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From the
Irradiation induces the production of superoxide radicals (O),
which play an important causative role in radiation damage. Manganese
superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in
scavenging O. This study examined MnSOD gene regulation by irradiation
in WI38 human fibroblasts. Unstimulated fibroblasts constitutively
expressed MnSOD activity and mRNA; irradiation markedly increased MnSOD
activity and mRNA levels. The increase in MnSOD transcripts by
irradiation was both time- and dose-dependent. WI38 fibroblasts
constitutively produce low levels of interleukin-1 (IL-1). The
induction of MnSOD mRNA by irradiation was partially blocked by
anti-IL-1 antibodies, and treatment of cells with IL-1 also increased
MnSOD mRNA levels. Inhibition of the cyclo-oxygenase pathway with
indomethacin augmented the induction MnSOD mRNA by irradiation and
prostaglandin E
Irradiation produces physical and chemical damage to tissues
that may lead to cell death or neoplastic transformation. In the
presence of oxygen, irradiation or some chemicals increase the
formation of superoxide radicals (O)(1, 2) , which are
important mediators of tissue damages(3, 4) . Superoxide
radicals also have been implicated as important pathologic mediators in
various disorders including cancer, inflammation, or
ischemia(5, 6) . The reaction of these radicals with DNA
results in DNA strand breaks, which play an important role in
radiation-induced carcinogenesis(6) . In response to these
stresses, cells induce the synthesis or activation of proteins with
protective capacities. Previous studies have shown that cytokines such
as granulocyte-macrophage colony-stimulating factor, tumor necrosis
factor (TNF),
Superoxide dismutases (SODs, EC 1.15.1.1) are metalloenzymes that
catalyze the dismutation of O to H
Fibroblasts constitute a major element of bone marrow stroma and
submucosal and subcutaneous tissues, where they are important for
repair of tissue injury. In this study, we examined the effects of
irradiation on expression of the MnSOD gene and found an intracellular
accumulation of MnSOD induced by irradiation in human fibroblasts. We
also explored the mechanisms for regulation of the MnSOD gene by
irradiation.
To investigate further the involvement of endogenously
produced IL-1 in the expression of MnSOD mRNA by irradiation, cells
were preincubated with antibody against either human IL-1
Many of the damaging effects of ionizing irradiation are
mediated by reactive free radicals(1, 4, 38) .
Irradiation increases the rate of O production, and O causes DNA
breakage, lipid peroxidation, and protein modification(39) . The
data presented here demonstrate that the level of MnSOD mRNA and its
activity can be modulated by irradiation in human fibroblasts. We also
found that accumulation of MnSOD mRNA after exposure to irradiation
occurs through, at least in part, indirect mechanisms: the activation
of IL-1 and the inhibition of prostaglandin production. Mitochondria
are thought to be a major intracellular target for oxidant damage such
as irradiation and chemical
drugs(40, 41, 42, 43) . A recent study
has shown that preferential oxidative damage is to mitochondrial DNA
rather than to nuclear DNA after irradiation(43) . Studies have
also shown that MnSOD is involved in resistance to irradiation and that
overexpression of MnSOD promotes the survival of cells exposed to
irradiation(4) . Our results thus suggest that the increase in
cellular accumulation of MnSOD may be an important biological response
to irradiation and may confer enhanced resistance to the lethal effects
of irradiation.
In the present study, both irradiation and CHX, a
protein synthesis inhibitor, increased levels of MnSOD RNA; irradiation
did not further enhance the level of MnSOD transcripts after treatment
with CHX. Taken together, our studies cannot determine whether the
induction of MnSOD mRNA expression by irradiation requires de novo protein synthesis. WI38 fibroblasts constitutively produced IL-1
at a low level, and, moreover, treatment of cells with either
anti-IL-1
Stimulators of several pathways of signal transduction
increase levels of MnSOD including inflammatory mediators such as IL-1
or TNF(16, 17, 20) . Prostaglandins play an
important role in the pathogenesis of the inflammation, and augmented
synthesis of prostaglandin by fibroblasts in response to various
stimuli is a prominent feature of inflammatory reactions. PGE
The steady-state level of mRNA in the cell is dependent on
the rates of both transcription and degradation. Transcriptional
regulation is one of the important mechanisms for mRNA accumulation.
Our results showed that irradiation increased the rate of MnSOD
transcription in human fibroblasts. A recent study reported that either
x-rays or UV irradiation increased the chloramphenicol
acetyltransferase gene containing the long terminal repeat of Moloney
murine sarcoma provirus (48). The human immunodeficiency virus long
terminal repeat-directed expression of reporter genes has also been
shown to be activated by UV irradiation in the absence of the Tat
transactivator(49, 50) . The promoter region of human
MnSOD gene lacks both the TATA and the CAT boxes(51) . On the
other hand, this region contains the AP-1 binding site (TGACTCA) and
eight repeats of the hexanuclotide core for binding transcriptional
factor Sp1 (GGGCGGG)(51) . These studies indicate the
possibility that irradiation-responsive element(s) may play a role in
activation of transcription by irradiation.
