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180 The nuclear pore complex is a target in the cellular stress response to ionizing radiation
$62
March 12 - 15
MSI, most studies on MSI have been carried out in Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and in a proportion of sp~r~clic colorectal tumors. In 1997, an International Workshop organized by the National Cancer Institute, identified a working "reference" panel of microsatellite markers, also called the Bethesda reference panel. Next to this panel of 5 markers (2 mononucleotide, 3 dinucleotide), a list of alternative loci was published. The optimal set of loci to diagnose MSI in cancer other than colorectal cancer was said likely to be different. Therefore, the aim of this project was to compare the sensitivity of a panel of 10 Bethesda markers with a panel of 12 markers with high reported frequencies of MSI and LOH in HNSCC. Material and methods: 82 patients with advanced HNSCC of different sites were included. Tumoral DNA was extracted from pretreatment fresh frozen tumoral biopsies, while pared normal DNA was extracted from either paraffin-embedded material or peripheral lymphocytes. Multiplex PCR reactions on pared normal and tumoral DNA were performed with the Bethesda markers as well as with the new panel. Fluorescent labeled markers were used and automatic fragmentanalysis was carried out using the ABI PRISM3100 genetic analyzer, thereby evaluating the fragments for MSI and LOH. Only those fragments in which both tumoral and normal DNA showed signals above background level, were considered assessable. Samples were run in duplicate to achieve higher reliability. Results: At this time point, analyses for the new panel are completed. MSI at one locus (MSI-Low) was found in 11 patients, while MSI at two or more loci (MSI-High) was detected in 7 cases. The highest MSI levels were found with markers D8S133 (9/57 assessable cases), D13S154 (5/71 cases), D8S264 (5/80 cases) and D17S799 (5/70 cases). LOH was seen at 1 locus in 12 patients, and at more than one locus in 28 patients. In accordance to literature, most LOH was found on chromosome arms 3p ( D 3 S 1 6 1 1 : 1 9 / 4 5 informative cases; D 3 S 1 2 8 4 : 7 / 2 0 cases), 8p ( D 8 S 1 3 3 : 8 / 2 9 cases), 9p (D9S171:18/42 cases; D 9 S 1 6 1 : 1 3 / 3 5 cases) and 17p ( D 1 7 S 7 9 9 : 1 1 / 3 8 cases). All PCR reactions with the Bethesda microsatellite markers have been carried out. However, these analyses are not completed yet. Conclusions: In this series of 82 patients with HNSCC a comparison was made of the sensitivity of the Bethesda reference markers versus a panel of literature-based makers to assess MSI and LOH. First results show that the new panel reveals MSI and LOH percentages in accordance to literature. At the time of the congress, the comparison with the Bethesda reference panel will be completed. If the literature-based microsatellite markers prove to be more sensitive in the detection of MSI and LOH than the Bethesda markers, these results might indicate that indeed tumor-specific sets are needed when assessing microsatellite alterations. 180 THE NUCLEAR PORE COMPLEX I S A TARGET I N THE CELLULAR STRESS RESPONSE TO I O N I Z I N G R A D I A T I O N
A. Broggini-Tenzer, A. Hollenstein, B. Hofstetter, D. Dickinson, M. Pruschy Department of Radiation Oncology, Universitiy Hospital ZQrich, Switzerland Objective: The identification of tumor-specific and therapyinduced processes reveals important information on the cellular phenotype and leads to potential novel targets for anticancer therapies. We previoulsy developed a novel phage display screening technique (Tenzer et al., Proteomics 2004) to profile the entire cellular proteome for treatment-induced proteolytic activities. The aim of this study was the characterization of a treatment-specific profile for enzyme activities regulated by ionizing radiation and to identify signaling processes which are co-regulated by these proteolytic activities. Material and methods: The screening was performed with a T7-phage-based peptide phage display library which was specifically constructed to detect posttranslational modifications of peptides. For this study we focused on the identification of peptides recognized by proteolytic enzyme activities. An iterative subtractive screening was performed with a highly diverse phage library (10xE9 different substrate peptides) to select for peptide recognition sequences specific for enzymes active under
