P. J. Abrahams

Enhanced reactivation and enhanced mutagenesis of herpes simplex virus in normal human and xeroderma pigmentosum cells.

Enhanced reactivation (ER) and enhanced mutagenesis (EM) of herpes simplex virus type 1 were studied simultaneously in UV-irradiated stationary cultures of diploid normal human and xeroderma pigmentosum (XP) fibroblasts. Mutagenesis was assayed with unirradiated herpes simplex virus type 1 as a probe in a forward mutation assay (resistance to iododeoxycytidine). Dose-response studies showed that ER increased with the UV dose given to the virus. Optimal reactivation levels were obtained when normal cells and XP variant cells were exposed to a UV dose of 8 J . m-2 and the virus was irradiated with 150 J . m-2. Repair-deficient XP cells of complementation groups A, C, and D showed optimal reactivation levels with a UV dose to the cells of 1.0 J . m-2 and a UV dose to the virus of 40 J . m-2. The time course of appearance of ER and EM was also studied, both in the normal and XP cells. In all cell types except the XP variant cells, EM followed similar kinetics of appearance as did ER. Maximal activities occurred when infection was delayed 1 or 2 days after cell treatment. In XP variant cells, however, maximal expression of the EM function was significantly delayed with respect to ER. The results indicate that ER and EM are transiently expressed in normal and repair-deficient XP cells. Although both phenomena may be triggered by the same cellular event, ER and EM appear to be separate processes that occur independently of each other.

Impaired DNA repair capacity in skin fibroblasts from various hereditary cancer-prone syndromes

Host-cell reactivation (HCR) of UV-C-irradiated herpes simplex virus type 1 (HSV-1) has been determined in skin fibroblasts from the following hereditary cancer-prone syndromes: aniridia (AN), dysplastic nevus syndrome (DNS), Von Hippel-Lindau syndrome (VHL), Li-Fraumeni syndrome (LFS) and a family with high incidence of breast and ovarian cancer. Cells from AN, DNS or VHL patients were found to exhibit heterogeneity in HCR. Cells from individuals belonging to an LFS family show reduced HCR in all cases where the cells were derived from persons carrying one mutated p53 allele, whereas cells derived from members with two wild-type alleles show normal HCR. LFS cells with reduced HCR also reveal reduced genome overall repair, and a slower gene-specific repair of the active adenosine deaminase (ADA) gene, but little if any repair of the inactive 754 gene. In the breast/ovarian cancer family, reduced HCR is observed in skin fibroblasts derived from both afflicted and unaffected individuals. In addition, these cells display lower survival after exposure to UV-C and exhibit higher levels of SCEs than those in normal cells. These observations indicate that various hereditary cancer-prone syndromes, carrying mutations in different tumor-suppressor genes, exhibit an unexplained impairment of the capacity to repair UV-damaged DNA.

Different regulation of p53 stability in UV-irradiated normal and DNA repair deficient human cells

The stabilization of p53 protein was studied after UV exposure of normal human skin fibroblasts and cells derived from patients suffering from xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). The data show that p53 is transiently stabilized both in UV-irradiated normal and repair deficient cells. However, particularly at later times after UV irradiation, stabilization of p53 persists much longer in repair deficient XP and TTD cells than in normal cells. The stabilization of p53 was found to be dose-dependent in normal and XP cells. These results indicate that unremoved DNA damage could possibly be responsible for the induction of transient stabilization of p53.

Differential Reactivation and Mutagenesis of Single- and Double-Stranded DNA Viruses in Irradiated Cells from Ataxia Telangiectasia Patients

Still much has to be learned about the cellular and molecular mechanisms involved in the induction of gene mutations in mammalian cells. The induction of mutations by UV-light and chemicals producing bulky DNA lesions in E.coli depends to a great extent on an inducible pathway belonging to the so-called SOS regulatory network. Thanks to numerous bacterial mutants, the SOS system has been well studied at the molecular, phenomenological and genetic levels (Walker, 1984). Bacteriophages proved particularly useful as probes to unravel repair and mutagenic processes in SOS-induced E.coli (Defais et al., 1983). The increase in the survival of damaged phages in bacteria treated with UV-light, ionizing radiation or various chemical mutagens prior to infection, a phenomenon denoted Weigle Reactivation, was among the first evidence for the inducibility of a repair component of the SOS system. Similarly, SOS mutagenesis was first revealed as an enhanced induction of phage mutations under Weigle Reactivation conditions, a response known as Weigle Mutagenesis.