Báo cáo khoa học: Photodynamic treatment and H2O2-induced oxidative stress result in different patterns of cellular protein oxidation ppt

Photodynamic treatment and H
2
O
2
-induced oxidative stress result
in different patterns of cellular protein oxidation
Dmitri V. Sakharov
1
, Anton Bunschoten
1
, Huib van Weelden
2
and Karel W. A. Wirtz
1
1
Department of Biochemistry of Lipids, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes,
Utrecht University, the Netherlands;
2
Department of Photodermatology, University Medical Center Utrecht, the Netherlands
Photodynamic treatment (PDT) is an emerging therapeutic
procedure for the management of cancer, based on the
use of photosensitizers, compounds that generate highly
reactive oxygen species (ROS) on irradiation with visible
light. The ROS generated may oxidize a variety of bio-
molecules within the cell, loaded with a photosensitizer.
The high reactivity of these ROS restricts their radius of
action to 5–20 nm from the site of their generation. We
studied oxidation of intracellular proteins during PDT
using the ROS-sensitive probe acetyl-tyramine-fluorescein
(acetylTyr-Fluo). This probe labels cellular proteins, which
become oxidized at tyrosine residues under the conditions

of oxidative stress in a reaction similar to dityrosine for-
mation. The fluorescein-labeled proteins can be visualized
after gel electrophoresis and subsequent Western blotting
using the antibody against fluorescein. We found that PDT
of rat or human fibroblasts, loaded with the photosensitizer
Hypocrellin A, resulted in labeling of a set of intracellular
proteins that was different from that observed on treatment
of the cells with H
2
O
2
. This difference in labeling patterns
was confirmed by 2D electrophoresis, showing that a lim-
ited, yet distinctly different, set of proteins is oxidized under
either condition of oxidative stress. By matching the
Western blot with the silver-stained protein map, we infer
that a-tubulin and b-tubulin are targets of PDT-induced
protein oxidation. H
2
O
2
treatment resulted in labeling of
endoplasmic reticulum proteins. Under conditions in which
the extent of protein oxidation was comparable, PDT
caused massive apoptosis, whereas H
2
O
2
treatment had no
effect on cell survival. This suggests that the oxidative stress

generated by PDT with Hypocrellin A activates apoptotic
pathways, which are insensitive to H
2
O
2
treatment. We
hypothesize that the pattern of protein oxidation observed
with Hypocrellin A reflects the intracellular localization of
the photosensitizer. The application of acetylTyr-Fluo may
be useful for characterizing protein targets of oxidation by
PDT with various photosensitizers.
Keywords: apoptosis; Hypocrellin A; photodynamic treat-
ment; protein oxidation; tubulin.
Photodynamic therapy is an emerging modality for the
treatment of cancer [1]. It is based on the killing of tumor
cells by light-activatable photosensitive compounds, or
photosensitizers. In the presence of oxygen, the combination
of visible light and a photosensitizer causes generation of
singlet oxygen and other cytotoxic reactive oxygen species
(ROS), such as superoxide anions and the extremely reactive
hydroxy radical [2]. The higher uptake of photosensitizers
by cancerous tissues compared with normal tissues, and the
possibility of local illumination of tumors are essential for
selective eradication of tumor cells with photodynamic
treatment (PDT). The mode of cell death by PDT may be
either apoptosis or necrosis, depending on the nature and
concentration of the photosensitizer and the amount of
irradiation [3,4].
Although the signaling pathways activated in response to
PDT are partly delineated and the sequence of apoptotic