The MnSOD mRNA levels
were also increased due to the enhanced stability of the transcripts.
The t of MnSOD RNA in fibroblasts was less than 1.5 h; irradiation
markedly stabilized MnSOD mRNA (t >4 h). Stabilization of mRNA is an
important mechanism for increasing the levels of the encoded proteins,
especially for cytokines and proto-oncogenes because these RNAs are
short-lived (52-54). Irradiation has been shown to stabilize
mRNAs coding various
proteins(7, 8, 10, 11) . Previously, we
found that irradiation stabilized granulocyte-macrophage
colony-stimulating factor mRNA through AU-rich sequences containing
AUUUA repeats(7, 8) . However, RNA coding MnSOD has no
AU-rich sequences containing AUUUA repeats in the 3`-untranslated
region(55) . How irradiation stabilizes MnSOD transcripts is
unknown and requires further investigation.
Mesenchymal cells,
including fibroblasts, produce a variety of factors in response to
various stimuli(56, 57) . Fibroblasts constitute a major
element of bone marrow stroma as well as submucosal and subcutaneous
tissue. We have shown that WI38 fibroblasts constitutively transcribe
the MnSOD gene and produce the increased level of MnSOD in response to
irradiation. Irradiation is immunosuppressive because it increases the
production of O; O causes DNA breakage and induces apoptosis. Thus, it
seems paradoxical that irradiation induces MnSOD in fibroblasts.
Because MnSOD protects cells exposed to oxidative stress including
irradiation, fibroblasts may be the cells primarily responsible for
irradiation in the repair of tissue injury.
We thank Ikuko Furusawa for assistance.
Volume 270,
Number 26,
Issue of June 30, pp. 15864-15869, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
POSSIBLE MECHANISMS FOR ITS ACCUMULATION (*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
inhibited the accumulation of MnSOD mRNA by
irradiation. Transcriptional run-on analysis showed that irradiation
increased the rate of MnSOD transcription 2-fold. Stability studies of
MnSOD mRNA in these cells showed that the half-life increased from
<1.5 h in unirradiated cells to >4 h in irradiated cells. These
results suggest that induction of the MnSOD gene after irradiation is
regulated, at least in part, by IL-1 production and that increased
levels of MnSOD transcripts also occur through a pathway of endogenous
prostaglandin E production. Our data indicate that the
increase in MnSOD mRNA observed after irradiation occurs through both
transcriptional and post-transcriptional mechanisms.
()and interleukin-1 (IL-1) are
produced after irradiation in various
cells(7, 8, 9, 10, 11, 12, 13) .
O (hydrogen
peroxide) and O (oxygen)(14, 15) . Thus,
SODs are important initial components in the cellular defense against O (14) and radiation-induced tissue damage. Three forms of SODs
with distinctive cellular distributions and metal requirements are
found; MnSOD is found in prokaryotes and in mitochondria of eukaryotes,
copper-zinc SOD occurs in eukaryotes, and iron SOD is located in the
cytosol of prokaryotes. Whereas production of copper-zinc SOD and iron
SOD is constitutive, MnSOD is inducible by various stimuli, such as
IL-1, TNF, lipopolysaccharide, interferon-, or
hypoxia(16, 17, 18, 19, 20, 21) .
Cells and Culture
Normal human embryonic lung
fibroblasts (WI38, obtained from American Type Culture Collection) were
cultured in 
-medium (Cosmo Bio Co. Ltd., Tokyo, Japan)
supplemented with 7% fetal calf serum (Mitsubishi Kasei Co., Tokyo,
Japan) in a humidified atmosphere containing 5% CO. Flasks
containing confluent cells were used for experiments. Conditioned media
from confluent cultures of either control or irradiated fibroblasts
were prepared by centrifuging the supernatants at 1000 g for 10 min and stored at -20 °C until use.
Irradiation
Cells were irradiated with rays
by a
Cs source emitting at a fixed dose rate of 12 Gy/min
as determined by dosimetry.
