This proposal is designed to investigate mechanism of radiation induced genetic instability (GI) in human cells. The overall hypothesis is that GI is associated with a critical structural alteration of a chromosome which is sensitive to further breakage and rearrangement, rather than a mutation of a specific gene. Furthermore, we postulate that chromosomal rearrangements involving the novel juxtaposition of euchromatin with centromeric (alpha) or pericentromeric (classical) heterochromatin is one such critical structural alteration. Sub-clonal analysis of persistent radiation induced instability will be utilized to test the hypothesis that elevated rates of delayed chromosomal rearrangements, mutation at the tk locus, and delayed reproductive cell death, varies among sub-clones from the same parent. We will specifically determine if sub-clones including complex chromosomal abnormalities will exhibit higher rates of persistent GI than other sub-clones which do not include a complex abnormality due to karyolytic heterogeneity in the parental clone. 2,6- diaminopurine (DAP) will be utilized as a reagent which induces highly preferential breakage in the alpha and classical heterochromatin of chromosomes 1, 9 and 16. The high specificity and frequency of DAP induced damage, and the availability of easily visualized fluorescent probes for the affected heterochromatic sequences, will enable a prospective analysis of the emergence of GI by focused examination of the fate of damaged heterochromatin. This system will also be utilized to test the hypothesis that persistent chromosomal instability will be influenced by cellular capacity for double strand break (dsb) repair. An APRT derivative of the DNA-dependent protein kinase catalytic sub unit (DNA-PK) Cho cell mutant, V-3, will be isolated and restored to radio resistance by instruction of the XRCC7 gene which encode DNA-PK. This cell, and its isogeneic non-transfected counterpart, will be used as recipients for the transfer of abnormal or control clonal copies of chromosome 16 from DAP treated TK6 cells, using a prt (16q24.3) as a selectable marker. The rate of instability of these chromosomes will be compared in the two recipient cell lines, and to the ongoing rate of instability of the same chromosomes in their native cellular environment.
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| Pongsaensook, Punnajit; Ritter, Linda E; Parks, Karyn K et al. (2004) Cis-acting transmission of genomic instability. Mutat Res 568:49-68 |
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