scholarly journals Genetic toxicity testing using human in vitro organotypic airway cultures: assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing

2021 ◽  
Author(s):  
Yiying Wang ◽  
Roberta A. Mittelstaedt ◽  
Rebecca Wynne ◽  
Ying Chen ◽  
Xuefei Cao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxicity Testing ◽  
Genetic Toxicity ◽  
Duplex Sequencing

Genetic instability of in vitro cell lines: Implications for genetic toxicity testing

2019 ◽  
Author(s):  
Jinpeng Li ◽  
Raja S. Settivari ◽  
Matthew J. LeBaron
Keyword(s):  
Cell Lines ◽  
Genetic Instability ◽  
Toxicity Testing ◽  
Genetic Toxicity ◽  
In Vitro Cell Lines

scholarly journals Duplex-Repair enables highly accurate sequencing, despite DNA damage

2021 ◽  
Author(s):  
Kan Xiong ◽  
Douglas Shea ◽  
Justin Rhoades ◽  
Timothy Blewett ◽  
Ruolin Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Accuracy ◽  
Base Pairs ◽  
Free Dna ◽  
Duplex Sequencing ◽  
Tumor Biopsies ◽  
The Impact ◽  
Formalin Fixed ◽  
Fixed Tumor

Abstract Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via ‘End Repair/dA-Tailing,’ may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7–17% and 32–57% of interior ‘duplex base pairs’ from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles’ heel in sequencing and offers a solution to restore high accuracy.

Follow-up actions from positive results of in vitro genetic toxicity testing

2010 ◽  
Vol 52 (3) ◽  
pp. 177-204 ◽  
Author(s):  
Kerry L. Dearfield ◽  
Véronique Thybaud ◽  
Michael C. Cimino ◽  
Laura Custer ◽  
Andreas Czich ◽  
...  
Keyword(s):  
Toxicity Testing ◽  
Genetic Toxicity ◽  
Positive Results

scholarly journals Duplex-Repair enables highly accurate sequencing, despite DNA damage

2021 ◽  
Author(s):  
Kan Xiong ◽  
Doug Shea ◽  
Justin Rhoades ◽  
Tim Blewett ◽  
Ruolin Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Accuracy ◽  
Base Pairs ◽  
Free Dna ◽  
Duplex Sequencing ◽  
Tumor Biopsies ◽  
The Impact ◽  
Formalin Fixed ◽  
Fixed Tumor

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via End Repair/dA-Tailing, may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior duplex base pairs from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles heel in sequencing and offers a solution to restore high accuracy.

Bestimmung der DNA-Schädigung in vitro

2010 ◽  
Vol 49 (S 01) ◽  
pp. S64-S68
Author(s):  
E. Dikomey
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Chromosome Aberrations ◽  
Genetic Factors ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cell Inactivation ◽  
Repair Mechanisms ◽  
Break Repair

SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

Synthesis and in vitro Cytotoxicity Studies of Certain Novel Heterocycles Derived from Bis 1, 2, 4-Triazoles and their DNA Damage Studies

2013 ◽  
Vol 9 (8) ◽  
pp. 1063-1072
Author(s):  
Madhusudan Purohit ◽  
Chandrashekhar Mangannavar ◽  
Mayur Yergeri
Keyword(s):  
Dna Damage ◽  
In Vitro Cytotoxicity ◽  
Cytotoxicity Studies

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

Prediction of chemical carcinogenicity from in vitro genetic toxicity

Mutagenesis ◽  
1988 ◽  
Vol 3 (4) ◽  
pp. 287-291 ◽  
Author(s):  
John A. Heddle
Keyword(s):  
Genetic Toxicity ◽  
Chemical Carcinogenicity

scholarly journals CX-5461 Enhances the Efficacy of APR-246 via Induction of DNA Damage and Replication Stress in Triple-Negative Breast Cancer

2021 ◽  
Vol 22 (11) ◽  
pp. 5782
Author(s):  
Ashwini Makhale ◽  
Devathri Nanayakkara ◽  
Prahlad Raninga ◽  
Kum Kum Khanna ◽  
Murugan Kalimutho
Keyword(s):  
Breast Cancer ◽  
Dna Damage ◽  
Triple Negative Breast Cancer ◽  
Combination Treatment ◽  
Triple Negative ◽  
Annexin V ◽  
Replication Stress ◽  
Breast Cancer Cell Lines ◽  
Combination Strategy

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking targeted therapy. Here, we evaluated the anti-cancer activity of APR-246, a P53 activator, and CX-5461, a RNA polymerase I inhibitor, in the treatment of TNBC cells. We tested the efficacy of individual and combination therapy of CX-5461 and APR-246 in vitro, using a panel of breast cancer cell lines. Using publicly available breast cancer datasets, we found that components of RNA Pol I are predominately upregulated in basal-like breast cancer, compared to other subtypes, and this upregulation is associated with poor overall and relapse-free survival. Notably, we found that the treatment of breast cancer cells lines with CX-5461 significantly hampered cell proliferation and synergistically enhanced the efficacy of APR-246. The combination treatment significantly induced apoptosis that is associated with cleaved PARP and Caspase 3 along with Annexin V positivity. Likewise, we also found that combination treatment significantly induced DNA damage and replication stress in these cells. Our data provide a novel combination strategy by utilizing APR-246 in combination CX-5461 in killing TNBC cells that can be further developed into more effective therapy in TNBC therapeutic armamentarium.

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