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DNA repair — A determinant for sensitivity to radiation in lungcancer?
Radiation
162
•6]
Role of epidermal growth factor receptor and its blockade in tumor radioresponse
T. Akimoto, N. Mitsuhashi, L. Milas. Gunma University, Maebashi,
Japan, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA Growth factors are essential regulators of cell growth and are involved in tumor and normal tissue response to cytotoxic agents. Our study investigated the involvement of epidermal growth factor receptor (EGFR) and its blockade in in vivo tumor radioresponse. Nine different murine carcinomas, that greatly differ in their radioresponse and in their susceptibility to radiation-induced apoptosis, were analyzed for EGFR expression using western blot analysis. The expression of EGFR varied among tumors by 21-fold and its magnitude positively correlated with poorer tumor radioresponse, determined by both tumor growth delay and tumor cure. These tumors also differed greatly in the expression of cyclin D1, a growth factor sensor. The level of cyclin D1 expression paralleled that of EGFR, and like EGFR, positively correlated with tumor resistance to radiation. Radiation affected transduction and other molecular processes involved in cell growth or cell death. A dose of 15 Gy activated EGFR phosphorylation and increased the activity of protein tyrosine kinase (PTK), but this occurred only in tumors with high EGFR expression. Also, radiation (15 Gy) had no influence on constitutive expression of cyclin D1 in radioresistant tumors, but led to its reduced expression in radiosensitive tumors. At the cellular level, radiation induced no significant apoptosis or change in the percentage of PCNA positive (proliferating) cells in tumors with high EGFR levels, but it induced significant apoptosis and a decrease in the percentage of proliferating cells in tumors with low EGFR expression. Also, regeneration of tumor cells that survived irradiation was more rapid in tumors with high EGFR expression. Using A431 human squamous cell carcinoma xenografts in nude mice, we observed that the blockade of EGFR with C225 anti-EGFR monoclonal antibody greatly enhanced tumor radioresponse by factors of 1.6 to 3.6. Overall, our findings suggest that EGFR expression is a major determinant of tumor radioresponse in vivo, which may have important clinical implications. The pretreatment assessment of EGFR expression could serve as a useful predictor of radiotherapy outcome and may assist in selecting an effective treatment modality, such as the blockade of EGFR receptor in combination with radiotherapy.
15•
DNA repair - A determinant for sensitivity to radiation in lungcancer?
R. Lewensohn, J. Ekedahl, B. Joseph, A. Nilsson, A. Polischouk, F. Sirzdn, B. Zhivotovsky. Cancer Centre Karolinska, Karolinska
Institutet, Stockholm, Sweden Small cell (SCLC) as opposed to non-small cell lung cancer (NSCLC) is known to be radiosensitive (RS). We have shown that when comparing SCLC and NSCLC cell types, SCLC cell lines in parallel to increased RS display less efficacy in repairing DNA double and single strand breaks induced by radiation. These data have been obtained using pulsed field gel electrophoresis and the comet assay. The DNAdependent protein kinase (DNA-PK), a serine/threonine kinase is an important component in the process of DNA-dsb repair. Analysing the lung carcinoma cell lines, here in order of increasing radioresistance, U-1285, U-1908, H-89, H-82 and U-1810 we found a correlation (r 2 = 0.818) with DNA-dsb rejoining. The RS was also correlated with DNA-PK activity and the content of the catalytic subunit of DNAPK (r 2 = 0.941 and r 2 = 0.944, respectively). DNA ligase activity was measured using a nicked substrate ([5 ,! -32P] Poly (dA) oligo (dT)). Comparison of ligase activities in two SCLC lines, U-1285 & U1906, and one NSCLC line, U-1810, showed that the U-1906 cell line displayed a significantly decreased DNA ligase activity as compared to the other two ceil lines. The lower activity was accompanied by lower phosphorylation of the ligase as assessed by western blotting. Interestingly, the U-lg06 cell line was the cell line most sensitive to DNA strand break generating drugs, such as VP16 and Topotecan. We conclude, that to explain sensitivities of the cell lines to agents causing
DNA strand breaks both the activity of DNA-PK as well as DNA ligases should be considered. Using X-ray doses of 2-8 Gy apoptosis was induced in the SCLC lines but not in NSCLC line, including U-1810. Analyses of caspase activation revealed in both the most radiosensitive (U-1285) as well as the radioresistant (U-1810) cell lines, that cytochrome c is released and caspase-9 and the executioner caspase3 are activated. However, cleavage of known nuclear substrates for caspase-3, as PARP, ICAD/DFF45 and DNA-PKcs, was observed only in the sensitive, but not in the resistant cells. Furthermore, the active caspase-3 relocalizes in the nucleus upon irradiation only in the SCLC cell line. It appears that in NSCLC the apoptotic process is inhibited downstream of mitochondrial changes and caspase activation, and most probably upstream of nuclear events. We suggest that increased sensitivity of SCLC (compared with NSCLC) cells to DNA strand breaks is related to a lowered activity of DNA repair proteins as well as to the nuclear presence of caspase-3 after irradiation, either augmenting DNA breakage or inactivating proteins involved in DNA repair.
