Molecular Cancer Therapeutics Molecular Diagnostics in Cancer Therapeutic Development: Fulfilling the Promise of Personalized Medicine Targeting the PI3-Kinase Pathway in Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wild, P. J.
Right arrow Articles by Knuechel, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wild, P. J.
Right arrow Articles by Knuechel, R.
Mol Cancer Ther. 2005;4:516-528
© 2005 American Association for Cancer Research

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

Peter J. Wild1, Rene C. Krieg4, Juergen Seidl1, Robert Stoehr2, Kerstin Reher2, Claudia Hofmann3, Jari Louhelainen5, André Rosenthal6, Arndt Hartmann1, Christian Pilarsky7, Anja K. Bosserhoff1 and Ruth Knuechel4

1 Institute of Pathology and 2 Departments of Urology and 3 Internal Medicine I, University of Regensburg, Regensburg, Germany; 4 Institute of Pathology, University of Aachen, Aachen, Germany; 5 Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, United Kingdom; 6 Signature Diagnostics AG, Potsdam, Germany; and 7 Department of Surgery, University Hospital Dresden, Dresden, Germany

Requests for reprints: Ruth Knuechel, Institute of Pathology, University of Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany. Phone: 49-241-8089280; Fax: 49-941-944-6634. E-mail: knuechel{at}pat.rwth-aachen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Photodynamic therapy using 5-aminolevulinic acid–induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line–specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 µg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.

Key Words: Genitourinary cancers:bladder • Gastrointestinal cancers: colorectal • Gene expression profilling • Mechanisms of Drug Action/New Molecular Targets/Therapeutics • Photobilogy/photodynamic therapy


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Photodynamic therapy is based on the tumor-selective accumulation of a photosensitizer. After irradiation with light of appropriate wavelength and in the presence of oxygen, this photosensitizer will induce cellular damage by generating singulett oxygen (Fig. 1A–C). Many photosensitizers are known but only a few are used in clinical practice. 5-Aminolevulinic acid–induced protoporphyrin IX is preferred to competitive porphyrin derivatives (1). In comparison with, e.g., Photofrin (2), the most obvious advantages of 5-aminolevulinic acid are the possibility of a topical application and the rapid clearance within 2 days. In addition, neither 5-aminolevulinic acid nor protoporphyrin IX shows a dark toxicity as they are both heme precursors and therefore physiologic substances. In contrast to the general toxicity pattern of chemotherapy regimens which are more toxic to low-differentiated tumor cells (3), a higher protoporphyrin IX content in differentiated compared with undifferentiated tumor cells has been 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 a tumor-selective treatment modality. Endoscopically accessible malignant lesions of the urinary bladder or the gastrointestinal tract have been successfully treated in clinical trials (5, 6). Attempts have been made to further optimize this method by derivatization of 5-aminolevulinic acid. The velocity of 5-aminolevulinic acid uptake has been shown to increase with esterification of 5-aminolevulinic acid, especially by using 5-aminolevulinic acid hexyl or benzylester derivatives (7). However, mechanisms leading to preferential protoporphyrin IX accumulation of tumor cells after 5-aminolevulinic acid incubation are still not known for sure, but assumed to be due to a different heme metabolism in the tumor compared with normal tissue. This could already be shown for different organs in vitro (8, 9).



View larger version (74K):
[in this window]
[in a new window]
  		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 (x100) 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, x40).

 
Because traditional therapy for endoscopically accessible precancerous lesions (e.g., carcinoma in situ of the bladder, dysplasia of Barrett's esophagus) and early-stage tumors is still insufficient, new treatment modalities are urgently needed. Understanding the mechanism of tumor-selective phototoxicity of photodynamic therapy with 5-aminolevulinic acid may also provide a basis for combinatory therapy regimens. Oligonucleotide microarrays, analyzing the RNA expression level of thousands of genes simultaneously, are promising for gaining insight into the complex pathway of cellular damage. To screen for molecules indicating the mechanisms of protoporphyrin IX–mediated toxicity, an in vitro study with one human normal cell line (bladder) and two human cancer cell lines (bladder and colon) was carried out. Cells were subjected to photodynamic therapy with cell-specific LD50 values and harvested for array analysis 0, 10, and 30 minutes after photodynamic therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Culturing
RT4 cells (well-differentiated bladder tumor cells), originally derived from a recurrent papillary G1 tumor (10), UROtsa cells (11), derived from normal urothelium and immortalized by a temperature-sensitive SV40 large T-antigen gene construct (provided by Dr. J.R.W. Masters, University College, London, United Kingdom), and moderately differentiated (G2) HT29, established from a colorectal adenocarcinoma (12), were used. Cell lines were maintained as monolayer cultures in RPMI 1640 (Biochrom, Berlin, Germany) and HT29 in DMEM (PAN BIOTECH GmbH, Aidenbach, Germany) without phenol red supplemented with 5% FCS (PAN BIOTECH), 1% (v/v) L-glutamine, and 1% (v/v) sodium pyruvate (Gibco, Eggenstein, Germany) at 37°C in a humidified atmosphere, containing 5% carbon dioxide. Cells were detached for subculturing and experiments using 0.05% trypsin/0.02% EDTA (Gibco) in PBS (Biochrom). All experiments were done using plateau phase cells. To reach plateau phase growth, defined cell densities were seeded into culture dishes and allowed to grow for the times indicated: RT4: 22,000 cells/cm2, 9 days; UROtsa: 5,600 cells/cm2, 10 days; HT29: 50,000 cells/cm2, 7 days.

