Page 131 - 2014 Printable Abstract Book
P. 131
(PS1-53) Distinct RNA transcript signatures in radiosensitive bone marrow stromal compared to
radioresistant hematopoietic cell lines from fanconi anemia (fa) (fancd2-/-) fvb/n mice. Ashwin Shinde;
Byung Han. Rhieu; Hebist Berhane; Michael W. Epperly; Hong Wang; Donna Shields; and Shaonan Xao,
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA
Purpose: We investigated the molecular mechanism of the recently reported radioresistance of C57Bl/6
Fancd2-/- mouse hematopoietic cells (Berhane, et al. Rad Res.181:76-89, 2014) using hematopoietic and
mesenchymal cell lines derived from a different FVB/N background strain of Fancd2-/- mice. Methods:
Long-term bone marrow cultures (LTBMCs) were established from Fancd2+/+, Fancd2+/-, and Fancd2-/-
mice (FVB/N background). Radiation survival curves of non-adherent hematopoietic cells and adherent
stromal cell lines and RT-PCR (for levels of 14 representative genes involved in irradiation damage
response, promoter binding factors, and apoptosis) were carried out. Results: Fancd2-/- mouse
mesenchymal (marrow stromal) cell lines were radiosensitive (D0 = 1.75 ± 0.09, ñ = 2.5 ± 0.1) compared
to those from Fancd2+/+ mice (D0 = 1.71 ± 0.03, ñ = 8.2 ± 0.5, p = 0.0078). In contrast, IL-3 dependent
hematopoietic progenitor cell lines derived from Fancd2-/- mice were radioresistant (D0 = 2.02 ± 0.11, ñ
= 3.73 ± 0.07) compared to those from Fancd2+/+ mice (D0 = 1.08 ± 0.06, ñ = 2.07 ± 0.05, p = 0.0027). This
data confirms, in the FVB/N strain, prior results of the effect of the Fancd2-/- genotype on the radiobiology
of hematopoietic compared to mesenchymal cell phenotypes derived from C57Bl/6 mice. Gene transcript
levels in Fancd2-/- and heterozygote Fancd2+/- stromal and hematopoietic cell lines demonstrated
significant elevation for TNFa (p ≤ 0.012), p21 (p ≤ 0.017) transcripts and suppression of SP1 (p ≤ 0.043)
compared to wild-type Fancd2+/+ cells However, differences between cell phenotypes included: stromal
cell suppression of PGC-1A (≤ -78%, p ≤ 0.001) but hematopoietic cell elevation of PGC-1A (≥ 400%, p ≤
0.011). In addition, NFKb was suppressed (≤ -64%, p ≤ 0.003) in Fancd2-/- and Fancd2+/- stromal cells, but
NFKb was elevated in hematopoietic cells (≥ 57, p ≤ 0.009) which also showed elevated AP1 (≥ 71%, p ≤
0.014), Nrf2 (≥ 34%, p ≤ 0.025) and Rad51 (≥ 444%, p ≤ 0.004).
Conclusions: Radioresistant Fancd2-/- mouse hematopoietic cells display distinct baseline gene transcript
levels and suggest a cell-phenotype specific role of the Fancd2 protein and bone marrow stromal cell
etiology of the radiosensitivity observed in some FA patients. Supported by NIAID/NIH U19-A 1068021
and the Fanconi Anemia Research Foundation.
