]CANCER RESEARCH53, 4505-4510, October 1, 1993]
Singlet Oxygen: A Primary Effector in the Ultraviolet A/Near-Visible Light
Induction of the H u m a n H e m e Oxygenase Gene 1
Sharmila Basu-Modak and Rex M. Tyrrell 2
Swiss Institute for Experimental Cancer Research, Physical Carcinogenesis Unit, CH-1066 Epalinges/Lausanne, Switzerland
ABSTRACT
protein by near-monochromatic UVA (365 nm) radiation. From this
Both singlet oxygen and the hydroxyl radical are generated in mammalian ccHs by UVA (~3Z0---~3{50rim) and possibly near-visible (~{50--420 nm)
radiation. We have modulated the cellular levels of these two reactive
oxygen species in order to compare their involvement in the induction of
Fenton reaction was involved in the induction of the H O gene. How-
the humanheme0xygenase(I40) geneby broadspectrumUVA/nearvisible light 0LIVA/NVL). Irradiation in deuterium oxide (in which singlet
oxygen has a longer half-life) enhances the broad spectrum U V A / N V L
induction of this gene. Sodium azide and L-histidine which are scavengers
of both singlet oxygen and the hydroxyl radical reduce the fluence-dependent accumulation of HO mRNA, while compounds which are only hydroxyl radical scavengers, namely, mannitol and dimethyl sulfoxide do
not. Rose Bengal, a known generator of singlet oxygen, also increases the
HO mRNA levels, and this induction is enhanced in deuterium oxide. We
conclude that the observed effects of deuterium oxide and singlet oxygen
scavengers on HO m R N A levels are not due to a nonspecific effect on
transcription but that singlet oxygen is a primary effector in the UVA/NVL
induction of the human heme oxygenase gene.
INTRODUCTION
Heine oxygenase (EC 1.14.99.3), the rate-limiting enzyme in the
heme degradation pathway, is ubiquitous in higher eukaryotes. Generally, the enzyme is induced in vivo and in vitro by diverse factors
such as heine compounds, therapeutic agents, heat shock, hormones,
bacterial toxins, sulfhydryl reagents, metal ions, etc. (1). In human
skin fibroblasts, HO 3 is induced by UVA (320-380 nm), a component
of sunlight clearly established as a carcinogen in animals (2). The
human H O gene is also induced by the oxidizing agent hydrogen
peroxide (3, 4) but not by heat shock (3, 5, 6). In rat liver, induction
of HO enzyme activity by either heroin, cadmium chloride or bromo-
benzene als0 caused an increase in the translatable H0 mRNA (7).
T h i s w a s t h e first i n d i c a t i o n t h a t t h e i n d u c t i o n o f n o
gene expression
was probably by transcriptional activation. Nuclear run-on transcription assays in human skin fibroblasts (8) showed that the gene is
induced by a transient increase in the transcription rate of HO mRNA,
reaching a maximum within 1 h after treatment and then declining
steadily to basal levels by 6 h. The initial increase in the rate of HO
mRNA transcription is also correlated with an accumulation of HO
mRNA to a maximum between 2 and 4 h, followed by a decrease to
basal level by 8 h. Thus, the H O gene induction by UVA involves
transcriptional activation, but the signal transduction events are not
yet well understood.
In a previous report (9), it was shown that the presence of iron
chelators, o-phenanthroline and desferrioxamine, lowered the H202
induction of both HO mRNA and protein by 50-80%. A similar effect
of these iron chelators was observed on the induction of the HO
study, it was concluded that the .OH generated by the iron catalyzed
ever, iron is also involved in other reactions, such as decomposition of
lipid hydroperoxides and autooxidation of a number of biomolecules.
Thus, in addition to the .OH, iron-catalyzed reactions could generate
a number of other active oxygen species such as the lipid peroxyl
radical and singlet oxygen (10). Furthermore, chemical modulation of
UVA inactivation of human fibroblasts (11) indicated that 10 2 generation plays a major role in the cytotoxicity of UVA. This prompted
us to address the question of the involvement of -OH and 102 in H O
gene induction by UVA in more detail.
