Introduction

Apoptosis is an active highly conserved process that plays an important role in the development, homeostasis and maintenance of multicellular organisms. Apoptotic mechanisms participate in the removal of toxicologically or genetically damaged cells. Inappropriate apoptosis is associated with many diseases, including cancer.1, 2 Apoptosis is triggered by precise signals that induce crucial biochemical changes in target cells, including activation of caspases, mitochondrial depolarization, nucleosomal DNA fragmentation and alterations in the pattern of protein expression and stability.3, 4

Recent evidence suggests that oxidative stress is a potential common mediator of apoptosis.5 Many of the chemical and physical treatments capable of inducing apoptosis also evoke oxidative stress.6 One of them is photodynamic therapy (PDT), an approved treatment for several types of cancer as well as for age-related macular degeneration.7 It utilizes a bimodal protocol including a combination of photosensitizer and visible light. The interaction of light with photosensitizer molecules in the presence of molecular oxygen catalyses the formation of reactive oxygen species (ROS) within cells to produce the cytotoxic effect.8, 9 Apoptotic and necrotic pathways are both involved in cell death after PDT.10, 11, 12 In cell culture, the oxidative stress induced by PDT triggers a series of events, including activation of phospholipase A2 and C,13 release of nitric oxide,14 activation of stress kinases,12 nuclear factor kB,15 and increased expression of early response genes16 and heat-shock proteins (HSP's).17 It has been reported that the apoptotic response depends on the nature of the photosensitizer, the subcellular localization of the sensitizer, the cell types used and the treatment conditions.12 However, the critical events and specific targets triggering apoptosis after PDT remain to be investigated.

Recent studies support the concept that oxidative damage to proteins during oxidative stress and ageing is selective, and that proteins with specific sensitivity to oxidation can regulate cellular signalling events, including apoptosis.18, 19, 20, 21, 22 Therefore, we hypothesized that oxidative stress associated with PDT induces selective oxidation of specific target proteins, which could play a key role in the induction of apoptosis. The most widely studied oxidative stress-induced modification of proteins is the formation of carbonyl groups in some amino-acid side chains.23 Oxidized proteins can be detected by Western blotting because the protein-bound carbonyl groups react with 2,4-dinitrophenylhydrazine (DNPH) and can be recognized by anti-2,4-dinitrophenol antibodies (anti-DNP).24 To test our hypothesis, we used cytofluorimetric analysis of dying cells, two-dimensional gel (2-D) electrophoresis coupled with immunoblotting proteins and mass spectrometry. The major goal of this study was to identify specific carbonylated proteins and to relate their function to apoptosis.

As photosensitizer we used Purpurin-18 (Pu-18), a chlorophyll derivative and photoactive pigment. We previously reported that Pu-18 in combination with red light induces apoptosis in HL60 cells via caspase-3.10 We were interested in evaluating Pu-18 because its anhydride group provides the possibility of coupling the photosensitizer to cell-targeting agents like antibodies. Our experiments were conducted on HL60 cells that are very sensitive to various stimuli including oxidative stress and PDT; hence, they are a good model to highlight the markers of apoptosis.

First, we used cytofluorimetric analysis of dying cells to monitor the relationships among the time course of ROS generation, mitochondrial transmembrane potential (Δψm), changes in glutathione (GSH) content and the degree of apoptosis. When the cells induced to apoptosis by PDT had been characterized for critical parameters of cellular redox status and the most representative conditions of response time had been established, we proceeded with the separation of proteins in normal and treated cells by high-resolution 2-D electrophoresis, as well as the evaluation of carbonylated proteins by Western blotting with specific antibodies.

Proteome analysis combined with immunoblotting with specific antibodies (functional proteomics) is a powerful tool to study a family of proteins with the same post-translational modification such as carbonylation.

We show here that apoptosis induced by PDT with Pu-18 is associated with ROS generation, changes in Δψm and GSH depletion. For the first time, we demonstrate the specific oxidation of a set of proteins in HL60 dying by PDT-induced apoptosis; these proteins include molecular chaperones, such as glucose-regulated protein-78 (Grp78), Hsp60, heat-shock protein cognate 71 (Hsc71), phosphate disulphide isomerase (PDI) and calreticulin, as well as other proteins such as β-actin, tubulin-α-1-chain and enolase-α. The peculiar function of the most oxidized proteins supports the idea that protein oxidation is a signalling mechanism for apoptosis induced by PDT.