[-~
The use of CT-simulation and digitally reconstructed radiographs (DRR's) in setup vedficaUon allows for smaller planning target volumes in lung cancer
S. Senan, J. van Sornsen de Koste, J. de Boer, B. Heijmen. University
Hospital Rotterdam, Rotterdam, The Netherlands Background: Significant setup deviations have been observed during treatment simulation for patients with lung cancer, relative to their position during the planning CT scan [De Boer Radiother Oncol 48 (S 1): 56, 1998]. This gives rise to systematic errors in setup verification as the simulator images commonly serve to define the reference setup. Aim: To minimize setup errors by using [a] DRRs as reference images in setup verification, and [b] a setup-correction protocol with an electronic portal imaging device (EPID). Methods: CT-scans were performed for 20 patients on a CTsimulator. An isocenter for treatment planning was defined with p~tients still on the scanner table, and demarcated using lead markers. A second scan was acquired in order to exclude displacement of the isocenter, which was registered using ink and tattoos. DRRs were generated and served as reference images for portal image analysis. Setup during treatment was studied using an EPID, and an off-line correction protocol with shrinking action level was implemented. Results: A total of 152 treatment fractions (7.6 fractions/patient) were analyzed. The random setup errors were 1.8, 2.0 and 1.9 mm (1 SD) for the lateral (L-R), superior-inferior (S-I) and anterior-posterior (A-P) directions, respectively. With a setup-correction protocol, the systematic errors were 1.5, 1.5 and 1.3 mm (1 SD). A limited number of corrections per patient (0.8) reflected the small random errors in our setup method. Had no correction protocol been applied, the systematic errors would have been 2.5, 3.4 and 2.4 mm. When digitized simulator films are used as reference images, the random errors are 2.0, 2.1 and 1.8 mm, and the corresponding systematic errors are 3.2, 3.6 and 1.7 mm [De Boer submitted]. The comparable systematic and random errors indicate that contour delineation and template matching on DRRs can be performed as accurately as with simulator images. With DRRs and a setup protocol, the required CTV-P'I'V margins were derived using coverage probability calculations [Stroom et al. Int J Radiat Oncol Biol Phys 1999; 43: 905]. Margins of 4, 5 and 4 mm, respectively, were needed for the L-R, S-I and A-P directions. Data from our previous study using simulator images (i.e. incorporating simulator set-up errors), indicated that margins of 10, 8 and 7 mm were required. Conclusion: The treatment planning margins necessary for external setup accuracy are improved by about 50% when a conventional simulation step for lung cancer patients is omitted, and when DRRs are used in an off-line correction protocol.
162
•6]
Role of epidermal growth factor receptor and its blockade in tumor radioresponse
T. Akimoto, N. Mitsuhashi, L. Milas. Gunma University, Maebashi,
Japan, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA Growth factors are essential regulators of cell growth and are involved in tumor and normal tissue response to cytotoxic agents. Our study investigated the involvement of epidermal growth factor receptor (EGFR) and its blockade in in vivo tumor radioresponse. Nine different murine carcinomas, that greatly differ in their radioresponse and in their susceptibility to radiation-induced apoptosis, were analyzed for EGFR expression using western blot analysis. The expression of EGFR varied among tumors by 21-fold and its magnitude positively correlated with poorer tumor radioresponse, determined by both tumor growth delay and tumor cure. These tumors also differed greatly in the expression of cyclin D1, a growth factor sensor. The level of cyclin D1 expression paralleled that of EGFR, and like EGFR, positively correlated with tumor resistance to radiation. Radiation affected transduction and other molecular processes involved in cell growth or cell death. A dose of 15 Gy activated EGFR phosphorylation and increased the activity of protein tyrosine kinase (PTK), but this occurred only in tumors with high EGFR expression. Also, radiation (15 Gy) had no influence on constitutive expression of cyclin D1 in radioresistant tumors, but led to its reduced expression in radiosensitive tumors. At the cellular level, radiation induced no significant apoptosis or change in the percentage of PCNA positive (proliferating) cells in tumors with high EGFR levels, but it induced significant apoptosis and a decrease in the percentage of proliferating cells in tumors with low EGFR expression. Also, regeneration of tumor cells that survived irradiation was more rapid in tumors with high EGFR expression. Using A431 human squamous cell carcinoma xenografts in nude mice, we observed that the blockade of EGFR with C225 anti-EGFR monoclonal antibody greatly enhanced tumor radioresponse by factors of 1.6 to 3.6. Overall, our findings suggest that EGFR expression is a major determinant of tumor radioresponse in vivo, which may have important clinical implications. The pretreatment assessment of EGFR expression could serve as a useful predictor of radiotherapy outcome and may assist in selecting an effective treatment modality, such as the blockade of EGFR receptor in combination with radiotherapy.
15•
DNA repair - A determinant for sensitivity to radiation in lungcancer?
