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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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Excision of 6-TG/UVA photoproducts. GM005 (filled circle) and GM2345 (open circle) cells were grown for 48 h in 1 μM 6-TG and irradiated with 5 kJ/m2 UVA. DNA extracted from 106 cells at the times shown was digested to 2′-deoxynucleosides which were separated by HPLC. 6-TGdR was quantified by A342 and dGSO3 by fluorescence. The initial dGSO3:6-TGdR ratio immediately after irradiation was set to 100%. In a separate experiment, the same cells were irradiated with 30 J/m2 UVC. DNA extracted at the times shown was analysed for 6–4 photoproducts by ELISA. GM005 (filled triangle) and GM2345 (open triangle).
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Figure 5: Excision of 6-TG/UVA photoproducts. GM005 (filled circle) and GM2345 (open circle) cells were grown for 48 h in 1 μM 6-TG and irradiated with 5 kJ/m2 UVA. DNA extracted from 106 cells at the times shown was digested to 2′-deoxynucleosides which were separated by HPLC. 6-TGdR was quantified by A342 and dGSO3 by fluorescence. The initial dGSO3:6-TGdR ratio immediately after irradiation was set to 100%. In a separate experiment, the same cells were irradiated with 30 J/m2 UVC. DNA extracted at the times shown was analysed for 6–4 photoproducts by ELISA. GM005 (filled triangle) and GM2345 (open triangle).

Mentions: GSO3 significantly reduces the stability of oligonucleotide duplexes. It is unable to form stable base pairs with canonical DNA bases (7) and is likely to cause significant distortion of duplex DNA. Since it is also a strong block to transcription and replication, we examined whether these UVA-induced photoproducts might be subject to repair by NER. To do this, we compared the persistence of DNA GSO3 in excision repair-proficient GM005 and GM2345 XP group A lymphoblasts. Following 48-h treatment with 1 μM 6-TG, the levels of DNA substitution by the thiobase were similar (1% of DNA G) as determined by HPLC analysis of digested DNA. UVA (5 kJ/m2) induced ∼2 × 105 DNA GSO3 lesions per cell. In DNA extracted at times up to 48 h after radiation, the amount of GSO3 remained effectively unchanged (Figure 5). Lesion persistence was independent of NER status and neither GM005 nor GM2345 cells excised GSO3 to a measurable extent. As a control, we examined by ELISA the introduction and persistence of UVC-induced 6–4 photoproducts. As expected, these were efficiently excised in an NER-dependent fashion. Approximately 90% were excised by 2 h post irradiation in the NER proficient GM005 cells whereas the levels remained unchanged in the XPA GM2345 cells (Figure 5). We conclude that the GSO3 photoproduct of DNA 6-TG is a poor substrate for NER.Figure 5.


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

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

Excision of 6-TG/UVA photoproducts. GM005 (filled circle) and GM2345 (open circle) cells were grown for 48 h in 1 μM 6-TG and irradiated with 5 kJ/m2 UVA. DNA extracted from 106 cells at the times shown was digested to 2′-deoxynucleosides which were separated by HPLC. 6-TGdR was quantified by A342 and dGSO3 by fluorescence. The initial dGSO3:6-TGdR ratio immediately after irradiation was set to 100%. In a separate experiment, the same cells were irradiated with 30 J/m2 UVC. DNA extracted at the times shown was analysed for 6–4 photoproducts by ELISA. GM005 (filled triangle) and GM2345 (open triangle).
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Figure 5: Excision of 6-TG/UVA photoproducts. GM005 (filled circle) and GM2345 (open circle) cells were grown for 48 h in 1 μM 6-TG and irradiated with 5 kJ/m2 UVA. DNA extracted from 106 cells at the times shown was digested to 2′-deoxynucleosides which were separated by HPLC. 6-TGdR was quantified by A342 and dGSO3 by fluorescence. The initial dGSO3:6-TGdR ratio immediately after irradiation was set to 100%. In a separate experiment, the same cells were irradiated with 30 J/m2 UVC. DNA extracted at the times shown was analysed for 6–4 photoproducts by ELISA. GM005 (filled triangle) and GM2345 (open triangle).
Mentions: GSO3 significantly reduces the stability of oligonucleotide duplexes. It is unable to form stable base pairs with canonical DNA bases (7) and is likely to cause significant distortion of duplex DNA. Since it is also a strong block to transcription and replication, we examined whether these UVA-induced photoproducts might be subject to repair by NER. To do this, we compared the persistence of DNA GSO3 in excision repair-proficient GM005 and GM2345 XP group A lymphoblasts. Following 48-h treatment with 1 μM 6-TG, the levels of DNA substitution by the thiobase were similar (1% of DNA G) as determined by HPLC analysis of digested DNA. UVA (5 kJ/m2) induced ∼2 × 105 DNA GSO3 lesions per cell. In DNA extracted at times up to 48 h after radiation, the amount of GSO3 remained effectively unchanged (Figure 5). Lesion persistence was independent of NER status and neither GM005 nor GM2345 cells excised GSO3 to a measurable extent. As a control, we examined by ELISA the introduction and persistence of UVC-induced 6–4 photoproducts. As expected, these were efficiently excised in an NER-dependent fashion. Approximately 90% were excised by 2 h post irradiation in the NER proficient GM005 cells whereas the levels remained unchanged in the XPA GM2345 cells (Figure 5). We conclude that the GSO3 photoproduct of DNA 6-TG is a poor substrate for NER.Figure 5.

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