Photodynamic Therapy and Cell Harvest
Stock solutions of 5-aminolevulinic acid (Synopharm, Barsbüttel, Germany) were prepared in deionized water (10 mg/mL) and stored at –20°C. For each experiment the stock solution was diluted in culture medium without FCS. Before 5-aminolevulinic acid incubation, the culture medium was removed and the cell monolayer was rinsed with PBS to remove remaining FCS. Cells were incubated with 5-aminolevulinic acid at a final concentration of 100 µg/mL in culture medium without FCS for 3 hours. A total volume of 250 µL per square centimeter of growth area was used. In subsequent handling, care was taken to avoid exposure of the cells to ambient light. After the incubation period, the 5-aminolevulinic acid solution was removed and fresh medium without FCS was added. Each cell line was then illuminated with a specific light dose leading to a cell survival of 50% (LD50). In vitro photodynamic therapy was done using a high-pressure Xenon arc lamp (Karl Storz GmbH, Tuttlingen, Germany). The wavelength ranges, overall light doses, and power densities were as follows: RT4: 400 to 700 nm, 0.8 J cm–2, 50 mW cm–2; UROtsa: 400 to 700 nm, 1.5 J cm–2, 50 mW cm–2; and HT29: 590 to 700 nm, 3.9 J cm–2, 40 mW cm–2. After photodynamic treatment, medium without FCS was removed and fresh culture medium with FCS was added. Zero, 10, and 30 minutes after photodynamic therapy the cellular monolayers were briefly rinsed with PBS and cells were lysed with guanidium thiocyanate buffer (provided with the PolyATract System 1000, Promega, Heidelberg, Germany) to stop all biological activity and to protect RNA integrity.

Preliminary Toxicity Testing
To study structural and functional changes of photodynamic therapy–treated cells, cell membrane integrity and mitochondrial activity were selected as variables as already described by Seidl et al. (13). Whereas the plasma membrane is the main site of sensitizer localization, protoporphyrin IX generation is located in mitochondria. Phototoxic effects were analyzed 0 to 24 hours after photodynamic therapy (0.5, 1, 2, 3, and 24 hours), with 0 hour after photodynamic therapy serving as untreated control.

Plasma Membrane Integrity
Cells seeded into six-well dishes were used for flow cytometric analysis of membrane integrity by using propidium iodide (Sigma, Deisenhofen, Germany) exclusion of viable cells. After 5-aminolevulinic acid incubation and illumination (see above), cells were detached, spun down at 200 x g, and resuspended in 1 mL PBS. From a stock solution of propidium iodide (1 mg/mL in PBS), 3 µL were added to the cell suspension to a final concentration of 3 µg/mL. After a few minutes of incubation (3–5 minutes at room temperature), the cell suspension was immediately measured on a FACSCalibur flow cytometer. The data were gated for forward versus right angle light scatter and the percentage of cells showing high red propidium iodide fluorescence (measured in channel FL3, 650 nm lp) was determined.

Mitochondrial Activity
For the determination of mitochondrial activity and their inner membrane potential, cells were seeded into six-well culture dishes as described above. A stock solution of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1; Molecular Probes, Leiden, the Netherlands) of 500 µg/mL in 70% methanol was prepared. JC-1 accumulates in mitochondria and forms multimeres (J aggregates) depending on the potential across the inner mitochondrial membrane. Detached cells were centrifuged at 200 x g, the supernatant was removed, cells were resuspended in 1 mL PBS, and 10 µL of the stock solution were added to a final concentration of 5 µg/mL. After a 15-minute incubation at room temperature, the cell suspension was measured without washing on a FACSCalibur flow cytometer. After 488 nm laser excitation the fluorescence of monomers (emission maximum, 527 nm) and aggregates (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 using suitable instrument settings (RT4 and UROtsa: FL1—monomer 310 V, FL2—aggregate 259 V, FL1—%FL2 2.0, FL2—%FL1 20.9; HT29: FL1—monomer 370 V, FL2—aggregate 279 V, FL1—%FL2 4.0, FL2—%FL1 5.1). Debris was excluded from further analysis by gating for forward versus right angle light scatter.

Statistical Analyses
The two-sided Mann-Whitney U test was used to compare results of cell membrane integrity and mitochondrial activity at certain time points. P values < 0.05 were considered statistically significant. Statistical analyses were completed using SPSS version 10.0 (SPSS, Chicago, IL).

Chip Design
The metg001A GeneChip (metaGen Pharmaceuticals GmbH, Berlin, Germany) consists of 6,117 probe sets representing roughly 3,000 human genes based on the annotation of the probe sets with the GoldenPath assembly.8 We selected two different sets of genes: first, genes known as involved in the progression of human tumors, including genes from several signal transduction pathways (e.g., TGFB, RAS, and WNT) and androgen receptor–regulated genes; and second, cDNA fragments differentially expressed in silico [i.e., Schmitt et al. (14) have systematically screened whole expressed sequence tag (EST) libraries for genes differentially expressed in normal and tumor tissues].

