Early host cell reactivation of an oxidatively damaged adenovirus-encoded reporter gene requires the Cockayne syndrome proteins CSA and CSB

Abstract

Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for normal reactivation.

Fig. 1

Reduced HCR of the MB + VL-treated reporter in SV40-transformed CSA and CSB fibroblasts compared to the SV40-transformed normal fibroblast GM637F. CSA-SV40 and CSB-SV40 fibroblasts demonstrate a reduced capacity to reactivate β-gal expression from the MB + VL-treated reporter gene (AdCA17) 44 h after infection compared to the normal SV40-transformed fibroblast GM637F. The above β-gal survival curve is from a representative experiment done in triplicate. Each point represents β-gal expression for the given VL dose ± standard error. The average relative D₃₇ values compared to G637F for CSA-SV40 and CSB-SV40 at 44 h (0.70 and 0.71, respectively) for three independent experiments were significantly decreased by one-sample two-tailed t-test (P < 0.05). A similar significant decrease in relative D₃₇ was observed for CSA-SV40 and CSB-SV40 compared to GM637F at 24 h after infection (data not shown).

Fig. 2

Time course of gene reactivation for MB + VL or UVC-damaged AdCA35 in SV40-transformed normal and CS fibroblasts. (A) HCR curves for β-gal expression from MB + VL-treated AdCA35 over time (3–44 h) in SV40-transformed human skin fibroblasts. Cells were infected with the MB + VL-treated (or mock treated) AdCA35 at an MOI of 100 pfu/cell and subsequently harvested for β-gal expression at 3, 6, 12, 24 and 44 h following infection. Representative survival curves for β-gal expression in SV40-transformed cells are shown. Each point is an average ± standard error (SE) of triplicate determinations. (B) Time course of gene reactivation (relative D₃₇ ± SE) for MB + VL-damaged AdCA35 in SV40-transformed normal and CS-deficient fibroblasts shows reduced reactivation of reporter gene expression in CSA- and CSB-deficient fibroblasts. The increase in gene reactivation is significantly reduced in CSA-SV40 (6–44 h, P = 0.04 and 12–44 h, P = 0.02) and CSB-SV40 (3–44 h, P = 0.03; 6–44 h, P = 0.03; 12–44 h, P = 0.01; 24–44 h, P = 0.009) compared to GM637F as determined by the χ² goodness of fit test. (C) Time course of gene reactivation (relative D₃₇ ± SE) of UV damaged AdCA35 in SV40-transformed normal and CS-deficient fibroblasts. The increase in gene reactivation is significantly reduced in CSA-SV40 (6–72 h, P = 0.03; 12–72 h, P = 0.01; 24–72 h, P = 0.006 and 44–72 h, P = 0.02) and CSB-SV40 (for all time ranges: 3–72 h, 6–72 h, 12–72 h, 24–72 h, 44–72 h, P < 0.0001) compared to GM637F as determined by the χ² goodness of fit test. For both (B and C), each point on the curves is the arithmetic average of at least three independent experiments done in triplicate determinations ± SE.

Fig. 3

Time course of gene reactivation for MB + VL-damaged AdCA35 in WT MEFs (A) HCR curves for β-gal expression over time in MEFs. The spontaneously transformed MEFs WT MEF and the SV40-transformed MEFs BC1-6 were infected with MB + VL-damaged (or mock treated) AdCA35 at an MOI of 100 pfu/cell and subsequently harvested for β-gal expression at 1, 2, 3, 6, 12, 24 and 44 h after infection. Representative survival curves for β-gal expression for both MEF cell lines examined 3–44 h after infection are shown. Each point is an average ± standard error (SE) of triplicate determinations. (B) Change in the D₃₇ value for each cell line. D₃₇ values for each cell line at each time point were normalised to the D₃₇ value obtained for WT MEF at 12 h for at least three independent experiments each done in triplicate. Each point is the average of the pooled results ± SE.

Fig. 4

Time course of gene reactivation for MB + VL-damaged AdCA35 in repair-proficient and repair-deficient CHO cells. (A) HCR curves for β-gal expression over time in CHO cells. CHO-AA8 and the CHO-UV61 cells were infected with MB + VL-damaged (or mock treated) AdCA35 at an MOI of 100 pfu/cell and subsequently harvested for β-gal expression at 3, 6, 9, 12, 24 and 44 h after infection. Representative survival curves for β-gal expression for both CHO cell lines examined 3–44 h after infection are shown. Each point is an average ± standard error (SE) of triplicate determinations. (B) Change in the D₃₇ value for each cell line. D₃₇ values for each cell line at each time point were normalised to the D₃₇ value obtained for CHO-AA8 at 12 h for at least three independent experiments each done in triplicate (n = 1 for 9-h time point, n = 2 for 24 and 44 h). Each point is the average of the pooled results ± SE. The increase in gene reactivation is significantly reduced in CHO-UV61 compared to CHO-AA8 cells of the time points examined (3–44 h, P = 0.0439; 6–44 h, P = 0.025; 12–44 h, P = 0.0301; 24–44 h, P = 0.0085) as determined by the χ² goodness of fit test.