Reduced global genomic repair of ultraviolet light–induced cyclobutane pyrimidine dimers in simian virus 40–transformed human cells

Abstract

The p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in human cells. Because the large T antigen (LTAg) of the simian virus 40 (SV40) binds p53 protein and can interfere with its function, it was of interest to study DNA repair in normal human fibroblasts that had been transformed by SV40 compared with that in their nontransformed parental counterparts and to determine whether such transformation attenuated global genomic repair (GGR) of CPDs. Three methods were used to measure GGR in UV-irradiated cells: (i) an immunoassay using monoclonal antibodies specific for CPDs or 6-4 photoproducts (6-4PPs), (ii) zone sedimentation in alkaline sucrose gradients to measure the average DNA strand size after specific nicking at CPD sites in duplex DNA with T4 endonuclease V (TEV), and (iii) Southern hybridization of TEV-treated DNA with strand-specific mRNA probes to assess removal of CPDs from either strand of a defined genetic sequence in an expressed gene. Whereas repair of 6-4PPs was very similar in paired SV40-transformed and primary fibroblasts, GGR of CPDs was significantly reduced in the SV40-transformed cells. In contrast, SV40 transformation did not appreciably affect the efficiency of transcription-coupled repair. These data support the hypothesis that SV40 transformation can result in reduced levels of GGR, most likely because of the inhibition of normal p53 function by LTAg.

Metabolic labeling of human cells with tritiated nucleosides results in activation of the ATM-dependent p53 signaling pathway and acceleration of DNA repair

“…Although in such techniques the cells are prelabeled with very low activities of [ 3 H]dThd, typically between 0.1 and 1 mCi/ml (Mirzayans et al, 1996;Ford et al, 1998;Hwang et al, 1999;Bowman et al, 2000;Smith et al, 2000), we have shown here that prelabeling of NHFs with 0.05 mCi/ml [ 3 H]dThd (stock specific activity, 5 Ci/mmol) results in nuclear accumulation of p53 and growth arrest (Figures 1 and 2). Our studies further demonstrate that, unlike labeling with [ 3 H]dThd, labeling of human cells with [ 14 C]dThd for 24 h does not produce any effect on proliferation (Figure 1), p53 protein levels (Figure 3 and data not shown), or CPD repair (data not shown).…”

“…Metabolic labeling of human cells with [ 3 H]dThd is commonly used in DNA repair assays (Mirzayans et al, 1996;Ford et al, 1998;Hwang et al, 1999;Bowman et al, 2000;Smith et al, 2000). Since we demonstrated here that [ 3 H] labeling induces nuclear accumulation of p53, and because p53 is known to influence nucleotide excision repair (Wang et al, 1995), we determined the impact of radiolabeling on the repair of UV-induced CPDs.…”

DNA nucleotide excision repair-dependent signaling to checkpoint activation

“…This difference may be due to the analysis of primary cell lines compared with transformed cells. In these immortalized cells, p53 function is compromised, affecting the removal of cyclobutane pyrimidine dimers from genomic DNA, in addition to other pathways (12,13). Cells from CS patients mutated in either the CSA or CSB gene (14) show no defect in Chk1 and p53 phosphorylation, whereas XP-A fibroblasts (15) are defective in activating the checkpoint after UV irradiation, compared with normal and CS fibroblasts (Fig.…”

Cell Sorting Experiments Link Persistent Mitochondrial DNA Damage with Loss of Mitochondrial Membrane Potential and Apoptotic Cell Death

“…In fact, a significant number of physiological changes after viral transformation have been reported. For example, loss of p53 function with consequent alteration in cell cycle control (24) decreased DNA repair (25,26), and diminished catalase activity (27) was found.…”