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Cocultivation of plasma and lymphocytes from ataxia telangiectasia (AT) patients with those of normal individuals resulted in a significant increase in chromosomal damage in the normal cells. Tissue culture medium used to cultivate AT skin fibroblasts also significantly increased chromosome breakage in PHA-stimulated normal lymphocytes. This clastogenic effect was maximal when the "conditioned medium" was 8-9 days old. A similar effect was not observed with medium derived from normal skin fibroblasts. These observations suggest the presence of a clastogenic factor in the plasma of AT patients which may also be produced by AT skin fibroblasts in culture.

Fiber autoradiograms prepared from radioactive DNA of diploid skin fibroblasts and lymphocytes from normal adult humans show patterns of replication of chromosomal DNA that are closely similar to those from other mammalian cells. The rate of replication fork movement is 0.64 micrometer/min and 0.42 micrometer/min for fibroblasts and lymphocytes respectively. In both types of cells, replication units show a median length of about 70 micrometer. Replication proceeds bidirectionally from a central origin in most units and adjacents units initiate synthesis synchronously. In skin fibroblasts from individuals with Fanconi's anemia or ataxia telangiectasia, the autoradiographic patterns of DNA replication are the same as in controls. This suggests that S phase DNA synthesis is normal in these disorders.

Chromosome analyses are reported for 14 lines of fibro-blasts derived from 8 ataxia-telangiectasia (ATT) patients and for 14 lines of control cells. An elevated incidence of chromosome damage including gaps and breaks, rings, dicentrics and fragments, and chromosome figures has been found to occur in ATT cells. Similar abnormalities present in different cell lines suggest that common break points occur in ATT fibroblast chromosomes. The SV40 virus transformation rates found for ATT cells lie within the range found for normal cells, and there is no direct correlation between the degree of chromosome damage exhibited by any ATT cell line and its transformation rate with SV40.

The autosomal recessive disorder, ataxia telangiectasia (AT) is characterised by cellular sensitivity to ionizing radiation. The molecular basis of this radiosensitivity is the subject of controversy. We report here that cultured fibroblasts from AT patients are also sensitive to the lethal effects of bleomycin. As with ionizing radiation, no defect has been observed in the overall rejoining of single or double-strand breaks produced by bleomycin. Since, however, only apyrimidinic (and to a lesser extent apurinic) sites and strand breaks are known to be produced by bleomycin, we tentatively suggest that AT cells are unable to rejoin a very small fraction of the total strand breaks. We attribute our inability to detect such unrejoined strand breaks to the relative insensitivity of the sucrose gradient procedures normally used to detect strand breaks.

We previously showed that radiation-sensitive fibroblasts from ataxia-telangiectasia (A-T) patients sustain less G2 delay after X-irradiation than normal fibroblasts (Scott and Zampetti-Bosseler, 1982). Caffeine is known to reduce the amount of X-ray-induced delay in various mammalian cell types. Painter and Young (1980) proposed that A-T cells have an altered chromatin structure, similar to that of caffeine-treated normal cells and that this results in a failure of A-T cells to delay their progression through the cell cycle to allow time for DNA repair. We now show that caffeine treatment after X-irradiation reduces G2 delay in both A-T and normal cells. We confirm the results previously obtained on lymphocytes that caffeine potentiates the chromosome-damaging effects of X-rays in both A-T and normal fibroblasts. These and other data suggest that the radiation responses of A-T cells and of caffeine-treated normal cells are caused by different mechanisms.

The effects of modification of poly(ADP-ribosyl)ation reactions have been examined in normal (F107) and ataxia telangiectasia (AT23) fibroblasts following damage by methyl methanesulphonate (MMS) and u.v. light. The technique of benzoylated DEAE (BD)-cellulose chromatography was utilized to estimate both the extent and nature of the damage to DNA induced by these agents and to examine the effects of an inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), on these parameters. Single strand breakage, determined by nucleoid sedimentation, and levels of poly ADP(ribose) synthesis were monitored. Increase in the proportion of DNA containing single-stranded regions, as measured by stepwise elution from BD-cellulose, was observed following MMS damage in both cell types. In the presence of 3AB, a further accumulation of DNA containing single-stranded regions occurred, with the effect being more prominent in AT23 fibroblasts. U.v. light damage did not induce increased binding to BD-cellulose in normal cells, and the increase observed in AT23 cells was much less than that seen following alkylation damage. Examination of the nature of single-stranded damage by caffeine gradient elution from BD-cellulose following MMS treatment revealed discrete structural lesions, which were enhanced in the presence of 3AB. A similar effect was exerted by arabinofuranosyl cytosine. The behaviour of these intermediates, which could be associated with repair, was not in accord with the suggestion that 3AB inhibits only the ligation stage of the repair process. Our results suggest that specific intermediate stages in DNA repair are sensitive to 3AB, and it seems likely that these stages occur prior to ligation.

