RNA Expression Profiling of Normal and Tumor Cells Following Photodynamic Therapy with 5-Aminolevulinic Acid–Induced Protoporphyrin IX in Vitro

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RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid–induced protoporphyrin IX in vitro

Peter J. Wild,1 Rene C. Krieg,4 Juergen Seidl,1 RNA was found continuously up-regulated over time in all Robert Stoehr,2 Kerstin Reher,2 three cell lines. Induction of DUSP1 protein expression was Claudia Hofmann,3 Jari Louhelainen,5 clearly shown after 1 hour using Western blot analysis. Andre´ Rosenthal,6 Arndt Hartmann,1 Interestingly, no changes of caspase-8 protein expression Christian Pilarsky,7 Anja K. Bosserhoff,1 but activation of catalytic activity was detected only in and Ruth Knuechel4 UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. 1 2 3 Institute of Pathology and Departments of Urology and Internal According to caspase-8, the active caspase 3 fragment Medicine I, University of Regensburg, Regensburg, Germany; 4Institute of Pathology, University of Aachen, Aachen, Germany; was found only in the normal urothelial cell line (UROtsa) 5Cancer Research UK Clinical Centre, St James’s University 1 hour after photodynamic therapy. Combined data analysis 6 Hospital, Leeds, United Kingdom; Signature Diagnostics AG, suggests that photodynamic therapy in vitro (LD50)leads 7 Potsdam, Germany; and Department of Surgery, University to apoptosis in UROtsa and to necrosis in the tumor cell Hospital Dresden, Dresden, Germany lines, respectively. RNA expression profiling of normal and Abstract tumor cell lines following photodynamic therapy with 5- aminolevulinic acid gave insight into the major molecular Photodynamic therapy using 5-aminolevulinic acid–induced mechanisms induced by photodynamic therapy. [Mol Cancer protoporphyrin IX synthesis as a photosensitizing reagent Ther 2005;4(4):516–28] is an encouraging modality for cancer treatment. Under- standing the mechanism of tumor phototoxicity is important Introduction to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell Photodynamic therapy is based on the tumor-selective lines (RT4, urothelial; HT29, colonic) were treated with cell accumulation of a photosensitizer. After irradiation with light of appropriate wavelength and in the presence of line–specific LD50 doses of light after exposure to 5- aminolevulinic acid (100 Ag/mL), and harvested for RNA oxygen, this photosensitizer will induce cellular damage by extraction 0, 10, and 30 minutes after irradiation. The RNA generating singulett oxygen (Fig. 1A–C). Many photo- was hybridized to the metg001A Affymetrix GeneChip sensitizers are known but only a few are used in clinical containing 2,800 genes, focusing on cancer-related and practice. 5-Aminolevulinic acid–induced protoporphyrin growth regulatory targets. Comparing the gene expression IX is preferred to competitive porphyrin derivatives (1). In profiles between the different samples, 40 genes (e.g., comparison with, e.g., Photofrin (2), the most obvious SOD2, LUC7A, CASP8,andDUSP1) were identified as advantages of 5-aminolevulinic acid are the possibility of significantly altered in comparison with the control samples, a topical application and the rapid clearance within 2 days. and grouped according to their gene ontology. We selected In addition, neither 5-aminolevulinic acid nor protopor- caspase-8 (CASP8) and dual specificity phosphatase 1 phyrin IX shows a dark toxicity as they are both heme (DUSP1) for further validation of the array findings, and precursors and therefore physiologic substances. In con- compared their expression with the expression of the trast to the general toxicity pattern of chemotherapy immediate early gene FOS by quantitative reverse transcrip- regimens which are more toxic to low-differentiated tumor tion-PCR. RNA expression of CASP8 stayed unchanged cells (3), a higher protoporphyrin IX content in differenti- whereas DUSP1 RNA was up-regulated in normal and tumor ated compared with undifferentiated tumor cells has been cells starting 30 minutes after irradiation. In contrast, FOS found (4). Due to the preferential protoporphyrin IX generation in tumor cells compared with normal cells after 5-aminolevulinic acid exposure, protoporphyrin IX phototoxicity is limited mainly to tumors cells, providing Received 6/7/04; revised 1/15/05; accepted 2/3/05. a tumor-selective treatment modality. Endoscopically Grant support: Deutsche Krebshilfe grant (R. Knuechel). accessible malignant lesions of the urinary bladder or The costs of publication of this article were defrayed in part by the the gastrointestinal tract have been successfully treated in payment of page charges. This article must therefore be hereby marked clinical trials (5, 6). Attempts have been made to further advertisement in accordance with 18 U.S.C. Section 1734 solely to optimize this method by derivatization of 5-aminolevulinic indicate this fact. acid. The velocity of 5-aminolevulinic acid uptake has been Note: P.J. Wild and R.C. Krieg contributed equally to this work. shown to increase with esterification of 5-aminolevulinic Requests for reprints: Ruth Knuechel, Institute of Pathology, University of Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany. Phone: 49-241- acid, especially by using 5-aminolevulinic acid hexyl or 8089280; Fax: 49-941-944-6634. E-mail: [email protected] benzylester derivatives (7). However, mechanisms leading Copyright C 2005 American Association for Cancer Research. to preferential protoporphyrin IX accumulation of tumor

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Figure 1. A, schematic representation of photodynamic diagnosis (PDD) and therapy (PDT) with 5-aminolevulinic acid (ALA) – induced protoporphyrin IX (PPIX) for the treatment of carcinoma in situ. B, fluorescence histology (Â100) of a papillary noninvasive low-grade bladder tumor after photodynamic diagnosis with 5-aminolevulinic acid in vivo; only the tumor shows red fluorescence while the adjacent normal urothelium is negative (white arrow). C, carcinoma in situ of the urinary bladder with complete ablation of atypical cells after photodynamic therapy with 5-aminolevulinic acid in vivo (black arrow); remaining suburothelial tissue shows chronic inflammation (HE, Â40).