Reagents
Human recombinant IL-1
was purchased
from Genzyme Corp. (Cambridge, MA). The neutralizing antibodies against
IL-1 (OCT-304K) and IL-1 (297) were polyclonal rabbit
anti-human IL-1 and IL-1 antibodies and were kindly provided
by Dr. Tsutomu Nishida (Otsuka Pharmaceutical Co. Ltd., Tokushima,
Japan). These antibodies neutralize more than 100 units of IL-1 at a
final dilution of 1:100 in the thymocyte co-stimulating assay. The
antibody against TNF was polyclonal rabbit anti-human TNF
antibody (Genzyme Corp.), and 1 µl of this antibody neutralizes
1000 units/ml of TNF in the L929 cell cytotoxic assay. Nitroblue
tetrazolium (NBT), indomethacin, actinomycin D, and cycloheximide were
purchased from Sigma.
Assay for MnSOD Activity
The MnSOD activity was
determined by NBT methods using the xanthine-xanthine oxidase as a
source of O as described previously(22, 23) . Untreated
or irradiated cells were collected and suspended in ice-cold potassium
phosphate buffer (0.05 mol/liter, pH 7.8) with
diethylenetriaminepentaacetic acid. After sonication on ice, the
homogenates were centrifuged, the supernatants were analyzed for MnSOD
activity, and a competitive inhibition was performed using
xanthine-xanthine oxidase-generated O to reduce NBT at a constant rate
(0.015-0.025 absorbance units/min). The rate of NBT reduction was
monitored by spectrophotometer at 560 nm. One unit of SOD was defined
as the amount of enzyme activity that inhibited the NBT reduction rate
by 50%. The activity of MnSOD was assayed in the presence of 5
mmol/liter sodium cyanide to inhibit copper-zinc SOD. Endogenous
activity of NBT reductase was subtracted. Protein levels in cell lysate
were measured by the Lowry method(24) .
[
Fibroblasts were exposed to cycloheximide or
indomethacin in culture dishes (Falcon, Becton Dickinson Labware,
Oxnard, CA) in triplicate per experimental point for 2 h. Cells were
pulsed with either 1 mCi of [
H]Uridine and
[S]Methionine
Incorporation
H]uridine (specific
activity, 43 Ci/mmol) (ICN, Irvine, CA) or 4 mCi of [S]methionine (specific activity, 200
µCi/mmol) for 1 h at 37 °C, washed twice with
phosphate-buffered saline, precipitated in 5% trichloroacetic acid in
30 mmol/liter Na
HPO
at 4 °C for 1 h,
filtered onto a glass microfiber membrane (GF/F, Whatman), washed with
3% trichloroacetic acid (30 mmol/liter NaHPO),
and heated at 80 °C for 1 h. Each sample was counted by a liquid
scintillation counter. Results were compared with those of unirradiated
cells.
Assay for Prostaglandin E
The
amount of PGE secreted by WI38 fibroblasts in culture
supernatants was measured by radioimmunoassay as described previously
(25). This assay is based upon the competition of cold PGE with labeled PGE for antibody (polyclonal rabbit
anti-human PGE, DuPont) binding. The cross-reactivity of
this antibody between PGE and PGD or PGE and PGF was less than 0.01%, and the limit of
detection by radioimmunoassay was 0.25 pg. Untreated or irradiated
cells were cultured for 8 h in -minimum essential medium without
fetal calf serum (Falcon 3001 culture dishes; Becton Dickinson
Labware). To each polypropylene tube were added 0.1 ml of
anti-PGE, 0.1 ml of I-PGE
, and
0.1 ml of either standard PGE or culture supernatant. Assay
tubes were incubated at 4 °C for 24 h. Immune complexes were
precipitated at 4 °C with 1 ml of a polyethylene glycol solution (M
6000) and centrifuged at 2000 g at 4 °C for 20 min. The supernatants were decanted, and the
radioactivity in the precipitate was counted on a
counter
(Pharmacia Biotech Inc.). The percentage of binding was compared with a
standard curve, and the amount of PGE
in the sample was
calculated.
Biological Activity of IL-1
IL-1 activity in
conditioned medium from WI38 cells was measured by enhanced
[H]thymidine incorporation by murine thymocytes
that had been exposed to phytohemagglutinin(26) . Potency of
samples was determined by comparison with human recombinant IL-1.
DNA Probes
Human MnSOD cDNA probe (EcoRI-EcoRI, 1.0 kilobase) was a gift from Dr.
Madsushi (National Cancer Institute, Bethesda, MD). Human IL-1 and
IL-1 cDNA were from pHL4 and pA-26,
respectively(27, 28) . -actin DNA probe (0.7
kilobase, EcoRI-BamHI) was from pHFb A-3`ut
plasmid(29) . These probes were P-labeled by a
random priming method(30) . The specific activity was 2
10
cpm/µg DNA.