events induced by PDT is well described [3–8], the specific
cellular targets of PDT and critical early events involved
in triggering PDT-induced apoptosis are not clear [4,9].
Different photosensitizers have different intracellular locali-
zations. Singlet oxygen and hydroxy radical, the most
reactive photodynamically generated species, have extre-
mely short lifetimes (less than 1 ls) in the intracellular
environment, and therefore their sphere of influence is very
small, not more than 20 nm from the site of their generation
[2]. In this way, the intracellular localization of the
photosensitizer determines the areas of its photodynamic
action. Molecular targets of oxidation may therefore also
vary depending on the localization of the photosensitizer
[2,3].
PDT may damage proteins, lipids, DNA, and a variety of
small molecules in the cell [2]. Recent data [10] suggest that
cellular proteins are a likely key target for toxicity mediated
by singlet oxygen. Oxidative assault may cause modifica-
tions of the side chains of amino acids within a protein. In
particular, the side chains of cysteine, histidine, methionine,
tryptophan and tyrosine are susceptible to ROS-induced
modifications [2,11,12]. Modifications of tyrosine are of
particular interest, because it is critically involved in intra-
cellular signal transduction via tyrosine phosphorylation.
Correspondence to D. V. Sakharov, CBLE, Utrecht University,
PO Box 80.054, 3508 TB Utrecht, the Netherlands.
Fax: + 31 30 2533151, Tel.: + 31 30 2532852,
E-mail:
Abbreviations: PDT, photodynamic treatment; ROS, reactive oxygen
species; acetylTyr-Fluo, acetyl-tyramine-fluorescein; ECL, enhanced

chemiluminescence.
(Received 29 July 2003, revised 8 October 2003,
accepted 21 October 2003)
Eur. J. Biochem. 270, 4859–4865 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03885.x
Interactions of tyrosine with ROS may result in generation
of tyrosyl radicals, which can dimerize to yield dityrosine
[13,14]. It has been shown that tyrosyl radicals, and
eventually dityrosine, are formed as a result of PDT of
tyrosine, probably in a reaction mediated by singlet oxygen
[15].
We have recently developed a technique that utilizes a
probe, acetyl-tyramine-fluorescein (acetylTyr-Fluo), which
allows detection and identification of intracellular proteins
that become oxidized at tyrosine residues under the
conditions of oxidative stress [16,17]. Using this technique,
we have shown that the proteins of the endoplasmic
reticulum are the major targets of oxidation induced by
treatment of cells with H
2
O
2
[18].
In this study, we used this technique in combination with
2D electrophoresis to assess the oxidation of proteins in cells
subjected to PDT with the photosensitizer Hypocrellin A.
Hypocrellins are stucturally related to polycyclic quinones.
They show extremely high phototoxicity towards tumors
and viruses and are being explored for a variety of
therapeutic applications [19–21]. We have found that PDT
with Hypocrellin A oxidizes a distinct set of cellular

proteins, including tubulins, which are not oxidized by
treatment of the cells with H
2
O
2
.
Experimental procedures
Materials
Hypocrellin A and Hoechst 33342 were purchased from
Molecular Probes (Leiden, the Netherlands). Rose Bengal,
carbonic anhydrase, H
2
O
2
, propidium iodide and tyramine
were from Sigma. Polyclonal antibody against fluorescein,
conjugated to horseradish peroxidase, was purchased from
Biogenesis (Poole, Dorset, UK). Tyramine-fluorescein (Tyr-
Fluo) and acetylTyr-Fluo were synthesized as described
elsewhere [16].
Photodynamic treatment
Specimens containing either cells or solutions of purified
components were illuminated with visible light from a slide
projector equipped with a 250 W tungsten lamp. The purple
and blue part of the light spectrum with k< 470 nm was
cut off by a short-cut filter. The fluence rate in the
irradiation area was 10 mWÆcm
)2
. To reach the fluence of
1JÆcm

)2
and 2 JÆcm
)2
, the specimens were irradiated for
100 s and 200 s, respectively. The fluence rate was measured
with a specially modified and calibrated photometer
(Waldmann AG, Schwenningen, Germany).
Assessment of dityramine formation caused by PDT
Photosensitizers at a final concentration of 10 l
M
were
added to a well of a plastic culture plate containing 1 m
M
tyramine in NaCl/P
i
, pH 7.4. The wells were irradiated with
visible light as described above. Dityramine formation was
assessed by measuring a characteristic fluorescence signal of
dityramine (excitation maximum at 315 nm, emission
maximum at 405 nm). Some of the samples were also
analyzed by electrospray MS using a Quattro Ultima mass
spectrometer.
Photodynamic labeling of a model protein
with the Tyr-Fluo probe
A solution containing 0.4 mgÆmL
)1
carbonic anhydrase and
10 l
M
Tyr-Fluo in NaCl/P