cells after 5-aminolevulinic acid incubation are still not rectal adenocarcinoma (12), were used. Cell lines were known for sure, but assumed to be due to a different maintained as monolayer cultures in RPMI 1640 (Bio- heme metabolism in the tumor compared with normal chrom, Berlin, Germany) and HT29 in DMEM (PAN tissue. This could already be shown for different organs BIOTECH GmbH, Aidenbach, Germany) without phenol in vitro (8, 9). red supplemented with 5% FCS (PAN BIOTECH), 1% (v/v) Because traditional therapy for endoscopically accessi- L-glutamine, and 1% (v/v) sodium pyruvate (Gibco, ble precancerous lesions (e.g., carcinoma in situ of the Eggenstein, Germany) at 37jC in a humidified atmosphere, bladder, dysplasia of Barrett’s esophagus) and early-stage containing 5% carbon dioxide. Cells were detached for tumors is still insufficient, new treatment modalities subculturing and experiments using 0.05% trypsin/0.02% are urgently needed. Understanding the mechanism of EDTA (Gibco) in PBS (Biochrom). All experiments were tumor-selective phototoxicity of photodynamic therapy done using plateau phase cells. To reach plateau phase with 5-aminolevulinic acid may also provide a basis for growth, defined cell densities were seeded into culture combinatory therapy regimens. Oligonucleotide micro- dishes and allowed to grow for the times indicated: RT4: arrays, analyzing the RNA expression level of thousands 22,000 cells/cm2, 9 days; UROtsa: 5,600 cells/cm2, 10 days; of genes simultaneously, are promising for gaining insight HT29: 50,000 cells/cm2, 7 days. into the complex pathway of cellular damage. To screen PhotodynamicTherapy and Cell Harvest for molecules indicating the mechanisms of protopor- Stock solutions of 5-aminolevulinic acid (Synopharm, phyrin IX–mediated toxicity, an in vitro study with one Barsbu¨ ttel, Germany) were prepared in deionized water human normal cell line (bladder) and two human cancer (10 mg/mL) and stored at À20jC. For each experiment the cell lines (bladder and colon) was carried out. Cells were stock solution was diluted in culture medium without FCS. subjected to photodynamic therapy with cell-specific Before 5-aminolevulinic acid incubation, the culture me- LD50 values and harvested for array analysis 0, 10, and dium was removed and the cell monolayer was rinsed with 30 minutes after photodynamic therapy. PBS to remove remaining FCS. Cells were incubated with 5- aminolevulinic acid at a final concentration of 100 Ag/mL Materials and Methods in culture medium without FCS for 3 hours. A total volume Cell Lines and Culturing of 250 AL per square centimeter of growth area was used. In RT4 cells (well-differentiated bladder tumor cells), origi- subsequent handling, care was taken to avoid exposure of nally derived from a recurrent papillary G1 tumor (10), the cells to ambient light. After the incubation period, the 5- UROtsa cells (11), derived from normal urothelium and aminolevulinic acid solution was removed and fresh immortalized by a temperature-sensitive SV40 large T- medium without FCS was added. Each cell line was then antigen gene construct (provided by Dr. J.R.W. Masters, illuminated with a specific light dose leading to a cell University College, London, United Kingdom), and mode- survival of 50% (LD50). In vitro photodynamic therapy rately differentiated (G2) HT29, established from a colo- was done using a high-pressure Xenon arc lamp (Karl Storz

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GmbH, Tuttlingen, Germany). The wavelength ranges, using suitable instrument settings (RT4 and UROtsa: FL1— overall light doses, and power densities were as follows: monomer 310 V, FL2—aggregate 259 V, FL1—%FL2 2.0, RT4: 400 to 700 nm, 0.8 J cmÀ2,50mWcmÀ2; UROtsa: 400 to FL2—%FL1 20.9; HT29: FL1—monomer 370 V, FL2— 700 nm, 1.5 J cmÀ2,50mWcmÀ2; and HT29: 590 to 700 nm, aggregate 279 V, FL1—%FL2 4.0, FL2—%FL1 5.1). Debris 3.9 J cmÀ2,40mWcmÀ2. After photodynamic treatment, was excluded from further analysis by gating for forward medium without FCS was removed and fresh culture versus right angle light scatter. medium with FCS was added. Zero, 10, and 30 minutes Statistical Analyses after photodynamic therapy the cellular monolayers were The two-sided Mann-Whitney U test was used to briefly rinsed with PBS and cells were lysed with guani- compare results of cell membrane integrity and mitochon- dium thiocyanate buffer (provided with the PolyATract drial activity at certain time points. P values < 0.05 were System 1000, Promega, Heidelberg, Germany) to stop all considered statistically significant. Statistical analyses were biological activity and to protect RNA integrity. completed using SPSS version 10.0 (SPSS, Chicago, IL). Preliminary Toxicity Testing Chip Design To study structural and functional changes of photody- The metg001A GeneChip (metaGen Pharmaceuticals namic therapy–treated cells, cell membrane integrity and GmbH, Berlin, Germany) consists of 6,117 probe sets mitochondrial activity were selected as variables as already representing roughly 3,000 human genes based on the described by Seidl et al. (13). Whereas the plasma mem- annotation of the probe sets with the GoldenPath assem- brane is the main site of sensitizer localization, protopor- bly.8 We selected two different sets of genes: first, genes phyrin IX generation is located in mitochondria. Phototoxic known as involved in the progression of human tumors, effects were analyzed 0 to 24 hours after photodynamic including genes from several signal transduction pathways therapy (0.5, 1, 2, 3, and 24 hours), with 0 hour after (e.g., TGFB, RAS, and WNT) and androgen receptor– photodynamic therapy serving as untreated control. regulated genes; and second, cDNA fragments differentially Plasma Membrane Integrity expressed in silico [i.e., Schmitt et al. (14) have systemati- Cells seeded into six-well dishes were used for flow cally screened whole expressed sequence tag (EST) libraries cytometric analysis of membrane integrity by using for genes differentially expressed in normal and tumor propidium iodide (Sigma, Deisenhofen, Germany) exclu- tissues]. sion of viable cells. After 5-aminolevulinic acid incubation Gene Expression Profiling and illumination (see above), cells were detached, spun Poly(A)+ RNA was isolated from the cells by magnetic down at 200 Â g, and resuspended in 1 mL PBS. From separation (PolyATract System 1000, Promega) according to a stock solution of propidium iodide (1 mg/mL in PBS), the recommendations of the manufacturer. Linear amplifi- 3 AL were added to the cell suspension to a final con- cation (two rounds) was done as described previously (15). centration of 3 Ag/mL. After a few minutes of incubation In brief, after priming with the Affymetrix T7-oligo-dT (3–5 minutes at room temperature), the cell suspension promoter-primer combination (5V-GGC CAG TGA ATT was immediately measured on a FACSCalibur flow GTA ATA CGA CTC ACT ATA GGG AGG CGG T24-3V cytometer. The data were gated for forward versus right at 100 mmol/L), first and second strand synthesis, and angle light scatter and the percentage of cells showing high in vitro transcription, the amplified RNA was again red propidium iodide fluorescence (measured in channel amplified in one subsequent round of cDNA synthesis FL3, 650 nm lp) was determined. and in vitro transcription. Within the last in vitro transcrip- Mitochondrial Activity tion, biotin-labeled nucleotides were incorporated into For the determination of mitochondrial activity and their the amplified RNA. Hybridization and detection of the inner membrane potential, cells were seeded into six-well labeled amplified RNA on the metg001A Affymetrix culture dishes as described above. A stock solution of GeneChip was done once according to the instructions of 5,5V,6,6V-tetrachloro-1,1V,3,3V-tetraethyl-benzimidazolyl-car- the manufacturer. bocyanine iodide (JC-1; Molecular Probes, Leiden, the Data Processing Netherlands) of 500 Ag/mL in 70% methanol was prepared. GeneChips were scanned using an Agilent GeneArray JC-1 accumulates in mitochondria and forms multimeres Scanner (Agilent Technologies, Inc., Palo Alto, CA) and (J aggregates) depending on the potential across the inner processed as described (16). In brief, raw intensity values mitochondrial membrane. Detached cells were centrifuged were extracted from the Cel files and a background at 200 Â g, the supernatant was removed, cells were correction was performed. The background-corrected resuspended in 1 mL PBS, and 10 AL of the stock solution probe intensity values were normalized by dividing them were added to a final concentration of 5 Ag/mL. After a 15- by the median value of all probes. A representative minute incubation at room temperature, the cell suspension expression value for each probe set (PMQ value) was was measured without washing on a FACSCalibur flow generated by using the 75% percentile of the perfect match cytometer. After 488 nm laser excitation the fluorescence of intensities. PMQ values (0, 10, and 30 minutes) of the monomers (emission maximum, 527 nm) and aggregates three cell lines were compared with the 5-aminolevulinic (emission maximum, 590 nm) was separated and detected in the FL1 (530/30 nm bp) and FL2 (585/42 nm bp) photomultiplier tubes on a FACSCalibur flow cytometer 8 http://genome.ucsc.edu/

