[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
Photodynamic therapy (PDT) is a local treatment of cancers. The
principle of PDT is the production of reactive oxygen species, in particular
singlet oxygen, by light activation of a photosensitizer introduced into the
target cells. The direct photochemical and subsequent redox reactions can
lead to cell death. This study sought to identify effects occurring during
PDT and some of their consequences in surviving cells. Using epithelial
cells in tissue culture and in tumors, several distinct PDT-mediated reactions were found, including global dephosphorylation of proteins, induced
phosphorylation of a 71-kDa protein, initiation of cellular stress responses,
structural modification and loss of epidermal growth factor receptor, and
cross-linking of proteins. Specific covalent cross-linking of nonactivated
signal transducer and activator of transcription (STAT)-3, and to a lesser
extent of STAT1 and STAT4, correlated with PDT dose. Cross-linked
STAT3 was primarily localized to the cytoplasm and failed to bind to
DNA. The combination of STAT cross-linking and inactivation of receptor
functions rendered PDT-treated cells refractory for at least 24 hours to
interleukin-6 and oncostatin M, cytokines known to be elevated at site of
tissue damage and inflammation. It is suggested that the loss of responsiveness to these inflammatory cytokines in the PDT-treated field assists
tumor cells in evading the growth-suppressive activity of these mediators
expected to be present at tissue sites after PDT.
INTRODUCTION
Photodynamic therapy (PDT) is designed to eliminate abnormal
tissue lesions, including tumors (1). Although it is desired to kill target
cells either directly by the photochemical reactions or indirectly by
PDT-induced secondary responses, invariably, a fraction of the malignant and normal cells within or adjacent to the treatment field
escape the lethal PDT effects. In the case of cancer treatment, the
surviving tumor cell population contributes to the recurrence of the
cancer. A survival process demands that the cells exposed to nonlethal
PDT are able to repair cellular damages and eventually to resume
proliferation.
Molecular studies of PDT damage have focused largely on the
mechanisms that explain necrosis and apoptosis of treated cells (2–5).
The relative levels of PDT effects may determine the difference
between lethal and nonlethal outcome, however, the biochemical
nature of these effects is not well understood. We and others have
identified PDT-dependent modifications of cellular proteins and have
evaluated the consequence of these modifications on the functions of
these proteins. The range of modifications includes oxidation, adduct
formation, protein cross-linking, and degradation (6 –11). Particularly
noted was the loss of enzymatic activities due to redox reactions such
as found for phosphatases (12) and kinases (6, 13–15). The PDTReceived 5/5/04; revised 6/20/04; accepted 7/16/04.
Grant support: NCI grants CA85580 (H. Baumann) and CA55791 (A. Oseroff) and
Roswell Park Cancer Support grant CA16056.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Heinz Baumann, Department of Molecular and Cellular
Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Fax:
716-845-5908; E-mail: [email protected].
©2004 American Association for Cancer Research.
dependent loss of immunodetectable epidermal growth factor (EGF)
receptor (EGFR) appeared as one of the prominent effects of PDT in
various epithelial cell types (6, 14). To gain a more comprehensive
picture of the processes initiated by PDT and to identify potential
quantitative markers for immediate PDT damage, we determined in
cells in culture and growing as tumors in vivo the effects of PDT on
profiles of protein modification and signaling functions.
The study indicates several novel responses that define PDT action:
(1) increased dephosphorylation of tyrosine-phosphorylated proteins,
(2) stimulation of a PDT- sensitive protein tyrosine kinase activity that
is detectable after inhibition of tyrosine phosphatases, and (3) a highly
specific cross-linking of nonactivated signal transducer and activator
of transcription (STAT)-3 and, to a lesser extent, STAT1 and STAT4.
The modification of STAT proteins, together with the attenuated
signaling by the cytokine receptors, correlates with reduced cellular
responsiveness to interleukin (IL)-6 cytokines.
MATERIALS AND METHODS
Cells. The following cells lines were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and antibiotics: FaDu (human
hypopharyngeal carcinoma), A549 (human lung carcinoma), H-35 (rat hepatoma), Colo26 (mouse colon carcinoma), and COS-1 (green monkey kidney
epithelial). Human prostate carcinoma PC3 cells were maintained in RPMI
1640 containing 10% fetal calf serum. Human pulmonary macrophages were
isolated from residual lung tissue obtained from Roswell Park Cancer Institute
Tissue Procurement Service under the approved Institutional Review Board
protocol CIC 00-17. Macrophages were mechanically extracted from minced
lung tissue and purified by Histopaque gradient centrifugation (Sigma Chemical Co., St. Louis, MO). The cells (1 ⫻ 106 per mL) were resuspended in
RPMI 1640 containing 10% fetal calf serum and 1 ␮g/mL lipopolysaccharides.
