Inhibition Of DNA Double-strand Break Repair Research Articles

Radiosensitization of Normal Human Cells by LY294002: Cell Killing and the Rejoining of DNA and Interphase Chromosome Breaks

The radiosensitizing effect of a phosphoinositide-3 kinase (PI3K) inhibitor, wortmannin, has been studied rather extensively, but there have been few studies on the radiosensitizing effect of another PI3K inhibitor, LY294002. In this report, we present the radiosensitizing effect of LY294002 using normal human cells. Clonogenic cell survival indicated that LY294002 enhanced the killing effect of gamma-rays in a dose-dependent manner, although this drug by itself did not affect the cell killing. To obtain a 10% cell survival, about one half of the radiation dose was needed when cells were treated with 50 microM LY294002 as compared to cells without the drug. A mild inhibition of repair of DNA double strand breaks (DSBs) was observed in irradiated normal human cells pre-treated with LY294002 (50 microM). At the interphase chromosome level, we also observed an increase in the number of residual breaks when irradiated cells were pre-treated with this drug (about 2-fold at 5 Gy). These results suggest that the inhibition of DSB repair mediated the radiosensitizing effect of LY294002 at the dose level that we used.

Inhibition of radiation-induced DNA-double strand break repair by various metal/metalloid compounds

In order to clarify the mechanism underlying the combined effects of radiation and metals, we have studied the cytotoxicity and inhibition of radiation-induced DNA double-strand-break (DNA-DSB) repair by various metals and metalloids. CHO cells were treated with NiCl 2, NaAsO 2, ZnCl 2, CdCl 2, CuCl 2, and potassium antimonyl tartrate for 2 h, and irradiated with γ-rays at a dose of 40 Gy to induce DNA-DSB. Then, the degree of DNA-DSB was determined by an electrophoresis technique immediately after irradiation and following a 30-min repair period. The DNA-DSB repair was significantly inhibited by exposure to Ni, Cu, Zn, As, Sb, and Cd compounds at concentrations of 200, 2.0, 0.4, 0.08, 0.55, and 1.0 mM, respectively. At these concentrations, the cell viability determined by trypan blue dye exclusion was over 50% for all the chemicals, suggesting that all of these compounds inhibited the repair of radiation-induced DNA-DSB at the concentrations where the acute cytotoxicity was relatively low. More significantly, the plating efficiencies for As, Sb, and Zn compounds at these concentrations were higher than 10%, suggesting that these chemicals inhibited DNA-DSB repair at relatively low concentrations where some of the cells sustained the ability of proliferation.

Heat-Induced Aggregation of XRCC5 (Ku80) in Nontolerant and Thermotolerant Cells

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.

Creation and Repair of Specific DNA Double-Strand Breaks in Vivo Following Infection with Adenovirus Vectors Expressing Saccharomyces cerevisiae HO Endonuclease

To study DNA double-strand break (DSB) repair in mammalian cells, the Saccharomyces cerevisiae HO endonuclease gene, or its recognition site, was cloned into the adenovirus E3 or E1 regions. Analysis of DNA from human A549 cells coinfected with the E3::HO gene and site viruses showed that HO endonuclease was active and that broken viral genomes were detectable 12 h postinfection, increasing with time up to ∼30% of the available HO site genomes. Leftward fragments of ∼30 kbp, which contain the packaging signal, but not rightward fragments of ∼6 kbp, were incorporated into virions, suggesting that broken genomes were not held together tightly after cleavage. There was no evidence for DSB repair in E3::HO virus coinfections. In contrast, such evidence was obtained in E1::HO virus coinfections of nonpermissive cells, suggesting that adenovirus proteins expressed in the permissive E3::HO coinfection can inhibit mammalian DSB repair. To test the inhibitory role of E4 proteins, known to suppress genome concatemer formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the E4 region. The results strongly suggest that the E4 protein(s) inhibits DSB repair.