A common approach to detect 102 involvement in reactions is the
use of scavengers. A disadvantage of this approach is that some of the
commonly used scavengers of ~O2 are also efficient scavengers of the
9OH as seen by the in vitro rate constants shown in Table 1 (12, 13).
Another approach is to replace water by D20 in which 102 has a
longer half-life (14). A combination of the two approaches as used in
this study will provide complementary information.
It has been shown that UVA irradiation of macromolecules in vitro
causes the generation of H202 and superoxide anion (15, 16). Furthermore, it has been proposed that the .OH could be generated by the
iron-catalyzed reduction of H202 by superoxide anion (17), and this
was demonstrated in vitro (18). Studies of prokaryotic and eukaryotic
cells indicate that the .OH could be generated in vivo by UVA irradiation (15, 16). On the other hand, a number of reactions capable of
generating 102 under conditions relevant in vivo have been proposed
(19). It has also been suggested that 102 is involved in the UVAinduced lipid peroxidation of liposomal membranes in vitro (20, 21),
and lipid peroxidation by UVA has been demonstrated recently in
culturedhumanfibr0blasts(22). Sinceboth ,OH and 10a are generatedin vivo b y E V A i r r a d i a t i o n , w e h a v e m o d u l a t e d t h e c e l l u l a r l e v e l s
of these active oxygen species by various chemical agents. We find
that 102 is a primary effector in the UVA/NVL induction of HO
mRNA.
MATERIALS AND M E T H O D S
Cell Culture. The normal human fibroblast cell line FEK4 (23) was cultured at 37~ in Earle's modified minimal essential medium supplemented
with penicillin, streptomycin, glutamine, sodium bicarbonate, and 15% fetal
calf serum. Cells were passaged at weekly intervals by trypsinization and used
for experiments between passages 8 and 17.
Chemical Treatments and UVA Irradiation. All test agents (100 mM
sodium azide, 10 mr~ L-histidine, 0.1 Mmannitol, and 4% DMSO) were dissolved in PBS. D20 buffer solution was prepared by dissolving 1 PBS tablet
(Oxoid, Basingstoke, England) in 100 ml of 100% D20. Cells were seeded (5
• 105/10-cm dish) in 10 ml of medium 3 days prior to the experiment. Prior
Received 11/4/92; accepted 7/28/93.
to treatment with the test agent and irradiation, the culture medium was
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
removed and kept aside. The cell monolayer was rinsed with PBS, 5 ml of the
18 U.S.C. Section 1734 solely to indicate this fact.
test agent in PBS (supplemented with Ca2+/Mg2+, 0.01% each) was added to
1 This work was supported by grants from the Swiss League against Cancer and the
each dish and incubated at 37~ for 15 min. Control dishes were incubated for
Swiss National Science Foundation (31.30880.91).
the same time in 5 ml of PBS containing Ca2§
2§ After pretreatment with
2 To whom reprint requests should be addressed, at Ch. des Boveresses 155, Ch-1066
Epalinges, Switzerland.
test agents, cells were irradiated with different fluences (0-1 MJ/m2) of UVA
3 The abbreviations used are: HO, Heine oxygenase 1; D20, deuterium oxide; DMSO,
using a broad spectrum (330-450 nm, with maximum energy between 360 and
dimethyl sulfoxide; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde phosphate
420 nm) UVASUN 3000 lamp (Mutzhas, Munich, Germany) at a distance
dehydrogenase; NVL, near-visible light; .OH, hydroxyl radical; 1Oa, singlet oxygen; RB,
which did not cause heating during irradiation. Although this lamp is considRose Bengal.
