM. A similar inhibitory
effect was observed after treatment of JP-1 cells with
oligonucleotide A 1 at 3 and 5 ~tM(data not shown), but no
inhibition of BK-3 cells treated with oligonucleotides A1
and S 1 at a concentration of 5 pM (Fig. 2) or with A2 or $2
at 5 ~tM was found (data not shown).
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Antisense oligonucleotides against M D V gene
0
60
120
I
I
I
180 kbp
J
I
I
I
I
24
I
48
I
72
t 107
~30
~---20
[TRL ! ~ UL1 DR,,~,
BamHIHI t
11 I
[
UL2
II
I,
II I I
[
I
FIRL
- - FIRs
q Vs FTRs
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I~
lu
~
11111 [i
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N 5
r.
r-
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I
S
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B
I
5.5 kbp
~
,.Q
2
E
2:
I
2
1
Incubation time (h)
Exon I
Exon II
I
S1
A1
$2
A2
5'ATG
3'TAC
5'GCT
3'CGA
TAT
ATA
CGG
GCC
GTG
CAC
CGA
GCT
I
96
TGG
ACA
GCA
CGT
GAG
CTC
TGT
ACA
AAA3'
TTT 5'
TCT 3'
AGA5'
Fig. 1. BamHI restriction map of MDV DNA, transcription map of the
1.8 kb gene family produced from the BamHI-H region and the
sequences of the oligonucleotides. Arrowheads indicate the two splice
donor regions of exon I of the l-8 kb mRNA. Restriction sites are
shown as A, AccI; B, BamHI; E, EcoRI; P, PstI and S, SmaI.
Fig. 3. The concentration-dependent effects of sense and antisense
oligonucleotides on cellular proliferation. Growth curves of transformed lymphoblastoid MSB-1 cells in the absence of oligonucleotide
(×), and in the presence of oligonucleotides A1 and S1 at
concentrations of I gM (O, for A1 ; O, for SI), 3 gM (Z~, for A1 ; A, for
SI) and 5~tM (D, for A1; II, for S1).
I
I
I
40
530
? 20
10
t
I
I
I
O
30
20
X
E
~10
Z
1
5
I
0
.e 2
Z
1
1
0
24
48
72
Incubation time (h)
96
Fig. 2. Effects on the proliferation of MSB-1 and BK-3 cells of
treatment with oligonucleotides. Growth curves of transformed
lymphoblastoid MSB-I cells in the absence of oligonucleotide ( x ), and
in the presence of oligonucleotides A1 (C)), S1 (O), A2 (z~) and $2 (A)
at a concentration of 5 ~tM.Growth curves of transformed lymphoblastoid BK-3 cells in the absence of oligonucleotide (O), and in the
presence of oligonucleotides A 1 ([-1) and S 1 (11) at a concentration of
5 ~tM.
To investigate whether inhibition of cellular proliferat i o n by t r e a t m e n t w i t h o l i g o n u c l e o t i d e A1 w a s r e v e r s i ble, M S B - 1 cells w e r e i n c u b a t e d for 70 h in t h e p r e s e n c e
o f 5 ~ M - o l i g o n u c l e o t i d e A 1, p e l l e t e d b y c e n t r i f u g a t i o n at
24
I
I
48
72
Incubation time (h)
I
96
Fig. 4. The growth curves of MSB-I cells after removal of
oligonucleotides from the medium. Transformed lymphoblastoid MSB1 cells were incubated for 70 h in the absence of oligonucleotide ( x ),
and in the presence of oligonucleotides A1 (©) and S1 (O) at a
concentration of 5 ~tM. After removal of oligonucleotides from the
medium, cells were incubated in oligonucleotide-free medium.
1000g, washed with PBS and resuspended with oligon u c l e o t i d e - f r e e m e d i u m at a d e n s i t y o f a p p r o x i m a t e l y
5 x 105 c e l l s / m l . A f t e r t h e o l i g o n u c l e o t i d e s h a d b e e n
w a s h e d o u t o f t h e m e d i u m , t h e s e M S B - 1 cells t r e a t e d
w i t h o l i g o n u c l e o t i d e A1 p r o l i f e r a t e d at a r a t e s i m i l a r to
t h a t o f c o n t r o l cells w h i c h h a d n o t b e e n t r e a t e d w i t h
oligonucleotide or which had been treated previously
w i t h o l i g o n u c l e o t i d e S1 (Fig. 4).
T o p r o v i d e f u r t h e r i n f o r m a t i o n c o n c e r n i n g t h e role o f
t h e 1-8 k b g e n e f a m i l y o f M D V in t h e m a i n t e n a n c e o f t h e
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M. Kawamura and others
1108
(a) 1
2
3
4
5
T a b l e 1. Efficiency o f colony formation by MSB-1 cells
treated with antisense oligonucleotide
Colony-forming efficiency of MSB-1 cells (~)
(_+ S.D.)
