Vol.
166,
No.
February
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 1095-1101
14, 1990
TUMOR NECXOSIS FACTOR ANTAGONIZES INHIBITORY EFFECT OF AZIDOTHYMIDINE HUMAN IMMUNODEFICIENCY VIRUS (HIV) REPLICATION IN VITRO Masahiko
ITO,
Masanori SATO,
Department
of
Bacteriology,
*Rega
Received
BABA, Shuichi MORI, Kazuhiro HIR$BAYASHI, Shiro SHIGETA and Erik DE CLERCQ
Fukushima Japan
Institute,
Katholieke
December
19,
Medical
College,
Universiteit Belgium
Akihiko
Fukushima
Leuven,
B-3000
ON
960-12,
Leuven,
1989
Tumor necrosis factor a (TNF-a) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-l) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-l, increased from 5.8 nM in the absence of TNF-a to > 125 @l in the presence of TNF-a (100 TNF-a also antagonized the anti-HIV-l activity of dideoxycytidine U/d). but did not markedly affect the anti-HIV-l activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-a. 01990 Academic Press, Inc.
The acquired rious
diseases
shown
to
for
that
inhibit
(HIV-l), dine
isnuune
the
mankind the
agent
(azidothymidine,
AZT)
use
(2).
On the
the
pathogenesis
in
other
the
hand, of this
expression
of
is
virus
replication
(5).
We have
acutely
in
therapeutic treatment the
combined
was
found
T4
modalities, of
retrovirus effects
to
completely
only are
disease.
Folks This
several
T-cell that
from
AIDS and AIDS-related
et
cells
(6). in
the
a critical
(3)
have
reported
(GM-CSF) (4).
HIV-l
with
complex that
shown
of
in
factor enhance
with
HIV-l
of HIV-l considered
agents,
interest
to
replication activity
gra-
up-regu-
to
infected been
in of
in
observed
replication have
approved
necrosis
been
antiviral
seemed
anti-HIV-l
1
role
been
Tumor
chronically enhances
on HIV-l
been
type
markedly
also
recently
cytokines it
se-
have
been
play
has
also
so far
to
with
combination
of AZT and TNF-a
has
phenomenon
Since
infections, reverse
al.
has
most
virus
that
lines
TNF-a
of compounds
drug
assumed
and TNF-a
the
3'-azido-2',3'-dideoxythymi-
factor
infected
one of
immunodeficiency
yet,
stimulating HIV-l.
is
A number
suffering
cytokine,
demonstrated
infected
the
(AIDS)
human
cytokines
acutely
a typical
of
is
colony
monocyte-macrophages (TNF)
now facing.
of AIDS (1);
patients
nulocyte-macrophage lates
is
syndrome
replication
causative
clinical
deficiency
in as
for
the
investigate vitro.
AZT in
TNF-a MOLT-4
cells. 0006-291X@CI $1.50
1095
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
166, No. 3. 1990
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS
RESEARCH COMMUNICATIONS
AND METHODS
Cells
and Virus The T4 lymphocyte cell line, MOLT-4 clone No. 8 (7), was used throughout the experiments. The cells were cultured and maintained in RPMI-1640 medium supplemented with 10% fetal calf medium, 100 U/ml penicillin G, and 100 kg/ml streptomycin. HIV-l was obtained from the culture supernatant of MOLT-4/HTLV-IIIB, as previously described (8). Compounds Recombinant TNF-a was obtained from Genzyme (Boston, MA). Dextran sulfate (molecular weight: 5,000) was purchased from Sigma Chemical Co. (St. Louis, MO). AZT and 2' ,3'-dideoxycytidine (DDC) were synthesized by Dr. P. Herdewijn (Rega Institute, Belgium). [!-fetby1-3H]AZT (33 Ci/mmol) was supplied by Moravek Biochemicals (Brea, CA). Antiviral assay Activity of the compounds against HIV-l replication was based on the inhibition of virus-induced cytopathogenicity in MOLT-4 cell cultures. Briefly, MOLT-4 cells were suspended at 1 x 10' cells/ml and infected with HIV-l at a multiplicity of infection (MOI) of 0.1. After 60-min virus adsorption, 100 ~1 of the cell suspension was placed into each well of a flat-bottomed microtiter tray containing various concentrations of TNF-a and the test compounds. After a 4-day incubation at 37%, the cells were subcultured with fresh medium containing appropriate concentrations of TNFa and the test compounds. On day 7, the number of viable cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli~ bromide (MTT) method, as previously described (9). The MTT method was also used to assess cytotoxicity of the compounds for mock-infected MOLT-4 cells. Analysis for metabolism of AZT Intracellular metabolism of AZT in the presence or absence of TNF-a was analyzed following a slight modification of the HPLC procedure described by Perno et al. (10). Briefly, 5 x lo6 MOLT-4 cells were incubated at 37°C in the presence or absence of TNF-a (100 U/ml) and exposed to After 24 h-incubation, the cells were washed three 1 pM [methyl-3H]AZT. times with ice-cold RPMI-1640 and immediately frozen in dry ice. The cells were then extracted with 60% (v/v) methanol, and the methanol extracts were further heated at 9S°C for 1.5 min. The extracts were clarified by centrifugation at 12.000 g for 6 min. and 100 yl-aliquots were loaded onto a Partisil-10 SAX column and eluted with ammonium phosphate (11). RESULTS When MOLT-4
cells
of any compounds the of
cells the
been
cells
to
(Fig.
