Page 114 - 2014 Printable Abstract Book
P. 114
Then, cells were either given a single 2 Gy PRT dose or a 2 Gy dose for 5 consecutive days and cell
proliferation measured by MTT assay two days following the last radiation treatment to determine the
effect of multiple PRT treatments. DNA double-strand breaks were measured by phosphorylation of
histone H2AX, cell cycle progression was measured using antiphospho-Histone H3 mitosis marker, and
apoptosis was scored using FITC-conjugated Monoclonal Active Caspase 3 Antibody Apoptosis Kit, and
then all parameters analyzed on a FACSCanto flow cytometer. Slower DNA repair was seen after PRT for
SK-N-BE (2) but not MC-IXC and SK-N-SH cells. This is consistent with a role for HRS, and overcoming HRS
with increasing dose, in cell death after PRT observed in our earlier tumor studies. In addition, VEGF, FGF-
basic, and Angiopoietin-L4 levels were measured using ELISA kits and radiation-induced changes in
expression were found to be cell line dependent. For example, MC-IXC cells exhibited a 20-fold increase
in angiopoietin-L4, while SK-N-BE (2) and SH-SY5Y cells showed 15-20 fold increases in FGF-basic.
Together, these data provide evidence that HRS-associated DNA repair and checkpoint mechanisms have
a role in the tumor response after PRT.
(PS1-20) NEK9 is a new gemcitabine sensitivity gene and a component of the replication stress response.
2
1
1
1
Aleksandra Petrova ; Scott C. Smith, PhD ; Matthew Z. Madden, BA ; Hongyan Wang, PhD ; Matthew D.
1
1
1
1
3
Warren, MS ; Claire W. Hardy, BS ; M. Hope Robinson, BA ; Jie Wang, PhD ; Ya Wang, PhD ; and David S.
2
Yu, MD, PhD, Emory University School of Medicine, Atlanta, GA ; University of Louseville, Louseville, KY ;
1
1
3
and Vanderbuilt University School of Medicine, Nashville, TN
Background: Never in mitosis gene A (NIMA)-related kinase 9 (NEK9) is a member of the NEK
family of serine/threonine kinases that are emerging as important regulators of the cell cycle and
checkpoint control. We identified NEK9 in as a gemcitabine sensitivity gene through a synthetic lethal
siRNA screen in human triple-negative breast cancer (TNBS) cells. In this study we characterize NEK9 as a
component of Replication Stress Response (RSR). Methods: MDA-MB-231 cells were transfected in 96-
well plates 25 nM siRNA from a custom siGenome siRNA library of 4,024 siRNAs corresponding to 1,006
unique human nuclear enzyme genes using a one-gene:one well format. Twenty-four hours later plates
were split 1:4, and then treated following another 24 hours with or without 13nM gemcitabine for 72
hours prior to assaying for cell proliferation using WST-1 reagent. Each plate contained two positive
controls (ATR and CHK1) and several negative controls (NT), and plate-to-plate variability was controlled
by normalizing the values on each plate to the average of the negative control values on that plate. A ratio
of gemcitabine treated/untreated viability was calculated and normalized to that of non-targeting (NT)
siRNA. Results: Three of four siRNAs targeting NEK9 caused gemcitabine hypersensitivity in MDA-MB-321
(breast cancer) cells and MIA PaCa-2 (pancreatic cancer) cells. Hypersensitivity correlated with decreased
NEK9 levels, confirmed by Western blot following siRNA knockdown. NEK9 knockdown also caused
hypersensitivity to mitomycin C, suggesting that NEK9 responds generally to replication stress. We found
that depletion of NEK9 in cells results in spontaneous accumulation of γH2AX, an early marker of DNA
damage, and RPA to foci. Cells with NEK9 knockdown showed significant delay in recovery from replication
arrest. We also found that NEK9 protein levels in cells increases in response to replication stress, caused
by hydroxyurea. Finally, we found that NEK9 forms complex with CHK1 and that NEK9 depletion in cells
impairs CHK1 Ser296 autophosphorylation and kinase activity in response to replication stress.
Conclusions: Our findings reveal a novel function for NEK9 as a RSR protein, which promotes the activity
of CHK1 in response to challenges to DNA replication.
