Yechiel Becker

Colony-forming ability of ataxia-telangiectasia skin fibroblasts is an indicator of their early senescence and increased demand for growth factors

Abstract A gradual decrease in colony-forming ability of human fibroblast strains was found to be an early sign of cellular senescence. This decrease occurs in fibroblast strains from patients with ataxia-telangiectasia (A-T) at significantly earlier passage levels than with normal strains; accordingly, the lifespan of A-T cells in culture is shorter than that of normal strains. The colony-forming efficiency of A-T cells can serve as a quantitative measure for serum quality: A-T cells fail to form colonies, or have a markedly reduced colony-forming efficiency, in some batches of calf serum and newborn calf serum, whereas in other batches their clonal growth is maximal. Normal fibroblasts show the same colony-forming efficiency with all batches of serum. Fibroblast growth factor (FGF) and epidermal growth factor (EOF), but not insulin, were able to correct this deficiency in the growth-promoting ability of certain serum batches. In addition, conditioning medium containing these sera on confluent unirradiated or irradiated monolayers of any other human fibroblast strain had the same effect. A-T cells seem to have an increased demand for certain FGF- or EGF-like growth factors that are present in variable amounts in different serum batches and also in conditioned medium.

Electron microscopy of herpes simplex virus DNA molecules isolated from infected cells by centrifugation in CsCl density gradients.

Herpes simplex virus (HSV) DNA molecules were isolated from infected BSC 1 cells and centrifuged in CsCl-ethidium bromide density gradients. Both newly labelled and mature virus DNA molecules were found to have a linear conformation. The morphology of virus DNA molecules at different stages of the virus growth cycle in BSC 1 cells, was studied by electron microscopy after separation of virus DNA from cellular DNA by centrifugation in CsCl gradients. In each sample, about 200 virus DNA molecules were photographed and the different morphological forms were studied. Four classes of virus DNA molecules were observed: (a) mature linear DNA molecules, 52-4 +/- 3-3 micronm in length, (b) DNA intermediates, (c) virus DNA molecules having one or more single-stranded filaments attached to them and (d) molecules with collapsed regions or with branches. A few circular molecules as well as linear DNA molecules longer than unit length were also observed. The virus DNA molecules resembling replicative intermediates gradually increased in number and reached a maximal amount of about 5% of the virus DNA population at 12 h after infection. The other forms of virus DNA were found to persist after the number of replicating DNA molecules decreased.

A low thymidine kinase-producing mutant of herpes simplex virus type 1 causes latent trigeminal ganglia infections in mice

SummaryThe wild type NIH strain of herpes simplex virus type 1 (HSV-1) has a mixed plaque morphology of both large and small plaques. From this virus we selected a large plaque isolate that was a high producer of thymidine kinase (TK) activity (designated TK+) and a small plaque isolate that produced 25 per cent of the TK activity of the large plaque mutant (designated TK 1/4). A TK− mutant of the large plaque virus was obtained after passage of the virus in the presence of BUdR. The pathogenicity of the TK 1/4 virus strain in relation to the TK+ and TK− strains was investigated in mice after inoculation of the virus into the eyes by corneal scarification. The TK+ strain was highly pathogenic, caused encephalitis and killed most of the mice, whereas the TK− strain did not cause latent infections in the trigeminal ganglia or kill the mice. The TK 1/4 virus strain replicated in the eyes within 24 hours after inoculation and entered the trigeminal ganglia, establishing a latent infection in almost all of the mice. By increasing the infectious dose tenfold, the TK 1/4 virus caused an active infection in the trigeminal ganglia (ganglionitis), migrated to the brain, and killed the mice. The results indicate that not only is a low level of TK required to establish latent infections in mice, but also the degree of virulence is determined by the amount of TK produced by the infecting virus.

Cells from patients with ataxia telangiectasia are abnormally sensitive to the cytotoxic effect of a tumor promoter, phorbol-12-myristate-13-acetate

Fibroblast strains from 6 patients with ataxia telangiectasia (A-T) were found to be markedly hypersensitive to the cytotoxic action of the tumor promoter phorbol-12-myristate-13-acetate (PMA), their D37 values being 5 times lower than those of two normal controls. Two A-T heterozygous strains were slightly hypersensitive to PMA, while a third one showed normal sensitivity. It is concluded that the DNA lesion which is critical in A-T cells is an important component of the damage caused by PMA-induced free radicals and may play a role both in the tumor-promoting activity of PMA and the cancer proneness of A-T patients.

Repair of potentially lethal and sublethal damage induced by neocarzinostatin in normal and ataxia-telangiectasia skin fibroblasts.