Results

PDT with Pu-18 induces ROS generation, depletion of cellular GSH, changes in Δψm and apoptosis

We reported previously that Pu-18 at nanomolar concentrations in combination with red light (1 J/cm2) induced rapid apoptotic cell death, as documented by subdiploid DNA content and phosphatidylserine (PS) exposure in the human leukaemia cell line (HL60) via caspase-3. The present studies were undertaken to further characterize these initial results.

In the first series of experiments, we evaluated the time course of ROS generation, GSH content and mitochondrial changes; these are critical parameters of the redox status of cells and are important for the investigation of oxidized proteins. We monitored the intracellular ROS formation in HL60 cells after PDT with Pu-18 by measuring the conversion of nonfluorescent 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to fluorescent DCF by flow cytometry. Preliminary experiments showed that it was possible to evaluate the time course of ROS production with adequate precision if the cells were preincubated with DCFH-DA 30 min before irradiation (to evaluate ROS production within 1 h after PDT) or at appropriate time points during the last 30 min before cell harvesting (for ROS production 2–4 h after PDT). Figure 1a shows an intense generation of ROS in HL60 cells incubated for 16 h with Pu-18 and then exposed to red light (1 J/cm2). The ROS generation proceeded in two phases: the first increase was recorded 15 min after PDT, with maximal levels at 1 h and a decrease after 2 h; the second increase occurred at 4 h after treatment.

Figure 1
figure 1

Flow cytometric analysis of the time course of ROS generation, GSH content, Δψm and cell cycle profiles after PDT with Pu-18. Cells were treated with 0.5 μM Pu-18 for 16 h and exposed to a light dose of 1 J/cm2. At the indicated times after irradiation, cells were collected and the different assays were performed as described in ‘Materials and Methods’. The number of cells is indicated on the Y-axis. Filled histograms correspond to control cells while open histograms represent PDT-treated cells. The logarithm of fluorescence intensity in the FL1-H channel (X-axis) is an indicator of ROS production (a), changes in GSH content (b) and changes in Δψm (c), while the fluorescence intensity in the FL2-H channel (X-axis) reflects DNA content. The figure is representative of four different experiments. (*) indicates the cells used for 2-D electrophoresis and immunoblotting

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GSH, an intracellular antioxidant defence, is the principal detoxifying system capable of scavenging ROS and maintaining the redox state of cellular thiols. Hence, ROS generation may lead to the depletion of GSH. Therefore, we used flow cytometry to measure the single-cell o-phtaldialdehyde (OPT) fluorescence in order to examine whether PDT with Pu-18 changes the GSH content. A large percentage of untreated cells had high levels of glutathione, whereas a small percentage had low GSH content; the latter population increased in number after PDT and after 4 h about 90% of all cells showed a decrease in intracellular GSH levels, indicating that PDT induces GSH depletion (Figure 1b).

We then used flow cytometry to evaluate if PDT with Pu-18 induces changes in Δψm, as shown by decreased fluorescence of Rhodamine 123 (Rhod123), a lipophilic cationic probe that becomes localized in the mitochondria of viable cells because of the relatively high electric potential across the mitochondrial inner membrane. Figure 1c illustrates the time course of changes in Δψm after irradiation. Control cells display a baseline level of fluorescence representing normal transmembrane potential. Early on (0–15 min), a subpopulation of cells gained green fluorescence (consistent with a higher Δψm), while at different times after PDT the proportion of cells with reduced Δψm increased in a time-dependent manner and remained high until 4 h after irradiation.

To correlate these biochemical parameters with apoptosis, we also report the degree of apoptosis at the same times, evaluated as the amount of subdiploid DNA (Figure 1d). The increase in the sub-G0/G1 peak at different times after PDT is correlated with the drop in Δψm. Analysis of the percentage of cells in the different cell cycle phases (by MODFIT) revealed no significant change at any time (data not shown). Light alone or Pu-18 in the absence of light did not induce changes in the biochemical parameters.

2-D reference map of HL60 cells

To identify particular changes associated with apoptosis induced by PDT with Pu-18, we separated the proteins in normal and treated cells by high-resolution 2-D electrophoresis. We considered 2 h after PDT an appropriate time to perform proteome analysis, since at this time the production of ROS was declining, most cells (75%) showed a GSH depletion and about 45% of them were apoptotic. The protein pattern of HL60 cells obtained by 2-D PAGE is shown in Figure 2. With 60 μg of total protein lysate, more than 1000 spots were resolved on silver-stained gels in the pI range from 3 to 10 and the molecular mass range from 10 to 250 kDa. We identified several spots, including Grp78, Hsc71, PDI, Hsp60, β-actin, Grp75, PDI-ER60 and triosephosphate isomerase, by matching them with HL60 cell reference maps available through the Swiss 2-D PAGE database (http://www.expasy.org).