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acid–incubated control (without irradiation) by calculating change folds for each gene. A P value (Wilcoxon rank test) smaller than 0.05 was mandatory for a gene to be considered expressed and therefore included into analysis. Z scores larger than 3 were classified as significant changes in gene expression. Change folds were then ranked in descending order, resulting in a list of 40 genes. Genes that were coexpressed at different time points after photodynamic therapy were of highest interest. To obtain clusters of related expression patterns among the 40 genes, an algorithm based on a self-organizing hierarchical neural network [self-organizing tree algorithm (SOTA)] was applied9 (refs. 17, 18). The comparison was based on a Pearson correlation coefficient. Additionally, hierarchical clustering with a linear correlation metrics was used. For each gene, the gene ontology biological process annotation according to the Gene Ontology Consortium10 was listed (19). Quantitative ReverseTranscription-PCR For verification of the differentially expressed cDNAs, quantitative reverse transcription-PCR analysis was done measuring duplicates of each cDNA. HDAC1 was used as housekeeping gene (20). First-strand cDNA was synthe- sized using 2 Ag of the isolated total RNA of photodynamic therapy–treated cells after 0, 15, and 30 minutes, 1 Agof random primer (Amersham Pharmacia Biotech, Frankfurt, Germany), 4 ALof5Â First Strand Buffer (Gibco), 2 ALof DTT 10 mmol/L, 1 AL of deoxynucleotide triphosphates (10 mmol/L), and 1 AL of Superscript Plus (Gibco) in a total volume of 20 AL. To quantify the expression of cDNAs, a LightCycler real- time PCR system (Roche, Mannheim, Germany) was used. For the RT-PCR, 1 to 3 AL of cDNA, 0.5 to 2.4 ALof 25 mmol/L MgCl2, 0.5 Amol/L of forward and reverse primers, and 2 AL of LightCycler-FastStart DNA Master SYBR Green I (Roche) in a total volume of 20 AL were applied. The following PCR program was done: 5 minutes j Figure 2. Structural and functional analysis of RT4 and UROtsa cell lines 95 C (initial denaturation), temperature transition rate after photodynamic therapy (LD50) with 5-aminolevulinic acid in vitro. A, 20jC/s, 95jC for 15 seconds, 10 seconds 58jC (annealing membrane integrity; percentage of membrane damaged cells after photo- temperature), 10 seconds 72jC, acquisition mode single, dynamic therapy; *, significant Mann-Whitney U test at 24 h. B, mitochondrial activity, percentage of mitochondrial damaged cells after repeated for 50 times (amplification). MgCl2 concentration photodynamic therapy; ns, nonsignificant Mann-Whitney U test at 24 h. was optimized for each primer set. Primer characteristics (name and 5V-3V sequence) are listed below. The PCR reaction was evaluated by melting curve analysis (0 second 95jC, 15 seconds 50jC, temperature j j HDAC1 f ACC GAA AAA TGG AAA TCT ATC transition rate 0.1 C/s up to 95 C) following the instruc- HDAC1 r AGT CCT CAC CAA CGT TGA ATC tions of the manufacturer and detecting the PCR products DUSP1 f GCG CAA GTC TTC TTC CTC AA on 1.8% agarose gels. The LightCycler Relative Quanti- DUSP1 r CGC TGT CAG GGA CGC TAG TA fication Software (Roche) was used for determining CASP8 f GGA GAT GGA AAG GGA ACT TCA calibrator-normalized target/reference ratios with auto- CASP8 r ATC CAG CAG GTT CAT GTC ATC mated efficiency-corrected quantification according to the FOS f TCT GCT TTG CAG ACC GAG ATT GCC recommendations of the manufacturer. FOS r CCA CTG AGG GCT TGG GCT CAG GGT Western Blot Analysis After photodynamic therapy, cells were washed twice with ice-cold PBS buffer and lysed in radioimmunoprecipi- tation assay buffer (50 mmol/L Tris pH 7.5, 150 mmol/L 9 http://bioinfo.cnio.es/Sotarray/ NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and a 10 http://www.geneontology.org protease inhibitor cocktail; Roche) for 15 minutes on ice.

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Insoluble fragments were removed by centrifugation at noprecipitation assay cell lysate were loaded per lane, 13,000 rpm for 10 minutes and the supernatant lysate was separated on SDS polyacrylamide gradient gels (Invitrogen immediately shock frozen and stored at À80jC. The protein GmbH, Karlsruhe, Germany), and subsequently blotted concentration of the cell lysate was determined using the onto a polyvinylidene difluoride membrane (Roche). Blots bicinchoninic acid protein assay reagent (Pierce Biotechno- for detection of caspase-8, caspase 3, dual specificity logy, Inc., Rockford, IL). Forty micrograms of radioimmu- phosphatase 1 (DUSP1), and h-actin were blocked with

Figure 3. A, dendrogram using hierarchical clustering with a linear correlation metrics. The colored matrix represents the results of clustering microarray experiments. In the matrix each row represents a gene, and each column represents the expression of that gene in a particular microarray hybridization. Photodynamic therapy – mediated experiments are displayed in the following order: HT29 0 min, HT29 10 min, HT29 30 min, RT4 0 min, RT4 30 min, UROtsa 0 min, UROtsa 10 min, UROtsa 30 min. The color scale for drawing profiles ranges from blue to light red. B, dendrogram using SOTA algorithm with a vari- ability threshold of 48.5%. The size of the ratio of circles is proportional to the amount of genes in that particular cluster. The regulation patterns of the cluster appear on the right of the circles as histograms. Photodynamic therapy – mediated experiments are displayed in the following order: HT29 0 min, HT29 10 min, HT29 30 min, RT4 0 min, RT4 30 min, UROtsa 0 min, UROtsa 10 min, UROtsa 30 min. Clustered genes are in identical order in A and B (see Table 2). FOS, DUSP1, and CASP8 were selected (arrows) for further validation of the array findings by quantitative real-time RT-PCR.

