Page 318 - 2014 Printable Abstract Book
P. 318
molecular alterations that accompany prostate development and progression. The orthotopic prostate
cancer will provide the idea model to mimic clinical therapy scenario for radiation-induced non-targeted
response, especially together with the application of an image-guided Small Animal Radiation Research
Platform (SARRP). Before start this model, the first question we asked is what is the contribution of the
immune system to radiation-induced non-targeted response? Using athymic nude mice, preliminary data
have shown that partial body irradiation (1cmx1cm area in lower abdomen) can increase cyclooxygenase-
2 (COX-2) expression and induce γ-H2AX formation in out of field lung tissues in a manner similar to those
reported recently using an inbreed mouse strain. The western blotting results confirm the increasing of
COX-2 expression in out of filed lung tissue. These findings suggest the suitability of using nude mice in
radiation induced non-targeted response study. As a continuation of this study, two human prostate cell
lines (PC3 and LNCaP) were orthotopically injected into the mouse dorsal glands and tumor growth was
monitored by the bioluminescent signals. Mice will be irradiated with focused X-ray beam using SARRP
6
once bioluminescence reaches 2-3x10 cps, which correlates with a tumor size of 0.2-0.5 grams. The
studies are on progressing, results from these studies will be discussed in the meeting.
(PS5-54) Balance shift between error-free and error prone DNA double-strand break repair as a novel
mechanism of radiosensitization by nucleoside analogs. Simon Magin and George Iliakis, PhD; Institute
of Medical Radiation Biology, Essen, Germany
Nucleoside analogs (NAs) represent a large group of anticancer drugs, several members of which
have been shown to sensitize cells to ionizing radiation (IR). Among the lesions induced in cells by IR, the
DNA double-strand break (DSB) is considered to be the most detrimental. Therefore we focused on
interactions between NAs and pathways of DSB repair. We opted for post-irradiation treatment with NAs
to avoid complications arising from redistribution of cells throughout the cycle prior to IR exposure. We
compared the effects of 4h treatments post-IR with vidarabine (ara-A), fludarabine (F-ara-A) or
gemcitabine (dFdC) on the survival of A549 lung carcinoma cells. We found that post-irradiation treatment
with ara-A and F-ara-A, but not dFdC, resulted in cell radiosensitization to killing. Radiosensitization by
ara-A was superior to that by F-ara-A. We employed immunofluorescence detection of IR-induced foci
and I-SceI-based reporter cell lines to identify the pathways of DSB repair that are inhibited by ara-A and
cause the observed radiosensitization. We found that the formation of IR-induced Rad51-foci was
compromised by ara-A suggesting inhibition of homologous recombination repair (HRR). Repair of I-SceI-
induced DSBs by HRR was also suppressed by ara-A. Furthermore, A549 cells deprived of Rad51 by RNAi
could not be efficiently radiosensitized by ara-A. On the other hand, we could not detect impairment by
ara-A of non-homologous end-joining (NHEJ), as measured by pulsed-field gel electrophoresis (PFGE) at
high doses of IR (20 Gy). Notably, we observed an increase in the frequency of mutagenic NHEJ events in
several I-SceI-based repair reporter systems, indicative of pro-cessing by error-prone repair pathways
such as alternative/backup NHEJ (B-NHEJ). In addition, we could show that treatment with ara-A induces
-/-
a dramatic increase in B-NHEJ in plateau phase Lig4 MEFs. Treatment with F-ara-A also inhibited HRR,
but was less proficient in promoting mutagenic NHEJ. We conclude that inhibition of HRR and promotion
of B-NHEJ represent two important mechanisms of radiosensitization by NAs. The strong impact of ara-A
on both of these repair pathways underpins its superior radiosensitizing potential. Targeting of both HRR
and B-NHEJ may provide a promising new strategy for radiosensitization.
316 | P a g e
cancer will provide the idea model to mimic clinical therapy scenario for radiation-induced non-targeted
response, especially together with the application of an image-guided Small Animal Radiation Research
Platform (SARRP). Before start this model, the first question we asked is what is the contribution of the
immune system to radiation-induced non-targeted response? Using athymic nude mice, preliminary data
have shown that partial body irradiation (1cmx1cm area in lower abdomen) can increase cyclooxygenase-
2 (COX-2) expression and induce γ-H2AX formation in out of field lung tissues in a manner similar to those
reported recently using an inbreed mouse strain. The western blotting results confirm the increasing of
COX-2 expression in out of filed lung tissue. These findings suggest the suitability of using nude mice in
radiation induced non-targeted response study. As a continuation of this study, two human prostate cell
lines (PC3 and LNCaP) were orthotopically injected into the mouse dorsal glands and tumor growth was
monitored by the bioluminescent signals. Mice will be irradiated with focused X-ray beam using SARRP
6
once bioluminescence reaches 2-3x10 cps, which correlates with a tumor size of 0.2-0.5 grams. The
studies are on progressing, results from these studies will be discussed in the meeting.
(PS5-54) Balance shift between error-free and error prone DNA double-strand break repair as a novel
mechanism of radiosensitization by nucleoside analogs. Simon Magin and George Iliakis, PhD; Institute
of Medical Radiation Biology, Essen, Germany
Nucleoside analogs (NAs) represent a large group of anticancer drugs, several members of which
have been shown to sensitize cells to ionizing radiation (IR). Among the lesions induced in cells by IR, the
DNA double-strand break (DSB) is considered to be the most detrimental. Therefore we focused on
interactions between NAs and pathways of DSB repair. We opted for post-irradiation treatment with NAs
to avoid complications arising from redistribution of cells throughout the cycle prior to IR exposure. We
compared the effects of 4h treatments post-IR with vidarabine (ara-A), fludarabine (F-ara-A) or
gemcitabine (dFdC) on the survival of A549 lung carcinoma cells. We found that post-irradiation treatment
with ara-A and F-ara-A, but not dFdC, resulted in cell radiosensitization to killing. Radiosensitization by
ara-A was superior to that by F-ara-A. We employed immunofluorescence detection of IR-induced foci
and I-SceI-based reporter cell lines to identify the pathways of DSB repair that are inhibited by ara-A and
cause the observed radiosensitization. We found that the formation of IR-induced Rad51-foci was
compromised by ara-A suggesting inhibition of homologous recombination repair (HRR). Repair of I-SceI-
induced DSBs by HRR was also suppressed by ara-A. Furthermore, A549 cells deprived of Rad51 by RNAi
could not be efficiently radiosensitized by ara-A. On the other hand, we could not detect impairment by
ara-A of non-homologous end-joining (NHEJ), as measured by pulsed-field gel electrophoresis (PFGE) at
high doses of IR (20 Gy). Notably, we observed an increase in the frequency of mutagenic NHEJ events in
several I-SceI-based repair reporter systems, indicative of pro-cessing by error-prone repair pathways
such as alternative/backup NHEJ (B-NHEJ). In addition, we could show that treatment with ara-A induces
-/-
a dramatic increase in B-NHEJ in plateau phase Lig4 MEFs. Treatment with F-ara-A also inhibited HRR,
but was less proficient in promoting mutagenic NHEJ. We conclude that inhibition of HRR and promotion
of B-NHEJ represent two important mechanisms of radiosensitization by NAs. The strong impact of ara-A
on both of these repair pathways underpins its superior radiosensitizing potential. Targeting of both HRR
and B-NHEJ may provide a promising new strategy for radiosensitization.
316 | P a g e
