Page 115 - 2014 Printable Abstract Book
P. 115
(PS1-22) Characterizing the DNA damage response by cell tracking algorithms and cell features
classification using high-content time lapse analysis. Walter Georgescu; Deepa Sridharan; Maria Rojec;
William C. Hines; Jonathan Tang; and Sylvain Costes, Lawrence Berkeley National Laboratory, Berkeley,
CA
Analysis of the behaviors of repair proteins tagged with fluorescent markers has greatly increased our
understanding of the molecular mechanisms responsible for repair of DNA damage, and in particular
double strand breaks (DSBs). However, detailed knowledge of the dynamics of the DNA damage response
(DDR) across multiple cell generations has been hampered by over-reliance on analyses of fixed cells. Here
we report on the dynamics of DSBs across multiple cell generations using live, non-malignant human
mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-
mCherry. Time-lapse images were obtained before and after exposure to different doses of X-rays without
removal from the microscope, using a custom-mounted MiniX 40kV X-ray tube. Movies were analyzed
using a custom image processing pipeline that tracks individual nuclei across four cell generations. After
irradiation, 53BP1-GFP is recruited to damage sites and forms radiation induced foci (RIF), whose
movement, resolution and inheritance were monitored using imaging classifiers that track cell cycle
phases, normal mitosis, mitotic catastrophe, and apoptosis up to 72 hours post-irradiation. We observe
that after doses greater than 1 Gy, RIF’s motion is constrained to few large nuclear domains of 1 to 3 µm
in diameter. Using automatic clustering and linear programming techniques we compute the kinetics of
foci aggregation and repair at different doses and show there is an enhanced clustering response at high
dose with the majority of foci resolving from super-large clusters.
(PS1-23) Erb-041 mediates photoprotection through bca2 inhibition. YUAN-HAO LEE and Komaraiah
Palle, Mitchell Cancer Institute, Mobile, AL
Purpose: The effect of an agonist of estrogen receptor β (ERβ), Erb-041, on UVB-induced
photocarcinogenesis has shown to suppress development of squamous cell carcinoma in SKH-1 hairless
mice. It was found that Erb-041 blocks the cell cycle propagation while inhibiting nuclear exit of p53. This
indicates a role of ERβ on launching cell cycle checkpoint for minimizing UV-induced carcinogenesis. ERβ
expression in breast cancer is lower than in the normal breast, and its mediated effects are often
antagonistic to that of UV-induced ERα signaling. Materials and methods: In this study, we applied
+/+
ER/BCA2 MCF-7 cells for investigating the expression of proteins involved in DNA damage and repair.
Erb-041 (40μM) modulatory effect on UVC-induced DNA damage responses was analyzed with Western
blot, RT-PCR, FACS and clonogenic assay. TLR after UVC irradiation was identified based on PCNA and
PAF15 polyubiquitination. To test whether BCA2 interact with Mdm2 and Rad18, BCA2-targeted siRNAs,
Flag-BCA2 and HA-Rad18 constructs were applied for immunoprecipitation. Statistical analyses were
performed using multiple comparisons with Bonferroni method in ProStat. Results: From RT-PCR and
Western blot, Erb-041 was found to reduce the mRNA level of BCA2 and the nuclear levels of FANCD2 and
Rad18 in presence or absence of UV irradiation. In addition, UV-induced ATM and Chk1 activation were
abrogated by Erb-041 pretreatment in parallel with reduced DNA DSBs, indicated by γH2AX. Moreover,
Erb-041 induced cell cycle arrest in G1 phase and promoted cell survival after irradiation as a result of p21
up-regulation and enhanced Rad18 expression, respectively. Concomitantly, UVC-induced double-
stranded breaks (DSBs) were decreased by BCA2 knockdown/down-regulation due to blockade of DNA
113 | P a g e
classification using high-content time lapse analysis. Walter Georgescu; Deepa Sridharan; Maria Rojec;
William C. Hines; Jonathan Tang; and Sylvain Costes, Lawrence Berkeley National Laboratory, Berkeley,
CA
Analysis of the behaviors of repair proteins tagged with fluorescent markers has greatly increased our
understanding of the molecular mechanisms responsible for repair of DNA damage, and in particular
double strand breaks (DSBs). However, detailed knowledge of the dynamics of the DNA damage response
(DDR) across multiple cell generations has been hampered by over-reliance on analyses of fixed cells. Here
we report on the dynamics of DSBs across multiple cell generations using live, non-malignant human
mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-
mCherry. Time-lapse images were obtained before and after exposure to different doses of X-rays without
removal from the microscope, using a custom-mounted MiniX 40kV X-ray tube. Movies were analyzed
using a custom image processing pipeline that tracks individual nuclei across four cell generations. After
irradiation, 53BP1-GFP is recruited to damage sites and forms radiation induced foci (RIF), whose
movement, resolution and inheritance were monitored using imaging classifiers that track cell cycle
phases, normal mitosis, mitotic catastrophe, and apoptosis up to 72 hours post-irradiation. We observe
that after doses greater than 1 Gy, RIF’s motion is constrained to few large nuclear domains of 1 to 3 µm
in diameter. Using automatic clustering and linear programming techniques we compute the kinetics of
foci aggregation and repair at different doses and show there is an enhanced clustering response at high
dose with the majority of foci resolving from super-large clusters.
(PS1-23) Erb-041 mediates photoprotection through bca2 inhibition. YUAN-HAO LEE and Komaraiah
Palle, Mitchell Cancer Institute, Mobile, AL
Purpose: The effect of an agonist of estrogen receptor β (ERβ), Erb-041, on UVB-induced
photocarcinogenesis has shown to suppress development of squamous cell carcinoma in SKH-1 hairless
mice. It was found that Erb-041 blocks the cell cycle propagation while inhibiting nuclear exit of p53. This
indicates a role of ERβ on launching cell cycle checkpoint for minimizing UV-induced carcinogenesis. ERβ
expression in breast cancer is lower than in the normal breast, and its mediated effects are often
antagonistic to that of UV-induced ERα signaling. Materials and methods: In this study, we applied
+/+
ER/BCA2 MCF-7 cells for investigating the expression of proteins involved in DNA damage and repair.
Erb-041 (40μM) modulatory effect on UVC-induced DNA damage responses was analyzed with Western
blot, RT-PCR, FACS and clonogenic assay. TLR after UVC irradiation was identified based on PCNA and
PAF15 polyubiquitination. To test whether BCA2 interact with Mdm2 and Rad18, BCA2-targeted siRNAs,
Flag-BCA2 and HA-Rad18 constructs were applied for immunoprecipitation. Statistical analyses were
performed using multiple comparisons with Bonferroni method in ProStat. Results: From RT-PCR and
Western blot, Erb-041 was found to reduce the mRNA level of BCA2 and the nuclear levels of FANCD2 and
Rad18 in presence or absence of UV irradiation. In addition, UV-induced ATM and Chk1 activation were
abrogated by Erb-041 pretreatment in parallel with reduced DNA DSBs, indicated by γH2AX. Moreover,
Erb-041 induced cell cycle arrest in G1 phase and promoted cell survival after irradiation as a result of p21
up-regulation and enhanced Rad18 expression, respectively. Concomitantly, UVC-induced double-
stranded breaks (DSBs) were decreased by BCA2 knockdown/down-regulation due to blockade of DNA
113 | P a g e
