Gene expression pattern following PTD

In order to explain mechanisms of the cell response to photodynamic treatment with aminolevulinic acid-induced protoporphyrin IX and consequently improve the therapy, the modulation of gene expression after treatment of the human tumor cell line A-43 1 shall be investigated by a high density cDNA array. Photodynamic Tumor Therapy (PDT) is a most promising novel approach in tumor treatment. It is based on the preferential uptake and retention of a photosensitizer by tumors and the production of reactive oxygen species (ROS) during irradiation of the accumulated photosensitizer in the tumor area with visible light. The resulting primary tumor damage occurs by mainly direct killing of the neoplastic cells. PDT is characterized by, both, high tumor specificity and low side effects in contrast to radiation- or chemotherapy. The photosensitizer protoporphyrin IX (PpIX), endogenously generated by the precursor aminolevulinic acid (ALA) in the heme biosynthetic pathway, is successfully applied in several clinical areas, when accumulated by external addition of ALA. In spite of very encouraging results of ALA-PDT reported until now the therapy could still be improved, if more information on basic mechanisms underlying the obvious cellular effects and the curative outcome would be available. The aim of the present project is therefore to analyze by a detailed, comprehensive and interlinked approach the modulation of gene expression following photodynamic treatment with ALA/PpIX and mediated by reactive oxygen species. The squamous cell carcinoma cell line A-43 1 will be used as a human tumor cell model. The major analysis tool will be expression analysis based on high density cDNA arrays which we already used in preliminary experiments. cDNAs from cells subjected to PDT and from control cells are hybridized onto filters containing PCR fragments representing approximately 30000 different human transcripts. Differentially expressed genes will be identified by a data analysis program and verified by Northern blot analysis or sub-arrays containing only the candidate genes. Based on our preliminary investigations we expect to find genes already described in the context of PDT, known genes not previously found with PDT and novel genes of unknown function.

Analysis of alterations in the gene expressions pattern of the human carcinoma cell line A-431 at different time points after photodynamic treatment with endogenous protoporphyrin IX by cDNA-array technique could show that the observed PDT-generated necrosis is not only a passive event but is accompanied by a considerable alteration in gene expression. Since necrosis has a strong immune stimulatory effect, the present results might have an impact on patient treatment. Photodynamic Therapy (PDT) is a relatively new modality in treatment of malignant and non-malignant diseases. In spite of encouraging clinical results, the basic mechanisms leading to cell death are not fully understood. We therefore set out to analyse alterations in the gene expressions pattern of the human squamous cell carcinoma cell line A-431 at different time points after photodynamic treatment (pt) with endogenous PpIX by cDNA-array technique. Like that we could yield expression kinetics for a wide range of genes which are relevant in carcinogenesis, proliferation, stress response and signal transduction. Cells were treated with the optimum protocol providing sufficient intact mRNA for analysis. RNA was isolated at 1.5, 3, 5 and 8 hours pt including controls, radioactively labeled by reverse transcription with 33P-dCTP and hybridized onto macroarray PCR filters containing PCR products of 2135 different genes. Verification of observed expression changes of genes of interest was carried out by Real-Time PCR. Due to the mainly necrotic cell death occurring during ALA-PDT (negative for apoptosis in assays) we found only two genes with up-regulated expression compared to the untreated control: the stress response genes c-fos and c-jun (up-regulation within 1.5 - 8 hrs pt; maximum 217-fold at 1.5 hrs and 51-fold at 8 hrs, respectively). The quantity of down-regulated genes showed a strong increase over time pt based on array results alone: 1 gene at 1.5 hours, 10 genes at 3 hours, 60 at 5 hours and 107 genes at 8 hours. By Real-Time PCR quantification 10 out of 15 selected down-regulated genes could be verified. Among these verified genes were the cell attachment gene fibronectin, which might hint to an active detachment as a consequence of PDT-stress, the proliferation- and cell-cycle-promoting genes proliferating cell nuclear antigen (PCNA), CDC-like kinase 2 (CLK2), retinoblastoma 1 (RB1) and cell division cycle 2(G1 to S and G2 to M) (CDC2), indicating an active deceleration of proliferation, as well as the apoptosis inducing genes c-myc and Fas-associated via death domain (FADD) showing a possible circumvention of pro-apoptotic pathways. From these results in can be concluded that PDT-generated necrosis is not only a passive event but is accompanied by a considerable alteration in gene expression. The occurrence of necrosis could be due to the localisation of PpIX in cellular membranes, which are thus damaged by PDT.