DNA damage and cell death assessment in patients with severe multiple trauma using comet assay

Objective: The objective of the present study was to investigate the growth inhibitory effect, apoptosis initiation and genotoxic effect of zerumbone (ZER), a phytochemical and cisplatin, a chemotherapeutic drug on human colorectal cancer cell line COLO205 and normal human lymphocytes.Methods: The antiproliferative activity of ZER and cisplatin (positive control) on COLO205 cells and lymphocytes was analysed by 3( 4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. Morphological analysis of the cells was studied by using inverted phase contrast microscope. Propidium iodide staining method was used to observe the apoptotic morphological changes in the treated cells. Finally comet assay was conducted to observe the extent of DNA damage induced by ZER and cisplatin on COLO205 and lymphocytes.Results: ZER and cisplatin exhibited growth inhibition in a dose and time dependent manner against COLO205 with no considerable effect on lymphocytes. The IC50 values of ZER on COLO205 for 24h, 48h and 72h were 19 µg/ml, 10 µg/ml and 5 µg/ml. Comparatively the IC50 values of cisplatin on COLO205 for 24h, 48h and 72h were 38 µg/ml, 24 µg/ml and 15 µg/ml. Morphological changes such as cell shrinkage, membrane blebbing and nuclear condensation were observed in COLO205 while no significant change was observed in lymphocytes. Fluorescence imaging studies confirmed apoptotic cell death in treated COLO205 cells while no significant cell death was observed in treated lymphocytes. Comet assay revealed significant DNA damage in treated COLO205 cells.Conclusion: The present study demonstrated the cytotoxic and genotoxic effect of ZER and cisplatin on COLO205 cells. These drugs showed no significant effect on lymphocytes.

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Cell survival after DNA damage in the comet assay

Archives of Toxicology ◽

10.1007/s00204-021-03164-3 ◽

2021 ◽

Author(s):

Ezgi Eyluel Bankoglu ◽

Carolin Schuele ◽

Helga Stopper

Keyword(s):

Dna Damage ◽

Cell Death ◽

Comet Assay ◽

Survival Data ◽

Topoisomerase Ii ◽

Basic Research ◽

Human Biomonitoring ◽

A Cell ◽

Tk6 Cells ◽

Genotoxicity Testing

AbstractThe comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H2O2) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20–30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H2O2 or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.

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The Comet Assay to Determine the Mode of Cell Death for the Ultrasonic Delivery of Doxorubicin to Human Leukemia (HL-60 Cells) from Pluronic P105 Micelles

Technology in Cancer Research & Treatment ◽

10.1177/153303460500400616 ◽

2005 ◽

Vol 4 (6) ◽

pp. 707-711 ◽

Cited By ~ 25

Author(s):

Ghaleb A. Husseini ◽

Kim L. O'Neill ◽

William G. Pitt

Keyword(s):

Dna Damage ◽

Cell Death ◽

Comet Assay ◽

Propidium Iodide ◽

Cell Killing ◽

Human Leukemia ◽

Dna Profile ◽

Fluorescent Microscope

This notes examines the mode of cell death of HL-60 cells exposed to 70 kHz and 1.3 W/cm2 ultrasound in the presence of 1% Pluronic P105 and 1.67 μg/ml doxorubicin (Dox). The cells were ultrasonicated for 30, 60, and 120 minutes. They were then lysed, electrophorised, stained using propidium iodide, and their DNA profile captured using a fluorescent microscope. The gradual DNA damage observed and the comet tails captured after one and two hours of insonation suggest that the mode of cell killing is apoptosis.

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Differential effects of selenium (Se) on normal and malignant cells treated with chemotherapy (CT) and radiation (RT).

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e13583 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e13583-e13583

Author(s):

Michael B. Jameson ◽

Richard J Lobb ◽

Gregory M Jacobson ◽

Ray T Cursons

Keyword(s):

Dna Damage ◽

Cell Death ◽

Stress Response ◽

Comet Assay ◽

Er Stress ◽

Stress Responses ◽

Mononuclear Cells ◽

Malignant Cells ◽

Er Stress Response ◽

Therapeutic Selectivity

e13583 Background: Preclinical work has demonstrated that Se compounds potentiate anticancer effects of CT and RT while reducing normal tissue toxicities. The molecular basis for the therapeutic selectivity has yet to be fully elucidated but includes modulation of intracellular glutathione (GSH) concentrations, endoplasmic reticulum (ER) stress responses, DNA repair, induction of apoptosis and cellular resistance to CT and RT. Our aim was to evaluate the dose-response relationship of the Se compound methylseleninic acid (MSA) on molecular pathways involved in the response of normal and malignant cells to CT and RT. Methods: Peripheral blood mononuclear cells (PBMC) obtained from healthy blood donors and malignant THP-1 human monocytic leukaemia cells were exposed in vitro to MSA 2.5, 5 or 15 µM in varying combinations with MSA, RT, cisplatin (Pt), doxorubicin (Dox) and cytosine arabinoside (Ara-C). GSH concentration was measured by ELISA, DNA damage and repair by COMET assay, cell viability by MTT assay and ER stress response protein expression by western blotting. Results: MSA was selectively toxic to THP-1 cells and induced a protective increase in GSH in PBMC but a decrease in high concentrations within THP-1 cells. DNA damage induced by Ara-C or Dox in the COMET assay was significantly reduced by MSA in PBMC but increased in THP-1 cells. Cell death after 2 Gy RT was increased by all doses of MSA in THP-1 cells but only by the highest dose in PBMC. The cytotoxicity of Dox and Ara-C at sublethal doses was significantly enhanced by MSA in THP-1 cells and to a lesser extent in PBMC, but MSA increased cell death from Pt only in THP-1 cells. MSA induced a protective ER stress response in PBMC exposed to Ara-C but an apoptotic response in THP-1 cells. Conclusions: MSA at clinically-relevant concentrations had a differential effect on cell survival and death responses to RT and CT with relative protection of PBMC and enhanced death of THP-1 cells. Several mechanisms mediated this therapeutic selectivity and the dose-dependence of the Se effect varied between malignant and normal cells. These assays could potentially be used in clinical trials to evaluate pharmacodynamic markers of Se effects in conjunction with CT and/or RT.

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Assessment of cell cytotoxicity and comet assay on HER2/neu positive cell line due to 111In Auger electrons as DNA-targeting radioimmunoconjugate

Current Radiopharmaceuticals ◽

10.2174/1874471014666210625115111 ◽

2021 ◽

Vol 14 ◽

Author(s):

Behnaz Piroozfar ◽

Behrouz Alirezapour ◽

Farahnaz Motamedi Sedeh ◽

Mohammad Mirzaii ◽

Amir Reza Jalilian ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Comet Assay ◽

Cell Viability ◽

Cell Line ◽

Cell Nuclei ◽

Auger Electrons ◽

Oncology Research ◽

Electron Therapy

Background: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab is a high affinity-binding monoclonal antibody against HER2/neu, which is overexpressed in breast tumors and used in radiopharmaceutical development. Objective: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab, and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. Methods: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA-NLS-trastuzumab were labeled with 111In, followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line) was used in the cell viability assessment assay for 11In, 111In-DTPA-trastuzumab, and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT, and comet assay was utilized by DNA damage detection. Results: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1 was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA-NLS-trastuzumab. Conclusion: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate that the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA, leading to cell death.

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