Isolation and Blotting of RNA
For isolation of
total cytoplasmic RNA, WI38 cells were suspended in hypotonic buffer
(10 mmol/liter Tris-HCl, pH 7.8, 150 mmol/liter NaCl, 1.5 mmol/liter
MgCl) and lysed with 0.65% Nonidet P-40. Cytoplasmic RNA
was extracted by a phenol/chloroform method as described previously
(31, 32). After denaturation at 65 °C, RNA was electrophoresed in a
formaldehyde-agarose gel (1%) and transferred to a nylon membrane
filter (Hybond-N, Amersham Corp.)(33) . Filters were hybridized
with P-labeled probe for 16-24 h at 42 °C in 50%
formamide, 2
SSC (1
SSC = 150 mmol/liter NaCl,
15 mmol/liter sodium citrate), 5
Denhardt's solution,
0.1% SDS, 10% dextran sulfate, and 100 µg/ml salmon sperm DNA.
Filters were washed to a stringency of 0.1
SSC for 10 min at 65
°C and exposed to x-ray film (RX, Fuji Photo Film Co. Ltd.,
Kanagawa, Japan). Blots were usually sequentially hybridized with
P-labeled MnSOD cDNA and
-actin DNA. The densities of
MnSOD mRNA bands in different lanes were determined with a Pharmacia
UltroScan XL Laser densitometer using multiple exposures of the blot.
-actin bands or the picture of the ethidium bromide-stained
formaldehyde gel before Northern blotting helped to confirm that each
lane had similar amounts of RNA.
Transcriptional Run-on Assay
WI38 fibroblasts were
exposed to irradiation and nuclei were isolated by suspension in an
ice-cold hypotonic buffer (10 mmol/liter Tris-HCl, pH 7.4, 10
mmol/liter KCl, 3 mmol/liter MgCl) for 30 min and then
lysis in a hyptonic buffer containing 0.5% Nonidet P-40. Nuclei were
harvested by centrifugation (500 g, 5 min), washed in
a hypotonic buffer, and resuspended in nuclear storage buffer (40%
glycerol, 50 mmol/liter Tris-HCl, pH 8.3, 5 mmol/liter
MgCl
, 0.1 mmol/liter EDTA). Nuclei were incubated for 30
min at 30 °C in a reaction buffer containing 150 mmol/liter KCl,
2.5 mmol/liter MgCl, 5 mmol/liter Tris-HCl, pH 8.0, 0.25
mmol/liter ATP, 0.25 mmol/liter GTP, 0.25 mmol/liter CTP, and 200
µCi of [-P]UTP (3000 Ci/mmol). The
reaction was terminated by adding DNase I (for 10 min at 30 °C).
The reaction mixture was digested with 400 µg/ml of proteinase K in
buffer (10 mmol/liter EDTA, 1% SDS) and followed by phenol/chloroform
extraction. The aqueous phase was precipitated at -70 °C with
50% isopropyl alcohol in the presence of 0.3 M sodium acetate.
The precipitate was collected by centrifugation and dissolved in TE
buffer (10 mmol/liter Tris-HCl, pH 8.0, 1 mmol/liter EDTA). After
denaturation in 0.3 N NaOH (ice-cold) and neutralization in
0.25 mol/liter HEPES, nuclear RNA was passed through a Sephadex G-50
spun column to remove unincorporated [
P]UTP.
Plasmid DNA containing the cDNA coding sequences was denatured by heat
and alkalization (0.3 N NaOH). Denatured plasmids (5 µg)
were bounded to nylon membranes (Hybond-N) using a slot blot apparatus
(BIO-DOT SF, Bio-Rad) and immobilized by UV cross-linker. Newly
elongated nuclear RNA was hybridized to the filters containing
plasmids. Hybridizations were performed with 10
cpm of P-labeled RNA/ml in 3
SSC, 5 mmol/liter EDTA, 0.1%
SDS, 10
Denhardt's solution, 50% formamide, 0.2 mg/ml
yeast tRNA, 10 mmol/liter NaH
P0, pH 7.0, and
100 mg/ml of salmon sperm DNA for 3 days at 42 °C. After
hybridization, filters were rinsed in 2 SSC at room temperature
and then in 2
SSC and 0.1
SSC at 42 °C. Lanes of
MnSOD and
-actin in untreated or irradiated cells were determined
by densitometry; relative density of MnSOD was compared by a ratio of
MnSOD/-actin.
Induction of MnSOD Activity by Irradiation in
Fibroblasts
Confluent WI38 fibroblasts were cultured for 16 h
after exposure to irradiation at different doses (10-80 Gy). As a
control, unirradiated cells were cultured for 16 h. Cells were
harvested and tested for MnSOD activity by the NBT reduction method.
