Photodynamic Therapy Causes Cross-Linking of Signal Transducer

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[CANCER RESEARCH 64, 6579–6587, September 15, 2004] Photodynamic Therapy Causes Cross-linking of Signal Transducer and Activator of Transcription Proteins and Attenuation of Interleukin-6 Cytokine Responsiveness in Epithelial Cells

Weiguo Liu,1,2 Allan R. Oseroff,2 and Heinz Baumann1 Departments of 1Molecular and Cellular Biology and 2Dermatology, Roswell Park Cancer Institute, Buffalo, New York

ABSTRACT dependent loss of immunodetectable epidermal growth factor (EGF) receptor (EGFR) appeared as one of the prominent effects of PDT in Photodynamic therapy (PDT) is a local treatment of cancers. The various epithelial cell types (6, 14). To gain a more comprehensive principle of PDT is the production of reactive oxygen species, in particular picture of the processes initiated by PDT and to identify potential singlet oxygen, by light activation of a photosensitizer introduced into the target cells. The direct photochemical and subsequent redox reactions can quantitative markers for immediate PDT damage, we determined in lead to cell death. This study sought to identify effects occurring during cells in culture and growing as tumors in vivo the effects of PDT on PDT and some of their consequences in surviving cells. Using epithelial profiles of protein modification and signaling functions. cells in tissue culture and in tumors, several distinct PDT-mediated reac- The study indicates several novel responses that define PDT action: tions were found, including global dephosphorylation of proteins, induced (1) increased dephosphorylation of tyrosine-phosphorylated proteins, phosphorylation of a 71-kDa protein, initiation of cellular stress responses, (2) stimulation of a PDT- sensitive protein tyrosine kinase activity that structural modification and loss of epidermal growth factor receptor, and is detectable after inhibition of tyrosine phosphatases, and (3) a highly cross-linking of proteins. Specific covalent cross-linking of nonactivated specific cross-linking of nonactivated signal transducer and activator signal transducer and activator of transcription (STAT)-3, and to a lesser of transcription (STAT)-3 and, to a lesser extent, STAT1 and STAT4. extent of STAT1 and STAT4, correlated with PDT dose. Cross-linked The modification of STAT proteins, together with the attenuated STAT3 was primarily localized to the cytoplasm and failed to bind to DNA. The combination of STAT cross-linking and inactivation of receptor signaling by the cytokine receptors, correlates with reduced cellular functions rendered PDT-treated cells refractory for at least 24 hours to responsiveness to interleukin (IL)-6 cytokines. interleukin-6 and oncostatin M, cytokines known to be elevated at site of tissue damage and inflammation. It is suggested that the loss of respon- MATERIALS AND METHODS siveness to these inflammatory cytokines in the PDT-treated field assists tumor cells in evading the growth-suppressive activity of these mediators Cells. The following cells lines were grown in Dulbecco’s modified Ea- expected to be present at tissue sites after PDT. gle’s medium containing 10% fetal calf serum and antibiotics: FaDu (human hypopharyngeal carcinoma), A549 (human lung carcinoma), H-35 (rat hepa- toma), Colo26 (mouse colon carcinoma), and COS-1 (green monkey kidney INTRODUCTION epithelial). Human prostate carcinoma PC3 cells were maintained in RPMI 1640 containing 10% fetal calf serum. Human pulmonary macrophages were Photodynamic therapy (PDT) is designed to eliminate abnormal isolated from residual lung tissue obtained from Roswell Park Cancer Institute tissue lesions, including tumors (1). Although it is desired to kill target Tissue Procurement Service under the approved Institutional Review Board cells either directly by the photochemical reactions or indirectly by protocol CIC 00-17. Macrophages were mechanically extracted from minced PDT-induced secondary responses, invariably, a fraction of the ma- lung tissue and purified by Histopaque gradient centrifugation (Sigma Chem- lignant and normal cells within or adjacent to the treatment field ical Co., St. Louis, MO). The cells (1 ϫ 106 per mL) were resuspended in escape the lethal PDT effects. In the case of cancer treatment, the RPMI 1640 containing 10% fetal calf serum and 1 ␮g/mL lipopolysaccharides. surviving tumor cell population contributes to the recurrence of the Conditioned medium was collected after 12 hours of incubation. cancer. A survival process demands that the cells exposed to nonlethal PDT Treatment. Cells at ϳ90% confluence were incubated for 4 hours at PDT are able to repair cellular damages and eventually to resume 37°C with growth medium containing 0 to 1 mmol/L aminolevulinic acid (16) ␮ proliferation. or0to1.2 mol/L 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH; ref. 17), or for 15 minutes with PBS containing 0 to 1.5 ␮mol/L Molecular studies of PDT damage have focused largely on the 2-carboxy-3,4,5,6-tetrachlorophenyl)-2,4,5,7-tetraiodo-3,6-dihydroxyxanthylium- mechanisms that explain necrosis and apoptosis of treated cells (2–5). dipotassium salt [Rose Bengal (RB); Sigma Chemical Co.; ref. 18]. Where The relative levels of PDT effects may determine the difference necessary, advance treatment for 2 hours with 1 mmol/L sodium vanadate or between lethal and nonlethal outcome, however, the biochemical for 15 minutes with 100 ng/mL oncostatin M (Amgen Inc., Seattle, WA) or nature of these effects is not well understood. We and others have EGF (Invitrogen Corp., Carlsbad, CA) was initiated by adding these reagents identified PDT-dependent modifications of cellular proteins and have to the medium containing the photosensitizer. Medium was replaced with evaluated the consequence of these modifications on the functions of fresh, photosensitizer-free medium containing 10% fetal calf serum and the these proteins. The range of modifications includes oxidation, adduct cytokines or EGF necessary to maintain signaling during the light-induced formation, protein cross-linking, and degradation (6–11). Particularly photoreactions. Light treatments were carried at room temperature by applying noted was the loss of enzymatic activities due to redox reactions such the following conditions: aminolevulinic acid–PDT used a halogen light at 400 to 700 nm and was delivered at a fluence rate of 18 mW/cm2 to a fluence as found for phosphatases (12) and kinases (6, 13–15). The PDT- ranging from 1 to 10 J/cm2; RB-PDT used the same light delivered to a fluence ranging from 0.5 to 1.5 J/cm2; and HPPH-PDT used a red-filtered arc light at Received 5/5/04; revised 6/20/04; accepted 7/16/04. 590 to 700 nm with a fluence rate of 14 mW/cm2 and fluence ranging from 0.5 Grant support: NCI grants CA85580 (H. Baumann) and CA55791 (A. Oseroff) and to 8 J/cm2. All experimental series included controls consisting of cultures that Roswell Park Cancer Support grant CA16056. The costs of publication of this article were defrayed in part by the payment of page were incubated with photosensitizer but not exposed to light. Immediately after charges. This article must therefore be hereby marked advertisement in accordance with PDT or after various length of time in growth medium and treatments with 18 U.S.C. Section 1734 solely to indicate this fact. cytokines or EGF, the cells were washed with PBS and processed one of two Requests for reprints: Heinz Baumann, Department of Molecular and Cellular ways. The cells were lysed in the culture dish with radioimmunoprecipitation Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Fax: 716-845-5908; E-mail: [email protected]. assay buffer for analysis of protein expression by immunoblotting, or the cells ©2004 American Association for Cancer Research. were extracted with high-salt buffer [0.4 mol/L NaCl, 20 mmol/L 4-(2- 6579

hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.6), 20 mmol/L NaF, 1 Electrophoretic Mobility Shift Assay. To determine DNA-binding activ-

mmol/L Na4P2O7, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothre- ity of STAT1 and STAT3, aliquots of whole-cell or nuclear extracts containing itol, 20% glycerol, 1 mmol/L sodium orthovanadate, 1 ␮g/mL leupeptin and 5to20␮g of proteins were reacted with a [32P]-labeled double-stranded aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride) for measuring DNA oligonucleotide representing the m67 sis-inducible element and analyzed for binding activity of STATs by electrophoretic shift assay. Most PDT experi- protein interaction by electrophoresis as described previously (19, 20). Incu- ments also included cultures that were subjected to the treatment and were used bation of extracts with anti-STAT3 (C20; Santa Cruz Biotechnology) before 24 hours later to determine the effect of PDT on cell survival. Adherent cells electrophoresis identified the presence of STAT3 in the sis-inducible element were washed free of material derived from dead cells with PBS and then were complexes by supershifting. released by trypsinization. Viable cells were counted based on trypan blue dye Immunoelectrophoretic Analysis. H-35 cell monolayers were treated for exclusion in a hematocytometer. 24 hours with 100 ng/mL human recombinant IL-6 (Genetics Institute, Cam- Cell Fractionation. After washing with PBS, the cells were allowed to bridge, MA) or one-tenth diluted conditioned medium of lipopolysaccharide- swell in 4 volumes of hypotonic buffer [20 mmol/L Tris (pH 7.4), 10 mmol/L activated human pulmonary macrophages in Dulbecco’s modified Eagle’s ␮ NaCl, and 2 mmol/L MgCl2] on ice and then were broken in a tight-fitted medium containing 0.5% fetal calf serum and 1 mol/L dexamethasone. The ␣ Dounce homogenizer. The homogenate was centrifuged for 5 minutes at amounts of thiostatin and 1-acid glycoprotein secreted into the culture me- 10,000 ϫ g to remove nuclei and large cell fragments. The supernatant (termed dium were determined by immunoelectrophoresis (21) and expressed in mil- “cytoplasmic fraction”) was centrifuged for 60 minutes at 100,000 ϫ g, and the ligrams per milliliter using purified plasma proteins as reference. resulting supernatant (termed “cytosol fraction”) was used for protein analysis. Transfection. Using FuGene 6 (Roche Diagnostics Corp., Indianapolis, To recover nuclei free of cytoplasmic contamination, cells were treated with IN), COS-1 cells were transfected with the pSPORT1 expression vectors for hypotonic buffer as described above and then homogenized in the presence of wild-type rat STAT1, STAT3, STAT5A, STAT5B, mouse STAT4 and 0.5% NP40. The nuclei were collected by centrifugation for 5 minutes at STAT6, COOH-terminally truncated rat STAT3⌬55C, (20), or rat STAT3 with

1,000 ϫ g. The nuclear pellet was resuspended in 1 mL of sucrose buffer [10 six tandem copies of the Myc epitope tag added to the NH2 terminus (provided mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 25 mmol/L KCl, by Dr. Tomek Kordula). After a 36-hour recovery period, the cells were

