Object's details: The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells, Sonata Bis 2019/34/E/NZ7/00056, Sonata Bis-9 (dataset)

Provider:Repozytorium Uniwersytetu Łódzkiego

Description

Title:
The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells, Sonata Bis 2019/34/E/NZ7/00056, Sonata Bis-9 (dataset)
Creator:
Rogalska, Aneta
Contributor:
Department of Medical Biophysics, Institute of Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland;
aneta.rogalska@biol.uni.lodz.pl
Description:
Files description: 1. Cell viability after treatment with PARPi, CHK1i, and ATRi in BRCAMUT (PEO-1) and BRCAWT (OV-90, SKOV-3) cells at a concentration of 4 µM for 24 and 48 h (raw data + methodology description) 2. BRCAMUT (PEO-1) and BRCAWT (OV-90, SKOV-3) cell doubling time calculated from the cell growth curve during the exponential growth phase using the formula, Td = t/log2 (Nt/N0) (raw data + methodology description) 3. Percentage of necrotic, early and late apoptotic SKOV-3, PEO-1 and OV-90 cells after 24 and 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (raw data + fluorescence images + methodology description) 4. Flow cytometry (Annexin V+propidium iodide staining) measurement of apoptotic and dead SKOV-3, PEO-1 and OV-90 cells after 24 and 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (raw data + methodology description) 5. Confocal microscope immunofluorescence staining of caspase-3 and gamma-H2AX in SKOV-3, PEO-1 and OV-90 cells after 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (fluorescence images + methodology description) 6. Western blot analysis of caspase-3 and gamma-H2AX expression in SKOV-3, PEO-1 and OV-90 cells after 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (raw data + Western blot images + methodology description) 7. Flow cytometry (Vybrant® Dye Cycle™ Violet and SYTOX® AADvanced™ staining) measurement of apoptotic and dead SKOV-3, PEO-1 and OV-90 cells after 24 and 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (raw data + methodology description) 8. Flow cytometry measurement of cell cycle distribution, S-phase progression and incorporation of BrdU in SKOV-3, PEO-1 and OV-90 cells after 24 and 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (raw data + methodology description) 9. Fluorescence images of EdU incorporation in SKOV-3, PEO-1 and OV-90 cells after 24 and 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (fluorescence images + methodology description) 10. Chromosome aberrations in SKOV-3, PEO-1 and OV-90 cells after 24 treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)). DNA damage effects were measured by metaphase chromosome spread. The mitotic index was calculated as the percent ratio between the number of dividing cells and the entire cell population. The quantification was determined by BrdU incorporation. Aberration index (M-phase aberrant cells) was calculated as the percent ratio between the number of cells showing chromosome aberra_tions and all mitotic cells. Nuclear phenotypes in PEO-1/OV-90/SKOV-3 cells were determined by Giemsa and by counterstaining with DAPI (0.1 mg/mL). Types of chromosomal aberrations were counted under the microscope (raw data + microscope images + methodology description) 11. Number of cells with micronuclei in OV-90, SKOV-3 and PEO-1 cell lines after 24 and 48 h treatment with PARPi (4 µM), ATRi (4 µM) or CHK1i (4 µM) alone or in combination (PARPi (4 µM)+ATRi (4 µM); PARPi (4 µM)+CHK1i (4 µM)) (raw data + microscope images + methodology description);
Olaparib is a poly (ADP-ribose) polymerase inhibitor (PARPi) that inhibits PARP1/2, leading to replication-induced DNA damage that requires homologous recombination repair. Olaparib is often insufficient to treat BRCA-mutated (BRCAMUT) and BRCA wild-type (BRCAWT) high-grade serous ovarian carcinomas (HGSOCs). The short-term (up to 48 h) efficacy of PARPi treatment on a DNA damage response pathway mediated by ATR and CHK1 kinases in BRCAMUT (PEO-1) and BRCAWT (SKOV-3 and OV-90) cells was examined. The combination of ATRi/CHK1i with PARPi was not more cytotoxic than ATR and CHK1 monotherapy. The combination of olaparib with inhibitors of the ATR/CHK1 pathway generated chromosomal abnormalities, independent on BRCAMUT status of cells and formed of micronuclei (MN). However, the beneficial effect of the PARPi:ATRi combination on MN was seen only in the PEO1 BRCAMUT line. Monotherapy with ATR/CHK1 inhibitors reduced BrdU incorporation due to a slower rate of DNA synthesis, which resulted from elevated levels of replication stress, while simultaneous blockade of PARP and ATR caused beneficial effects only in OV-90 cells. Inhibition of ATR/CHK1 increased the formation of double-strand breaks as measured by increased γH2AX expression at collapsed replication forks, resulting in increased levels of apoptosis. Our findings indicate that ATR and CHK1 inhibitors provoke premature mitotic entry, leading to genomic instability and ultimately cell death.;
This research was funded by the National Science Centre, Poland (Project grant number: Sonata Bis 2019/34/E/NZ7/00056)
Object availability:
free access
Rights:
Uznanie autorstwa 4.0 Międzynarodowe;
http://creativecommons.org/licenses/by/4.0
Date:
2022.07.26;
2022
Type:
Dataset
Format:
application/octet-stream
Subject:
ATR inhibitor;
CHK1 inhibitor;
ovarian cancer;
PARP inhibitor;
replication stress;
targeted therapy
Identifier: http://hdl.handle.net/11089/42567
Data provider:
Repozytorium Uniwersytetu Łódzkiego

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Object's details: The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells, Sonata Bis 2019/34/E/NZ7/00056, Sonata Bis-9 (dataset)

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