In 1986, an accident at the nuclear power plant in Chernobyl, USSR, led to the release of high levels of radioisotopes. Ten years later, the incidence of childhood papillary thyroid cancer (chPTC) near Chernobyl had risen by 2 orders of magnitude, most likely as a consequence of increased exposure to ionizing radiation. The routes and mechanisms by which radiation generated these additional thyroid cancers remain mysterious. More than 10 years ago, we and our collaborators in Germany began to archive thyroid tumor tissues from patients who underwent thyroidectomy near the site of the power plant, among them 214 specimens from children. Several tumors from this collection aberrantly expressed ret tyrosine kinase transcripts due to a ret/PTC1 or ret/PTC3 chromosomal rearrangements involving chromosome 10. However, many other chPTC tumors have phenotypes not attributable to aberrant ret expression. Instead, most cells showed a normal G-banded karyotype. Moreover, even within a ret-positive chPTC tumor, not all cells express ret or contain a rearranged chromosome 10. We hypothesize that these other classes of tumors may inappropriately express a different oncogene or have lost function of a tumor suppressor as a result of chromosomal rearrangements and that knowledge of the kind of genetic alterations leading to chPTC may facilitate the early detection and staging of tumors as well as provide guidance for therapeutic intervention. To test this hypothesis, we propose to map the sites of chromosomal breakpoints in 29 cases of radiation-induced chPTC and identify genes with abnormal pattern of expression. Using a sensitive, genome-wide screening technique termed 'BAC-FISH', which we developed recently, we will localize the breakpoints in 10 radiation-induced tumors (9 chPTC cases and 1 adult case) for which we have cell lines as well as G-banding and Spectral Karyotyping results. With the breakpoints grossly determined, we will prepare breakpoint-spanning BAC contigs and high- resolution physical maps of the rearrangements. Matrigel-based cell invasion assays will provide us with subsets of invasive cells for further cytogenetic studies investigating the presence or absence of structural abnormalities in invasive cells as well as the expression of candidate genes. Next, we will determine the presence or absence of these translocations in 20 additional cases of chPTC for which we already archived metaphase spreads. Control groups will be comprised of children who developed thyroid cancer without prior radiation exposure as well as tumors in adult patients who underwent radiotherapy as children or adults. Additional controls samples will be comprised of thyroid cancer cell lines from patients without a known history of exposure to ionizing radiation. Interphase cell preparations from the control groups will be studied with DNA probes specific for candidate loci to detect rearrangements. For the six most common rearrangements, we will identify genes that map at or near the chromosomal breakpoints using publicly available databases and bioinformatics tools. Finally, we will examine the levels and intracellular localization of potentially oncogenic transcripts (mRNAs) from candidate genes and expressed sequences in the breakpoint regions using multi-target in situ hybridization analyses.
Our innovative screening assay for cryptic translocations is at least one order of magnitude more sensitive than existing tumor karyotyping procedures and might thus provide a wealth of information for a better risk assessment, diagnosis or individualized disease management. Specifically, the proposed studies will help to enhance our understanding of complex biological systems such as the thyroid- specific carcinogenicity of radionuclides. We expect this research to lead to improved control of PTC with regard to early disease detection and better disease management and, thus, to enhance public health services.
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