Replicative DNA synthesis in normal human fibroblasts was inhibited by 50% when they were X-irradiated (8 Gy) and made permeable 30 min later, whereas only a slight inhibition (20%) was observed in similarly treated ataxia-telangiectasia cells. Treatment of irradiated normal cells with caffeine (2 mM) before permeabilization reversed the inhibitory effects of X-rays, buf caffeine had no effect on DNA synthesis in permeable ataxia-telangiectasia cells. Diadenosine tetraphosphate (0.1 mM) did not affect DNA synthesis in permeable normal fibroblasts.

In the present study we examined cells from several patients clinically diagnosed as having ataxia-telangiectasia (AT), for the capacity of their cells to inhibit DNA synthesis following exposure to gamma irradiation, and for the rate of spontaneous or bleomycin-induced chromosomal aberrations. Cells from two patients showed normal inhibition of DNA synthesis and levels of induced chromosomal aberrations intermediate between normal and AT cells. These two patients had only minimal immunologic impairment. These findings appear to define one distinct subset of AT.

Peripheral blood lymphocytes from four patients with ataxia telangiectasia (AT), an inherited disorder showing, among other features, radiosensitivity and a high frequency of cancers, were shown to be cytogenetically more sensitive to bleomycin than were lymphocytes from both normal individuals and a single patient with xeroderma pigmentosum. With cell survival techniques, a biphasic dose-response curve was seen for both normal and AT fibroblasts, although the AT cells showed a much lower survival. The increased sensitivity to bleomycin in AT cells might be expected since it is a radiomimetic drug, but more importantly the known action of bleomycin in producing DNA strand scission suggests that AT cells might be defective in rejoining a proportion of DNA strand breaks.

The effect of gamma-radiation on the synthesis of short fragments of DNA was studied in control and ataxia telangiectasia lymphoblastoid cell lines. Cells were pulsed with [3H]thymidine for 90 sec, which resulted in labelling of DNA fragments ranging from 0.1-2.1 x 10(7) daltons in size in both control and ataxia telangiectasia cells. After exposure to 800 rad of gamma-rays, a decrease in the formation of DNA over this molecular weight range was observed in four control cell lines, 30 min post-irradiation. Four ataxia telangiectasia cell lines however exhibited no reduction in the synthesis of sub-replicon size DNA.

An assay has been developed to measure the ability of human lymphocytes to repair damage to DNA. In this assay, purified human lymphocytes are exposed to graded doses of radiation and then stimulated with phytohemagglutinin to undergo DNA replication. The rate of incorporation of thymidine in irradiated lymphocytes during the second and subsequent rounds of DNA replication is taken to be indicative of the ability of the cells to repair damage to DNA. In lymphocytes from normal individuals, X-irradiation with doses of 100 to 800 rads was found to inhibit phytohemagglutinin-stimulated thymidine incorporation proportionally to the dose of radiation without curtailing the induction of DNA polymerase. The response to phytohemagglutinin of lymphocytes from a patient with xeroderma pigmentosum after exposure to graded doses of X-irradiation was found to be similar to that of the normal controls, whereas the response after ultraviolet irradiation was markedly impaired. In contrast, lymphocytes from patients with ataxia telangiectasia were hypersensitive to X-irradiation. The data on these clinical syndromes support the idea that this assay measures DNA repair and indicates the feasibility of using this method for screening individuals for genetic deficits in DNA repair.

Cell lines established from donors with the inherited disorder ataxia-telangiectasia (A-T) exhibit exceptional sensitivity to ionizing radiation and chemicals known to produce increased levels of intracellular H2O2, suggesting a deficiency in glutathione-dependent detoxication reactions. Glutathione (GSH) biosynthesis in fibroblast and lymphoblast cultures derived from individuals known to be clinically unaffected, homozygous, or heterozygous for A-T was assessed. Following GSH depletion by diethylmaleate, fibroblasts (GM 3492) from a clinically unaffected individual resynthesized GSH at a rate approximately twice that observed in fibroblasts from known heterozygotes (GM 3488 and GM 3489). Unrelated A-T homozygote fibroblast lines GM 3487B and GM 5823 resynthesized GSH only very slowly. GM 3492 cells repleted intracellular GSH by 6 h after depletion, the heterozygote lines by 18 h. The A-T homozygotes replaced only 30% of the intracellular GSH pool by 24 h. A lymphoblast cell line from the A-T homozygote (GM 3189) also exhibited slow resynthesis after depletion. However, if these cells were permeabilized by treatment with digitonin, GSH synthesis proceeded at a rate exceeding synthesis in permeabilized or untreated normal lymphoblasts (GM 3323). The first enzyme in GSH synthesis, gamma-glutamylcysteine synthetase, was found to be elevated about 2.7-fold in A-T homozygote fibroblasts, suggesting that a substrate for GSH synthesis may be rate limiting. A-T homozygote lymphoblasts contained about 2-fold more gamma-cystathionase activity over other cell lines tested suggesting increased flux through the transsulfuration pathway for cysteine production in response to reduced cysteine supply. Transport of cysteine and cystine was found to be 8- and 5-fold slower in A-T homozygotes that did not affect fibroblasts while glutamate and methionine transport Vmax did not differ among the cell lines tested. These experiments demonstrate that cells from A-T homozygotes are deficient in cysteine transport, thus limiting GSH resynthesis after a depleting challenge such as radiation or GSH-depleting xenobiotic compounds.