5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L spermidine, 1 mmol/L phen- extracted by three cycles of freeze-thaw in 3 volumes of PBS containing a ylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 10% glycerol, and 2 mol/L mixture of inhibitors for proteases and phosphatases. Cell debris was removed sucrose], loaded onto a cushion of 4 mL of sucrose buffer, and centrifuged for by centrifugation at 15,000 ϫ g for 20 minutes. The cytosolic fraction was 60 minutes at 100,000 ϫ g. The nuclei were solubilized by boiling in SDS obtained by subsequent centrifugation at 100,000 ϫ g for 1 hour. buffer. Tumor. FaDu cells were released by trypsin and washed with PBS, and 1 ϫ 106 were resuspended in 50 ␮L of serum-free medium and injected RESULTS subcutaneously on the dorsal sides of both hind flanks of scid/scid BALB/c mice. Similarly, Colo26 cells (1 ϫ 106) were injected into normal BALB/c PDT Causes Several Immediate Biochemical Reactions. Be- mice. After the tumors had reached a size of ϳ6 mm in diameter, the animals cause biological consequences of PDT often are only evident hours received a tail vein injection of 0.4 ␮mol/kg HPPH. After 24 hours, one tumor after treatment (22), we asked what cell reactions describe the initial on each animal was treated with 665 nm of light using an argon-dye laser PDT effects in epithelial cells. To broaden the assessment, we also system (Spectra Physics, Mountain View, CA; fluence rate, 28 mW/cm2; determined the effects of PDT on signaling by the receptors for fluence, 48 J/cm2). The contralateral tumor was kept protected from light oncostatin M and EGF, two factors that prominently act on normal exposure and served as control. After PDT, the treated and untreated tumors and on many transformed epithelial cells. We used the human hypo- were excised, rinsed with PBS, and immediately homogenized in radioimmu- pharyngeal cell line FaDu as an experimental system because this line noprecipitation assay buffer. expresses abundant amounts of receptors for oncostatin M, IL-6, Gel Electrophoresis. Depending on the molecular size of the antigens, leukemia inhibitory factor, and EGF and responds to these factors aliquots of extracts (5–30 ␮g of protein) were separated on 6 to 12% SDS- comparably with normal epithelial cells (6). We examined two sec- polyacrylamide gels. Proteins were transferred to protean membranes (Schleicher & Schuell, Riviera Beach, FL). Wherever possible, replicates of ond-generation photosensitizers that currently are in clinical trials: the the samples were electrophoresed for identification of multiple antigens. This prodrug aminolevulinic acid that is converted into the endogenous approach circumvented the problem of incomplete removal of antibodies photosensitizer protoporphyrin IX (16), and the exogenous photosen- during sequential immunoblotting of the same membrane (6). The membranes sitizer HPPH (17). Both agents are believed to target primarily mito- were reacted with antibodies to EGFR; leukemia inhibitory factor receptor-␣; chondria. To evaluate the relationship of subcellular distribution of the gp130; STAT1; STAT3 (C20 and H-190); STAT4; STAT5; STAT6; extracel- photochemical reaction and pattern of outcome, we also treated cells lular signal-regulated kinase (ERK)-1/-2; caveolin-1; heat shock protein with RB, a more hydrophilic cationic photosensitizer that shows a (HSP)-90 and HSP70 (Santa Cruz Biotechnology, Santa Cruz, CA); poly- broad cytoplasmic and membrane distribution (18). (ADP-ribose) polymerase (Cell Signaling Technology, Beverly, MA); phos- The PDT effects were assessed by determining the level of phosphotyrosine-STAT1, phosphotyrosine-STAT3, or P-ERK-1/-2 (New England phorylation of total cell proteins, expression of receptors, and modi- Biolabs, Inc. Beverly, MA); glucose-regulated protein-58 (provided by Dr. Naveen Banjia); oncostatin M receptor ␤ (Amgen, Inc.); phosphotyrosine fication of downstream signaling proteins. Key PDT responses are (Upstate Biotechnology, Lake Placid, NY); human fibrinogen (Dako, Carpin- summarized by the representative experiment shown in Fig. 1. Data ␣ from more than 30 independent series of PDT treatments of FaDu teria, CA); rat thiostatin; and 1-acid glycoprotein (Department of Laboratory Animal Resources at Roswell Park Cancer Institute). Reaction with secondary cells support the stated findings. The results, in part, confirmed antibodies (ICN Biomedicals, Inc., Aurora, OH) occurred in PBS containing reactions reported previously (6, 14). Using an aminolevulinic acid– 0.1% Tween 20, 5% milk, or 3% albumin. Immune complexes were visualized PDT dose that led to ϳ60% killing of the cells during a 24-hour by enhanced chemiluminescence (ECL) reaction (Amersham Biosciences, post-treatment period, five distinct immediate PDT effects were con- Piscataway, NJ). To quantify the immunodetectable signals, 2-fold serially sistently observed. (1) EGFR changed to a slower electrophoretic diluted extracts were subjected to Western blotting. The ECL images were mobility. (2) The level of EGFR protein was reduced. (3) Tyrosine recorded on X-ray films by various length of exposure to ensure recovery of phosphorylation of many cellular proteins was decreased. Because the signals that lay in the linear range of detection by digital scanning. The presentation of data in the figures used longer exposed images. The net pixel basal level of protein tyrosine phosphorylation in FaDu cells was low, values of each band were determined by integration using the ImageQuant a longer ECL exposure of the immunoblot was necessary to reveal this program (Amersham Biosciences). The linear range of the signals within the PDT effect (Fig. 1, left panel at the top). The loss of phosphorylation dilution series was used to calculate the relative amounts of antigens among also was associated with a lower action of EGFR and oncostatin M samples. Statistically significant differences were evaluated by Student’s t test. receptor, as was evident by the reduced phosphorylation of EGFR, 6580

