The comet assay: A new method to examine heterogeneity associated with solid tumors

Olive, Peggy L.; Johnston, Peter J.; Banáth, Judit P.; Durand, Ralph E.

doi:10.1038/nm0198-103

Cited by 69 publications

(44 citation statements)

References 13 publications

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“…Under the conditions we used, with a pH of the clectrophoresis buffer Ͼ12.5, DNA single-strand breaks, as well as DNA double-strand breaks are detected. 25,26 In contrast to the epipodophyllotoxins, anthracyclines are able to induce DNA damage in several ways in addition to the inhibition of topo II. One of them is the intracellular production of free oxygen radicals, which had been held responsible for the cumulative cardiotoxicity of anthracyclines.…”

Section: Discussionmentioning

confidence: 99%

Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage

Gieseler¹,

Bauer

Nuessler³

et al. 1999

Leukemia

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We examined the cellular effects of topo II inhibitors in two human myeloid cell lines, HL-60 and KG-1 cells, with the purpose of finding molecular markers for the sensitivity of leukemia cells to topo II inhibitors. These cell lines are widely used, well characterized and they differ in their sensitivities to topo II inhibitors. Despite the fact that HL-60 cells are p53-negative, they are much more sensitive than KG-1 cells. Three different topo II inhibitors with distinct molecular ways of action have been used. Daunorubicin and aclarubicin are DNA intercalators that secondarily interact with topo II; etoposide, on the other hand, directly binds to the enzyme. In contrast to daunorubicin, which induces protein-associated DNA double-strand breaks due to the blockage of topo II action, aclarubicin inhibits the access of DNA by topo II. No correlation could be established between the drug-induced DNA damage and apoptosis. In fact, the amount and pattern of DNA damage examined with the 'comet assay' was characteristic for each drug in both cell lines. The DNA binding of daunorubicin was slightly higher in HL-60 cells, but there was no notable variance between the cell lines for aclarubicin. The most striking difference could be found for the nuclear topo II activity, which was about half in KG-1 cells and, additionally, less than 1% of the nuclear topo II activity was bound to the DNA in KG-1 cells when compared to HL-60 cells. This fraction of topo II interacts with the inhibitors; subsequently these findings might well explain the variance in the cellular sensitivity. Additional factors are alterations of the apoptotic pathways, eg loss of p53 in HL-60 cells. Although we found no differences in the quantity of DNA damage between the cell lines after drug treatment, the quality of DNA damage appeared to be distinct for each topo II inhibitor. The morphological appearance of the comet tails after treatment was characteristic for each drug. Further studies are necessary to decide whether these in vitro data are compatible with the clinical situation.

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Section: Discussionmentioning

confidence: 99%

Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage

Gieseler¹,

Bauer

Nuessler³

et al. 1999

Leukemia

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“…A total of 100 cells were evaluated per specimen and dose, 50 cells per duplicate slide. The Olive tail moment (OTM) was calculated as the product of the percentage DNA in the comet tail and the distance between the centres of gravity for DNA in the head and tail [35] and the data were presented as mean ±S.E. Oxaliplatin or satraplatin crosslinks were concluded as a decrease in percentage of the OTM compared to that of hydrogen peroxide (H 2 O 2 ) [36].…”

