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Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions.

Brem R, Li F, Karran P - Nucleic Acids Res. (2009)

Bottom Line: In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory.In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells.Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK.

ABSTRACT
Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.

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Transcription in cells containing DNA 6-TG following UVA irradiation. (A) Activation of p53. Extracts were prepared at the times indicated from cells grown for 24 h in 0.8 μM 6-TG and irradiated with 10 kJ/m2 UVA. Phosphorylation of p53Ser15 was analysed by western blotting. Control cells were treated for 18 h with 50 μM DRB. (B) RNA synthesis. CCRF-CEM cells were grown in the presence or absence of 6-TG for 24 h before irradiation with 10 kJ/m2 UVA as indicated. They were then returned to normal medium. Incorporation of [3H-uridine] into nascent RNA was measured for 15 min at the indicated times. Values are expressed as percentage of the incorporation by non-6-TG-treated, non-irradiated cells. Each measurement was performed in duplicate and the mean values of three independent experiments are shown.
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Figure 1: Transcription in cells containing DNA 6-TG following UVA irradiation. (A) Activation of p53. Extracts were prepared at the times indicated from cells grown for 24 h in 0.8 μM 6-TG and irradiated with 10 kJ/m2 UVA. Phosphorylation of p53Ser15 was analysed by western blotting. Control cells were treated for 18 h with 50 μM DRB. (B) RNA synthesis. CCRF-CEM cells were grown in the presence or absence of 6-TG for 24 h before irradiation with 10 kJ/m2 UVA as indicated. They were then returned to normal medium. Incorporation of [3H-uridine] into nascent RNA was measured for 15 min at the indicated times. Values are expressed as percentage of the incorporation by non-6-TG-treated, non-irradiated cells. Each measurement was performed in duplicate and the mean values of three independent experiments are shown.

Mentions: We have previously shown that the interaction between DNA 6-TG and UVA causes inhibition of replication, and provokes the p53 DNA damage response. In that study, we noted that at higher levels of photochemical damage, induction of the transcriptionally activated p21 protein was attenuated and suggested that this might reflect global inhibition of transcription by DNA 6-TG photoproducts (7). To investigate this point further, we compared p53 phosphorylation after 6-TG/UVA and after exposure to 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), a known inhibitor of transcription. CCRF-CEM cells were cultured in the presence of 0.8 μM 6-TG for 24 h to allow substitution of DNA by the thiobase, then irradiated with 10 kJ/m2 UVA. Phosphorylation of p53 Ser15 was determined by western blotting. Figure 1A shows that combined 6-TG/UVA induced p53 phosphorylation. This was detectable 1 h after radiation and by 4 h, phosphorylation had increased substantially in parallel with stabilization of the p53 protein. The level of Ser15 phosphorylation was comparable to that following overnight treatment with the transcription inhibitor DRB. Neither 6-TG nor UVA alone induced detectable p53 phosphorylation.Figure 1.


Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions.

Brem R, Li F, Karran P - Nucleic Acids Res. (2009)

Transcription in cells containing DNA 6-TG following UVA irradiation. (A) Activation of p53. Extracts were prepared at the times indicated from cells grown for 24 h in 0.8 μM 6-TG and irradiated with 10 kJ/m2 UVA. Phosphorylation of p53Ser15 was analysed by western blotting. Control cells were treated for 18 h with 50 μM DRB. (B) RNA synthesis. CCRF-CEM cells were grown in the presence or absence of 6-TG for 24 h before irradiation with 10 kJ/m2 UVA as indicated. They were then returned to normal medium. Incorporation of [3H-uridine] into nascent RNA was measured for 15 min at the indicated times. Values are expressed as percentage of the incorporation by non-6-TG-treated, non-irradiated cells. Each measurement was performed in duplicate and the mean values of three independent experiments are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2665240&req=5

Figure 1: Transcription in cells containing DNA 6-TG following UVA irradiation. (A) Activation of p53. Extracts were prepared at the times indicated from cells grown for 24 h in 0.8 μM 6-TG and irradiated with 10 kJ/m2 UVA. Phosphorylation of p53Ser15 was analysed by western blotting. Control cells were treated for 18 h with 50 μM DRB. (B) RNA synthesis. CCRF-CEM cells were grown in the presence or absence of 6-TG for 24 h before irradiation with 10 kJ/m2 UVA as indicated. They were then returned to normal medium. Incorporation of [3H-uridine] into nascent RNA was measured for 15 min at the indicated times. Values are expressed as percentage of the incorporation by non-6-TG-treated, non-irradiated cells. Each measurement was performed in duplicate and the mean values of three independent experiments are shown.
Mentions: We have previously shown that the interaction between DNA 6-TG and UVA causes inhibition of replication, and provokes the p53 DNA damage response. In that study, we noted that at higher levels of photochemical damage, induction of the transcriptionally activated p21 protein was attenuated and suggested that this might reflect global inhibition of transcription by DNA 6-TG photoproducts (7). To investigate this point further, we compared p53 phosphorylation after 6-TG/UVA and after exposure to 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), a known inhibitor of transcription. CCRF-CEM cells were cultured in the presence of 0.8 μM 6-TG for 24 h to allow substitution of DNA by the thiobase, then irradiated with 10 kJ/m2 UVA. Phosphorylation of p53 Ser15 was determined by western blotting. Figure 1A shows that combined 6-TG/UVA induced p53 phosphorylation. This was detectable 1 h after radiation and by 4 h, phosphorylation had increased substantially in parallel with stabilization of the p53 protein. The level of Ser15 phosphorylation was comparable to that following overnight treatment with the transcription inhibitor DRB. Neither 6-TG nor UVA alone induced detectable p53 phosphorylation.Figure 1.

Bottom Line: In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory.In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells.Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK.

ABSTRACT
Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.

Show MeSH
Related in: MedlinePlus