Conditioned medium was collected after 12 hours of incubation.
PDT Treatment. Cells at ⬃90% confluence were incubated for 4 hours at
37°C with growth medium containing 0 to 1 mmol/L aminolevulinic acid (16)
or 0 to 1.2 ␮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
2-carboxy-3,4,5,6-tetrachlorophenyl)-2,4,5,7-tetraiodo-3,6-dihydroxyxanthyliumdipotassium salt [Rose Bengal (RB); Sigma Chemical Co.; ref. 18]. Where
necessary, advance treatment for 2 hours with 1 mmol/L sodium vanadate or
for 15 minutes with 100 ng/mL oncostatin M (Amgen Inc., Seattle, WA) or
EGF (Invitrogen Corp., Carlsbad, CA) was initiated by adding these reagents
to the medium containing the photosensitizer. Medium was replaced with
fresh, photosensitizer-free medium containing 10% fetal calf serum and the
cytokines or EGF necessary to maintain signaling during the light-induced
photoreactions. Light treatments were carried at room temperature by applying
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
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
590 to 700 nm with a fluence rate of 14 mW/cm2 and fluence ranging from 0.5
to 8 J/cm2. All experimental series included controls consisting of cultures that
were incubated with photosensitizer but not exposed to light. Immediately after
PDT or after various length of time in growth medium and treatments with
cytokines or EGF, the cells were washed with PBS and processed one of two
ways. The cells were lysed in the culture dish with radioimmunoprecipitation
assay buffer for analysis of protein expression by immunoblotting, or the cells
were extracted with high-salt buffer [0.4 mol/L NaCl, 20 mmol/L 4-(2-
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PDT-MEDIATED MODIFICATION OF STATS
hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.6), 20 mmol/L NaF, 1
mmol/L Na4P2O7, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 20% glycerol, 1 mmol/L sodium orthovanadate, 1 ␮g/mL leupeptin and
aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride) for measuring DNA
binding activity of STATs by electrophoretic shift assay. Most PDT experiments also included cultures that were subjected to the treatment and were used
24 hours later to determine the effect of PDT on cell survival. Adherent cells
were washed free of material derived from dead cells with PBS and then were
released by trypsinization. Viable cells were counted based on trypan blue dye
exclusion in a hematocytometer.
Cell Fractionation. After washing with PBS, the cells were allowed to
swell in 4 volumes of hypotonic buffer [20 mmol/L Tris (pH 7.4), 10 mmol/L
NaCl, and 2 mmol/L MgCl2] on ice and then were broken in a tight-fitted
Dounce homogenizer. The homogenate was centrifuged for 5 minutes at
10,000 ⫻ g to remove nuclei and large cell fragments. The supernatant (termed
“cytoplasmic fraction”) was centrifuged for 60 minutes at 100,000 ⫻ g, and the
resulting supernatant (termed “cytosol fraction”) was used for protein analysis.
To recover nuclei free of cytoplasmic contamination, cells were treated with
hypotonic buffer as described above and then homogenized in the presence of
0.5% NP40. The nuclei were collected by centrifugation for 5 minutes at
1,000 ⫻ g. The nuclear pellet was resuspended in 1 mL of sucrose buffer [10
mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 25 mmol/L KCl,
5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L spermidine, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 10% glycerol, and 2 mol/L
sucrose], loaded onto a cushion of 4 mL of sucrose buffer, and centrifuged for
60 minutes at 100,000 ⫻ g. The nuclei were solubilized by boiling in SDS
buffer.
Tumor. FaDu cells were released by trypsin and washed with PBS, and
1 ⫻ 106 were resuspended in 50 ␮L of serum-free medium and injected
subcutaneously on the dorsal sides of both hind flanks of scid/scid BALB/c
mice. Similarly, Colo26 cells (1 ⫻ 106) were injected into normal BALB/c
mice. After the tumors had reached a size of ⬃6 mm in diameter, the animals
received a tail vein injection of 0.4 ␮mol/kg HPPH. After 24 hours, one tumor
on each animal was treated with 665 nm of light using an argon-dye laser
system (Spectra Physics, Mountain View, CA; fluence rate, 28 mW/cm2;
fluence, 48 J/cm2). The contralateral tumor was kept protected from light
exposure and served as control. After PDT, the treated and untreated tumors
were excised, rinsed with PBS, and immediately homogenized in radioimmunoprecipitation assay buffer.
