Alastair P.W. Waugh

Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest.

Cell lines from Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) patients show defective S phase checkpoint arrest. In contrast, only A-T but not NBS cells are significantly defective in radiation-induced G1/S arrest. Phosphorylation of some ATM substrates has been shown to occur in NBS cells. It has, therefore, been concluded that Nbs1 checkpoint function is S phase specific. Here, we have compared NBS with A-T cell lines (AT-5762ins137) that express a low level of normal ATM protein to evaluate the impact of residual Nbs1 function in NBS cells. The radiation-induced cell cycle response of these NBS and ‘leaky’ A-T cells is almost identical; normal G2/M arrest after 2 Gy, intermediate G1/S arrest depending on the dose and an A-T-like S phase checkpoint defect. Thus, the checkpoint assays differ in their sensitivity to low ATM activity. Radiation-induced phosphorylation of the ATM-dependent substrates Chk2, RPAp34 and p53-Ser15 are similarly impaired in AT-5762ins137 and NBS cells in a dose dependent manner. In contrast, NBS cells show normal ability to activate ATM kinase following irradiation in vitro and in vivo. We propose that Nbs1 facilitates ATM-dependent phosphorylation of multiple downstream substrates, including those required for G1/S arrest.

Possible association between mutant frequency in peripheral lymphocytes and domestic radon concentrations.

To investigate whether previously found geographical correlations between leukaemia incidence and exposure to radon are reflected in a detectable mutagenic effect on individuals, the frequency of mutations in the hypoxanthine guanine phosphoribosyl transferase gene (hprt) in peripheral blood T lymphocytes was measured in subjects with known domestic radon concentrations. These concentrations were measured in December, 1989, in houses in Street, Somerset, UK, by passive alpha-track radon detectors. 20 non-smoking subjects aged 36-55 years were selected from the patient list at the local health centre on the basis of the radon concentrations in their homes--the range selected varied by a factor of ten. Blood samples for preparation of T lymphocytes were taken in July, 1990. There was a significant association between the log mutant frequency and radon concentration (t = 3.47, p less than 0.01). A second analysis of a further set of radon measurements (October, 1990, to January, 1991), in both living rooms and bedrooms, and repeated mutant frequency determinations also showed a significant relation, which remained significant even after exclusion of the highest frequency and adjustment for subjects age and cloning efficiency. These data must be regarded as preliminary and further more extensive studies should be done to determine whether the observed association is causal.

Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.

Abstract— By using specific monoclonal antibodies in situ and a computer‐assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad‐spectrum UVB and narrow‐spectrum UVB. The lamps produced these lesions in different proportions, with broad‐spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow‐spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad‐ or narrow‐spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad‐spectrum or narrow‐spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T‐lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T‐lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.

Effect of deoxyribonucleosides on the hypersensitivity of human peripheral blood lymphocytes to UV-B and UV-C irradiation.

We have previously shown that non-cycling (unstimulated) human lymphocytes from normal donors show extreme hypersensitivity to UV-B irradiation, and are killed by an excisable lesion which is not a pyrimidine dimer or 6-4 photoproduct. In this paper we show that addition of the 4 deoxyribonucleosides to the medium, each at 10(-5) M, substantially increased the survival of non-cycling normal human T-lymphocytes following UV-B irradiation and substantially reduced the frequency of excision-related strand breaks in human mononuclear cells. Addition of ribonucleosides to the medium did not enhance excision-break rejoining. The survival of fibroblasts, of cycling T-lymphocytes and of unstimulated xeroderma pigmentosum T-lymphocytes was not enhanced by deoxyribonucleosides. This suggests that the hypersensitivity is due to reduced rejoining of excision breaks as a consequence of low intracellular deoxyribonucleotide pools and that it can be redressed by supplementation of the medium with deoxyribonucleosides or upregulation of ribonucleotide reductase following mitogen stimulation. We suggest that UV-B forms an additional DNA lesion which is not a pyrimidine dimer or 6-4 photoproduct, which is relatively common, and at which incision is particularly efficient. In fibroblasts, repair of this lesion is completed with high efficiency, whereas in normal unstimulated T-lymphocytes, rapid incision exacerbates the effects of the reduced rate of strand rejoining and leads to cell death.

Molecular analysis of mutations in the hprt gene in circulating lymphocytes from normal and DNA-repair-deficient donors

Circulating lymphocytes from patients with the DNA-repair-deficient disorders, xeroderma pigmentosum (XP) and ataxia telangiectasia (A-T) have elevated frequencies of mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus. We have analysed the DNA sequence of the hprt gene in mutants from normal donors, and compared them with mutants from XP and A-T individuals. In normal donors we found a range of mutations including principally transitions (40%), transversions (32%) and small deletions (20%). In an excision-deficient XP donor from complementation group C the mutation spectrum was similar to that from normal donors, whereas in an XP variant there was a significantly higher frequency (44%) of small deletions. In the two A-T donors, there was a high frequency of large deletions (22 and 75%) compared with only 4% in normal donors.

Lack of evidence for an association between the frequency of mutants or translocations in circulating lymphocytes and exposure to radon gas in the home

Radon measurements in the living room and main bedroom of 41 houses in the town of Street, Somerset, England have been made. Exposure levels, weighted using the formula of the UK National Radiological Protection Board, of 19-484 Bq m-3 (about half > 100 Bq m-3) were found. Blood samples were obtained from a total of 66 occupants in these homes, and the frequency of genetic alterations in lymphocytes was estimated using two different end points. Gene mutations at the hypoxanthine guanine phosphoribosyl transferase locus were determined in T lymphocytes for 65 subjects using a clonal assay, and the frequency of the BCL-2 t(14;18) translocation, a chromosomal event associated with leukemia/lymphoma, was estimated in lymphocytes using a polymerase chain reaction-based technique for 64 subjects. In neither case was a significant correlation with radon levels in the home found, in contrast to our earlier observation with a smaller series.