1B).
U/ml),
AZT could
hence,
the
anti-HIV-l
cells
activity the
activity bility
AZT
next
more
no
longer
were
of
cells
the
if from
Thus,
formed
cell
the
the
HIV-l-infected giant
were
of
and death cells
cell at
cells
absence
and most
formation
destruction
infected
suppress
destroyed.
were
Giant
in
had
formation
and
a concentration exposed
to
TNF-a
giant cell formation (Fig. TNF-a seemed to annihilate
lC), the
of AZT. set
of experiments,
HIV-l-infected of
and cultured
inhibited
cells
when
of AZT was monitored
concentrations
rapidly
completely
MOLT-4
(100
In
giant 1A).
However,
and,
HIV-l
multinuclear
much
TNF-a.
with
by day 7 (Fig.
HIV-l-infected
1 Ql
infected
TNF-a,
destroyed occurred
exposed
protected of
or
were
were
AZT with
the
by the
MOLT-4
effect
of TNF-a
MTT procedure.
cells
cultured
or without
TNF-a.
1096
in At
Fig. the the
on the
anti-HIV-l
2 shows
presence concentrations
the of
via-
various used,
Vol.
166,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 1. MOLT-4 cells following infection with HIV-l and incubation for 7 days in the absence of AZT and TNF-a (panel A). in the presence of 1 @-l AZT (panel B), and in the presence of 1 @-! AZT plus 100 U/ml TNF-a (panel C). Giant cells are apparent in panels A and C, but not in panel B.
TNF-a
reduced
U/ml)
or
of
the
30% (Fig.
killed
dose-dependent of
activity
cells
(not 2B).
cells
and
100 U/ml
of TNF-a,
was apparent or 7 days 1 shows
cytotoxic
AZT is
0.0058
pM).
HIV-l
activity TNF-a
topathogenicity of AZT
the (Fig.
the
for
viability
an extremely
at
the
of AZT,
from
the
protective viability
of
presence
antagonistic was
in a
a concentrathe
of 10
effect tested
evaluated
concentration
DDC and
inhibitor
100
it
could
with
(EC50: not
at a concentration
3.8
achieve as high
cells
dextran
of HIV-l
AZT was combined
MOLT-4
cells
effective
by 650-fold
mock-infected
destruction
Ql AZT the
This
by
had been
of (0.32
at
4 days
infection.
decreased U/ml,
2B).
cells
reduced
0.2
infection,
of AZT concentrations
of
potent
when
even
markedly of
range
2B) after
against
5% (10
was reduced
all
was achieved
(Fig.
entire
(CC50)
However,
cells
than
virus
100% to 25 and 5% in
50% antiviral
concentration
cells.
with
the
whether
2A)
the
TNF-a
respectively
over
Table
infection,
protection
from
after
AZT or TNF-a)
after
at a concentration
was reduced
by no more
4 days
to either
7 days
However,
so that
infected
At
AZT protected
2B).
the
(Fig.
exposed at
cells
shown).
complete
p&l (Fig.
nM - 125 @i),
not
However,
(Fig.
of AZT,
TNF-a
(data
fashion:
0.2
of mock-infected
U/ml)
ZA).
by HIV-l
tion
viability
20% (100
viability only
the
was not
1097
(EC50) sulfate
in
in MOLT-4
TNF-a @l).
MOLT-4
cells
(10 U/ml), When AZT was
50% inhibition
and 50%
of
(EC50:
its
anti-
combined virus
cy-
as 125 @-l. The cytotoxicity affected
by TNF-a
(CC50:
7
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
concentration
RESEARCH COMMUNICATIONS
(PM)
Figure 2. Dose-response curves for the inhibitory effect of AZT. in combination with TNF-a at either 0, 10 or 100 U/ml, on HIV-l-induced cytopathogenicity in MOLT-4 cells. The cells were infected with HIV-1 at a MO1 of 0.1 and cultured in the presence of various concentrations of AZT and TNF-a. On day 4 (panel A) and 7 (panel B) after infection, the number of viable cells was determined by the MTT method and expressed as percent of the number of mock-infected control cells. Concentrations of AZT: as indicated on the x-axis; concentrations of TNF-a: n , 0 U/ml: 0, 10 U/ml; 0. 100 U/ml. Data represent mean values for two separate experiments. 125 p&f throughout). a. with
but
to a lesser
TNF-a
at 0,
TABLE 1. Effect
Compound
The anti-HIV-l extent
than
10 or 100 U/ml,
activity that
of DDC was also
of AZT.