Unirradiated fibroblasts had constitutive MnSOD activity (Fig. 1). A significant increase of MnSOD activity was observed
at 10 Gy of irradiation (p < 0.05); the increase of MnSOD
activity occurred in a dose-dependent fashion. At 80-Gy irradiation,
MnSOD activity was approximately 4 times greater than that from
unirradiated cells (p < 0.025).
Figure 1:
Increased levels of MnSOD activity in
WI38 human fibroblasts exposed to irradiation. Cells were cultured for
16 h after irradiation with 0-80 Gy. Cells were harvested and
assayed for MnSOD activity by the NBT reduction method as described
under ``Materials and Methods.'' Results represent the means
and standard errors of triplicate assays.
Effect of Irradiation on Levels of MnSOD mRNA in
Fibroblasts
Fibroblasts were cultured for 4 h after 0-80
Gy of irradiation. To determine the effect of irradiation on MnSOD gene
expression, Northern blot analysis was performed with cytoplasmic RNA
using P-labeled MnSOD cDNA probe. Untreated fibroblasts
contained a low but detectable amount of MnSOD mRNA, which was
increased by irradiation with a dose of 10 Gy. The induction of MnSOD
RNA was increased in a dose-dependent fashion of irradiation, and the
induction of the MnSOD gene was maximal after exposure to 80 Gy
(8-fold) (Fig. 2, left).
Figure 2:
Left, dose-dependent effect of irradiation
on levels of MnSOD mRNA in fibroblasts. Cells were cultured for 4 h
after irradiation. Cytoplasmic RNA (15 µg/lane) was prepared and
analyzed by formaldehyde-agarose gel electrophoresis and transferred to
a nylon membrane as described under ``Materials and
Methods.'' Hybridization was with P-labeled MnSOD
cDNA (4.0-kilobase bands of hybridization). Right,
time-dependent effect of irradiation on levels of MnSOD mRNA in
fibroblasts. Cell were cultured for various durations (0-24 h)
after irradiation at 40 Gy. Northern blot analysis of mRNA was
performed by blotting cytoplasmic RNA (15 µg/lane). C,
control; kb, kilobases.
For kinetics of MnSOD mRNA
induction by irradiation, fibroblasts were irradiated at 40 Gy and
harvested sequentially at several different time points. Northern blot
analysis showed that a significant increase in MnSOD mRNA was first
observed after 4 h and that it continued to increase until 24 h after
irradiation (Fig. 2, right).
Effect of Irradiation on Expression of MnSOD mRNA in the
Absence of Protein Synthesis
Fibroblasts cultured with
cycloheximide (CHX) (0.2, 2, or 20 µg/ml) decreased protein
synthesis by 74, 85, or 93%, respectively, as compared with that of
untreated cells. Pretreatment with CHX (20 µg/ml, 0.5 h) did not
affect the accumulation of MnSOD RNA by irradiation (80 Gy, 4 h),
although irradiation or treatment with CHX alone significantly
increased the level of MnSOD transcripts (Fig. 3). An experiment
using 5 µg/ml CHX produced similar results (data not shown).
Figure 3:
Effect of inhibitor of protein synthesis,
CHX, on expression of MnSOD mRNA after irradiation. Cells were left
untreated, treated with either irradiation (80 Gy for 4 h) or CHX (20
µg/ml), or pretreated with CHX for 0.5 h and then irradiated for 4
h (CHX+Irradiation). Analysis was performed by blotting
and hybridizing cytoplasmic RNA (15 µg/lane). kb,
kilobases.
Effect of IL-1 Production on Expression of MnSOD mRNA
after Irradiation
WI38 fibroblasts constitutively produce IL-1
protein at low levels(34) . We measured IL-1 bioactivity using a
thymocyte proliferation assay. Conditioned medium of WI38 cells
cultured for 3 days contained less than 0.2 unit/ml of IL-1 activity.
Northern blot analysis showed that WI38 cells contained very low levels
of IL-1 and IL-1 mRNAs and that there was no significant
increase of levels of IL-1 RNAs after irradiation in these cells (data
not shown).