Gel Electrophoresis. Depending on the molecular size of the antigens,
aliquots of extracts (5–30 ␮g of protein) were separated on 6 to 12% SDSpolyacrylamide gels. Proteins were transferred to protean membranes
(Schleicher & Schuell, Riviera Beach, FL). Wherever possible, replicates of
the samples were electrophoresed for identification of multiple antigens. This
approach circumvented the problem of incomplete removal of antibodies
during sequential immunoblotting of the same membrane (6). The membranes
were reacted with antibodies to EGFR; leukemia inhibitory factor receptor-␣;
gp130; STAT1; STAT3 (C20 and H-190); STAT4; STAT5; STAT6; extracellular signal-regulated kinase (ERK)-1/-2; caveolin-1; heat shock protein
(HSP)-90 and HSP70 (Santa Cruz Biotechnology, Santa Cruz, CA); poly(ADP-ribose) polymerase (Cell Signaling Technology, Beverly, MA); phosphotyrosine-STAT1, phosphotyrosine-STAT3, or P-ERK-1/-2 (New England
Biolabs, Inc. Beverly, MA); glucose-regulated protein-58 (provided by Dr.
Naveen Banjia); oncostatin M receptor ␤ (Amgen, Inc.); phosphotyrosine
(Upstate Biotechnology, Lake Placid, NY); human fibrinogen (Dako, Carpinteria, CA); rat thiostatin; and ␣1-acid glycoprotein (Department of Laboratory
Animal Resources at Roswell Park Cancer Institute). Reaction with secondary
antibodies (ICN Biomedicals, Inc., Aurora, OH) occurred in PBS containing
0.1% Tween 20, 5% milk, or 3% albumin. Immune complexes were visualized
by enhanced chemiluminescence (ECL) reaction (Amersham Biosciences,
Piscataway, NJ). To quantify the immunodetectable signals, 2-fold serially
diluted extracts were subjected to Western blotting. The ECL images were
recorded on X-ray films by various length of exposure to ensure recovery of
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
values of each band were determined by integration using the ImageQuant
program (Amersham Biosciences). The linear range of the signals within the
dilution series was used to calculate the relative amounts of antigens among
samples. Statistically significant differences were evaluated by Student’s t test.
Electrophoretic Mobility Shift Assay. To determine DNA-binding activity of STAT1 and STAT3, aliquots of whole-cell or nuclear extracts containing
5 to 20 ␮g of proteins were reacted with a [32P]-labeled double-stranded
oligonucleotide representing the m67 sis-inducible element and analyzed for
protein interaction by electrophoresis as described previously (19, 20). Incubation of extracts with anti-STAT3 (C20; Santa Cruz Biotechnology) before
electrophoresis identified the presence of STAT3 in the sis-inducible element
complexes by supershifting.
Immunoelectrophoretic Analysis. H-35 cell monolayers were treated for
24 hours with 100 ng/mL human recombinant IL-6 (Genetics Institute, Cambridge, MA) or one-tenth diluted conditioned medium of lipopolysaccharideactivated human pulmonary macrophages in Dulbecco’s modified Eagle’s
medium containing 0.5% fetal calf serum and 1 ␮mol/L dexamethasone. The
amounts of thiostatin and ␣1-acid glycoprotein secreted into the culture medium were determined by immunoelectrophoresis (21) and expressed in milligrams per milliliter using purified plasma proteins as reference.
Transfection. Using FuGene 6 (Roche Diagnostics Corp., Indianapolis,
IN), COS-1 cells were transfected with the pSPORT1 expression vectors for
wild-type rat STAT1, STAT3, STAT5A, STAT5B, mouse STAT4 and
STAT6, COOH-terminally truncated rat STAT3⌬55C, (20), or rat STAT3 with
six tandem copies of the Myc epitope tag added to the NH2 terminus (provided
by Dr. Tomek Kordula). After a 36-hour recovery period, the cells were
extracted by three cycles of freeze-thaw in 3 volumes of PBS containing a
mixture of inhibitors for proteases and phosphatases. Cell debris was removed
by centrifugation at 15,000 ⫻ g for 20 minutes. The cytosolic fraction was
obtained by subsequent centrifugation at 100,000 ⫻ g for 1 hour.