Gene Expression Profiling
Poly(A)+ RNA was isolated from the cells by magnetic separation (PolyATract System 1000, Promega) according to the recommendations of the manufacturer. Linear amplification (two rounds) was done as described previously (15). In brief, after priming with the Affymetrix T7-oligo-dT promoter-primer combination (5'-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG T24-3' at 100 mmol/L), first and second strand synthesis, and in vitro transcription, the amplified RNA was again amplified in one subsequent round of cDNA synthesis and in vitro transcription. Within the last in vitro transcription, biotin-labeled nucleotides were incorporated into the amplified RNA. Hybridization and detection of the labeled amplified RNA on the metg001A Affymetrix GeneChip was done once according to the instructions of the manufacturer.

Data Processing
GeneChips were scanned using an Agilent GeneArray Scanner (Agilent Technologies, Inc., Palo Alto, CA) and processed as described (16). In brief, raw intensity values were extracted from the Cel files and a background correction was performed. The background-corrected probe intensity values were normalized by dividing them by the median value of all probes. A representative expression value for each probe set (PMQ value) was generated by using the 75% percentile of the perfect match intensities. PMQ values (0, 10, and 30 minutes) of the three cell lines were compared with the 5-aminolevulinic 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 Reverse Transcription-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 synthesized using 2 µg of the isolated total RNA of photodynamic therapy–treated cells after 0, 15, and 30 minutes, 1 µg of random primer (Amersham Pharmacia Biotech, Frankfurt, Germany), 4 µL of 5x First Strand Buffer (Gibco), 2 µL of DTT 10 mmol/L, 1 µL of deoxynucleotide triphosphates (10 mmol/L), and 1 µL of Superscript Plus (Gibco) in a total volume of 20 µL.

To quantify the expression of cDNAs, a LightCycler real-time PCR system (Roche, Mannheim, Germany) was used. For the RT-PCR, 1 to 3 µL of cDNA, 0.5 to 2.4 µL of 25 mmol/L MgCl2, 0.5 µmol/L of forward and reverse primers, and 2 µL of LightCycler-FastStart DNA Master SYBR Green I (Roche) in a total volume of 20 µL were applied. The following PCR program was done: 5 minutes 95°C (initial denaturation), temperature transition rate 20°C/s, 95°C for 15 seconds, 10 seconds 58°C (annealing temperature), 10 seconds 72°C, acquisition mode single, repeated for 50 times (amplification). MgCl2 concentration was optimized for each primer set. Primer characteristics (name and 5'-3' sequence) are listed below.


View this table:
[in this window]
[in a new window]
 
 
The PCR reaction was evaluated by melting curve analysis (0 second 95°C, 15 seconds 50°C, temperature transition rate 0.1°C/s up to 95°C) following the instructions of the manufacturer and detecting the PCR products on 1.8% agarose gels. The LightCycler Relative Quantification Software (Roche) was used for determining calibrator-normalized target/reference ratios with automated efficiency-corrected quantification according to the recommendations of the manufacturer.

Western Blot Analysis
After photodynamic therapy, cells were washed twice with ice-cold PBS buffer and lysed in radioimmunoprecipitation assay buffer (50 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail; Roche) for 15 minutes on ice. Insoluble fragments were removed by centrifugation at 13,000 rpm for 10 minutes and the supernatant lysate was immediately shock frozen and stored at –80°C. The protein concentration of the cell lysate was determined using the bicinchoninic acid protein assay reagent (Pierce Biotechnology, Inc., Rockford, IL). Forty micrograms of radioimmunoprecipitation assay cell lysate were loaded per lane, separated on SDS polyacrylamide gradient gels (Invitrogen GmbH, Karlsruhe, Germany), and subsequently blotted onto a polyvinylidene difluoride membrane (Roche). Blots for detection of caspase-8, caspase 3, dual specificity phosphatase 1 (DUSP1), and ß-actin were blocked with 5% nonfat dry milk in TBS with 0.5% Tween 20 (TBST) for 1 hour at room temperature and then incubated for 16 hours at 4°C with the following primary antibodies: anti-caspase-8 (1:1,000, Cell Signaling, Beverly, MA), anti-caspase 3 (1:3,000, BD Transduction Laboratories, San Jose, CA), anti-DUSP1 (1:1,000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti–ß-actin (1:5,000, Sigma). The membranes were then washed in TBST or PBS, incubated for 1 hour at room temperature with alkaline phosphotase–conjugated antibodies, and diluted in blocking buffer. Finally, immunoreactions were visualized by nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (Zymed Laboratories, Inc., South San Francisco, CA) staining. As a positive control for the induction of apoptosis, cells were cultured in 5 mL medium with 5% FCS and treated with 1 µmol/L staurosporine for 3 hours.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To understand differential cell death response to photodynamic therapy and the mechanism of toxicity, a normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with LD50 doses of light after exposure to 5-aminolevulinic acid, and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The gene expression profiles between the different samples were compared by Affymetrix GeneChip analysis, focusing on cancer-related and growth regulatory targets.