The EC50 of DDC, when 0.15
of TNF-a on anti-HIV-l activity and cytotoxicity DDC, and dextran sulfate in MOLT-4 cells
of AZT,
0.20
EC50a (WLM)
0 10 100
DDC
0 10 100
0.026 0.15 0.20
0 10 100
1.1 2.2 3.1
sulfate
and
combined
respectively
AZT
Dextran
by TNF-
pM,
TNF-a (U/ml)
was 0.016,
reduced
0.0058 3.8 > 125
'50% Antiviral effective concentration, based on the inhibition induced cytopathogenicity in MOLT-4 cells. b50% Cytotoxic concentration, based on the reduction of viability infected MOLT-4 cells. Data represent mean values for two separate experiments. 1098
CCSOb (IN > 125 > 125 > 125 0.43 0.38 0.27 > 600 > 600 > 600 of HIV-lof mock-
Vol.
166, No. 3, 1990
(Table
1).
BIOCHEMICAL
In contrast
with
When DDC was
combined
on the
of
ratio
these
conditions,
cation.
The
fluenced
with
CC.30 to
anti-HIV-l in
the
to
Therefore,
4 cells
(100
had
been
U/ml).
of AZT and
had been
exposed
does
alter
not
in
the
were
or not
exposed
di-
intracellular
only
(Table
of AZT may metabolism
These
phosphorylation
or
without
intracellular between
results
of
of AZT in MOLT-
AZT with
in the
triphosphate
in-
on HIV-l
1).
pattern of
repli-
slightly
activity
presence
to TNF-a.
under
HIV-l
intracellular
detected or
based
that
effect
anti-HIV-l the
phosphorylation
incubated mono-,
was
inhibitory
of TNF-a
on the
to
cells.
index
indicates
sulfate
with
the
selectivity
which
its
of TNF-a
its
the
dextran
of TNF-a
to MOLT-4
inhibitory
retained
No differences
trations
its
1.3,
of 100 U/ml
we investigated
which
TNF-a
of
presence
effect
to
toxicity
selectively
sulfate
interference
high
of TNF-a,
dropped
activity
The antagonizing AZT.
100 U/ml ECS0,
Dextran
even
be ascribed
DDC showed
DDC was no longer
by TNF-a.
replication
AZT,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
concen-
the
cells
that
that
TNF-a
indicate
pattern
of AZT.
DISCUSSION After is
initial
maintained
fase
of
infection as proviral
the
infection
mechanism
by which
elucidated,
various
tion
of
TNF-a now
and
last
active factors
demonstrate
HIV-l
another
replication
cytokine, in
both
a monocytic
The results
hibitory
to
replication
replication
HIV-l in
1 activity
primary
resulting
Although
is
induced
has
that
of
activates
We have
previously
infected
MOLT-4
latent the
not
as triggers
anti-HIV-l
increase
treated
with
production The
primary
in
exact
yet
been
the
induc-
the
latent
shown cells
activity
TNF-a,
combined cell
that
(6), of
and
AZT in
CEM cells,
monocytes
line
U-937 in
cells
for
and primary that
but
its
(4,lO).
stimulatory
has cells
The effect
line
to
been
observed if
on
monocyte to
the of
another.
little,
effect
GM-CSF proved
GM-CSF potentiated
one cell
in MT-4
examined
any,
HIV-l
anti-HIV-
cytokines
For in
in-
on
example,
MOLT-4
increase
a
cells
in virus
(6).
effects
lines.
U-937
production
whereas
been
inconsistent
However,
from
virus
was detected
activity
in
may vary
cell
were
monocytes.
of AZT in
replication
marked
in
The
(12).
the
has recently
(4,10,14).
several
cell.
factors
acutely
RNA genome
years
TNF-a.
annihilates
cultures
HIV-l
viral
recognized such
of AIDS is TNF-a
the
cells. GM-CSF,
HIV-l
been
in
HIV-l,
infected
several
One of
replication
that
the
with
replication
have
(13).
HIV-l
cells
for
HIV-l
progression
enhances
MOLT-4
DNA in can
HIV expression
infection
of the
of AZT and TNF-a
In .MOLT-4
of AZT.
TNF-a
albeit
to
cells,
also
a lesser
TNF-a
diminished extent 1099
have
also
been
completely the than
reversed
anti-HIV-l in
investigated
MOLT-4
activity cells
in
the
antiof AZT
(data
not
Vol.
166, No. 3, 1990
BIOCHEMICAL
I
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A
B
UT
h
AZTDP
AZTTP \ZT
lo2 0
20
60
40
20
40
60
t (min) Figure 3. Ion exchange (Partisil-10 SAX) HPLC elution methanol extract of MOLT-4 cells incubated for 24 h with @J) in the absence (A) or presence of TNF-a (100 U/ml) (B).
shown).
In
presence 100
CHM cells,
of
U/ml,
stant
10 U/ml to
0.24
of
The
mechanism
enhances
intracellular
cate
that
TNF-a
3). formation) their cD4+
cells
fated
are
TNF-a
TNF-a and
fore,
these
tion.