RESULTS
PDT Causes Several Immediate Biochemical Reactions. Because biological consequences of PDT often are only evident hours
after treatment (22), we asked what cell reactions describe the initial
PDT effects in epithelial cells. To broaden the assessment, we also
determined the effects of PDT on signaling by the receptors for
oncostatin M and EGF, two factors that prominently act on normal
and on many transformed epithelial cells. We used the human hypopharyngeal cell line FaDu as an experimental system because this line
expresses abundant amounts of receptors for oncostatin M, IL-6,
leukemia inhibitory factor, and EGF and responds to these factors
comparably with normal epithelial cells (6). We examined two second-generation photosensitizers that currently are in clinical trials: the
prodrug aminolevulinic acid that is converted into the endogenous
photosensitizer protoporphyrin IX (16), and the exogenous photosensitizer HPPH (17). Both agents are believed to target primarily mitochondria. To evaluate the relationship of subcellular distribution of the
photochemical reaction and pattern of outcome, we also treated cells
with RB, a more hydrophilic cationic photosensitizer that shows a
broad cytoplasmic and membrane distribution (18).
The PDT effects were assessed by determining the level of phosphorylation of total cell proteins, expression of receptors, and modification of downstream signaling proteins. Key PDT responses are
summarized by the representative experiment shown in Fig. 1. Data
from more than 30 independent series of PDT treatments of FaDu
cells support the stated findings. The results, in part, confirmed
reactions reported previously (6, 14). Using an aminolevulinic acid–
PDT dose that led to ⬃60% killing of the cells during a 24-hour
post-treatment period, five distinct immediate PDT effects were consistently observed. (1) EGFR changed to a slower electrophoretic
mobility. (2) The level of EGFR protein was reduced. (3) Tyrosine
phosphorylation of many cellular proteins was decreased. Because the
basal level of protein tyrosine phosphorylation in FaDu cells was low,
a longer ECL exposure of the immunoblot was necessary to reveal this
PDT effect (Fig. 1, left panel at the top). The loss of phosphorylation
also was associated with a lower action of EGFR and oncostatin M
receptor, as was evident by the reduced phosphorylation of EGFR,
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PDT-MEDIATED MODIFICATION OF STATS
Fig. 1. PDT mediates several types of immediate cell reactions. FaDu cells were treated
with 0.8 mmol/L aminolevulinic acid for 4 hours. Where indicated at the top, pretreatments with 1 mmol/L sodium vanadate (2 hours) and EGF or oncostatin M (OSM; 15
minutes) were carried out. Cells were subjected to light treatment (10 J/cm2) and lysed
immediately after PDT. A duplicate set of control cells, with and without PDT treatment,
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 immunoblotting for the antigens indicated at the left. The ECL exposure of the film was limited
by the patterns with highest signal. Longer exposures permitted identification of changes
in samples with low immune signals. PDT-mediated changes in basal level tyrosinephosphorylated proteins are demonstrated by the 45-minute ECL exposure of the control
samples at the left of the top panel (WB: PY). Conversely, the reduction in immunodetectable 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
antigens, the cross-linked complexes, and the molecular size markers are indicated at the
right. The open arrow at the top panel points to the position of the 71-kDa tyrosinephosphorylated protein detected in PDT-treated cells, the phosphatase activities of which
were inhibited by vanadate. Circles in the phosphotyrosine-STAT3 (PY-STAT3) patterns
indicate the position of cross-linked STAT3 complexes containing phosphorylated
STAT3.
STAT3, and ERK. (4) The level of phosphorylated (activated) c-junNH2-terminal kinase (JNK) was increased. Activation of the p38
stress mitogen-activated protein kinase was also observed in FaDu
cells (see ref. 6), however, the available antibodies yielded only a
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 ⬃15%. HPPH-PDT resulted in a
consistently higher conversion of ⬃30%. 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 ⬃95% (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
level of STAT3 cross-linking appeared to be an indicator of the
severity of the photoreaction. Moreover, PDT conditions could be
chosen that yielded a sufficient number of surviving cells that permitted determination of the relationship of STAT cross-linking and
STAT function.
The analysis of different STAT proteins in FaDu and other epithelial cell types, including A549 lung epithelial cells that have a particularly high level expression of STAT1 and STAT3, indicated that
PDT-mediated STAT cross-linking was not restricted to FaDu cells.
Most effectively cross-linked was STAT3, less was STAT1, and not
appreciably was STAT5 (Fig. 2C, left panel). When cells were pretreated with oncostatin M to increase tyrosine phosphorylation of
STAT3 and STAT1, only a minimal amount of phosphorylated
STAT3 and none of phosphorylated STAT1 were detectably associated with cross-linked STAT proteins (Fig. 1; data not shown). PDT
treatment of the human prostate cell line PC3, which lacks STAT3,
did not result in the appearance of protein forms that reacted with
anti-STAT3 (data not presented). This finding ruled out the possibility
that an epitope was generated by the PDT reaction that cross-reacted
with anti-STAT3 but was unrelated to STAT3.