i
was irradiated with visible light
in either the presence or absence of a photosensitizer (Rose
Bengal or Hypocrellin A, 10 l
M
). The samples were
subjected to SDS/PAGE and Western blotting with anti-
body against fluorescein.
Cell culture and PDT
Rat-1 fibroblasts or adult normal human dermal fibro-
blasts were cultured in Dulbecco’s modified Eagle’s
medium with 7.5% fetal bovine serum at 5% CO
2
(v/v)
in the presence of penicillin and streptomycin. The
experiments were performed with 70–80% confluent cells
growing in 10 cm Petri dishes. For the experiments
involving microscopy, the cells were grown in glass-
bottomed 3.5-cm dishes (Willco Wells, Amsterdam, the
Netherlands). Most of the experiments were performed
with Rat-1 fibroblasts, which are easier to culture. Key
experiments, in particular those involving 2D-PAGE, were
also repeated with human fibroblasts, because their
detailed protein map has been published. Hypocrellin A
was loaded into the cells in the culture medium for 3 h.
Then the medium with photosensitizer was removed, and
the cells were incubated for 15 min with acetylTyr-Fluo
(5 l
M
)inNaCl/P

i
supplemented with 0.9 m
M
CaCl
2
,
0.5 m
M
MgCl
2
,and5m
M
glucose (NaCl/P
i
+). After
removal of NaCl/P
i
+ containing acetylTyr-Fluo, fresh
NaCl/P
i
+ was added and the cells were irradiated with
visible light as described above. Immediately after irradi-
ation, the cells were rinsed with a salt-free isotonic buffer
(0.25
M
sucrose, 1 m
M
EDTA, and 20 m
M
Tris/HCl,

pH 7.4) and lysed in buffer containing 20 m
M
Tris/HCl
(pH 7.4), 1 m
M
EDTA, 1% Triton X-100 and a cocktail
of protease inhibitors (Sigma P-8340) diluted 1 : 40.
In some experiments, cells loaded with acetylTyr-Fluo as
described above were treated with H
2
O
2
in NaCl/P
i
+for
15 min and lysed.
Detection of cellular proteins susceptible to oxidation
Cell lysates were subjected to either SDS/PAGE under redu-
cing conditions in 10% polyacrylamide gels or 2D-PAGE.
Isoelectrofocusing, the first step of the 2D-PAGE, was per-
formed on 11 cm-long Bio-Rad IPG strips (ReadyStrip
TM
),
pH 3–10, according to the manufacturer’s instructions,
using a Protean IEF Cell. SDS/PAGE in the second
direction was run under reducing conditions in 15%
polyacrylamide gel with 0.08% bisacrylamide. 1D PAGE
gels were blotted on to a nitrocellulose membrane and
subjected to Western blotting with peroxidase-conjugated
antibody against fluorescein to detect the Tyr-Fluo-labeled

proteins. An enhanced chemiluminescence (ECL) kit from
Bio-Rad was used to visualize the labeled spots. 2D gels
were either stained with silver or subjected to Western
blotting, as described above for 1D gels. After blotting of
the two-dimensional gels (before blocking of the membrane
and application of the antibody), the membranes were
stained with Ponceau Red and scanned.
4860 D. V. Sakharov et al.(Eur. J. Biochem. 270) Ó FEBS 2003
To colocalize the labeled spots on the ECL film with the
spots on the silver-stained gels, a composite image file was
created, containing the spots labeled with fluorescein
(oxidized proteins, detected by ECL after Western blotting)
and 7–10 major spots visible on Ponceau-stained mem-
branes.
PDQUEST
software was used to edit the images of
silver-stained gels and spots from the ECL films. Adobe
Photoshop software was used to rescale the images and fit
the major spots of the silver-stained gel to the corresponding
Ponceau-stained spots on the membrane. In this way, it was
possible to match the ECL-detected spots to the corres-
ponding spots on the silver-stained gels.
MS (peptide mass fingerprints of trypsin digests of the
spots of interest obtained with MALDI-TOF, followed by a
database search with the Mascot software for peptide
mapping result) and matching of our protein maps to the
published protein maps of human fibroblasts, available at
the Human 2D-PAGE Databases of the Danish Centre for
Human Genome Research (),
were used to identify the protein spots of interest in the