4505
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SINGLET OXYGEN INDUCTION OF THE HUMAN HO 1 GENE
Table 1 Rate constants for reaction of singlet oxygen and the hydroxyl radical (12, 13)
RESULTS
Rate constant (M-is -1)
Test agent
10 2
.OH
Azide
2.2 x 10 9
1.1 x 101~
L-Histidine
3.2 • 10 7
(pn 7.1)
(pn 6-7)
DMSO
3.4 >( 10 4
7.1 x 10 9
<103
(pH 7.0)
2.7 X 109
Modulation of UVA/NVL Induction of Heme Oxygenase mRNA
b y D e u t e r i u m Oxide. U V A / N V L irradiation o f h u m a n skin fibroblasts induces accumulation of H O m R N A (Fig. 1, A and B). Since
both "OH and 102 are generated in UVA-irradiated cells, w e studied
the fluence-dependent accumulation o f H O m R N A in cells irradiated
with U V A / N V L in the presence o f D 2 0 , a solvent in w h i c h 10 2 is
(pn 9.0)
Mannitol
5 x 10 9
known (14) to have a longer half-life. Cells were incubated for 15 min
in buffer prepared with 100k D20 prior to irradiation with UVA/NVL
(pH 7,0)
fluences ran~in~ f r o m 0--0.5 M J / m =. We find (Fi~. 1~ A and B ) that in
ered primarily as a UVA-emitting lamp, we cannot exclude the possibility that
the near-visible (380-420 nm) component contributes to the effects observed;
hence, the term UVA/NVL is used for the radiation emitted by this lamp. After
irradiation, the PBS containing test agent was aspirated, cells were washed
with fresh PBS, the original medium was added back, and the cells were
incubated for 3 h at 37~ prior to the extraction of total RNA.
RNA Isolation and Analysis. Total cellular RNA was isolated by the acid
guanidinium thiocyanate-phenol-chloroform extraction method (24), electrophoresed (15 ~g/well) in a 2.2 M formaldehyde-l.3% agarose gel (25) and
transferred onto a Gene Screen nylon membrane (NEN Research Products) by
capillary blotting (conditions proposed by the manufacturer were used for RNA
transfer and hybridization). Baked blots were hybridized to the larger (1
kilobase) EcoRI fragment of a fun-length HO cDNA clone [clone 2/10; (4)]
which was 32p labeled by the random-priming DNA-labeling method (26).
After autoradiography, the HO probe was stripped off by boiling in 0.1•
standard sodium citrate (15 mM sodium chloride, 1.5 mM sodium citrate) and
0.1% sodium dodecyl sulfate (twice for 20 min each time), and the blots were
reprobed with the 32p-labeled PstI fragment (1300 base pairs) of rat GAPDH
cDNA. The GAPDH-RNA signal was used as an internal control for the
loading error between samples since the constitutive levels of this RNA are
unaffected by the test agents used.
For autoradiography, films were preflashed, and blots were exposed at
-70~ Radioactive signals were quantified by densitometry on a Elscript 400
(Hirschmann) densitometer. The HO mRNA signals (corrected for the loading
error with GAPDH) was expressed as a relative (fold) increase above basal
level and plotted as a function of fluence.
The ratio of the corrected HO mRNA signal area of treated sample and the
corresponding control was also calculated for each sample, and mean values of
3-5 experiments for each test agent were plotted as a function of fiuence. These
plots represent the general pattern of the fluence-dependent effect of the test
agents.
RNA Synthesis in Cells. Cells were cultured in 12-well tissue culture
dishes (104 cells/2 ml/well) for 24 h and labeled with [14C]thymidine (10
nCi/wen of 57 mCi/mmol specific activity) for another 24 h. After the cells
were treated with test agent in PBS (1 ml/well) and UVA/NVL as described
above, 1.5 ml of the original culture medium supplemented with 3 p.Ci of
[3H]uridine (specific activity, 46 Ci/mmol) was added back to each well and
incubated for 3 h at 37~ Cells were lysed in 100 p.1 of lysis buffer [2%
sodium dodecyl sulfate-0.1 mg/ml bovine serum albumin-20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.3] and spotted onto GF/C
discs prewetted with 1 N HC1 (27). Discs were washed with 100% ethanol and
air dried, and radioactivity was determined in a liquid scintillation counter. The
ratio of 3I"I/14C cpm was used as a measure of RNA synthesis.
Modulation of HO mRNA Levels by a Photosensitizer. A stock solution
of Rose Bengal (1 mM), a known generator of 10~, was prepared in DMSO.