Neocarzinostatin is a radiomimetic antibiotic with a potent cytotoxic effect which elicits a hypersensitive response in human cells homozygous or heterozygous for the gene for ataxia-telangiectasia. The extent and the time course of potentially lethal damage repair and sublethal damage repair following neocarzinostatin treatment were investigated in human skin fibroblast strains and were found to be remarkably similar to those obtained following X-irradiation. Ataxia-telangiectasia homozygous cells essentially lacked potentially lethal damage repair, but were able to perform some degree of sublethal damage repair following neocarzinostatin treatment. Ataxia-telangiectasia heterozygous cells which show an intermediate degree of neocarzinostatin sensitivity could perform both processes but with somewhat reduced efficiency as compared to normal cells. These observations provide further evidence for a DNA repair defect in ataxia-telangiectasia cells.

Reduced inhibition of replicon initiation and chain elongation by neocarzinostatin in skin fibroblasts from patients with ataxia telangiectasia.

Cells from patients with the genetic disease ataxia telangiectasia are hypersensitive to the DNA-breaking agents X-rays, bleomycin and neocarzinostatin, and show reduced inhibition of DNA synthesis after treatment with these agents, as compared to normal cells. The rate of replicon initiation and chain elongation was measured shortly after brief exposure of two normal and two ataxia telangiectasia fibroblast strains to low doses (0.10-0.30 microgram/ml) of neocarzinostatin, by means of alkaline sucrose gradient analysis. Neocarzinostatin was found to inhibit both initiation and elongation, and both components of DNA synthesis were more resistant to this inhibition in the A-T strains.

Similar repair of O6-methylguanine in normal and ataxia-telangiectasia fibroblast strains. Deficient repair capacity of lymphoblastoid cell lines does not reflect a genetic polymorphism.

The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.

Further characterization of the ataxia-telangiectasia clastogenic factor by reversed-phase liquid chromatography

Abstract The clastogenic factor present in medium conditioned by ataxia-telangiectasia (A-T) fibroblast cultures was chromatographed on LiChrosorb RP-8 columns and was eluted with a solution of 20% methanol in 0.005 M NH 4 HCO 3 . Based on this property, the A-T clastogenic factor was isolated from a C 8 column by high-performance liquid chromatography (HPLC). A specific fraction of the HPLC eluate contained the clastogenic factor. This method makes possible the purification of the A-T clastogenic factor for further analysis.

Chromosome Breakage and Sensitivity to DNA Breaking Agents in Ataxia-Telangiectasia and their Possible Association with Predisposition to Cancer

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by increased spontaneous chromosomal breakage and a predisposition to cancer in both A-T patients and their heterozygous relatives. We have demonstrated the presence of a clastogenic factor in plasma of A-T patients, in medium conditioned by A-T fibroblasts, and in amniotic fluid of an affected A-T fetus. Our A-T fibroblast strains were found to be as sensitive as normal strains to the cytotoxic effect of methylating carcinogens. We confirmed their hypersensitivity to ionizing radiation and found that they were also markedly sensitive to treatment with the antitumor antibiotic neocarzinostatin. Two A-T heterozygote strains had intermediate sensitivity to neocarzinostatin which was distinct from that of the homozygote and the normal cells. The intrinsic hypersensitivity to DNA-breaking agents, coupled with the presence of a clastogenic factor, may account for the high rate of chromosomal breakage in vivo and the predisposition to cancer.

Analysis of Herpes Simplex Virus DNA Synthesized in Infected Nuclei by Chromatography on Benzoylated Naphthoylated DEAE Cellulose Columns

The nature of the DNA molecules synthesized in nuclei of herpes simplex virus (HSV)-infected cells in vivo and in vitro was studied by chromatography on BND-cellulose columns after shearing to DNA fragments of 10 to 20 X 10(6) daltons. The incorporation of labelled precursors occurs in the DNA fragments containing single-stranded regions, presumably the replication forks. Prolongation of DNA synthesis leads to the accumulation of labelled DNA fragments that lack single-stranded sequences. Analysis of the isolated DNA fragments by density centrifugation in CSCl gradients revealed that most of the labelled DNA molecules are of virus specificity and the minority are cellular DNA fragments. Double-stranded virus DNA fragments and virus DNA fragments containing single-stranded sequences band in CSCl gradients at a density of 1-718 g/ml, the density of virion DNA. This suggests that the replicating HSV DNA molecules have the same density as the virion DNA and contain relatively little single-stranded DNA. The synthesis of HSV DNA molecules under in vitro conditions in isolated nuclei occurs by incorporation of the precursors into DNA fragments with single-stranded regions. The synthesis of cellular DNA in nuclei from hydroxyurea and cytosine arabinoside treated cells also occurs by elongation of nascent DNA chains.