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Table 1. Clustering of expression microarray experiments using the SotaTree Server, and gene ontology annotation (biological process)

SOTA UniGene Sequence Symbol Name Gene ontology biological cluster cluster accession ID process according to the Gene Ontology Consortium*

1 Hs.410900 NM_181353 ID1 inhibitor of DNA binding 1, regulation of transcription dominant negative from Pol II promotor, helix-loop-helix protein development Hs.296638 NM_004864 GDF15 prostate differentiation factor cell-cell signaling, transforming growth factor h receptor signaling pathway, signal transduction Hs.25647 NM_005252 FOS v-fos FBJ murine regulation of transcription from Pol II osteosarcoma viral promotor, cell growth and/or oncogene homologue maintenance, DNA methylation, inflammatory response — EST, AP000318.1 2 Hs.331202 NM_172178 MRPL42 mitochondrial ribosomal protein biosynthesis protein L42 Hs.356721 NM_031157 HNRPA1 heterogeneous nuclear ribonucleoprotein A1 3 Hs.81665 NM_000222 KIT v-kit Hardy-Zuckerman 4 protein amino acid de-/phosphorylation, feline sarcoma viral transmembrane receptor protein oncogene homologue tyrosine kinase activity/signaling pathway, cell growth and/or maintenance, signal transduction 4 Hs.384944 NM_000636 SOD2 superoxide dismutase 2, response to oxidative stress, mitochondrial superoxide metabolism Hs.166254 NM_030938 VMP1 likely orthologue of rat vacuole membrane protein 1 Hs.127811 Homo sapiens cDNA FLJ43544 fis, clone PROST2009388 5 Hs.287558 NM_000700 ANXA1 annexin A1 cell motility, lipid metabolism, cell surface receptor linked signal transduction, inflammatory response Hs.232400 NM_031243 HNRPA2B1 heterogeneous nuclear RNA processing ribonucleoprotein A2/B1 Hs.71465 NM_003129 SQLE squalene epoxidase sterol biosynthesis, electron transport, aromatic compound metabolism 6 Hs.326035 NM_001964 EGR1 early growth response 1 regulation of transcription Hs.171695 NM_004417 DUSP1 dual specificity phosphatase 1 protein amino acid dephosphorylation, response to oxidative stress, cell cycle 7 — EST, AP000349.1 8 Hs.6686 NM_030800 hypothetical DKFZp564O1664 protein Hs.374854 NM_173852 KRTCAP2 keratinocytes associated protein 2 Hs.297304 NM_152932 AD-017 glycosyltransferase AD-017 carbohydrate biosynthesis Hs.243491 NM_033357 CASP8 caspase 8, apoptosis-related apoptotic program, proteolysis cysteine protease and peptidolysis Hs.464708 — EST, BF001177.1 — 18S rRNA — 18S rRNA — EST, AC004547 9 Hs.446678 NM_006540 NCOA2 nuclear receptor coactivator 2 signal transduction, regulation of transcription Hs.406114 NM_018011 hypothetical protein FLJ10154 Hs.14891 NM_003953 MPZL1 myelin protein zero-like 1 transmembrane receptor protein tyrosine kinase signaling pathway, cell-cell signaling

* http://www.geneontology.org Continued on following page

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Table 1. Clustering of expression microarray experiments using the SotaTree Server, and gene ontology annotation (biological process) (Cont’d)

SOTA UniGene Sequence Symbol Name Gene ontology biological cluster cluster accession ID process according to the Gene Ontology Consortium*

— EST, LOC188788 — EST, AC005036.1 10 Hs.357637 NM_001439 EXTL2 exostoses (multiple)-like 2 Hs.130293 NM_016424 LUC7A cisplatin resistance–associated RNA splicing overexpressed protein 11 Hs.64056 NM_002576 PAK1 p21/Cdc42/Rac1-activated c-jun-NH2-kinase cascade, kinase 1 (STE20 protein amino acid phosphorylation homologue, yeast) Hs.194679 NM_003881 WISP2 WNT1 inducible signaling cell-cell signaling, regulation of cell pathway protein 2 growth, cell adhesion, signal transduction Hs.286192 NM_032192 PPP1R1B protein phosphatase 1, regulatory signal transduction (inhibitor) subunit 1B (DARPP-32) Hs.79136 NM_012319 SLC39A6 solute carrier family 39 metal ion transport (zinc transporter), member 6 12 Hs.737 NM_004907 ETR101 immediate early protein Hs.2256 NM_002423 MMP7 matrix metalloproteinase 7 collagen catabolism (matrilysin, uterine) 13 — 28S rRNA 14 Hs.79474 NM_006761 YWHAE tyrosine 3-monooxygenase/ intracellular signaling cascade tryptophan 5-monooxygenase activation protein, epsilon polypeptide 15 Hs.446546 NM_133436 ASNS asparagine synthetase asparagine biosynthesis, metabolism, glutamine metabolism

5% nonfat dry milk in TBS with 0.5% Tween 20 (TBST) Preliminary Toxicity Testing for 1 hour at room temperature and then incubated for Irradiation with LD50 was based on the assumption 16 hours at 4jC with the following primary antibodies: that intracellular alterations are determined by differen- anti-caspase-8 (1:1,000, Cell Signaling, Beverly, MA), anti- tial gene expression, and not completely covered by fast caspase 3 (1:3,000, BD Transduction Laboratories, San Jose, necrotic cell death. Of note, LD50 for UROtsa cells was CA), anti-DUSP1 (1:1,000, Santa Cruz Biotechnology, identical to LD100 for RT4, given the same incubation Inc., Santa Cruz, CA), and anti–h-actin (1:5,000, Sigma). conditions. Before defining time points for RNA isolation The membranes were then washed in TBST or PBS, after photodynamic therapy, mitochondrial activity and incubated for 1 hour at room temperature with alkaline membrane integrity were determined by flow cytometry phosphotase–conjugated antibodies, and diluted in block- over 24 hours using urothelial cell lines (UROtsa and ing buffer. Finally, immunoreactions were visualized by RT4). The trend of the two variables investigated for the nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phos- two cell lines is shown in Fig. 2A and B. phate (Zymed Laboratories, Inc., South San Francisco, Membrane Integrity CA) staining. As a positive control for the induction of RT4 and UROtsa showed a clear reduction of viable apoptosis, cells were cultured in 5 mL medium with 5% cells within 24 hours. Whereas RT4 cells reached their FCS and treated with 1 Amol/L staurosporine for 3 hours. level of maximum membrane damage 3 hours after photodynamic therapy, the percentage of dead or propi- Results dium iodide–positive UROtsa cells increased until the To understand differential cell death response to photody- end of the experimental period. Furthermore, no recovery namic therapy and the mechanism of toxicity, a normal cell of membrane integrity was detectable within 24 hours line (UROtsa, urothelial) and two tumor cell lines (RT4, after treatment. This is in agreement with earlier experi- urothelial; HT29, colonic) were treated with LD50 doses of ments, indicating that the rate of membrane damage in light after exposure to 5-aminolevulinic acid, and harvested urothelial cells increased up to 48 hours or even later for RNA extraction 0, 10, and 30 minutes after irradiation. after irradiation (13). At 24 hours after photodynamic The gene expression profiles between the different samples therapy, the percentage of dead UROtsa cells was signi- were compared by Affymetrix GeneChip analysis, focusing ficantly lower than the percentage of RT4 cells (P = 0.03; on cancer-related and growth regulatory targets. Fig. 2A).

