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culture plastic and supplemented with full culture media. Reattached cells were fixed with 70% EtOH and stained with crystal violet solution (0.2% in MeOH) 1 day later. Soft agar colony formation assays were also performed in these cells using standard method. For in vivo tumor formation studies, HaCaTp65 or HaCaT mock-transfected control single cell suspensions were injected subcutaneously into the right hind limbs of 8-week old athymic NCR NUM mice. Tumor growth was monitored every 2 days for 2 months. MTT and Clonogenic survival assays were performed in HaCaTEGFR and HaCaTp65 cells to investigate their roles in radiation response. MTT assays were performed 7 days after irradiation (2–10 Gy) by incubating cultures with MTT solution at 37oC for 4 hours. MTT solubilization solution (10% Triton X-100/0.1N HCL in isopropanol) was added and the cultures were incubated for additional 12 hours at 37oC before absorbance was spectrophotometrically assessed at 570 nm (ref. 690 nm). Clonogenic survival assays were carried out 14 days after irradiation (2–10 Gy). Only colonies containing 50 cells were scored. Results: Previous studies from our group demonstrated that EGFR-dependent signal transduction supports anchorage- independent HaCaT cell survival. Here we show that overexpression of the EGFR in HaCaT cells fails to induce anchorage- independent growth in soft agar colony formation assay. Conversely, ectopic expression of NF-Bp65 causes anchorage- independent survival, colony formation in soft agar and tumorigenicity of HaCaT cells in nude mice. To investigate the radiation response of HaCaT cells over-expressing EGFR and NF-B, we performed MTT assays and clonogenic survival assays after exposure to ionizing radiation. HaCaT cells overexpressing the EGFR were more radioresistant than mock-transfected controls. In contrast, forced NF-Bp65 expression did not result in enhanced radioresistance of HaCaT cells. Experiments are underway to study the tumorigenicity of EGFR over-expressing HaCaT cells in nude mice. Conclusions: Over-expression of NF-B rendered human keratinocytes HaCaT tumorigenic, but did not enable radioresistance of these cells. These studies highlight distinct contributions of EGFR and NF-B signaling to tumor progression and resistance to radiation therapy. Serra Kamer is a visiting physician from Dept. of Radiation Oncology, EGE Univ. Med. School, Izmir, Turkey. Author Disclosure: Q. Ren, None; C. Kari, None; M.R.D. Quadros, None; Y.P. Sui, None; S. Kamer, None; A.P. Dicker, None; U. Rodeck, None. 2649 Ultrasound Imaging and Spectrosocopy of Cancer Therapy Effects W. Chu 1 , M. C. Kolios 2,3 , G. J. Czarnota 2,4 1 Department of Radiation Oncology, Sunnybrook Health Sciences Centre and University of Toronto, Toronto, ON, Canada, 2 Department of Physics, Ryerson University, Toronto, ON, Canada, 3 Department of Medical Biophysics, University of Toronto and Ontario Cancer Institute, Toronto, ON, Canada, 4 Department of Radiation Oncology, Sunnybrook Health Sciences Centre, and Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Purpose/Objective(s): To non-invasively monitor the apoptotic response of tumours exposed to increasing doses of radiation with spectroscopic, high-frequency ultrasound imaging. Materials/Methods: Human PC3 prostate cancer tumours were grown as xenotransplants in SCID mice. Animals were subjected to 0, 2, 4 and 8 Gy of single fraction, 100 kV X-ray radiation. Ultrasound analyses were carried out 24 hours after irradiation. Animals were sacrificed for histological analysis using standard haematoxylin and eosin staining, and TdT-analysis to detect apoptotic cell death. Results: Ultrasound analysis showed an increase in backscatter for the irradiated prostate tumours compared to the control animals. Mid-band fit and spectral slope were -12 dBr and -0.12 dBr MHz-1 for 8Gy samples, compared to -29 dBr and -0.05 dBr MHz-1 for controls. Image analysis demonstrated a correlation between radiation dose and the number and size of high intensity patches in irradiated tumours. The mean number (SD) of high intensity patches per image was 1.3 1.1, 5.4 2.2, 3.9 2.1 and 5.3 2.5 for the 0, 2, 4 and 8 Gy irradiated tumours, respectively. The average size of these patches (length x width (SD) in millimeters) was (0.36 0.33 x 0.25 0.18), (0.55 0.48 x 0.49 0.38), (0.65 0.53 x 0.58 0.43) and (1.10 1.18 x 0.69 0.55) for the 0, 2, 4 and 8 Gy irradiated tumours, respectively. The size and location of these areas correlated with histological areas of increased apoptosis. Conclusions: In summary, spectroscopic ultrasound analyses can detect dose-dependent, radiation-induced cell death. Ultra- sound results correlate with histological evidence of increased apoptosis with higher doses of radiation. Therefore, high- frequency ultrasound may be used to non-invasively monitor effects of radiation at the cellular and molecular level in animal models of human tumours. Interestingly, PC3 cells not exhibit apoptosis with radiation exposure in vitro. We postulate that radiation affects the tumour vasculature in vivo, leading to secondary apoptosis. Author Disclosure: W. Chu, None; M.C. Kolios, None; G.J. Czarnota, None. 2650 Intravenous Injection of Manganese Superoxide Dismutase Plasmid-Liposome (MnSOD-PL) Complex or MnSOD Mimetic EUK-134 Protects Mice From Whole Body Irradiation (WBI) M. W. Epperly, Y. Niu, J. S. Greenberger University of Pittsburgh Cancer Inst., Pittsburgh, PA Purpose/Objective(s): Local organ specific administration of manganese superoxide dismutase-plasmid/liposomes (MnSOD- PL) has been shown to protect the lungs, esophagus, bladder, intestine and oral cavity from irradiation damage. The goal of the present studies was to determine whether intravenous systemic delivery of MnSOD-PL protects the bone marrow from whole body irradiation (WBI) damage. Materials/Methods: C57BL/6NHsd female mice were injected intravenously (I.V.) with MnSOD-PL (100 g plasmid DNA) or EUK-134 (30 mg/kg). The mice were irradiated 24 hours after injection of MnSOD-PL or 15 minutes after injection of EUK-134. Mice were irradiated to doses of 9, 9.5, or 9.75 Gy WBI. Mice were then followed for survival. Results: Control mice irradiated to 9 Gy had a survival rate of 80% 30 days following irradiation while mice injected with MnSOD-PL prior to 9 Gy had a survival rate of 93.3%. Following irradiation to 9.5 Gy, control mice had a survival rate of 53% S571 Proceedings of the 48th Annual ASTRO Meeting
Transcript of 2649
culture plastic and supplemented with full culture media. Reattached cells were fixed with 70% EtOH and stained with crystalviolet solution (0.2% in MeOH) 1 day later. Soft agar colony formation assays were also performed in these cells using standardmethod. For in vivo tumor formation studies, HaCaTp65 or HaCaT mock-transfected control single cell suspensions wereinjected subcutaneously into the right hind limbs of 8-week old athymic NCR NUM mice. Tumor growth was monitored every2 days for 2 months. MTT and Clonogenic survival assays were performed in HaCaTEGFR and HaCaTp65 cells to investigatetheir roles in radiation response. MTT assays were performed 7 days after irradiation (2–10 Gy) by incubating cultures withMTT solution at 37oC for 4 hours. MTT solubilization solution (10% Triton X-100/0.1N HCL in isopropanol) was added andthe cultures were incubated for additional 12 hours at 37oC before absorbance was spectrophotometrically assessed at 570 nm(ref. 690 nm). Clonogenic survival assays were carried out 14 days after irradiation (2–10 Gy). Only colonies containing � 50cells were scored.
Results: Previous studies from our group demonstrated that EGFR-dependent signal transduction supports anchorage-independent HaCaT cell survival. Here we show that overexpression of the EGFR in HaCaT cells fails to induce anchorage-independent growth in soft agar colony formation assay. Conversely, ectopic expression of NF- Bp65 causes anchorage-independent survival, colony formation in soft agar and tumorigenicity of HaCaT cells in nude mice. To investigate the radiationresponse of HaCaT cells over-expressing EGFR and NF- B, we performed MTT assays and clonogenic survival assays afterexposure to ionizing radiation. HaCaT cells overexpressing the EGFR were more radioresistant than mock-transfected controls.In contrast, forced NF- Bp65 expression did not result in enhanced radioresistance of HaCaT cells. Experiments are underwayto study the tumorigenicity of EGFR over-expressing HaCaT cells in nude mice.
Conclusions: Over-expression of NF- B rendered human keratinocytes HaCaT tumorigenic, but did not enable radioresistanceof these cells. These studies highlight distinct contributions of EGFR and NF- B signaling to tumor progression and resistanceto radiation therapy.