Cells were seeded in 10-cm dishes and prepared for chemical treatment as
described above. For chemical treatment, 5 ml of PBS (supplemented with
Ca2+/Mg 2+) containing 1 /xM RB was added to each dish, and cells were
incubated in the dark for 30 min at 37~ Control cells were incubated similarly in Ca2+/Mg2+-PBS without the photosensitizer. After the pretreatment,
cells were irradiated with a range of fluences (0-1500 J/m 2) of broad spectrum
(400-700 nm) visible light by placing the culture dishes on a fluorescent light
box. After irradiation, the PBS-containing RB was aspirated, the monolayer
was washed with fresh PBS, and the original medium was added back to
incubate for 3 h at 37~ prior to RNA extraction.
control cells irradiated with U V A / N V L the H O m R N A signal intensity
increases steadily to 17-fold above basal level at the m a x i m u m fluence
used. While treatment with D 2 0 alone does not change the basal level
o f H O m R N A significantly, the fluence-dependent accumulation o f
this message is e n h a n c e d (Fig. 1, A and B). In D20-treated cells, the
H O m R N A levels first increase to a m a x i m u m of 24-fold at 0.2
M J / m 2, and at higher fluences these levels decrease rapidly, reaching
basal levels at 0.5 MJ/m:. Ratios o f the H O m R N A signal intensities
in DeO-treated and control cells (Fig. 1C) s h o w the general pattern o f
the fluence-dependent DzO effect. We find that the m a x i m u m enh a n c e m e n t o f the H O m R N A levels is at 0.1 M J / m 2. At higher fluences, the effect declines rapidly, and at 0.5 M J / m 2 the H O m R N A
levels in DzO-treated cells are only 10% o f the control levels.
A
0
MJm .2
D20
"-
0.1
4- " ' -
0.2
4- " "
0.3
4- " ' -
0.5
4- " "
4.
"
HO
~
B
.~e
C
30
~em
Control
---O-.--- D20
~00
"
s
Eo
o~
o~
~
0
9~ 9
0.0
0.1
0.2
0.3
0.4
0.5
0.6
FIuence ( MJm "2 )
Fig. 1. Effect of 020 on the HO mRNA accumulation induced by UVA/near-visible
light. Human skin fibroblasts were irradiated with increasing fluences of UVA/NVLin the
presence (+) or absence (-) of 100% DzO.A, Northern blot of total cellular RNA was first
probed for HO mRNA and then for GAPDH mRNA. B, HO and GAPDH mRNA signals
were quantified by densitometry. The GAPDH signal was used as an internal loading
control. The HO mRNA signal, corrected with GAPDH for difference in loading between
samples, was expressed as a relative increase above basal level and plotted as a function
of UVA/NVLfluence. C, ratio of the HO mRNA signals (corrected for loading error) in
D20-treated and control cells was calculated for each sample. A mean of 3-5 experiments
was plotted as a function of UVA/NVLfluence. Point (bar), mean (-+SD).
4506
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SINGLET OXYGEN INDUCTION OF THE HUMAN HO I GENE
These results indicate that it is primarily the 10 2 generated by
UVA/NVL irradiation which is involved in the induction of the H O
gene.
Effect of Sodium Azide and L-Histidine. To further test the involvement of 102, we studied the effect of sodium azide (28) and
L-histidine (29) which are known to scavenge 102 almost as effectively as the .OH (Table 1). Monolayer cultures were preincubated
with 100 mM sodium azide or 10 mM L-histidine for 15 min and then
irradiated with a similar range of UVA/NVL fluences in the presence
of the scavenger. The fluence-dependent increase in HO mRNA levels
in cells treated with these agents is shown in Fig. 2, A, B, D, and E.
These scavengers alone have little effect on the basal level of HO
mRNA. In cells treated with sodium azide, the fluence-dependent HO
message induction lags initially and reaches corresponding control
levels only at 0.4 MJ/m 2 (Fig. 2B). L-Histidine also suppresses the
fluence-dependent accumulation of HO mRNA (Fig. 2E). The ratios
of HO mRNA signal intensity between cells treated with 102 scavenger and control cells (Fig. 2, C and F) indicate that in the presence
of sodium azide the HO mRNA levels are reduced to 40% of the
corresponding control at 0.2 MJ/m 2 (Fig. 2C). In histidine-treated
cells (Fig. 2F), the HO mRNA levels decrease to 50% of the controls
at 0.3 MJ/m 2. At higher fluences, the HO mRNA levels in cells treated
with either scavenger approach that in the corresponding controls.
