Thesis (PhD)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The factors which control radiosensitivity are of vital importance for the
understanding of cell inactivation and for cancer therapy. Cell cycle blocks, total
induced DNA damage, DNA repair, apoptosis and chromatin structure are likely
to playa role in the responses leading to cell death.
I have examined aspects of irradiation-induced G2/M blocks in DNA damage
and repair. In HT29, L132 and ATs4 cells the total amount of induced DNA
damage by isodoses of 4.5 Gy, 5 Gy and 2 Gy was found to be 14 %, 14 % and
12 % respectively. Most of the DNA repair was completed before the G2/M
maximum and only 3 % of DNA damage remains to be restored in the G2/M
block.
The radiosensitivity in eleven cell lines was found to range from SF2 of 0.02 to
0.61. By FADU assay the undamaged DNA at 5 Gy was found to range from
56% 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 lines
emerged. The group 1 cell lines restored undamaged DNA to a level ranging
from 94 % to 98 %. The group 2 cell lines restored the undamaged DNA to a
level ranging from 77 % to 82 %. No correlation was seen between residual
DNA damage remaining after 12 hours repair and radiosensitivity.
In CHO-K1 cells chromatin condensation induced by Nocodazole was found to
marginally increase the radiosensitivity as shown by the change of the mean
inactivation dose (D) from 4.446 to 4.376 Gy. Nocodazole also increased the initial DNA damage, induced by 5 Gy, from 7 % to 13 %. In xrs1 cells these
conditions increased the radiosensitivity from D of 1.209 to 0.7836 Gy and the
initial DNA damage from 43 % to 57 %. Disruption of chromatin structure with a
hypertonic medium was found to increase radiosensitivity in CHO-K1 cells from
D of 4.446 to 3.092 Gy and the initial DNA damage from 7 % to 15 %. In xrs1
cells these conditions caused radiosensitivity to decrease from D of 1.209 to
1.609 Gy and the initial DNA damage from 43 % to 36 %.
Repair inhibition by Wortmannin increased the radiosensitivity in CHO-K1 from
a D of 5.914 Gy in DMSO controls to a D 3.043 Gy. In xrs1 cells repair inhibition
had no effect on radiosensitivity. Significant inhibition of repair was seen in
CHO-K1 at 2 hours (p<0.0001) and at 20 hours (p=0.0095). No inhibition of
repair 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/M
block seen in p53 mutants reaches a maximum only 12 hours post-irradiation
when most of the repair is completed. As the G2/M block resolves and cells reenter
cycle 28 hours after the G2 maximum it appears that repair processes
cannot be the only reason for the G2IM cell cycle arrest. At low doses of
irradiation initial DNA damage correlates with radiosensitivity. This suggests
that the initial DNA damage is a determinant for radiosensitivity. Repair of DNA
double-strand breaks by the non-homologous end joining (NHEJ) mechanism,
identified by inhibition with Wortmannin, was shown to influence residual DNA
damage and cell survival. Both the initial DNA damage and DNA repair were
found to be influenced by chromatin structure. Chromatin structure was modulated by high salt and by Nocodazole, and has heen identified as a
parameter which influences radiosensitivity. / AFRIKAANSE OPSOMMING: Die faktore wat betrokke is in die meganisme van stralings-sensitisering is van
hoogs belang vir die begrip van sel inaktiveering en kanker terapie. Sel siklus
blokke, totale geïnduseerde DNS skade, DNS herstel, apoptose en chromatien
struktuur is moontlike rol vertolkers in die sellulêre response wat ly tot seldood.
Ek het die aspekte van stralings-geïnduseerde G2/M blokke in DNS skade en
DNS herstelondersoek. Die hoeveelheid geïnduseerde DNS skade, deur
ooreenstemmende stralings-dosisse, in HT29, L132 en ATs4 selle is 14 %, 14
% en 12 %. Meeste van die DNS herstel is klaar voordat die G2/M maksimum
beryk word en net 3 % DNS skade blyoor om herstel te word in die G2/M blok.
Die stralings-sensitiwiteit in elf sel lyne varieer tussen 'n SF2 van 0.02 en 0.61.
Deur die gebruik van die FADU metode is gevind dat die onbeskadigde DNS na
5 Gy bestraling varieer tussen 56 % en 93 %. Die totale geïnduseerde DNS
skade en stralings-sensitiwiteit was hoogs gekorreleer (r2=0.81). Na 5 Gy
bestraling en 12 ure herstel kan die sel lyne in twee groepe gegroepeer word.
Die groep 1 sellyne herstel die onbeskadigde DNS terug na 'n vlak wat varieer
tussen 94 % en 98 %. Die groep 2 sel lyne herstel die onbeskadigde DNS terug
tot op 'n vlak wat varieer tussen 77 % en 82 %. Geen korrelasie is gesien
tussen oorblywende DNS skade en stralings-sensitiwiteit na 12 ure herstel nie.
In die CHO-K1 sel lyn, chromatien kompaksie geïnduseer deur Nocodazole,
vererger die stralings- sensitiwiteit soos gesien deur die gemiddelde
inaktiveerings dosis (D) wat verlaag het van 4.446 tot 4.376. Nocodazole het
ook die totale DNS skade verhoog van 7 % tot 13 %. Onder dieselfde kondisies, in die xrs1 sel lyn, is 'n verergering van stralings-sensitiwiteit (D) gesien van
1.209 tot 0.7836 en verhoog ONS skade van 43 % tot 57 %. Die ontwrigting van
die chromatien struktuur deur die gebruik van hipertoniese medium het die
stralings-sensitiwiteit (D) vererger in CHO-K1 selle van 4.446 tot 3.092. Die
totale ONS skade is verhoog van 7 % tot 15 %. Onder dieselfde kondisies, in
die xrs1 sellyn, verbeter die stralings-sensitiwiteit (D) van 1.209 tot 1.609 en die
totale ONS skade verminder van 43 % tot 36 %. ONS herstel inaktiveering in die
teenwoordigheid van Wortmannin het die stralings-sensitiwiteit (D) in CHO-K1
selle vererger van 5.914 in DMSO verwysings kondisies tot 3.043. Die ONS
herstel inaktiveering in xrs1 selle het geen uitwerking gehaat op stralingssensitiwiteit
nie. Noemenswaardige inaktiveering van ONS herstel is gesien in
CHO-K1 selle na 2 ure (p<0.0001) en na 20 ure (p=0.0095). Geen inaktiveering
is gesien in xrs1 selle na 2 ure (p=0.6082) of na 20 ure (p=0.6069) nie.
TerwylONS herstel moet plaasvind na die bestralings periode, beryk die G2/M
blok in p53 gemuteerde selle sy maksimum 12 ure na bestraling terwyl meeste
van die ONS herstel alreeds voltooi is. Aangesien die G2/M blok eers 28 ure
later begin sirkuleer moet die G2/M blok nog 'n funksie vervul anders as ONS
herstel. By lae dosisse van bestraling korreleer die totale geïnduseerde ONS
skade met stralings-sensitiwiteit. Dit dui daarop dat die totale ONS skade 'n
bepalende faktor moet wees in stralings-sensitiwiteit. Die herstel van ONS
skade deur die nie-homoloë eindpunt samevoeging (NHES) meganisme,
geïdentifiseer deur inaktiveering deur Wortmann in, het 'n invloed op
oorblywende ONS skade en sellulêre oorlewing. Beide die totale ONS skade en
ONS herstel was beïnvloed deur die chromatien struktuur. Chromatien struktuur was gemoduleer deur hoë sout konsentrasies en deur Nocodazole, en is
geïdentifiseer as a belangrike parameter wat stralings-sensitiwiteit beïnvloed.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52540 |
| Date | 12 1900 |
| Creators | Roos, Wynand Paul |
| Contributors | Bohm, E. L .J F., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Imaging and Clinical Oncology. Radiodiagnosis. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 97 p. : ill. |
| Rights | Stellenbosch University |
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