Gene targeting is enhanced in human cells overexpressing hRAD51.
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The ideal therapy for single gene disorders would be repair of the mutated disease genes. Homologous recombination is one of several cellular mechanisms for the repair of DNA damage. Recombination between exogenous DNA and homologous chromosomal loci (gene targeting) can be used to repair an endogenous gene, but the low efficiency of this process is a serious barrier to its therapeutic potential. Recent progress in the isolation and characterisation of mammalian genes and proteins involved in DNA recombination has raised the possibility that the cellular biochemistry of recombination can be manipulated to improve the efficiency of gene targeting. As an initial test of this approach, we have overexpressed the gene encoding hRAD51, a protein with homologous DNA pairing and strand exchange activities, in human cells and measured its effect on gene targeting. We report a two- to three-fold increase in gene targeting, and enhanced resistance to ionising radiation in hRAD51-overexpressing cells with no obvious detrimental effects. These observations provide valuable genetic evidence for the involvement of hRAD51 in both gene targeting and DNA repair in human cells. Our data also establish overexpression of recombination genes as a viable approach to improving gene targeting efficiencies. (+info)
Whole-genome shotgun optical mapping of Deinococcus radiodurans.
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A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation. (+info)
A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy.
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Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction. (+info)
Genetic alterations on chromosome 17p associated with response to radiotherapy in bulky cervical cancer.
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Chromosome 17 alterations are found in more cancers than those of any other chromosome, and frequently involve the p53 gene on 17p13. The aim of this study was to identify the correlations between the presence of loss of heterozygosity (LOH) and microsatellite instability (MI) on chromosome 17p13 in patients with cervical cancer and the patients' response to radiotherapy. A total of 50 patients were treated with definitive radiotherapy. We performed biopsies and took specimens from the tumour and venous blood of all patients. Tumour and normal DNAs were analysed by polymerase chain reaction for genetic losses and instability at three polymorphic microsatellite loci mapped to 17p13. Nineteen of the 50 tumours (38%) displayed a genetic alteration (GA) on 17p13, 16 (32%) were found to have LOH, and three (6%) showed MI. The sizes of the tumours of the GA-positive patients were significantly greater than those of the GA-negative patients (P = 0.009). The mean tumour diameter of all patients was 6 +/- 2.4 cm. We divided the patients into those with tumours smaller than 6 cm in diameter (n = 26) and those with tumours equal to or greater than 6 cm in diameter (n = 24). The former group survived significantly longer compared to the latter group (P = 0.0002). Among the patients with < 6 cm tumours, all six GA-positive patients are alive with no evidence of disease (NED), whereas of the 20 GA-negative patients, 18 have NED and two are alive with disease (AWD) or suffered cancer-caused death (CD). Thus, there was no correlation between GA and radiotherapy response in the tumours smaller than 6 cm. However, among the patients with > or = 6 cm tumours, two of the GA-positive patients have NED and 11 are AWD/CD, whereas seven of the GA-negative patients have NED and four are AWD/CD. Among the patients with > or = 6 cm tumours, the response to radiotherapy of the GA-positive patients were significantly poorer than those of the GA-negative patients (P = 0.02). In addition, the GA-negative patients survived significantly longer compared to the GA-positive patients (P = 0.026). The results of this study suggest that GA increases with tumour growth. Improved success in the management of bulky cervical cancer requires a better understanding of its biological behaviour. (+info)
Altered telomere nuclear matrix interactions and nucleosomal periodicity in ataxia telangiectasia cells before and after ionizing radiation treatment.
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Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene. (+info)
Effect of estradiol on radiation-induced chromosome aberrations in human lymphocytes.
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As a part of studies on physiological factors that affect radiosensitivity, we examined the in vitro effect of estradiol (E2) on the yield of radiation-induced chromosome aberrations in human peripheral lymphocytes. Lymphocytes were cultured for 3 days in the medium containing E2 at 0-100,000 ng/ml. On the second day, they were irradiated by X-rays at 3 Gy, and then 2% phytohemagglutinin and 0.05 microgram/ml colcemid were added to the medium. After further 48 h, mitotic indices and the yields of chromosome aberrations were examined at various E2 concentrations. E2 treatment at concentrations above 1000 ng/ml resulted in dose-related inhibition of mitosis. Repeated experiments showed that the yield of dicentrics plus centric rings in the culture containing E2 at 100 ng/ml was significantly higher than the yields at 0 ng/ml. Similarly, the yield of total chromosome breaks in the culture containing E2 at 100 ng/ml was significantly higher than that at 1 ng/ml. This study provides the direct evidence in human that radiosensitivity may vary in relation to hormonal conditions. (+info)
Radioprotective effects of sodium tungstate on hematopoietic injury by exposure to 60Co gamma-rays in Wistar rats.
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Radioprotective effects of sodium tungstate (ST) on 60Co gamma-ray induced decrease in hematocrit value and in survival rate in Wistar strain male rats were examined. A long-term administration of ST (less than 150 mg/kg body weight/day) for 60-300 days had no significant effects on body and organs weights and survival days. The LD50/60 in 20 weeks old rats was 220 mg/kg body weight/day. Daily administration of 38, 75 or 150 mg from 7 days before and after irradiation to 60 days significantly mitigated the decrease in hematocrit values, especially at 23 days after irradiation (P < 0.05). The highest mitigation rate of the decrease in hematocrit value was observed in rats administered at a dose of 38 mg ST/day. Simultaneously, a dose of 38 mg ST/day inhibited lethal effect of 60Co gamma-rays significantly. The dose-reduction factor for survival of 38 mg ST administered rats was 1.14. (+info)
Chromosome breakage and cell lethality in human hepatoma cells irradiated with X rays and carbon-ion beams.
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Prediction of radiosensitivity would be valuable for heavy-ion radiotherapy. Premature chromosome condensation (PCC) technique has been a potential predictive assay in photon radiotherapy, but has not been investigated for hepatomas receiving heavy ions. Two human hepatoma cell lines, i.e., HLE and HLF, were irradiated with either 290 MeV/u carbon ions or 200 kVp X rays. Cell lethality was assayed by colony formation and compared with the unrejoined fraction of chromatin breaks as measured by PCC technique. Carbon ions at linear energy transfer (LET) of 76 keV/micron produced cell death more effectively than those of 13 keV/micron and X rays. For the cell killing, the relative biological effectiveness (RBE) of 13 and 76 keV/micron carbon ions compared with X rays was 1.10-1.24 and 2.57-2.59, respectively. Mean number of chromosomes in HLE and HLF cells was similar to each other, i.e., 60.48 and 60.28. RBEs for chromatin breaks of 13 and 76 keV/micron carbon ions were 1.30-1.31 and 2.64-2.79, respectively. A strong correlation between unrejoined chromatin breaks and cell killing for human hepatoma cells was observed irrespective of radiation quality. We conclude that PCC provides a potential predictor for the radiosensitivity of individual hepatoma that are treated with photon as well as heavy ion irradiation. (+info)