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

Photodynamic treatment and H2O2-induced oxidative stress resultin different patterns of cellular protein oxidation Dmitri V.. We found that PDT of rat or human fibroblasts, loaded with th

Photodynamic treatment and H2O2-induced oxidative stress result

in different patterns of cellular protein oxidation

Dmitri V Sakharov1, Anton Bunschoten1, Huib van Weelden2and Karel W A Wirtz1

1

Department of Biochemistry of Lipids, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes,

Utrecht University, the Netherlands;2Department 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 H2O2 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 H2O2 treatment resulted in labeling of endoplasmic reticulum proteins Under conditions in which the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas H2O2treatment 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 H2O2 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,

POBox 80.054, 3508 TB Utrecht, the Netherlands.

Fax: + 31 30 2533151, Tel.: + 31 30 2532852,

E-mail: d.sakharov@chem.uu.nl

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)

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 H2O2[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 H2O2

Experimental procedures

Materials

Hypocrellin A and Hoechst 33342 were purchased from

Molecular Probes (Leiden, the Netherlands) Rose Bengal,

carbonic anhydrase, H2O2, 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

1 JÆcm)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 lM were

added to a well of a plastic culture plate containing 1 mM

tyramine in NaCl/Pi, 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)1carbonic anhydrase and

10 lMTyr-Fluo in NaCl/Piwas irradiated with visible light

in either the presence or absence of a photosensitizer (Rose Bengal or Hypocrellin A, 10 lM) 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% CO2 (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 lM) in NaCl/Pi supplemented with 0.9 mM CaCl2, 0.5 mM MgCl2, and 5 mM glucose (NaCl/Pi+) After removal of NaCl/Pi+ containing acetylTyr-Fluo, fresh NaCl/Pi+ 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 (0.25M sucrose, 1 mM EDTA, and 20 mM Tris/HCl,

pH 7.4) and lysed in buffer containing 20 mM Tris/HCl (pH 7.4), 1 mM 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 H2O2in NaCl/Pi+ 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 (ReadyStripTM),

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

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.PDQUESTsoftware 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 (http://cancer.proteomics.dk),

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 C for

5 min, permeabilized with 0.1% (v/v) Triton X-100 in NaCl/Pifor 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 A solution of 1 m 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)1carbonic 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

1 JÆcm)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.

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 specspec-trum

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 H2O2

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 H2O2treatment, 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 H2O2, 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 staining is shown in (C) and (D) in blue 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.

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 lMHypocrellin 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 lMHypocrellin 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

O n PDT at 1 lMHypocrellin 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 H2O2did 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.

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

H2O2 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 lMHypocrellin A, irradiation at

1 JÆcm)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 H2O2 treatment), the relationships between overall

protein oxidation and cell death were dramatically different

For instance, treatment with 200 lM H2O2 resulted in

profound protein oxidation, but caused no cell death PDT

with 1 lM 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 H2O2, 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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