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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

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

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/

⁹ http://bioinfo.cnio.es/Sotarray/

¹⁰ http://www.geneontology.org

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