Page 128 - 2014 Printable Abstract Book
P. 128
(PS1-46) Poly (ADP-ribose) glycohydrolase enhances cellular sensitivity to hyperthermia and
hyperthermia induced radiosensitization. Junko Maeda, DVM; Justin J. Bell; Jeremy Haskins; and
Takamitsu A. Kato, PhD, Colorado State University, Fort Collins, CO
Hyperthermia has been shown to sensitize mammalian cells to subsequent radiation treatment.
Radiosensitization mechanisms can be explained by inhibition of DNA repair pathways. We investigated
Poly (ADP-ribose) glycohydrolase (PARG) role in hyperthermia induced radiosensitization. PARG
knockdown cells (A549 lung cancer cells with siRNA, LN428 with shRNA) showed marked sensitization to
ionizing radiation when it combined with hyperthermia. PARG knockdown cells also showed sensitization
to hyperthermia in relatively low temperature (less than 44C). On the other hand, PARG overexpressed
cells showed resistant phenotype to hyperthermia treatment. PARG knockdown cells showed
hyperthermia induced radiosensitization in lower temperature. Control needed 42C. But knockdown cells
needed only 40C. We observed hyperthermia induced poly (ADP-ribose) formation in PARG knockdown
cells but not in normal cells. We conclude PARG knockdown disrupt poly (ADP-ribose) metabolism trigger
by hyperthermia.
(PS1-48) Regulation of non-homologous end joining (NHEJ) pathway to repair radiation induced DNA
double strand breaks in human PBMCs by administration of Podophyllum hexandrum active principles.
Nitya Nand. Srivastava, MSc (Biochemistry), M.Phil (life Sciences); Sandeep Kumar. Shukla, MSc, Ph.D; M
H. Yashavarddhan, MSc (Biotechnology); and Manju Lata. Gupta, MSc, Ph.D, Institute of Nuclear Medicine
and Allied Sciences, Defence Research and Development Organization, Delhi, India
Radioprotective property of active principles of Podophyllum hexandrum (G-002M) against
radiation has been proved time and again in our repeated studies. Mechanistically also G-002M has shown
protection to cellular macromolecules. Present study has been undertaken to further delineate its mode
of action. Peripheral blood mononuclear cells (PBMCs) isolated from the human blood were divided into
three groups 1) radiation only, 2) G-002M+R and 3) untreated controls. Samples were processed to
evaluate phosphorylated form of H2AX, 53BP1, ATM, DNA-PKCs, NBS1 in differentially treated PBMCs.
Time (0-90min) and radiation dose (0.5-5Gy) responses of ionizing radiation induced foci (IRIF) formation
of γ-H2AX and P53BP1 in PBMCs was also studied. To reaffirm the phosphorylation status of ATM, DNA-
PKcs, Ku80 besides XRCCIV and SMC1, flow cytometry analysis was also performed. Radiation induced cell
death frequency was measured by AnnexinV-PI method. Results revealed that IRIF of γ-H2AX and P53BP1
were countable up to 3Gy and beyond this the foci lost their identity because of overlapping. The time
response kinetics of both the biomarker indicated that maximum IRIF formation was at 60 min and
decreased thereafter. G-002M pretreated PBMCs attenuated the DNA double strand break (DSBs)
signaling molecules γ-H2AX, P53BP1, pATM, pSMC1, pDNA-PKcs when compared with radiation only
group. Ku80 and XRCCIV protein levels were found significantly up-regulated in this group. Radiation (5Gy)
exposure could induce apoptosis in PBMCs upto 20% within 8 hr however, at 96 hr increase was upto 83%,
whereas G-002M pre-treatment attenuated apoptosis from 20% to 1% at 8 hr and 83% to 35% at 96 hr.
Observations revealed that phosphorylation of H2AX, 53BP1, ATM, NBS1 and DNA-PKcs in G-002M treated
PBMCs was significantly less and NHEJ pathway also got regulated by enhancing Ku80 and XRCCIV protein
levels. G-002M had also shown regulated SMC1 phosphorylation, known to play lead role in maintaining
chromatin structure, besides protecting cells undergoing apoptosis. The study conveys that G-002M has
significantly protected DNA against radiation by ROS scavenging and also repaired damaged DNA by up-
126 | P a g e
hyperthermia induced radiosensitization. Junko Maeda, DVM; Justin J. Bell; Jeremy Haskins; and
Takamitsu A. Kato, PhD, Colorado State University, Fort Collins, CO
Hyperthermia has been shown to sensitize mammalian cells to subsequent radiation treatment.