This
thus
the of
It
would
dideoxynucleosides sulfate)
(AZT, (Table
In
the
nM and,
in
the
presence
of
the
or
con-
quasi
presence
anti-HIV-l
(10
or
activity
of
to
form
well-known
that
formation
HIV-l
sulfate
replication of
prone
to
gp120 giant
why TNF-a
counteracts
DDC)
not
but
1). 1100
that
of
(Fig.
role
AZT (giant
in
the
cell loss
dideoxynucleosides
(expressing
expression
indi-
syncytia
an important
cell
studies
metabolism
cells
as dextran
be more
Our metabolic
giant cells
such
explain
nM.
absence
(10).
plays
is to
up-regulates
cells
5.1
intracellular the
infection
inhibitory
would
the
annihilates
of AZT
alter
HIV-l
enhances
was
The mechanism by which GM-CSF of AZT may be attributed to an increase in
and HIV-l-infected
cells.
(dextran
not
HIV-l
EC50 of AZT remained
of
60% (1
shown).
propensity
polysaccharides
(X,16).
the
8.0
of
to be elucidated.
to AZT. not
up to
cells,
activity
does
sensitivity AZT
not
remains
the
following
ding
went
phosphorylation
Presumably,
AZT against
irrespective
by which
anti-HIV-l
of
it
MT-4
(data
cells
the
In @I),
of TNF-a
AZT in MOLT-4
EC50
TNF-a
@I.
(0.0019-0.0026
100 U/ml)
the
the
profile [methyl-3H]AZT
between
gp120).
In
interfere in
on the cell
formation
the of
cell anti-HIV-l
sulfated
inclu(uninfected)
contrast,
with
acutely
of
this
sulprocess
infected
MOLT-4
surface.
There-
and destrucactivity polysaccharides
of
Vol.
BIOCHEMICAL
166, No. 3, 1990 It
to the act,
remains in and
vivo even
to
be established
situation.
dideoxynucleosides
in
dideoxynucleotides
with
whether the
However,
annihilate,
AND BIOPHYSICAL
the
the fact
present that
anti-HIV-l
vitro
cautions
TNF in
the
RESEARCH COMMUNICATIONS
TNF-a
activity against
treatment
findings is of
combination
also
able AZT
extend
to counterand therapy
other of
of AIDS patients.
REFERENCES 1. 2.
3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16.
De Clercq, E. (1989) Antiviral Res. 12, l-20. Fischl, M.A., Richman, D.D., Grieco, M.H., Gottlieb, M.S., Volberding, P.A., Laskin. O.L., Leedom, J.M., Groopman, J.E., Mildvan, D., Schooley, R.T., Jackson, G.G., Durack, D.T., King, D., and the AZT Collaborative Working Group (1987) New Engl. J. Med. 317, 185-191. Folks, T.M., Justement, J., Kinter, A., Dinarello, C.A., and Fauci, A.S. (1987) Science 238, 800-802. Koyanagi, Y.. O'Brien, W.A., Zhao, J.Q., Golde, D.W., Gasson, J.C., and Chen, I.S.Y. (1988) Science 241, 1673-1675. Matsuyama, T., Hamamoto, Y., Soma, G., Mizuno, D., Yamamoto, N., and Kobayashi, N. (1989) J. Virol. 63, 2504-2509. Ito, M., Baba, M., Sato, A., Hirabayashi, K., Tanabe, F., Shigeta, S., and De Clercq, E. (1989) Biochem. Biophys. Res. Commun. 158, 307-312. Kikukawa, R., Koyanagi, Y.. Harada, S., Kobayashi, N., Hatanaka, M., and Yamamoto, N. (1986) J. Virol. 57, 1159-1162. Harada, S., Koyanagi, Y.. and Yamamoto, N. (1985) Virology 146, 272281. Balzarini, J., Baba, M., Snoeck, R.. Schols, D., Pauwels, R., Herdewijn, P., Desmyter, J., and De Clercq, E. (1988) J. Virol. Methods 20, 309-321. Perno, C.-F., Yarchoan, R., Cooney, D.A., Hartman, N.R., Webb, D.S.A., Hao, Z., Mitsuya, H., Johns, D.G., and Broder, S. (1989) J. Exp. Med. 169, 933-951. Cooney, D.A., Dalal, M., Mitsuya, H., McMahon, J.B., Nadkarni, M., Balzarini, J., Broder, S., and Johns, D.G. (1986) Biochem. Pharmacol. 35. 2065-2068. Melbye, M., Bigger, R.J., Ebbesen, P., Neuland, C., Goedert, J., Faber, V., Lorenzen, I., Skinhoj, P., Gallo, R.C., and Blattner, W.A. (1986) Ann. Intern. Med. 104, 496-500. Rosenberg, Z.F., and Fauci, A.S. (1989) AIDS Res. Human Retrovir 5, l4. Hammer, S.M.. and Gillis, J.M. (1987) Antimicrob. Agents Chemother. 31, 1046-1050. Mitsuya, H., Looney, D.J., Kuno, S., Ueno, R., Wong-Staal, F., and Broder, S. (1988) Science 240, 646-649. Baba, M., Schols, D., Pauwels, R., Nakashima, H., and De Clercq, E. (1990). J. Acquir. Immun. Defic. Syndr., in press.
1101
166,
No.
February
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 1095-1101
14, 1990
TUMOR NECXOSIS FACTOR ANTAGONIZES INHIBITORY EFFECT OF AZIDOTHYMIDINE HUMAN IMMUNODEFICIENCY VIRUS (HIV) REPLICATION IN VITRO Masahiko
ITO,
Masanori SATO,
Department
of
Bacteriology,
*Rega
Received
BABA, Shuichi MORI, Kazuhiro HIR$BAYASHI, Shiro SHIGETA and Erik DE CLERCQ
Fukushima Japan
Institute,
Katholieke
December
19,
Medical
College,
Universiteit Belgium
Akihiko
Fukushima
Leuven,
B-3000
ON
960-12,
Leuven,
1989
Tumor necrosis factor a (TNF-a) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-l) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-l, increased from 5.8 nM in the absence of TNF-a to > 125 @l in the presence of TNF-a (100 TNF-a also antagonized the anti-HIV-l activity of dideoxycytidine U/d). but did not markedly affect the anti-HIV-l activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-a. 01990 Academic Press, Inc.
The acquired rious
diseases
shown
to
for
that
inhibit
(HIV-l), dine
isnuune
the
mankind the
agent
(azidothymidine,
AZT)
use
(2).
On the
the
pathogenesis
in
other
the
hand, of this
expression
of
is
virus
replication
(5).
We have
acutely
in
therapeutic treatment the
combined
was
found
T4
modalities, of
retrovirus effects
to
completely
only are
disease.
Folks This
several
T-cell that
from
AIDS and AIDS-related
et
cells
(6). in
the
a critical
(3)
have
reported
(GM-CSF) (4).
HIV-l
with
complex that
shown
of
in
factor enhance
with
HIV-l
of HIV-l considered
agents,
interest
to
replication activity
gra-
up-regu-
to
infected been
in of
in
observed
replication have
approved
necrosis
been
antiviral
seemed
anti-HIV-l
1
role
been
Tumor
chronically enhances
on HIV-l
been
type
markedly
also
recently
cytokines it
se-
have
been
play
has
also
so far
to
with
combination
of AZT and TNF-a
has
phenomenon
Since
infections, reverse
al.
has
most
virus
that
lines
TNF-a
of compounds
drug
assumed
and TNF-a
the
3'-azido-2',3'-dideoxythymi-
factor
infected
one of
immunodeficiency
yet,
stimulating HIV-l.
is
A number
suffering
cytokine,
demonstrated
infected
the
(AIDS)
human
cytokines
acutely
a typical
of
is
colony
monocyte-macrophages (TNF)
now facing.
of AIDS (1);
patients
nulocyte-macrophage lates
is
syndrome
replication
causative
clinical
deficiency
in as
for
the
investigate vitro.
AZT in
TNF-a MOLT-4
cells. 0006-291X@CI $1.50
1095
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
166, No. 3. 1990
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS
RESEARCH COMMUNICATIONS
AND METHODS
Cells
and Virus The T4 lymphocyte cell line, MOLT-4 clone No. 8 (7), was used throughout the experiments. The cells were cultured and maintained in RPMI-1640 medium supplemented with 10% fetal calf medium, 100 U/ml penicillin G, and 100 kg/ml streptomycin. HIV-l was obtained from the culture supernatant of MOLT-4/HTLV-IIIB, as previously described (8). Compounds Recombinant TNF-a was obtained from Genzyme (Boston, MA). Dextran sulfate (molecular weight: 5,000) was purchased from Sigma Chemical Co. (St. Louis, MO). AZT and 2' ,3'-dideoxycytidine (DDC) were synthesized by Dr. P. Herdewijn (Rega Institute, Belgium). [!-fetby1-3H]AZT (33 Ci/mmol) was supplied by Moravek Biochemicals (Brea, CA). Antiviral assay Activity of the compounds against HIV-l replication was based on the inhibition of virus-induced cytopathogenicity in MOLT-4 cell cultures. Briefly, MOLT-4 cells were suspended at 1 x 10' cells/ml and infected with HIV-l at a multiplicity of infection (MOI) of 0.1. After 60-min virus adsorption, 100 ~1 of the cell suspension was placed into each well of a flat-bottomed microtiter tray containing various concentrations of TNF-a and the test compounds. After a 4-day incubation at 37%, the cells were subcultured with fresh medium containing appropriate concentrations of TNFa and the test compounds. On day 7, the number of viable cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli~ bromide (MTT) method, as previously described (9). The MTT method was also used to assess cytotoxicity of the compounds for mock-infected MOLT-4 cells. Analysis for metabolism of AZT Intracellular metabolism of AZT in the presence or absence of TNF-a was analyzed following a slight modification of the HPLC procedure described by Perno et al. (10). Briefly, 5 x lo6 MOLT-4 cells were incubated at 37°C in the presence or absence of TNF-a (100 U/ml) and exposed to After 24 h-incubation, the cells were washed three 1 pM [methyl-3H]AZT. times with ice-cold RPMI-1640 and immediately frozen in dry ice. The cells were then extracted with 60% (v/v) methanol, and the methanol extracts were further heated at 9S°C for 1.5 min. The extracts were clarified by centrifugation at 12.000 g for 6 min. and 100 yl-aliquots were loaded onto a Partisil-10 SAX column and eluted with ammonium phosphate (11). RESULTS When MOLT-4
cells
of any compounds the of
cells the
been
cells
to
(Fig.