silver-stained gels.
Fluorescence/confocal microscopy
Nikon Eclipse TE2000-U microscope, equipped with both
conventional fluorescence appliances and confocal laser
scanning C1 unit, was used in this study. Hypocrellin A
distribution before and after PDT was assessed using the
confocal mode with excitation at 543 nm from a HeNe
laser. For the immunofluorescence detection of tubulin, the
cells subjected to PDT were briefly incubated with propi-
dium iodide for 3 min, fixed with methanol at )20 °Cfor
5 min, permeabilized with 0.1% (v/v) Triton X-100 in
NaCl/P
i
for 15 min, and stained with Cy3-labeled tubulin
antibody (Sigma). For assessment of the viability, the cells
were stained with a mixture of Hoechst 33342 and
propidium iodide (both at 2 lgÆmL
)1
in the culture
medium), and fluorescence images were taken using the
conventional fluorescence mode. Cell morphology was
documented by differential interference contrast.
Results
In this study we focused on the detection of tyrosine
oxidation of the intracellular proteins on oxidative stress
induced by PDT of the cells. A tyrosine analogue,
tyramine, coupled covalently to fluorescein (Tyr-Fluo),
was used as a probe to label the cellular proteins
susceptible to this type of oxidative modification. On
oxidation of the tyramine moiety by ROS, tyramine is

converted into a tyrosyl radical that can form crosslinks
Fig. 1. Photosensitized formation of dityramine. Asolutionof1m
M
tyramine was irradiated with visible light (2 JÆcm
)2
) in either the
presence or absence of a photosensitizer (Rose Bengal or Hypocrel-
lin A, 10 l
M
). Formation of dityramine was assessed by measuring
fluorescence with the characteristic spectra of dityramine (excitation
maximum at 315 nm, emission maximum at 405 nm). 1, Rose Bengal
with light; 2, Hypocrellin A with light; 3, Rose Bengal without light;
4, Hypocrellin A without light; 5, no photosensitizer with light.
Fig. 2. Photosensitized labeling of carbonic anhydrase with tyramine-
fluorescein. A solution containing 0.4 mgÆmL
)1
carbonic anhydrase
and 10 l
M
Tyr-Fluo was irradiated with visible light (2 JÆcm
)2
)in
either the presence or absence of a photosensitizer (Rose Bengal or
Hypocrellin A, 10 l
M
). The samples were subjected to SDS/PAGE
and Western blotting with an antibody against fluorescein. Lane 1, no
photosensitizer; 2, no photosensitizer with light; 3, Rose Bengal; 4,
Rose Bengal with light; 5, Hypocrellin A; 6, Hypocrellin A with light.

Fig. 3. Labeling of cellular proteins on PDT and treatment with H
2
O
2
.
Lane 1, control cells loaded with acetylTyr-Fluo, no treatment; 2, cells
were loaded with acetylTyr-Fluo and irradiated with visible light at
1JÆcm
)2
(no photosensitizer control). Lanes 3 and 4, cells were loaded
with Hypocrellin A (1 l
M
and 2 l
M
, respectively), then with acetyl-
Tyr-Fluo, and were finally irradiated with visible light (1 JÆcm
)2
).
Lane 5, cells were loaded with 2 l
M
Hypocrellin A, then with acetyl-
Tyr-Fluo, and were not irradiated (no light control). Lane 6, cells were
loaded with acetylTyr-Fluo and then treated with 200 l
M
H
2
O
2
.Cell
lysates were subjected to SDS/PAGE and Western blotting with

antibody against fluorescein.
Ó FEBS 2003 Protein oxidation on photodynamic treatment (Eur. J. Biochem. 270) 4861
with oxidized tyrosine residues in a target protein. In the
first experiments, we assessed whether PDT can cause
dityrosine (dityramine) formation, and the covalent coup-
ling of the Tyr-Fluo to a model protein.
Figure 1 shows that dityramine is formed on PDT of a
solution of tyramine with either Rose Bengal or Hypocrel-
lin A as photosensitizer. Dityramine formation was docu-
mented by generation of a fluorescent signal with a
characteristic spectrum (maximum of the excitation spec-
trum at 315 nm and a maximum of the emission spectrum
at 405 nm). MS (not shown) also confirmed generation of
dityramine on PDT with Hypocrellin A. No dityramine was
formed in the absence of either light or photosensitizer.
Figure 2 shows that PDT in the presence of either Rose
Bengal or Hypocrellin A causes labeling of carbonic
anhydrase with Tyr-Fluo. Labeling was dependent on the
concentration of the photosensitizer used (not shown). Rose
Bengal caused stronger labeling than Hypocrellin A. In
further experiments, Hypocrellin A was used because Rose
Bengal does not accumulate in the cell.
Irradiation of rat fibroblasts, loaded with both Hypo-
crellin A and acetylTyr-Fluo, resulted in the labeling of
cellular proteins, as shown in Fig. 3. PDT-induced protein
labeling was dependent on the concentration of the
photosensitizer (Fig. 3, lanes 3 and 4) and the dose of
irradiation (not shown). The pattern of labeling in the cells
treated with PDT was different from that obtained with the
cells treated with H