P r o f f e r e d Papers
normal conditions and in irradiated cells respectively (human radioresistant adenocarcinoma cell line SW480, 10Gy). The identified substrate peptide sequences were validated in vitro by plaque-assays with phage mono-cultures and by ELISAassays with synthetic peptides. The role of the selected peptide sequences in vivo was investigated with synthetic cell permeable peptide substrates (internalized HIV-TAT-proteins) but also linked to potential endogenous counterparts on the basis of datasearch analysis. Classical biochemical techniques (proliferation assays, western blot, retroviral transfected cells, siRNA) were used to identify the relevance of these candidate proteins in the response to ionizing radiation and to dissect related signaling processes. Results: In this study we applied the subtractive phage display screening method for the first time in a discovery-oriented approach and used the radiationresistant adenocarcinoma cell line SW480 to identify radiationspecific substrate peptides sequences. Multiple peptide sequences were selected that are specifically recognized and cleaved by radiation-dependent proteolytic enzyme activities and one substrate sequence was investigated in detail based of its high homology to a peptide domain within an endogenous component of the nuclear pore complex. Substrate specificity was validated with synthetic peptides using cell extracts derived from control and irradiated SW480 cells and the exact cleavage site was determined by MALDITOF. Treatment-dependent endogenous cleavage (in vivo) of the selected sequences and effects on the cellular phenotype were also determined using internalized substrate-peptides (H IV-TAT-fusion-peptides). Interestingly, cells with internalized TAT-peptide displayed a more radioresistant phenotype. A putative substrate sequence with high homology to the peptide identified in this study is present in the human nucleoporin protein Nup50 (Npap60), which is a relevant component of protein shuttling machinery through the nuclear pore complex. This sequence is part of an important binding site for additional elements involved in the nuclear shuttling process (importin a/b). Western blotting experiments revealed that the endogenous protein Nup50 carrying the homologous peptide sequence is also a substrate for ionizing radiationregulated serine proteases in vivo. Subsequently we investigated therelevance of functionally intact Nup50 in the cellular stress response to irradiation. Interestingly, cells with siRNA-silenced Nup50 resulted in a more radiosensitive phenotype while Nup50-overexpressing cells displayed an increased radiation resistance. Conclusions: The novel subtractive peptide phage display technique represents a complementary genome-wide screening approach for the identification of treatment-induced posttranslational peptide modifications. Based on a specific substrate peptide sequence and its endogenous counterpart we discovered a novel ionzing radiation-regulated function of the nuclear protein shuttling machinery and its effect on the cellular radiosensitivity. 181 CELL ADHESION TO FIBRONECTIN STRONGLY ATTENUATES PHARMACOLOGICAL EGF-RECEPTOR T Y R O S I N E KINASE I N H I B I T I O N I N AN I N T E G R I N LINKED K I N A S E - I N D E P E N D E N T MANNER
I. Eke, A. Mischkus, M. Baumann, N. Cordes OncoRay - Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany Objective: Specific integrins tightly interact with the epidermal growth factor receptor (EGF-R) through a protein complex consisting of integrin-linked kinase (ILK), PINCH-1 and Nck-2 to control cell survival and proliferation. As the EGF-R has emerged as target in anticancer therapies, this study elucidates the impact of cell adhesion to fibronectin (FN) on the pharmacological inhibition of the EGF-R tyrosine kinase in unirradiated and irradiated human FaDu squamous cell carcinoma cells stably transfected with ILK contructs. Material and methods: Experiments were performed in cells stably transfected with wild-type (wt) or constitutive active (ca) ILK. Cells were grown on FN or poly-I-lysine (poly-L) under serum presence or depletion. Colony formation and growth curve assays, flow cytometric cell cycle analysis
