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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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The effects of RNA and DNA 6-TG. (A) 6-TG substitution. CCRF-CEM cells were treated with 0.8 μM 6-TG for 24 h. DNA and RNA were extracted and digested to nucleosides and analysed by HPLC. 6-TG nucleosides were detected by A342. (B) 6-TG in DNA versus RNA as inhibitors of transcription. CCRF-CEM cells treated as indicated for 2 h with 1 mM HU were grown further in the presence or absence of 0.8 μM 6-TG and/or 1 mM HU for 24 h. Following UVA irradiation (10 kJ/m2) and an additional 1 h incubation in the presence or absence of HU, cells were labelled with 1 μCi/ml [3H-uridine] or 1 μCi/ml [3H-thymidine] for 15 min and incorporation of radioactivity into TCA-insoluble material was determined by scintillation counting.
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Figure 2: The effects of RNA and DNA 6-TG. (A) 6-TG substitution. CCRF-CEM cells were treated with 0.8 μM 6-TG for 24 h. DNA and RNA were extracted and digested to nucleosides and analysed by HPLC. 6-TG nucleosides were detected by A342. (B) 6-TG in DNA versus RNA as inhibitors of transcription. CCRF-CEM cells treated as indicated for 2 h with 1 mM HU were grown further in the presence or absence of 0.8 μM 6-TG and/or 1 mM HU for 24 h. Following UVA irradiation (10 kJ/m2) and an additional 1 h incubation in the presence or absence of HU, cells were labelled with 1 μCi/ml [3H-uridine] or 1 μCi/ml [3H-thymidine] for 15 min and incorporation of radioactivity into TCA-insoluble material was determined by scintillation counting.

Mentions: 6-TG is incorporated into DNA and RNA and interference with transcription might be mediated by 6-TG photoproducts in either transcribed DNA or the RNA product. We examined these two possibilities. First, the extent of 6-TG incorporation into DNA and RNA of CCRF-CEM cells was measured directly. DNA and RNA extracted from cells grown in 0.8 µM 6-TG for 24 h, was digested to nucleosides which were separated by HPLC. 2′-deoxyribo- and ribo-6-TG were detected and quantified by their absorbance at 342 nm. DNA was ∼20-fold more heavily substituted with 6-TG than RNA. 0.06% of RNA guanine was substituted by 6-TG whereas 6-TG replaced 1.1% of guanine in DNA from the same cells (Figure 2A).Figure 2.


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

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

The effects of RNA and DNA 6-TG. (A) 6-TG substitution. CCRF-CEM cells were treated with 0.8 μM 6-TG for 24 h. DNA and RNA were extracted and digested to nucleosides and analysed by HPLC. 6-TG nucleosides were detected by A342. (B) 6-TG in DNA versus RNA as inhibitors of transcription. CCRF-CEM cells treated as indicated for 2 h with 1 mM HU were grown further in the presence or absence of 0.8 μM 6-TG and/or 1 mM HU for 24 h. Following UVA irradiation (10 kJ/m2) and an additional 1 h incubation in the presence or absence of HU, cells were labelled with 1 μCi/ml [3H-uridine] or 1 μCi/ml [3H-thymidine] for 15 min and incorporation of radioactivity into TCA-insoluble material was determined by scintillation counting.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: The effects of RNA and DNA 6-TG. (A) 6-TG substitution. CCRF-CEM cells were treated with 0.8 μM 6-TG for 24 h. DNA and RNA were extracted and digested to nucleosides and analysed by HPLC. 6-TG nucleosides were detected by A342. (B) 6-TG in DNA versus RNA as inhibitors of transcription. CCRF-CEM cells treated as indicated for 2 h with 1 mM HU were grown further in the presence or absence of 0.8 μM 6-TG and/or 1 mM HU for 24 h. Following UVA irradiation (10 kJ/m2) and an additional 1 h incubation in the presence or absence of HU, cells were labelled with 1 μCi/ml [3H-uridine] or 1 μCi/ml [3H-thymidine] for 15 min and incorporation of radioactivity into TCA-insoluble material was determined by scintillation counting.
Mentions: 6-TG is incorporated into DNA and RNA and interference with transcription might be mediated by 6-TG photoproducts in either transcribed DNA or the RNA product. We examined these two possibilities. First, the extent of 6-TG incorporation into DNA and RNA of CCRF-CEM cells was measured directly. DNA and RNA extracted from cells grown in 0.8 µM 6-TG for 24 h, was digested to nucleosides which were separated by HPLC. 2′-deoxyribo- and ribo-6-TG were detected and quantified by their absorbance at 342 nm. DNA was ∼20-fold more heavily substituted with 6-TG than RNA. 0.06% of RNA guanine was substituted by 6-TG whereas 6-TG replaced 1.1% of guanine in DNA from the same cells (Figure 2A).Figure 2.

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