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Comet assay research into skin cancer

About Cornwall Dermatology Research

Cornwall Dermatology Research (CDR) was formed in 1997 to conduct high quality research in dermatology in the south west of England. In 2005 the research group was incorporated into the Institute of Biomedical and Clinical Science, at the newly formed Peninsula Medical School.

CDR is located in Truro in the south west of England a region which exhibits one of the highest rates of skin cancer in the UK. Research is focussed on the action of a range of naturally occurring compounds in causing or preventing DNA damage in association with a light (UV) insult. Compounds with properties that prevent irradiation induced DNA damage have the potential to be incorporated into sunscreens to prevent the damage at source and may help reduce skin cancer incidence.

In addition the research group also conducts research into Photodynamic therapy – a novel treatment therapy for patients with non-melanoma skin cancers such as basal cell carcinoma, Bowen’s disease and actinic keratoses. Photodynamic Therapy (PDT) involves the application of a cream to skin tumours. The cream is taken up and metabolized by tumour cells which convert the cream into a light sensitive drug called a photosensitizer. Exposure of the photosensitzer to light results in the production of reactive oxygen species which lead to tumour cell death. It is particularly successful for the treatment of cancers which are superficial in nature, in problematic areas or in regions where excellent cosmesis is desirable. CDR is investigating modifications to the standard PDT therapy using novel compounds.

Use of the comet assay

The comet assay has been used at CDR for almost 10 years and a key investigator is Dr. Andrew Pye, Postdoctoral Research Fellow and Laboratory Manager. Andrew is responsible for setting up the comet assay and training his team which includes two researchers, two PhD students and one MSc student.

Present research is aimed at establishing whether environmental radon gas acts synergistically with UV radiation to cause an increase in DNA damage in human skin cells.

To date the comet assay has contributed significantly to three major fields of research:   

Investigation into environmental causes of DNA damage that may lead to skin cancer. including UVA and UVB radiation, mercury and arsenic. 

Prevention of DNA damage that may lead to skin cancer by various antioxidant compounds including those found in green tea.   

Investigation of novel modifications to dermatological Photodynamic Therapy (PDT) to treat skin cancer.

Comet Assay protocol used at CDR

The alkaline version of the comet assay is used which allows for the detection of double strand breaks, single strand breaks, alkali labile sites, and transient repair sites. The particular protocol is based on that reported by Singh et al which was further optimised in the CDR laboratory.

Briefly, DNA damage is assessed microscopically in individual cells by the determination of DNA migration induced by electrophoresis. Smaller DNA fragments and loops are able to move through the electrophoresis gel more readily than the main body of DNA. This results in the typical comet shaped distribution of DNA from which the assay derives its name, the smaller fragments of DNA forming a ‘tail’ while the main body of DNA forms the ‘head’.

Cell suspensions are mixed with molten agarose and spread on to pre-heated proprietary comet slides (supplied by Trevigen). After chilling, cells are irradiated and then lysed. Slides are then transferred to electrophoresis tanks where electrophoresis under alkaline conditions allows the DNA to relax and unwind. After electrophoresis slides are removed from the tank and placed in a bath of ethanol to dehydrate and left overnight to dry.

Comet slides are stained using ethidium bromide and examined under excitation with a fluorescence microscope (CETI, Belgium). A Perceptive Instruments Comet Assay IV system is used to quantify the extent of DNA damage. A charge couple device (CCD) camera attached to the microscope relays a live image directly to the host computer. The % DNA in the tail region of the ‘comets’ is chosen as the parameter to determine the level of DNA damage in the cells. Sixty comets are scored from each sample area giving a total of 120 comets scored per slide. Data processing is carried out using Microsoft Excel.

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