Figure 2
figure 2

2-D electrophoretic pattern of HL60 cells, obtained using a non-linear Immobiline pH gradient 3–10 followed by 9–16% SDS-PAGE. Proteins (60 μg) were detected by silver staining. Whole-cell extracts from HL60 cells were prepared as described in ‘Materials and Methods’. Proteins are indicated in Table 1

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MALDI-TOF mass spectrometry confirmed the assignments based on gel matching and allowed the identification, for the first time, of other spots corresponding to seven proteins (Figure 2, Table 1): mitofilin, KH-type splicing regulatory protein, pyruvate kinase, enolase-α, phosphoglycerate kinase, fructose-biphosphate aldolase A, tubulin-α-1-chain. Figure 3 shows a typical peptide mass fingerprint spectrum obtained from the 2-D separated spot corresponding to mitofilin.

Table 1 List of proteins identified by gel matching and mass spectrometry
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Figure 3
figure 3

Peptide mass fingerprinting spectrum of mitofilin (mitochondrial inner membrane protein Q16891) obtained with an Ettan MALDI-ToF Pro as described in ‘Materials and Methods’

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Differential analysis by 2-D gel electrophoresis

Computer analysis performed with Melanie 3 showed that PDT with Pu-18 induced only very limited qualitative and quantitative changes in the protein pattern of HL60 lysates obtained at 2 h after treatment (Figure 4a and b). We repeated the same experiment four times; only the changes observed in all the experiments, and only when the spots changed by almost three times their volume, were considered significant. No changes in protein pattern occurred when the treatment with Pu-18 occurred in the dark (data not shown). At 2 h after irradiation, only 19 of more than 1000 protein spots resolved were found to be modified during PDT-induced apoptosis: the expression of at least two proteins, mitofilin and spot 6, decreased; two unidentified proteins appeared increased (spots 4–5) and four emerged (spots 7–10) during apoptosis (Figure 4; Table 2).

Figure 4
figure 4

Differences in protein patterns from control HL60 cells (a) and Pu-18-treated HL60 cells (b). Proteins not yet identified are indicated by numbers. Whole-cell extracts from HL60 cells obtained 2 h after PDT with Pu-18 were prepared as described in ‘Materials and Methods’. Arrows: protein spots with changes of almost three times the volume in all experiments; Circles: spots emerging in apoptotic cells; Squares: spots disappearing in apoptotic cells; Triangles: spots displaying increasing intensity in apoptotic cells; Convergent Arrows: spots with a pI shift. We repeated the same experiment four times and the figure represents a typical experiment

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Table 2 (a) Protein alterations with respect to control in apoptotic cells 2 h after PDT with Pu-18 and (b) Frequency of modifications among 19 modified spots in apoptotic cells 2 h after PDT with Pu-18
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The most striking feature of the proteome maps of apoptotic cells is the appearance of additional or increased amounts of acidic isoforms of nine proteins: Grp78, Hsc71, PDI, Hsp60, β-actin, tubulin-α-1-chain (the identity of these proteins was confirmed by mass spectrometry) plus other unidentified proteins (indicated by numbers 1–2–3) (Figure 4; Table 2). This suggests that post-translational modifications occurred in the apoptotic cells. We then investigated if the appearance of acidic isoforms also occurred after a classical model of apoptosis-inducing oxidative stress, i.e. treatment with hydrogen peroxide (H2O2) at a final concentration 150 μM for 4 h. The acidic spots did not appear in these conditions (data not shown), suggesting that the particular pattern is associated with PDT. When we used necrosis-inducing doses of PDT, massive cell death occurred after only 1 h and the accompanying cell lysis (documented by loss of proteins) precluded a correct 2-D analysis (data not shown).