While these results support those obtained in D20, the possible
involvement of the .OH as a primary effector needed further experimental consideration since the 10 z scavengers mentioned above are
also excellent scavengers of the .OH as predicted by their in vitro rate
constants (Table 1).
Hydroxyl Radical Scavengers. In order to eliminate the possibility that the observed effect of sodium azide or L-histidine on HO
induction was really due to scavenging of the -OH radical, we used
DMSO (30) and mannitol which scavenge the .OH far more specifically because of their low rate constants for reaction with 102 (Table
1). We find that these agents themselves have little effect on the basal
level of HO mRNA, and the fluence-dependent increase of this message in cells treated with either 4% DMSO or 0.1 Mmannitol is similar
to that observed in the corresponding control cells (Fig. 3, A, B, D, and
E). Plots of fluence-dependent change in ratios (Fig. 3, C and F)
indicate that the HO mRNA levels in the .OH scavenger-treated cells
are either almost equal to the corresponding controls (in mannitol) or
slightly higher (in DMSO) than the levels in the corresponding untreated cells.
Thus, scavengers of the .OH do not decrease the fluence-dependent
accumulation of HO mRNA, and at least in cells treated with DMSO
cause a slight enhancement of the HO mRNA levels.
Effect of Test Agents on Total RNA Synthesis. In order to determine whether the modulation of HO mRNA levels by the various test
agents is due to a general effect on transcription, we measured the total
[3H]uridine incorporation during 3 h in cells which had been pretreated with D20, sodium azide, or DMSO and then irradiated with
increasing fluences of UVA/NVL in the presence of these agents. In
control cells, the RNA synthesis is inhibited with increasing fluences
and reaches almost zero at 1 MJ/m 2 (Fig. 4A). In cells treated with
D20, there is further suppression of transcriptional activity at fluences
above 0.25 MJ/m 2. In contrast, sodium azide seems to protect against
the toxic effects of UVA on transcription, as seen by the higher
incorporation of [3H]uridine in cells irradiated in the presence of azide
(Fig. 4B). Finally, the .OH scavenger DMSO does not have a significant effect on the fluence-dependent decrease in transcription (Fig.
4C).
Fig. 2. Effect of sodium azide (NaN3) and L-histidine on UVA/NVL-induced HO mRNA accumulation. Northern blot analysis of HO and GAPDH mRNA in the presence ( + ) or
absence ( - ) of either 100 mM sodium azide (A) or 10 mM L-histidine (D). B and E, HO mRNA signals quantified and corrected for loading error and expressed as a relative increase
over basal levels. C and F, ratios of the HO signal intensity in scavenger-treated and control cells plotted as a function of UVA/NVL fluence. Point (bar), mean (•
4507
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Research.
SINGLET OXYGEN INDUCTION OF THE HUMAN HO 1 GENE
Fig. 3. UVA/NVL-induced HO mRNA accumulation in the presence of hydroxyl radical scavengers. Northern blot analysis of HO and GAPDH mRNA in the presence (+) and
absence (-) of 4% DMSO (A) and 0.1 M mannitol (D). B and E, relative increase in HO mRNA accumulation quantified by densitometry of autoradiographs. C and F, ratios of the
signal intensities of HO mRNA (corrected for loading error with GAPDH) in hydroxyl radical scavenger-treated and control cells. Point (bar), mean (_+SD).
mum concentrations as determined in a previous study (11) on radical
involvement in UVA inactivation of human fibroblasts.
