This method is designed to evaluate changes in DNA thymidine dimer content in human skin tissue equivalents after exposure to UVB.  Typically the tissues are treated topically overnight with either test materials,  Trolox (positive control), or left untreated (negative control).  On the following day, the tissues are exposed to UVB.  Following the exposures, the DNA is extracted from the tissues and assayed for thymine dimer content.  For the assay, samples of the DNA are immobilized on a solid membrane support and incubated with an antibody that recognizes thymidine dimers in double stranded DNA. The primary antibody is then detected using a secondary antibody conjugated to an alkaline phosphatase enzyme followed by the addition of a suitable substrate that the alkaline phosphatase enzyme can use to generate a chemiluminescent signal.  The light generated by this reaction is captured using film with the intensity of the light signal being proportional to the amount of the thymine dimers present in the sample.

	UVB + Trolox  UVB + PBS  Non-UVB Exposed

p53 Induction

p53 is a 53 kD protein that functions to inhibit cell proliferation.  Under normal conditions cells express very low levels of p53 however in response to DNA damaging conditions, such as those caused by UVB exposure, p53 expression can increase dramatically.  p53 functions as a transcription factor and when it binds to DNA it promotes the expression of p21.  p21 in turn can prevent the activation of cylcin-Cdk complexes that are required to drive the cell past the G1 phase of the cell cycle.  Tissue p53 can be measured via Western Analysis. 

DNA Laddering

If the DNA damage that occurs in a cell after UVB irradiation can not be repaired, then the cell will undergo the process of programmed cell death also known as apoptosis.  One of the hallmark indicators of cells undergoing apoptosis is the formation of DNA ladders.  DNA ladders are formed as the DNA within the cell is progressively broken down into fragments that are roughly multiples of 200 base pairs.  When DNA samples from apoptotic cells are resolved on an agarose gel and stained with ethidium bromide the fragments of DNA produce a characteristic DNA laddering pattern.