weak and often close to nondetectable immunoblot signal for p38. Thus, the analysis of stress mitogen-activated protein kinases was restricted to JNK. Under condition of vanadate- inhibited phosphatase activities, a PDT-dependent tyrosine phosphorylation of a 71-kDa protein was detected. This protein, the identity of which remains to be established, also was a target for phosphorylation by EGFR signaling. The vanadate treatment also helped to maintain the receptor-mediated phosphorylation of many, but not all, signaling proteins during PDT. (5) Specific cross-linked products were evident in addition to a low level “nonspecific” cross-linking of proteins that appeared as smears of immunodetectable proteins at the higher molecular size region. Of note was the shift of the nonphosphorylated, but not the oncostatin M–activated (phosphorylated), STAT3 to discrete slower mobility forms (labeled as I, II, and III). Due to the strong immunoblot signal for STAT3, the proportional reduction of non-cross–linked, monomeric STAT3 proteins was only evident on short ECL exposures of immunoblots (Fig. 1, panel at the left of the third immunoblot). A cross-linking of the EGFR dimer was also detectable in EGF-treated and PDT-exposed cells. Because of the critical role of STAT3 in mediating the signaling of cytokines and growth factors, the specificity of STAT3 cross-linking prompted an additional characterization of its process and its consequence for cellular receptor functions in surviving cells that rely on STAT3. Cross-linking of STAT Proteins Is a Function of the PDT Photoreaction. Cross-linking of STAT3 was proportional to the photosensitizer dose (Fig. 2A) and fluence (Fig. 2B, two left panels) for aminolevulinic acid–PDT and HPPH-PDT. Both PDT regimens generated qualitatively similar conversion into forms migrating with apparent mass of 178 kDa (form I), 190 kDa (form II), and 200 kDa (form III). Maximal conversion of monomeric to cross-linked STAT3 was with aminolevulinic acid–PDT ϳ15%. HPPH-PDT resulted in a consistently higher conversion of ϳ30%. Although aminolevulinic acid–PDT and HPPH-PDT were targeted to mitochondria, the reactive intermediates seemed to reach the cytoplasmic sites where STAT3 resides. By using RB, a cross-linking was obtained that on average was 1.5-fold higher than with HPPH-PDT (Fig. 2B, right panel). PDT reactions, which produced maximal STAT3 cross-linking, led to killing of FaDu cells ranging from 80 to 90% (aminolevulinic acid–PDT) to ϳ95% (HPPH) and 100% (RB) when checked for viable cells 24 hours after PDT (see also ref. 6). Interestingly, although somewhat variable from experiment to experiment and among the photosensitizers used, half-maximal cross-linking of STAT3 by the PDT reaction correlated with killing 40 to 90% of the cultures. Thus, the relative Fig. 1. PDT mediates several types of immediate cell reactions. FaDu cells were treated level of STAT3 cross-linking appeared to be an indicator of the with 0.8 mmol/L aminolevulinic acid for 4 hours. Where indicated at the top, pretreat- ments with 1 mmol/L sodium vanadate (2 hours) and EGF or oncostatin M (OSM;15 severity of the photoreaction. Moreover, PDT conditions could be minutes) were carried out. Cells were subjected to light treatment (10 J/cm2) and lysed chosen that yielded a sufficient number of surviving cells that per- immediately after PDT. A duplicate set of control cells, with and without PDT treatment, mitted determination of the relationship of STAT cross-linking and was maintained for an additional 24 hours in culture to determine the percentage of viable cells (40% in this example). Equal amounts of cell lysates were analyzed by immuno- STAT function. blotting for the antigens indicated at the left. The ECL exposure of the film was limited The analysis of different STAT proteins in FaDu and other epithe- by the patterns with highest signal. Longer exposures permitted identification of changes in samples with low immune signals. PDT-mediated changes in basal level tyrosine- lial cell types, including A549 lung epithelial cells that have a par- phosphorylated proteins are demonstrated by the 45-minute ECL exposure of the control ticularly high level expression of STAT1 and STAT3, indicated that samples at the left of the top panel (WB: PY). Conversely, the reduction in immunode- PDT-mediated STAT cross-linking was not restricted to FaDu cells. tectable monomeric STAT3 due to cross-linking is shown by a 1-second ECL exposure of the control samples at the left of the third panel (WB: STAT3). Position of the nonmodified Most effectively cross-linked was STAT3, less was STAT1, and not antigens, the cross-linked complexes, and the molecular size markers are indicated at the appreciably was STAT5 (Fig. 2C, left panel). When cells were pre- right. The open arrow at the top panel points to the position of the 71-kDa tyrosine- treated with oncostatin M to increase tyrosine phosphorylation of phosphorylated protein detected in PDT-treated cells, the phosphatase activities of which were inhibited by vanadate. Circles in the phosphotyrosine-STAT3 (PY-STAT3) patterns STAT3 and STAT1, only a minimal amount of phosphorylated indicate the position of cross-linked STAT3 complexes containing phosphorylated STAT3 and none of phosphorylated STAT1 were detectably associ- STAT3. ated with cross-linked STAT proteins (Fig. 1; data not shown). PDT treatment of the human prostate cell line PC3, which lacks STAT3, STAT3, and ERK. (4) The level of phosphorylated (activated) c-jun- did not result in the appearance of protein forms that reacted with

NH2-terminal kinase (JNK) was increased. Activation of the p38 anti-STAT3 (data not presented). This finding ruled out the possibility stress mitogen-activated protein kinase was also observed in FaDu that an epitope was generated by the PDT reaction that cross-reacted cells (see ref. 6), however, the available antibodies yielded only a with anti-STAT3 but was unrelated to STAT3. 6581

Fig. 2. Cross-linking of STAT proteins is PDT dose dependent. A. Duplicate cultures of FaDu cells were treated with increasing concentrations of aminolevulinic acid (ALA)or HPPH and then exposed to light (10 J/cm2 for aminolevulinic acid–PDT and 3 J/cm2 for HPPH-PDT). B. FaDu cells were treated with 0.8 mmol/L aminolevulinic acid, 0.8 ␮mol/L HPPH, or the indicated concentrations of RB. The cells were exposed to the light to reach the indicated fluence. Cells in A and B were extracted immediately after light treatment. Equal aliquots of cell extracts were separated on 6% PAGE and immunoblotted for STAT3. C. A549 cells were treated with HPPH-PDT (0.8 ␮mol/L; 3 J/cm2) or RB-PDT (at indicated concentration; 0.5 J/cm2). Extracts prepared immediately after PDT were immunoblotted for the STAT isoforms indicated at the top. A549 cells treated with RB-PDT were also homogenized, and cytosolic (C) and nuclear fractions (N) were prepared. From each fraction, aliquots containing 20 ␮g of protein were immunoblotted. D. Duplicate aliquots of high-salt extracts from control A549 cells or A549 cells treated with RB-PDT (9 ␮mol/L; 0.5 J/cm2; PDT), each containing 20 ␮g, or from cells treated for 15 minutes with oncostatin M (OSM), containing 5 ␮g of protein, were reacted with [32P]-labeled sis-inducible element. One set of the reactions also received 1 ␮g of anti- STAT3. The STAT-sis-inducible element (STAT3-SIE) complexes were subjected to electrophoretic mobility shift assay. The lanes representing the oncostatin M samples were exposed to the X-ray film for 6 hours, and the lanes representing the control and PDT samples were exposed for 24 hours. The picture represents a composite of these exposures. The position of the sis-inducible element complexes containing the indicated STAT1 and/or STAT3 dimers is marked on the right.