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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 antiSTAT3. 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
FaDu cells treated with aminolevulinic acid–PDT, HPPH-PDT, or
RB-PDT, only trace amounts of cross-linked STAT3 and none for
STAT1 were detected in the nuclear fraction. In cell lines with high
levels of STAT proteins, such as A549, the PDT reaction caused a
conversion of up to 70% of STAT3 epitopes appearing in cross-linked
complexes (Fig. 2C, center panel). In RB-PDT–treated A549 cells,
cross-linked STAT3 was also more abundant in the nuclear fraction
(Fig. 2C, right panel). Of note is that the amount of cross-linked
STAT3 relative to monomeric STAT3 in nuclei was 2- to 5-fold lower
than in the cytoplasmic fraction from the same cells. The cross-linked
nuclear STAT3 failed to react with antibodies against tyrosine phosphorylated STAT3. These results suggest that cross-linking of STAT3
is mainly a cytoplasmic event with the cross-linked STAT proteins
remaining largely localized to the cytoplasm and/or that reactive
oxygen species, which are responsible for cross-linking, did not
efficiently reach the nuclear compartment (23).
The relatively high level of cross-linked STAT3 complexes as
found in RB-PDT–treated A549 cells allowed us also to determine
whether cross-linked STAT3 gained DNA-binding activity, such as to
the high-affinity substrate sis-inducible element. Such a DNA-binding
activity was found with STAT3 dimers generated by oncostatin M
treatment through phosphorylation or with nonphosphorylated dimers
formed by the A661C and N663C mutations in STAT3-C (24).
However, electrophoretic mobility shift assay analysis with extracts
from RB-PDT–treated A549 cells indicated no PDT-induced sisinducible element-binding by STAT3 (Fig. 2D). In fact, PDT lowered
the basal activity of STAT3 binding probably in part by PDT-dependent dephosphorylation of STAT3 (Fig. 1). From these findings, we
concluded that PDT- dependent cross-linking inactivated STAT3 and
thus removed it from the pool of signal-transducing proteins.
Cross-linked STAT Proteins Are Stable. Time-course analysis
of the post-PDT reactions indicated that protein cross-linking by
either aminolevulinic acid–PDT or HPPH-PDT was limited to the
light treatment period (Fig. 3A), suggesting that it was solely a
function of the light-dependent photoreaction. In contrast, other PDTinitiated reactions continued to increase during post-PDT period, such
as activation (phosphorylation) of JNK, dephosphorylation of ERK,
loss of EGFR, and appearance of markers for apoptosis, such as the
delayed degradation of phosphorylated JNK and poly(ADP-ribose)
polymerase. Cross-linked STAT3 protein forms were detectable in the
surviving cell population for several days, indicating an apparent
half-life of 18 hours (Fig. 3B).
Nature of the Cross-linked STAT Proteins. PDT-dependent
cross-linking of STAT3 was primarily through formation of covalent
linkages that were noncleavable by reducing agents (Fig. 4A). In three
separate experiments, we determined that consistently less than 25%
of the cross-linked STAT3 contained within forms I, II, and III was
susceptible to reduction by 2-mercaptoethanol. This was in contrast to
PDT-cross–linked EGFR dimers, of which ⬃50% were cleaved by
reduction (Fig. 4B).
The molecular size of 177 kDa for the major cross-linked form I is
compatible with a homodimer of STAT3. It has been shown previously that noncovalently linked homodimers of nonactivated STAT1
and STAT3 are present in cytoplasmic cell extracts (25–27). Thus, we
hypothesized that the same preformed dimers of STAT3 also exist in
intact cells and that PDT covalently cross-linked these to yield form
I. Forms II and III may include additional accessory proteins. Prior
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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 posttreatment 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 numerical 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 SDSPAGE under nonreducing [⫺␤-mercaptoethanol (⫺␤MeOH)] and reducing [⫹␤-mercaptoethanol (⫹␤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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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, glucoseregulated protein-58, or caveolin-1 was integrated into the crosslinked 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 immunoprecipitation 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 overexpressed 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 wildtype STAT3 was prepared and subjected to HPPH-PDT. The same
cross-linked STAT3 forms I to III were generated as found in PDTtreated 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 decreasing order: STAT3⬎STAT1⬎STAT4⬎⬎STAT5B⬎STAT5B⬃STAT6.
The results for STAT1, STAT3, and STAT5 were comparable with the
specificity of cross-linking endogenous STATs in FaDu and A549 (Fig.