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harvest were selected for RNA expression analysis after incubation with 5-aminolevulinic acid (100 Ag/mL for 3 hours) 0, 10, and 30 minutes after irradiation with LD50. RNA Expression after PhotodynamicTherapy The simultaneous use of these three cell lines not only enabled the comparison of tumor and normal cells within a particular organ but also allowed the comparison of tumor cells of different origin (bladder versus colon). Comparing the gene expression profiles between the different samples, we identified 40 significantly altered genes. Correlation in gene expression patterns among the regulated genes was evaluated by hierarchical and SOTA clustering, respectively (Fig. 3A and B). The data set of 40 genes was reduced to only 15 clusters of genes, which are listed in Table 1. For each gene the UniGene code, the sequence accession identification number, the gene symbol, and the gene ontology annotation (biological process) are listed.10 Comparing the gene ontology annotations in each gene cluster, gene products could not be grouped according their biological process. However, genes involved in the same biological process were regulated after photodynamic therapy in vitro [Table 1; e.g., response to oxidative stress (DUSP1 and SOD2), signal transduction (GDF15, KIT, NCOA2,and WISP2), and inflammatory response (FOS and ANXA1)]. Interestingly, LUC7A RNA was down-regulated in the colon cancer cell line HT29 after photodynamic therapy and not regulated in the two bladder cancer cell lines (Fig. 4A). The related protein was cloned from cisplatin- resistant cell lines by differential display, and was therefore designated cisplatin resistance–associated protein (21). In response to oxidative stress, superoxide dismutase 2 (SOD2) was up-regulated in RT4 cells, not regulated in HT29, and slightly down-regulated in UROtsa (Fig. 4B). Further- more, the immediate early gene ETR101 (22) was up- regulated after 30 minutes in HT29, but down-regulated in RT4 (Fig. 4C). The matrix metalloproteinase 7 (MMP7) was not regulated in any of the three cell lines after photodynamic therapy (Fig. 4D). Figure 4. Gene expression (single PMQ values, normalization to control) Validation of Expression Profiles by Quantitative Re- of LUC7A (A), SOD2 (B), ETR101 (C), and MMP7 (D)following photodynamic therapy with 5-aminolevulinic acid. Blue rhombuses, verseTranscription-PCR HT29; red squares, RT4; green triangles, UROtsa. Quantitative RT-PCR supported the results of the oligonucleotide arrays. In detail, FOS, DUSP1,and Mitochondrial Activity CASP8 were selected for further validation of the array RT4 and UROtsa showed an increasing number of cells findings (Fig. 5A–C). Expression of the immediate early with loss of mitochondrial function within the first few gene FOS was continuously up-regulated over 30 minutes hours after photodynamic therapy. Maximum mitochon- in all cell lines, whereas DUSP1 mRNA was up-regulated drial damage was visible 2 to 3 hours after photodynamic in normal and tumor cells starting 30 minutes after therapy for both cell lines, with a maximum rate of f50%. irradiation. DUSP1 is known as induced by oxidative and A beginning but nonsignificant (P = 0.2) recovery process heat stress (23). In contrast, CASP8 mRNA was found from photodamage could be observed in UROtsa cells at unaltered in the three cell lines over time, comparing array 24 hours after photodynamic therapy. In contrast, the and RT-PCR results. The related caspase-8 protein is a percentage of RT4 cells with damaged mitochondria protease involved in the FAS/APO1- and p55 tumor remained at maximum until the end of the experimental necrosis factor receptor–induced signaling cascades (24). period (Fig. 2B). The initial down-regulation of CASP8 RNA at 0 minute With the maximum mitochondrial damage being after photodynamic therapy in UROtsa cells could not be reached after 2 to 3 hours, shorter periods for cell confirmed in independent RT-PCR runs (Fig. 5C).

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Figure 5. Comparison of array results (single PMQ values, normalization to control; blue lines) with LightCycler quantitative RT-PCR experiments (mean calibrator-normalized target/reference ratios, normalization to control; red lines) regarding FOS (A), DUSP1 (B), and CASP8 (C). Bar, SD of quantitative RT-PCR results.

Western Blot Analysis photodynamic therapy are dependent on the photosensi- In contrast to the DUSP1 gene expression data, induction tizer and its localization in the cell, the illumination of DUSP1 protein expression was only shown after 1 hour conditions (25, 26), the oxygenation status of the tissue, in UROtsa cells (Fig. 6). The expression levels of DUSP1 and the type of cells (27–29) involved. The involvement of protein in the HT29 and RT4 cell lines were obviously different subcellular targets, such as mitochondria, lyso- not high enough to be detected by Western blot analysis. somes, and the plasma membrane, in cell death and Using relative quantification, DUSP1 expression was survival is reviewed in detail by Moor (30). Cells can highest in UROtsa cells after normalization to DUSP1 respond to photodynamic therapy by initiating a rescue expression in HT29 cells (data not shown). For caspase-8, response and/or by undergoing cell death either in an no changes in protein expression but activation of catalytic apoptotic or in a necrotic fashion (2, 31–33). Signaling activity was detected only in UROtsa cells starting 1 hour pathways influenced by photodynamic therapy have not after photodynamic therapy, whereas no changes were been fully elucidated although a number of studies have seen in both tumor cell lines. In contrast to UROtsa cells, addressed this issue. However, the interpretation of the enforcement of apoptosis by use of staurosporin was data has been complicated due to different models used absent in HT29 and weak in RT4 cells. Because caspase 3 and the utilization of many different sensitizers. is considered another important effector in apoptosis, Photodynamic therapy in vitro with LD50 was based on caspase 3 activation was characterized after photodyna- the assumption that intracellular alterations are determined mic therapy in vitro (Fig. 7) using Western blot analysis. by differential gene expression rather than by fast necrotic According to caspase-8, the active caspase 3 fragment cell death. Besides the light dosage, the time point of cell (17 kDa) was found only in the normal urothelial cell line harvest after the applied stimulus is crucial for gaining (UROtsa) starting 1 hour after photodynamic therapy. insight into the cellular response after photodynamic Data on differential toxicity between normal and tumor therapy. Before defining time points for RNA isolation cells indicate that photodynamic therapy in vitro (LD50) after photodynamic therapy, mitochondrial activity and leads to apoptosis in UROtsa and to necrosis in the tumor membrane integrity following photodynamic therapy cell lines (HT29 and RT4), respectively. in vitro (LD50) have been quantified by flow cytometry over 24 hours (13, 34). With the maximum mitochondrial damage being reached after 2 to 3 hours, the time points 0, Discussion 10, and 30 minutes after photodynamic therapy were Photodynamic therapy is an experimental treatment mo- considered optimal for cell harvest, evaluating photody- dality which is under development for application in both namic therapy–dependent fast cellular response on the neoplastic and nonneoplastic diseases (2). Responses to RNA level.