Cross-linked STAT Complexes Are Primarily Cytoplasmic. In concluded that PDT- dependent cross-linking inactivated STAT3 and FaDu cells treated with aminolevulinic acid–PDT, HPPH-PDT, or thus removed it from the pool of signal-transducing proteins. RB-PDT, only trace amounts of cross-linked STAT3 and none for Cross-linked STAT Proteins Are Stable. Time-course analysis STAT1 were detected in the nuclear fraction. In cell lines with high of the post-PDT reactions indicated that protein cross-linking by levels of STAT proteins, such as A549, the PDT reaction caused a either aminolevulinic acid–PDT or HPPH-PDT was limited to the conversion of up to 70% of STAT3 epitopes appearing in cross-linked light treatment period (Fig. 3A), suggesting that it was solely a complexes (Fig. 2C, center panel). In RB-PDT–treated A549 cells, function of the light-dependent photoreaction. In contrast, other PDT- cross-linked STAT3 was also more abundant in the nuclear fraction initiated reactions continued to increase during post-PDT period, such (Fig. 2C, right panel). Of note is that the amount of cross-linked as activation (phosphorylation) of JNK, dephosphorylation of ERK, STAT3 relative to monomeric STAT3 in nuclei was 2- to 5-fold lower loss of EGFR, and appearance of markers for apoptosis, such as the than in the cytoplasmic fraction from the same cells. The cross-linked delayed degradation of phosphorylated JNK and poly(ADP-ribose) nuclear STAT3 failed to react with antibodies against tyrosine phos- polymerase. Cross-linked STAT3 protein forms were detectable in the phorylated STAT3. These results suggest that cross-linking of STAT3 surviving cell population for several days, indicating an apparent is mainly a cytoplasmic event with the cross-linked STAT proteins half-life of 18 hours (Fig. 3B). remaining largely localized to the cytoplasm and/or that reactive Nature of the Cross-linked STAT Proteins. PDT-dependent oxygen species, which are responsible for cross-linking, did not cross-linking of STAT3 was primarily through formation of covalent efficiently reach the nuclear compartment (23). linkages that were noncleavable by reducing agents (Fig. 4A). In three The relatively high level of cross-linked STAT3 complexes as separate experiments, we determined that consistently less than 25% found in RB-PDT–treated A549 cells allowed us also to determine of the cross-linked STAT3 contained within forms I, II, and III was whether cross-linked STAT3 gained DNA-binding activity, such as to susceptible to reduction by 2-mercaptoethanol. This was in contrast to the high-affinity substrate sis-inducible element. Such a DNA-binding PDT-cross–linked EGFR dimers, of which ϳ50% were cleaved by activity was found with STAT3 dimers generated by oncostatin M reduction (Fig. 4B). treatment through phosphorylation or with nonphosphorylated dimers The molecular size of 177 kDa for the major cross-linked form I is formed by the A661C and N663C mutations in STAT3-C (24). compatible with a homodimer of STAT3. It has been shown previ- However, electrophoretic mobility shift assay analysis with extracts ously that noncovalently linked homodimers of nonactivated STAT1 from RB-PDT–treated A549 cells indicated no PDT-induced sis- and STAT3 are present in cytoplasmic cell extracts (25–27). Thus, we inducible element-binding by STAT3 (Fig. 2D). In fact, PDT lowered hypothesized that the same preformed dimers of STAT3 also exist in the basal activity of STAT3 binding probably in part by PDT-depen- intact cells and that PDT covalently cross-linked these to yield form dent dephosphorylation of STAT3 (Fig. 1). From these findings, we I. Forms II and III may include additional accessory proteins. Prior 6582

Fig. 3. PDT induces cellular stress reactions. A. FaDu cultures were incubated with 0.4 mmol/L aminolevulinic acid or 0.6 ␮mol/L HPPH and then exposed to light to the indicated fluence. Note, the light treatment period was 26 minutes for aminolevulinic acid–PDT and 3 minutes for HPPH-PDT. This time difference is reflected in the time course of the cellular stress response in the two series. Both PDT treatments yielded 40% of viable cells at the 24-hour time point. Cells from the entire cultures (adherent and floating) were collected at the times in hours indicated at the bottom and extracted. Replicate aliquots of the extracts containing equal amounts of protein were analyzed by immunoblotting for the indicated proteins. A long exposure (2 hours) of the chemiluminescence image of phsophorylated ERK was chosen to demonstrate basal level phosphorylation and PDT-induced changes. B. In three separate experimental series, FaDu cells were treated with aminolevulinic acid–PDT as indicated in A. During a 96-hour post- treatment period, the adherent (viable) cells were collected and extracted. The levels of immunodetectable cross-linked STAT3 complexes I, II, and III in equal number of viable cells were determined and expressed relative to the value for complex I in the extract from the 0-hour post-PDT culture of each series (defined as 100%). The numer- ical values for each time point from the three experimental series are indicated. OSMR␤, oncostatin M receptor ␤. n.s., non-specific.

