Repair of Potentially Lethal Damage in X-Irradiated HeLa Cells

Phillips, R. A.; Tolmach, L. J.

doi:10.2307/3572022

Cited by 323 publications

(85 citation statements)

References 35 publications

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“…It has been observed that the fraction of cells surviving a given dose of IR increases if a time interval is allowed between irradiation and replating because during this interval potentially lethal damage is repaired (53)(54)(55). As shown in Fig.…”

Section: Rad51c Sirna Treatment Alters Cell Cycle Progression and Incmentioning

confidence: 86%

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Lio

Schild

Brenneman

et al. 2004

Journal of Biological Chemistry

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The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, Rad51C was found to be a central component present in two complexes, Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2. We have shown previously that the human Rad51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G 2 /M phases of the cell cycle but not in G 1 phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.In mammalian cells, DNA double strand breaks (DSBs) 1 are repaired primarily by two distinct mechanisms, non-homologous end joining (NHEJ), a non-templated, potentially errorprone process in which nucleotide alternations are tolerated at the site of rejoining, and homologous recombination (HR), a largely error-free process in which a sister chromatid or homologous chromosome is used as a template for repair (for review, see Refs. 1 and 2). Homologous recombinational repair (HRR) provides high fidelity in repairing DNA damage and is therefore essential and critical for the maintenance of genome stability and tumor avoidance (for review, see Refs. 3 and 4). The Rad51 protein plays a key role in HR, functioning to mediate homologous DNA pairing and strand exchange (5, 6). Five vertebrate Rad51 paralogs are expressed in mitotically growing cells: Rad51B (7-9), Rad51C (10), Rad51D (9, 11, 12), XRCC2 (13-15), and XRCC3 (13,16,17). These proteins share 20 -30% sequence identity with Rad51 and with each other. Only vertebrates appear to contain all five of these Rad51 paralogs. In human cells, Rad51C participates in various paralog complexes, including Rad51B-Rad51C, Rad51C-XRCC3, and Rad51B-Rad51C-Rad51D-XRCC2 (18 -22). In terms of proteinprotein interactions, Rad51C apparently has a central role, interacting directly with Rad51B, Rad51D, and XRCC3 and also weakly with Rad51 (23,24). However, the functional significance of these complexes is not yet clear.Mutant studies provide a direct means for identifying the function of genes. A knock-out ...

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Section: Rad51c Sirna Treatment Alters Cell Cycle Progression and Incmentioning

confidence: 86%

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Lio

Schild

Brenneman

et al. 2004

Journal of Biological Chemistry

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“…The presence of potentially lethal damage (PLD) caused by ionizing radiation is usually implied by the change in survival when the cultivation conditions are altered after irradiation (Phillips and Tolmach, 1966;Little, 1971). Information regarding heavy~ion induced PLD is limited in the literature.…”

Section: Repair Of Potentially Lethal Damage a Delayed Platingmentioning

confidence: 99%

Heavy-Ion Radiobiology: Cellular Studies

Blakely

Ngo

Curtis

et al. 1984

Advances in Radiation Biology

199

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“…This can be observed after a single exposure to radiation, when the irradiated cells are subject to conditions which inhibit the normal progression of cells through the proliferative cycle (Phillips and Tolmach, 1966). This type of radiation repair is not normally found in exponentially growing cells.…”

Section: Resultsmentioning

confidence: 99%

Interaction of ICRF 159 with radiation, and its effect on sub-lethal and potentially lethal radiation damage in vitro

Taylor¹,

Bleehen²

1977

Br J Cancer

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Summary.-ICRF 159 has been shown to increase the X-radiation sensitivity of exponentially growing EMT6 mouse tumour cells in vitro. This was found only with ICRF 159 exposure times longer than 10 h and only when the drug was given before irradiation. The increase in radiation sensitivity was expressed as a reduction of the shoulder of the radiation survival curve.As ICRF 159 was shown to have no effect on the repair of sub-lethal radiation damage, it was concluded that the drug reduced the capacity to accumulate such damage. ICRF 159 was also shown to have no effect on the repair of potentially lethal radiation damage in late plateau cells.PRIOR treatment for 24 h with the antimitotic agent ICRF 159 was found to increase the effects of X-rays on EMT6 cells grown in vitro (Taylor and Bleehen, 1977). This potentiation was seen to be dependent on the number of proliferating cells in the drug-treated cell population. Cells in the exponential phase of growth when treated with ICRF 159, were found to have a much reduced radiation survivalcurve shoulder.In this paper we have examined the time course of this effect and investigated interactions between ICRF 159 and repair processes associated with cellular radiation damage. MATERIALS AND METHODSThe cell line.-The cells used in this study were EMT6/M/CC. Cells were cultured in 35 ml plastic flasks (Nunc U.K. Ltd) containing 5 ml of Eagles MEM supplemented with 20% calf serum and gassed with a mixture of 95% air and 5% Co2. Flasks for exponentialphase cultures were seeded with 3-5 x 104 cells and allowed to grow, undisturbed, for 48 h before treatment began. In all experiments to be described, exponential-phase cultures were used unless otherwise stated.The in vitro proliferation kinetics of this cell line have been fully described (Twentyman et al., 1975).Radiation treatment.-Irradiations were carried out using 250 kV X-rays from a Pantak machine, with a dose rate of about 63 rad/min. The cells were irradiated at room temperature whilst covered with medium and attached to the surface of the flasks. In the split-dose experiments the cells were returned to the incubator between irradiations.ICRF 159 treatment.-A quantity of ICRF 159 was dissolved in 0-4M HCI to produce a solution 50 x the final concentration required in the culture flasks. This stock solution was sterilized by millipore filtration and 01 ml was then added to each of the experimental flasks. 01 ml of 0-4M HCI was added to control flasks. In all experiments to be described a drug dose of 200 jug/ml was used.Survival assay.-Immediately after final irradiation or ICRF 159 treatment, the cells were removed from the surface of the flask by 15 min incubation with 0-075% trypsin solution. Two washes were used to ensure adequate removal of the drug. Following resuspension in medium, the cells were counted in a haemocytometer. After the appropriate dilutions were made, the cells were plated into 50 mm plastic culture dishes (Sterilin). These were incubated at 37°C for 10 days in plastic boxes gassed with 95% air

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Repair of Potentially Lethal Damage in X-Irradiated HeLa Cells

Cited by 323 publications

References 35 publications

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Heavy-Ion Radiobiology: Cellular Studies

Interaction of ICRF 159 with radiation, and its effect on sub-lethal and potentially lethal radiation damage in vitro

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