2
O
2
.
2D-PAGE in combination with Western blotting was
applied to resolve the difference in the protein labeling.
These experiments were performed with both rat (not
shown) and human fibroblasts with similar results.
2D-PAGE images obtained with human fibroblasts are
presented in Fig. 4. Only a limited number of proteins were
labeled on PDT and H
2
O
2
treatment, but the patterns of
protein labeling were distinctly different (Fig. 4A,B).
Matching the blot with the protein map shows that PDT
caused labeling of a-tubulin and b-tubulin (spots 1 and 2).
The minor spot 3 probably reflects labeling of a small
fraction of actin. The rest of the spots remain to be
identified. We could not detect any labeled spots in the
control samples obtained from cells either loaded with
the photosensitizer but not irradiated or irradiated in the
absence of the photosensitizer. As for treatment with H
2
O
2
,
the labeling pattern agreed with the results of our previous
study [18], which showed labeling of endoplasmic reticulum

proteins (Bip, spot 4; PDI, spot 5; GPP58, spot 6).
Careful assessment of the general changes of the protein
map on PDT was beyond the scope of this study. Under the
conditions of the experiment presented in Fig. 4, the protein
map did not change dramatically, although some of the
spots in the silver-stained gels were upregulated or down-
regulated in PDT-treated samples. PDT at higher concen-
trations of the photosensitizer had a dramatic effect on the
protein map (not shown): many spots either disappeared or
were spread along the horizontal axis of the gel. This was
probably a result of photodynamic crosslinking of proteins
[22,23]. Under these conditions, PDT resulted in rapid cell
death (not shown).
Figure 5A shows the subcellular localization of Hypo-
crellin A in rat fibroblasts before irradiation. In agreement
with other studies (reviewed in [19]), Hypocrellin A locali-
zed mainly in lysosomes. We observed that it was also
present throughout the cytoplasm, although to a lesser
extent. Some of the photosensitizers have been shown to
rapidly redistribute within the cell under irradiation, for
instance to leak from lysosomes to cytosol [24,25]. It was not
the case under the conditions used in this study. Under the
conditions used in the experiment presented in Fig. 4, the
distribution of Hypocrellin A did not change during and
immediately after irradiation (not shown), implying that
oxidation of cytoskeletal proteins is not a result of acute
Fig. 4. 2D-PAGE detection of oxidized pro-
teins in cells treated with PDT or H
2
O

2
. (A,C)
Human fibroblasts were loaded with 1 l
M
Hypocrellin A, then with acetylTyr-Fluo, and
were finally irradiated with visible light
(1 JÆcm
)2
); (B,D) Cells were loaded with ace-
tylTyr-Fluo and exposed to 200 l
M
H
2
O
2
for
10 min. Cell lysates were subjected to 2D-
PAGE and either analyzed for the presence of
oxidized proteins by Western blotting with
antibody against fluorescein, or stained with
silver. Oxidized proteins detected by Western
blotting are shown in (A) and (B). Silver
stainingisshownin(C)and(D)inblue
superimposed with the spots of oxidized pro-
teins shown in red. Protein labels: 1, a-tubulin;
2, b-tubulin;3,actin;4,PDI;5,BiP;6,
GRP58.
4862 D. V. Sakharov et al.(Eur. J. Biochem. 270) Ó FEBS 2003
leakage of the photosensitizer from the sites of its primary
localization into the cytosol.