Section: The Comet Assay and Data Analysismentioning

confidence: 99%

<i>In Vitro</i> Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients

Alotaibi¹,

Baumgartner²,

Najafzadeh³

et al. 2012

JCT

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Exposure to toxic chemicals, especially chemotherapeutic drugs, may induce several DNA lesions, including DNA interstrand crosslinks. These crosslinks are considered toxic lesions to the dividing cells since they can induce mutations, chromosomal rearrangements, and cell death. Many DNA interstrand crosslinks lesions can be generated by platinum-based chemotherapeutic agents. Satraplatin is a novel orally administered platinum-based chemotherapeutic agent. In the present study, we investigated DNA interstrand crosslinks lesions induced by oxaliplatin and satraplatin in lymphocytes obtained from colorectal cancer patients and healthy volunteers. Satraplatin demonstrated an increase in interstrand crosslinks in a dose-dependent manner in the Comet assay (p < 0.001). In addition, satraplatin and oxaliplatin increased significantly the number of sister chromatid exchanges up to 8.5-fold and 5.1-fold (p < 0.001) respectively, when treated with 2 µM concentration in comparison to untreated colorectal cancer cells. Further, the γH2AX foci formation was investigated by an immunofluorescence assay with oxaliplatin and satraplatin. The γH2AX foci formation rate was increased by approximately 9-fold when lymphocytes were treated with 2 μM oxaliplatin. Satraplatin was found to significantly induce the number of γH2AX foci by 8.5-fold and 11-fold with both 0.2 μM and 2.0 μM, respectively, compared to the control volunteers that may indicate the repair system in cancer cells experiences a loss of ability to cope with the repair of DSBs. In conclusion, oxaliplatin and satraplatin effectively induced DNA interstrand crosslinks in lymphocytes obtained from colorectal cancer patients and healthy volunteers in vitro. Here, to the best of our knowledge we report for the first time evidence of DNA double strand breaks formation as a possible molecular mechanism of action for satraplatin.

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“…Methods for assessing the radiobiological hypoxic fraction include the classical paired survival curve assay and, more recently, the comet assay (Olive et al, 1998). Most clinical data have been obtained with the oxygen micro-electrodes.…”

Section: Hypoxiamentioning

confidence: 99%

Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas

et al. 2000

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The relevance of blood flow for the outcome of radiation treatments was already described by Schwartz in 1909. He noticed that skin reactions were less severe when the radium applicator was firmly pressed to the skin. It was Cramer in 1935 who gave the first suggestion of an 'oxygen effect'. He found that mouse mammary tumours with a well developed stroma and vascular network were more radiosensitive than tumours with a more delicate stromal component. He suggested that the radiosensitive tumours had a better blood supply and, consequently, were better oxygenated. Later, Gray et al (1953) established the importance of tumour oxygenation for radiotherapy.The tumour micro-environment is a key factor in tumour biology and has great impact on clinical radiotherapy as well as other cancer treatments. Single parameters such as polarographic pO 2 measurements and microvascular density have been demonstrated to be prognostic indicators in a variety of tumour types, including carcinomas of the head and neck (Gatenby et al, 1987;Dray et al, 1995;Zätterström et al, 1995;Jenssen et al, 1996;Nordsmark et al, 1996;Brizel et al, 1997). However, considering the complexity of the micro-environmental system, it is unlikely that a single parameter can reliably predict treatment outcome on an individual basis. Multiple parameter analysis will be necessary to acquire a better understanding of the tumour micro-environment and to obtain a 'predictive profile' which can guide the clinician in the selection of patients for new treatment strategies.A computerized image analysis system for multi-parameter analysis has been developed in our institute (Rijken et al, 1995). The method involves computer controlled microscopic scanning of immunohistochemically stained tissue sections. It allows quantitative and simultaneous analysis of vascular parameters, oxygenation status and proliferation parameters. We have recently published our experience with this method in xenografted human tumours (Bussink et al, 1998(Bussink et al, , 1999.We now apply this method also to tumour biopsies from patients with squamous cell carcinomas of the head and neck. Before a biopsy was taken, the patients were injected with Hypoxyprobe-1 (pimonidazole hydrochloride) as a hypoxia marker. Pimonidazole can easily be dissolved in saline, has a low toxicity, and an efficient tumour uptake. So far pimonidazole has proven to be an effective hypoxia marker for human tumours of the cervix and head and neck (Kennedy et al, 1997;Raleigh et al, 1998;. In the present study we report the results of the analysis of biopsy material from 21 patients with squamous cell carcinoma of the head and neck. MATERIALS AND METHODS PatientsPatients with primary laryngeal, oropharyngeal (stage III-IV) or hypopharyngeal (stage II-IV) squamous cell carcinomas were Summary Tumour oxygenation and vasculature are determinants for radiation treatment outcome and prognosis in patients with squamous cell carcinomas of the head and neck. In this study we visualized and quantified these factors which m...

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The comet assay: A new method to examine heterogeneity associated with solid tumors

Cited by 69 publications

References 13 publications

Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage

Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage

<i>In Vitro</i> Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients

Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas

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The comet assay: A new method to examine heterogeneity associated with solid tumors

Cited by 69 publications

References 13 publications

Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage

Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage

&lt;i&gt;In Vitro&lt;/i&gt; Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients

Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas

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<i>In Vitro</i> Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients