Background

The genomes of all cells are continually damaged and this disrupts normal cellular metabolism, leading to mutations and, potentially, cell death. Thus, all cells contain a range of repair pathways that have evolved to optimise their survival following damage to their DNA (1). As our understanding of DNA repair improves, it is apparent that damage to DNA is important in the development of some human diseases and in determining the rate of natural ageing in many mammals (1,2). The link between DNA damage to disease is becoming particularly strong for problems associated with ageing, such as cataracts (1). This project will use a range of biochemical, molecular and cell biological experiments that are ideally suited to identify the activities and regulation of DNA repair pathways within the human lens. The unique biology of the lens and its anatomical situation make it an excellent experimental tool to identify causal factors in the disease and natural ageing processes.

Experimental Programme.

To assess links between cataracts of the eye and DNA damage, experiments will be performed on lens tissue obtained from human donor eyes, which will be received from the East Anglian Eye Bank with full ethical approval. To assess the impact of DNA damage on cataract development we will use from lenses from people of different ages, which will be compared directly to healthy, clear lenses of similar ages. Preliminary experiments have optimised assays to allow testing of the effects of specific types of DNA damage on the physiology of lens using the cell line FHL124.

Using the various cells and tissues, we will assess the impact of a range of environmental stresses on the samples, particularly at the level of DNA damage. These will include examination of stresses known to cause DNA damage, such as UV light, and compare with stresses induced by alterations in cellular physiology, such as the response to various concentrations of glucose. The experiments will identify the types and extent of DNA damage present in the different samples using a variety of techniques that are available in the collaborator's laboratories (3). The amount of DNA strand breaks will be detected using the Comet assay. Commercial antibodies will be used to determine levels of specific types of DNA damage. Alongside these experiments we will use commercial antibodies and assays to determine the level of expression, biochemical activity and location of various DNA repair proteins. To provide a general indicator of cell death and damage in the samples, we will also determine levels of lactate dehydrogenase and the extent of apoptosis.

Student's contribution and Training:

Training will provide a wide ranging skill set, including cell and tissue culture of human lenses, PCR and gel electrophoresis, fluorescence microscopy, RT-PCR and comet assays (3). Initially experiments will be performed under direct supervision, but the student is expected to be independent at the end of year 1.General and transferable skills training will be provided through the Science Graduate School at UEA and will be tailored towards the student involved.

(i) Jackson, S.P. and Bartek, J. (2009) The DNA-damage response in human biology and disease. Nature, 461, 1071-1078

(ii) Hoeijmakers, J.H. (2009) DNA damage, aging, and cancer. N Engl J Med, 361, 1475-1485.

(iii) Wang, L., Eldred, J.A., Sidaway, P., Sanderson, J., Smith, A.J., Bowater, R.P., Reddan, J.R. and Wormstone, I.M. (2012) Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens. Mech Ageing Dev., in press