660: In vitro model for DNA double-strand break (DSB) repair analysis in cells derived from breast cancer specimens identifies new prognostic markers

Abstract Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1–10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.

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Biochemical and structural characterization of analogs of MRE11 breast cancer-associated mutant F237C

Scientific Reports ◽

10.1038/s41598-021-86552-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Samiur Rahman ◽

Mahtab Beikzadeh ◽

Michael P. Latham

Keyword(s):

Breast Cancer ◽

Structural Changes ◽

Pyrococcus Furiosus ◽

Strand Break ◽

Double Strand Break ◽

Vital Role ◽

Dna Double Strand Break ◽

Cancer Tumor

AbstractThe MRE11–RAD50–NBS1 (MRN) protein complex plays a vital role in DNA double strand break sensing, signaling, and repair. Mutation in any component of this complex may lead to disease as disrupting DNA double strand break repair has the potential to cause translocations and loss of genomic information. Here, we have investigated an MRE11 mutation, F237C, identified in a breast cancer tumor. We found that the analogous mutant of Pyrococcus furiosus Mre11 diminishes both the exonuclease and endonuclease activities of Mre11 in vitro. Solution state NMR experiments show that this mutant causes structural changes in the DNA-bound Mre11 for both exo- and endonuclease substrates and causes the protein to become generally more rigid. Moreover, by comparing the NMR data for this cancer-associated mutant with two previously described Mre11 separation-of-nuclease function mutants, a potential allosteric network was detected within Mre11 that connects the active site to regions responsible for recognizing the DNA ends and for dimerization. Together, our data further highlight the dynamics required for Mre11 nuclease function and illuminate the presence of allostery within the enzyme.

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A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo

10.1101/2020.03.30.015271 ◽

2020 ◽

Author(s):

Jasper Che-Yung Chien ◽

Elie Tabet ◽

Kelsey Pinkham ◽

Cintia Carla da Hora ◽

Jason Cheng-Yu Chang ◽

...

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Non Invasive ◽

Longitudinal Tracking ◽

Dna Double Strand Break ◽

Significant Difference ◽

Therapeutic Development

ABSTRACTTracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.

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β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

Molecular and Cellular Biology ◽

10.1128/mcb.00672-17 ◽

2018 ◽

Vol 38 (9) ◽

Cited By ~ 11

Author(s):

Kazi Mokim Ahmed ◽

Raj K. Pandita ◽

Dharmendra Kumar Singh ◽

Clayton R. Hunt ◽

Tej K. Pandita

Keyword(s):

Breast Cancer ◽

Homologous Recombination ◽

Molecular Mechanisms ◽

Ectopic Expression ◽

Strand Break ◽

Double Strand Break ◽

Β1 Integrin ◽

Integrin Expression ◽

Dsb Repair ◽

Dna Double Strand Break

ABSTRACT The molecular mechanisms underlying resistance to radiotherapy in breast cancer cells remain elusive. Previously, we reported that elevated β1-integrin is associated with enhanced breast cancer cell survival postirradiation, but how β1-integrin conferred radioresistance was unclear. Ionizing radiation (IR) induced cell killing correlates with the efficiency of DNA double-strand break (DSB) repair, and we found that nonmalignant breast epithelial (S1) cells with low β1-integrin expression have a higher frequency of S-phase-specific IR-induced chromosomal aberrations than the derivative malignant breast (T4-2) cells with high β1-integrin expression. In addition, there was an increased frequency of IR-induced homologous recombination (HR) repairosome focus formation in T4-2 cells compared with that of S1 cells. Cellular levels of Rad51 in T4-2 cells, a critical factor in HR-mediated DSB repair, were significantly higher. Blocking or depleting β1-integrin activity in T4-2 cells reduced Rad51 levels, while ectopic expression of β1-integrin in S1 cells correspondingly increased Rad51 levels, suggesting that Rad51 is regulated by β1-integrin. The low level of Rad51 protein in S1 cells was found to be due to rapid degradation by the ubiquitin proteasome pathway (UPP). Furthermore, the E3 ubiquitin ligase RING1 was highly upregulated in S1 cells compared to T4-2 cells. Ectopic β1-integrin expression in S1 cells reduced RING1 levels and increased Rad51 accumulation. In contrast, β1-integrin depletion in T4-2 cells significantly increased RING1 protein levels and potentiated Rad51 ubiquitination. These data suggest for the first time that elevated levels of the extracellular matrix receptor β1-integrin can increase tumor cell radioresistance by decreasing Rad51 degradation through a RING1-mediated proteasomal pathway.

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The Human Breast Cancer Cell DNA Synthesome Can Serve as a Novel in Vitro Model System for Studying the Mechanism of Action of Anticancer Drugs

10.21236/ada384124 ◽

1999 ◽

Author(s):

Haiyan Jiang

Keyword(s):

Breast Cancer ◽

Human Breast Cancer ◽

In Vitro Model ◽

Model System ◽

Human Breast Cancer Cell ◽

Human Breast ◽

Dna Synthesome ◽

In Vitro Model System ◽

Vitro Model

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The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo

Blood ◽

10.1182/blood-2010-01-265769 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4578-4587 ◽

Cited By ~ 189

Author(s):

Victoria J. Weston ◽

Ceri E. Oldreive ◽

Anna Skowronska ◽

David G. Oscier ◽

Guy Pratt ◽

...

Keyword(s):

Cell Line ◽

Tumor Cells ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Prolymphocytic Leukemia ◽

Dna Double Strand Break ◽

Break Repair

Abstract The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

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Quantification of DNA double-strand break induced by radiation in cervix cancer cells: in vitro study

Journal of Radiotherapy in Practice ◽

10.1017/s1460396918000456 ◽

2018 ◽

Vol 18 (1) ◽

pp. 52-54

Author(s):

Sothing Vashum ◽

Rabi Raja Singh I ◽

Saikat Das ◽

Mohammed Azharuddin KO ◽

Prabhakaran Vasudevan

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Strand Break ◽

Double Strand Break ◽

Ionising Radiation ◽

Dependent Manner ◽

Survival Fraction ◽

Dna Double Strand Break ◽

Cervical Cancer Cells

AbstractAimDNA double-strand break (DSB) results in the phosphorylation of the protein, H.2AX histone. In this study, the effect of radiotherapy and chemotherapy on DNA DSB in cervical cancer cells is analysed by the phosphorylation of the protein.MethodsThe cervical cancer cells (HeLa cells) were cultured and exposed to ionising radiation. Radiation sensitivity was measured by clonogenic survival fraction after exposing to ionising radiation. Since the phosphorylation of H.2AX declines with time, the DNA damage was quantified at different time points: 1 hour, 3 hours and 1 week after exposed to the radiation. The analysis of γ-H.2AX was done by Western-blot technique. The protein expression was observed at different dose of radiation and combination of both radiation and paclitaxel.ResultsLow-dose hypersensitivity was observed. By 1 week after radiation at 0·5, 0·8 and 2 Gy, there was no expression of phosphorylated H.2AX. Previous experiments on the expression of phosphorylated H.2AX (γ-H.2AX) in terms of foci analysis was found to peak at 1 hour and subsequently decline with time. In cells treated with the DNA damaging agents, the expression of phosphorylated H.2AX decreases in a dose-dependent manner when treated with radiation alone. However, when combined with paclitaxel, at 0·5 Gy, the expression peaked and reduces at 0·8 Gy and slightly elevated at 2 Gy.FindingsIn this study, the peak phosphorylation was observed at 3 hour post irradiation indicating that DSBs are still left unrepaired.

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