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Mouse Rad1 deletion enhances susceptibility for skin tumor development.

Han L, Hu Z, Liu Y, Wang X, Hopkins KM, Lieberman HB, Hang H - Mol. Cancer (2010)

Bottom Line: Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals.Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells.The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Road Datun, Beijing 100101, China.

ABSTRACT

Background: Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown.

Results: To address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells.

Conclusion: These data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

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Skin tumor induction by DMBA-TPA treatment. A, Papillomas induced by DMBA-TPA treatment in Mrad1+/+ mouse skin (left) and treated Mrad1+/- mouse skin (right). B, Incidence of papilloma-free mice after DMBA-TPA treatment. Kaplan-Meier plot of tumor-free state as a function of time after DMBA painting followed by TPA treatment (blue, Mrad1+/+; red, Mrad1+/-). Mrad1+/+ and Mrad1+/- mice (n = 38) were initially treated once with DMBA at week 1 on the skin topically starting at ages 7 to 8 weeks, and TPA twice weekly for 17 weeks. There was a significant difference in papilloma formation between Mrad1+/+ and Mrad1+/- mice (P = 0.003). C, Average numbers of papillomas on each mouse (blue, Mrad1+/+; red, Mrad1+/-). Only papillomas larger than 1 mm diameter were counted. There was a significant difference in the number of papillomas per mouse between two the genotypes at the 17-week end point (P = 0.010). D, Size distribution of papillomas. The length of a papilloma was used to represent its size. E, H & E staining for papillomas. A typical papilloma was shown, with connective tissues extending into the tumor. F, Keratin 14 staining for keratinocytes. The same tumor sample in E was also stained for Keratin 14, and it was thus shown to be derived from keratinocytes.
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Figure 3: Skin tumor induction by DMBA-TPA treatment. A, Papillomas induced by DMBA-TPA treatment in Mrad1+/+ mouse skin (left) and treated Mrad1+/- mouse skin (right). B, Incidence of papilloma-free mice after DMBA-TPA treatment. Kaplan-Meier plot of tumor-free state as a function of time after DMBA painting followed by TPA treatment (blue, Mrad1+/+; red, Mrad1+/-). Mrad1+/+ and Mrad1+/- mice (n = 38) were initially treated once with DMBA at week 1 on the skin topically starting at ages 7 to 8 weeks, and TPA twice weekly for 17 weeks. There was a significant difference in papilloma formation between Mrad1+/+ and Mrad1+/- mice (P = 0.003). C, Average numbers of papillomas on each mouse (blue, Mrad1+/+; red, Mrad1+/-). Only papillomas larger than 1 mm diameter were counted. There was a significant difference in the number of papillomas per mouse between two the genotypes at the 17-week end point (P = 0.010). D, Size distribution of papillomas. The length of a papilloma was used to represent its size. E, H & E staining for papillomas. A typical papilloma was shown, with connective tissues extending into the tumor. F, Keratin 14 staining for keratinocytes. The same tumor sample in E was also stained for Keratin 14, and it was thus shown to be derived from keratinocytes.

Mentions: To determine if Mrad1 is important for tumorigenesis, the skin of mice with Mrad1+/+ and Mrad1+/- genotypes was treated with DMBA plus TPA. A total of 38 mice were divided into 2 groups, each with the Mrad1+/+ or Mrad1+/- genotype. The 2 groups were from 19 litters, each litter consisting of 2 mice with Mrad1+/+ and Mrad1+/- genotypes, and identical sex, either female (14 liters) or male (5 liters). Under this setting, Log-Rank Test in the Kaplan-Meier PL method can be used for statistical analysis on the significance of differences of tumor development between two groups of animals [23]. DMBA was used to initiate skin tumorigenesis, and TPA was used to promote skin tumor growth. Mice were first painted with 15 μg DMBA in 100 μl acetone. One week after DMBA treatment, mice were painted with 2 μg TPA in 100 μl acetone twice a week (Monday and Thursday) for 17 weeks. Only skin tumors larger than 1 mm were recorded. After 7 weeks of TPA treatment, Mrad1+/- mice began to develop skin tumors in the treated area. It took 13 weeks of TPA treatment for the first Mrad1+/+ mouse to develop skin tumors (Fig. 3A and 3B). At the 17th week of TPA treatment, when the experiment ended, 14 Mrad1+/- mice had skin tumors while only 7 Mrad1+/+ mice, treated in the same fashion, developed skin tumors (Fig. 3B). Kaplan-Meier PL method [23] was used for comparison of the relative risk of tumor development induced by DMBA plus TPA between mice with different genotypes. The rate of tumor development in Mrad1+/- mice was significantly higher than in Mrad1+/+ mice (P = 0.003, Log-Rank Test). Additionally, the average number of tumors in each of Mrad1+/- mice that developed tumors was significantly higher (P = 0.010) than that in tumor-bearing Mrad1+/+ mice (Fig. 3C). We also measured the size of tumors on mouse skin after 17 weeks of TPA treatment. Tumors larger than 6 mm in diameter only appeared in Mrad1+/- mouse skin (Fig. 3D; data not shown). We made skew analysis on the tumor size distribution and found that G1 values were 0.40528 and 1.84488 for the wild type and Mrad1 heterozygous mice, respectively, suggesting that Mrad1 heterozygous mice bore significantly larger tumors. Staining skin specimens with H&E or anti-keratin 14 indicated that the tumors were derived from keratinocytes and possessed characteristics of papillomas (Fig. 3E and 3F). All these data suggest that Mrad1 plays an important role in the prevention of skin papilloma development.


