Page 126 - 2014 Printable Abstract Book
P. 126
(PS1-43) Automated analysis of the effects of Bcl-2 and Bcl-XL expression on the radiation response of
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MCF-10A cells. Caitlin E. Mills, PhD ; David W. Andrews, PhD ; and Douglas R. Boreham, PhD
1
Sunnybrook Research Institute, Toronto, Canada and Northern Ontario School of Medicine, Sudbury,
2
Canada
The effects of Bcl-2 and Bcl-XL expression on the radiation response of human mammary epithelial
MCF-10A cells were investigated. Stable cell lines in which Bcl-2 or Bcl-XL were exogenously expressed, or
Bcl-XL expression was knocked down were established. Spectral karyotyping and array genomic
hybridization were used to confirm the identity and stability of the MCF-10A cells used. The anti-apoptotic
effects of the exogenous proteins against known apoptotic stimuli were confirmed using traditional
assays. The influence of Bcl-2 and Bcl-XL expression on the immediate and short term effects of radiation
exposure was investigated using cell growth, flow cytometry, and automated image based assays for DNA
double strand breaks (53BP1) and changes in nuclear morphology. The automation of traditional assays
using a Thermo SP-Workcell fully contained robotic liquid handling platform for sample preparation and
a high-throughput Opera confocal microscope for imaging enabled unbiased concurrent investigation of
the effects of numerous doses at many time points in multiple cell lines. The throughput of the automated
assays was also exploited to directly examine the effects of doses as low as those associated with
mammography. The potential for Bcl-2 or -XL expression to promote transformation post-irradiation was
evaluated using the soft agar assay. Bcl-2 and -XL expression levels were not found to significantly
influence the effects of ionizing radiation in MCF-10A cells despite resulting in drastic differential effects
when challenged with other apoptotic stimuli. The initial number of radiation-induced 53BP1foci in the
dose range examined was linear up to 1 Gy and then began to plateau regardless of Bcl-2 or -XL expression.
Likewise, repair kinetics based on the resolution of foci were found to be independent of Bcl-2/XL
expression levels as were growth post-irradiation, and cell cycle perturbations. Radiation exposure in
conjunction with the overexpression of Bcl-2 or -XL was not sufficient to promote growth in soft agar. This
work contributes to the understanding of the response of non-tumourigenic breast epithelial cells to the
effects of clinically relevant radiation exposures. Furthermore, it suggests that Bcl-2 and Bcl-XL do not
potentiate radiation effects in these cells.
(PS1-44 ) Modulating DNA repair mechanisms to enhance hyperthermic radiosensitization of breast
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cancer cells. Nicholas B. Dye, M.S. ; Rishabh Chaudhari, MD ; Juong G. Rhee, MD ; and Zeljko Vujaskovic,
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MD, PhD, University of Maryland School of Medicine, Baltimore, MD and George Washington School of
2
Medicine and Health Sciences, Washington, DC
Introduction: Hyperthermia is known to increase the radiosensitivity of cancer cells through various
mechanisms including degradation of the BRCA2 protein required for repairing DNA double strand breaks
(DSB’s) through homologous recombination (HR). Loss of HR relegates cells to more error-prone
mechanisms of repair resulting in susceptibility to DNA damage. A number of drugs are under
investigation to further disrupt a cell’s response to DNA damage including heat shock protein (HSP)
inhibitors and poly-ADP-ribose polymerase (PARP) inhibitors. If a cancer cell’s ability to repair its DNA can
be disrupted using a combination of heat, HSP inhibitors, and PARP inhibitors the efficacy of
radiatiotherapy may be optimized. Methods: MDA-MB-453 “parent” cells were transfected with a
plasmid-encoding GFP under control of the Oct-3/4 promoter to create a “stem-like” phenotype. Parent
and stem-like cells were irradiated at 2-6 Gy and subjected to heating (42ºC) for 1 hour either before or
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1
2
MCF-10A cells. Caitlin E. Mills, PhD ; David W. Andrews, PhD ; and Douglas R. Boreham, PhD
1
Sunnybrook Research Institute, Toronto, Canada and Northern Ontario School of Medicine, Sudbury,
2
Canada
The effects of Bcl-2 and Bcl-XL expression on the radiation response of human mammary epithelial
MCF-10A cells were investigated. Stable cell lines in which Bcl-2 or Bcl-XL were exogenously expressed, or
Bcl-XL expression was knocked down were established. Spectral karyotyping and array genomic
hybridization were used to confirm the identity and stability of the MCF-10A cells used. The anti-apoptotic
effects of the exogenous proteins against known apoptotic stimuli were confirmed using traditional
assays. The influence of Bcl-2 and Bcl-XL expression on the immediate and short term effects of radiation
exposure was investigated using cell growth, flow cytometry, and automated image based assays for DNA
double strand breaks (53BP1) and changes in nuclear morphology. The automation of traditional assays
using a Thermo SP-Workcell fully contained robotic liquid handling platform for sample preparation and
a high-throughput Opera confocal microscope for imaging enabled unbiased concurrent investigation of
the effects of numerous doses at many time points in multiple cell lines. The throughput of the automated
assays was also exploited to directly examine the effects of doses as low as those associated with
mammography. The potential for Bcl-2 or -XL expression to promote transformation post-irradiation was
evaluated using the soft agar assay. Bcl-2 and -XL expression levels were not found to significantly
influence the effects of ionizing radiation in MCF-10A cells despite resulting in drastic differential effects
when challenged with other apoptotic stimuli. The initial number of radiation-induced 53BP1foci in the
dose range examined was linear up to 1 Gy and then began to plateau regardless of Bcl-2 or -XL expression.
Likewise, repair kinetics based on the resolution of foci were found to be independent of Bcl-2/XL
expression levels as were growth post-irradiation, and cell cycle perturbations. Radiation exposure in
conjunction with the overexpression of Bcl-2 or -XL was not sufficient to promote growth in soft agar. This
work contributes to the understanding of the response of non-tumourigenic breast epithelial cells to the
effects of clinically relevant radiation exposures. Furthermore, it suggests that Bcl-2 and Bcl-XL do not
potentiate radiation effects in these cells.
(PS1-44 ) Modulating DNA repair mechanisms to enhance hyperthermic radiosensitization of breast
2
1
1
cancer cells. Nicholas B. Dye, M.S. ; Rishabh Chaudhari, MD ; Juong G. Rhee, MD ; and Zeljko Vujaskovic,
1
1
MD, PhD, University of Maryland School of Medicine, Baltimore, MD and George Washington School of
2
Medicine and Health Sciences, Washington, DC
Introduction: Hyperthermia is known to increase the radiosensitivity of cancer cells through various
mechanisms including degradation of the BRCA2 protein required for repairing DNA double strand breaks
(DSB’s) through homologous recombination (HR). Loss of HR relegates cells to more error-prone
mechanisms of repair resulting in susceptibility to DNA damage. A number of drugs are under
investigation to further disrupt a cell’s response to DNA damage including heat shock protein (HSP)
inhibitors and poly-ADP-ribose polymerase (PARP) inhibitors. If a cancer cell’s ability to repair its DNA can
be disrupted using a combination of heat, HSP inhibitors, and PARP inhibitors the efficacy of
radiatiotherapy may be optimized. Methods: MDA-MB-453 “parent” cells were transfected with a
plasmid-encoding GFP under control of the Oct-3/4 promoter to create a “stem-like” phenotype. Parent
and stem-like cells were irradiated at 2-6 Gy and subjected to heating (42ºC) for 1 hour either before or
124 | P a g e
