Limits...
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

Chromosomal damage after 6-TG/UVA treatmentA) Induction of MN by exposure to 6-TG/UVA. Number of binucleated cells with MN in WT (white circles) and Mutyh−/− (black circles) MEFs following 6-TG/UVA treatment (48h growth at various doses of 6-TG followed by UVA irradiation). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). °P<0.05; °°P<0.001 (χ² test: treated vs control). B) Nuclear division index relative to WT and Mutyh−/− MEFs cultivated in increasing 6-TG concentrations and UVA irradiated. C) Induction of NPBs by exposure to 6-TG/UVA. Number of binucleated cells with NPBs (light gray) and total number of NBPs (dark gray) in WT and Mutyh−/− cells MEFs exposed to 6-TG/UVA (experimental conditions as described in A) **P<0.001 (χ² test: WT vs Mutyh−/−). °P<0.001 (χ² test: treated vs control). D) Number of apoptotic binucleated cells in WT (white bars) and Mutyh−/− (black bars) MEFs following 6-TG/UVA (experimental conditions as described in C). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). E) A representative image of MN (indicated by arrows) in the cytoplasm of two binucleated cells. F) A representative image of an NPB connecting two nuclei in the same cell (arrowhead). In the same binucleated cell a MN is also visible (arrow). G) A representative image of an apoptotic binucleated cell containing five MN (arrows) and one NPB (arrowhead). All images are X 1000.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480694&req=5

Figure 5: Chromosomal damage after 6-TG/UVA treatmentA) Induction of MN by exposure to 6-TG/UVA. Number of binucleated cells with MN in WT (white circles) and Mutyh−/− (black circles) MEFs following 6-TG/UVA treatment (48h growth at various doses of 6-TG followed by UVA irradiation). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). °P<0.05; °°P<0.001 (χ² test: treated vs control). B) Nuclear division index relative to WT and Mutyh−/− MEFs cultivated in increasing 6-TG concentrations and UVA irradiated. C) Induction of NPBs by exposure to 6-TG/UVA. Number of binucleated cells with NPBs (light gray) and total number of NBPs (dark gray) in WT and Mutyh−/− cells MEFs exposed to 6-TG/UVA (experimental conditions as described in A) **P<0.001 (χ² test: WT vs Mutyh−/−). °P<0.001 (χ² test: treated vs control). D) Number of apoptotic binucleated cells in WT (white bars) and Mutyh−/− (black bars) MEFs following 6-TG/UVA (experimental conditions as described in C). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). E) A representative image of MN (indicated by arrows) in the cytoplasm of two binucleated cells. F) A representative image of an NPB connecting two nuclei in the same cell (arrowhead). In the same binucleated cell a MN is also visible (arrow). G) A representative image of an apoptotic binucleated cell containing five MN (arrows) and one NPB (arrowhead). All images are X 1000.

Mentions: Aza induces chromosomal aberrations [30]. To examine whether MUTYH influences 6-TG/UVA-induced chromosomal damage, we compared micronucleus (MN) formation in 6-TG/UVA treated wild-type and Mutyh−/− MEFs (Figure 5A). 6-TG/UVA treatment increased MN frequency in both cells. The effect was, however, much more pronounced in wild-type compared to Mutyh−/− cells. MN frequencies were significantly increased at 15 nM and maximal at 30nM 6-TG. In Mutyh−/− cells, the increase in MN frequency was only significant at the higher 6-TG doses (30 and 60 nM) (Figure 5A). Nuclear division indexes confirmed that MN were scored in cell populations showing similar proliferation rates (Figure 5B).


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)

Chromosomal damage after 6-TG/UVA treatmentA) Induction of MN by exposure to 6-TG/UVA. Number of binucleated cells with MN in WT (white circles) and Mutyh−/− (black circles) MEFs following 6-TG/UVA treatment (48h growth at various doses of 6-TG followed by UVA irradiation). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). °P<0.05; °°P<0.001 (χ² test: treated vs control). B) Nuclear division index relative to WT and Mutyh−/− MEFs cultivated in increasing 6-TG concentrations and UVA irradiated. C) Induction of NPBs by exposure to 6-TG/UVA. Number of binucleated cells with NPBs (light gray) and total number of NBPs (dark gray) in WT and Mutyh−/− cells MEFs exposed to 6-TG/UVA (experimental conditions as described in A) **P<0.001 (χ² test: WT vs Mutyh−/−). °P<0.001 (χ² test: treated vs control). D) Number of apoptotic binucleated cells in WT (white bars) and Mutyh−/− (black bars) MEFs following 6-TG/UVA (experimental conditions as described in C). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). E) A representative image of MN (indicated by arrows) in the cytoplasm of two binucleated cells. F) A representative image of an NPB connecting two nuclei in the same cell (arrowhead). In the same binucleated cell a MN is also visible (arrow). G) A representative image of an apoptotic binucleated cell containing five MN (arrows) and one NPB (arrowhead). All images are X 1000.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Chromosomal damage after 6-TG/UVA treatmentA) Induction of MN by exposure to 6-TG/UVA. Number of binucleated cells with MN in WT (white circles) and Mutyh−/− (black circles) MEFs following 6-TG/UVA treatment (48h growth at various doses of 6-TG followed by UVA irradiation). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). °P<0.05; °°P<0.001 (χ² test: treated vs control). B) Nuclear division index relative to WT and Mutyh−/− MEFs cultivated in increasing 6-TG concentrations and UVA irradiated. C) Induction of NPBs by exposure to 6-TG/UVA. Number of binucleated cells with NPBs (light gray) and total number of NBPs (dark gray) in WT and Mutyh−/− cells MEFs exposed to 6-TG/UVA (experimental conditions as described in A) **P<0.001 (χ² test: WT vs Mutyh−/−). °P<0.001 (χ² test: treated vs control). D) Number of apoptotic binucleated cells in WT (white bars) and Mutyh−/− (black bars) MEFs following 6-TG/UVA (experimental conditions as described in C). *P<0.05, **P≤0.001 (χ² test: WT vs Mutyh−/−). E) A representative image of MN (indicated by arrows) in the cytoplasm of two binucleated cells. F) A representative image of an NPB connecting two nuclei in the same cell (arrowhead). In the same binucleated cell a MN is also visible (arrow). G) A representative image of an apoptotic binucleated cell containing five MN (arrows) and one NPB (arrowhead). All images are X 1000.
Mentions: Aza induces chromosomal aberrations [30]. To examine whether MUTYH influences 6-TG/UVA-induced chromosomal damage, we compared micronucleus (MN) formation in 6-TG/UVA treated wild-type and Mutyh−/− MEFs (Figure 5A). 6-TG/UVA treatment increased MN frequency in both cells. The effect was, however, much more pronounced in wild-type compared to Mutyh−/− cells. MN frequencies were significantly increased at 15 nM and maximal at 30nM 6-TG. In Mutyh−/− cells, the increase in MN frequency was only significant at the higher 6-TG doses (30 and 60 nM) (Figure 5A). Nuclear division indexes confirmed that MN were scored in cell populations showing similar proliferation rates (Figure 5B).

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