or
IL-1, which neutralizes 100 units/ml of IL-1 for 1 h. Then cells
were irradiated with 40 Gy in the presence of either anti-IL-1 or
anti-IL-1 antibody. After 4 h, cells were harvested, and levels of
MnSOD mRNA were compared with levels in control cells that were
cultured in medium alone. In order to compare clearly the constitutive
levels of MnSOD mRNA in untreated cells (basal levels) and the levels
in cells treated with anti-IL-1 antibodies and also to show the
decrease more clearly, the blot with MnSOD probe was exposed to x-ray
film longer than usual (96 h). Neutralization of the endogenous IL-1
with anti-IL-1 antibodies resulted in a prominent decrease in levels of
constitutive expression of MnSOD mRNA in untreated cells;
anti-IL-1 antibody decreased levels by 48% and anti-IL-1
decreased expression by 73%. Treatment with anti-IL-1 and
anti-IL-1 antibodies also blocked the increase in
irradiation-induced MnSOD transcripts by 52 and 47%, respectively (Fig. 4A). However, treatment with anti-TNF
antibody, which neutralizes 1000 units/ml of TNF (used as
nonspecific antibody), did not affect the constitutive level of MnSOD
mRNA or the induction of MnSOD RNA by irradiation in these cells (Fig. 4B, left). Furthermore, WI38 fibroblasts
were cultured with different concentrations of IL-1 for 4 h
(1-100 units/ml). Treatment of cells with IL-1 clearly induced
MnSOD mRNA in these cells in a dose-dependent fashion (Fig. 4B, right).
Figure 4:
Effect of IL-1 production on expression of
MnSOD mRNA in fibroblasts exposed to irradiation. Cells were pretreated
for 1 h either with anti-IL-1 or anti-IL-1 antibody at a
concentration that neutralizes 100 units/ml either IL-1 or
IL-1 (A) or with anti-TNF antibody that neutralizes
1000 units/ml TNF (B, left). These cells were
then irradiated at 40 Gy in the presence of the antibody. After 4 h,
unirradiated and irradiated cells were harvested, and levels of MnSOD
mRNA were determined. The blot was exposed to x-ray film for 96 h. B (right), cells were cultured with 1, 10, or 100
units/ml IL-1 for 4 h, and cytoplasmic RNA was extracted. Levels
of MnSOD mRNA were determined by Northern blotting. kb,
kilobases.
Effect of PGE
Previous studies have shown
that cyclo-oxygenase metabolite PGEon Expression of
MnSOD mRNA after Irradiation is a potent regulator
of cytokine production and that prostaglandins are synthesized by
fibroblasts(25, 35, 36, 37) . In order
to determine whether prostaglandins are involved in the regulation of
the MnSOD gene by irradiation, induction of MnSOD mRNA by irradiation
was examined in the presence of a cyclo-oxygenase inhibitor,
indomethacin (Fig. 5, left). WI38 cells were initially
cultured with different concentrations of indomethacin
(10-10
M) for 0.5 h and
then exposed to 40 Gy of irradiation. After 4 h of cultures, cells were
harvested and MnSOD mRNA levels were determined. Treatment of cells
with indomethacin enhanced the induction of MnSOD mRNA by irradiation.
We also tested levels of PGE
in culture supernatant by
radioimmunoassay. Irradiation with 10 Gy inhibited PGE synthesis more than 50%; a 70% decrease of PGE production was observed with 40 Gy of irradiation (data not
shown). Furthermore, addition of PGE blocked the induction
of MnSOD mRNA by irradiation in a dose-dependent manner (Fig. 5, right).
Figure 5:
Effects of PGE synthesis on
the induction of MnSOD transcripts exposed to irradiation in
fibroblasts. Cells were pretreated with varying concentrations of
indomethacin (10 or 10
mol/liter,
0.5 h)(left). Cells were irradiated in the presence of
indomethacin and then cultured for 4 h. Cytoplasmic RNA was extracted
and each blot (15 µg/lane) was sequentially hybridized. Cells were
irradiated in the presence of PGE
and then cultured for 4 h (right).
Transcriptional and Post-transcriptional Regulation of
MnSOD in Irradiated Fibroblasts
The steady-state level of mRNAs
in the cell is dependent on the rates of both transcription and
degradation. To investigate the mechanisms for accumulation of MnSOD
transcripts by irradiation in these cells, transcriptional run-on
assays were performed. MnSOD was constitutively transcribed at a low
level in untreated WI38 cells (Fig. 6). Exposure of the cells to
irradiation (40 Gy, 4 h) increased the transcriptional rate of MnSOD
2-fold. To examine post-transcriptional regulation of MnSOD mRNA in
irradiated fibroblasts, unirradiated or 40 Gy-irradiated fibroblasts
were cultured for 4 h, and then actinomycin D (5 µg/ml) was added
to the cultures. Cells were cultured for an additional 0.5-4.0 h
and were sequentially harvested and examined for MnSOD mRNA levels (Fig. 7). The half-life (t) of steady-state MnSOD mRNA in
unirradiated fibroblasts was <1.5 h, whereas t of MnSOD mRNA after
irradiation was >4 h.
Figure 6:
Transcriptional run-on analysis of MnSOD
in irradiated fibroblasts. WI38 cells were either left untreated or
irradiated at 40 Gy, and 4 h later nuclei were isolated as described
under ``Materials and Methods.'' Newly elongated P-labeled transcripts were hybridized to the plasmid
containing inserts of either MnSOD,
-actin, or the control
plasmid, pUC118.
