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Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair.

Larin M, Gallo D, Tamblyn L, Yang J, Liao H, Sabat N, Brown GW, McPherson JP - Nucleic Acids Res. (2014)

Bottom Line: Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair.This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity.Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada.

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Response of FkoMko cells to DNA damaging agents. (a-c) Sensitivity of immortalized fibroblasts to mitomycin-C (a), cisplatin (b) and Ara-C (c) by clonogenic assay. Data represent mean percentage of colony forming units compared to control (untreated FhetMhet) per dose assessed ±SD. For (a), ***P < 0.001 for FkoMko versus all genotypes, ±±P < 0.01 for FhetMko versus FhetMhet, +++P < 0.001 for FkoMhet or FkoMko versus FhetMko and FhetMhet. For (b), ***P < 0.001 for all genotypes versus each other, †††P < 0.001 for FkoMko versus all genotypes. For (c), ***P < 0.001 for FhetMhet versus all genotypes. All data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. (d, e) Percentage of primary fibroblasts with elevated γH2AX 0, 24 or 36 h after mitomycin-C exposure for 24 h (UT, untreated) with data grouped according to time point (d) or genotype (e). Data represent mean % cells ± SD with elevated γH2AX (n = 3) assessed by two-way ANOVA followed by Bonferroni posthoc test. For (d), ***P < 0.001 for FkoMko versus all genotypes or FkoMhet versus FhetMhet or FhetMko, **P < 0.01 for FkoMko versus all genotypes, +++P < 0.001 for FkoMhet versus FhetMhet and FhetMko, ††P < 0.01 for FkoMko versus FhetMhet or FhetMko. For (e), ***P < 0.001 for 36 h versus 24 h and 0 h but not UT, +++P < 0.005 for 36 h versus 24 h, 0 h and UT. (f) Replication fork rate in primary fibroblasts. Whiskers represent 1.5 × interquartile range. *P < 0.001 for FhetMhet versus FkoMhet or FkoMko, **P = 0.04 for FhetMhet versus FhetMko, ***P = 0.018 for FhetMko versus FkoMhet. Data were assessed using Mann–Whitney U-test for non-parametric data.
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Figure 6: Response of FkoMko cells to DNA damaging agents. (a-c) Sensitivity of immortalized fibroblasts to mitomycin-C (a), cisplatin (b) and Ara-C (c) by clonogenic assay. Data represent mean percentage of colony forming units compared to control (untreated FhetMhet) per dose assessed ±SD. For (a), ***P < 0.001 for FkoMko versus all genotypes, ±±P < 0.01 for FhetMko versus FhetMhet, +++P < 0.001 for FkoMhet or FkoMko versus FhetMko and FhetMhet. For (b), ***P < 0.001 for all genotypes versus each other, †††P < 0.001 for FkoMko versus all genotypes. For (c), ***P < 0.001 for FhetMhet versus all genotypes. All data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. (d, e) Percentage of primary fibroblasts with elevated γH2AX 0, 24 or 36 h after mitomycin-C exposure for 24 h (UT, untreated) with data grouped according to time point (d) or genotype (e). Data represent mean % cells ± SD with elevated γH2AX (n = 3) assessed by two-way ANOVA followed by Bonferroni posthoc test. For (d), ***P < 0.001 for FkoMko versus all genotypes or FkoMhet versus FhetMhet or FhetMko, **P < 0.01 for FkoMko versus all genotypes, +++P < 0.001 for FkoMhet versus FhetMhet and FhetMko, ††P < 0.01 for FkoMko versus FhetMhet or FhetMko. For (e), ***P < 0.001 for 36 h versus 24 h and 0 h but not UT, +++P < 0.005 for 36 h versus 24 h, 0 h and UT. (f) Replication fork rate in primary fibroblasts. Whiskers represent 1.5 × interquartile range. *P < 0.001 for FhetMhet versus FkoMhet or FkoMko, **P = 0.04 for FhetMhet versus FhetMko, ***P = 0.018 for FhetMko versus FkoMhet. Data were assessed using Mann–Whitney U-test for non-parametric data.

Mentions: To ascertain sensitivity to DNA crosslink damage, clonogenic assays with immortalized fibroblasts of each genotype were conducted in the presence of mitomycin-C or cisplatin. As expected, FhetMko and FkoMhet cells were hypersensitive to mitomycin-C compared to FhetMhet, with FkoMhet cells showing a greater sensitivity to this agent at the 50–100-nM doses (P < 0.001) than FhetMko cells (Figure 6A). FkoMko cells were remarkably more sensitive than cells from all other genotypes at 5–50-nM doses (P < 0.001). A similar ranking of sensitivity by genotype was observed when cells were exposed to cisplatin (FkoMko > FkoMhet > FhetMko > FhetMhet) with FkoMko cells more sensitive than cells from all other genotypes at all doses (P < 0.001; Figure 6B). To determine if this sensitivity ranking was specific for crosslink damage or also included agents that generate replication-fork associated DNA damage, cells of all genotypes were exposed to Ara-C. At all doses tested, FkoMko, FkoMhet and FhetMko cells were significantly more sensitive than FhetMhet cells (P < 0.001; Figure 6C). In contrast to results with crosslinking agents, FkoMko cells showed equivalent sensitivity to cells deficient in either Mus81 or FancC. Our findings indicate FA signaling and Mus81 operate in parallel pathways with respect to crosslink resistance, but in the same pathway with respect to replication fork-associated DNA damage.


Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair.