The results presented in Fig. 4 indicate that tubulin is a
direct target of oxidation on PDT. To follow the fate of the
microtubule network, we used immunofluorescence. Micro-
tubule organization was already disturbed 5 min after
PDT. At 1 l
M
Hypocrellin A, the tubulin network became
less regular and less sharp (Fig. 5C) than in control cells
(Fig. 5B). At a higher concentration of the photosensitizer,
the microtubules were completely destroyed (Fig. 5D).
Under the latter conditions (2 l
M
Hypocrellin A), the cells
were not yet dead 5 min after PDT, as judged by the
absence of staining with propidium iodide, but after 1 h
most of the cells were dead through necrosis.
OnPDTat1l
M
Hypocrellin A (conditions used in the
experiment presented in Figs 4 and 5C), most cells became
apoptotic 4 h after irradiation (Fig. 6A,C,E). Quantitative
analysis of three independent experiments showed that only
6 ± 4% (mean ± SD) of the cells remained alive (normal
cellular and nuclear morphology, no propidium iodide
staining), 68 ± 28% were apoptotic (blebbing, condensed
or fragmented nucleus, no propidium iodide staining),
and 26 ± 16% were necrotic (characteristic necrotic
morphology, propidium iodide staining of the nucleus). In
the light-only and photosensitizer-only controls, there were
practically no dead cells (less than 2% necrotic, no apoptotic

cells). In contrast with PDT, treatment with H
2
O
2
did not
result in noticeable cell death after 4 h (Fig. 6B,D,F) or 24 h
(not shown).
Discussion
Oxidative stress induced by PDT can affect several types of
biomacromolecules including proteins, lipids, and DNA [2].
A substantial body of evidence indicates that the cellular
proteins are the key target of ROS-mediated toxicity
[11,12,26] including singlet oxygen-mediated toxicity
[10,26]. Oxidation of cellular proteins in response to PDT
may be crucially involved in the mechanisms of PDT-
induced cell death.
Although a number of particular intracellular proteins
have been shown to be modified as a result of PDT [27–29],
little work has been done at the level of the whole cellular
proteome in response to PDT. In the only available paper,
Grebenova et al. [30] showed that a number of protein spots
in the proteomic map of the HL60 cell lysates are
significantly reduced after subjection of the cells to PDT
Fig. 5. Distribution of Hypocrellin A in Rat-1 fibroblasts and effect of
PDT on the microtubule network. (A) Rat-1 fibroblasts were loaded
with Hypocrellin A under the conditions described in the legend to
Fig. 4. The confocal image shows Hypocrellin A distribution before
irradiation. No considerable change in the localization of Hypocrel-
lin A was observed after irradiation (not shown). (B–D) Cells were
loaded with 0 l

M
(B), 1 l
M
(C), or 2 l
M
(D) Hypocrellin A, irradiated
with visible light (1 JÆcm
)2
), fixed with cold methanol 5 min after
irradiation and stained with Cy3-labeled antibody against tubulin. Bar:
20 lm.
Fig. 6. PDT, but not H
2
O
2
treatment, induces apoptosis. Rat-1 fibro-
blasts were treated with either PDT (A,C,E) or H
2
O
2
(B,D,F) under
the conditions described in the legend to Fig. 4, incubated in a CO
2
incubator for 4 h and stained with a mixture of Hoechst 33342 and
propidium iodide. Differential interference contrast images (A,B) show
apoptotic morphology (blebbing) in the most of the cells treated with
PDT (A), but not in the cells treated with H
2
O
2

(B). Hoechst 33342
staining (C,D) allows the distinction between normal cells (large evenly
stained nucleus, indicated with No) and apoptotic cells (condensed or
fragmented nucleus, indicated with Ap). Staining with propidium
iodide (E,F) indicates dead cells with permeabilized plasma membrane.
Bar: 20 lm.
Ó FEBS 2003 Protein oxidation on photodynamic treatment (Eur. J. Biochem. 270) 4863
with 5-aminolevulinic acid. In our study, we combined the
proteomics approach with detection of proteins oxidized
in response to PDT. We used a technique that utilizes an
intracellular oxidation-sensitive probe, acetylTyr-Fluo,
which labels proteins susceptible to oxidation at tyrosine
residues.
In a purified system we have shown that dityramine
formation, the reaction essential for Tyr-Fluo labeling of
proteins, can be induced by PDT of tyramine solution with
the photosensitizers Hypocrellin A and Rose Bengal. Fur-
thermore, a model protein was labeled with Tyr-Fluo by
PDT with the same photosensitizers. Furthermore, in the
cells, protein oxidation was observed, which was dependent
on the concentration of the photosensitizer and on the
illumination. 2D electrophoresis was further applied to
determine which proteins are oxidized on PDT.
We have previously shown that treatment of cells with
H
2
O
2
causes oxidation of proteins localized in the endo-
plasmic reticulum. This has been suggested to be a

