Page 309 - 2014 Printable Abstract Book
P. 309
to compensate for impaired gas exchange. Development of radiation induced lung injury is associated
with acute pulmonary distress linked with changes in lung function. We hypothesized that Ti to Te ratio
would be significantly different in irradiated mice compared with controls and could potentially identify
animals that would develop severe respiratory injury. Methods: One-hundred twenty CBA/J mice were
exposed to 6 doses of radiation (0, 12, 12.75, 13.5, 14.25, 15Gy). Respiratory function was measured every
2 weeks using whole body plethysmography system. During the 6 months of follow-up time, Ti/Te was
compared between irradiated and non-irradiated animals using t-test. Mean + 1standard deviation of
Ti/Te in each time point was used as a cut-off point to determine abnormality in Ti/Te ratio. Probit analysis
was performed for Ti/Te ratio for weeks 14 and 16 after radiation. Effective dose to causes an increase in
Ti/Te ratio in 50 % of animal (ED50) was calculated and compared to the lethal dose in 180 days
(LD50/180). Results: There were significant time and dose changes in Ti/Te in different follow-up time
points which were significantly different between irradiated and control animals. The threshold dose for
significant change in Ti/Te was 13.5 Gy and the difference between irradiated and control animals was
first detected in weeks 14 (P=0.02) followed in week16 (P=0.005). In week 14, Ed50 was 14.03 and in week
16, ED50 was 13.49 Gy which was almost the same as LD50/180 value of 13.56 Gy. Conclusion: The ratio
of time of inspiration to time of expiration can be used as a screening test to detect early respiratory
distress following whole thorax irradiation in CBA/J mice that precedes mortality. ED50 in 16 weeks is the
same as LD50/180 days suggesting that longitudinal measurements of Ti/Te over time after irradiation
can be used as a predictor of respiratory distress and mortality. Acknowledgements: This project has been
funded with Federal funds from the National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Department of Health and Human Services, Contract No. HHSN277201000046C.
(PS5-40) ROS mediated radiosensitization by mTOR inhibition. Jae Ho Kim; Andrew J.J. Kolozsvary; Karen
Lapanowski; Shuo Wang; Kenneth A. Jenrow; and Stephen L. Brown, Henry Ford Hospital, Detroit, MI
Purpose: To test the hypothesis that enhanced radiosensitization by mTOR inhibition is at least in
part mediated by damaging reactive oxygen species, ROS. Methods: A549 lung cancer cells and 9L rat
gliosarcoma cells were grown in culture and in the hind limbs of athymic mice and exposed to radiation
alone (5 or 10 Gy for cell culture; 15 Gy for animal studies), mTOR inhibitor (Everolimus, 10mM for in vitro
studies and 5 mg/kg by gavage for animal studies) or the combination. In vitro endpoints included
clonogenic assay, ROS levels assessed using flow cytometry measured DCFDA and DHE fluorescence
(DCFDA and DHE reflect different ROS). In vivo endpoint was tumor growth delay. Results: Significant
radiosensitization was observed by mTOR inhibition both in vitro and in vivo. The mean lethal dose, Do,
was significantly reduced, from 3 Gy to 2 Gy when mTOR inhibitor was administered after radiation (either
immediately, 1 or 6 hours after radiation) compared with Everolimus given 24 hours before radiation. In
vivo, radiosensitization was observed when Everolimus was given once daily starting 3 days before
radiation. ROS were significantly increased 1 hour after Everolimus exposure assessed by flow cytometry
of cultured cells using DCFDA. A different array of ROS were significantly increased 24 hours after radiation
exposure assessed by flow cytometry of cultured cells using DHE. In each case, ROS were reduced to near
control levels when treated cells were co-incubated with N-acetyl-cysteine. Discussion and Conclusion:
Previously, some investigators have shown radiosensitization by mTOR inhibitor in vitro regardless of the
timing and sequence of drug and radiation. Radiosensitization has been attributed to arrest of
proliferating cells in a radiosensitive phase of the cell cycle. Other investigators have shown no