Preliminary Toxicity Testing
Irradiation with LD50 was based on the assumption that intracellular alterations are determined by differential gene expression, and not completely covered by fast necrotic cell death. Of note, LD50 for UROtsa cells was identical to LD100 for RT4, given the same incubation conditions. Before defining time points for RNA isolation after photodynamic therapy, mitochondrial activity and membrane integrity were determined by flow cytometry over 24 hours using urothelial cell lines (UROtsa and RT4). The trend of the two variables investigated for the two cell lines is shown in Fig. 2A and B.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Structural and functional analysis of RT4 and UROtsa cell lines after photodynamic therapy (LD50) with 5-aminolevulinic acid in vitro. A, membrane integrity; percentage of membrane damaged cells after photodynamic therapy; *, significant Mann-Whitney U test at 24 h. B, mitochondrial activity, percentage of mitochondrial damaged cells after photodynamic therapy; ns, nonsignificant Mann-Whitney U test at 24 h.

 
Membrane Integrity
RT4 and UROtsa showed a clear reduction of viable cells within 24 hours. Whereas RT4 cells reached their level of maximum membrane damage 3 hours after photodynamic therapy, the percentage of dead or propidium iodide–positive UROtsa cells increased until the end of the experimental period. Furthermore, no recovery of membrane integrity was detectable within 24 hours after treatment. This is in agreement with earlier experiments, indicating that the rate of membrane damage in urothelial cells increased up to 48 hours or even later after irradiation (13). At 24 hours after photodynamic therapy, the percentage of dead UROtsa cells was significantly lower than the percentage of RT4 cells (P = 0.03; Fig. 2A).

Mitochondrial Activity
RT4 and UROtsa showed an increasing number of cells with loss of mitochondrial function within the first few hours after photodynamic therapy. Maximum mitochondrial damage was visible 2 to 3 hours after photodynamic therapy for both cell lines, with a maximum rate of ~50%. A beginning but nonsignificant (P = 0.2) recovery process from photodamage could be observed in UROtsa cells at 24 hours after photodynamic therapy. In contrast, the percentage of RT4 cells with damaged mitochondria remained at maximum until the end of the experimental period (Fig. 2B).

With the maximum mitochondrial damage being reached after 2 to 3 hours, shorter periods for cell harvest were selected for RNA expression analysis after incubation with 5-aminolevulinic acid (100 µg/mL for 3 hours) 0, 10, and 30 minutes after irradiation with LD50.

RNA Expression after Photodynamic Therapy
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). Furthermore, 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).



View larger version (22K):
[in this window]
[in a new window]
  		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 variability 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.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Clustering of expression microarray experiments using the SotaTree Server, and gene ontology annotation (biological process)

 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. Gene expression (single PMQ values, normalization to control) of LUC7A (A), SOD2 (B), ETR101 (C), and MMP7 (D) following photodynamic therapy with 5-aminolevulinic acid. Blue rhombuses, HT29; red squares, RT4; green triangles, UROtsa.

 
Validation of Expression Profiles by Quantitative Reverse Transcription-PCR
Quantitative RT-PCR supported the results of the oligonucleotide arrays. In detail, FOS, DUSP1, and CASP8 were selected for further validation of the array findings (Fig. 5A–C). Expression of the immediate early gene FOS was continuously up-regulated over 30 minutes in all cell lines, whereas DUSP1 mRNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. DUSP1 is known as induced by oxidative and heat stress (23). In contrast, CASP8 mRNA was found unaltered in the three cell lines over time, comparing array and RT-PCR results. The related caspase-8 protein is a protease involved in the FAS/APO1- and p55 tumor necrosis factor receptor–induced signaling cascades (24). The initial down-regulation of CASP8 RNA at 0 minute after photodynamic therapy in UROtsa cells could not be confirmed in independent RT-PCR runs (Fig. 5C).



View larger version (29K):
[in this window]
[in a new window]
  		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
In contrast to the DUSP1 gene expression data, induction of DUSP1 protein expression was only shown after 1 hour in UROtsa cells (Fig. 6). The expression levels of DUSP1 protein in the HT29 and RT4 cell lines were obviously not high enough to be detected by Western blot analysis. Using relative quantification, DUSP1 expression was highest in UROtsa cells after normalization to DUSP1 expression in HT29 cells (data not shown). For caspase-8, no changes in protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. In contrast to UROtsa cells, enforcement of apoptosis by use of staurosporin was absent in HT29 and weak in RT4 cells. Because caspase 3 is considered another important effector in apoptosis, caspase 3 activation was characterized after photodynamic therapy in vitro (Fig. 7) using Western blot analysis. According to caspase-8, the active caspase 3 fragment (17 kDa) was found only in the normal urothelial cell line (UROtsa) starting 1 hour after photodynamic therapy.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 6. Protein expression of CASP8, DUSP1, and ACTB in RT4 (A), HT29 (B), and UROtsa (C) cells at certain time points after photodynamic therapy in vitro. As a positive control for the induction of apoptosis, cells were treated with staurosporine.

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 7. Protein expression of CASP3 in RT4, HT29, and UROtsa cells at certain time points after photodynamic therapy in vitro.

