(Received for publication, November 14, 1994; and in revised form, December 20, 1994)
From the
Reactive oxygen intermediates like hydrogen peroxide
(HO
) have been shown to serve as messengers in
the induction of NF-
B and, then, in the activation and replication
of human immunodeficiency virus (HIV)-1 in human cells. Because
H
O
can be converted into the highly reactive
OH
at various locations inside the cells, we started
to investigate the generation of Reactive oxygen intermediates by
photosensitization. This technique is based on the use of a
photosensitizer which is a molecule absorbing visible light and which
can be located at various sites inside the cell depending on its
physicochemical properties. In this work, we used proflavine (PF), a
cationic molecule having a high affinity for DNA, capable of
intercalating between DNA base pairs. Upon visible light irradiation,
intercalated PF molecules oxidize guanine residues and generate DNA
single-strand breaks. In lymphocytes or monocytes latently infected
with HIV-1 (ACH-2 or U1, respectively), this photosensitizing treatment
induced a cytotoxicity, an induction of NF-
B, and a reactivation
of HIV-1 in cells surviving the treatment. NF-
B induction by
PF-mediated photosensitization was not affected by the presence of N-acetyl-L-cysteine while strong inhibition was
recorded when the induction was triggered by H
O
or by phorbol 12-myristate 13-acetate. Another transcription
factor like AP-1 is less activated by this photosensitizing treatment.
In comparison with other inducing treatments, such as phorbol
12-myristate 13-acetate or tumor necrosis factor
, the activation
of NF-
B is slow, being optimal 120 min after treatment. These
kinetic data were obtained by following, on the same samples, both the
appearance of NF-
B in the nucleus and the disappearance of
I
B-
in cytoplasmic extracts. These data allow us to postulate
that signaling events, initiated by DNA oxidative damages, are
transmitted into the cytoplasm where the inactive NF-
B factor is
resident and allow the translocation of p50/p65 subunits of NF-
B
to the nucleus leading to HIV-1 gene expression.
Considerable interest has been focused recently on the role that
some host transcriptional factors may play in the initial activation of
human immunodeficiency virus (HIV-1) ()gene expression by
interacting with the long terminal repeat (LTR) of the integrated
provirus(1) . Transcriptional activation of the LTR depends
largely on a major enhancer made of two directly repeated sequences
able to respond to the transcription factor
NF-
B(2, 3) . The consensus recognition site for
this factor is a decamer with two pentameric half-sites, each of which
participates in the recognition and stabilization of binding of the
NF-
B dimer (for a review, see Refs. 4 to 6). For this DNA binding
to occur and HIV-1 transcription to be initiated, NF-
B usually
associating p50 and p65 subunits must be translocated into the nucleus
from the cytoplasm where it is normally retained by interaction with
its inhibitory subunit named I
B(7, 8) . Related
B enhancers are present in the regulatory region of various
cellular genes like interleukin-2 (IL-2), IL-2 receptor-
,
interleukin-6 (IL-6), tumor necrosis factor-
(TNF-
) and have
further been shown to be functionally involved in the transcriptional
induction by various T-cell stimulants(9) . Functionally active
NF-
B complexes are induced after cellular activation in one of the
following ways: through the CD3-T-cell receptor complex in
T-lymphocytes, in response to antigen recognition (10, 11) or to anti-CD3 antibodies (12) , or
following stimulation with other inducers such as phorbol esters,
selected cytokines, and lipopolysaccharide in both lymphocytes and
monocytes-macrophages(13, 14, 15) .
Treatment of T-lymphocytes with hydrogen peroxide
(HO
) induces NF-
B DNA binding activity and
nuclear appearance of this factor (16) followed by a
transcriptional activation of the proviral DNA in cells latently
infected with HIV-1 (17) . The activation of NF-
B by
treatment of T-cells with H
O
appears to be a
specific event because it occurs at low extracellular concentrations,
and other DNA-binding proteins do not seem to be affected(16) .
