Cellular responses to ionizing radiation and cisplatin.
Creators: name: Dolling, Jo-Anna.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1998
Subjects: subject: Biology, Genetics.

description: This thesis tested the hypothesis that the synergistic effect of cisplatin in combination with radiation was due to cisplatin inhibition of repair of radiation-induced DNA damage. Two model cell systems were used. DNA repair inhibition caused by cisplatin and the fundamental mechanism of DNA double strand break repair were studied in human fibroblast cells. The effect of cisplatin on stress response and the induction of radiation resistance was studied in yeast. It was shown that normal human fibroblasts cells (AG1522), irradiated in the presence or absence of cisplatin adducts have biphasic DNA repair curves with a fast and slow component. When cells were treated with cisplatin immediately before irradiation, fast repair was not affected but slow repair was inhibited. The rate of the slow repair in the presence of cisplatin adducts was ten fold less than the rate in the absence of cisplatin adducts. Cisplatin treatment 24 hours prior to radiation resulted in inhibition of both fast and slow repair. Yeast experiments tested whether defects in DNA repair mechanisms would amplify the radiosensitizing effect of cisplatin. It was shown that cisplatin sensitized cells with a competent recombinational repair mechanism, but did not sensitize cells defective in recombinational repair. It was also shown that under certain circumstances cisplatin did not radiosensitize normal, repair competent cells but induced stress response and cellular radiation resistance. Repair proficient wild type yeast cells became thermal tolerant and radiation resistant two hours after a sublethal cisplatin treatment. Cisplatin pretreatment also suppressed mutations caused by N-methyl-N$\sp\prime$-nitro-N-nitrosoguanidine (MNNG) treatment, a response previously shown in wild type cells with radiation pretreatment. Like radiation, cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52). These results support the idea that cisplatin crosslinks in DNA can induce a stress response which subsequently confers radiation resistance, thermal tolerance, and mutation resistance in yeast. The mechanism of this cisplatin-induced resistance was determined to be dependent on an error free recombinational repair pathway. The results obtained in this thesis indicated that error free recombinational repair plays a major role in the interaction between cisplatin and radiation. To further examine error-free recombinational repair in mammalian cells, confocal microscopy in conjunction with fluorescence in situ hybridization was used to show that the distance between homologous chromosome domains was reduced after radiation. The mechanism for homologous chromosome rearrangement following exposure to radiation is not known, however, it is plausible that a recombinational repair process is responsible. Overall, this work has shown that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving recombinational repair. However, it was also shown that, under specific conditions, cisplatin treatment induced a stress response that conferred cellular radiation resistance. Therefore, the effect of cisplatin in combination with radiation was variable depending upon the cellular processes influenced by cisplatin.