Fig. 4. Nature of PDT cross-linked proteins. A and B. Duplicate cultures of FaDu cells were incubated with 0.8 ␮mol/L HPPH. Fifteen minutes before light treatment, the set of cultures in B was treated with EGF. The cells were exposed for 3 minutes to light yielding a fluence of 3 J/cm2. Cells were immediately extracted. Aliquots were subjected to SDS- PAGE under nonreducing [Ϫ␤-mercaptoethanol (Ϫ␤MeOH)] and reducing [ϩ␤-mercaptoethanol (ϩ␤MeOH)] condition and immunoblotted for STAT3 (A) and EGFR (B). C. COS-1 cells were transfected with the expression vectors for Myc-STAT3, STAT3D55C (STAT3), or the combination of the two as indicated at the bottom. The cells were subjected to HPPH-PDT (0.8 ␮mol/L; 3 J/cm2) and immediately extracted. Equal amounts of extracts were immunoblotted for STAT3 and Myc epitopes. Open circles to the right of the PDT lanes indicate the position of heteromers formed by the STAT3 isoforms. D. Wild-type STAT3 was expressed in COS-1 cells. The cells were extracted in isotonic buffer, and the cytosolic fraction was prepared. Five-␮L aliquots (containing 5 ␮g of protein) were placed on a parafilm sheet on an ice-cooled support. To two samples, 40 mmol/L N-acetyl-cysteine (NAC) was added. Then, each sample received 0.75 ␮L of 6.73 ␮mol/L HPPH and was immediately illuminated to a fluence of 3 J/cm2. Aliquots of 0.25 ␮L of the samples were immunoblotted for STAT3.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. PDT-MEDIATED MODIFICATION OF STATS analysis of STAT3 contained in subcellular fractions had suggested a coassociation of STAT3 with caveolin-1 and stress proteins (28). Therefore, by coimmunoprecipitation and immunoblotting, we assessed in PDT-treated FaDu cells whether HSP90, HSP70, glucose- regulated protein-58, or caveolin-1 was integrated into the cross- linked STAT complexes. Data not shown indicated that none of the accessory proteins could be coimmunoprecipitated with any of the PDT-cross–linked STAT3 forms. Due to technical limitations, we were unable to recover by immuno- precipitation and SDS gelelectrophoresis sufficient quantity of the cross-linked STAT3 forms to identify by mass spectrometry proteins contained in these complexes. Therefore we applied an alternative approach to test whether the cross-linked STAT3 form I could represent STAT3 homodimers. This approach used recombinant STAT proteins that were specifically tagged. To obtain recombinant proteins, we used COS-1 cells that yielded high expression of transfected genes under the control of the SV40 promoter/enhancer. We overex- pressed in COS-1 cells two electrophorectically distinct forms of STAT3, Myc-STAT3 and the truncated STAT3⌬55C (labeled as STAT3␤ in Fig. 4C), alone or in combination. These cells were treated with HPPH-PDT. Each STAT3 form produced a cross-linked component that migrated with a size of a dimer. Cells coexpressing both Myc-STAT3 and STAT3⌬55C also indicated two similarly sized bands with intermediate mobility consistent with that predicted for a cross-linked heterodimers of the two STAT3 species. The biochemical nature for the two size forms is, however, still unknown. Based on the fact that STAT3 dimers are stable in cell extract (27), we reasoned that PDT in cell-free solution should also cross-link STAT3. The cytosolic fraction of COS-1 cells overexpressing wild- type STAT3 was prepared and subjected to HPPH-PDT. The same cross-linked STAT3 forms I to III were generated as found in PDT- treated cells (Fig. 4D). The involvement of an oxidative process was demonstrated by the loss of cross-linking in the presence of the reactive oxygen species scavenger N-acetyl-cysteine (Fig. 4D). In experiments similar to those shown in Fig. 4C and D,we extended the analysis to other STAT isoforms. The specificity of STAT cross-linking by PDT was determined in intact cells and in cell-free extracts using COS-1 cells overexpressing transfected STAT1, STAT3, STAT4, STAT5A, STAT5B, or STAT6. The results (not shown) indicated a preference for cross-linking in following decreas- ing order: STAT3ϾSTAT1ϾSTAT4ϾϾSTAT5BϾSTAT5BϳSTAT6. The results for STAT1, STAT3, and STAT5 were comparable with the specificity of cross-linking endogenous STATs in FaDu and A549 (Fig. Fig. 5. Recovery of cytokine responsiveness after PDT is a slow process. A. FaDu cells were subjected to aminolevulinic acid–PDT (0.8 mmol/L; 10 J/cm2). This treatment 2C). PDT-mediated cross-linking of STAT4 was in part predicted by the resulted in a survival of 40% cells when determined after 24 hours. Immediately after previous finding that nonphosphorylated STAT4, like STAT1 and PDT, or at successive 24-hour intervals, the adherent cells were treated with EGF or STAT3, forms homodimers (29). When using cytoplasmic extracts from oncostatin M (OSM) for 15 minutes and then extracted. The extracts were analyzed by Western blotting for the proteins indicated at the left. B. Cultures of H-35 cells were cells expressing the combination of STAT1 and STAT3, we detected treated with HPPH-PDT (0.6 ␮mol/L HPPH; 3 J/cm2). Immediately after PDT and at cross-linking of the appropriate homodimeric STAT complexes, but not subsequent 24-hour intervals, replicate cultures were treated for 24 hours with IL-6 [induction of thiostatin (TST)] or conditioned medium from lipopolysaccharide-activated of heterodimeric STAT1/STAT3 complexes. To test cross-linking of ␣ lung macrophages [induction of 1-acid glycoprotein (AGP)]. The number of viable cells phosphorylated STAT1 and STAT3, we applied PDT treatment to high- after cytokine treatment was determined for each culture. Aliquots of conditioned medium ␣ salt extracts from STAT-transfected COS-1 cells that had been treated for were analyzed by immunoelectrophoresis for the amount of 1-acid glycoprotein and thiostatin. The level of plasma protein production was normalized to the number of viable ,ء .minutes with oncostatin M. Although each extract contained a high cells in these cultures. Values represent mean and SD of four separate measurements 15 level of phosphorylated STAT protein, which also showed strong DNA values that are significantly different from those of the control cultures (P Ͻ 0.01). C. (sis-inducible element) binding, we did not detect appreciable cross- A549 cells, like the cells in B, were exposed to HPPH PDT. Immediately after PDT, or at successive 24-hour post-treatment intervals, the adherent cells were treated for 24 hours linking of these proteins. This supported the conclusion that activated with oncostatin M. The number of viable cells present at the end of oncostatin M treatment STATs are not substrates for the oxidative cross-linking reactions caused was determined and indicated a survival rate of 25%. Aliquots of cell lysates, representing by PDT. equivalent amounts of viable cells at the end of treatment, were used for immunoblot detection of fibrinogen. Functional Consequence of PDT on Cytokine- and STAT- Dependent Processes. Besides cross-linking of STAT3, PDT also altered levels of membrane receptors and their responses to ligands ately after PDT, partially recovered by 24 hours, and fully restored by (Fig. 1; refs. 6, 13, and 14). In three separate experimental series, we 48 hours (Fig. 5A). This recovery process coincided temporarily with consistently observed that in the surviving FaDu cell population, the disappearance and restoration of EGFR protein expression and STAT3 activation by oncostatin M was drastically reduced immedi- responsiveness to EGF (Fig. 5A). 6584