Serra Kamer is a visiting physician from Dept. of Radiation Oncology, EGE Univ. Med. School, Izmir, Turkey.
Author Disclosure: Q. Ren, None; C. Kari, None; M.R.D. Quadros, None; Y.P. Sui, None; S. Kamer, None; A.P. Dicker, None;U. Rodeck, None.
2649 Ultrasound Imaging and Spectrosocopy of Cancer Therapy Effects
W. Chu1, M. C. Kolios2,3, G. J. Czarnota2,4
1Department of Radiation Oncology, Sunnybrook Health Sciences Centre and University of Toronto, Toronto, ON, Canada,2Department of Physics, Ryerson University, Toronto, ON, Canada, 3Department of Medical Biophysics, University ofToronto and Ontario Cancer Institute, Toronto, ON, Canada, 4Department of Radiation Oncology, Sunnybrook HealthSciences Centre, and Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada
Purpose/Objective(s): To non-invasively monitor the apoptotic response of tumours exposed to increasing doses of radiationwith spectroscopic, high-frequency ultrasound imaging.
Materials/Methods: Human PC3 prostate cancer tumours were grown as xenotransplants in SCID mice. Animals weresubjected to 0, 2, 4 and 8 Gy of single fraction, 100 kV X-ray radiation. Ultrasound analyses were carried out 24 hours afterirradiation. Animals were sacrificed for histological analysis using standard haematoxylin and eosin staining, and TdT-analysisto detect apoptotic cell death.
Results: Ultrasound analysis showed an increase in backscatter for the irradiated prostate tumours compared to the controlanimals. Mid-band fit and spectral slope were -12 dBr and -0.12 dBr MHz-1 for 8Gy samples, compared to -29 dBr and -0.05dBr MHz-1 for controls. Image analysis demonstrated a correlation between radiation dose and the number and size of highintensity patches in irradiated tumours. The mean number (�SD) of high intensity patches per image was 1.3 �1.1, 5.4 �2.2,3.9 �2.1 and 5.3 �2.5 for the 0, 2, 4 and 8 Gy irradiated tumours, respectively. The average size of these patches (length xwidth (�SD) in millimeters) was (0.36 �0.33 x 0.25 �0.18), (0.55 �0.48 x 0.49 �0.38), (0.65 �0.53 x 0.58 �0.43) and (1.10�1.18 x 0.69 �0.55) for the 0, 2, 4 and 8 Gy irradiated tumours, respectively. The size and location of these areas correlatedwith histological areas of increased apoptosis.
Conclusions: In summary, spectroscopic ultrasound analyses can detect dose-dependent, radiation-induced cell death. Ultra-sound results correlate with histological evidence of increased apoptosis with higher doses of radiation. Therefore, high-frequency ultrasound may be used to non-invasively monitor effects of radiation at the cellular and molecular level in animalmodels of human tumours. Interestingly, PC3 cells not exhibit apoptosis with radiation exposure in vitro. We postulate thatradiation affects the tumour vasculature in vivo, leading to secondary apoptosis.
Author Disclosure: W. Chu, None; M.C. Kolios, None; G.J. Czarnota, None.
2650 Intravenous Injection of Manganese Superoxide Dismutase Plasmid-Liposome (MnSOD-PL) Complex orMnSOD Mimetic EUK-134 Protects Mice From Whole Body Irradiation (WBI)
M. W. Epperly, Y. Niu, J. S. Greenberger
University of Pittsburgh Cancer Inst., Pittsburgh, PA
Purpose/Objective(s): Local organ specific administration of manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) has been shown to protect the lungs, esophagus, bladder, intestine and oral cavity from irradiation damage. The goal of thepresent studies was to determine whether intravenous systemic delivery of MnSOD-PL protects the bone marrow from wholebody irradiation (WBI) damage.
Materials/Methods: C57BL/6NHsd female mice were injected intravenously (I.V.) with MnSOD-PL (100 �g plasmid DNA)or EUK-134 (30 mg/kg). The mice were irradiated 24 hours after injection of MnSOD-PL or 15 minutes after injection ofEUK-134. Mice were irradiated to doses of 9, 9.5, or 9.75 Gy WBI. Mice were then followed for survival.
Results: Control mice irradiated to 9 Gy had a survival rate of 80% 30 days following irradiation while mice injected withMnSOD-PL prior to 9 Gy had a survival rate of 93.3%. Following irradiation to 9.5 Gy, control mice had a survival rate of 53%
S571Proceedings of the 48th Annual ASTRO Meeting