Mouse Rad1 deletion enhances susceptibility for skin tumor development.

Han L, Hu Z, Liu Y, Wang X, Hopkins KM, Lieberman HB, Hang H - Mol. Cancer (2010)

Skin tumor induction by DMBA-TPA treatment. A, Papillomas induced by DMBA-TPA treatment in Mrad1+/+ mouse skin (left) and treated Mrad1+/- mouse skin (right). B, Incidence of papilloma-free mice after DMBA-TPA treatment. Kaplan-Meier plot of tumor-free state as a function of time after DMBA painting followed by TPA treatment (blue, Mrad1+/+; red, Mrad1+/-). Mrad1+/+ and Mrad1+/- mice (n = 38) were initially treated once with DMBA at week 1 on the skin topically starting at ages 7 to 8 weeks, and TPA twice weekly for 17 weeks. There was a significant difference in papilloma formation between Mrad1+/+ and Mrad1+/- mice (P = 0.003). C, Average numbers of papillomas on each mouse (blue, Mrad1+/+; red, Mrad1+/-). Only papillomas larger than 1 mm diameter were counted. There was a significant difference in the number of papillomas per mouse between two the genotypes at the 17-week end point (P = 0.010). D, Size distribution of papillomas. The length of a papilloma was used to represent its size. E, H & E staining for papillomas. A typical papilloma was shown, with connective tissues extending into the tumor. F, Keratin 14 staining for keratinocytes. The same tumor sample in E was also stained for Keratin 14, and it was thus shown to be derived from keratinocytes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Skin tumor induction by DMBA-TPA treatment. A, Papillomas induced by DMBA-TPA treatment in Mrad1+/+ mouse skin (left) and treated Mrad1+/- mouse skin (right). B, Incidence of papilloma-free mice after DMBA-TPA treatment. Kaplan-Meier plot of tumor-free state as a function of time after DMBA painting followed by TPA treatment (blue, Mrad1+/+; red, Mrad1+/-). Mrad1+/+ and Mrad1+/- mice (n = 38) were initially treated once with DMBA at week 1 on the skin topically starting at ages 7 to 8 weeks, and TPA twice weekly for 17 weeks. There was a significant difference in papilloma formation between Mrad1+/+ and Mrad1+/- mice (P = 0.003). C, Average numbers of papillomas on each mouse (blue, Mrad1+/+; red, Mrad1+/-). Only papillomas larger than 1 mm diameter were counted. There was a significant difference in the number of papillomas per mouse between two the genotypes at the 17-week end point (P = 0.010). D, Size distribution of papillomas. The length of a papilloma was used to represent its size. E, H & E staining for papillomas. A typical papilloma was shown, with connective tissues extending into the tumor. F, Keratin 14 staining for keratinocytes. The same tumor sample in E was also stained for Keratin 14, and it was thus shown to be derived from keratinocytes.
Mentions: To determine if Mrad1 is important for tumorigenesis, the skin of mice with Mrad1+/+ and Mrad1+/- genotypes was treated with DMBA plus TPA. A total of 38 mice were divided into 2 groups, each with the Mrad1+/+ or Mrad1+/- genotype. The 2 groups were from 19 litters, each litter consisting of 2 mice with Mrad1+/+ and Mrad1+/- genotypes, and identical sex, either female (14 liters) or male (5 liters). Under this setting, Log-Rank Test in the Kaplan-Meier PL method can be used for statistical analysis on the significance of differences of tumor development between two groups of animals [23]. DMBA was used to initiate skin tumorigenesis, and TPA was used to promote skin tumor growth. Mice were first painted with 15 μg DMBA in 100 μl acetone. One week after DMBA treatment, mice were painted with 2 μg TPA in 100 μl acetone twice a week (Monday and Thursday) for 17 weeks. Only skin tumors larger than 1 mm were recorded. After 7 weeks of TPA treatment, Mrad1+/- mice began to develop skin tumors in the treated area. It took 13 weeks of TPA treatment for the first Mrad1+/+ mouse to develop skin tumors (Fig. 3A and 3B). At the 17th week of TPA treatment, when the experiment ended, 14 Mrad1+/- mice had skin tumors while only 7 Mrad1+/+ mice, treated in the same fashion, developed skin tumors (Fig. 3B). Kaplan-Meier PL method [23] was used for comparison of the relative risk of tumor development induced by DMBA plus TPA between mice with different genotypes. The rate of tumor development in Mrad1+/- mice was significantly higher than in Mrad1+/+ mice (P = 0.003, Log-Rank Test). Additionally, the average number of tumors in each of Mrad1+/- mice that developed tumors was significantly higher (P = 0.010) than that in tumor-bearing Mrad1+/+ mice (Fig. 3C). We also measured the size of tumors on mouse skin after 17 weeks of TPA treatment. Tumors larger than 6 mm in diameter only appeared in Mrad1+/- mouse skin (Fig. 3D; data not shown). We made skew analysis on the tumor size distribution and found that G1 values were 0.40528 and 1.84488 for the wild type and Mrad1 heterozygous mice, respectively, suggesting that Mrad1 heterozygous mice bore significantly larger tumors. Staining skin specimens with H&E or anti-keratin 14 indicated that the tumors were derived from keratinocytes and possessed characteristics of papillomas (Fig. 3E and 3F). All these data suggest that Mrad1 plays an important role in the prevention of skin papilloma development.

Bottom Line: Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals.Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells.The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Road Datun, Beijing 100101, China.

ABSTRACT

Background: Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown.

Results: To address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells.

Conclusion: These data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

Show MeSH
Related in: MedlinePlus