PDT with Pu-18 induces carbonylation of specific proteins

To evaluate the oxidized proteins after PDT, we used 2-D electrophoresis combined with immunoblotting with specific antibodies. Oxidized proteins can be detected by Western blotting because the protein-bound carbonyl groups react with DNPH and can be recognized by anti-DNP antibodies. Analysis of the oxidized proteins revealed that the oxidation was not random but specific and occurred systematically 2 h after PDT in four experiments. Typical results are shown in Figure 5a and b. In comparison with control cells, there is an evident increase in the number of carbonylated spots appearing in a relatively small area of the immunostained gel, ranging from Mr 78 KD to 41 kDa and from pH 4.3 to 6.7. A slight degree of carbonylation, which increased after PDT, occurred in control cells only for tubulin-α-1-chain, but this is not surprising since the generation of ROS occurs only in physiological conditions. We detected another seven carbonylated proteins in treated cells: β-actin, Hsc71, Grp78, Hsp60, PDI, calreticulin and enolase-α and the unidentified spot 3. Interestingly, seven of the carbonylated proteins (Grp78, Hsc71, Hsp60, PDI, tubulin-α-1-chain, β-actin and spot 3) also showed the pI shift, while the other two, calreticulin and enolase-α, lacked an isoelectric shift (Table 2).

Figure 5
figure 5

Immunostaining with an anti-DNP antibody of proteins from control HL60 cells (a) and Pu-18-treated HL60 cells (b), separated by 2-D electrophoresis and derivatized by DNPH as described in ‘Materials and Methods’. The immunoblotting for control and treated cells corresponds to the 2-D maps reported in Figure 4. The arrows indicate the carbonylated proteins. We repeated the same experiment four times and the figure represents a typical experiment

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Discussion

In a previous study, we demonstrated that HL60 cells treated with Pu-18 and light exhibited DNA laddering, activation of caspase-3 and an increase in subdiploid DNA and phosphatidylserine externalization, indicating DNA fragmentation and loss of membrane phospholipid asymmetry. The PS exposure and the nuclear features of apoptosis were prevented by the treatment of the cells before illumination with the caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp- fluoromethylketone (z-DEVD-FMK).10

In this study, we have shown that several inter-related events occur in HL60 cells during apoptosis induced by PDT with Pu-18. In the first experiments, the time course (0–4 h) of ROS generation, glutathione content, mitochondrial changes and apoptosis were monitored by flow cytometry. We showed that apoptosis induced by PDT with Pu-18 was preceded by a transient hyperpolarization of Δψm, an increase of ROS generation and GSH depletion. These events were followed by disruption of Δψm and cell death. The time course of ROS generation showed that PDT increased the level of ROS, with two peaks at 1 and 4 h. It has been reported that PDT produces singlet oxygen8, 12 and DCF does not detect O.2, but rather reacts with hydroperoxides. Therefore, the initial increase of ROS generation is likely due to the reactivity of O.2 formed early after PDT and its ability to induce the formation of other ROS species that can be detected by DCF. The late increase of ROS production, detected 4 h after PDT, is likely a downstream event associated with the drop in Δψm.

The early transient increase of Δψm is consistent with recent studies showing an early increase of Δψm followed by a drop in Δψm as a late event in apoptosis.25, 26

ROS generation in the first hour after PDT was correlated with GSH depletion. At later times after PDT, the percentage of cells with glutathione depletion increased dramatically until 4 h when most cells were GSH depleted. Flow cytometric analysis of DNA content showed that apoptotic cell death rapidly increased beginning at 1 h after PDT and was nearly complete after 6 h, as reported previously.10 The drop in Δψm was simultaneous with the increase of the sub-G0/G1 peak and occurred over a similar time course, even though it appeared to be restricted to a smaller number of cells. The time-course studies suggested that the high levels of ROS, associated with reduced GSH content and mitochondrial changes, can perturb the normal redox balance and shift HL60 cells into a state of oxidative stress that induces apoptosis. Our results are consistent with recent reports showing that the changes in cellular redox potential due to enhanced ROS generation and depletion of reduced glutathione are sufficient to induce the opening of mitochondrial megachannels, leading to the disruption of the Δψm and finally the release of cell death-promoting factors, including cytochrome c and apoptosis-inducing factor (AIF).27, 28 Moreover, the loss of Δψm is an apoptotic event reported for several photosensitizers whose principal site of localization is the mitochondrion,9, 29 such as Pu-18.30