 
Data on differential toxicity between normal and tumor cells indicate that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines (HT29 and RT4), respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Photodynamic therapy is an experimental treatment modality which is under development for application in both neoplastic and nonneoplastic diseases (2). Responses to photodynamic therapy are dependent on the photosensitizer and its localization in the cell, the illumination conditions (25, 26), the oxygenation status of the tissue, and the type of cells (27–29) involved. The involvement of different subcellular targets, such as mitochondria, lysosomes, and the plasma membrane, in cell death and survival is reviewed in detail by Moor (30). Cells can respond to photodynamic therapy by initiating a rescue response and/or by undergoing cell death either in an apoptotic or in a necrotic fashion (2, 31–33). Signaling pathways influenced by photodynamic therapy have not been fully elucidated although a number of studies have addressed this issue. However, the interpretation of the data has been complicated due to different models used and the utilization of many different sensitizers.

Photodynamic therapy in vitro with LD50 was based on the assumption that intracellular alterations are determined by differential gene expression rather than by fast necrotic cell death. Besides the light dosage, the time point of cell harvest after the applied stimulus is crucial for gaining insight into the cellular response after photodynamic therapy. Before defining time points for RNA isolation after photodynamic therapy, mitochondrial activity and membrane integrity following photodynamic therapy 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, 10, and 30 minutes after photodynamic therapy were considered optimal for cell harvest, evaluating photodynamic therapy–dependent fast cellular response on the RNA level.

We selected FOS, DUSP1, and CASP8 for further validation of the array findings by quantitative real-time RT-PCR. The increased photodynamic therapy–induced expression of the immediate early gene FOS in all three cell lines is in accordance with gene expression results of 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, cell proliferation, and cell survival.

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.

The simultaneous use of the three cell lines, HT29, RT4, and UROtsa, not only enabled the comparison of tumor and normal cells but also allowed the comparison of tumor cells of different origin (bladder versus colon). 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 LUC7A protein was cloned by Nishii et al. (21) from cisplatin-resistant cell lines by differential display, and was therefore designated cisplatin resistance–associated protein. The fact that cisplatin resistance was not induced after photodynamic therapy may provide a basis for combinatory therapy regimens. Lottner et al. (39) have already combined the cytostatic activity of cisplatin/oxaliplatin and the photodynamic effect of hematoporphyrin in the same molecule. Synergistic antiproliferative effects of hematoporphyrin-platinum(II) conjugates were found in vitro against J82 bladder cancer cells and UROtsa.

Ferrario et al. (40) have evaluated the role of Photofrin-mediated photodynamic therapy in eliciting expression of MMPs in a mouse mammary tumor model. Administration of the MMP inhibitor prinomastat significantly improved photodynamic therapy–mediated tumor response, linking our gene expression results to anticancer drugs. Prinomastat is an anticancer drug and belongs to the group of angiogenesis inhibitors. Immunohistochemical analyses indicated that infiltrating inflammatory and endothelial cells were the primary source of MMP expression. According to our in vitro results, Ferrario et al. (40) observed negligible expression of MMPs in tumor cells.

Photodynamic therapy can trigger both modes of cell death, apoptosis and necrosis (41). For complete tumor eradication the desired apoptosis/necrosis ratio should be adjusted. In case of cancer therapy for instance, photodynamic therapy causing necrotic cell death is preferred because the immune reaction elicited by the inflammation (Fig. 1C) secondary to tumor necrosis could be useful in killing additional tumor cells. As previously reported for other apoptotic stimuli (26), the type of cell death induced by photodynamic therapy switches from apoptosis to necrosis with the increase of the intensity of the insult (42). According to Almeida et al. (42), who have reviewed intracellular signaling mechanisms in photodynamic therapy, two major apoptotic pathways have been characterized, the death receptor mediated and the mitochondria mediated. In both pathways, the activation of initiator caspases (caspase-8 or caspase 9) leads to the activation of effector caspases (caspase 3, caspase 6, and caspase 7). To be able to compare the intracellular signaling mechanisms in normal and tumor cells, both were treated with their individual LD50. Given the same incubation conditions, LD50 for UROtsa cells would be identical to LD100 for the tumor cell lines.

RNA expression of CASP8 was unchanged (Fig. 5C). The related protein is a protease involved in apoptosis (24). For caspase-8 protein, strong but delayed activation of catalytic activity was detected only in UROtsa cells starting 1 hour after treatment, whereas no changes were seen in both tumor cell lines (Fig. 6). In agreement with Granville et al. (43), a delayed activation of CASP8 was shown in UROtsa cells, concluding that activation of the CASP8 pathway may serve as a secondary way for the cell to ensure demise in case of damage. Accordingly, enforcement of apoptosis by use of staurosporin resulted in strong expression of activated caspase-8 protein in UROtsa cells (Fig. 6). With HT29 and RT4 cells showing no expression of activated caspase-8 protein even with staurosporin, we concluded that 5-aminolevulinic acid–induced photodynamic therapy in vitro (LD50) may lead to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. According to caspase-8, the active caspase 3 fragment (17 kDa) was found only in the normal urothelial cell line (UROtsa), starting 1 hour after photodynamic therapy. Cleavage of pro-caspase 3 after photodynamic therapy with 5-aminolevulinic acid has already been reported by Grebenova et al. (44) in HL60 leukemia cells. Of note, apoptosis can principally be induced in the two cancer cell lines using bile salts for HT29 (45) and Adriamycin for RT4 cells (46).