Biomonitoring of possible human exposure to environmental genotoxic chemicals: Lessons from a study following the wreck of the oil tanker Braer

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mono‐nuclear cell fraction by the butanol modification of the 32P‐postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved “in house” samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to “outlier” results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large‐scale study is likely to be capable of yielding useful information. Environ. Mol. Mutagen. 30:97–111, 1997.

Correlated mutagenesis of bcl2 and hprt loci in blood lymphocytes

In vivo measurement of human somatic mutations may be a valuable biodosimeter of exposure to carcinogens and of cancer risk. We have surveyed translocations of the bcl2 locus in B lymphocytes, and mutations of hprt in T lymphocytes, in 120 individuals with varying exposure to radon and cigarette smoke. bcl2 t(14: 18) translocation is the commonest chromosomal alteration observed in non‐Hodgkins lymphoma (NHL). We observed a significantly larger range of bcl2 translocation frequency (range: 0–372 × 10−6, median: 1.9 × 10−6) than of hprt mutation frequency (range: 0–76.4 × 10−6, median: 11.1 × 10−6), which is likely the result of clonal proliferation of deathless B cell mutants. We observed that the frequencies of these two distinct lymphocytic mutations are significantly correlated. Although some of the correlated variation is explained by age, a significant correlation of bcl2 mutagenesis persists after age adjustment. Correlated mutagenesis at distinct loci in distinct cell types could be explained by the existence of a mutator phenotype or by variation in exposure to environmental mutagens. NHL is commoner in men than in women, and our data indicate a trend toward higher bcl2 mutagenesis in males than females. There is mounting epidemiological evidence for a worldwide increase in NHL, which may have an environmental basis; molecular epidemiological analysis of bcl2 mutagenesis in exposed populations might be especially relevant to the identification of putative environmental causes. Given the relative ease of the bcl2 assay versus the hprt assay, and the consistency with which data are reproduced from laboratory to laboratory, it is likely that the bcl2 assay will be soon added to the array of assays used in human mutational surveillance. Environ. Mol. Mutagen. 29:36–45, 1997

Comparative Human Cellular Radiosensitivity: IV. The Increased Sensitivity of Human Neonatal Cord Blood Lymphocytes to γ-irradiation Compared with Lymphocytes from Children and Adults

We have compared the gamma-irradiation survival of G0 peripheral blood lymphocytes from 18 neonatal cord blood samples in a cloning assay with results from 21 controls (age range 1-65 years and consisting of 20 adults and one child). Using mean inactivation dose as the discriminating parameter, the cord blood cells showed a significantly greater radiosensitivity (mean inactivation dose for pooled data = 1.54 Gy) than the normal controls (mean inactivation dose for pooled data = 1.90 Gy, p less than 0.001). These results confirm and extend earlier work suggesting that T-lymphocytes in newborn children are more radiosensitive than normal controls, and this may have implications for the radiation protection of the unborn child.

Protective effect of deoxyribonucleosides on UV-irradiated human peripheral blood T-lymphocytes: possibilities for the selective killing of either cycling or non-cycling cells

Non-cycling human T-lymphocytes from normal subjects show a 10-fold greater sensitivity than fibroblasts to UV-B (280-315 nm) irradiation from a Westinghouse FS20 lamp, but only a 2.7-fold greater sensitivity to UV-C (254 nm) irradiation. Hypersensitivity is associated with a deficiency in the rejoining of excision breaks. Non-cycling T-lymphocytes have extremely low deoxyribonucleotide pools. Addition to the medium of the four deoxyribonucleosides, each at a concentration of 10(-5) M, substantially increases survival and reduces the persistence of excision-related strand breaks following UV-B or UV-C irradiation (Yew and Johnson (1979) Biochim. Biophys. Acta 562, 240-241; Green et al. (1994) Mutation Res., 315, 25-32). UV-resistance of T-lymphocytes is also increased by stimulating the cells into cycle. The addition of deoxyribonucleosides does not further enhance survival of cycling cells and they do not reach the level of resistance achieved by non-cycling cells in the presence of deoxyribonucleosides. We suggest that two opposing effects are in operation. Cells out of cycle can show increased resistance to DNA damage in the absence of division but they also have reduced deoxyribonucleotide pools, which may limit DNA repair. With UV-B irradiation, the exceptionally low dNTP pools in non-cycling T-lymphocytes cause this second effect to predominate. In contrast, with ionising radiation, which forms highly cytotoxic double-strand breaks, non-cycling human T-lymphocytes are slightly more resistant than fibroblasts. Non-cycling cells such as T-lymphocytes should be especially sensitive to agents which produce a high proportion of read excisable damage, but should show normal resistance to agents which highly toxic lesions. It may be possible by choice of DNA damaging agent and manipulation of cellular deoxyribonucleotide pools, to choose regimes which will selectively kill either cycling or non-cycling cells and to improve the efficacy of standard therapeutic procedures. Conditions favouring selective killing of non-dividing T-lymphocytes but sparing stem cells may be of value in bone marrow transplantation. Conditions favouring selective killing of dividing cancer cells but sparing non-dividing normal tissue may be of value in cancer therapy.