(PS1-54) Participation of exosomes in radiation-induced bystander effects in normal stem cells. Nicholas
Colangelo; Sonia de Toledo, PhD; and Edouard Azzam, PhD, Rutgers University-New Jersey Medical School,
Newark, NJ
Traditionally, it was assumed that ionizing radiation produced genetic damage and other
important biological responses only in cells where the radiation directly traversed the nucleus. However,
it is now understood that the surrounding non-irradiated cells are also significantly affected through
intercellular communication with nearby irradiated cells. The repercussions of this bystander effect
impact both radiation health risks and therapeutic treatments. Several mechanisms involving junctional
channels and secreted molecules have been implicated in this bystander phenomenon. Recently,
attention has been given to the role exosomes play in this response. With their diverse protein and nucleic
acid contents, exosomes have the potential to alter the normal functioning of recipient cells. Using
glioblastoma cells (T98G, U87) as a model system, the effects of exosomes harvested 24 h after exposure
137
to an absorbed dose of 10 Gy of Cs rays are being studied. Initial results have demonstrated exosomes
129 | P a g e
radioresistant hematopoietic cell lines from fanconi anemia (fa) (fancd2-/-) fvb/n mice. Ashwin Shinde;
Byung Han. Rhieu; Hebist Berhane; Michael W. Epperly; Hong Wang; Donna Shields; and Shaonan Xao,
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA
Purpose: We investigated the molecular mechanism of the recently reported radioresistance of C57Bl/6
Fancd2-/- mouse hematopoietic cells (Berhane, et al. Rad Res.181:76-89, 2014) using hematopoietic and
mesenchymal cell lines derived from a different FVB/N background strain of Fancd2-/- mice. Methods:
Long-term bone marrow cultures (LTBMCs) were established from Fancd2+/+, Fancd2+/-, and Fancd2-/-
mice (FVB/N background). Radiation survival curves of non-adherent hematopoietic cells and adherent
stromal cell lines and RT-PCR (for levels of 14 representative genes involved in irradiation damage
response, promoter binding factors, and apoptosis) were carried out. Results: Fancd2-/- mouse
mesenchymal (marrow stromal) cell lines were radiosensitive (D0 = 1.75 ± 0.09, ñ = 2.5 ± 0.1) compared
to those from Fancd2+/+ mice (D0 = 1.71 ± 0.03, ñ = 8.2 ± 0.5, p = 0.0078). In contrast, IL-3 dependent
hematopoietic progenitor cell lines derived from Fancd2-/- mice were radioresistant (D0 = 2.02 ± 0.11, ñ
= 3.73 ± 0.07) compared to those from Fancd2+/+ mice (D0 = 1.08 ± 0.06, ñ = 2.07 ± 0.05, p = 0.0027). This
data confirms, in the FVB/N strain, prior results of the effect of the Fancd2-/- genotype on the radiobiology
of hematopoietic compared to mesenchymal cell phenotypes derived from C57Bl/6 mice. Gene transcript
levels in Fancd2-/- and heterozygote Fancd2+/- stromal and hematopoietic cell lines demonstrated
significant elevation for TNFa (p ≤ 0.012), p21 (p ≤ 0.017) transcripts and suppression of SP1 (p ≤ 0.043)
compared to wild-type Fancd2+/+ cells However, differences between cell phenotypes included: stromal
cell suppression of PGC-1A (≤ -78%, p ≤ 0.001) but hematopoietic cell elevation of PGC-1A (≥ 400%, p ≤
0.011). In addition, NFKb was suppressed (≤ -64%, p ≤ 0.003) in Fancd2-/- and Fancd2+/- stromal cells, but
NFKb was elevated in hematopoietic cells (≥ 57, p ≤ 0.009) which also showed elevated AP1 (≥ 71%, p ≤
0.014), Nrf2 (≥ 34%, p ≤ 0.025) and Rad51 (≥ 444%, p ≤ 0.004).
Conclusions: Radioresistant Fancd2-/- mouse hematopoietic cells display distinct baseline gene transcript
levels and suggest a cell-phenotype specific role of the Fancd2 protein and bone marrow stromal cell
etiology of the radiosensitivity observed in some FA patients. Supported by NIAID/NIH U19-A 1068021
and the Fanconi Anemia Research Foundation.
(PS1-54) Participation of exosomes in radiation-induced bystander effects in normal stem cells. Nicholas
Colangelo; Sonia de Toledo, PhD; and Edouard Azzam, PhD, Rutgers University-New Jersey Medical School,
Newark, NJ
Traditionally, it was assumed that ionizing radiation produced genetic damage and other
important biological responses only in cells where the radiation directly traversed the nucleus. However,
it is now understood that the surrounding non-irradiated cells are also significantly affected through
intercellular communication with nearby irradiated cells. The repercussions of this bystander effect
impact both radiation health risks and therapeutic treatments. Several mechanisms involving junctional
channels and secreted molecules have been implicated in this bystander phenomenon. Recently,
attention has been given to the role exosomes play in this response. With their diverse protein and nucleic
acid contents, exosomes have the potential to alter the normal functioning of recipient cells. Using
glioblastoma cells (T98G, U87) as a model system, the effects of exosomes harvested 24 h after exposure
137
to an absorbed dose of 10 Gy of Cs rays are being studied. Initial results have demonstrated exosomes
129 | P a g e