Figure 7:
Stability of steady-state MnSOD mRNA in
fibroblasts exposed to irradiation. Untreated cells or cells irradiated
at 40 Gy were cultured with actinomycin D (5 µg/ml) for 0.5-4
h. Cytoplasmic RNA (30 µg/lane in untreated cells and 15
µg/lane in irradiated cells) was extracted and analyzed by RNA
blotting as described under ``Materials and Methods.'' The
intensity of hybridization was determined by densitometry of several
different exposures of the autoradiograms. Untreated cells were assumed
to have 100% activity. kb,
kilobases.
or anti-IL-1 antibody decreased the constitutive
expression of MnSOD mRNA. Moreover, IL-1 stimulated expression of MnSOD
mRNA in fibroblasts. These results suggest that IL-1 stimulates these
cells to produce MnSOD in an autocrine manner. Furthermore, this
constitutive expression of IL-1 could be augmented after appropriate
stimulation (34). Other investigators have demonstrated that
irradiation induced IL-1 (9, 13, 44) and that UV
irradiation also induced the release of IL-1 in human
keratinocytes(45) . In this study, no significant change could
be detected in the levels of IL-1 transcripts. However, anti-IL-1
antibodies blocked induction of MnSOD gene by irradiation as compared
with the level of MnSOD mRNA in untreated cells (anti-IL-1
antibody, anti-IL-1 antibody, or both; data partly not shown).
Irradiation has been reported to modify some proteins at
post-translational levels (46). Our results indicate that irradiation
may induce the expression of MnSOD mRNA through the autocrine pathway
involving the activation of IL-1. On the other hand, irradiation
increased levels of MnSOD transcripts in the presence of IL-1
antibodies when compared with treatment with antibodies alone. This
induction may be through the autocrine loops in the endoplasmic
reticulum, because exogenously added antibodies can only interrupt an
autocrine loop on the exterior of the cells. Further studies are
required.
is synthesized by cyclo-oxygenase metabolism of arachidonic acid
and transduces information via the second messenger cAMP. Studies have
shown that PGE is a potent regulator of cytokine
production(35, 36, 37) . UV irradiation
increases PGE release in human skin
keratinocytes(47) . In the present study, however, inhibition of
cyclo-oxygenase enhanced the induction of MnSOD mRNA by irradiation.
Furthermore, PGE blocked the induction of MnSOD mRNA by
irradiation, and irradiation decreased PGE production (data
not shown). Our data suggest that induction of MnSOD mRNA by
irradiation is likely to be regulated through the cyclo-oxygenase
pathway. Treatment with 1 mmol/liter of indomethacin slightly inhibited
the accumulation of MnSOD mRNA mediated by irradiation. However, 1
mmol/liter of indomethacin also decreased RNA synthesis by more than
50% as measured with [H]uridine incorporation
(data not shown). In view of the relatively short half-life of MnSOD
RNA, we believe that the slightly decreased accumulation of MnSOD RNA
in the presence of 1 mmol/liter indomethacin is probably a nonspecific
effect.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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  C. Ngo, C. Chereau, C. Nicco, B. Weill, C. Chapron, and F. Batteux Reactive Oxygen Species Controls Endometriosis Progression Am. J. Pathol., July 1, 2009; 175(1): 225 - 234. [Abstract] [Full Text] [PDF]
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 J. Kim, J.-W. Park, and K. M. Park Increased superoxide formation induced by irradiation preconditioning triggers kidney resistance to ischemia-reperfusion injury in mice Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1202 - F1211. [Abstract] [Full Text] [PDF]
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 S. K. Dhar, Y. Xu, T. Noel, and D. K. St Clair Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate represses sod2 induction in vivo: the negative role of p50 Carcinogenesis, December 1, 2007; 28(12): 2605 - 2613. [Abstract] [Full Text] [PDF]
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 X. G. Luo, S. F. Li, L. Lu, B. Liu, X. Kuang, G. Z. Shao, and S. X. Yu Gene Expression of Manganese-Containing Superoxide Dismutase as a Biomarker of Manganese Bioavailability for Manganese Sources in Broilers Poult. Sci., May 1, 2007; 86(5): 888 - 894. [Abstract] [Full Text] [PDF]
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 Y. Han, S.-J. Son, M. Akhalaia, A. Platonov, H.-J. Son, K.-H. Lee, Y.-S. Yun, and J.-Y. Song Modulation of Radiation-Induced Disturbances of Antioxidant Defense Systems by Ginsan Evid. Based Complement. Altern. Med., December 1, 2005; 2(4): 529 - 536. [Abstract] [Full Text] [PDF]
