Ionizing radiation (IR) is a known human carcinogen. However, the precise molecular mechanisms by which IR induces cancer are very poorly understood. Recent major advances in generating immortal, but otherwise normal cell lines from a variety of specialized human tissues has now made possible the study of the genetics of stages in the development of IR-induced malignancy, using clinically relevant cell system. In this project, these techniques will be combined with advanced molecular cytogenetic methods and proven genetic complementation (i.e. functional) approaches to identify, map and isolate novel genes involved in the process of IR carcinogenesis. Human radiation-associated thyroid cancer cells will be investigated in parallel to in vitro generated tumour cells. A detailed molecular description of chronomosal instability mechanism driving IR-induced malignant development will also be provided.
Two main approaches were adopted to study the process of malignant transformation of human cells in which a causal role for ionizing radiation in the process could be confidently assumed. These were:
(i) the study of human cancers associated with radiation exposure, viz. thyroid cancers from Belarus and, from the clinic, secondary tumours that had developed in the radiation field following radiotherapy for an unrelated primary tumour, and
(ii) the development and molecular characterization of human cell culture models responsive to malignant transformation by IR.
With regard to the latter, we exploited the use of the gene (hTERT) encoding the catalytic sub-unit of the human telomerase (telomerase is activated during the development of 90% of all human cancers) to generate immortalized human cell clones, with normal chromosomes and growth characteristics, from clinically relevant tissues such as the human breast. Such cultures were then used as target systems for malignant conversion by ionizing radiation. Two cell culture models were established, one based on human breast epithelial cells, in which additional transformation events could be induced following exposure to gamma and X-radiation. Interestingly, the most effective treatment regimes were those involving multiple fractionated radiation doses. Exhaustive characterization of chromosome changes in cell lines transformed in this way revealed the presence of several common alterations associated with transformation (eg deletions involving chromosome-13 and gene amplifications involving chromosome-10) and a candidate novel oncogene was identified. Substantial progress was made in cloning two additional radiation-induced breakpoint-associated genes localized to chromosomes-17 and X, and in localizing specific genes involved in two further aberrations (chromosomes 3 and 11) identified in radiation-transformed human breast epithelial cells. Similarly, in thyroid cancers from Belarus, breakpoint cloning resulted in the identification of the gene involved in a critical cancer-associated chromosome translocation. Cytogenetic and molecular analysis of secondary radiation-induced cancers (following radiotherapy of the eye orbit for an inherited eye cancer) produced a number of novel results, including direct demonstration of the involvement of two tumour suppressor gene-inactivating alterations and the identification of a possible radiation-specific molecular signature. Collectively, these results shed much new light on the mechanisms by which ionizing radiation acts during the malignant transformation of human cells.
The identification of tumour suppressor genes central to the development of human breast cancer, an important tumour type in the context of ionizing radiation, produced two sets of major findings. First, the gene responsible for the tumour suppressive activity of human chromosome-1 in breast cancer cells (located in the frequently lost 1p35 region) was identified unequivocally as that encoding the epithelial-specific marker and growth suppressor 14-3-3 sigma; this gene was subsequently shown to be involved in our in vitro breast cell transformation system, suggesting a role in radiation-induced transformation. Second, major advances were made in understanding the genetics and molecular biology of telomerase repression in normal human cells (which must be overcome for human cancers to develop). We showed that the all-important repressor gene acts via transcriptional repression of the gene encoding a component of telomerase (known as hTERT) by a mechanism that involves repackaging of the gene. These results, recently published in high-impact journals, will be extremely valuable in facilitating the identification and isolation of the repressor gene itself, which we have recently been successful in locating to an extremely small region of normal human chromosome 3 (within band p21).
The importance of telomerase activation in radiation-induced cancer was determined in both in vitro (cell culture) and in vivo (genetically manipulated mouse models). In the latter, clear experimental evidence was obtained (resulting in a number of high-impact publications) that telomerase substantially enhances susceptibility to cancer induction, and that its absence acts to suppress tumour development. The role of telomerase in human epithelial carcinogenesis was established using cell culture models based on breast epithelial cells, and cultures derived from human head & neck cancers and premalignant conditions. The latter investigation identified loss of a key tumour suppressor gene (known as p16) and telomerase activation as co-operating early events in the immortalization process during cancer formation. In associated studies, additional molecular events that are likely to contribute to human cells immortalization were identified.
Funding SchemeCSC - Cost-sharing contracts
92265 Fontenay Aux Roses
2300 RA Leiden
KY16 9TS St Andrews