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ETR101 is another immediate early gene, of which mRNA levels were up-regulated in HT29 cells after photodynamic therapy and down-regulated in RT4 30 minutes after photodynamic therapy (Fig. 4C). As shown by Shimizu et al. (22), ETR101 was expressed on induction by 12-O-tetradecanoylphorbol-13-acetate in the human leukemia cell line HL60. Other proteins involved in cellular stress response have been shown to be induced after photodynamic therapy (30); for instance, heat shock protein 1 was found to be phosphorylated after photodynamic therapy of mouse lymphoma L5178Y cells with phthalocyanine Pc 4 (37). In our study, DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after illumination (Fig. 4B). DUSP1 is known as induced by oxidative and heat stress (23). Induction of DUSP1 protein expression was clearly shown after 1 hour in UROtsa cells. With relative expression levels of DUSP1 RNA after photodynamic therapy being significantly lower for tumor cells (RT4 and HT29), the expression levels for DUSP1 protein in the tumor cell lines were obviously not high enough to be detected by Western blot analysis. SOD2 was up-regulated in RT4 cells (Fig. 3B), not regulated in HT29, and slightly down-regulated in UROtsa. Golab et al. (38) have shown in vitro that 2-methoxyestradiol, an inhibitor of SODs, is capable of potentiating the antitumor effects of photodynamic therapy. The observed differential SOD2 gene expression after photodynamic therapy provides a rationale for the clinical use of SOD inhibitors. Figure 6. Protein expression of CASP8, DUSP1, and ACTB in RT4 (A), The simultaneous use of the three cell lines, HT29, HT29 (B), and UROtsa (C) cells at certain time points after photodynamic RT4, and UROtsa, not only enabled the comparison of therapy in vitro. As a positive control for the induction of apoptosis, cells tumor and normal cells but also allowed the comparison were treated with staurosporine. of tumor cells of different origin (bladder versus colon). Interestingly, LUC7A RNA was down-regulated in the colon We selected FOS, DUSP1,andCASP8 for further cancer cell line HT29 after photodynamic therapy and validation of the array findings by quantitative real-time not regulated in the two bladder cancer cell lines (Fig. 4A). RT-PCR. The increased photodynamic therapy–induced The LUC7A protein was cloned by Nishii et al. (21) from expression of the immediate early gene FOS in all three cell cisplatin-resistant cell lines by differential display, and lines is in accordance with gene expression results of was therefore designated cisplatin resistance–associated Verwanger et al. (35) who have already reported gene expression patterns following photodynamic therapy with endogenous protoporphyrin IX of the squamous cell carcinoma cell line A-431. Verwanger and colleagues have shown increased expression of the heat shock protein 70 and the immediate early genes FOS and JUN. Accordingly, Luna et al. (36) have shown the photodynamic therapy– mediated induction of the early response gene FOS through protein kinase–mediated signal transduction pathways. In agreement, we found continuous up-regulation of FOS in the normal and the two tumor cell lines. FOS and JUN play a role in cell proliferation, apoptosis, and stress response (35). The JUN and FOS proteins form, together with activating transcription factor, the activator protein-1 transcription factor complex, which binds to a common DNA site, the activator protein-1 binding site. Activator protein-1 has a function in apoptosis modulation, Figure 7. Protein expression of CASP3 in RT4, HT29, and UROtsa cells at cell proliferation, and cell survival. certain time points after photodynamic therapy in vitro.

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protein. The fact that cisplatin resistance was not induced in vitro (LD50) may lead to apoptosis in UROtsa and after photodynamic therapy may provide a basis for com- to necrosis in the tumor cell lines, respectively. According to binatory therapy regimens. Lottner et al. (39) have already caspase-8, the active caspase 3 fragment (17 kDa) was found combined the cytostatic activity of cisplatin/oxaliplatin only in the normal urothelial cell line (UROtsa), starting 1 hour and the photodynamic effect of hematoporphyrin in after photodynamic therapy. Cleavage of pro-caspase 3 after the same molecule. Synergistic antiproliferative effects photodynamic therapy with 5-aminolevulinic acid has of hematoporphyrin-platinum(II) conjugates were found already been reported by Grebenova et al. (44) in HL60 in vitro against J82 bladder cancer cells and UROtsa. leukemia cells. Of note, apoptosis can principally be Ferrario et al. (40) have evaluated the role of Photofrin- induced in the two cancer cell lines using bile salts for mediated photodynamic therapy in eliciting expression of HT29 (45) and Adriamycin for RT4 cells (46). MMPs in a mouse mammary tumor model. Administration Proskuryakov et al. (47) have described necrosis as of the MMP inhibitor prinomastat significantly improved another specific form of programmed cell death, with photodynamic therapy–mediated tumor response, linking mitochondria being the key players in determination of the our gene expression results to anticancer drugs. Prinoma- pathway of cell suicide. Gene expression profiling is not the stat is an anticancer drug and belongs to the group of method of first choice to investigate the molecular scenario angiogenesis inhibitors. Immunohistochemical analyses of necrotic cell death, as reviewed by Proskuryakov et al. indicated that infiltrating inflammatory and endothelial (47). Many of the players are not regulated on the gene cells were the primary source of MMP expression. Accor- expression level but are a matter of faster cellular reactions ding to our in vitro results, Ferrario et al. (40) observed (massive DNA breaks, rapid efflux of cell constituents into negligible expression of MMPs in tumor cells. extracellular space, etc.). In our preliminary tests to define Photodynamic therapy can trigger both modes of cell time points for cell harvest, an initial but nonsignificant death, apoptosis and necrosis (41). For complete tumor (P = 0.2) mitochondrial recovery process from photo- eradication the desired apoptosis/necrosis ratio should be damage could be observed in UROtsa cells (Fig. 2B). In adjusted. In case of cancer therapy for instance, photody- contrast, the percentage of RT4 cells with damaged mito- namic therapy causing necrotic cell death is preferred chondria remained at maximum until the end of the expe- because the immune reaction elicited by the inflammation rimental period. Levels of maximum damage were reached (Fig. 1C) secondary to tumor necrosis could be useful in within the first 2 to 3 hours. However, even in vitro, killing additional tumor cells. As previously reported for apoptosis finally leads to plasma membrane permeabili- other apoptotic stimuli (26), the type of cell death induced zation (‘‘secondary’’ necrosis; Fig. 2A), but it does not by photodynamic therapy switches from apoptosis to occur in vivo because apoptotic cells are digested by necrosis with the increase of the intensity of the insult macrophages or by surrounding cells before their plasma (42). According to Almeida et al. (42), who have reviewed membrane becomes disrupted (47). intracellular signaling mechanisms in photodynamic the- Another gene found to be regulated following photo- rapy, two major apoptotic pathways have been characte- dynamic therapy encoded for GDF15 (PLAB). The cell line rized, the death receptor mediated and the mitochondria UROtsa with apoptotic behavior following photodynamic mediated. In both pathways, the activation of initiator therapy showed no regulation of this gene. RT4 as well as caspases (caspase-8 or caspase 9) leads to the activation of HT29 with a clear necrotic response to photodynamic effector caspases (caspase 3, caspase 6, and caspase 7). To therapy showed a strong activation of GDF15 RNA be able to compare the intracellular signaling mechanisms expression levels. The corresponding protein growth and in normal and tumor cells, both were treated with their differentiation factor 15 (GDF15) is an interesting factor in individual LD50. Given the same incubation conditions, cellular response to injuries and seems to be expressed in LD50 for UROtsa cells would be identical to LD100 for the an organ-independent manner. Subramaniam et al. found tumor cell lines. GDF15 levels to be overexpressed in neurons following RNA expression of CASP8 was unchanged (Fig. 5C). The injury. Furthermore, they analyzed GDF15 to inhibit related protein is a protease involved in apoptosis (24). For apoptosis by direct interference with AKT and extracellu- caspase-8 protein, strong but delayed activation of catalytic lar signal–regulated kinase. This raises the question activity was detected only in UROtsa cells starting 1 hour whether or not strong apoptotic stimuli might then result after treatment, whereas no changes were seen in both in necrosis. Hsiao et al. (49) and his group were able to tumor cell lines (Fig. 6). In agreement with Granville et al. show a strong up-regulation of GDF15 in liver following (43), a delayed activation of CASP8 was shown in UROtsa chemical and surgical trauma both in vivo and in vitro. cells, concluding that activation of the CASP8 pathway may GDF5, another member of the GDF family, is known to serve as a secondary way for the cell to ensure demise in enhance bone repair in vivo when administered following case of damage. Accordingly, enforcement of apoptosis by a per se necrotic traumatic stimulus (50, 51). In general, use of staurosporin resulted in strong expression of GDF15 seems to be an important factor which is expressed activated caspase-8 protein in UROtsa cells (Fig. 6). With after a severe deadly stimulus as a cellular response and in HT29 and RT4 cells showing no expression of activated attempt to survive. caspase-8 protein even with staurosporin, we concluded In summary, one normal cell line (UROtsa, urothelial) that 5-aminolevulinic acid–induced photodynamic therapy and two tumor cell lines (RT4, urothelial; HT29, colonic)