March 12 - 15
MSI, most studies on MSI have been carried out in Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and in a proportion of sp~r~clic colorectal tumors. In 1997, an International Workshop organized by the National Cancer Institute, identified a working "reference" panel of microsatellite markers, also called the Bethesda reference panel. Next to this panel of 5 markers (2 mononucleotide, 3 dinucleotide), a list of alternative loci was published. The optimal set of loci to diagnose MSI in cancer other than colorectal cancer was said likely to be different. Therefore, the aim of this project was to compare the sensitivity of a panel of 10 Bethesda markers with a panel of 12 markers with high reported frequencies of MSI and LOH in HNSCC. Material and methods: 82 patients with advanced HNSCC of different sites were included. Tumoral DNA was extracted from pretreatment fresh frozen tumoral biopsies, while pared normal DNA was extracted from either paraffin-embedded material or peripheral lymphocytes. Multiplex PCR reactions on pared normal and tumoral DNA were performed with the Bethesda markers as well as with the new panel. Fluorescent labeled markers were used and automatic fragmentanalysis was carried out using the ABI PRISM3100 genetic analyzer, thereby evaluating the fragments for MSI and LOH. Only those fragments in which both tumoral and normal DNA showed signals above background level, were considered assessable. Samples were run in duplicate to achieve higher reliability. Results: At this time point, analyses for the new panel are completed. MSI at one locus (MSI-Low) was found in 11 patients, while MSI at two or more loci (MSI-High) was detected in 7 cases. The highest MSI levels were found with markers D8S133 (9/57 assessable cases), D13S154 (5/71 cases), D8S264 (5/80 cases) and D17S799 (5/70 cases). LOH was seen at 1 locus in 12 patients, and at more than one locus in 28 patients. In accordance to literature, most LOH was found on chromosome arms 3p ( D 3 S 1 6 1 1 : 1 9 / 4 5 informative cases; D 3 S 1 2 8 4 : 7 / 2 0 cases), 8p ( D 8 S 1 3 3 : 8 / 2 9 cases), 9p (D9S171:18/42 cases; D 9 S 1 6 1 : 1 3 / 3 5 cases) and 17p ( D 1 7 S 7 9 9 : 1 1 / 3 8 cases). All PCR reactions with the Bethesda microsatellite markers have been carried out. However, these analyses are not completed yet. Conclusions: In this series of 82 patients with HNSCC a comparison was made of the sensitivity of the Bethesda reference markers versus a panel of literature-based makers to assess MSI and LOH. First results show that the new panel reveals MSI and LOH percentages in accordance to literature. At the time of the congress, the comparison with the Bethesda reference panel will be completed. If the literature-based microsatellite markers prove to be more sensitive in the detection of MSI and LOH than the Bethesda markers, these results might indicate that indeed tumor-specific sets are needed when assessing microsatellite alterations. 180 THE NUCLEAR PORE COMPLEX I S A TARGET I N THE CELLULAR STRESS RESPONSE TO I O N I Z I N G R A D I A T I O N
A. Broggini-Tenzer, A. Hollenstein, B. Hofstetter, D. Dickinson, M. Pruschy Department of Radiation Oncology, Universitiy Hospital ZQrich, Switzerland Objective: The identification of tumor-specific and therapyinduced processes reveals important information on the cellular phenotype and leads to potential novel targets for anticancer therapies. We previoulsy developed a novel phage display screening technique (Tenzer et al., Proteomics 2004) to profile the entire cellular proteome for treatment-induced proteolytic activities. The aim of this study was the characterization of a treatment-specific profile for enzyme activities regulated by ionizing radiation and to identify signaling processes which are co-regulated by these proteolytic activities. Material and methods: The screening was performed with a T7-phage-based peptide phage display library which was specifically constructed to detect posttranslational modifications of peptides. For this study we focused on the identification of peptides recognized by proteolytic enzyme activities. An iterative subtractive screening was performed with a highly diverse phage library (10xE9 different substrate peptides) to select for peptide recognition sequences specific for enzymes active under
