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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

Survival, skin DNA 8-oxoG levels and tumor onset in WT and mice treated with Aza/UVAA) Schematic representation of the treatment. B) Summary of survival data of WT and Mutyh−/− mice treated with Aza, UVA or the combined treatment as indicated in A). C) Kaplan–Meier curves of Aza and Aza/UVA treated WT (solid line) and Mutyh−/− (dotted line) mice. D) DNA 6-TG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza or Aza/UVA combined treatment. Data are mean ± SE of 11-19 animals/genotype. E) DNA 8-oxoG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza, UVA or combined treatment. Data are compared to historical controls (CTR) and they are mean ± SE of 16-25 animals/genotype. *P≤0.05 (Student's t-test). F - H) Representative histological images of SCCs from two Aza/UVA treated Mutyh−/− mice. F) Well-differentiated Grade I SCC showing full thickness epidermal atypia and involvement of hair follicles. Keratinization-induced “pearl-like” structures (dermal nests of keratinocytes attempting to mature in a layered fashion containing keratinizing cells and horny pearls) are indicated by an arrow. G) Poorly-differentiated Grade III/IV SCC. H) A higher magnification of the tumor shown in G).
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Figure 6: Survival, skin DNA 8-oxoG levels and tumor onset in WT and mice treated with Aza/UVAA) Schematic representation of the treatment. B) Summary of survival data of WT and Mutyh−/− mice treated with Aza, UVA or the combined treatment as indicated in A). C) Kaplan–Meier curves of Aza and Aza/UVA treated WT (solid line) and Mutyh−/− (dotted line) mice. D) DNA 6-TG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza or Aza/UVA combined treatment. Data are mean ± SE of 11-19 animals/genotype. E) DNA 8-oxoG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza, UVA or combined treatment. Data are compared to historical controls (CTR) and they are mean ± SE of 16-25 animals/genotype. *P≤0.05 (Student's t-test). F - H) Representative histological images of SCCs from two Aza/UVA treated Mutyh−/− mice. F) Well-differentiated Grade I SCC showing full thickness epidermal atypia and involvement of hair follicles. Keratinization-induced “pearl-like” structures (dermal nests of keratinocytes attempting to mature in a layered fashion containing keratinizing cells and horny pearls) are indicated by an arrow. G) Poorly-differentiated Grade III/IV SCC. H) A higher magnification of the tumor shown in G).

Mentions: To analyse the effects of 6-TG/UVA in vivo, wild-type and Mutyh-defective C57BL6 mice were divided in three groups: UVA alone (Group I, 8 animals/genotype), Aza alone (Group II, 15 and 16 wild-type and Mutyh−/− mice, respectively) and Aza/UVA (Group III, 15 animals/genotype). Group II mice were given 15 mg/kg Aza i.p. All irradiated animals received 150 kJ/m2 UVA on shaved dorsal skin. Group III mice were irradiated 1 h after each Aza injection (Figure 6A). All procedures were repeated three times/week for 12 months. Immunosuppression in Aza-treated animals was verified by mixed lymphocyte reaction after 4-weeks treatment (data not shown). As previously reported (32) repeated dosage with 15 mg/kg Aza was well-tolerated and caused only low-level toxicity in both Mutyh−/− and wild-type animals (Figure 6B and C). In contrast, in Groups III (Aza/UVA) we observed a significant difference in survival between Mutyh−/− and wild-type mice (p=0.019 by Log-rank test) (Figure 6B and D). At 12 months, 14/15 (>90%) Mutyh-defective animals were still alive compared to 7/15 (46.7%) wild-type mice. As expected (33), UVA alone did not cause any discernable skin damage (sunburn cells) in either genotype and 12-month survival was 100% in both Groups I (Figure 6B).


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)

Survival, skin DNA 8-oxoG levels and tumor onset in WT and mice treated with Aza/UVAA) Schematic representation of the treatment. B) Summary of survival data of WT and Mutyh−/− mice treated with Aza, UVA or the combined treatment as indicated in A). C) Kaplan–Meier curves of Aza and Aza/UVA treated WT (solid line) and Mutyh−/− (dotted line) mice. D) DNA 6-TG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza or Aza/UVA combined treatment. Data are mean ± SE of 11-19 animals/genotype. E) DNA 8-oxoG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza, UVA or combined treatment. Data are compared to historical controls (CTR) and they are mean ± SE of 16-25 animals/genotype. *P≤0.05 (Student's t-test). F - H) Representative histological images of SCCs from two Aza/UVA treated Mutyh−/− mice. F) Well-differentiated Grade I SCC showing full thickness epidermal atypia and involvement of hair follicles. Keratinization-induced “pearl-like” structures (dermal nests of keratinocytes attempting to mature in a layered fashion containing keratinizing cells and horny pearls) are indicated by an arrow. G) Poorly-differentiated Grade III/IV SCC. H) A higher magnification of the tumor shown in G).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Survival, skin DNA 8-oxoG levels and tumor onset in WT and mice treated with Aza/UVAA) Schematic representation of the treatment. B) Summary of survival data of WT and Mutyh−/− mice treated with Aza, UVA or the combined treatment as indicated in A). C) Kaplan–Meier curves of Aza and Aza/UVA treated WT (solid line) and Mutyh−/− (dotted line) mice. D) DNA 6-TG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza or Aza/UVA combined treatment. Data are mean ± SE of 11-19 animals/genotype. E) DNA 8-oxoG levels in the skin of WT (white bars) and Mutyh−/− (black bars) mice following Aza, UVA or combined treatment. Data are compared to historical controls (CTR) and they are mean ± SE of 16-25 animals/genotype. *P≤0.05 (Student's t-test). F - H) Representative histological images of SCCs from two Aza/UVA treated Mutyh−/− mice. F) Well-differentiated Grade I SCC showing full thickness epidermal atypia and involvement of hair follicles. Keratinization-induced “pearl-like” structures (dermal nests of keratinocytes attempting to mature in a layered fashion containing keratinizing cells and horny pearls) are indicated by an arrow. G) Poorly-differentiated Grade III/IV SCC. H) A higher magnification of the tumor shown in G).
Mentions: To analyse the effects of 6-TG/UVA in vivo, wild-type and Mutyh-defective C57BL6 mice were divided in three groups: UVA alone (Group I, 8 animals/genotype), Aza alone (Group II, 15 and 16 wild-type and Mutyh−/− mice, respectively) and Aza/UVA (Group III, 15 animals/genotype). Group II mice were given 15 mg/kg Aza i.p. All irradiated animals received 150 kJ/m2 UVA on shaved dorsal skin. Group III mice were irradiated 1 h after each Aza injection (Figure 6A). All procedures were repeated three times/week for 12 months. Immunosuppression in Aza-treated animals was verified by mixed lymphocyte reaction after 4-weeks treatment (data not shown). As previously reported (32) repeated dosage with 15 mg/kg Aza was well-tolerated and caused only low-level toxicity in both Mutyh−/− and wild-type animals (Figure 6B and C). In contrast, in Groups III (Aza/UVA) we observed a significant difference in survival between Mutyh−/− and wild-type mice (p=0.019 by Log-rank test) (Figure 6B and D). At 12 months, 14/15 (>90%) Mutyh-defective animals were still alive compared to 7/15 (46.7%) wild-type mice. As expected (33), UVA alone did not cause any discernable skin damage (sunburn cells) in either genotype and 12-month survival was 100% in both Groups I (Figure 6B).

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