Fig. 6. PDT reactions in tumor tissue are comparable with those seen in cultured cells. FaDu tumors in scid BALB/c mice (A) and Colo26 tumors in BALB/c mice (B) were subjected to HPPH-PDT. Treated and the contralateral control tumors were extracted. Aliquots of the extracts, containing equal amounts of protein, were analyzed by immunoblotting for the proteins indicated at the side. Note, the levels of EGFR and phosphoSTAT3 in Colo26 cells were below detection level.

To assess the functional consequence of PDT effects on processes process has proven to be a new and sensitive marker for PDT action downstream of STAT3, such as induction of STAT3-sensitive genes, in cultured cells and in tumors. Cross-linking of STATs and PDT- we needed appropriate gene targets. Because for FaDu cells, no mediated reduction in signaling by cytokine receptors result in a STAT3-responsive genes were known that were amenable to simple 24-hour and longer period of reduced responsiveness to IL-6 cyto- analysis, we applied cells with known STAT3-responsive plasma kines in surviving cells. protein genes, the expression of which was determined by the level of Three aspects regarding the consequences of PDT demand discus- IL-6 cytokines. We chose as a model the induction of STAT3- sion: (1) the biochemical reactions that alter cellular proteins and, at sensitive plasma protein genes by IL-6 in H-35 cells (thiostatin and the same time, inform us about the biology of the affected proteins; (2) ␣ 1-acid glycoprotein; Fig. 5B) and by oncostatin M in A549 cells the biological responses to PDT; and (3) the potential role of the PDT (fibrinogen; Fig. 5C). These gene regulatory systems were chosen reactions in the physiology of tumor tissues. because the expression of the plasma protein genes is detectable only Fundamental to the biochemistry of PDT is the photosensitizer- in the presence of the IL-6 cytokines. We applied to H-35 cells an dependent and subcellular site–restricted activity of singlet oxygen HPPH-PDT treatment that led to 50 to 60% killing of the cells within that has an approximately 10- to 100-nanosecond life span and thus a 24 hours after PDT. The surviving cells showed strongly attenuated very restricted range of action (ϳ10–20 nm; ref. 2). Although amin- induction of the plasma protein production. By 48 to 72 hours, the ␣ olevulinic acid–PDT and HPPH-PDT are believed primarily to act cells regained full cytokine inducibility of 1-acid glycoprotein and thiostatin. However by 72 hours after PDT, the cellular proliferation within the mitochondria, it is unlikely that the direct photochemical rate had not yet returned to the normal 24-hour doubling time (Fig. oxidation and cross-linking of STAT3 at cytoplasmic sites and EGFR 5B). A549 cells showed a slower recovery of oncostatin M-inducible at the plasma membrane is due to singlet oxygen emanating from fibrinogen production (Fig. 5C), with no sign of cell proliferation over mitochondria. However, the photosensitizers are very lipophillic, and 72 hours (not shown). low levels of broadly distributed protoporphyrin IX exported from Cross-linking of STAT3 Is a Maker for PDT Action in Tumors mitochondria in aminolevulinic acid–treated cells or low levels of In vivo. To relate the data describing PDT action in FaDu cells in HPPH maintained in cytoplasmic and plasma membrane structures tissue culture with corresponding events in tumors, FaDu cells were could contribute to the generation of singlet oxygen throughout the grown as xenografts in scid/scid BALB/c mice. HPPH-PDT of the cells, particularly near nonpolar sites. Moreover, generation of longer tumors led to reactions (Fig. 6A) that were very similar to those seen lived reactive oxygen species by singlet oxygen (31) at the mitochon- in vitro (see Fig. 1): STAT3 was cross-linked with detectable forms I dria and other subcellular sites may account for some of the cell-wide to III; the electrophoretic mobility of EGFR was decreased; and the molecular modifications. The ensuing peroxidation reactions may lead immunodetectable amount of EGFR was reduced, as was phospho- to adduct formation involving, among others, lipids and proteins (31), rylated STAT3. An equivalent PDT effect on STAT3 cross-linking which could result in the low level of cross-linked molecules with was also observed in HPPH-PDT–treated Colo26 tumors grown in broad size distributions that were found. BALB/c mice (Fig. 6B). More interesting is that PDT-initiated reactions effectively cross- From these data, we concluded that cells in a tumor were subject to link defined substrates, such as STATs and EGFR with nonreducible the same modifications that we have detected in tissue culture. This and reducible bonds (Fig. 4A). Previous studies (25–27, 29) have suggested that surviving cells in PDT-treated tumors would be tran- indicated that nonactivated STAT1, STAT3, and STAT4, but not siently insensitive to IL-6 cytokines, the mediators that are prominent STAT5, are found as noncovalently bound homodimeric forms in cell during the post-PDT inflammatory reaction at the site of treatment extracts. The PDT results show that dimers for these STAT isoforms (30). are also formed in cells, probably in or near a nonpolar environment that would contain photosensitizers. The dimeric complexes of non- DISCUSSION activated STAT1, STAT3, or STAT4 must have residues that are able This study demonstrates several mechanistic aspects of PDT activ- to form covalent linkages (cysteine, methionine, tryptophane, tyro- ity in cells. The data indicate that PDT causes a set of immediate sine, and histidine) in close proximity (11, 32). The fact that fully modifications of cellular proteins that includes loss of EGFR, dephos- tyrosine phosphorylated and SH-2 domain-interacted STAT1 or phorylation of many cellular proteins, increased kinase action leading STAT3 dimers cannot be cross-linked by PDT suggests that the to the phosphorylation of a 71-kDa protein, activation of cellular activated STAT dimers differ structurally from the nonactivated stress responses, and cross-linking of nonactivated STAT3. The last dimers. The low level of cross-linked STAT complexes reacting with 6585