Proskuryakov et al. (47) have described necrosis as another specific form of programmed cell death, with mitochondria being the key players in determination of the pathway of cell suicide. Gene expression profiling is not the method of first choice to investigate the molecular scenario of necrotic cell death, as reviewed by Proskuryakov et al. (47). Many of the players are not regulated on the gene expression level but are a matter of faster cellular reactions (massive DNA breaks, rapid efflux of cell constituents into extracellular space, etc.). In our preliminary tests to define time points for cell harvest, an initial but nonsignificant (P = 0.2) mitochondrial recovery process from photodamage could be observed in UROtsa cells (Fig. 2B). In contrast, the percentage of RT4 cells with damaged mitochondria remained at maximum until the end of the experimental period. Levels of maximum damage were reached within the first 2 to 3 hours. However, even in vitro, apoptosis finally leads to plasma membrane permeabilization ("secondary" necrosis; Fig. 2A), but it does not occur in vivo because apoptotic cells are digested by macrophages or by surrounding cells before their plasma membrane becomes disrupted (47).

Another gene found to be regulated following photodynamic therapy encoded for GDF15 (PLAB). The cell line UROtsa with apoptotic behavior following photodynamic therapy showed no regulation of this gene. RT4 as well as HT29 with a clear necrotic response to photodynamic therapy showed a strong activation of GDF15 RNA expression levels. The corresponding protein growth and differentiation factor 15 (GDF15) is an interesting factor in cellular response to injuries and seems to be expressed in an organ-independent manner. Subramaniam et al. found GDF15 levels to be overexpressed in neurons following injury. Furthermore, they analyzed GDF15 to inhibit apoptosis by direct interference with AKT and extracellular signal–regulated kinase. This raises the question whether or not strong apoptotic stimuli might then result in necrosis. Hsiao et al. (49) and his group were able to show a strong up-regulation of GDF15 in liver following chemical and surgical trauma both in vivo and in vitro. GDF5, another member of the GDF family, is known to enhance bone repair in vivo when administered following a per se necrotic traumatic stimulus (50, 51). In general, GDF15 seems to be an important factor which is expressed after a severe deadly stimulus as a cellular response and in attempt to survive.

In summary, one normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated in vitro with LD50 dosages of light after exposure to 5-aminolevulinic acid, and harvested for gene expression profiling 0, 10, and 30 minutes after irradiation to better understand tumor phototoxicity of photodynamic therapy. Fifteen clusters of related expression patterns among 40 differentially expressed genes were obtained. Three of the genes (LUC7A, SOD2, and MMP7) have already been described in the context of tumor treatment, providing a molecular basis for combinatory therapy regimens. Combined data analysis of gene expression profiling, quantitative RT-PCR, and Western blot analysis suggested that photodynamic therapy with 5-aminolevulinic acid (LD50) in vitro leads to apoptosis in the normal urothelial cell line UROtsa and to necrosis in the tumor cell lines, respectively.


   Acknowledgments
 
We thank Sabine Dietrich, Nina Nießl, and Rudolf Jung for excellent technical assistance, and metaGen Pharmaceuticals for providing the oligonucleotide arrays.


   Footnotes
 
Grant support: Deutsche Krebshilfe grant (R. Knuechel).

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.

Note: P.J. Wild and R.C. Krieg contributed equally to this work.