radiosensitization by mTOR inhibitor in vitro. In vivo, radiosensitization has been demonstrated previously
307 | P a g e
with acute pulmonary distress linked with changes in lung function. We hypothesized that Ti to Te ratio
would be significantly different in irradiated mice compared with controls and could potentially identify
animals that would develop severe respiratory injury. Methods: One-hundred twenty CBA/J mice were
exposed to 6 doses of radiation (0, 12, 12.75, 13.5, 14.25, 15Gy). Respiratory function was measured every
2 weeks using whole body plethysmography system. During the 6 months of follow-up time, Ti/Te was
compared between irradiated and non-irradiated animals using t-test. Mean + 1standard deviation of
Ti/Te in each time point was used as a cut-off point to determine abnormality in Ti/Te ratio. Probit analysis
was performed for Ti/Te ratio for weeks 14 and 16 after radiation. Effective dose to causes an increase in
Ti/Te ratio in 50 % of animal (ED50) was calculated and compared to the lethal dose in 180 days
(LD50/180). Results: There were significant time and dose changes in Ti/Te in different follow-up time
points which were significantly different between irradiated and control animals. The threshold dose for
significant change in Ti/Te was 13.5 Gy and the difference between irradiated and control animals was
first detected in weeks 14 (P=0.02) followed in week16 (P=0.005). In week 14, Ed50 was 14.03 and in week
16, ED50 was 13.49 Gy which was almost the same as LD50/180 value of 13.56 Gy. Conclusion: The ratio
of time of inspiration to time of expiration can be used as a screening test to detect early respiratory
distress following whole thorax irradiation in CBA/J mice that precedes mortality. ED50 in 16 weeks is the
same as LD50/180 days suggesting that longitudinal measurements of Ti/Te over time after irradiation
can be used as a predictor of respiratory distress and mortality. Acknowledgements: This project has been
funded with Federal funds from the National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Department of Health and Human Services, Contract No. HHSN277201000046C.
(PS5-40) ROS mediated radiosensitization by mTOR inhibition. Jae Ho Kim; Andrew J.J. Kolozsvary; Karen
Lapanowski; Shuo Wang; Kenneth A. Jenrow; and Stephen L. Brown, Henry Ford Hospital, Detroit, MI
Purpose: To test the hypothesis that enhanced radiosensitization by mTOR inhibition is at least in
part mediated by damaging reactive oxygen species, ROS. Methods: A549 lung cancer cells and 9L rat
gliosarcoma cells were grown in culture and in the hind limbs of athymic mice and exposed to radiation
alone (5 or 10 Gy for cell culture; 15 Gy for animal studies), mTOR inhibitor (Everolimus, 10mM for in vitro
studies and 5 mg/kg by gavage for animal studies) or the combination. In vitro endpoints included
clonogenic assay, ROS levels assessed using flow cytometry measured DCFDA and DHE fluorescence
(DCFDA and DHE reflect different ROS). In vivo endpoint was tumor growth delay. Results: Significant
radiosensitization was observed by mTOR inhibition both in vitro and in vivo. The mean lethal dose, Do,
was significantly reduced, from 3 Gy to 2 Gy when mTOR inhibitor was administered after radiation (either
immediately, 1 or 6 hours after radiation) compared with Everolimus given 24 hours before radiation. In
vivo, radiosensitization was observed when Everolimus was given once daily starting 3 days before
radiation. ROS were significantly increased 1 hour after Everolimus exposure assessed by flow cytometry
of cultured cells using DCFDA. A different array of ROS were significantly increased 24 hours after radiation
exposure assessed by flow cytometry of cultured cells using DHE. In each case, ROS were reduced to near
control levels when treated cells were co-incubated with N-acetyl-cysteine. Discussion and Conclusion:
Previously, some investigators have shown radiosensitization by mTOR inhibitor in vitro regardless of the
timing and sequence of drug and radiation. Radiosensitization has been attributed to arrest of
proliferating cells in a radiosensitive phase of the cell cycle. Other investigators have shown no
radiosensitization by mTOR inhibitor in vitro. In vivo, radiosensitization has been demonstrated previously
307 | P a g e