R. Lewensohn, J. Ekedahl, B. Joseph, A. Nilsson, A. Polischouk, F. Sirzdn, B. Zhivotovsky. Cancer Centre Karolinska, Karolinska
Institutet, Stockholm, Sweden Small cell (SCLC) as opposed to non-small cell lung cancer (NSCLC) is known to be radiosensitive (RS). We have shown that when comparing SCLC and NSCLC cell types, SCLC cell lines in parallel to increased RS display less efficacy in repairing DNA double and single strand breaks induced by radiation. These data have been obtained using pulsed field gel electrophoresis and the comet assay. The DNAdependent protein kinase (DNA-PK), a serine/threonine kinase is an important component in the process of DNA-dsb repair. Analysing the lung carcinoma cell lines, here in order of increasing radioresistance, U-1285, U-1908, H-89, H-82 and U-1810 we found a correlation (r 2 = 0.818) with DNA-dsb rejoining. The RS was also correlated with DNA-PK activity and the content of the catalytic subunit of DNAPK (r 2 = 0.941 and r 2 = 0.944, respectively). DNA ligase activity was measured using a nicked substrate ([5 ,! -32P] Poly (dA) oligo (dT)). Comparison of ligase activities in two SCLC lines, U-1285 & U1906, and one NSCLC line, U-1810, showed that the U-1906 cell line displayed a significantly decreased DNA ligase activity as compared to the other two ceil lines. The lower activity was accompanied by lower phosphorylation of the ligase as assessed by western blotting. Interestingly, the U-lg06 cell line was the cell line most sensitive to DNA strand break generating drugs, such as VP16 and Topotecan. We conclude, that to explain sensitivities of the cell lines to agents causing
DNA strand breaks both the activity of DNA-PK as well as DNA ligases should be considered. Using X-ray doses of 2-8 Gy apoptosis was induced in the SCLC lines but not in NSCLC line, including U-1810. Analyses of caspase activation revealed in both the most radiosensitive (U-1285) as well as the radioresistant (U-1810) cell lines, that cytochrome c is released and caspase-9 and the executioner caspase3 are activated. However, cleavage of known nuclear substrates for caspase-3, as PARP, ICAD/DFF45 and DNA-PKcs, was observed only in the sensitive, but not in the resistant cells. Furthermore, the active caspase-3 relocalizes in the nucleus upon irradiation only in the SCLC cell line. It appears that in NSCLC the apoptotic process is inhibited downstream of mitochondrial changes and caspase activation, and most probably upstream of nuclear events. We suggest that increased sensitivity of SCLC (compared with NSCLC) cells to DNA strand breaks is related to a lowered activity of DNA repair proteins as well as to the nuclear presence of caspase-3 after irradiation, either augmenting DNA breakage or inactivating proteins involved in DNA repair.
[-~
The use of CT-simulation and digitally reconstructed radiographs (DRR's) in setup vedficaUon allows for smaller planning target volumes in lung cancer
S. Senan, J. van Sornsen de Koste, J. de Boer, B. Heijmen. University
Hospital Rotterdam, Rotterdam, The Netherlands Background: Significant setup deviations have been observed during treatment simulation for patients with lung cancer, relative to their position during the planning CT scan [De Boer Radiother Oncol 48 (S 1): 56, 1998]. This gives rise to systematic errors in setup verification as the simulator images commonly serve to define the reference setup. Aim: To minimize setup errors by using [a] DRRs as reference images in setup verification, and [b] a setup-correction protocol with an electronic portal imaging device (EPID). Methods: CT-scans were performed for 20 patients on a CTsimulator. An isocenter for treatment planning was defined with p~tients still on the scanner table, and demarcated using lead markers. A second scan was acquired in order to exclude displacement of the isocenter, which was registered using ink and tattoos. DRRs were generated and served as reference images for portal image analysis. Setup during treatment was studied using an EPID, and an off-line correction protocol with shrinking action level was implemented. Results: A total of 152 treatment fractions (7.6 fractions/patient) were analyzed. The random setup errors were 1.8, 2.0 and 1.9 mm (1 SD) for the lateral (L-R), superior-inferior (S-I) and anterior-posterior (A-P) directions, respectively. With a setup-correction protocol, the systematic errors were 1.5, 1.5 and 1.3 mm (1 SD). A limited number of corrections per patient (0.8) reflected the small random errors in our setup method. Had no correction protocol been applied, the systematic errors would have been 2.5, 3.4 and 2.4 mm. When digitized simulator films are used as reference images, the random errors are 2.0, 2.1 and 1.8 mm, and the corresponding systematic errors are 3.2, 3.6 and 1.7 mm [De Boer submitted]. The comparable systematic and random errors indicate that contour delineation and template matching on DRRs can be performed as accurately as with simulator images. With DRRs and a setup protocol, the required CTV-P'I'V margins were derived using coverage probability calculations [Stroom et al. Int J Radiat Oncol Biol Phys 1999; 43: 905]. Margins of 4, 5 and 4 mm, respectively, were needed for the L-R, S-I and A-P directions. Data from our previous study using simulator images (i.e. incorporating simulator set-up errors), indicated that margins of 10, 8 and 7 mm were required. Conclusion: The treatment planning margins necessary for external setup accuracy are improved by about 50% when a conventional simulation step for lung cancer patients is omitted, and when DRRs are used in an off-line correction protocol.