consequence of the specific redox status of the endoplasmic
reticulum, facilitating local generation of radicals capable of
inducing tyrosyl radical formation [31]. In this study, we
observed a different pattern of protein labeling on PDT of
cells loaded with Hypocrellin A. We hypothesize that this
pattern reflects the cellular localization of Hypocrellin A.
Hypocrellin A is a moderately hydrophobic substance,
which localizes mainly to the membranes of various
organelles. Labeling of cytoskeletal proteins (a-tubulin
and b-tubulin, and slight labeling of actin) suggests that
the cytoplasmic compartment is exposed to the oxidative
stress generated by PDT with Hypocrellin A. This is in
agreement with the partial presence of the photosensitizer
throughout the cytoplasm (Fig. 5A).
In a number of papers [32–36], deleterious effects of PDT
on the microtubules have been documented. Under our
experimental conditions (1 l
M
Hypocrellin A, irradiation at
1JÆcm
)2
), the microtubules were partly depolymerized
immediately after PDT (Fig. 5C). Inactivation of the
microtubules leads to the inability of the photosensitized
cells to form functional mitotic spindles and finally results in
the arrest at the G2/M phase of the cell cycle and subsequent
apoptosis [32]. It has been hypothesized that the micro-
tubules may be damaged within the radius of action of
singlet oxygen in close proximity to the organelles in which
photosensitizers accumulate (lysosomes, mitochondria,

endoplasmic reticulum) [32,34]. Alternatively, an indirect
mechanism has been suggested involving release of calcium
caused by photodynamic insult and subsequent calcium-
induced microtubule depolymerization [36]. In this paper,
we show that PDT with Hypocrellin A results in direct
oxidative modification of tubulin, and we hypothesize that
this modification may be responsible for the PDT-induced
impairment of microtubules. Further studies, including
those in a purified system (reconstituted microtubules), will
be needed to determine the sites of the oxidative modifica-
tions within the tubulin molecule, and to elucidate the role
of these modifications in the functional damage to tubulin.
Interestingly, for the two modes of oxidative stress (PDT
and H
2
O
2
treatment), the relationships between overall
protein oxidation and cell death were dramatically different.
For instance, treatment with 200 l
M
H
2
O
2
resulted in
profound protein oxidation, but caused no cell death. PDT
with 1 l
M
Hypocrellin A and illumination at 1 JÆcm

)2
resulted in comparable protein oxidation (Figs 3 and 4), but
the cells became massively apoptotic. This implies that the
total degree of protein oxidation is not a critical determinant
for the onset of apoptosis. Oxidation of endoplasmic
reticulum proteins, occurring on treatment with H
2
O
2
,
appears not to be critical for cell survival. Rather, oxidation
of particular proteins in particular subcellular sites deter-
mines the onset of apoptosis. Oxidation of other biomol-
ecules, for instance lipid peroxidation, may also trigger cell
death, mostly through rather unspecific mechanisms invol-
ving damage to the cellular membranes. In contrast,
oxidation of particular proteins may activate specific
signaling pathways that regulate cell death or survival
[27,29], which may be important at sublethal doses of PDT.
In conclusion, we have shown for the first time that the
pattern of intracellular protein oxidation depends on the
kind of oxidative stress exerted. The methodology described
here offers the possibility to identify the proteins oxidized
under various forms of oxidative stress, including PDT with
various photosensitizers localized to different cellular com-
partments. It is hoped that this will allow the identification of
photosensitizer-specific protein targets and will help to
further elucidate the mechanisms of PDT-induced cell death.
Acknowledgements
The study was supported by NWO/ZON MW grant No 901-03-097.

We are grateful to E. Romijn and C. Versluis for performing MS
measurements, and to C. L. H. Guikers for assistance with PDT
experiments.
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Ó FEBS 2003 Protein oxidation on photodynamic treatment (Eur. J. Biochem. 270) 4865