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radiation effect of this Auger-electron emitter HeLa and V79 cells were incubated either with Cs131
(Auger-electron emitter) or Cs137 (gamma-emitter) or were exposed to external gamma-rays (Co-60).
Radiation induced DNA damage was quantified by measuring histone H2X phosphorylation (γ-H2AX), p53
binding protein No. 1 (53BP1) foci formation, micronuclei formation and clonogenic cell survival.
In order to calculate the dose delivered, we first needed to estimate the Cs131 cell concentration and the
duration of the exposure. This required measuring the cell uptake and release of Cs131 by counting the
activity in the medium and the cells using a scintillation counter. Results from these internal exposures to
densely and sparsely ionizing radiation were compared to external γ-radiation. Our results show that
Cs131 is likely to be taken up by the Na+/K+ ATPase in a concentration dependent manner. However Cs131
is also released from the cells probably passively over the cell membrane. Cells incubated with Cs131
showed in general a higher level of damage compared to cells incubated with Cs137 or exposure to
external γ-radiation.
(PS1-08) Polynucleotide kinase/phosphatase, PNKP, as a target for enhanced cancer therapy. Zahra
Shire and Michael Weinfeld, University of Alberta, Edmonton, Canada
The radioresistance and chemoresistance of tumors are major obstacles that may lead to the failure of
cancer therapy. Reducing DNA repair in cancer cells is emerging as a new strategy for improving cancer
treatment. One approach is to identify small molecules inhibitors of DNA repair enzymes. Human
polynucleotide kinase/phosphatase (hPNKP), which possesses DNA 5’-kinase and 3’-phosphatase
activities, plays an essential role in DNA strand break repair by rendering strand-break termini suitable for
DNA polymerases and ligases. Previous work in our lab has shown that reduced expression of PNKP
sensitizes cells to ionizing radiation, alkylating agents and the topoisomerase I poison camptothecin. We
have developed a rapid fluorescence-based assay to identify small molecule inhibitors of the 3’-
phosphatase activity of PNKP. The main components of this assay are two oligonucleotide hairpin probes,
PNKP enzyme, and T4 DNA polymerase. T4 DNA polymerase has a powerful 3’-5’ exonuclease activity that
digests oligonucleotides that have a 3’-OH terminus, but is blocked by the presence of a 3’-phosphate
terminus. We detect the inhibition of PNKP by monitoring the fluorescence signal resulting from the
release of 2-aminopurine (2 Ap) embedded in the stem of the hairpin probes. The fluorescence of 2 Ap is
highly quenched when it is incorporated in duplex DNA. As a proof of principle, the novel assay was used
to test 200 compounds selected from three chemical libraries of natural derivative compounds, and two
chemically related compounds were identified that inhibit the phosphatase activity of hPNKP. Currently,
we are examining the mechanism of inhibition by these compounds and their ability to sensitize cancer
cells to radio/chemo-therapy.
(PS1-09)Ionizing radiation decreased Beclin1/LC3B and increased oxidative stress and mTOR via
PI3K/Akt to inhibit autophagy and promote intestinal cell proliferation in C57BL/6J mice.
Shubhankar Suman, PhD; Albert J. Fornace Jr., MD; and Kamal Datta, MD, Department of Biochemistry
and Molecular & Cellular Biology, Georgetown University, Washington, DC
Purpose: Ionizing radiation exposure is a recognized risk factor for intestinal pathologies. While
acute effects of toxic radiation doses on intestine are well established, we have yet to acquire a complete
spectrum of radiation-induced chronic intestinal perturbations at the molecular level. The purpose of this
106 | P a g e
(Auger-electron emitter) or Cs137 (gamma-emitter) or were exposed to external gamma-rays (Co-60).
Radiation induced DNA damage was quantified by measuring histone H2X phosphorylation (γ-H2AX), p53
binding protein No. 1 (53BP1) foci formation, micronuclei formation and clonogenic cell survival.
In order to calculate the dose delivered, we first needed to estimate the Cs131 cell concentration and the
duration of the exposure. This required measuring the cell uptake and release of Cs131 by counting the
activity in the medium and the cells using a scintillation counter. Results from these internal exposures to
densely and sparsely ionizing radiation were compared to external γ-radiation. Our results show that
Cs131 is likely to be taken up by the Na+/K+ ATPase in a concentration dependent manner. However Cs131
is also released from the cells probably passively over the cell membrane. Cells incubated with Cs131
showed in general a higher level of damage compared to cells incubated with Cs137 or exposure to
external γ-radiation.
(PS1-08) Polynucleotide kinase/phosphatase, PNKP, as a target for enhanced cancer therapy. Zahra
Shire and Michael Weinfeld, University of Alberta, Edmonton, Canada
The radioresistance and chemoresistance of tumors are major obstacles that may lead to the failure of
cancer therapy. Reducing DNA repair in cancer cells is emerging as a new strategy for improving cancer
treatment. One approach is to identify small molecules inhibitors of DNA repair enzymes. Human
polynucleotide kinase/phosphatase (hPNKP), which possesses DNA 5’-kinase and 3’-phosphatase
activities, plays an essential role in DNA strand break repair by rendering strand-break termini suitable for
DNA polymerases and ligases. Previous work in our lab has shown that reduced expression of PNKP
sensitizes cells to ionizing radiation, alkylating agents and the topoisomerase I poison camptothecin. We
have developed a rapid fluorescence-based assay to identify small molecule inhibitors of the 3’-
phosphatase activity of PNKP. The main components of this assay are two oligonucleotide hairpin probes,
PNKP enzyme, and T4 DNA polymerase. T4 DNA polymerase has a powerful 3’-5’ exonuclease activity that
digests oligonucleotides that have a 3’-OH terminus, but is blocked by the presence of a 3’-phosphate
terminus. We detect the inhibition of PNKP by monitoring the fluorescence signal resulting from the
release of 2-aminopurine (2 Ap) embedded in the stem of the hairpin probes. The fluorescence of 2 Ap is
highly quenched when it is incorporated in duplex DNA. As a proof of principle, the novel assay was used
to test 200 compounds selected from three chemical libraries of natural derivative compounds, and two
chemically related compounds were identified that inhibit the phosphatase activity of hPNKP. Currently,
we are examining the mechanism of inhibition by these compounds and their ability to sensitize cancer
cells to radio/chemo-therapy.
(PS1-09)Ionizing radiation decreased Beclin1/LC3B and increased oxidative stress and mTOR via
PI3K/Akt to inhibit autophagy and promote intestinal cell proliferation in C57BL/6J mice.
Shubhankar Suman, PhD; Albert J. Fornace Jr., MD; and Kamal Datta, MD, Department of Biochemistry
and Molecular & Cellular Biology, Georgetown University, Washington, DC
Purpose: Ionizing radiation exposure is a recognized risk factor for intestinal pathologies. While
acute effects of toxic radiation doses on intestine are well established, we have yet to acquire a complete
spectrum of radiation-induced chronic intestinal perturbations at the molecular level. The purpose of this
106 | P a g e