The UVA/NVL fluence-dependent increase in HO mRNA levels is
initially enhanced in DEO (Fig. 1). Although the enhancement of
lifetime of 102 in vitro by D20 is by a factor of 10-15, this is not
reflected in the enhancement of HO mRNA levels in vivo presumably
due to the interaction of this active oxygen intermediate with biomolecules. Furthermore, deuteration of biological antioxidants could contribute to lowering the threshold of UVA/NVL fluence required to
induce HO gene in the presence of DzO. At fluences higher than 0.2
MJ/m z, the HO mRNA levels decrease rapidly (Fig. 1, A and B)
probably due to a further suppression of the UVA/NVL fluencedependent decrease in total RNA synthesis in the presence of DzO
(Fig. 4A). We have observed in untreated cells (data not shown) that
at fluences above 0.5 MJ/m 2, the HO mRNA levels also decline,
indicating that severe oxidant stress shuts off transcription of this
gene, probably because of a general inhibitory effect on total RNA
synthesis in cells. In the presence of sodium azide and L-histidine, the
fluence-dependent induction of HO mRNA is significantly (50--60%)
lower than the controls, and this is not due to an inhibitory effect on
transcription (Figs. 2 and 4B). Thus, in the fluence range in which
DISCUSSION
D20 has no effect on total RNA synthesis, it enhances the UVA/NVL
In the present study, we have compared the relative involvement of induction of HO mRNA. On the other hand, sodium azide protects to
102 and .OH in the UVA/NVL induction of the HO gene by chemi- some extent against the the toxic effects of UVA/NVL on total RNA
cally modulating the cellular concentration of these active oxygen synthesis in cells but decreases the UVA/NVL fluence-dependent HO
species, and we find that 102 is a primary effector in this induction. An mRNA accumulation up to fluences of 0.3 MJ/m 2. At higher fluences,
accompanying observation is that 102 also appears to be the active these agents are no longer effective, probably because of the generaintermediate involved in the UVA/NVL fluence-dependent inhibition tion of radicals by the scavengers themselves. Since sodium azide and
of overall transcription. Since it was not possible to measure effective L-histidine scavenge both the .OH and 1 0 2 with equal efficiency as
intracellular concentrations of the test agents used, we used the opti- predicted by their in vitro rate constants (Table 1), our results could be
4508
These results indicate that the enhancement of HO mRNA accumulation in D20 and suppression of this accumulation in sodium azide
are not due to a general effect of these agents on transcription.
Modulation of HO mRNA Levels by Rose Bengal. It is known
(31) that the photosensitizer RB generates 102 when illuminated with
visible light. Therefore, we studied the effect of this photosensitizer on
HO mRNA accumulation. We find that irradiation with a range of
fluences of visible light alone does not induce HO (Fig. 5, A and C).
In cells treated with 1 /XM RB, the HO mRNA levels increase to
29-fold above basal levels at the maximum fluence of visible light
used. In the presence of D20, the RB/visible light-induced increase in
HO mRNA levels is enhanced; the HO message levels increase to
82-fold above basal level at 1 kJ/m 2 (Fig. 5, B and C). At higher
fluences, the HO mRNA levels decline rapidly to basal levels. Thus,
the enhancement of HO mRNA accumulation by RB/visible light in
the presence of D20 is similar to that observed in the UVA/NVLinduced accumulation of this message.
These results are entirely consistent with the conclusion that it is the
102 which is a primary effector in the UVA/NVL induction of the
human heme oxygenase gene.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1993 American Association for Cancer
Research.
SINGLET
9.0 i
T
OXYGEN
INDUCTION
OF
THE
HUMAN
HO
1
GENE
0.5
0
kJm -2
-----On Control
f
~
m
D20
1.5
71
- +
Rose Bengal
3.06"0 ~
1.0
II
I
+ - +
HO
A
GAPDH
c
E
tiO
0.0
9.0
A
"t'~.'~
T
T
~
9
.o [,
Control
NaN3
e
[
GAPDH I--~
6.0
-
.E
"o
'r..
-I m-
__,____..,...] B
C
Control
B e ~ RoseBengal
- - - ~ D20- control
m A ~ D20. RoseBengal
3.0
B
0.0 . . . . .
9.O 1
Control
~
6.0 ~
~
80
~
60
*
._c Z
._>
3.0
0.0
c
. . . . . . . . . . .