Presumably, the mechanism involves a passive diffusion of
H
O
through the cell membrane where it would
trigger indirectly I
B phosphorylation and its controlled
proteolytic degradation, through a cytoplasmic chymotrypsin-like
protease, providing therefore an irreversible NF-
B
activation(18) .
Eukaryotic and prokaryotic cells produce
reactive oxygen intermediates (ROIs) continuously as side products of
the mitochondrial electron transfer chain reaction but also upon
exposition to solar radiations (UV and visible light) or -rays
(reviewed in (19) and (20) ). Most inducers of
NF-
B seem to rely on the production of ROIs as evidenced by the
inhibitory effect of antioxidants such as cysteine derivatives (16, 21) , metal chelators and
dithiocarbamates(16, 22, 23) , vitamin E and
-lipoic acid(24) . It has then been proposed that ROIs
serve as common messengers in the activation of NF-
B (16) and that NF-
B is primarily an oxidative
stress-responsive transcription factor.
The molecular pathways
leading to the critical dissociation of NF-B heterodimers from
I
B are not yet fully elucidated, especially the nature of the
biochemical events capable of initiating the redox controlled pathway.
With UV, several authors have postulated that DNA damages can trigger
the events leading to the activation of NF-
B and then to HIV-1
gene expression. The demonstration that the initial event in UV
activation of HIV-1 could well be lesions induced to DNA has been
obtained by (i) determining the action spectrum of UV-induced HIV-1
gene expression(25) , (ii) demonstrating that the HIV-1 LTR is
activated by much lower doses of UV when it is resident in a cell from
the repair-deficient disease, xeroderma pigmentosum, than when it is in
a repair-proficient cell(25) , and (iii) by abrogation of the
viral gene activation when cells are fused with liposomes encapsulating
T4 endonuclease V(26) . Thus, a signal which leads to HIV-1
gene expression could be initiated in the nucleus by UV. It would
migrate to the cytoplasm where it activates NF-
B by releasing its
inhibitory subunit I
B and allowing p50/p65 subunits to translocate
to the nucleus where they bind to their DNA responsive elements
situated in the LTR of the HIV-1 provirus. However, it has also been
shown(27, 28) that the mammalian UV response is
triggered by the activation of tyrosine kinases situated at the plasma
membrane level, and, also, cells anucleated by cytochalasin B treatment
are still fully responsive to UV in terms of NF-
B induction. Thus,
HIV-1 activation by UV-C could well involve two distinct pathways, one
being initiated by UV-products in DNA such as pyrimidine dimers, the
other through oxidative damages induced in membranes. In this paper, we
show that oxidative DNA modifications like 7,8-dihydro-oxyoguanine and
single-stranded breaks, generated by a photosensitizer which
intercalates between DNA base pairs(29, 30) , can
activate NF-
B and HIV-1 reactivation, supporting the idea that
oxidative DNA damages would be molecular intermediates able to trigger
a signaling pathway to the cytoplasm.
An oxidative stress mediated by HO
can lead to the HIV-1 reactivation (17) through the
activation of the cellular factor
NF-
B(16, 22, 36) . Several authors
speculated that H
O
can passively cross the
plasma membrane and, within the cell, is either converted by catalases
into H
O and O
, or, by the Fenton reaction into
hydroxyl radicals (OH
). Because H
O
can be converted into the highly reactive OH radical at various
cellular localizations, it was unclear from these data what type of
cellular oxidative damage triggers NF-
B activation and then HIV-1
reactivation. To try to clarify whether or not DNA oxidative damages
can participate in the NF-
B activation pathway, we have used
photosensitization reactions to generate ROIs in the DNA of lymphocytic
(ACH-2) or monocytic (U1) cell lines latently infected by HIV-1. The
photosensitizer used in this work is proflavine (PF) (Fig. 1A) which is a water-soluble cationic chromophore
having an important affinity for DNA (37, see (38) for
review). PF intercalates between DNA base pairs, and, when mixed with
ACH-2 or U1, it binds to DNA and is localized in the cell nucleus (Fig. 1B). No chromophore fluorescence was detected
outside the cell nucleus either by classical fluorescence microscopy or
by confocal microscopy using a highly sensitive camera even in the
presence of a large molar excess of PF compared to the concentrations
used in the following experiments (Fig. 1B). No PF
fluorescence was detected in the cytoplasm or in membrane and the
nuclear restriction of PF is particularly striking when compared with a
membrane-associated chromophore such as merocyanine 540 (Fig. 1B).