2C). PDT-mediated cross-linking of STAT4 was in part predicted by the
previous finding that nonphosphorylated STAT4, like STAT1 and
STAT3, forms homodimers (29). When using cytoplasmic extracts from
cells expressing the combination of STAT1 and STAT3, we detected
cross-linking of the appropriate homodimeric STAT complexes, but not
of heterodimeric STAT1/STAT3 complexes. To test cross-linking of
phosphorylated STAT1 and STAT3, we applied PDT treatment to highsalt extracts from STAT-transfected COS-1 cells that had been treated for
15 minutes with oncostatin M. Although each extract contained a high
level of phosphorylated STAT protein, which also showed strong DNA
(sis-inducible element) binding, we did not detect appreciable crosslinking of these proteins. This supported the conclusion that activated
STATs are not substrates for the oxidative cross-linking reactions caused
by PDT.
Functional Consequence of PDT on Cytokine- and STATDependent Processes. Besides cross-linking of STAT3, PDT also
altered levels of membrane receptors and their responses to ligands
(Fig. 1; refs. 6, 13, and 14). In three separate experimental series, we
consistently observed that in the surviving FaDu cell population,
STAT3 activation by oncostatin M was drastically reduced immedi-
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
resulted in a survival of 40% cells when determined after 24 hours. Immediately after
PDT, or at successive 24-hour intervals, the adherent cells were treated with EGF or
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
treated with HPPH-PDT (0.6 ␮mol/L HPPH; 3 J/cm2). Immediately after PDT and at
subsequent 24-hour intervals, replicate cultures were treated for 24 hours with IL-6
[induction of thiostatin (TST)] or conditioned medium from lipopolysaccharide-activated
lung macrophages [induction of ␣1-acid glycoprotein (AGP)]. The number of viable cells
after cytokine treatment was determined for each culture. Aliquots of conditioned medium
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
cells in these cultures. Values represent mean and SD of four separate measurements. ⴱ,
values that are significantly different from those of the control cultures (P ⬍ 0.01). C.
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
with oncostatin M. The number of viable cells present at the end of oncostatin M treatment
was determined and indicated a survival rate of 25%. Aliquots of cell lysates, representing
equivalent amounts of viable cells at the end of treatment, were used for immunoblot
detection of fibrinogen.
ately after PDT, partially recovered by 24 hours, and fully restored by
48 hours (Fig. 5A). This recovery process coincided temporarily with
the disappearance and restoration of EGFR protein expression and
responsiveness to EGF (Fig. 5A).
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PDT-MEDIATED MODIFICATION OF STATS
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
downstream of STAT3, such as induction of STAT3-sensitive genes,
we needed appropriate gene targets. Because for FaDu cells, no
STAT3-responsive genes were known that were amenable to simple
analysis, we applied cells with known STAT3-responsive plasma
protein genes, the expression of which was determined by the level of
IL-6 cytokines. We chose as a model the induction of STAT3sensitive plasma protein genes by IL-6 in H-35 cells (thiostatin and
␣1-acid glycoprotein; Fig. 5B) and by oncostatin M in A549 cells
(fibrinogen; Fig. 5C). These gene regulatory systems were chosen
because the expression of the plasma protein genes is detectable only
in the presence of the IL-6 cytokines. We applied to H-35 cells an
HPPH-PDT treatment that led to 50 to 60% killing of the cells within
24 hours after PDT. The surviving cells showed strongly attenuated
induction of the plasma protein production. By 48 to 72 hours, the
cells regained full cytokine inducibility of ␣1-acid glycoprotein and
thiostatin. However by 72 hours after PDT, the cellular proliferation
rate had not yet returned to the normal 24-hour doubling time (Fig.
5B). A549 cells showed a slower recovery of oncostatin M-inducible
fibrinogen production (Fig. 5C), with no sign of cell proliferation over
72 hours (not shown).
Cross-linking of STAT3 Is a Maker for PDT Action in Tumors
In vivo. To relate the data describing PDT action in FaDu cells in
tissue culture with corresponding events in tumors, FaDu cells were
grown as xenografts in scid/scid BALB/c mice. HPPH-PDT of the
tumors led to reactions (Fig. 6A) that were very similar to those seen
in vitro (see Fig. 1): STAT3 was cross-linked with detectable forms I
to III; the electrophoretic mobility of EGFR was decreased; and the
immunodetectable amount of EGFR was reduced, as was phosphorylated STAT3. An equivalent PDT effect on STAT3 cross-linking
was also observed in HPPH-PDT–treated Colo26 tumors grown in
BALB/c mice (Fig. 6B).