1B).
U/ml),
AZT could
hence,
the
anti-HIV-l
cells
activity the
activity bility
AZT
next
more
no
longer
were
of
cells
the
if from
Thus,
formed
cell
the
the
HIV-l-infected giant
were
of
and death cells
cell at
cells
absence
and most
formation
destruction
infected
suppress
destroyed.
were
Giant
in
had
formation
and
a concentration exposed
to
TNF-a
giant cell formation (Fig. TNF-a seemed to annihilate
lC), the
of AZT. set
of experiments,
HIV-l-infected of
and cultured
inhibited
cells
when
of AZT was monitored
concentrations
rapidly
completely
MOLT-4
(100
In
giant 1A).
However,
and,
HIV-l
multinuclear
much
TNF-a.
with
by day 7 (Fig.
HIV-l-infected
1 Ql
infected
TNF-a,
destroyed occurred
exposed
protected of
or
were
were
AZT with
the
by the
MOLT-4
effect
of TNF-a
MTT procedure.
cells
cultured
or without
TNF-a.
1096
in At
Fig. the the
on the
anti-HIV-l
2 shows
presence concentrations
the of
via-
various used,
Vol.
166,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 1. MOLT-4 cells following infection with HIV-l and incubation for 7 days in the absence of AZT and TNF-a (panel A). in the presence of 1 @-l AZT (panel B), and in the presence of 1 @-! AZT plus 100 U/ml TNF-a (panel C). Giant cells are apparent in panels A and C, but not in panel B.
TNF-a
reduced
U/ml)
or
of
the
30% (Fig.
killed
dose-dependent of
activity
cells
(not 2B).
cells
and
100 U/ml
of TNF-a,
was apparent or 7 days 1 shows
cytotoxic
AZT is
0.0058
pM).
HIV-l
activity TNF-a
topathogenicity of AZT
the (Fig.
the
for
viability
an extremely
at
the
of AZT,
from
the
protective viability
of
presence
antagonistic was
in a
a concentrathe
of 10
effect tested
evaluated
concentration
DDC and
inhibitor
100
it
could
with
(EC50: not
at a concentration
3.8
achieve as high
cells
dextran
of HIV-l
AZT was combined
MOLT-4
cells
effective
by 650-fold
mock-infected
destruction
Ql AZT the
This
by
had been
of (0.32
at
4 days
infection.
decreased U/ml,
2B).
cells
reduced
0.2
infection,
of AZT concentrations
of
potent
when
even
markedly of
range
2B) after
against
5% (10
was reduced
all
was achieved
(Fig.
entire
(CC50)
However,
cells
than
virus
100% to 25 and 5% in
50% antiviral
concentration
cells.
with
the
whether
2A)
the
TNF-a
respectively
over
Table
infection,
protection
from
after
AZT or TNF-a)
after
at a concentration
was reduced
by no more
4 days
to either
7 days
However,
so that
infected
At
AZT protected
2B).
the
(Fig.
exposed at
cells
shown).
complete
p&l (Fig.
nM - 125 @i),
not
However,
(Fig.
of AZT,
TNF-a
(data
fashion:
0.2
of mock-infected
U/ml)
ZA).
by HIV-l
tion
viability
20% (100
viability only
the
was not
1097
(EC50) sulfate
in
in MOLT-4
TNF-a @l).
MOLT-4
cells
(10 U/ml), When AZT was
50% inhibition
and 50%
of
(EC50:
its
anti-
combined virus
cy-
as 125 @-l. The cytotoxicity affected
by TNF-a
(CC50:
7
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
concentration
RESEARCH COMMUNICATIONS
(PM)
Figure 2. Dose-response curves for the inhibitory effect of AZT. in combination with TNF-a at either 0, 10 or 100 U/ml, on HIV-l-induced cytopathogenicity in MOLT-4 cells. The cells were infected with HIV-1 at a MO1 of 0.1 and cultured in the presence of various concentrations of AZT and TNF-a. On day 4 (panel A) and 7 (panel B) after infection, the number of viable cells was determined by the MTT method and expressed as percent of the number of mock-infected control cells. Concentrations of AZT: as indicated on the x-axis; concentrations of TNF-a: n , 0 U/ml: 0, 10 U/ml; 0. 100 U/ml. Data represent mean values for two separate experiments. 125 p&f throughout). a. with
but
to a lesser
TNF-a
at 0,
TABLE 1. Effect
Compound
The anti-HIV-l extent
than
10 or 100 U/ml,
activity that
of DDC was also
of AZT.
