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MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation.

Grasso F, Ruggieri V, De Luca G, Leopardi P, Mancuso MT, Casorelli I, Pichierri P, Karran P, Bignami M - Oncotarget (2015)

Bottom Line: Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh- cells failed to arrest in S-phase at late time points.Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG.Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

View Article: PubMed Central - PubMed

Affiliation: Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT
The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-mouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh- MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh- cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh- cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

No MeSH data available.


Related in: MedlinePlus

Strand breaks and check-point activation after 6-TG/UVA treatmentA) A representative western blotting of ɣH2AX and Lamin proteins in WT and Mutyh−/− cell lines at various time points after 6-TG/UVA treatment (48h growth in 60nM or 300nM 6-TG followed by UVA irradiation). Under the blot values of normalized ɣH2AX expression as ɣH2AX/Lamin ratio are shown. B) A representative western blotting of p-Chk1, total Chk1 and Lamin proteins in WT and Mutyh−/− MEFs at various time points after 6-TG/UVA treatment (48h growth in 0.6μM 6-TG followed by UVA irradiation). Under the blot values of normalized p-Chk1 expression as p-Chk1/Chk1 or p-Chk1/Lamin ratio are shown.
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Figure 4: Strand breaks and check-point activation after 6-TG/UVA treatmentA) A representative western blotting of ɣH2AX and Lamin proteins in WT and Mutyh−/− cell lines at various time points after 6-TG/UVA treatment (48h growth in 60nM or 300nM 6-TG followed by UVA irradiation). Under the blot values of normalized ɣH2AX expression as ɣH2AX/Lamin ratio are shown. B) A representative western blotting of p-Chk1, total Chk1 and Lamin proteins in WT and Mutyh−/− MEFs at various time points after 6-TG/UVA treatment (48h growth in 0.6μM 6-TG followed by UVA irradiation). Under the blot values of normalized p-Chk1 expression as p-Chk1/Chk1 or p-Chk1/Lamin ratio are shown.

Mentions: 6-TG/UVA treatment causes DNA single and double strand breaks (DSBs) [24,29]. We investigated whether the presence of MUTYH influenced DNA break formation. Cells grown for 24h in 6-TG (60nM or 300nM) were UVA irradiated and the phosphorylation of histone H2AX (γH2AX) was analysed by western blotting. In wild-type cells, γH2AX was detectable immediately after UVA irradiation, plateaued between 3h and 6h and decreased thereafter (Figure 4A). A similar trend was observed in Mutyh−/− MEFs, although the extent of H2AX phosphorylation was clearly diminished in comparison to wild-type cells (Figure 4A). We also noticed that the basal level of γH2AX appeared to be lower in the Mutyh−/− MEFs. A similar analysis of G396D-expressing MEFs treated with the low dose of 6-TG, indicated that γH2AX levels were comparable to those in wild-type cells. At higher 6-TG doses, DNA breaks were more persistent in cells expressing mutant MYH than in wild-type MEFs (Figure S1).


MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation.

Grasso F, Ruggieri V, De Luca G, Leopardi P, Mancuso MT, Casorelli I, Pichierri P, Karran P, Bignami M - Oncotarget (2015)

Strand breaks and check-point activation after 6-TG/UVA treatmentA) A representative western blotting of ɣH2AX and Lamin proteins in WT and Mutyh−/− cell lines at various time points after 6-TG/UVA treatment (48h growth in 60nM or 300nM 6-TG followed by UVA irradiation). Under the blot values of normalized ɣH2AX expression as ɣH2AX/Lamin ratio are shown. B) A representative western blotting of p-Chk1, total Chk1 and Lamin proteins in WT and Mutyh−/− MEFs at various time points after 6-TG/UVA treatment (48h growth in 0.6μM 6-TG followed by UVA irradiation). Under the blot values of normalized p-Chk1 expression as p-Chk1/Chk1 or p-Chk1/Lamin ratio are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480694&req=5

Figure 4: Strand breaks and check-point activation after 6-TG/UVA treatmentA) A representative western blotting of ɣH2AX and Lamin proteins in WT and Mutyh−/− cell lines at various time points after 6-TG/UVA treatment (48h growth in 60nM or 300nM 6-TG followed by UVA irradiation). Under the blot values of normalized ɣH2AX expression as ɣH2AX/Lamin ratio are shown. B) A representative western blotting of p-Chk1, total Chk1 and Lamin proteins in WT and Mutyh−/− MEFs at various time points after 6-TG/UVA treatment (48h growth in 0.6μM 6-TG followed by UVA irradiation). Under the blot values of normalized p-Chk1 expression as p-Chk1/Chk1 or p-Chk1/Lamin ratio are shown.
Mentions: 6-TG/UVA treatment causes DNA single and double strand breaks (DSBs) [24,29]. We investigated whether the presence of MUTYH influenced DNA break formation. Cells grown for 24h in 6-TG (60nM or 300nM) were UVA irradiated and the phosphorylation of histone H2AX (γH2AX) was analysed by western blotting. In wild-type cells, γH2AX was detectable immediately after UVA irradiation, plateaued between 3h and 6h and decreased thereafter (Figure 4A). A similar trend was observed in Mutyh−/− MEFs, although the extent of H2AX phosphorylation was clearly diminished in comparison to wild-type cells (Figure 4A). We also noticed that the basal level of γH2AX appeared to be lower in the Mutyh−/− MEFs. A similar analysis of G396D-expressing MEFs treated with the low dose of 6-TG, indicated that γH2AX levels were comparable to those in wild-type cells. At higher 6-TG doses, DNA breaks were more persistent in cells expressing mutant MYH than in wild-type MEFs (Figure S1).

Bottom Line: Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh- cells failed to arrest in S-phase at late time points.Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG.Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

View Article: PubMed Central - PubMed

Affiliation: Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT
The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-mouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh- MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh- cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh- cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

No MeSH data available.


Related in: MedlinePlus