Anne Priestley

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks.

We have studied the intrinsic radiosensitivity, repair of potentially lethal damage (PLD) and the repair rate of radiation-induced DNA double-strand breaks (DSB) in 11 non-transformed human fibroblast cell lines, four of which were homozygous for the A-T mutation and two that were heterozygous (A-TH). All the experiments were done on cells in plateau phase of growth (97-99% of cells in G0/G1). With a dose of 30 Gy delivered at 4 degrees C, the A-T cell lines had faster repair rates of up to 6 h, after which the repair curve crossed that of the control so that the residual damage at 24 h was higher in the A-T cells. Irradiation at 37 degrees C at low dose rate 1 cGy.min-1) produced even more marked differences between the A-T cells and controls: the residual DSB level was always higher in A-T cells than controls at doses of 5-40 Gy, due to defective repair of a small fraction of DSB in A-T cells. The two protocols showed DSB repair rates for the A-TH cell lines that were intermediate between those of the A-T and control cells. There was a quantitative relationship between the residual DSB after irradiation at 37 degrees C and the intrinsic radiosensitivity, and with the extent of PLD repair. There were very few apoptotic cells in the non-transformed control and A-T cell line, both before and after irradiation. In combination, these result support the contention that the defective repair of DSB is a mechanism of the hypersensitivity linked to the A-T mutation.

Molecular and biochemical characterization of xrs mutants defective in Ku80.

The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.

Comparative Human Cellular Radiosensitivity: I. The Effect of SV40 Transformation and Immortalisation on the Gamma-irradiation Survival of Skin Derived Fibroblasts from Normal Individuals and from Ataxia-telangiectasia Patients and Heterozygotes

We have compared cell killing following 60Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures. We have examined material from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. We have confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40 virus but the immortal cells are more gamma radiation resistant than the corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that it is expression of SV40 T-antigen, rather than immortalization per se which is responsible for the change. We use D0, obtained from a straight line fit, and D, estimated from a multitarget curve, as parameters to compare radiosensitivity. We suggest that both have their advantages; D0 is perhaps more reproducible, but D is more realistic when comparing shouldered and non-shouldered data.

DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage

Damage to DNA in the cell activates the tumour-suppressor protein p53 (ref. 1), and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53 (refs 2, 3), but the evidence for its involvement in this pathway is controversial,. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.

Comparative Human Cellular Radiosensitivity: II. The Survival Following Gamma-irradiation of Unstimulated (G0) T-lymphocytes, T-lymphocyte Lines, Lymphoblastoid Cell Lines and Fibroblasts from Normal Donors, from Ataxia-telangiectasia Patients and from Ataxia-telangiectasia Heterozygotes

We have measured clonal survival following gamma-irradiation of unstimulated (G0) T-lymphocytes from 35 donors, of 11 T-lymphocyte cell lines, of six lymphoblastoid cell lines, and of nine primary fibroblast strains for which we have G0 T-lymphocyte material from the same donor. Amongst the G0 lymphocytes we have results from nine normal donors, from eight cord bloods, from seven ataxia-telangiectasia (A-T) patients and from nine A-T heterozygotes. Although there is some variation between samples, G0 T-lymphocytes from normal donors appear to be slightly more radioresistant than T-lymphocyte lines, with a more shouldered survival curve. From our limited sample, lymphoblastoid cell lines appear to be slightly more radiosensitive than T-lymphocytes. The overall radiosensitivity of primary fibroblasts appears to be broadly similar to that of G0 T-lymphocytes. In nine instances, five A-Ts and four A-T heterozygotes, both G0 T-lymphocytes and primary fibroblasts from the same donor were tested. In five cases there was closely similar radiosensitivity in the two cell types, but in four cases there was some discrepancy. Further work, especially with normal donors, will be required in order to establish how reliably radiosensitivity in other cell types can be predicted from that of G0 T-lymphocytes. In all cell types the hypersensitivity of A-T cells was confirmed. Furthermore, the marginally greater sensitivity of A-T heterozygotes, when compared as a group with normals, was confirmed with G0 T-lymphocytes. Our results also suggest a slightly increased radiosensitivity in G0 T-lymphocytes from some, but not all, cord blood samples.

Comparative Human Cellular Radiosensitivity: III. γ-radiation Survival of Cultured Skin Fibroblasts and Resting T-lymphocytes from the Peripheral Blood of the Same Individual

Skin and blood samples were obtained from 34 donors, for whom there was no indication of abnormal radiosensitivity. From these, in 33 cases both fibroblast and T-lymphocyte cultures were obtained and in 26 cases at least three fibroblast and at least two G0 (resting) T-lymphocyte survival assays were possible. Within this set of results, differences in radiosensitivity between donors were significant for fibroblasts but not T-lymphocytes, although the range of radiosensitivity was similar for the two cell types (D 0.90-1.68 Gy for fibroblasts; 1.26-2.15 Gy for T-lymphocytes). Furthermore, there was little evidence for a correlation in radiosensitivity between the two cell types. These results suggest limitations in the predictive value of conventional measurement of cell survival.

SV40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.