The role of G2-block abrogation, DNA double-strand break repair and apoptosis in the radiosensitization of melanoma and squamous cell carcinoma cell lines by pentoxifylline

Purpose : To examine the role of G2-block abrogation, DNA repair inhibition and apoptosis in the enhancement of radiotoxicity by pentoxifylline. Materials and methods : The influence of pentoxifylline on radiotoxicity was assessed by colony assay in TP53 wild-type Be11 and mutant MeWo melanoma, and in TP53 wild-type 4197 and mutant 4451 squamous cell carcinoma (SCC) cell lines. G2-block abrogation was assessed by flow cytometry. Induction of DNA damage and repair was measured over a dose range of 0-100Gy by constant field gel electrophoresis (CFGE). The Annexin-V binding assay was used to identify apoptotic cells. Results : Pentoxifylline, when combined with irradiation, significantly increased radiotoxicity in the TP53 mutant MeWo and 4451 cell lines by radiotoxicity enhancement factors of 3 and 14.5 respectively. No radiosensitization was seen in the TP53 wild-type Be11 and 4197 cells. When the drug was added after irradiation at the time of maximum G2-block expression, no radiosensitization was seen in any of the four cell lines. CFGE analyses showed that pentoxifylline effectively suppressed DNA double-strand break (DSB) repair in all four cell lines, as indicated by 20 h repair inhibition factors of 1.4-2.4. Pentoxifylline did not increase apoptosis in any of the four cell lines. Conclusion : These data suggest that radiosensitization by pentoxifylline is not a consequence of G2-block abrogation alone, but that inhibition of DSB repair plays a role in certain cell types.

Involvement of G 2-dependent DNA double-strand break repair in the formation of ultraviolet light B-induced chromosomal aberrations

Wortmannin, an inhibitor of DNA double-strand break (DSB) repair added 19 h before harvest enhanced the incidence of ultraviolet light B (UVB)-induced chromatid aberrations in Chinese hamster V79 cells. Posttreatment with wortmannin for last 3 h of culture also enhanced the yield of breakage-type chromatid aberrations and suppressed the yield of exchange-type chromatid aberrations almost completely. Thus, the inhibition of DSB repair in the G 2 phase stimulated the breakage-type aberration formation, while suppressing the exchange-type aberration formation. We propose the model of UVB-induced chromatid-type aberration formation which might be fully related to G 2-dependent DSB repair pathway.

In Vitroandin VivoPotentiation of Radiosensitivity of Malignant Gliomas by Antisense Inhibition of the RAD51 Gene

The mammalianRAD51gene is a homologue of the yeastRAD51andE. coli RecAgenes, which are related to the repair of DNA double-strand breaks and are also involved in recombination repair and various SOS responses to DNA damage by γ-irradiation and alkylating reagents. In this study, we investigated bothin vitroandin vivowhether inhibition of theRAD51gene by antisense oligonucleotides (ODNs) enhances the radiosensitivity of mouse malignant gliomas. A volume of 100 nM ofRAD51antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70%. Treatment of mouse 203G glioma cells with 100 nM ofRAD51antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with sense or scrambled ODNs. When the glioma cells were implanted in the cisterna magna of mice followed by treatment withRAD51antisense ODNs, the survival time of the mice was markedly prolonged compared to that of the untreated group (p<0.001, logrank test). In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing mice much longer than could be achieved with radiation alone (p<0.0001, logrank test). These results suggest that inhibition ofRAD51can be expected to serve as a novel potentiator for radiation therapy in malignant gliomas by inhibiting DNA double-strand break repair.

Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells.

Wortmannin, an inhibitor of p110 PI 3-kinase, also inhibits DNA-dependent protein kinase, which is known to mediate DNA double strand break repair. It was recently demonstrated that wortmannin sensitized cells to ionizing radiation (IR) (Price and Youmell, Cancer Res., 56, 246-250, 1996). Wortmannin was used to determine if the potentiation of IR-induced cytotoxicity in Chinese hamster ovary cells could be accounted for by an inhibition of DNA double strand break (DSB) repair. Wortmannin, at concentrations which were non-toxic per se (5 and 20 microM), increased IR cytotoxicity with dose enhancement factors at 10% survival of 2.7+/-0.28 (5 microM) and 5.3+/-0.86 (20 microM). The effects of wortmannin on DSB levels were assessed by neutral elution. The effects of wortmannin on the kinetics of DSB repair were evaluated over a 3 h time course. Wortmannin (50 microM) completely inhibited DSB repair over this period, without having any effect on DSB levels itself. The concentration-dependent effects of wortmannin on DSB levels showed that inhibition of DSB repair was significant at 1 microM, and near-maximal at 20 microM. In marked contrast, it exerted no effect on the kinetics of single strand break (SSB) repair as assessed by alkaline elution, even at concentrations as high as 50 microM. There was an excellent correlation between the concentration-dependence and exposure time of wortmannin required to enhance IR cytotoxicity and inhibit DSB repair. These data implicate inhibition of DNA-dependent protein kinase, and the consequent inhibition of DSB repair, as the mechanism whereby wortmannin potentiates the cytotoxicity of IR.