Oligonucleotide
None
S1
A1
B
6
7
P
pGem-H
S
(b) 1
8
2
9
Exon I
Exon II
3
5
4
6
7
8
9
10
11
12
13
1
3
5
15-7 + 9-4*
15-6 + 6.1
2.5 + 0-5
15.7 + 9.4
23.7 + 15.3
0.3 + 0-3
15.7 + 9.4
14-1 + 8.2
0.0 + 0.0
* Each value represents the average of four to six separate
experiments, each performed in triplicate.
10
p1P.,. P2
Concentration of oligonucleotide 0tM)
B
M
Fig. 5. Northern blot hybridization and PCR analysis of cellular RNA
prepared from MSB-1 cells treated with oligonucleotide A1. (a) MSB-I
cells were incubated with or without oligonucleotides for 70 h. Cellular
RNA (20 ~tg)prepared from MSB-1 cells in the absence of oligonucleotide (lanes 1 and 6) and in the presence of oligonucleotides A 1 (lanes 2
and 7), S1 (lanes 3 and 8), A2 (lanes 4 and 9) and $2 (lanes 5 and 10), at a
concentration of 5 ~tM.Lanes 1, 2, 3, 4 and 5 were hybridized with an
RNA probe synthesized from pGem-H by T7 RNA polymerase. Lanes
t r a n s f o r m e d p h e n o t y p e o f MSB-1 cells, t h e effects o f
o l i g o n u c l e o t i d e s o n c o l o n y f o r m a t i o n i n soft a g a r w e r e
i n v e s t i g a t e d ( T a b l e 1). T h e c o l o n y - f o r m i n g efficiency o f
MSB-1 cells was 15 + 9 ~ i n t h e a b s e n c e o f o l i g o n u c l e o tide a n d t h a t o f cells t r e a t e d w i t h o l i g o n u c l e o t i d e S1 w a s
s i m i l a r . H o w e v e r , t h e efficiency m a r k e d l y d e c r e a s e d i n
t h e p r e s e n c e o f o l i g o n u c l e o t i d e A 1, e v e n at a c o n c e n t r a t i o n o f 1 ~tM.
W h e n c e l l u l a r R N A p r e p a r e d f r o m MSB-1 cells w a s
a n a l y s e d b y N o r t h e r n blot h y b r i d i z a t i o n u s i n g a n R N A
p r o b e s y n t h e s i z e d f r o m the B a m H I - H r e g i o n o f M D V
D N A u s i n g T 7 R N A p o l y m e r a s e , t h e 1.8 k b m R N A w a s
n o t d e t e c t e d if MSB-1 cells h a d b e e n t r e a t e d w i t h 5 ~M o
o l i g o n u c l e o t i d e A1 for 70 h (Fig. 5a), w h e r e a s it w a s
d e t e c t e d if MSB-1 cells h a d b e e n t r e a t e d w i t h 5 ~tMo l i g o n u c l e o t i d e A2, S1 or $2. T o o b t a i n m o r e i n f o r m a t i o n c o n c e r n i n g t h e s y n t h e s i s o f t h e 1.8 k b m R N A i n
MSB-1 cells t r e a t e d w i t h o l i g o n u c l e o t i d e A1, c e l l u l a r
RNA was analysed by the PCR using two oligonucleotides c o m p l e m e n t a r y to s e q u e n c e s w i t h i n e x o n I I as
p r i m e r s . W e c o u l d n o t d e t e c t t h e 239 b p f r a g m e n t i n t h e
a m p l i f i e d p r o d u c t f r o m 5 ~tM-oligonucleotide A l - t r e a t e d
MSB-1 cells (Fig. 5b).
6, 7, 8, 9 and 10 were rehybridized with/~-actin DNA as a probe after
dehybridization of the filter. Chicken 28S and 18S rRNAs were used as
size markers. Restriction sites in pGem-H are shown by B, BamHI; P,
Psfl; S, SmaI. P1 and P2 indicate the sites of primers for PCR. T7
indicates the promoter region of T7 RNA polymerase. (b) cDNAs from
MSB-1 cells in the absence of oligonucleotide (lane 2) and in the
presence of oligonucleotides A1 (lane 3) and S1 (lane 4) at a
concentration of 5 ~tM, and from BK-3 cells (lane 5) in the absence of
oligonucleotide, were amplified by PCR. A cloned BamHI-H fragment
was amplified using the same primers (lane 1). Amplified products
were analysed by 1.6~ agarose gel electrophoresis. HaeIII-digested
~bX174 DNA was used as a size marker (lane M). After denaturation,
1 ~tg (slots 6, 7, 8 and 9) and 5 ~tg (slots 10, 11, 12 and 13) of cellular
RNA prepared from MSB-1 cells in the absence of oligonucleotide
(slots 6 and 10), and in the presence of oligonucleotides A 1 (slots 7 and
11) and S1 (slots 8 and 12) at a concentration of 5 p.ta, and from BK-3
cells in the absence of oligonucleotide (slots 9 and 13), were spotted onto
the nitrocellulose membrane by using a slot blotting apparatus (BRL),
and hybridized with ~-actin as a probe.
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Antisense oligonucleotides against M D V gene
I
(a)
I
t
1
2
I 109
3
kbp
c)
e~
L.
8
23.1-9.4
r.
6"7--
I
24
!
48
Incubation time (h)
~
Q
:~:
~:
--BamHI-D
g
~
--BamHI-H
4.4--
!
72
2"3~
2-0--
(c)
(b)
1
2
3
4
5
1
2
3
4
5
-18-mer
Fig. 6. Incorporation of oligonucleotides into cells. (a) After incubation
of MSB-1 cells with radiolabelled 5ktM-oligonucleotide A1, the
radioactivity in the cell lysate was counted. Each point was an average
of four separate experiments; bars indicate standard deviation. (b and
c) After incubation of cells with radiolabelled 5 ~tM-oligonucleotideAt
for 0 (lane 1), 24 (lane 2), 48 (lane 3) and 72 (lane 4) h, the radioactive
compounds in the medium (b) and cell lysate (c) were analysed by 4 ~
agarose gel electrophoresis. Control oligonucleotide (lane 5) was not
incubated in the medium.
To investigate the stability of the oligonucleotide in
cells, oligonucleotide A 1 was radiolabelled and incorporated into cells (Fig. 6a). After incubation for 24, 48 and
72 h, radiolabelled compounds were analysed by 4 ~
Nusieve agarose gel electrophoresis. After incubation,
no change in the size of oligonucleotide A1 in the
medium, compared to that of a control oligonucleotide
(Fig. 6b), was observed. When the oligonucleotide in the
cell lysate was analysed (Fig. 6c), a slow migrating band
and a smear smaller than an 18-mer oligonucleotide were
observed.
To investigate whether the copy number of MDV
genomes in transformed lymphoblastoid MSB-1 cells
was decreased by treatment with oligonucleotide A1,
Fig. 7. Southern blot hybridization analysis of M D V D N A from
MSB-1 cells treated with antisense oligonucleotide. Cellular D N A was
extracted from transformed lymphoblastoid MSB-1 cells incubated in
the absence of oligonucleotide (lane 1), and in the presence of 5 ~tMoligonucleotides A1 (lane 2) and S1 (lane 3) for 70 h, digested with
BamHI and then hybridized with the BamHI-H fragment of M D V
D N A as a probe.
cellular DNA was extracted from MSB-1 cells which had
been incubated with oligonucleotide A1 for 70 h, and
analysed by Southern blot hybridization using the
B a m H I - H fragment of MDV DNA as a probe (Fig. 7).
The copy number of MDV DNA in oligonucleotide A1treated MSB-I cells was almost the same as that in
control cells without oligonucleotide or in cells treated
with oligonucleotide S1.
Discussion
Previous studies implicate the amplification of regions
contained in IR E and TRL, within the B a m H I - H and -D
fragments of MDV DNA respectively, as a critical event
associated with the loss of viral pathogenicity (Nazerian,
1970; Fukuchi et al., 1985b; Silva & Witter, 1985).
Previous reports predicted the existence of a gene (or
genes) within this region that was responsible for the
initiation or maintenance of the tumorigenic state
(Bradley et al., 1989a, b).
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1110
M. Kawamura and others
Although the molecular mechanisms underlying the
phenomenon are still unclear, the potential use of
antisense oligonucleotides in regulating the expression of
specific gene products and thus facilitating the analysis
of their function has been demonstrated (MarcusSekura, 1988; Stein & Cohen, 1988). To gain insight into
the biological role of the 1.8 kb BamHI-H gene family in
maintenance of the tumorigenic state of MDV-derived
transformed lymphoblastoid MSB-1 cells, we investigated the effects of treatment of MSB-1 cells with
oligonucleotides complementary to the 1-8 kb m R N A on
cellular proliferation and colony formation in soft agar.
The antiviral effects of oligonucleotides complementary to the splice junctions of herpes simplex virus type 1
immediate early pre-mRNAs 4 and 5 have been reported
(Smith et al., 1986) and therefore the splice sites might be
adequate targets for the study of inhibition of gene
expression by antisense oligonucleotides. We could not
detect the 1-8 kb mRNA from the BamHI-H region of
MDV DNA in cellular R N A from MSB-t cells treated
for 70 h with oligonucleotide A1, complementary to the
splice donor sequence of exon I of the 1.8 kb transcript;
synthesis of the 1-8 kb mRNA was not inhibited by
treatment of the cells with oligonucleotides S1, A2 or $2.