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 M Akashi Role of infection and bleeding in multiple organ involvement and failure Br. J. Radiol., January 1, 2005; Supplement_27(1): 69 - 74. [Abstract] [Full Text] [PDF]
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 S. K. Dhar, B. C. Lynn, C. Daosukho, and D. K. St. Clair Identification of Nucleophosmin as an NF-{kappa}B Co-activator for the Induction of the Human SOD2 Gene J. Biol. Chem., July 2, 2004; 279(27): 28209 - 28219. [Abstract] [Full Text] [PDF]
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Z. Guo, G. H. Boekhoudt, and J. M. Boss Role of the Intronic Enhancer in Tumor Necrosis Factor-mediated Induction of Manganous Superoxide Dismutase J. Biol. Chem., June 20, 2003; 278(26): 23570 - 23578. [Abstract] [Full Text] [PDF]
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R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, and K. Kishi M-LP, Mpv17-like Protein, Has a Peroxisomal Membrane Targeting Signal Comprising a Transmembrane Domain and a Positively Charged Loop and Up-regulates Expression of the Manganese Superoxide Dismutase Gene J. Biol. Chem., February 14, 2003; 278(8): 6301 - 6306. [Abstract] [Full Text] [PDF]
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 Y. Takada, M. Hachiya, S.-H. Park, Y. Osawa, T. Ozawa, and M. Akashi Role of Reactive Oxygen Species in Cells Overexpressing Manganese Superoxide Dismutase: Mechanism for Induction of Radioresistance Mol. Cancer Res., December 1, 2002; 1(2): 137 - 146. [Abstract] [Full Text] [PDF]
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 L. Naderi-Hachtroudi, T. Peters, P. Brenneisen, C. Meewes, C. Hommel, Z. Razi-Wolf, L. A. Schneider, J. Schuller, M. Wlaschek, and K. Scharffetter-Kochanek Induction of Manganese Superoxide Dismutase in Human Dermal Fibroblasts: A UV-B-Mediated Paracrine Mechanism With the Release of Epidermal Interleukin 1{alpha}, Interleukin 1{beta}, and Tumor Necrosis Factor {alpha} Arch Dermatol, November 1, 2002; 138(11): 1473 - 1479. [Abstract] [Full Text] [PDF]
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 H. Oubrahim, E. R. Stadtman, and P. B. Chock Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells PNAS, August 1, 2001; (2001) 181319898. [Abstract] [Full Text] [PDF]
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H.-P. Kim, J.-H. Roe, P. B. Chock, and M. B. Yim Transcriptional Activation of the Human Manganese Superoxide Dismutase Gene Mediated by Tetradecanoylphorbol Acetate J. Biol. Chem., December 24, 1999; 274(52): 37455 - 37460. [Abstract] [Full Text] [PDF]
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J. Wenk, P. Brenneisen, M. Wlaschek, A. Poswig, K. Briviba, T. D. Oberley, and K. Scharffetter-Kochanek Stable Overexpression of Manganese Superoxide Dismutase in Mitochondria Identifies Hydrogen Peroxide as a Major Oxidant in the AP-1-mediated Induction of Matrix-degrading Metalloprotease-1 J. Biol. Chem., September 3, 1999; 274(36): 25869 - 25876. [Abstract] [Full Text] [PDF]
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 J. Hollander, R. Fiebig, M. Gore, J. Bejma, T. Ookawara, H. Ohno, and L. L. Ji Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training Am J Physiol Regulatory Integrative Comp Physiol, September 1, 1999; 277(3): R856 - R862. [Abstract] [Full Text] [PDF]
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L. A. Esposito, S. Melov, A. Panov, B. A. Cottrell, and D. C. Wallace Mitochondrial disease in mouse results in increased oxidative stress PNAS, April 27, 1999; 96(9): 4820 - 4825. [Abstract] [Full Text] [PDF]
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S. Kuo, S. E. Chesrown, J. K. Mellott, R. J. Rogers, J.-L. Hsu, and H. S. Nick In Vivo Architecture of the Manganese Superoxide Dismutase Promoter J. Biol. Chem., February 5, 1999; 274(6): 3345 - 3354. [Abstract] [Full Text] [PDF]
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J. A. Melendez and K. J.A. Davies Manganese Superoxide Dismutase Modulates Interleukin-1alpha Levels in HT-1080 Fibrosarcoma Cells J. Biol. Chem., August 2, 1996; 271(31): 18898 - 18903. [Abstract] [Full Text] [PDF]
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H. Oubrahim, E. R. Stadtman, and P. B. Chock Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells PNAS, August 14, 2001; 98(17): 9505 - 9510. [Abstract] [Full Text] [PDF]
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