Limits...
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.

Show MeSH

Related in: MedlinePlus

Modification of RNA polymerase II subunits following UVA irradiation of cells containing DNA 6-TG. CCRF-CEM cells were grown in 0.8 μM 6-TG for 24 h before 10 kJ/m2 UVA irradiation. Cell extracts were prepared at the indicated times after irradiation. (A) The three largest subunits of RNAPII analyzed by western blotting. (B) Rpb1 immunoprecipitated from extracts of treated cells was analysed by western blotting with an anti-ubiquitin antibody.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665240&req=5

Figure 3: Modification of RNA polymerase II subunits following UVA irradiation of cells containing DNA 6-TG. CCRF-CEM cells were grown in 0.8 μM 6-TG for 24 h before 10 kJ/m2 UVA irradiation. Cell extracts were prepared at the indicated times after irradiation. (A) The three largest subunits of RNAPII analyzed by western blotting. (B) Rpb1 immunoprecipitated from extracts of treated cells was analysed by western blotting with an anti-ubiquitin antibody.

Mentions: Since UVA activation of DNA 6-TG can cause protein modification (9) and arrest at UVC-induced DNA lesions triggers polyubiquitylation and degradation of the Rpb1 subunit of RNAPII (17,18), we examined the effect of UVA/6-TG on the transcription machinery itself. Extracts of 6-TG-treated and UVA irradiated CCRF-CEM cells were analysed by immunoblotting (Figure 3A). Between 1 and 4 h after irradiation, increasing levels of a slowly migrating form of Rpb1were noted in extracts of cells treated with both 6-TG and UVA. This form was not observed after UVA or 6-TG treatment alone. The behaviour of the modified Rpb1 was consistent with polyubiquitylation and this was confirmed by immunoprecipitation followed by western blotting with a ubiquitin-specific antibody (Figure 3B). In addition to the ubiquitylation of Rpb1, 6-TG/UVA induced a rapid modification of Rpb2, whereas Rpb3 was stable and remained apparently unaltered over 4 h.Figure 3.


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

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

Modification of RNA polymerase II subunits following UVA irradiation of cells containing DNA 6-TG. CCRF-CEM cells were grown in 0.8 μM 6-TG for 24 h before 10 kJ/m2 UVA irradiation. Cell extracts were prepared at the indicated times after irradiation. (A) The three largest subunits of RNAPII analyzed by western blotting. (B) Rpb1 immunoprecipitated from extracts of treated cells was analysed by western blotting with an anti-ubiquitin antibody.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Modification of RNA polymerase II subunits following UVA irradiation of cells containing DNA 6-TG. CCRF-CEM cells were grown in 0.8 μM 6-TG for 24 h before 10 kJ/m2 UVA irradiation. Cell extracts were prepared at the indicated times after irradiation. (A) The three largest subunits of RNAPII analyzed by western blotting. (B) Rpb1 immunoprecipitated from extracts of treated cells was analysed by western blotting with an anti-ubiquitin antibody.
Mentions: Since UVA activation of DNA 6-TG can cause protein modification (9) and arrest at UVC-induced DNA lesions triggers polyubiquitylation and degradation of the Rpb1 subunit of RNAPII (17,18), we examined the effect of UVA/6-TG on the transcription machinery itself. Extracts of 6-TG-treated and UVA irradiated CCRF-CEM cells were analysed by immunoblotting (Figure 3A). Between 1 and 4 h after irradiation, increasing levels of a slowly migrating form of Rpb1were noted in extracts of cells treated with both 6-TG and UVA. This form was not observed after UVA or 6-TG treatment alone. The behaviour of the modified Rpb1 was consistent with polyubiquitylation and this was confirmed by immunoprecipitation followed by western blotting with a ubiquitin-specific antibody (Figure 3B). In addition to the ubiquitylation of Rpb1, 6-TG/UVA induced a rapid modification of Rpb2, whereas Rpb3 was stable and remained apparently unaltered over 4 h.Figure 3.

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