Larin M, Gallo D, Tamblyn L, Yang J, Liao H, Sabat N, Brown GW, McPherson JP - Nucleic Acids Res. (2014)

Response of FkoMko cells to DNA damaging agents. (a-c) Sensitivity of immortalized fibroblasts to mitomycin-C (a), cisplatin (b) and Ara-C (c) by clonogenic assay. Data represent mean percentage of colony forming units compared to control (untreated FhetMhet) per dose assessed ±SD. For (a), ***P < 0.001 for FkoMko versus all genotypes, ±±P < 0.01 for FhetMko versus FhetMhet, +++P < 0.001 for FkoMhet or FkoMko versus FhetMko and FhetMhet. For (b), ***P < 0.001 for all genotypes versus each other, †††P < 0.001 for FkoMko versus all genotypes. For (c), ***P < 0.001 for FhetMhet versus all genotypes. All data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. (d, e) Percentage of primary fibroblasts with elevated γH2AX 0, 24 or 36 h after mitomycin-C exposure for 24 h (UT, untreated) with data grouped according to time point (d) or genotype (e). Data represent mean % cells ± SD with elevated γH2AX (n = 3) assessed by two-way ANOVA followed by Bonferroni posthoc test. For (d), ***P < 0.001 for FkoMko versus all genotypes or FkoMhet versus FhetMhet or FhetMko, **P < 0.01 for FkoMko versus all genotypes, +++P < 0.001 for FkoMhet versus FhetMhet and FhetMko, ††P < 0.01 for FkoMko versus FhetMhet or FhetMko. For (e), ***P < 0.001 for 36 h versus 24 h and 0 h but not UT, +++P < 0.005 for 36 h versus 24 h, 0 h and UT. (f) Replication fork rate in primary fibroblasts. Whiskers represent 1.5 × interquartile range. *P < 0.001 for FhetMhet versus FkoMhet or FkoMko, **P = 0.04 for FhetMhet versus FhetMko, ***P = 0.018 for FhetMko versus FkoMhet. Data were assessed using Mann–Whitney U-test for non-parametric data.
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Figure 6: Response of FkoMko cells to DNA damaging agents. (a-c) Sensitivity of immortalized fibroblasts to mitomycin-C (a), cisplatin (b) and Ara-C (c) by clonogenic assay. Data represent mean percentage of colony forming units compared to control (untreated FhetMhet) per dose assessed ±SD. For (a), ***P < 0.001 for FkoMko versus all genotypes, ±±P < 0.01 for FhetMko versus FhetMhet, +++P < 0.001 for FkoMhet or FkoMko versus FhetMko and FhetMhet. For (b), ***P < 0.001 for all genotypes versus each other, †††P < 0.001 for FkoMko versus all genotypes. For (c), ***P < 0.001 for FhetMhet versus all genotypes. All data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. (d, e) Percentage of primary fibroblasts with elevated γH2AX 0, 24 or 36 h after mitomycin-C exposure for 24 h (UT, untreated) with data grouped according to time point (d) or genotype (e). Data represent mean % cells ± SD with elevated γH2AX (n = 3) assessed by two-way ANOVA followed by Bonferroni posthoc test. For (d), ***P < 0.001 for FkoMko versus all genotypes or FkoMhet versus FhetMhet or FhetMko, **P < 0.01 for FkoMko versus all genotypes, +++P < 0.001 for FkoMhet versus FhetMhet and FhetMko, ††P < 0.01 for FkoMko versus FhetMhet or FhetMko. For (e), ***P < 0.001 for 36 h versus 24 h and 0 h but not UT, +++P < 0.005 for 36 h versus 24 h, 0 h and UT. (f) Replication fork rate in primary fibroblasts. Whiskers represent 1.5 × interquartile range. *P < 0.001 for FhetMhet versus FkoMhet or FkoMko, **P = 0.04 for FhetMhet versus FhetMko, ***P = 0.018 for FhetMko versus FkoMhet. Data were assessed using Mann–Whitney U-test for non-parametric data.
Mentions: To ascertain sensitivity to DNA crosslink damage, clonogenic assays with immortalized fibroblasts of each genotype were conducted in the presence of mitomycin-C or cisplatin. As expected, FhetMko and FkoMhet cells were hypersensitive to mitomycin-C compared to FhetMhet, with FkoMhet cells showing a greater sensitivity to this agent at the 50–100-nM doses (P < 0.001) than FhetMko cells (Figure 6A). FkoMko cells were remarkably more sensitive than cells from all other genotypes at 5–50-nM doses (P < 0.001). A similar ranking of sensitivity by genotype was observed when cells were exposed to cisplatin (FkoMko > FkoMhet > FhetMko > FhetMhet) with FkoMko cells more sensitive than cells from all other genotypes at all doses (P < 0.001; Figure 6B). To determine if this sensitivity ranking was specific for crosslink damage or also included agents that generate replication-fork associated DNA damage, cells of all genotypes were exposed to Ara-C. At all doses tested, FkoMko, FkoMhet and FhetMko cells were significantly more sensitive than FhetMhet cells (P < 0.001; Figure 6C). In contrast to results with crosslinking agents, FkoMko cells showed equivalent sensitivity to cells deficient in either Mus81 or FancC. Our findings indicate FA signaling and Mus81 operate in parallel pathways with respect to crosslink resistance, but in the same pathway with respect to replication fork-associated DNA damage.

Bottom Line: Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair.This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity.Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada.

Show MeSH
Related in: MedlinePlus