From these data, we concluded that cells in a tumor were subject to
the same modifications that we have detected in tissue culture. This
suggested that surviving cells in PDT-treated tumors would be transiently insensitive to IL-6 cytokines, the mediators that are prominent
during the post-PDT inflammatory reaction at the site of treatment
(30).
DISCUSSION
This study demonstrates several mechanistic aspects of PDT activity in cells. The data indicate that PDT causes a set of immediate
modifications of cellular proteins that includes loss of EGFR, dephosphorylation of many cellular proteins, increased kinase action leading
to the phosphorylation of a 71-kDa protein, activation of cellular
stress responses, and cross-linking of nonactivated STAT3. The last
process has proven to be a new and sensitive marker for PDT action
in cultured cells and in tumors. Cross-linking of STATs and PDTmediated reduction in signaling by cytokine receptors result in a
24-hour and longer period of reduced responsiveness to IL-6 cytokines in surviving cells.
Three aspects regarding the consequences of PDT demand discussion: (1) the biochemical reactions that alter cellular proteins and, at
the same time, inform us about the biology of the affected proteins; (2)
the biological responses to PDT; and (3) the potential role of the PDT
reactions in the physiology of tumor tissues.
Fundamental to the biochemistry of PDT is the photosensitizerdependent and subcellular site–restricted activity of singlet oxygen
that has an approximately 10- to 100-nanosecond life span and thus a
very restricted range of action (⬃10 –20 nm; ref. 2). Although aminolevulinic acid–PDT and HPPH-PDT are believed primarily to act
within the mitochondria, it is unlikely that the direct photochemical
oxidation and cross-linking of STAT3 at cytoplasmic sites and EGFR
at the plasma membrane is due to singlet oxygen emanating from
mitochondria. However, the photosensitizers are very lipophillic, and
low levels of broadly distributed protoporphyrin IX exported from
mitochondria in aminolevulinic acid–treated cells or low levels of
HPPH maintained in cytoplasmic and plasma membrane structures
could contribute to the generation of singlet oxygen throughout the
cells, particularly near nonpolar sites. Moreover, generation of longer
lived reactive oxygen species by singlet oxygen (31) at the mitochondria and other subcellular sites may account for some of the cell-wide
molecular modifications. The ensuing peroxidation reactions may lead
to adduct formation involving, among others, lipids and proteins (31),
which could result in the low level of cross-linked molecules with
broad size distributions that were found.
More interesting is that PDT-initiated reactions effectively crosslink defined substrates, such as STATs and EGFR with nonreducible
and reducible bonds (Fig. 4A). Previous studies (25–27, 29) have
indicated that nonactivated STAT1, STAT3, and STAT4, but not
STAT5, are found as noncovalently bound homodimeric forms in cell
extracts. The PDT results show that dimers for these STAT isoforms
are also formed in cells, probably in or near a nonpolar environment
that would contain photosensitizers. The dimeric complexes of nonactivated STAT1, STAT3, or STAT4 must have residues that are able
to form covalent linkages (cysteine, methionine, tryptophane, tyrosine, and histidine) in close proximity (11, 32). The fact that fully
tyrosine phosphorylated and SH-2 domain-interacted STAT1 or
STAT3 dimers cannot be cross-linked by PDT suggests that the
activated STAT dimers differ structurally from the nonactivated
dimers. The low level of cross-linked STAT complexes reacting with
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PDT-MEDIATED MODIFICATION OF STATS
anti-phosphoSTAT3 (Fig. 1) may represent cross-linked STAT dimers
with one of the STAT3 molecules phosphorylated.
A multimolecular structure that incorporates the nonactivated cytoplasmic STAT3 has been proposed (28, 33). Analysis of that complex suggested an interaction of STAT3 with caveolin-1, HSP90, and
other chaperone proteins (28). However, we could not demonstrate
PDT-mediated cross-linking of those accessory proteins to STAT3 as
part of the pattern of covalent STAT3 complexes. Thus, the biochemical nature of the larger cross-linked complexes for wild-type STAT3
(forms II and III) remains to be explained. It is conceivable that they
contain interacting proteins that are not associated with the cell
membrane (both forms are also found in the PDT-treated cytosol
fraction; Fig. 4D) but may still represent part of the preformed
statosome.
Although our study has highlighted the cross-linking reaction of
PDT, a number of other biochemical reactions accompany the PDT
response, some of which are mediated by singlet oxygen and reactive
oxygen species. A general loss of tyrosine phosphorylation from
cellular proteins is evident (Fig. 1). Loss of phosphates can reflect an
increased action of phosphatases and/or decreased action of kinases.
