The influence of DNA damage, DNA repair and chromatin structure on radiosensitivity

[摘要] ENGLISH ABSTRACT:The factors which control radiosensitivity are of vital importance for theunderstanding of cell inactivation and for cancer therapy. Cell cycle blocks, totalinduced DNA damage, DNA repair, apoptosis and chromatin structure are likelyto playa role in the responses leading to cell death.I have examined aspects of irradiation-induced G2/M blocks in DNA damageand repair. In HT29, L132 and ATs4 cells the total amount of induced DNAdamage by isodoses of 4.5 Gy, 5 Gy and 2 Gy was found to be 14 %, 14 % and12 % respectively. Most of the DNA repair was completed before the G2/Mmaximum and only 3 % of DNA damage remains to be restored in the G2/Mblock.The radiosensitivity in eleven cell lines was found to range from SF2 of 0.02 to0.61. By FADU assay the undamaged DNA at 5 Gy was found to range from56% to 93%. The initial DNA damage and radiosensitivity were highly correlated(r2=0. 81). After 5 Gy irradiation and 12 hours repair two groups of cell linesemerged. The group 1 cell lines restored undamaged DNA to a level rangingfrom 94 % to 98 %. The group 2 cell lines restored the undamaged DNA to alevel ranging from 77 % to 82 %. No correlation was seen between residualDNA damage remaining after 12 hours repair and radiosensitivity.In CHO-K1 cells chromatin condensation induced by Nocodazole was found tomarginally increase the radiosensitivity as shown by the change of the meaninactivation dose (D) from 4.446 to 4.376 Gy. Nocodazole also increased theinitial DNA damage, induced by 5 Gy, from 7 % to 13 %. In xrs1 cells theseconditions increased the radiosensitivity from D of 1.209 to 0.7836 Gy and theinitial DNA damage from 43 % to 57 %. Disruption of chromatin structure with ahypertonic medium was found to increase radiosensitivity in CHO-K1 cells fromD of 4.446 to 3.092 Gy and the initial DNA damage from 7 % to 15 %. In xrs1cells these conditions caused radiosensitivity to decrease from D of 1.209 to1.609 Gy and the initial DNA damage from 43 % to 36 %.Repair inhibition by Wortmannin increased the radiosensitivity in CHO-K1 froma D of 5.914 Gy in DMSO controls to a D 3.043 Gy. In xrs1 cells repair inhibitionhad no effect on radiosensitivity. Significant inhibition of repair was seen inCHO-K1 at 2 hours (p<0.0001) and at 20 hours (p=0.0095). No inhibition ofrepair was seen in xrs1 cells at 2 hours (p=0.6082) or 20 hours (p=0.6069).While DNA repair must be allocated to the post-irradiation period, the G2/Mblock seen in p53 mutants reaches a maximum only 12 hours post-irradiationwhen most of the repair is completed. As the G2/M block resolves and cells reentercycle 28 hours after the G2 maximum it appears that repair processescannot be the only reason for the G2IM cell cycle arrest. At low doses ofirradiation initial DNA damage correlates with radiosensitivity. This suggeststhat the initial DNA damage is a determinant for radiosensitivity. Repair of DNAdouble-strand breaks by the non-homologous end joining (NHEJ) mechanism,identified by inhibition with Wortmannin, was shown to influence residual DNAdamage and cell survival. Both the initial DNA damage and DNA repair werefound to be influenced by chromatin structure. Chromatin structure wasmodulated by high salt and by Nocodazole, and has heen identified as aparameter which influences radiosensitivity.

[发布机构] Stellenbosch University