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. PDT-MEDIATED MODIFICATION OF STATS anti-phosphoSTAT3 (Fig. 1) may represent cross-linked STAT dimers advantageous for normal cells in that it would favor their survival and with one of the STAT3 molecules phosphorylated. the contribution of the cells to subsequent tissue repair. In the context A multimolecular structure that incorporates the nonactivated cy- of cancer, however, the same benefit that the loss of cytokine respon- toplasmic STAT3 has been proposed (28, 33). Analysis of that com- siveness provides to tumor cells will assist in tumor survival and plex suggested an interaction of STAT3 with caveolin-1, HSP90, and recurrence. other chaperone proteins (28). However, we could not demonstrate The covalent protein modification in cells surviving the PDT treat- PDT-mediated cross-linking of those accessory proteins to STAT3 as ment may conceivably have far reaching physiologic consequences. part of the pattern of covalent STAT3 complexes. Thus, the biochem- PDT-cross–linked or otherwise modified proteins at the treatment site ical nature of the larger cross-linked complexes for wild-type STAT3 may introduce neoantigens in the midst of an inflammatory reaction (forms II and III) remains to be explained. It is conceivable that they and may thereby facilitate an antitumor immune response. Cross- contain interacting proteins that are not associated with the cell linked STAT3 appears to be a stable compound that is detectable in membrane (both forms are also found in the PDT-treated cytosol PDT-surviving cells for more than 24 hours and thus is available for fraction; Fig. 4D) but may still represent part of the preformed presentation to the immune cells. Considering that STAT3 under statosome. normal conditions has a half-life of only few hours (38, 39), it seems Although our study has highlighted the cross-linking reaction of that cross-linking has enhanced the stability of the STAT proteins or PDT, a number of other biochemical reactions accompany the PDT that cross-linked STATs are subjected to a different degradation response, some of which are mediated by singlet oxygen and reactive mechanism than normal STATs. A positive immune response may oxygen species. A general loss of tyrosine phosphorylation from become relevant for patients that are subjected to multiple rounds of cellular proteins is evident (Fig. 1). Loss of phosphates can reflect an PDT treatments. Regardless of the site of treatment, the ubiquitous increased action of phosphatases and/or decreased action of kinases. STAT3 (or other analogous proteins) will be cross-linked and may The fact that vanadate treatment prevents in part the PDT-dependent provide the necessary immunogen for a host antitumor response. dephosphorylation of cellular proteins (Fig. 1) suggests that PDT Although we do not yet have evidence that cross-linked proteins are enhances the activity of vanadate-sensitive phosphatase(s). As sug- able to elicit a specific and effective immune response (40), if effec- gested by Kochevar et al. (14), PDT-resistant and even reactive tive, one could envision that it assists in eliminating cells bearing or oxygen species-inducible phosphatases may account for the net de- presenting the neoantigen during the post-PDT period. phosphorylation evident in PDT-treated cells. Although PDT action is associated with reactive oxygen species, the PDT effects on protein phosphorylation and oxidation differ drastically from those found in ACKNOWLEDGMENTS cells subjected to strong redox reactions, such as treatment with We are greatly indebted to Drs. Barbara Henderson and Ravindra K. Pandey hydrogen peroxide (34). In H2O2-treated cells, cysteine residues at the for their generous intellectual and material contributions and to the team of the active site of phosphatases are oxidized, and the enzymes are revers- PDT Center for help in the application of optical instruments. We thank Lurine ibly inactivated (12). The resulting loss of phosphatase activity shifts Vaughan and Sherry Davis for assistance in animal works and PDT treatments the balance of phosphorylation/dephosphorylation reactions and raises of tumors, Dr. Yanping Wang and Erin Tracy for help in experimental the level of tyrosine-phosphorylated proteins in cells, the opposite of procedures, Dr. Tomek Kordula (Cleveland State University, OH) for the what is found with PDT. expression vector of Myc-STAT3, and Dr. Naveen Banjia (Department of Besides activation of phosphatases, an immediate loss of protein Immunology, Roswell Park Cancer Institute) for glucose-regulated protein-58 tyrosine kinase activities can also contribute to the change in phos- antiserum. phorylation status of the cellular proteins (13, 15, 35). The complexity of PDT-dependent changes of enzymatic activities is further evident REFERENCES by the PDT effects in vanadate-inhibited cells (Fig. 1). Although the 1. Dougherty TJ. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. Photodynamic Therapy Causes Cross-linking of Signal Transducer and Activator of Transcription Proteins and Attenuation of Interleukin-6 Cytokine Responsiveness in Epithelial Cells

Weiguo Liu, Allan R. Oseroff and Heinz Baumann

Cancer Res 2004;64:6579-6587.

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