Treatment of human tumors with ionizing radiation has been an effective treatment of cancer for over 50 years. However, the continued limitation of this therapy is the inherent radiation resistance of some tumor types compared with others. The molecular basis of this difference in resistance is unknown. Throughout evolution, organisms have evolved a conserved mechanism of resistance. Induction of DNA damage signals a surveillance mechanism which arrests cells in the G2 phase of the cell cycle before entering mitosis where residual DNA breaks result in chromosome aberrations. During arrest in G2, DNA breaks are repaired and when repair is completed, the cells resume cycling. Two of the genes, Rad9 and Mec1, which control this arrest have been isolated in the budding yeast S. cerevisiae. This group of genes has been named checkpoints. We propose to isolate human homologues of these checkpoint genes and study their role radiation resistance. A simple genetic strategy has been developed to select for inhuman clones able to complement mutations in the yeast checkpoint genes. Human cDNAs which are obtained will be expressed in radiosensitive mammalian cell lines and increases in resistance determined. A survey of expression at the RNA and protein level in clinical specimens from categories of radiation sensitive and resistant tumors will be undertaken. In addition, a method to select for cDNA clones which interfere with Rad9 function in yeast has been developed. The selection will be used onto screen mutagenized Rad9 cDNAs and human cDNA libraries for dominant negative clones of Rad9. These clones will be expressed in mammalian cells and increases in radiation sensitivity monitored. cDNAs which can decrease the radiation resistance of tumors would be an unique resource and have potential clinical benefit in the future.
