Development and validation of mechanisms-based in vitro transformation assays for carcinogen screening

Humans are exposed to many man-made and naturally occurring chemicals at levels never before encountered during our evolution. A significant number of these can be expected to possess some degree of carcinogenic activity, either because they are able to interact with our DNA causing gene mutations or chromosome aberrations, or through some non-mutational mechanism (for example, epigenetic changes in gene expression, forced cell proliferation). It is therefore of vital importance for human health that we have in place reliable comprehensive screening procedures to establish which chemicals have carcinogenic potential, and to regulate human exposure to them. Currently approved systems are centred on laboratory animal tumour-induction tests, usually involving live rats or mice. These test are often complemented, or preceded by, short-term in vitro assays in bacteria or mammalian cells in culture that measure the ability of a chemical to induce genetic change. There is now enormous public support for reducing the numbers of live animals used in research, particularly in the area of toxicology. Advances in our understanding of the cell and molecular biology of cancer have over the past 25 years meant that we are now able to study individual steps in the cancer process using human or rodent cells grown in tissue culture. These cell-transformation models now offer enormous, largely untapped, potential for carcinogenicity screening. In this project, we aim to develop such cell transformation systems, based on hamster and human cells in culture, and to provide sufficient mechanistic underpinning confirming that the transformation endpoints (for example, cell immortalization, focus formation) measured, involve events that are directly relevant to the molecular mechanisms driving human carcinogenesis. In preliminary work we have used telomerase hTERT expression vectors to immortalise normal human breast epithelial cells, generating minimally transformed human cell systems responsive to the induction of later transformation events by carcinogens. We have also started to produce a complete molecular genetic description of existing assays based on Syrian hamster cells, particularly with respect to identifying the role of carcinogen-induced p16 damage in the all-important immortalisation step in transformation. We now propose to complete the cellular and molecular characterisation of transformation phenotypes in both systems and, further, to refine our assays and validate them using panels of human and rodent carcinogens representing a range of known mechanisms of action. By these means, a new generation of in vitro tests will be produced that can be incorporated into existing carcinogenicity screening regimes.

• Further Funding: NC3Rs Strategic Grant, Development of human epithelial cell transformation models for carcinogen screening, employing defined phenotypic endpoints mechanistically representative of rate-limiting events in human carcinogenesis, March 2012, £503,020