0
0.2
0.4
0.6
0.8
1.0
Fluence ( MJm -2)
Fig. 4. Cells were labeled with [14C]thymidinefor 24 h, followedby incubation in
100% D20, 100 mM sodium azide, or 4% DMSO for 15 min at 37~ first and then
irradiation with a range (0-1 MJ/m2) of UVA/NVLfluences prior to incubation with
[3H]uridine for 3 h to measure total RNA synthesis. Because the [14C]thymidinecpm
(used here as an indicatorof cell number)was relativelyconsistentin all samples,the ratio
of 3H/14Ccpm was used as a measure of total RNA synthesis. Ratios of [3H]uridine/
[14C]thymidinecpm are plottedas a functionof UVA/NVLfluencefor cells treated with
D20 (A), sodium azide (B), and DMSO (C). Point (bar), mean (•
I~
a:._c
~
40
~176
1
|
0
9
i
9
|
500
1000
Fluence (Jm "2)
,
|
1500
Fig. 5. Modulationof HO mRNAlevelsby Rose Bengaland visiblelight. HO mRNA
levels were inducedin cells by Rose Bengal and visible light.A, Northernblots of total
cellular RNA from cells treated with increasingfluencesof visible light in the presence
(+) and absence(-) of 1 p.MRB probed for HO and GAPDH.B, Northernblot analysis
of total RNA from cells induced with RB/visible light in the presence of D20. C,
autoradiographsquantifiedby densitometryand the relativeincreasesin HO mRNAlevels
expressed as a functionof fluence.
class of endogenous photodynamic sensitizers, cause oxidative stress
due to scavenging of either one or both of these species. However,
DMSO and mannitol which preferentially scavenge the .OH (Table 1) by generating 102 (34) and are present in cell membranes. Moreover,
it has been demonstrated in Propionibacterium acnes (35) that the
have little or no effect on either the fluence-dependent increase in HO
maximum sensitivity to blue light (380-440 nm) was in the region of
mRNA levels or total RNA synthesis in cells (Figs. 3 and 4C). The
415 nm, which corresponded to the absorption maxima of porphyrins
possibility exists that the -OH scavengers do not reach the target
in cells. A porphyrin involvement has also been suggested (36) in near
molecules within the cell. However, for UVA/NVL induction of the
UV (300-400 nm) inactivation events in Escherichia coll. It has been
HO gene, the cell membrane is a more likely site for triggering a
shown in bacteria that RB accumulates in the cell membrane (37) and
cascade of signal transduction events since UVB and UVC radiations
that the photodynamic inactivation of bacteria by this dye is similar to
(3, 32), which cause more DNA damage than UVA radiation, do not
induce this gene. Any .OH generated by UVA/NVL at or close to cell that caused by use of chemically pure 102 on bacterial cells (38).
Thus, the RB-mediated photodynamic inactivation seems to be a
membranes would be expected to be easily accessible to scavengers.
membrane event in bacteria, probably because of the generation of
Use of RB (which is known to generate 102) to modulate HO mRNA
102. If we assume that RB also accumulates in the cell membrane of
levels supports the idea of 10 2 involvement. Interpretation of results
mammalian cells, the HO induction in the presence of this dye and
obtained using RB is complicated by the fact that it can generate
radicals (type I reactions) in addition to 102 (type II reactions). To visible light could be interpreted to be a membrane event. There is
ample evidence that UVA can cause lipid peroxidation of cell memavoid this problem, we have also induced HO with RB/visible light
branes in human skin fibroblasts (22) and, based on experiments with
in the presence of D20 in which type I reactions are presumably una wide variety of scavengers, this process is believed to be mediated
affected (33). The D20 enhancement of the increase in HO mRNA
levels by RB/visible light confirms the effectiveness of 102 in the by 10 2 (20, 21).
Thus, we propose that 102 may be involved as a crucial intermeinduction of the HO gene. Furthermore, in the presence of L-histidine,
which has little reactivity toward the superoxide anion (28), there is a diate at an early step in the signal transduction events that lead to the
decrease in the HO m R N A levels induced by RB/visible light (data not
UVA/NVL induction of the human heme oxygenase gene.
shown). Taken together, these results are consistent with the conclusion that it is the 102 which is the primary effector in the UVA/NVL
ACKNOWLEDGMENTS
induction of this gene in human skin fibroblasts.
An indication that a crucial early step in HO gene induction could
We thank Drs. Glenn Vile and Sohan Modak for useful discussions during
the course of this work.
be a membrane event is that porphyrins, which constitute an important
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SINGLET OXYGEN INDUCTION OF THE HUMAN ttO 1 GENE
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Research.
Singlet Oxygen: A Primary Effector in the Ultraviolet
A/Near-Visible Light Induction of the Human Heme Oxygenase
Gene
Sharmila Basu-Modak and Rex M. Tyrrell
Cancer Res 1993;53:4505-4510.
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