Figure 1:
A, chemical structure of proflavine
(PF). B, localization of proflavine in ACH-2 cells. Cells were
mixed with 2 µM PF in the dark and mounted on slides
before being observed by confocal fluorescence microscopy
( = 450 nm). The two upper panels show cells mixed with 2 µM PF observed under
fluorescence (left panels) or by phase contrast (right
panels). The two lower panels show cells mixed with PF at
2 µM (left panels) or with merocyanine 540 at 5
µM (right panels).
Figure 2: Damage profiles induced either by proflavine (PF) or rose bengal (RB) in promonocytic cells (U397 cells) treated with 1.0 µM PF and 2.5 µM rose bengal, respectively, and lymphocytic cells (T-cells) treated with 0.25 µM PF and 1.0 µM rose bengal, respectively. These cells were irradiated for 20 s with a 1000-watt halogen lamp emitting visible light. Columns indicate the number of DNA single strand breaks (open bars) and formamidopyrimidine-DNA glycosylase-sensitive sites (filled bars) as determined by alkaline elution. Data are means of two independent experiments.
Figure 3:
A, cytotoxic effect induced 48 h after the
oxidative stress induced in ACH-2 cells (filled symbols) or in
U1 cells (open symbols) photoreacted with proflavine during
various irradiation times (PF, 1.0 mM for U1 and 0.25
µM for ACH-2 cells, respectively). Percentage of cell
survival (log) is plotted versus irradiation time (min). B, cytotoxic effect induced 48 h after the
oxidative stress induced in ACH-2 cells (filled symbols) or in
U1 cells (open symbols) photoreacted with increasing PF
concentrations and irradiated with visible light for 10 min. Percentage
of cell survival (log
) is plotted versus PF
concentration (µM).
Among the cells surviving PF photosensitization, HIV-1 reactivation can be detected by measuring reverse transcriptase activity in the cell supernatant 24 and 48 h after the phototreatment (Fig. 4). After 24 h, the level of reverse transcriptase activity is still relatively low (data not shown), but becomes maximal 48 h after photosensitization. Two important results can be deduced from reverse transcriptase measurements: (i) reverse transcriptase values increase as a function of stress intensity, and (ii) for similar survival fractions, reverse transcriptase activities are somewhat higher for ACH-2 than for U1 cells. These results indicate also that the cells have to be cultured for at least 48 h after the stress to release virus particles in the supernatant. 30% survival seems to correspond to a stress intensity leading to the optimal virus reactivation. In other words, when the survival fraction is too low, there are not enough viable cells in culture to proliferate and to reactivate HIV-1.
Figure 4: Induction of HIV-1 reactivation in ACH-2 cells (filled symbols) or in U1 cells (open symbols) mediated by PF (concentrations are the same than in Fig. 3A). Supernatant fluids, corresponding to an identical cell number, were taken 48 h after the photosensitization to determine reverse transcriptase activities as described by (29) . The stimulation of the reverse transcriptase activity is the ratio between the activities measured for various PF concentrations and the initial value. Stimulation of reverse transcriptase is plotted versus PF concentration (µM).