To define the role of changes in HL60 redox status after PDT from another point of view, we recorded protein changes and oxidation using the proteomics approach, which is particularly suited to studies of the integrated cellular response to oxidative stress, for example, during PDT. The proteome analysis was performed at 2 h after PDT when the production of ROS was declining, most cells (75%) showed GSH depletion and about 45% of them were apoptotic. Since all cells were dead by apoptosis after 24 h as reported previously,10 it is reasonable to suppose that all cells were committed to apoptosis at 2 h. Of the more than 1000 protein spots separated on the HL60 cell protein maps, only six unidentified spots were enhanced and two were suppressed following treatment with Pu-18. Interestingly mitofilin, a mitochondrion-associated protein, is down-regulated in apoptotic cells. Four spots related to mitofilin appeared to be decreased in HL60 undergoing PDT-induced apoptosis. Gieffers et al.31 demonstrated that mitofilin is a transmembrane protein of the inner mitochondrial membrane, expressed as two isoforms and transported into the mitochondrion in a reaction that depends on the Δψm.32 As PDT with Pu-18 caused the loss of Δψm and the function of this ubiquitous mitochondrial protein is currently unknown, it is tempting to speculate that mitofilin plays a role in apoptosis induced by PDT with Pu-18.

After a detailed look at the maps of apoptotic cells, the most striking aspect was the appearance of acidic isoforms of structural proteins (β-actin, tubulin-α-1-chain), several proteins with chaperone functions (Grp78, Hsp60, Hsc71, PDI) and unidentified proteins (spots 1, 2 and 3), possibly reflecting selective post-translational modifications. It is interesting that a proteome analysis by Gerner et al.33 demonstrated that two protein families, cytoskeletal proteins (like β-actin) and chaperone proteins, were predominantly affected in Fas-induced apoptosis. Moreover, Grebenova et al.34 reported that three ER chaperones involved in calcium homeostasis (calreticulin, PDI and ERp57) were downregulated in HL60 cells treated with ALA-PDT. The increase of acidic isoforms of some proteins seems to be peculiar to PDT. When we used mild doses of H2O2 as a source of ROS, there was no increase of acidic isoforms of specific proteins, while Raggiaschi et al.35 reported an extension towards the acidic side of some proteins in Candida albicans after PDT with cationic tetrasubstituted phthalocyanines. The most widely studied oxidative stress-induced modification of proteins is the formation of carbonyl groups, which can occur by different mechanisms, including direct oxidation of side chains of lysine, arginine, proline and threonine residues. The analysis of stress-induced carbonylated proteins is very interesting because the carbonylation is increased after oxidative stress and is involved in the etiology and progression of many human pathologies, such as cancer, rheumatoid arthritis and pulmonary disease, as well as other physiological conditions such as ageing and inflammation.18, 36, 37 Several studies have also shown increases in protein carbonyls in Alzheimer's38 and Parkinson's diseases.39 Using 2-D electrophoresis combined with immunoblotting with anti-DNP, we demonstrated that only a few proteins were particularly sensitive to oxidative stress. Interestingly, the carbonylated proteins were the same ones that showed the pI shift, plus calreticulin and α-enolase. The presence of carbonylated mitochondrial, cytosolic and endoplasmic reticulum (ER) proteins support the idea of a generalized intracellular state of oxidative stress after PDT, and suggests that only certain proteins are the targets of PDT-generated ROS. It is evident that the degree of carbonylation was not correlated to the amount of proteins: in fact, some proteins that appeared carbonylated were not among the largest spots. The finding that specific proteins become carbonylated clearly demonstrates that protein carbonylation is selective, confirming our hypothesis. The specific oxidation of β-actin and tubulin-α-1-chain in association with oxidative stress has been reported in the brain of Alzheimer patients.38 Oxidation of β-actin during oxidative stress may affect actin filament architecture and lead to disorganization of the cytoskeleton to form apoptotic blebs typical of apoptosis.

The specific oxidation of Hsp's is interesting and very exciting. The HSP's also known as molecular chaperones, help guide protein transport and folding under physiological conditions; they also prevent aggregation and aberrant folding during stress and are involved in the immune response.40 Their expression increases the survival of cells exposed to numerous types of stimuli that lead to apoptosis, including oxidative stress, treatment with apoptosis-inducing agents and PDT.17 In humans, the HSP70 multigene family consists of four members with high homology: Hsc70, Hsp70, Grp78 and Hsp75.41 HSP70 multigene family proteins play a role in protein assembly, but the level of single chaperone proteins in response to stress can vary. Hsp70.1 is highly stress -induced, while Hsc71 is the constitutive isoform, recruited by the cell as a primary defence against unfavourable conditions.41