The EC50 of DDC, when 0.15
of TNF-a on anti-HIV-l activity and cytotoxicity DDC, and dextran sulfate in MOLT-4 cells
of AZT,
0.20
EC50a (WLM)
0 10 100
DDC
0 10 100
0.026 0.15 0.20
0 10 100
1.1 2.2 3.1
sulfate
and
combined
respectively
AZT
Dextran
by TNF-
pM,
TNF-a (U/ml)
was 0.016,
reduced
0.0058 3.8 > 125
'50% Antiviral effective concentration, based on the inhibition induced cytopathogenicity in MOLT-4 cells. b50% Cytotoxic concentration, based on the reduction of viability infected MOLT-4 cells. Data represent mean values for two separate experiments. 1098
CCSOb (IN > 125 > 125 > 125 0.43 0.38 0.27 > 600 > 600 > 600 of HIV-lof mock-
Vol.
166, No. 3, 1990
(Table
1).
BIOCHEMICAL
In contrast
with
When DDC was
combined
on the
of
ratio
these
conditions,
cation.
The
fluenced
with
CC.30 to
anti-HIV-l in
the
to
Therefore,
4 cells
(100
had
been
U/ml).
of AZT and
had been
exposed
does
alter
not
in
the
were
or not
exposed
di-
intracellular
only
(Table
of AZT may metabolism
These
phosphorylation
or
without
intracellular between
results
of
of AZT in MOLT-
AZT with
in the
triphosphate
in-
on HIV-l
1).
pattern of
repli-
slightly
activity
presence
to TNF-a.
under
HIV-l
intracellular
detected or
based
that
effect
anti-HIV-l the
phosphorylation
incubated mono-,
was
inhibitory
of TNF-a
on the
to
cells.
index
indicates
sulfate
with
the
selectivity
which
its
of TNF-a
its
the
dextran
of TNF-a
to MOLT-4
inhibitory
retained
No differences
trations
its
1.3,
of 100 U/ml
we investigated
which
TNF-a
of
presence
effect
to
toxicity
selectively
sulfate
interference
high
of TNF-a,
dropped
activity
The antagonizing AZT.
100 U/ml ECS0,
Dextran
even
be ascribed
DDC showed
DDC was no longer
by TNF-a.
replication
AZT,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
concen-
the
cells
that
that
TNF-a
indicate
pattern
of AZT.
DISCUSSION After is
initial
maintained
fase
of
infection as proviral
the
infection
mechanism
by which
elucidated,
various
tion
of
TNF-a now
and
last
active factors
demonstrate
HIV-l
another
replication
cytokine, in
both
a monocytic
The results
hibitory
to
replication
replication
HIV-l in
1 activity
primary
resulting
Although
is
induced
has
that
of
activates
We have
previously
infected
MOLT-4
latent the
not
as triggers
anti-HIV-l
increase
treated
with
production The
primary
in
exact
yet
been
the
induc-
the
latent
shown cells
activity
TNF-a,
combined cell
that
(6), of
and
AZT in
CEM cells,
monocytes
line
U-937 in
cells
for
and primary that
but
its
(4,lO).
stimulatory
has cells
The effect
line
to
been
observed if
on
monocyte to
the of
another.
little,
effect
GM-CSF proved
GM-CSF potentiated
one cell
in MT-4
examined
any,
HIV-l
anti-HIV-
cytokines
For in
in-
on
example,
MOLT-4
increase
a
cells
in virus
(6).
effects
lines.
U-937
production
whereas
been
inconsistent
However,
from
virus
was detected
activity
in
may vary
cell
were
monocytes.
of AZT in
replication
marked
in
The
(12).
the
has recently
(4,10,14).
several
cell.
factors
acutely
RNA genome
years
TNF-a.
annihilates
cultures
HIV-l
viral
recognized such
of AIDS is TNF-a
the
cells. GM-CSF,
HIV-l
been
in
HIV-l,
infected
several
One of
replication
that
the
with
replication
have
(13).
HIV-l
cells
for
HIV-l
progression
enhances
MOLT-4
DNA in can
HIV expression
infection
of the
of AZT and TNF-a
In .MOLT-4
of AZT.
TNF-a
albeit
to
cells,
also
a lesser
TNF-a
diminished extent 1099
have
also
been
completely the than
reversed
anti-HIV-l in
investigated
MOLT-4
activity cells
in
the
antiof AZT
(data
not
Vol.
166, No. 3, 1990
BIOCHEMICAL
I
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A
B
UT
h
AZTDP
AZTTP \ZT
lo2 0
20
60
40
20
40
60
t (min) Figure 3. Ion exchange (Partisil-10 SAX) HPLC elution methanol extract of MOLT-4 cells incubated for 24 h with @J) in the absence (A) or presence of TNF-a (100 U/ml) (B).
shown).
In
presence 100
CHM cells,
of
U/ml,
stant
10 U/ml to
0.24
of
The
mechanism
enhances
intracellular
cate
that
TNF-a
3). formation) their cD4+
cells
fated
are
TNF-a
TNF-a and
fore,
these
tion.