Figure 5:
The effect of a photoreaction mediated by
PF on B-DNA binding activities in ACH-2 cells. A, rapid
induction of a nuclear
B enhancer DNA-binding protein by treatment
of ACH-2 cells with increasing concentrations of PF and irradiated for
10 min with visible light. Nuclear extracts were prepared 240 min after
the reaction with equal amounts of protein and mixed with a
P-labeled probe encompassing the
B elements of the
HIV-1 enhancer. Samples were loaded on 6% native polyacrylamide gels
and electrophoresed at 150 V. An autoradiogram of the gel is shown, and
the arrows indicate the position of the specific complex and
of the free probe. B, amount of
B DNA binding activity
detected in the nucleus of ACH-2 cells is plotted as a function of the
PF concentrations. C, appearance of a nuclear
B enhancer
DNA-binding protein by treatment of ACH-2 cells with PF (0.2
µM) and irradiated with visible light between 0 and 15
min. The sample noted - corresponds to cells treated with PF and
nonirradiated. Nuclear extracts were prepared in equal protein amounts
240 min after the reaction and mixed with a
P-labeled
probe encompassing the
B elements of the enhancer of the HIV-1
LTR. Samples were loaded on 6% native polyacrylamide gels and
electrophoresed at 150 V. Autoradiogram of the gel is shown, and the
arrows indicate the position of the specific complex and of the free
probe.
The level of NF-B
activation by the PF-mediated photosensitization was compared to those
induced by well-characterized agents such as H
O
and PMA. EMSA shows that NF-
B DNA binding activity is
stimulated at comparable levels by PF and H
O
(data not shown). Indeed, the stimulation level reached by the
PF-mediated treatment at 1.0 µM is similar to the one
obtained after treatment of ACH-2 cells with 250 µM H
O
, demonstrating that PF-mediated
photosensitization is very efficient in inducing NF-
B activity.
PMA also induces comparable levels of stimulation (data not shown).
The identity of the PF-activated DNA-protein complex with a
NF-B probe was further investigated by several different ways.
First, by competition analysis (Fig. 6A) and by the use
of antisera specific for the DNA-binding p50 and p65 subunits of
NF-
B (Fig. 6B). Nuclear extracts were prepared 240
min after PF-mediated photosensitization and were incubated with a
P-labeled NF-
B probe alone or in the presence of
increasing amounts of two different unlabeled competitor
oligonucleotides. Competition with either a 25- or 250-fold molar
excess of an oligonucleotide encompassing the same NF-
B elements
of HIV-1 eliminated the formation of the radioactive protein-DNA
complex induced by PF-mediated photosensitization. Competition
experiments carried out with this competitor mutated at the NF-
B
motifs did not abolish the binding, demonstrating unambiguously the
B-specific DNA binding of the PF-activated factor. The binding of
one minor activity to the DNA probe was only weakly influenced at a
high molar excess of both competitors demonstrating that this DNA
binding activity was not sequence-specific (Fig. 6A).
Figure 6:
Characterization of the PF-induced DNA
binding activity as being NF-B. A, competition analysis:
nuclear extracts from ACH-2 cells photoreacted for 10 min with 3.5
µM PF were prepared 240 min after the stress and analyzed
by EMSA as described in Fig. 5. Nuclear extracts were either
mixed directly with the
P-labeled probe or with the
labeled probe in the presence of 25 (+) or 250 (++)
molar excess of wild type or mutated
B site of the HIV-1
enhancer(77) . B, immunoreactivity of the PF-inducible
protein-
B enhancer complex. Nuclear extracts from ACH-2 cells
photoreacted with PF (1 µM) for 10 min were either mixed
directly with the
P-labeled
B probe or incubated with
antisera specific for p50 or p65 before being mixed with the
P-labeled
B probe(34) . Samples were then
loaded on a 4% polyacrylamide native gel and electrophoresed as in Fig. 5. C, the effect of cycloheximide (CHX)
on the induction of
B DNA-binding proteins. ACH-2 cells were left
untreated, were phototreated for 10 min with PF (0.25 µM),
were incubated with 50 µg/ml CHX alone, or phototreated 10 min with
PF (0.5 µM) and treated with 50 µg/ml
CHX.
Next, we have examined whether the PF-activated protein-DNA complex
could react with antisera raised against the DNA-binding p50 and p65
subunits of NF-B. These two sera clearly gave rise to a
characteristic supershift of the retarded complex demonstrating
unambiguously that the PF-activated factor is p50- and p65-containing
NF-
B (Fig. 6B).