8 http://genome.ucsc.edu/ Back

9 http://bioinfo.cnio.es/Sotarray/ Back

10 http://www.geneontology.org Back

Received 6/ 7/04; revised 1/15/05; accepted 2/ 3/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stapleton M, Rhodes LE. Photosensitizers for photodynamic therapy of cutaneous disease. J Dermatolog Treat 2003;14:107–12.[CrossRef][Medline]
  2. Dougherty TJ, Gomer CJ, Henderson BW, et al. Photodynamic therapy. J Natl Cancer Inst 1998;90:889–905.[Abstract/Free Full Text]
  3. Capella MA, Capella LS. A light in multidrug resistance: photodynamic treatment of multidrug-resistant tumors. J Biomed Sci 2003;10:361–6.[CrossRef][Medline]
  4. Ortel B, Sharlin D, O'Donnell D, Sinha AK, Maytin EV, Hasan T. Differentiation enhances aminolevulinic acid-dependent photodynamic treatment of LNCaP prostate cancer cells. Br J Cancer 2002;87:1321–7.[CrossRef][Medline]
  5. Ackroyd R, Kelty CJ, Brown NJ, Stephenson TJ, Stoddard CJ, Reed MW. Eradication of dysplastic Barrett's oesophagus using photodynamic therapy: long-term follow-up. Endoscopy 2003;35:496–501.[CrossRef][Medline]
  6. Shackley DC, Briggs C, Whitehurst C, et al. Photodynamic therapy for superficial bladder cancer. Expert Rev Anticancer Ther 2001;1:523–30.[CrossRef][Medline]
  7. Krieg RC, Uihlein D, Murthum T, et al. Improving the use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) for the gastrointestinal tract by esterification—an in vitro study. Cell Mol Biol (Noisy-le-grand) 2002;48:917–23.
  8. Hinnen P, de Rooij FW, van Velthuysen ML, et al. Biochemical basis of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation: a study in patients with (pre)malignant lesions of the oesophagus. Br J Cancer 1998;78:679–82.[Medline]
  9. Krieg RC, Messmann H, Rauch J, Seeger S, Knuechel R. Metabolic characterization of tumor cell-specific protoporphyrin IX accumulation after exposure to 5-aminolevulinic acid in human colonic cells. Photochem Photobiol 2002;76:518–25.[CrossRef][Medline]
  10. Rigby CC, Franks LM. A human tissue culture cell line from a transitional cell tumour of the urinary bladder: growth, chromosone pattern and ultrastructure. Br J Cancer 1970;24:746–54.[Medline]
  11. Petzoldt JL, Leigh IM, Duffy PG, Sexton C, Masters JR. Immortalisation of human urothelial cells. Urol Res 1995;23:377–80.[CrossRef][Medline]
  12. Thomas DR, Philpott GW, Jaffe BM. Prostaglandin E (PGE) control of cell proliferation in vitro: characteristics of HT-29. J Surg Res 1974;16:463–5.[CrossRef][Medline]
  13. Seidl J, Rauch J, Krieg RC, Appel S, Baumgartner R, Knuechel R. Optimization of differential photodynamic effectiveness between normal and tumor urothelial cells using 5-aminolevulinic acid-induced protoporphyrin IX as sensitizer. Int J Cancer 2001;92:671–7.[CrossRef][Medline]
  14. Schmitt AO, Specht T, Beckmann G, et al. Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues. Nucleic Acids Res 1999;27:4251–60.[Abstract/Free Full Text]
  15. Wissmann C, Wild PJ, Kaiser S, et al. WIF1, a component of the Wnt pathway, is down-regulated in prostate, breast, lung, and bladder cancer. J Pathol 2003;201:204–12.[CrossRef][Medline]
  16. Grutzmann R, Foerder M, Alldinger I, et al. Gene expression profiles of microdissected pancreatic ductal adenocarcinoma. Virchows Arch 2003;443:508–17.[CrossRef][Medline]
  17. Dopazo J, Carazo JM. Phylogenetic reconstruction using an unsupervised growing neural network that adopts the topology of a phylogenetic tree. J Mol Evol 1997;44:226–33.[CrossRef][Medline]
  18. Herrero J, Valencia A, Dopazo J. A hierarchical unsupervised growing neural network for clustering gene expression patterns. Bioinformatics 2001;17:126–36.[Abstract/Free Full Text]
  19. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000;25:25–9.[CrossRef][Medline]
  20. Warrington JA, Nair A, Mahadevappa M, Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics 2000;2:143–7.[Abstract/Free Full Text]
  21. Nishii Y, Morishima M, Kakehi Y, et al. CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line. FEBS Lett 2000;465:153–6.[CrossRef][Medline]
  22. Shimizu N, Ohta M, Fujiwara C, et al. Expression of a novel immediate early gene during 12-O-tetradecanoylphorbol-13-acetate-induced macrophagic differentiation of HL-60 cells. J Biol Chem 1991;266:12157–61.[Abstract/Free Full Text]
  23. Keyse SM, Emslie EA. Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase. Nature 1992;359:644–7.[CrossRef][Medline]
  24. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 1996;85:803–15.[CrossRef][Medline]
  25. Luo Y, Kessel D. Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol 1997;66:479–83.[Medline]
  26. Ben-Hur E, Oetjen J, Horowitz B. Silicon phthalocyanine Pc 4 and red light causes apoptosis in HIV-infected cells. Photochem Photobiol 1997;65:456–60.[Medline]
  27. Laukka MA, Wang KK, Bonner JA. Apoptosis occurs in lymphoma cells but not in hepatoma cells following ionizing radiation and photodynamic therapy. Dig Dis Sci 1994;39:2467–75.[CrossRef][Medline]
  28. He XY, Sikes RA, Thomsen S, Chung LW, Jacques SL. Photodynamic therapy with photofrin II induces programmed cell death in carcinoma cell lines. Photochem Photobiol 1994;59:468–73.[Medline]