Hsp60 is a mitochondrial chaperone and its overexpression protects mitochondrial function and prevents apoptosis induced by ischaemia.42 Calreticulin is one of the major calcium-binding proteins in the lumen of the ER.43 Several of these proteins negatively interfere with apoptosis: in particular, Hsp70 protects against apoptosis by inhibiting caspase cascade activation and can inhibit apoptosis by neutralizing and interacting with AIF, a mitochondrial flavoprotein that translocates to the nucleus via the cytosol after the induction of apoptosis.41, 44, 45 The induction of HSP's is a well-known response to PDT, but the cellular heat-shock response after PDT can vary significantly among different photosensitizers, cell types and treatment conditions.16, 17

That oxidation of particular chaperones is an important event during oxidative stress is supported by the most recent literature, showing the specific oxidation of different chaperones depending on the particular stress condition: ER chaperones (PDI, Bip, calreticulin) in aged mouse liver,46 hsc71 in the Alzheimer brain,47 Hsp60 and vimentin in neuronal cells exposed to mild oxidative stress.22 Moreover, molecular chaperones, such as Hsp60 in Saccharomyces cerevisiae and DnaK in Escherichia coli, were previously detected as major targets during oxidative stress, and it was suggested that these chaperones act as shields protecting proteins against oxidative damage.19, 20

Why are chaperones particularly sensitive to oxidative stress, including PDT? Does their particular function make them susceptible to oxidative stress? It is now thought that molecular chaperones closely interact with misfolded or damaged proteins, producing effects that can be likened to ‘protein massage’. An intriguing hypothesis to explain our results is that when the chaperones interact with damaged protein targets to assist their refolding, a protein–protein radical transfer can occur from oxidized proteins to chaperones. This leads to the idea that certain chaperones trying to reduce the toxic effect of oxidative stress were themselves oxidized. As protein oxidation often results in the loss of protein function, the oxidation of Hsp proteins may counteract their protective effect against oxidative stress and could provide a signal capable of triggering the suicide mechanism. Moreover, the chaperones could function as sentinels to recognize the amount of cellular damage and the extent of their oxidation could provide a pivotal decisional checkpoint to determine the cell's fate after oxidative stress. Hence, their carbonylation could result from their particular function. We suggest here that the specific carbonylation of some chaperones and their consequent inactivation could be the mechanism by which PDT kills otherwise resistant tumour cells.

The specific oxidation of β-actin, enolase-α and tubulin-α-1-chain in addition to that of chaperones suggests that the selective damage could be a consequence of the proteins’ structural relationship in addition to their function. Thus, the susceptibility to oxidative stress could depend on the structure of the proteins (the particular sequence motifs, linked sugars or bound metal atoms). Only a few of the carbonylated proteins are glycoproteins and only two (Grp78 and calreticulin) have sites to bind calcium.48 The specific oxidation of enolase-α, the only glycolytic pathway enzyme to be carbonylated, could be related to the structure of this protein. The specific sensitivity of enolase-α has been reported by others in different conditions of oxidative stress.19, 47

A link between these two families of proteins, structural proteins and chaperones, could be their ATPase activity. All members of the Hsp70 chaperone class possess two distinct domains: a highly conserved N-terminal ATPase domain and a more different C-terminal domain. Interestingly, the N-terminal ATP domain of Hsp70 is similar to that of actin in skeletal muscle despite the low sequence similarity and the analogous positions of arginine 177 of actin and lysine 71 of Hsp, suggesting that the two proteins might utilize a similar mechanism for ATP hydrolysis.49 This is supported by the abolition of ATPase activity by ADP -ribosylation of R177 by bacterial toxins; moreover, the lysine (K71) was found to be an essential catalytic residue for nucleotide hydrolysis by the 44-kDa N-terminal ATP domain of Hsp70. These structural relationships suggest that the carbonylation can occur on side chains of these critical residues (arginine 177 of actin and lysine 71 of Hsc71). The subsequent lack of a positive charge could be responsible for the lower pI observed on the gels for β-actin and Hsp71. We can speculate that the oxidative stress induced by PDT could lead to the addition of carbonyl groups in a residue of a particular sequence of target proteins. Further studies are in progress to identify the residues in which carbonylation occurs after PDT.