Radiosensitization mechanisms can be explained by inhibition of DNA repair pathways. We investigated
Poly (ADP-ribose) glycohydrolase (PARG) role in hyperthermia induced radiosensitization. PARG
knockdown cells (A549 lung cancer cells with siRNA, LN428 with shRNA) showed marked sensitization to
ionizing radiation when it combined with hyperthermia. PARG knockdown cells also showed sensitization
to hyperthermia in relatively low temperature (less than 44C). On the other hand, PARG overexpressed
cells showed resistant phenotype to hyperthermia treatment. PARG knockdown cells showed
hyperthermia induced radiosensitization in lower temperature. Control needed 42C. But knockdown cells
needed only 40C. We observed hyperthermia induced poly (ADP-ribose) formation in PARG knockdown
cells but not in normal cells. We conclude PARG knockdown disrupt poly (ADP-ribose) metabolism trigger
by hyperthermia.
(PS1-48) Regulation of non-homologous end joining (NHEJ) pathway to repair radiation induced DNA
double strand breaks in human PBMCs by administration of Podophyllum hexandrum active principles.
Nitya Nand. Srivastava, MSc (Biochemistry), M.Phil (life Sciences); Sandeep Kumar. Shukla, MSc, Ph.D; M
H. Yashavarddhan, MSc (Biotechnology); and Manju Lata. Gupta, MSc, Ph.D, Institute of Nuclear Medicine
and Allied Sciences, Defence Research and Development Organization, Delhi, India
Radioprotective property of active principles of Podophyllum hexandrum (G-002M) against
radiation has been proved time and again in our repeated studies. Mechanistically also G-002M has shown
protection to cellular macromolecules. Present study has been undertaken to further delineate its mode
of action. Peripheral blood mononuclear cells (PBMCs) isolated from the human blood were divided into
three groups 1) radiation only, 2) G-002M+R and 3) untreated controls. Samples were processed to
evaluate phosphorylated form of H2AX, 53BP1, ATM, DNA-PKCs, NBS1 in differentially treated PBMCs.
Time (0-90min) and radiation dose (0.5-5Gy) responses of ionizing radiation induced foci (IRIF) formation
of γ-H2AX and P53BP1 in PBMCs was also studied. To reaffirm the phosphorylation status of ATM, DNA-
PKcs, Ku80 besides XRCCIV and SMC1, flow cytometry analysis was also performed. Radiation induced cell
death frequency was measured by AnnexinV-PI method. Results revealed that IRIF of γ-H2AX and P53BP1
were countable up to 3Gy and beyond this the foci lost their identity because of overlapping. The time
response kinetics of both the biomarker indicated that maximum IRIF formation was at 60 min and
decreased thereafter. G-002M pretreated PBMCs attenuated the DNA double strand break (DSBs)
signaling molecules γ-H2AX, P53BP1, pATM, pSMC1, pDNA-PKcs when compared with radiation only
group. Ku80 and XRCCIV protein levels were found significantly up-regulated in this group. Radiation (5Gy)
exposure could induce apoptosis in PBMCs upto 20% within 8 hr however, at 96 hr increase was upto 83%,
whereas G-002M pre-treatment attenuated apoptosis from 20% to 1% at 8 hr and 83% to 35% at 96 hr.
Observations revealed that phosphorylation of H2AX, 53BP1, ATM, NBS1 and DNA-PKcs in G-002M treated
PBMCs was significantly less and NHEJ pathway also got regulated by enhancing Ku80 and XRCCIV protein
levels. G-002M had also shown regulated SMC1 phosphorylation, known to play lead role in maintaining
chromatin structure, besides protecting cells undergoing apoptosis. The study conveys that G-002M has
significantly protected DNA against radiation by ROS scavenging and also repaired damaged DNA by up-
126 | P a g e