P r o f f e r e d Papers
normal conditions and in irradiated cells respectively (human radioresistant adenocarcinoma cell line SW480, 10Gy). The identified substrate peptide sequences were validated in vitro by plaque-assays with phage mono-cultures and by ELISAassays with synthetic peptides. The role of the selected peptide sequences in vivo was investigated with synthetic cell permeable peptide substrates (internalized HIV-TAT-proteins) but also linked to potential endogenous counterparts on the basis of datasearch analysis. Classical biochemical techniques (proliferation assays, western blot, retroviral transfected cells, siRNA) were used to identify the relevance of these candidate proteins in the response to ionizing radiation and to dissect related signaling processes. Results: In this study we applied the subtractive phage display screening method for the first time in a discovery-oriented approach and used the radiationresistant adenocarcinoma cell line SW480 to identify radiationspecific substrate peptides sequences. Multiple peptide sequences were selected that are specifically recognized and cleaved by radiation-dependent proteolytic enzyme activities and one substrate sequence was investigated in detail based of its high homology to a peptide domain within an endogenous component of the nuclear pore complex. Substrate specificity was validated with synthetic peptides using cell extracts derived from control and irradiated SW480 cells and the exact cleavage site was determined by MALDITOF. Treatment-dependent endogenous cleavage (in vivo) of the selected sequences and effects on the cellular phenotype were also determined using internalized substrate-peptides (H IV-TAT-fusion-peptides). Interestingly, cells with internalized TAT-peptide displayed a more radioresistant phenotype. A putative substrate sequence with high homology to the peptide identified in this study is present in the human nucleoporin protein Nup50 (Npap60), which is a relevant component of protein shuttling machinery through the nuclear pore complex. This sequence is part of an important binding site for additional elements involved in the nuclear shuttling process (importin a/b). Western blotting experiments revealed that the endogenous protein Nup50 carrying the homologous peptide sequence is also a substrate for ionizing radiationregulated serine proteases in vivo. Subsequently we investigated therelevance of functionally intact Nup50 in the cellular stress response to irradiation. Interestingly, cells with siRNA-silenced Nup50 resulted in a more radiosensitive phenotype while Nup50-overexpressing cells displayed an increased radiation resistance. Conclusions: The novel subtractive peptide phage display technique represents a complementary genome-wide screening approach for the identification of treatment-induced posttranslational peptide modifications. Based on a specific substrate peptide sequence and its endogenous counterpart we discovered a novel ionzing radiation-regulated function of the nuclear protein shuttling machinery and its effect on the cellular radiosensitivity. 181 CELL ADHESION TO FIBRONECTIN STRONGLY ATTENUATES PHARMACOLOGICAL EGF-RECEPTOR T Y R O S I N E KINASE I N H I B I T I O N I N AN I N T E G R I N LINKED K I N A S E - I N D E P E N D E N T MANNER
I. Eke, A. Mischkus, M. Baumann, N. Cordes OncoRay - Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany Objective: Specific integrins tightly interact with the epidermal growth factor receptor (EGF-R) through a protein complex consisting of integrin-linked kinase (ILK), PINCH-1 and Nck-2 to control cell survival and proliferation. As the EGF-R has emerged as target in anticancer therapies, this study elucidates the impact of cell adhesion to fibronectin (FN) on the pharmacological inhibition of the EGF-R tyrosine kinase in unirradiated and irradiated human FaDu squamous cell carcinoma cells stably transfected with ILK contructs. Material and methods: Experiments were performed in cells stably transfected with wild-type (wt) or constitutive active (ca) ILK. Cells were grown on FN or poly-I-lysine (poly-L) under serum presence or depletion. Colony formation and growth curve assays, flow cytometric cell cycle analysis