Although our protein carbonylation analysis was based on a single dose of PDT and a single time after treatment, we believe that the carbonylation of several chaperones is not only a marker of oxidative damage but could be an important event in the signalling pathway of apoptosis by PDT with Pu-18, just like specific protein phosphorylation events reported to occur during apoptosis.

In conclusion, our results indicate that oxidative stress induced by PDT evokes an orchestrated response affecting mitochondria and ER chaperones, and provide additional information on cellular targets in the apoptotic responses to oxidative stress associated with PDT.

Materials and Methods

Cell treatment

HL60 human leukaemia cells, originating from the American Type Culture Collection (Rockville, MD, USA), were kindly provided by Professor Marcella Cintorino (Istituto di Anatomia Patologica, Policlinico Le Scotte, Università degli Studi di Siena, Siena, Italy).

HL60 cells were grown in RPMI medium containing 10% foetal calf serum, 100 U/ml penicillin G and 100 μg/ml streptomycin, at 37°C in a controlled humified incubator in 5% CO2. For the experiments, the cells at concentration of 5 × 105/ml were incubated in the dark with Pu-18 (Porphyrin Product) 0.5 μM for 16 h. After incubation, the cells were collected by centrifugation, resuspended in fresh medium at concentration of 1 × 106/ml, exposed to 1 J/cm2 of red light and maintained in Petri dishes at 37°C until further analysis. Irradiation was carried out at room temperature with a 150 W halogen lamp using a broad spectrum 600–700 nm filter. Pu-18 was dissolved in dimethylsulphoxide (DMSO) as a 20 mM stock solution and stored at −20°C. Working solutions were prepared in culture medium immediately before use. Cells treated with vehicle (DMSO) only and light served as controls. The final DMSO concentration never exceeded 0.1%.

Flow cytometric assays

The formation of intracellular ROS was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, Milan, Italy). By this method, it is possible to measure the amount of H2O2 generated by increased oxidative metabolism. Viable cells can deacetylate DCFH-DA to 2′,7′-dichlorofluorescein; the latter is not fluorescent but can react quantitatively with oxygen species within the cell to produce 2′,7′-dichlorofluorescein (DCF), which is fluorescent and is trapped inside the cell. The cytofluorimetric measurement of the DCF produced can provide an index of intracellular oxidation.25 However, DCF can leak out of cells, making it difficult to detect H2O2 production at later times. Therefore, to monitor the ROS production over time, we incubated the cells with DCFH-DA (10 μM final concentration) for 30 min either before irradiation or during the last 30 min of incubation,50 at appropriate time points after irradiation. Before analysis, 10 μl of a propidium iodide (PI, Sigma-Aldrich, Milan, Italy) stock solution (10 μg/ml final concentration) were added. For measurement of the intensity of DCF fluorescence, the cells were kept in ice until analysis with fluorescence-activated cell sorter (FACS) Calibur flow cytometer (Becton Dickinson) equipped with an excitation laser line at 488 nm and Cell Quest software (Becton Dickinson). The DCF (green fluorescence) was collected in a log scale through a 530±20 band pass filter. Monoparametric histograms of the fluorescence distribution were plotted for the estimation of ROS production.

Δψm was assessed by the uptake of the cationic lipophilic dye Rhod123 and PI using a commercial product (Sigma-Aldrich, Milan, Italy) according to the method described previously.25 Briefly, at different times after PDT, approximately 5 × 105 cells for each sample were washed twice in phosphate-buffered saline (PBS) and the pellet was resuspended in 500 μl of PBS. Rhod123 (4 μl) stock solution were added to the cell suspension (1 μg/ml final concentration) and incubated for 30 min at 37°C in the dark. Then the cells were washed in PBS and resuspended in 200 μl of binding buffer plus 10 μl of the PI stock solution (10 μg/ml final concentration). The cells, kept in ice, were analysed with a FACSCalibur. The Rhod123 (green fluorescence) and the PI (red fluorescence) were both collected in a log scale through a 530±20 and 575±15 nm band pass filter, respectively.

GSH content was evaluated using o-phthaldialdehyde (OPT) (Sigma-Aldrich, Milan, Italy) as described by Treumer and Valet.51 Briefly, the OPT method is based on reaction with both glutathione amino- and sulphhydryl groups, yielding a cyclic highly fluorescent product. The OPT-GSH fluorescent product appears in the green fluorescence channel. For staining, 1 × 106 cells were resuspended in 500 μl HEPES N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulphonic acid]-buffered saline pH 7.4 and OPT was added (final concentration 1 mM of a stock solution 0.1 M in dimethylformamide). After 5 min of incubation at room temperature, the suspension was immediately washed and the cells were analysed with a FACSCalibur. The OPT-GSH (green fluorescence) was collected in a log scale through a 530±20 band pass filter. Monoparametric histograms of the fluorescence distribution were plotted for the estimation of GSH content.