It was shown that oligonucleotide A1, at concentrations of 3 and 5 ~tM,inhibited cellular proliferation (Fig. 2
and 3). The inhibitory effect of oligonucleotide A1 was
sequence-specific because oligonucleotide S1 and control
oligonucleotides ( G A T A G A T A G A T A G A T A and
CATCATCATCATCAT, data not shown) showed no
inhibitory effect. When MSB-1 cells were incubated with
5 ~tM-oligonucleotide A1, approximately 1X of the
oligonucleotide was incorporated into cells in 24 h (Fig.
6) and radioactivity in the cells increased with incubation time. This result was in good agreement with the
recent report by Loke et al. (1989). The efficiency of
colony formation in soft agar also decreased markedly in
the presence of oligonucleotide A1 (Table 1). Although
the inhibitory effects on cellular proliferation were not
observed with 1 ~tM-oligonucleotide A1, the efficiency of
colony formation was significantly reduced. These
results suggest that expression of the 1.8 kb transcript is
essential for the continuous proliferation of transformed
lymphoblastoid MSB-1 cells and colony formation in soft
agar. The inhibitory effect was observed within 48 h of
treatment with oligonucleotide A1. This observation
suggests that the gene product of the BamHI-H region of
MDV DNA might turn over rapidly in transformed cells.
Exponential growth of MSB-1 cells was observed
when oligonucleotide A 1 was washed out of the medium
(Fig. 4). This result indicates that the inhibition of
cellular proliferation by treatment with oligonucleotide
A1 was reversible. Since no change in the size of
oligonucleotide A1 was observed, the oligonucleotide
remained intact in the medium during the 72 h incubation period (Fig. 6b). In contrast, smear products smaller
than the 18-met oligonucleotide were observed in cell
lysates after the incubation. These smear products
appear to be derived from degradation of the oligonucleotide. This result suggests that oligonucleotide A1
might react rapidly with the 1.8 kb m R N A and be
degraded. Therefore, continuous incorporation of oligonucleotide A1 into cells may be required for inhibition
of cellular proliferation, consistent with the observation
that removal of oligonucleotide from the medium results
in the growth of MSB-1 cells recovering rapidly.
A slowly migrating band also appeared in the cell
lysate, the character of which is not yet clear. Preliminary experiments showed that the mobility of the band
does not change after treatment with proteinase K, and
therefore it might be an intermediate product of the
reaction between oligonucleotide and mRNA, which
could be degraded by nucleases such as RNase H.
The transformed lymphoblastoid cell line, MSB-1, is
virus-producing and contains multiple copies of the
MDV genome. The copy number of MDV DNA in
oligonucleotide Al-treated MSB-1 cells was not reduced
compared to that in untreated control cells (Fig. 7). This
result shows that replication of the MDV genome in
MSB-1 cells is not inhibited by treatment with oligonucleotide A1.
No inhibitory effect of oligonucleotides complementary to mRNA transcribed from the BamHI-H region of
MDV DNA on the proliferation of ALL-induced
lymphoblastoid BK-3 cells was observed. This result is
consistent with the result showing that the 239 bp band
could not be detected in amplified products from the
cDNA of BK-3 cells. The BamHI-H fragment of MDV
DNA did not hybridize to cellular DNA prepared from
uninfected chicken fibroblasts (data not shown). Thus,
the inhibitory effect of oligonucleotide A1 is specific for
transformed lymphoblastoid cells derived from MDVinduced lymphoma and the possibility that oligonucleotide A1 inhibited expression of a chicken gene essential
for cellular proliferation can be excluded.
It has been reported that transformed cells treated
with c-fos antisense RNA show restored densitydependent growth arrest and reduced tumorigenicity
compared to control untreated cells (Mercola et al.,
1988), and that T lymphocyte proliferation was inhibited
by treatment with a c-myb antisense oligonucleotide
(Gewirtz et al., 1989). In the present study, we have
shown directly that the expression of the 1-8 kb mRNA
from the BamHI-H region of MDV DNA is essential for
the maintenance of the transformed phenotype of the
MDV-induced lymphoblastoid cell line, MSB-1.
The reason why oligonucleotide A2 did not show an
inhibitory effect remains unclear. A site 260 bp 3' to the
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Antisense oligonucleotides against MD V gene
Sinai site might be a major splice donor site of the
functional MDV 1.8 kb m R N A in transformed lymphoblastoid MSB-1 cells. Further analysis of the gene
product associated with transformation is in progress.
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