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were treated in vitro with LD dosages of light after 16. Grutzmann R, Foerder M, Alldinger I, et al. Gene expression profiles of 50 microdissected pancreatic ductal adenocarcinoma. Virchows Arch exposure to 5-aminolevulinic acid, and harvested for gene 2003;443:508 – 17. expression profiling 0, 10, and 30 minutes after irradiation 17. Dopazo J, Carazo JM. Phylogenetic reconstruction using an unsuper- to better understand tumor phototoxicity of photodynamic vised growing neural network that adopts the topology of a phylogenetic therapy. Fifteen clusters of related expression patterns tree. J Mol Evol 1997;44:226 – 33. 18. Herrero J, Valencia A, Dopazo J. A hierarchical unsupervised growing among 40 differentially expressed genes were obtained. neural network for clustering gene expression patterns. Bioinformatics Three of the genes (LUC7A, SOD2, and MMP7) have 2001;17:126 – 36. already been described in the context of tumor treatment, 19. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the providing a molecular basis for combinatory therapy unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25 – 9. regimens. Combined data analysis of gene expression 20. Warrington JA, Nair A, Mahadevappa M, Tsyganskaya M. Compar- profiling, quantitative RT-PCR, and Western blot analysis ison of human adult and fetal expression and identification of 535 suggested that photodynamic therapy with 5-aminolevu- housekeeping/maintenance genes. Physiol Genomics 2000;2:143 – 7. linic acid (LD50) in vitro leads to apoptosis in the normal 21. Nishii Y, Morishima M, Kakehi Y, et al. CROP/Luc7A, a novel serine/ urothelial cell line UROtsa and to necrosis in the tumor arginine-rich nuclear protein, isolated from cisplatin-resistant cell line. FEBS Lett 2000;465:153 – 6. cell lines, respectively. 22. Shimizu N, Ohta M, Fujiwara C, et al. Expression of a novel immediate early gene during 12-O-tetradecanoylphorbol-13-acetate- Acknowledgments induced macrophagic differentiation of HL-60 cells. J Biol Chem 1991; 266:12157 – 61. We thank Sabine Dietrich, Nina Nießl, and Rudolf Jung for excellent technical assistance, and metaGen Pharmaceuticals for providing the 23. Keyse SM, Emslie EA. Oxidative stress and heat shock induce a oligonucleotide arrays. human gene encoding a protein-tyrosine phosphatase. Nature 1992; 359:644 – 7. References 24. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and 1. Stapleton M, Rhodes LE . Photosensitizers for photodynamic therapy of TNF receptor-induced cell death. Cell 1996;85:803 – 15. cutaneous disease. J Dermatolog Treat 2003;14:107 – 12. 25. Luo Y, Kessel D. Initiation of apoptosis versus necrosis by 2. Dougherty TJ, Gomer CJ, Henderson BW, et al. Photodynamic therapy. photodynamic therapy with chloroaluminum phthalocyanine. Photochem J Natl Cancer Inst 1998;90:889 – 905. Photobiol 1997;66:479 – 83. 3. Capella MA, Capella LS. A light in multidrug resistance: photody- 26. Ben-Hur E, Oetjen J, Horowitz B. Silicon phthalocyanine Pc 4 and red namic treatment of multidrug-resistant tumors. J Biomed Sci 2003;10: light causes apoptosis in HIV-infected cells. Photochem Photobiol 361 – 6. 1997;65:456 – 60. 4. Ortel B, Sharlin D, O’Donnell D, Sinha AK, Maytin EV, Hasan T. 27. Laukka MA, Wang KK, Bonner JA. Apoptosis occurs in lymphoma Differentiation enhances aminolevulinic acid-dependent photodynamic cells but not in hepatoma cells following ionizing radiation and photody- treatment of LNCaP prostate cancer cells. Br J Cancer 2002;87:1321 – 7. namic therapy. Dig Dis Sci 1994;39:2467 – 75. 5. Ackroyd R, Kelty CJ, Brown NJ, Stephenson TJ, Stoddard CJ, Reed 28. He XY, Sikes RA, Thomsen S, Chung LW, Jacques SL. Photodynamic MW. Eradication of dysplastic Barrett’s oesophagus using photodynamic therapy with photofrin II induces programmed cell death in carcinoma cell therapy: long-term follow-up. Endoscopy 2003;35:496 – 501. lines. Photochem Photobiol 1994;59:468 – 73. 6. Shackley DC, Briggs C, Whitehurst C, et al. Photodynamic therapy for 29. Noodt BB, Berg K, Stokke T, Peng Q, Nesland JM. Apoptosis and superficial bladder cancer. Expert Rev Anticancer Ther 2001;1:523 – 30. necrosis induced with light and 5-aminolaevulinic acid-derived protopor- 7. Krieg RC, Uihlein D, Murthum T, et al. Improving the use of 5- phyrin IX. Br J Cancer 1996;74:22 – 9. aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) for the 30. Moor AC. Signaling pathways in cell death and survival after gastrointestinal tract by esterification—an in vitro study. Cell Mol Biol photodynamic therapy. J Photochem Photobiol B 2000;57:1 – 13. (Noisy-le-grand) 2002;48:917 – 23. 31. Gomer CJ, Ferrario A, Hayashi N, Rucker N, Szirth BC, Murphree AL. 8. Hinnen P, de Rooij FW, van Velthuysen ML, et al. Biochemical basis of Molecular, cellular, and tissue responses following photodynamic therapy. 5-aminolaevulinic acid-induced protoporphyrin IX accumulation: a study in Lasers Surg Med 1988;8:450 – 63. patients with (pre)malignant lesions of the oesophagus. Br J Cancer 1998; 78:679 – 82. 32. Agarwal ML, Larkin HE, Zaidi SI, Mukhtar H, Oleinick NL. Phospho- 9. Krieg RC, Messmann H, Rauch J, Seeger S, Knuechel R. Metabolic lipase activation triggers apoptosis in photosensitized mouse lymphoma characterization of tumor cell-specific protoporphyrin IX accumulation cells. Cancer Res 1993;53:5897 – 902. after exposure to 5-aminolevulinic acid in human colonic cells. Photochem 33. Oleinick NL, Evans HH. The photobiology of photodynamic therapy: Photobiol 2002;76:518 – 25. cellular targets and mechanisms. Radiat Res 1998;150:S146 – 56. 10. Rigby CC, Franks LM. A human tissue culture cell line from a 34. Krieg RC, Messmann H, Schlottmann K, et al. Intracellular localization transitional cell tumour of the urinary bladder: growth, chromosone pattern is a cofactor for the phototoxicity of protoporphyrin IX in the gastrointes- and ultrastructure. Br J Cancer 1970;24:746 – 54. tinal tract: in vitro study. Photochem Photobiol 2003;78:393 – 9. 11. Petzoldt JL, Leigh IM, Duffy PG, Sexton C, Masters JR. Immortal- 35. Verwanger T, Sanovic R, Aberger F, Frischauf AM, Krammer B. isation of human urothelial cells. Urol Res 1995;23:377 – 80. Gene expression pattern following photodynamic treatment of the 12. Thomas DR, Philpott GW, Jaffe BM. Prostaglandin E (PGE) control of carcinoma cell line A-431 analysed by cDNA arrays. Int J Oncol 2002; cell proliferation in vitro: characteristics of HT-29. J Surg Res 1974;16: 21:1353 – 9. 463 – 5. 36. Luna MC, Wong S, Gomer CJ. Photodynamic therapy mediated 13. Seidl J, Rauch J, Krieg RC, Appel S, Baumgartner R, Knuechel R. induction of early response genes. Cancer Res 1994;54:1374 – 80. Optimization of differential photodynamic effectiveness between normal 37. Xue LY, He J, Oleinick NL. Rapid tyrosine phosphorylation of HS1 in and tumor urothelial cells using 5-aminolevulinic acid-induced protopor- the response of mouse lymphoma L5178Y-R cells to photodynamic phyrin IX as sensitizer. Int J Cancer 2001;92:671 – 7. treatment sensitized by the phthalocyanine Pc 4. Photochem Photobiol 14. Schmitt AO, Specht T, Beckmann G, et al. Exhaustive mining of EST 1997;66:105 – 13. libraries for genes differentially expressed in normal and tumour tissues. 38. Golab J, Nowis D, Skrzycki M, et al. Antitumor effects of Nucleic Acids Res 1999;27:4251 – 60. photodynamic therapy are potentiated by 2-methoxyestradiol. A superox- 15. Wissmann C, Wild PJ, Kaiser S, et al. WIF1, a component of the Wnt ide dismutase inhibitor. J Biol Chem 2003;278:407 – 14. pathway, is down-regulated in prostate, breast, lung, and bladder cancer. 39. Lottner C, Knuechel R, Bernhardt G, Brunner H. Combined J Pathol 2003;201:204 – 12. chemotherapeutic and photodynamic treatment on human bladder cells