DNA content was analysed using PI as described previously.10 The percentages of cells in the G1, S and G2 phases were determined using the MODFIT program (Verity Software, Topahsm, ME, USA).

At least 20 000 events were collected for each sample using Cell Quest software; debris was excluded from the analysis by an appropriate morphological gate of forward scatter versus side scatter.

2-D electrophoresis

The HL60 cells were dissolved in lysis buffer (8 M urea, 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1propanesulphonate), 40 mM Tris base, 65 mM dithioerythritol (DTE) and trace amounts of bromophenol blue). The protein concentration was determined by Biorad protein assay according to Bradford52 and the samples were diluted in lysis buffer so that 350 μl of sample contained 60 μg of proteins.

2-D gel electrophoresis was performed essentially as described previously.53, 54 The first-dimension separation was carried out on a nonlinear wide-range immobilized pH gradient (pH 3.5-10, 18 cm long IPG strips, Amersham Biosciences). Strips were swollen in 350 μl of lysis buffer containing 60 μg of proteins and 0.8% carrier ampholytes (Resolyte pH 4–8, BDH) and a trace of bromophenol blue. The voltage was increased linearly from 300 to 3500 V during the first 3 h, then stabilized at 5000 V for 22 h (total 110 kVh). After electrophoresis, IPG strips were equilibrated for 12 min in 6 M urea, 30% w/v glycerol, 2% w/v sodium dodecyl sulphate (SDS), 0.05 M Tris-HCl, pH 6.8, 2% w/v DTE, and then for 5 min in the same urea/SDS/Tris buffer solution except that 2% DTE was replaced with 2.5% w/v iodoacetamide. For immunodetection of carbonyl groups, strips were treated after IEF with 10 mM DNPH in 5% trifluoroacetic acid and then neutralized in 0.15 M Tris pH 6.8, 8 M urea, 1% SDS, 20% glycerol, before the steps of equilibration as described.24 The second-dimension run was carried out on 9–16% polyacrylamide linear gradient gels (18 cm × 20 cm × 1.5 mm), at 40 mA/gel constant current and 10°C, until the dye front reached the bottom of the gel. Gels were stained with ammoniacal silver nitrate as described.55, 56

Electrophoretogram images were obtained with a computing densitometer (Molecular Dynamics 300S) and processed with Melanie 3 (GeneBio).

Immunoblotting

Proteins were electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Biosciences) using 25 mM Tris, 192 mM glycine and 20% (v/v) methanol, according to Towbin.57. Protein transfer was carried out using a tank apparatus (trans-blot cell, BioRad) for a total of 2 Å.

Before immunodetection, the membranes were stained in 0.2% w/v Ponceau S in 3% w/v trichloroacetic acid for 3 min and the spot position was marked to facilitate computer-assisted matching on the silver-stained gel.58 Immunodetection was performed as described previously58 using anti-DNP IgG (Sigma, dilution 1 : 10 000). The immunoreactive spots were detected using rabbit anti-mouse IgGs conjugated with peroxidase and a chemiluminescence detection system (Amersham Biosciences).

Protein identification of 2-D separated spots by mass spectrometry

Protein identification was carried out by MALDI-TOF and ESI-Ion Trap mass spectrometry. After visualization with Coomassie blue staining, electrophoretic spots were excised and protein digestion was carried out as described.59, 60 MS spectra were acquired using an Ettan MALDI-TOF mass spectrometer (Amersham Biosciences, Uppsala, Sweden) and peptide sequencing was carried out using an LCQ DECA ion trap mass spectrometer (Finnigan, San Jose, CA, USA). Mass fingerprinting and MS/MS database searching were carried out using ProFound (www.proteometrics.com) and Mascot (Matrix Science Ltd., London, UK, http://www.matrixscience.com) on-line-available software and TurboSEQUEST software (Finnigan, San Jose, CA, USA).

Database searching criteria were limited by the following parameters: less than 150 ppm Δmass between experimental and theoretical peptide masses, more than 20% of protein sequence coverage required and Z-score higher than 1.66.