The objectives are to compare available human transformation systems in terms of their ability to address radiation protection problems particularly radiation quality and low dose rate effects; and to develop new human epithelial systems capable of looking at initiation of carcinogenic damage, particularly by target specific radionuclides on the target organ in culture.
A human papillomavirus immortalised human keratinocyte (HPV-G) line has been established and tested for radiosensitivity and suitability for transformation assays. The tumorigenicity of the line in nude mice is being determined. Another immortalised keratinocyte line (Ha Cat) derived from a subject with skin cancer and Hela x human fibroblast CGLI cells have also been established and tested. The former has a slow growth rate and is unsuitable for transformation experiments while the latter look promising in transformation assays. The CGLI line, which can become malignant as a result of a deletion in the region of an oncosuppressor gene, expresses a protein marker, P75, when transformated.
In experiments where risk of initiation by radiation is important, normal human urothelial cells have been used. Attempts have been made to link enhanced proliferation/extended life span of progeny with radiation dose. Waves of enhanced growth were seen at relatively low doses but very cytocidal doses were required to induce a truly non senescent population. There was a significant increase in number of foci with time post irradiation at 10 Gy. Non significant effects occurred at 5 Gy. Ultrastructural and immunochemical analyses of the foci confirmed a number of nuclear and cytoplasmic characteristics associated with malignant cells and that they expressed high levels of cmyc which was not detected in non focal cells. Expression of P75 protein was also found.
It is important to develop suitable human epithelial cell lines to study potential carcinogens and to investigate the multistage mechanisms of carcinogenesis. A number of human epithelial cell lines have been established to investigate the mechanisms of radiation carcinogenesis. The human urothelial cell lines studied proved extremely resistant to radiation transformation. A human thyroid epithelial cell line could be effectively transformed following exposure to bothe single and multiple doses of gamma irradiation and after single exposure to alpha particle irradiation. A number of tumour cell lines were developed, allowing a comparison of changes occuring from the parent cell line. A first estimate of the RBE for tumour transformation of epithelial cells following exposure to alpha particles from plutonium 238 was obtained. This model will thus be useful in studies of carcinogenesis using human epithelial cellls.
Computerized image analysis techniques have been used to quantify several morphometric and densitometric parameters of human urothelial cells treated with carcinogenic agents or radiation. Significant differences in nuclear area and amount of deoxyribonucleic acid (DNA) per cell have been found in cells treated with carcinogenic agents compared to controls. In cells treated with radiation there was a reduction in the distribution of their nuclear areas, the reduction increasing with dose. Further analysis is being performed on transformant cells.
There is a need for a relevant in vitro assay for studying transformation of human cells. In this way the molecular mechanisms and dose response relationships of carcinogenesis in humans can be studied. As about 85 to 90% of human tumours are of epithelial origin, it is important to utilize cultures of normal human diploid epithelial cells in transformation studies. The systems which have been most extensively studied to date employ the:
BHK 21 line of Syrian hamster kidney cells;
primary Syrian hamster embryo cells;
BALB/C 3T3 mouse fibroblast cell line;
C3H 10T1/2 mouse fibroblast cell line.
However, extrapolation from one species to another may not be valid and C3H 1OT1/2 cells for example, are aneuploid and cannot be considered normal. Rodent fibroblasts behave differently from human cells in culture, exhibit spontaneous chromosome rearrangements and form permanent cell lines. The background transformation frequency assessed from experiments on fibroblastic lines and ability of agents to transform are apparently quite different for rodent cells and human cells. Human cells exhibit chromosome stability and do not spontaneously transform in culture.
Recent improvements in cell culture methodology have seen the development of human epithelial cell cultures.
It is proposed to collaborate on a systematic study of radiation induced oncogenic transformation using these different human epithelial cell lines. It has proved extremely difficult to transform primary cultures of normal human epithelial cells and thus this approach provides the next logical step in developing a full understanding of radiation-induced transformation of human epithelial cells. It must not be forgotten, however, that initiation of transformation by radiation is a problem urgently in need of investigation. Much effort has gone into this field and with little success.
The adoption of useful systems from the chemical carcinogenesis field may be profitable, for example the system developed by Thomassen for rat tracheal epithelium might adapt well to human epithelium, or the human buccal cell system of Grafstrom and Harris which shows enhanced proliferation in response to chemical carcinogens. Enhanced proliferation of epithelial and endothelial cells in mixed cell type outgrowths from primary explants has been detected. The problem with initiation studies is that unless immortalization is achieved, the meaning of the changes is hard to establish.
One of the main problems in defining the role of ionizing radiation in oncogenic transformation is that radiation induces different types of lesion in DNA in exposed cells. It has now been shown that exposure of permeabilized cells to type II restriction endonucleases which induce double strand breaks in DNA at specific sites leads to chromosomal aberrations, mutations and cell death. Recently it has been shown that oncogenic transformation also occurs as a result of this treatment, thus it may be possible to shed more light on the basic molecular mechanism of oncogenic transformation using this approach.
Funding SchemeCSC - Cost-sharing contracts
KY16 9AH St Andrews