Another characteristic of
NF-B is its activation by a post-translational mechanism involving
the release of the inhibitory subunit I
B from a cytoplasmic
inactive form(7) . In order to investigate whether the
activation of NF-
B by PF-mediated photosensitization involved a
post-translational mechanism, the photosensitization reaction was
performed in the presence of cycloheximide (CHX), a protein synthesis
inhibitor (Fig. 6C). When ACH-2 cells alone were
treated with 50 µg/ml CHX, only a very weak activation of NF-
B
was seen. On the other hand, NF-
B activity induced by PF
photosensitization was not affected by the presence of CHX indicating
that, in these conditions, NF-
B is activated by a
post-translational mechanism. Because several cytokines such as IL-1
and TNF-
are efficiently processed from precursors only under
inflammatory conditions(48, 49) , an autocrine
mechanism involving IL-1 or TNF-
release could be rather unlikely
in this case.
Figure 7:
NF-B inducibility in ACH-2 cells
grown in the absence (control) or in the presence of 30 mM NAC. NF-
B activation was performed by PF (0.2, 0.4, or 0.8
µM) photosensitization as described above or by
H
O
(25 or 50 µM) or by PMA (0.1
µM). NF-
B DNA binding activities were determined by
EMSA as reported in Fig. 5and the intensities of the
B
bands were quantitated by phosphorimaging. The amount of NF-
B
activities observed in the presence of NAC were compared directly to
those obtained in the absence of NAC (determined as being equal to
100%).
Figure 8:
Disappearance of IB and appearance of
NF-
B after the oxidative stress mediated by PF. A,
evolution of the NF-
B complex induced in ACH-2 cells by
phototreatment with PF (3.5 µM) for 10 min. Cells were
taken at various times after the photoreaction (from 0 to 240 min) and
used to prepare nuclear extracts to be analyzed by EMSA as described in Fig. 4. B, fate of I
B-
in ACH-2 cells
photoreacted exactly as described in A. Cytoplasmic proteins
(15 µg) from the same extracts as above were analyzed by
SDS-polyacrylamide gel electrophoresis and transferred on nylon
membrane followed by Western blot analysis using an anti-I
B-
IgG.
Figure 9:
Kinetics of induction of AP-1 DNA binding
activities in ACH-2 cells phototreated during increasing times (0 to 15
min) with 0.25 µM PF. Nuclear extracts were prepared 30
min (A), 60 min (B), and 90 min (C) after
the photoreaction. The nuclear extracts were mixed with a P-labeled probe encompassing the TRE elements of the type
I collagenase gene promoter.
Treatment of T-lymphocytes latently infected by HIV-1 with a
photosensitizer having a high affinity for DNA induces, upon visible
light irradiation, DNA oxidative damage, cytotoxicity, HIV-1
reactivation in cells surviving the treatment as well as the binding to
DNA, and nuclear appearance of a cellular transcription factor.
Evidence that this factor is NF-B is based on its
B-specific
DNA binding, its immunoreactivity with antisera raised against the p50
and p65 subunits of NF-
B, and the post-translational induction of
the DNA binding activity. The stimulation of the NF-
B DNA binding
activity can be detected at very low treatment intensity and it is very
high in comparison with the induction of another transcription factor
like AP-1. Moreover, NF-
B induction with ROIs generated in DNA
appears to be rather slow when compared with other inducing events like
phorbol ester treatment(18) . All these features allow us to
conclude that a photosensitization reaction capable of generating DNA
oxidative damages can induce specifically NF-
B.
Similar
induction of NF-B has already been described by several authors in
the case of
H
O
(16, 22, 23) .
