Development and validation of mechanisms-based in vitro transformation assays for carcinogen screening
Lead Research Organisation:
Brunel University London
Department Name: Sch of Health Sciences and Social Care
Abstract
Humans are exposed to many man-made and naturally occurring chemicals at levels we have never before encountered. A significant number of these can be expected to cause cancer. It is therefore of vital importance for human health that we have in place reliable screening procedures to establish which chemicals have cancer-causing potential, and to regulate human exposure to them. Currently approved systems are centred on laboratory animal cancer tests, usually involving live rats &/or mice. However, there is now enormous public support for reducing the numbers of live animals used in research, particularly in the testing of chemicals for harmful effects. This project has been designed to develop alternatives to animal cancer testing, using instead animal and human cells grown in culture to measure cancer-causing effects. Recent work by us at Brunel University, working with colleagues in the drug company AstaZeneca, has resulted in the development of promising cell culture models for cancer, using human breast and hamster cell cultures. Early results indicate that these new systems reproduce some of the important steps known to be critical in human cancer development. In this project, these assays will be improved further using modern genetic techniques, and the relative advantages and disadvantages of each of the new human and hamster assays determined. By this means, a new generation of cell culture-based tests will be produced that can be incorporated into existing screening programmes. This in turn is expected to lead to a reduction in the numbers of animals that need to be used in cancer testing. The results of the study will be communicated to the general public through press conferences, articles in the popular media, and via appropriate websites.
Technical Summary
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 (eg 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 last 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 (eg cell immortalization, focus formation) measured, involve events that are directly relevant to the molecular mechanisms driving human carcinogenesis. In preliminary work we have employed telomerase hTERT expression vectors to immortalize 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 immortalization step in transformation. We now propose to complete the cellular and molecular characterization 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.
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 last 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 (eg cell immortalization, focus formation) measured, involve events that are directly relevant to the molecular mechanisms driving human carcinogenesis. In preliminary work we have employed telomerase hTERT expression vectors to immortalize 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 immortalization step in transformation. We now propose to complete the cellular and molecular characterization 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.