MolCancerTher2005;4(4).April2005

by hematoporphyrin-platinum(II) conjugates. Cancer Lett 2004;203: Rogler G. Characterization of bile salt-induced apoptosis in colon cancer 171 – 80. cell lines. Cancer Res 2000;60:4270 – 6. 40. Ferrario A, Chantrain CF, von Tiehl K, et al. The matrix metal- 46. Bilim VN, Tomita Y, Kawasaki T, Takeda M, Takahashi K. Adriamycin loproteinase inhibitor prinomastat enhances photodynamic therapy res- (ADM) induced apoptosis in transitional cell cancer (TCC) cell lines ponsiveness in a mouse tumor model. Cancer Res 2004;64:2328 – 32. accompanied by p21 WAF1/CIP1 induction. Apoptosis 1997;2:207 – 13. 41. Plaetzer K, Kiesslich T, Krammer B, Hammerl P. Characterization of 47. Proskuryakov SY, Konoplyannikov AG, Gabai VL. Necrosis: a specific the cell death modes and the associated changes in cellular energy form of programmed cell death? Exp Cell Res 2003;283:1 – 16. supply in response to AlPcS4-PDT. Photochem Photobiol Sci 2002; 48. Subramaniam S, Strelau J, Unsicker K. Growth differentiation factor- 1:172 – 7. 15 prevents low potassium-induced cell death of cerebellar granule 42. Almeida RD, Manadas BJ, Carvalho AP, Duarte CB. Intracellular neurons by differential regulation of Akt and ERK pathways. J Biol Chem signaling mechanisms in photodynamic therapy. Biochim Biophys Acta 2003;278:8904 – 12. 2004;1704:59 – 86. 49. Hsiao EC, Koniaris LG, Zimmers-Koniaris T, Sebald SM, Huynh TV, 43. Granville DJ, Carthy CM, Jiang H, Shore GC, McManus BM, Hunt DW. Lee SJ. Characterization of growth-differentiation factor 15, a trans- Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed forming growth factor superfamily member induced following liver injury. by Bap31 cleavage in HeLa cells treated with photodynamic therapy. FEBS Mol Cell Biol 2000;20:3742 – 51. Lett 1998;437:5 – 10. 50. Simank HG, Manggold J, Sebald W, et al. Bone morphogenetic 44. Grebenova D, Kuzelova K, Smetana K, et al. Mitochondrial and protein-2 and growth and differentiation factor-5 enhance the healing of endoplasmic reticulum stress-induced apoptotic pathways are activated necrotic bone in a sheep model. Growth Factors 2001;19:247 – 57. by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia 51. Simank HG, Sergi C, Jung M, et al. Effects of local application of cells. J Photochem Photobiol B 2003;69:71 – 85. growth and differentiation factor-5 (GDF-5) in a full-thickness cartilage 45. Schlottmann K, Wachs FP, Krieg RC, Kullmann F, Scholmerich J, defect model. Growth Factors 2004;22:35 – 43.

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