These authors have postulated that H
O
penetrates the cell before being converted into highly reactive
OH radicals through a Fenton-like reaction. Since this conversion can
take place at various cellular locations where iron is available to
catalyze this reaction, it is not possible from these works to
determine what are the primary cellular targets responsible for the
initiation of the signaling pathway leading to NF-
B activation and
then to HIV-1 reactivation. To provide insights to this question, we
have used photosensitization to generate ROIs. Photosensitization is
based on the use of a chromophore absorbing visible light which in turn
transfers the absorbed energy either to molecular oxygen or directly to
a substrate (for review, see (40) and (55) ). In this
work, PF has been used as photosensitizer because it is an
acridine-derived molecule having a very high affinity for
DNA(29, 56) . The interaction of small planar acridine
molecules with DNA has been studied in detail (for review, see (39) and (57) ) to show that these molecules can draw
DNA base pairs apart from their normal 3.4-Å spacing to 6.8
Å causing the DNA to become longer and stiffer. At high molar
ratios between acridine and DNA, when all the intercalation sites are
filled, acridines can stack along the phosphate-sugar
backbone(37, 38, 57) . With chromatin, the
analysis of the binding isotherms shows that high affinity sites are
clustered on the nucleosomal DNA and at a very high intercalating drug
concentration it could cause dissociation of the nucleosome core
particle as observed with ethidium bromide(58, 59) .
The confirmation that acridines localize in cell nuclei has been
brought by numerous techniques such as fluorescence
microscopy(60) , electron miscrocopy (61) , and
electron spin resonance combined with the use of a spin-labeled
acridine derivative(62) . All these data confirm our
observation made using fluorescence microscopy to localize PF inside
lymphocytes or monocytes. Although no PF fluorescence can be detected
in the cytoplasm of these cells, intercalation into mitochondrial DNA
cannot be totally ruled out. However, the absence of PF fluorescence in
the cytoplasm would lead us to conclude that the vast majority of PF
molecules are localized in the cell nucleus. It should also be pointed
out that the lack of NF-
B inhibition by cell preincubation with
NAC can be used as an argument to exclude mitochondria as an
intermediate in the activation pathway initiated by PF-mediated
photosensitization.
Photosensitization reactions mediated by PF in
solution or bound to DNA have also been studied in detail (see (63) and (55) for review). Both type I (electron
transfer to DNA base) and type II (O
generation) reactions have been shown to take place. With
isolated DNA bases, these reactions occurred with a yield of 0.55 and
0.45 for types I and II, respectively(63) . The first mechanism
(type I) occurred only when PF was in contact with a substrate like
DNA, but the second pathway (type II) generated
O
with PF either free in solution or bound to DNA. Both
photochemical reactions have been shown to produce
7,8-dihydro-8-oxoguanine in DNA efficiently(64, 65) .
O
is a rather long-lived species in aqueous
solution (2 µs in water); however, in cells, its lifetime is very
short (between 5 and 100 ns for L1210 and erythrocyte ghosts,
respectively) because the intracellular concentration of substrates
which are readily oxidizable by this species is very high(66) .
Then, generated by PF inside the DNA of ACH-2 cells,
O2
could not diffuse easily outside DNA because its lifetime is too short
and the amount of oxidizable substrates too high. This interpretation
is in accordance with the observation that pretreatment of cells with
NAC does not affect the level of NF-
B induction by PF-mediated
photosensitization. Indeed, NAC itself or GSH does not interact with
DNA, then cannot interfere with a process generating ROIs inside DNA
explaining why no protection can be conferred by NAC against NF-
B
induction by PF photosensitization. On the other hand, NAC was shown to
be very effective when NF-
B induction was performed by
H
O
or PMA because NAC, in that case, localizes
in cell compartments where it can efficiently scavenge ROIs (16, 22, 23, 36) .
Oxidative stress mediated by PF induces DNA damage at purines, single-strand breaks, and cytotoxicity in ACH-2 cells. Among the induced DNA modifications, it has been shown that 7,8-dihydro-8-oxoguanine is not capable of blocking DNA replication (67) while other modifications, if not repaired, can inhibit DNA replication (68) and transcription; therefore, they are possibly responsible for the lethal effects observed with this photosensitizer. Thus, unrepaired purine oxidation products can well be suspected to play an important role in the lethal effect and in the HIV-1 reactivation which is induced among cells surviving the treatment. This hypothesis is supported by the observation that the cytotoxic effect induced by the photosensitization mediated by PF is always more intense 48 h after the reaction than at earlier times.