This
thus
the of
It
would
dideoxynucleosides sulfate)
(AZT, (Table
In
the
nM and,
in
the
presence
of
the
or
con-
quasi
presence
anti-HIV-l
(10
or
activity
of
to
form
well-known
that
formation
HIV-l
sulfate
replication of
prone
to
gp120 giant
why TNF-a
counteracts
DDC)
not
but
1). 1100
that
of
(Fig.
role
AZT (giant
in
the
cell loss
dideoxynucleosides
(expressing
expression
indi-
syncytia
an important
cell
studies
metabolism
cells
as dextran
be more
Our metabolic
giant cells
such
explain
nM.
absence
(10).
plays
is to
up-regulates
cells
5.1
intracellular the
infection
inhibitory
would
the
annihilates
of AZT
alter
HIV-l
enhances
was
The mechanism by which GM-CSF of AZT may be attributed to an increase in
and HIV-l-infected
cells.
(dextran
not
HIV-l
EC50 of AZT remained
of
60% (1
shown).
propensity
polysaccharides
(X,16).
the
8.0
of
to be elucidated.
to AZT. not
up to
cells,
activity
does
sensitivity AZT
not
remains
the
following
ding
went
phosphorylation
Presumably,
AZT against
irrespective
by which
anti-HIV-l
of
it
MT-4
(data
cells
the
In @I),
of TNF-a
AZT in MOLT-4
EC50
TNF-a
@I.
(0.0019-0.0026
100 U/ml)
the
the
profile [methyl-3H]AZT
between
gp120).
In
interfere in
on the cell
formation
the of
cell anti-HIV-l
sulfated
inclu(uninfected)
contrast,
with
acutely
of
this
sulprocess
infected
MOLT-4
surface.
There-
and destrucactivity polysaccharides
of
Vol.
BIOCHEMICAL
166, No. 3, 1990 It
to the act,
remains in and
vivo even
to
be established
situation.
dideoxynucleosides
in
dideoxynucleotides
with
whether the
However,
annihilate,
AND BIOPHYSICAL
the
the fact
present that
anti-HIV-l
vitro
cautions
TNF in
the
RESEARCH COMMUNICATIONS
TNF-a
activity against
treatment
findings is of
combination
also
able AZT
extend
to counterand therapy
other of
of AIDS patients.
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3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16.
De Clercq, E. (1989) Antiviral Res. 12, l-20. Fischl, M.A., Richman, D.D., Grieco, M.H., Gottlieb, M.S., Volberding, P.A., Laskin. O.L., Leedom, J.M., Groopman, J.E., Mildvan, D., Schooley, R.T., Jackson, G.G., Durack, D.T., King, D., and the AZT Collaborative Working Group (1987) New Engl. J. Med. 317, 185-191. Folks, T.M., Justement, J., Kinter, A., Dinarello, C.A., and Fauci, A.S. (1987) Science 238, 800-802. Koyanagi, Y.. O'Brien, W.A., Zhao, J.Q., Golde, D.W., Gasson, J.C., and Chen, I.S.Y. (1988) Science 241, 1673-1675. Matsuyama, T., Hamamoto, Y., Soma, G., Mizuno, D., Yamamoto, N., and Kobayashi, N. (1989) J. Virol. 63, 2504-2509. Ito, M., Baba, M., Sato, A., Hirabayashi, K., Tanabe, F., Shigeta, S., and De Clercq, E. (1989) Biochem. Biophys. Res. Commun. 158, 307-312. Kikukawa, R., Koyanagi, Y.. Harada, S., Kobayashi, N., Hatanaka, M., and Yamamoto, N. (1986) J. Virol. 57, 1159-1162. Harada, S., Koyanagi, Y.. and Yamamoto, N. (1985) Virology 146, 272281. Balzarini, J., Baba, M., Snoeck, R.. Schols, D., Pauwels, R., Herdewijn, P., Desmyter, J., and De Clercq, E. (1988) J. Virol. Methods 20, 309-321. Perno, C.-F., Yarchoan, R., Cooney, D.A., Hartman, N.R., Webb, D.S.A., Hao, Z., Mitsuya, H., Johns, D.G., and Broder, S. (1989) J. Exp. Med. 169, 933-951. Cooney, D.A., Dalal, M., Mitsuya, H., McMahon, J.B., Nadkarni, M., Balzarini, J., Broder, S., and Johns, D.G. (1986) Biochem. Pharmacol. 35. 2065-2068. Melbye, M., Bigger, R.J., Ebbesen, P., Neuland, C., Goedert, J., Faber, V., Lorenzen, I., Skinhoj, P., Gallo, R.C., and Blattner, W.A. (1986) Ann. Intern. Med. 104, 496-500. Rosenberg, Z.F., and Fauci, A.S. (1989) AIDS Res. Human Retrovir 5, l4. Hammer, S.M.. and Gillis, J.M. (1987) Antimicrob. Agents Chemother. 31, 1046-1050. Mitsuya, H., Looney, D.J., Kuno, S., Ueno, R., Wong-Staal, F., and Broder, S. (1988) Science 240, 646-649. Baba, M., Schols, D., Pauwels, R., Nakashima, H., and De Clercq, E. (1990). J. Acquir. Immun. Defic. Syndr., in press.
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