An aberration in gamma-ray-enhanced reactivation of irradiated adenovirus in ataxia telangiectasia fibroblasts.

Ataxia telangiectasia (AT) is a rare human genetic disorder, whose numerous clinical hallmarks include a predisposition to lymphoreticular cancers and a hypersensitivity to conventional radiotherapy. Furthermore, AT cells in vitro exhibit a hypersensitivity to ionising radiation that appears to be correlated with an increased frequency of chromosomal aberrations, a resistance of de novo DNA synthesis to inhibition by radiation-induced DNA damage, a reduced mitotic delay and possible defects in DNA repair. In this study, a sensitive viral assay has been used to investigate the capacity of gamma-irradiated AT cells to support the replication of undamaged virus, as well as the extent to which the survival of radiation-damaged virus was affected by gamma-irradiation of these host cells prior to infection. The expression of such enhanced reactivation (ER) of both u.v.-irradiated (u.v. dose = 1.2 X 10(3) J/m2) and gamma-irradiated (dose = 2 Mrad) adenovirus type 2 (Ad2) was examined in a variety of normal and AT human fibroblast strains. Unirradiated and gamma-irradiated fibroblasts were infected with unirradiated or irradiated Ad2, either immediately or at different times after cell monolayer irradiation, and at 48 h after infection cultures were examined by indirect immunofluorescence to determine the number of cells in which Ad2 viral structural antigen (Vag) was expressed. For immediate infection of normal human fibroblasts, both a decrease in unirradiated virus expression and an increase in ER were observed with increasing gamma-ray dose to the cells. In contrast, AT fibroblasts were found to be deficient in gamma-ray ER of irradiated Ad2, and this defect appeared to be related to a marked relative radioresistance of unirradiated virus expression in AT compared to normal cells. The potential significance of these results is discussed in the context of mammalian ER, which is believed to be, at least in part, an expression of a mutagen-inducible (and possibly error-prone) DNA repair mechanism.

U.V. enhanced reactivation of U.V.-and gamma-irradiated adenovirus in normal human fibroblasts.

An enhanced reactivation (UVER) of U.V.-irradiated as well as of gamma-irradiated human adenovirus type 2 (Ad 2) was detected following infection of normal human fibroblasts which had been pre-irradiated with U.V. light. U.V.-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or irradiated Ad 2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Results obtained using 5 different normal fibroblast strains showed that irradiation of host monolayers with 10J/m2 immediately prior to infection gave a U.V. enhanced reactivation (UVER) factor +/- standard error equal to 3 . 1 +/- 1 . 2 for virus U.V.-irradiated with 1 . 2 x 10(3) J/m2, and 2 . 1 +/- 0 . 5 for virus gamma-irradiated with 2 x 10(4) Gy. For a fixed survival of about 5 . 9 x 10(-2) for irradiated virus, the efficiency of UVER for gamma-irradiated virus was about 0 . 18, slightly less than the value of about 0 . 24 obtained for U.V.-irradiated virus. The results of time course experiments indicated that while U.V.-irradiation of normal host monolayers prior to infection gave rise to an increased rate of Vag formation for infection by unirradiated Ad 2, U.V.-irradiation of the cells increased the proportion of cells able to repair U.V.-damaged virus as well as allowing an earlier onset and/or increased rate of synthesis of Vag from a U.V.-damaged template. Similar experiments involving gamma-ray enhanced reactivation (gamma-RER) of irradiated Ad 2 indicated that gamma-RER and UVER may operate, in part at least, by different mechanisms in normal human cells.

U.V. enhanced reactivation of U.V.-and gamma-irradiated adenovirus in Cockayne syndrome and Xeroderma pigmentosum fibroblasts.

U.V.-enhanced reactivation (UVER) of both U.V.-irradiated and gamma-irradiated human adenovirus type 2 (Ad 2) was examined following the infection of a variety of Cockayne Syndrome (CS) and Xeroderma pigmentosum (XP) fibroblast strains which had been pre-irradiated with U.V. light. U.V.-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or irradiated Ad2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Normal levels of UVER (i.e. 2-4 fold) of U.V.- and of gamma-irradiated Ad 2 were detected in 2 CS strains (CS IBE and CS 3BE), 2 XP complementation group A strains (XP 12BE and XP 25RO), and 2 XP complementation group D strains (XP 5BE and XP 6BE), although the U.V. doses to these mutant cells which resulted in peak UVER values (0 . 2 Jm-2 for XP 25RO, 0 . 14 Jm-2 for XP 12BE, 0 . 8 Jm-2 for XP 5BE and XP 6BE, and 1 . 6-5 . 0 Jm-2 for CS 1BE and CS 3BE) were considerably lower than those yielding peak UVER in normal strains (10-15 Jm-2). XP variant strains (XP 4BE and XP 115LO), however, showed substantially lower levels of UVER than normal strains.

Gamma-ray-enhanced reactivation of irradiated adenovirus in Xeroderma pigmentosum and Cockayne syndrome fibroblasts.

A gamma-ray-enhanced reactivation (gamma RER) of uv-irradiated as well as of gamma-irradiated human adenovirus type 2 (Ad 2) was detected following infection of normal, Xeroderma pigmentosum (XP), and Cockayne syndrome (CS) fibroblasts that had been preirradiated with gamma rays. Gamma-irradiated or nonirradiated fibroblasts were infected with either nonirradiated or irradiated Ad 2, and 48 hr after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Results obtained using seven different normal fibroblast strains showed that irradiation of host monolayers with 1 krad immediately prior to infection resulted in a gamma RER factor +/- SE of 4.5 +/- 1.6 for uv-irradiated virus and 4.3 +/- 1.3 for gamma-irradiated virus. CS fibroblasts, as well as excision repair-deficient XP fibroblasts from complementation groups A and D, were all found to be capable of expressing gamma RER of irradiated Ad 2. XP variant cells expressed lower levels of gamma RER compared to most normal strains, suggesting a possible role for cellular postreplication repair in the mechanism responsible for ER in human cells. An excision-deficient XP fibroblast strain belonging to complementation group A, but derived from a patient afflicted with the severe De Sanctis-Cacchione form of XP, although proficient in gamma RER of gamma-irradiated Ad 2, yielded barely detectable levels of gamma RER for uv-irradiated Ad 2.