The fact that vanadate treatment prevents in part the PDT-dependent
dephosphorylation of cellular proteins (Fig. 1) suggests that PDT
enhances the activity of vanadate-sensitive phosphatase(s). As suggested by Kochevar et al. (14), PDT-resistant and even reactive
oxygen species-inducible phosphatases may account for the net dephosphorylation 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
cells subjected to strong redox reactions, such as treatment with
hydrogen peroxide (34). In H2O2-treated cells, cysteine residues at the
active site of phosphatases are oxidized, and the enzymes are reversibly inactivated (12). The resulting loss of phosphatase activity shifts
the balance of phosphorylation/dephosphorylation reactions and raises
the level of tyrosine-phosphorylated proteins in cells, the opposite of
what is found with PDT.
Besides activation of phosphatases, an immediate loss of protein
tyrosine kinase activities can also contribute to the change in phosphorylation status of the cellular proteins (13, 15, 35). The complexity
of PDT-dependent changes of enzymatic activities is further evident
by the PDT effects in vanadate-inhibited cells (Fig. 1). Although the
responsible enzymes still remain to be defined, a prominent PDTstimulated tyrosine kinase action is detected by the phosphorylation of
a 71-kDa protein. This activity is efficiently quenched in cells not
exposed to vanadate. This suggests that vanadate-sensitive tyrosine
phosphatases are effective in reversing the effects of the PDT-stimulated protein kinase activity. Surveying data derived from different
cell types and PDT treatments, we have to conclude, however, that
dephosphorylation prevails. The enhanced phosphatase activity in
PDT-treated cells may also account in part for the attenuated, or even
lost signaling function of cytokine and growth factor receptors.
One of the striking biological consequences of PDT is that surviving cells transiently lose responsiveness to cytokines such as oncostatin M. Oncostatin M is one of the cytokines that are expected to be
high at site of tissue injury as caused by PDT. Although in vivo PDT
may injure or eliminate host cytokine-producing cells that are located
within the treatment field, immigrating leukocytes will be activated by
the milieu of the damaged tissue (30, 36). Cytokines from these cells
including, among others, tumor necrosis factor, IL-1, IL-6, and oncostatin M act in a paracrine fashion on cells within and adjacent to
the PDT-treated area. These cytokines are known to suppress epithelial cell growth (37). Thus, those cells surviving PDT, whether normal
or tumor cells, are refractory to these components and temporarily
sheltered from the inhibitory cytokine actions. This scenario appears
advantageous for normal cells in that it would favor their survival and
the contribution of the cells to subsequent tissue repair. In the context
of cancer, however, the same benefit that the loss of cytokine responsiveness provides to tumor cells will assist in tumor survival and
recurrence.
The covalent protein modification in cells surviving the PDT treatment may conceivably have far reaching physiologic consequences.
PDT-cross–linked or otherwise modified proteins at the treatment site
may introduce neoantigens in the midst of an inflammatory reaction
and may thereby facilitate an antitumor immune response. Crosslinked STAT3 appears to be a stable compound that is detectable in
PDT-surviving cells for more than 24 hours and thus is available for
presentation to the immune cells. Considering that STAT3 under
normal conditions has a half-life of only few hours (38, 39), it seems
that cross-linking has enhanced the stability of the STAT proteins or
that cross-linked STATs are subjected to a different degradation
mechanism than normal STATs. A positive immune response may
become relevant for patients that are subjected to multiple rounds of
PDT treatments. Regardless of the site of treatment, the ubiquitous
STAT3 (or other analogous proteins) will be cross-linked and may
provide the necessary immunogen for a host antitumor response.
Although we do not yet have evidence that cross-linked proteins are
able to elicit a specific and effective immune response (40), if effective, one could envision that it assists in eliminating cells bearing or
presenting the neoantigen during the post-PDT period.
ACKNOWLEDGMENTS
We are greatly indebted to Drs. Barbara Henderson and Ravindra K. Pandey
for their generous intellectual and material contributions and to the team of the
PDT Center for help in the application of optical instruments. We thank Lurine
Vaughan and Sherry Davis for assistance in animal works and PDT treatments
of tumors, Dr. Yanping Wang and Erin Tracy for help in experimental
procedures, Dr. Tomek Kordula (Cleveland State University, OH) for the
expression vector of Myc-STAT3, and Dr. Naveen Banjia (Department of
Immunology, Roswell Park Cancer Institute) for glucose-regulated protein-58
antiserum.
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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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