National Cancer Institute®
Last Modified: November 21, 2001
UI - 21460697
AU - Brammer I; Zoller M; Dikomey E
TI - Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts.
SO - Int J Radiat Biol 2001 Sep;77(9):929-38
AD - Institute of Biophysics and Radiobiology, University of Hamburg, Martinistr. 52, D-20246, Hamburg, Germany. firstname.lastname@example.org
PURPOSE: To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. MATERIALS AND METHODS: Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. RESULTS: The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7+/-0.2 to 5.0+/-0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. CONCLUSIONS: The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.
UI - 21010702
AU - Asaad NA; Zeng ZC; Guan J; Thacker J; Iliakis G
TI - Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants.
SO - Oncogene 2000 Nov 23;19(50):5788-800
AD - Department of Radiation Oncology of Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.
UI - 21374349
AU - Klein C; Stewart GS; Quinn NP; Taylor AM
TI - Ataxia without telangiectasia revisited: update on genetic findings in two brothers with an ataxia-telangiectasia-like disorder.
SO - Mov Disord 2001 Jul;16(4):788-9
UI - 21398709
AU - Pincheira J; Bravo M; Navarrete MH; Marcelain K; Lopez-Saez JF; de la
TI - Torre C Ataxia telangiectasia: G2 checkpoint and chromosomal damage in proliferating lymphocytes.
SO - Mutagenesis 2001 Sep;16(5):419-22
AD - Programa de Genetica Humana and Departamento de Pediatria y Cirugia Infantil, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
There is a checkpoint pathway in eukaryotic cells that depends on ATM (ataxia telangiectasia mutated) kinase which activates the processes leading to the repair of DNA damage and also lengthens the G(2) stage of the cell cycle. In cells from ataxia telangiectasia patients, due to their lack of active ATM kinase, an increase in chromosomal aberrations and a failure to induce G(2) lengthening could be expected. However, the basal G(2) timing in ataxia telangiectasia cells was longer than in controls and was further extended after X-ray irradiation (0.4 Gy), although to a lesser extent than in controls. Moreover, in control cells caffeine shortened G(2) and increased chromosomal damage 7-fold, while in ataxia telangiectasia cells caffeine only trebled aberration yield without shortening G(2). As caffeine is an inhibitor of ATM kinase, these results suggest the existence of some redundant ATM-independent checkpoint in G(2) of ataxia telangiectasia cells. The differential response to caffeine of ataxia telangiectasia and control lymphocytes may be explained by the presence of two different subpathways in the G(2) checkpoint: one regulating the processing and repair of damaged DNA and the other controlling G(2) timing. While in controls both subpathways may be mediated by ATM kinase, in ataxia telangiectasia cells caffeine-sensitive ATR kinase and the caffeine-insensitive DNA-PK kinases might be responsible for DNA repair and the G(2) delay subpathways, respectively. Confirmation of this model in ataxia telangiectasia cells with another cell type in which both subpathways are mediated by DNA-PK should define whether a metylxanthine such as caffeine may also have an additional direct inhibitory effect on DNA repair.
UI - 21398713
AU - Menendez D; Mora G; Salazar AM; Ostrosky-Wegman P
TI - ATM status confers sensitivity to arsenic cytotoxic effects.
SO - Mutagenesis 2001 Sep;16(5):443-8
AD - Instituto de Investigaciones Biomedicas, UNAM, Apartado Postal 70228, D.F., Mexico City 04510, Mexico.
Arsenic (As), a human carcinogen, represents a worldwide health problem due to the high number of people exposed to this element in their drinking water. Previously our group has demonstrated that As can impair lymphocyte cell proliferation in vitro and in vivo and can increase the level of P53 protein, with different responses to these effects between individuals. Recently it has been shown that ATM protein, responsible for the autosomal recessive disorder ataxia telangiectasia (AT), regulates P53. In this study the induced response of P53 was evaluated following exposure to As in human lymphoblastoid cell lines normal (+/+), heterozygous (+/-) or homozygous (-/-) for the mutant ATM gene. After 24 h As treatment we found a dose-dependent induction of P53 in normal and heterozygous cell lines, although differences between cell lines were observed. An increase in P21(WAF) protein, a main effector of P53 activation, was also observed in the same cell lines. In contrast, neither P53 nor P21 induction was detected in homozygous cells. The ATM (+/-) and (-/-) genotypes confer more sensitivity to As cytotoxic effects than the normal allelic condition. Paradoxically, ATM heterozygous cells were more sensitive to As, leading us to propose that this might be related to activation of apoptosis and removal of non-repairable cells. In contrast, in AT cells in which ATM is absent or mutated activation of P53 and its target genes is abrogated, allowing cells to replicate with damage in the presence of As, with cell death ensuing by a pathway different from P53.
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