Rad51 and BRCA2 - New Molecular Targets for Sensitizing Glioma Cells to Alkylating Anticancer Drugs

First line chemotherapeutics for brain tumors (malignant gliomas) are alkylating agents such as temozolomide and nimustine. Despite growing knowledge of how these agents work, patients suffering from this malignancy still face a dismal prognosis. Alkylating agents target DNA, forming the killing lesion O6-alkylguanine, which is converted into DNA double-strand breaks (DSBs) that trigger apoptosis. Here we assessed whether inhibiting repair of DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioma cells to alkylating agents. For down-regulation of HR in glioma cells, we used an interference RNA (iRNA) approach targeting Rad51 and BRCA2, and for NHEJ we employed the DNA-PK inhibitor NU7026. We also assessed whether inhibition of poly(ADP)ribosyltransferase (PARP) by olaparib would enhance the killing effect. The data show that knockdown of Rad51 or BRCA2 greatly sensitizes cells to DSBs and the induction of cell death following temozolomide and nimustine (ACNU). It did not sensitize to ionizing radiation (IR). The expression of O6-methylguanine-DNA methyltransferase (MGMT) abolished all these effects, indicating that O6-alkylguanine induced by these drugs is the primary lesion responsible for the formation of DSBs and increased sensitivity of glioma cells following knockdown of Rad51 and BRCA2. Inhibition of DNA-PK only slightly sensitized to temozolomide whereas a significant effect was observed with IR. A triple strategy including siRNA and the PARP inhibitor olaparib further improved the killing effect of temozolomide. The data provides evidence that down-regulation of Rad51 or BRCA2 is a reasonable strategy for sensitizing glioma cells to killing by O6-alkylating anti-cancer drugs. The data also provide proof of principle that a triple strategy involving down-regulation of HR, PARP inhibition and MGMT depletion may greatly enhance the therapeutic effect of temozolomide

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

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

An investigation of the membrane-binding and aggregation of ovine liver cytochrome b5

Thesis (M. Sc.) -- University of Stellenbosch, 1997.One copy microfiche.Full text to be digitised and attached to bibliographic record

Role of DNA-PK dependent NHEJ and PARP dependent BER on sensitivity of glioma cells knockdown for Rad51 towards TMZ.

<p>(<b>A</b>) Inhibition of DNA-PK activity with NU7026. DNA-PK activity was determined in cell extracts in the presence and absence of NU7026. (<b>B</b>) Clonogenic survival of stable Rad51 knocked-down glioma cells (LN-229-Rad51sh-4) compared to empty vector-transfected cells (LN-229-pS-empty) after TMZ or ionizing radiation (IR) treatment in the presence or absence of the DNA-PKcs inhibitor NU7026. Equitoxic TMZ and ionizing radiation (IR) doses producing about 30 to 40% toxicity in the absence of DNA-PK inhibition were used [TMZ/IR doses: LN-229-pS-empty: 2.7 µM/2.7Gy; LN-229-Rad51sh-4: 1.5 µM/1.9 Gy]. *, p<0.05, significance levels were calculated using the Mann-Whitney U test (n = 5). (<b>C</b>) Clonogenic survival of control (LN-229-pS-empty-2) and Rad51 knockdown (LN-229-Rad51sh-23) glioma cells as a function of olaparib concentration. Cells were co-treated with TMZ or not. TMZ doses producing 30 to 40% toxicity in the absence of PARP inhibition were selected for LN-229-Rad51sh-23 (0.8 µM) and control cells (2.5 µM).</p

Knockdown of the HR protein Rad51 sensitizes glioma cells to O⁶AA chemotherapeutics.

<p>(<b>A</b>) Rad51 protein expression in knockdown clones and control assessed by western blot, quantified, corrected for loading (Erk-2) and expressed relative to control. (<b>B</b>) Clonogenic survival for stable Rad51 knockdown glioma cells (LN229-Rad51sh) compared to empty vector transfected cells (LN-229-pS-empty) following TMZ treatment. (<b>C</b>) Clonogenic survival for stable Rad51 knockdown glioma cells compared to empty vector transfected cells following ACNU treatment. (<b>D</b>) Correlation between the relative Rad51 expression, determined from A, and TMZ concentration that kills 95% of glioma cells, determined from B. Line was fitted using the equation y = minimum + (maximum-minimum)×(1-exp(-kx)). (<b>E</b>) Clonogenic survival for stable Rad51 knockdown glioma cells compared to empty vector transfected cells following ionizing radiation (IR).</p

HR downregulation sensitizes to O⁶-alkylguanine lesions in glioma cells.

<p>(<b>A</b>) Western blot analysis of MGMT and Rad51 protein levels of glioma cells stably transfected to express MGMT (LN-229-MGMT) and cells stably expressing MGMT and knockdown for Rad51 (LN-229-MGMT+Rad51sh). Erk-2 was used for loading control. (<b>B</b>) Clonogenic survival for cells stably transfected to express MGMT (LN-229-MGMT) and cells stably expressing MGMT and knockdown for Rad51 (LN-229-MGMT+Rad51sh) after TMZ treatment. (<b>C</b>) Apoptosis in T98G glioma cells that were transiently knocked-down for Rad51 (or transfected with non-sense siRNA) treated with TMZ. Apoptosis was determined by Sub-G1 flow cytometry 144 h after TMZ addition.</p

Correlations between Rad51 expression level and γH2AX foci (A) and the amount of γH2AX foci and cell death (B).

<p>Relative Rad51 expression was determined from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027183#pone-0027183-g001" target="_blank">Fig.1A</a> and the doses required to kill 95% of glioma Rad51 knockdown cell lines cells are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027183#pone-0027183-g001" target="_blank">Fig.1B</a>.</p

Co-localization of 53BP1 and γH2AX.

<p>53BP1 was immuno-detected as a second marker for DSB. Representative micrographs are shown.</p

Knockdown of the HR protein BRCA2 sensitizes glioma cells to O⁶AA chemotherapeutics.

<p>(<b>A</b>) BRCA2 protein expression following knockdown was assessed by western blot. (<b>B</b>) Clonogenic survival following TMZ treatments in transient BRCA2 or non-sense (n.s.) siRNA transfected glioma cells. (<b>C</b>) Clonogenic survival following ACNU treatments in transient BRCA2 or non-sense (n.s.) siRNA transfected glioma cells. (<b>D</b>) Clonogenic survival following ionizing radiation (IR) in transient BRCA2 or non-sense (n.s.) siRNA transfected glioma cells.</p

Apoptosis induction after TMZ treatment in Rad51 kd glioma cells.

<p>(<b>A</b>) Apoptosis determined by annexin V/PI double-staining 144 h after 10 µM TMZ in stable Rad51 knockdown clones of the cell line LN229. (<b>B</b>) Western blot analysis of transient Rad51 knockdown in U87MG and T98G cells, or transfected with non-sense siRNA. Erk-2 was used as loading control. (<b>C</b>) Apoptosis induction after TMZ treatment in Rad51 transient knocked-down glioma cells U87MG and T98G, as well as in the stable Rad51 knockdown clone LN-229-Rad51-sh8 and the empty vector control cell line LN-229-pS-empty. Apoptosis was assessed by Sub-G1 analysis performed 144 h after 10 µM TMZ treatment. For MGMT depletion 10 µM O⁶BG was added 1 h before TMZ. * p<0.05, significance level determined using the t-student test (n = 3).</p