  29. Noodt BB, Berg K, Stokke T, Peng Q, Nesland JM. Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX. Br J Cancer 1996;74:22–9.[Medline]
  30. Moor AC. Signaling pathways in cell death and survival after photodynamic therapy. J Photochem Photobiol B 2000;57:1–13.[CrossRef][Medline]
  31. Gomer CJ, Ferrario A, Hayashi N, Rucker N, Szirth BC, Murphree AL. Molecular, cellular, and tissue responses following photodynamic therapy. Lasers Surg Med 1988;8:450–63.[Medline]
  32. Agarwal ML, Larkin HE, Zaidi SI, Mukhtar H, Oleinick NL. Phospholipase activation triggers apoptosis in photosensitized mouse lymphoma cells. Cancer Res 1993;53:5897–902.[Abstract/Free Full Text]
  33. Oleinick NL, Evans HH. The photobiology of photodynamic therapy: cellular targets and mechanisms. Radiat Res 1998;150:S146–56.[Medline]
  34. Krieg RC, Messmann H, Schlottmann K, et al. Intracellular localization is a cofactor for the phototoxicity of protoporphyrin IX in the gastrointestinal tract: in vitro study. Photochem Photobiol 2003;78:393–9.[CrossRef][Medline]
  35. Verwanger T, Sanovic R, Aberger F, Frischauf AM, Krammer B. Gene expression pattern following photodynamic treatment of the carcinoma cell line A-431 analysed by cDNA arrays. Int J Oncol 2002;21:1353–9.[Medline]
  36. Luna MC, Wong S, Gomer CJ. Photodynamic therapy mediated induction of early response genes. Cancer Res 1994;54:1374–80.[Abstract/Free Full Text]
  37. Xue LY, He J, Oleinick NL. Rapid tyrosine phosphorylation of HS1 in the response of mouse lymphoma L5178Y-R cells to photodynamic treatment sensitized by the phthalocyanine Pc 4. Photochem Photobiol 1997;66:105–13.[Medline]
  38. Golab J, Nowis D, Skrzycki M, et al. Antitumor effects of photodynamic therapy are potentiated by 2-methoxyestradiol. A superoxide dismutase inhibitor. J Biol Chem 2003;278:407–14.[Abstract/Free Full Text]
  39. Lottner C, Knuechel R, Bernhardt G, Brunner H. Combined chemotherapeutic and photodynamic treatment on human bladder cells by hematoporphyrin-platinum(II) conjugates. Cancer Lett 2004;203:171–80.[CrossRef][Medline]
  40. Ferrario A, Chantrain CF, von Tiehl K, et al. The matrix metalloproteinase inhibitor prinomastat enhances photodynamic therapy responsiveness in a mouse tumor model. Cancer Res 2004;64:2328–32.[Abstract/Free Full Text]
  41. Plaetzer K, Kiesslich T, Krammer B, Hammerl P. Characterization of the cell death modes and the associated changes in cellular energy supply in response to AlPcS4-PDT. Photochem Photobiol Sci 2002;1:172–7.[CrossRef][Medline]
  42. Almeida RD, Manadas BJ, Carvalho AP, Duarte CB. Intracellular signaling mechanisms in photodynamic therapy. Biochim Biophys Acta 2004;1704:59–86.[Medline]
  43. Granville DJ, Carthy CM, Jiang H, Shore GC, McManus BM, Hunt DW. Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy. FEBS Lett 1998;437:5–10.[CrossRef][Medline]
  44. Grebenova D, Kuzelova K, Smetana K, et al. Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells. J Photochem Photobiol B 2003;69:71–85.[CrossRef][Medline]
  45. Schlottmann K, Wachs FP, Krieg RC, Kullmann F, Scholmerich J, Rogler G. Characterization of bile salt-induced apoptosis in colon cancer cell lines. Cancer Res 2000;60:4270–6.[Abstract/Free Full Text]
  46. Bilim VN, Tomita Y, Kawasaki T, Takeda M, Takahashi K. Adriamycin (ADM) induced apoptosis in transitional cell cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction. Apoptosis 1997;2:207–13.[CrossRef][Medline]
  47. Proskuryakov SY, Konoplyannikov AG, Gabai VL. Necrosis: a specific form of programmed cell death? Exp Cell Res 2003;283:1–16.[CrossRef][Medline]
  48. Subramaniam S, Strelau J, Unsicker K. Growth differentiation factor-15 prevents low potassium-induced cell death of cerebellar granule neurons by differential regulation of Akt and ERK pathways. J Biol Chem 2003;278:8904–12.[Abstract/Free Full Text]
  49. Hsiao EC, Koniaris LG, Zimmers-Koniaris T, Sebald SM, Huynh TV, Lee SJ. Characterization of growth-differentiation factor 15, a transforming growth factor superfamily member induced following liver injury. Mol Cell Biol 2000;20:3742–51.[Abstract/Free Full Text]
  50. Simank HG, Manggold J, Sebald W, et al. Bone morphogenetic protein-2 and growth and differentiation factor-5 enhance the healing of necrotic bone in a sheep model. Growth Factors 2001;19:247–57.[Medline]
  51. Simank HG, Sergi C, Jung M, et al. Effects of local application of growth and differentiation factor-5 (GDF-5) in a full-thickness cartilage defect model. Growth Factors 2004;22:35–43.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
S. Battah, S. Balaratnam, A. Casas, S. O'Neill, C. Edwards, A. Batlle, P. Dobbin, and A. J. MacRobert
Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates
Mol. Cancer Ther., March 1, 2007; 6(3): 876 - 885.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
P. G. Febbo and P. W. Kantoff
Noise and Bias in Microarray Analysis of Tumor Specimens
J. Clin. Oncol., August 10, 2006; 24(23): 3719 - 3721.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wild, P. J.
Right arrow Articles by Knuechel, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wild, P. J.
Right arrow Articles by Knuechel, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics