The role of Rad51 protein in radioresistance of spheroid model of DU145 prostate carcinoma cell line (original) (raw)
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Nucleic Acids Research, 2001
The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to γ-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of ∼2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the α parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.
Annals of Biomedical Engineering, 2008
Molecular mechanisms for the gamma-ionizing radiation (IR) resistance of human prostate cancer cells, PC-3, are not quite clear. Since the low-LET-IR effects are primarily manifested by the generation of reactive oxygen species (ROS), the IR-induced expressions both of ROSmetabolizing antioxidant enzymes, such as Mn-and CuZn superoxide dismutases (SODs) and catalase (Cat), and of the transcriptional nuclear factor-kappaB (NF-jB) were explored. A substantial increase in the concentrations of SODs was observed in the cells irradiated by 10 and 20 Gy relative to those irradiated by 0 and 2 Gy, while the Cat and NF-jB expressions were found to be fairly stable. A system biology model was developed to shed more light on how MnSOD affects the biological state of cells depending upon the production of H 2 O 2 . By raising the initial presence of MnSOD in the 0.7-10 lM concentration range, the timedependent concentrations of H 2 O 2 for various initial levels of MnSOD were contrasted. The radioresistance of PC-3 cells is suggested to be associated with the positive, feed-forward vicious circle established between the H 2 O 2 -mediated elevation of MnSOD expression.
Irish Journal of Medical Science
Aims To evaluate the potential role of key DNA repair proteins in the sensitivity of normal human fibroblasts to ionising radiations. Methods Radiosensitivity of six human fibroblast strains established from skin biopsies of women who had undergone conservative breast surgery and received a curative breast conserving radiotherapy was measured by colony-formation assay. The expression level of RAD51, BRCA1 and p53 proteins were studied using western blot analysis. Results The six fibroblast strains represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy (SF3.5) ranging from 0.21 to 0.40. We found that these differences in cell survival did not correlate with the expression of RAD51, BRCA1 nor p53 in the tested normal human fibroblast strains. Conclusions We conclude that measurement of protein expression of the three tested genes (RAD51, BRCA1 and p53) did not reflect sensitivity of normal fibroblasts to IR.
FEBS Letters, 2013
In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.
A radiosensitizing effect of RAD51 inhibition in glioblastoma stem-like cells
BMC Cancer, 2016
Background: Radioresistant glioblastoma stem cells (GSCs) contribute to tumor recurrence and identification of the molecular targets involved in radioresistance mechanisms is likely to enhance therapeutic efficacy. This study analyzed the DNA damage response following ionizing radiation (IR) in 10 GSC lines derived from patients. Methods: DNA damage was quantified by Comet assay and DNA repair effectors were assessed by Low Density Array. The effect of RAD51 inhibitor, RI-1, was evaluated by comet and annexin V assays. Results: While all GSC lines displayed efficient DNA repair machinery following ionizing radiation, our results demonstrated heterogeneous responses within two distinct groups showing different intrinsic radioresistance, up to 4Gy for group 1 and up to 8Gy for group 2. Radioresistant cell group 2 (comprising 5 out of 10 GSCs) showed significantly higher RAD51 expression after IR. In these cells, inhibition of RAD51 prevented DNA repair up to 180 min after IR and induced apoptosis. In addition, RAD51 protein expression in glioblastoma seems to be associated with poor progression-free survival. Conclusion: These results underscore the importance of RAD51 in radioresistance of GSCs. RAD51 inhibition could be a therapeutic strategy helping to treat a significant number of glioblastoma, in combination with radiotherapy.
Cellular Redistribution of Rad51 in Response to DNA Damage: NOVEL ROLE FOR Rad51C
Journal of Biological Chemistry, 2009
Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damageinduced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damageinduced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.
Rad51 Accumulation at Sites of DNA Damage and in
2000
Rad51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase (Tashiro, S., N. Kotomura, A. Shinohara, K. Tanaka, K. Ueda, and N. Kamada. 1996. Oncogene. 12:2165-2170). However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. Here, we report on ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. We also demonstrate the preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin.
Radiation Research, 2004
It has been reported that the loss of function of Trp53 protein is associated with a reduction in the expression of radiation-induced potentially lethal damage (PLD). These studies, however, were carried out using either transformed or transfected cell lines, and other factors may have existed that could interfere with PLD repair. In this study, we used isogenic fibroblasts derived from Trp53 knockout mice to study radiation sensitivity, PLD repair, and repair of DNA double-strand breaks (DSBs). Experiments were carried out using wild-type (Trp53 ؉/؉), heterozygous (Trp53 ؉/Ϫ) and homozygous mutant (Trp53 Ϫ/Ϫ) cells. This is an ideal system because the only difference in the three cell strains is the status of the Trp53 protein. DSB repair was measured by pulsed-field-gel electrophoresis (PFGE), while radiosensitivity and PLD repair were studied using the clonogenic survival assay. Cells were irradiated in plateau phase and then trypsinized and plated either immediately or 24 h later to allow for PLD repair. The results of Western blot analyses showed that Trp53 Ϫ/Ϫ cells expressed a putative mutant form of Trp53 that was unable to transcriptionally activate Cdkn1a (p21) protein in response to irradiation. The Trp53 Ϫ/Ϫ cells were significantly more radioresistant than the Trp53 ؉/؉ cells, and this was associated with a moderate reduction in PLD repair. DNA repair experiments showed no difference in DSB rejoining capability between the two cell lines. In conclusion, our results show that loss of wildtype Trp53 leads to increased radioresistance with consequent reduction in PLD repair but with no effect on DNA DSB repair.
Frontiers of Medicine in China, 2009
DNA double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions after radiation. Ku80, DNA-PK catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) proteins are major DSB repair proteins. In this study, survival fraction at 2Gy (SF2) values of eight human tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation assay, and western blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein. The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis. We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference. The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2 (r ＝0.723, P = 0.043), but Ku80 and ATM expression had no correlation with SF2 (P > 0.05). These findings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.