112 | P a g e
proliferation measured by MTT assay two days following the last radiation treatment to determine the
effect of multiple PRT treatments. DNA double-strand breaks were measured by phosphorylation of
histone H2AX, cell cycle progression was measured using antiphospho-Histone H3 mitosis marker, and
apoptosis was scored using FITC-conjugated Monoclonal Active Caspase 3 Antibody Apoptosis Kit, and
then all parameters analyzed on a FACSCanto flow cytometer. Slower DNA repair was seen after PRT for
SK-N-BE (2) but not MC-IXC and SK-N-SH cells. This is consistent with a role for HRS, and overcoming HRS
with increasing dose, in cell death after PRT observed in our earlier tumor studies. In addition, VEGF, FGF-
basic, and Angiopoietin-L4 levels were measured using ELISA kits and radiation-induced changes in
expression were found to be cell line dependent. For example, MC-IXC cells exhibited a 20-fold increase
in angiopoietin-L4, while SK-N-BE (2) and SH-SY5Y cells showed 15-20 fold increases in FGF-basic.
Together, these data provide evidence that HRS-associated DNA repair and checkpoint mechanisms have
a role in the tumor response after PRT.
(PS1-20) NEK9 is a new gemcitabine sensitivity gene and a component of the replication stress response.
2
1
1
1
Aleksandra Petrova ; Scott C. Smith, PhD ; Matthew Z. Madden, BA ; Hongyan Wang, PhD ; Matthew D.
1
1
1
1
3
Warren, MS ; Claire W. Hardy, BS ; M. Hope Robinson, BA ; Jie Wang, PhD ; Ya Wang, PhD ; and David S.
2
Yu, MD, PhD, Emory University School of Medicine, Atlanta, GA ; University of Louseville, Louseville, KY ;
1
1
3
and Vanderbuilt University School of Medicine, Nashville, TN
Background: Never in mitosis gene A (NIMA)-related kinase 9 (NEK9) is a member of the NEK
family of serine/threonine kinases that are emerging as important regulators of the cell cycle and
checkpoint control. We identified NEK9 in as a gemcitabine sensitivity gene through a synthetic lethal
siRNA screen in human triple-negative breast cancer (TNBS) cells. In this study we characterize NEK9 as a
component of Replication Stress Response (RSR). Methods: MDA-MB-231 cells were transfected in 96-
well plates 25 nM siRNA from a custom siGenome siRNA library of 4,024 siRNAs corresponding to 1,006
unique human nuclear enzyme genes using a one-gene:one well format. Twenty-four hours later plates
were split 1:4, and then treated following another 24 hours with or without 13nM gemcitabine for 72
hours prior to assaying for cell proliferation using WST-1 reagent. Each plate contained two positive
controls (ATR and CHK1) and several negative controls (NT), and plate-to-plate variability was controlled
by normalizing the values on each plate to the average of the negative control values on that plate. A ratio
of gemcitabine treated/untreated viability was calculated and normalized to that of non-targeting (NT)
siRNA. Results: Three of four siRNAs targeting NEK9 caused gemcitabine hypersensitivity in MDA-MB-321
(breast cancer) cells and MIA PaCa-2 (pancreatic cancer) cells. Hypersensitivity correlated with decreased
NEK9 levels, confirmed by Western blot following siRNA knockdown. NEK9 knockdown also caused
hypersensitivity to mitomycin C, suggesting that NEK9 responds generally to replication stress. We found
that depletion of NEK9 in cells results in spontaneous accumulation of γH2AX, an early marker of DNA
damage, and RPA to foci. Cells with NEK9 knockdown showed significant delay in recovery from replication
arrest. We also found that NEK9 protein levels in cells increases in response to replication stress, caused
by hydroxyurea. Finally, we found that NEK9 forms complex with CHK1 and that NEK9 depletion in cells
impairs CHK1 Ser296 autophosphorylation and kinase activity in response to replication stress.
Conclusions: Our findings reveal a novel function for NEK9 as a RSR protein, which promotes the activity
of CHK1 in response to challenges to DNA replication.
112 | P a g e