A central event in
the HIV-1 reactivation from ACH-2 cells photosensitized with PF is the
induction of NF-B. This activation occurs through a
post-translational mechanism leading to the release of the inhibitory
subunit I
B from its complex with p65 and p50 in the cytoplasm (for
review, see (4, 5, 6) ). Release of I
B
allows translocation to the nucleus of NF-
B and its DNA binding.
The reactions that abolish binding of I
B to p65 have been
elucidated. When the stimulation is due to phorbol esters, the
degradation seems to occur in two
steps(18, 51, 52, 53, 69) ,
(i) transfer of phosphoryl groups onto I
B by a protein kinase
which causes its release and (ii) degradation of I
B through a
cytoplasmic chymotrypsin-like protease (18) or
proteasome(70, 71) . With PMA, TNF-
, or
IL-1
, these events appear soon after the induction (less than 5
min), but turn out to be much slower with the reaction mediated by PF.
These results allow us to postulate that the I
B release from p65
and p50 after the oxidative stress induced by PF involves other
additional intermediates. The signaling pathway from the nucleus to the
cytoplasm would then be more complex and slower than the one initiated
at the plasma membrane level by PMA, TNF-
, or IL-1
. A similar
signaling pathway has been postulated by several authors in the case of
the UV-mediated activation of AP-1 (25, 72, 73) and
NF-
B(25, 26) . These authors postulate that DNA
modifications, i.e. pyrimidine dimers, induced by UV would be
responsible for the initiation of a signaling pathway from the nucleus
to the cytoplasm. However, Devary et al.(28) have
shown that UV activation of NF-
B can also occur without cell
nucleus implying that another pathway is likely to be initiated from
other cellular targets, e.g. membranes, growth factor
receptors (74) . The existence of a pathway initiated at the
membrane level has found a support in the demonstration that the
breakdown of phosphatidylcholine by the addition of exogenous
phospholipase C activates NF-
B and increases HIV-1 replication in
human monocytes and T-lymphocytes(75) . On the other hand, DNA
modifications have been shown to be directly responsible for the
transcriptional activation of gadd153 gene(76) . A
large variety of DNA modifications can lead to this activation like
those induced by UV, UV-mimetic, DNA cross-linking, and alkylating
agents but also by intercalators and inhibitors of topoisomerases.
These authors postulate that the induction of gadd153 gene
does not occur by chromatin decondensation as proposed by Valerie and
Rosenberg (77) and by Verdin et al.(78) but
through the activation of cellular factors like AP-1(79) .
Moreover, the activation of AP-1 binding activity and gadd153 promoter transcription activation after treatment of WI-38 human
fibroblasts with either UV or alkylating agents is diminished in late
passaged cells compared to early passages(79) .
In the case
of the NF-B activation by the photosensitization mediated by PF,
the pathway is very likely to be initiated by oxidative damage induced
in DNA because (i) the photosensitizer exhibits a high DNA affinity and
can be visualized in the cell nucleus and (ii) the ROIs generated by
the photoreaction induce DNA damages. Looking at the level of NF-
B
stimulation which is quite similar to the one induced by
H
O
for identical survival fractions, we think
that it is rather unlikely that NF-
B induced in PF-treated cells
would be due to a very small fraction of PF which would not be
intercalated into DNA. We postulate thus that oxidative base damage,
probably in genomic DNA, is the event initiating the signaling pathway
leading to the cytoplasmic release of I
B and in turn to NF-
B
translocation to the nucleus. Experiments are now in progress to try to
identify the nature of the intermediates involved in this pathway
paying special attention to the activation of DNA-activated protein
kinase (80) or to kinase homologs of a yeast protein kinase
encoded by the DUN1 gene which is known to control the DNA
damage response in yeast(81) .