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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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Expression of cell cycle checkpoint genes. A. Expression of p21 and p53 in Mrad1+/+ and Mrad1+/- keratinocytes. Western blotting analysis of p21 and p53 protein levels in keratinocytes incubated for four days after isolation. The first two lanes are the protein levels in cells without DMBA treatment, and the last two lanes are proteins from cells treated with 0.15 μg/ml DMBA for 24 hours. The data in the figure represent results from three independent western blotting experiments. +/+ and +/- indicate Mrad1+/+ and Mrad1+/- genotypes, respectively. B. Mrad9 and Mhus1 expression levels in Mrad1+/+ and Mrad1+/- keratinocytes. The gene expression levels were analyzed with real-time quantitative RT-PCR. Each result is the average ratio of the PCR results of an indicated gene relative to β-actin level for three independent samples, and each PCR result is the mean of triplicate PCR of the same sample. The difference of the Mrad1 expression levels between Mrad1+/- and Mrad1+/- cells is statistically significant (n = 3, P = 0.022). Either Mrad9 or Mhus1 expression levels were similar in both Mrad1+/+ and Mrad1+/- keratinocytes. C. Mrad1 expression levels in Mrad1+/+ and Mrad1+/- tumors. The expression levels of Mrad1 were analyzed by PCR as in B in this figure legend, and no statistical significance (n = 3, P = 0.34).
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Figure 5: Expression of cell cycle checkpoint genes. A. Expression of p21 and p53 in Mrad1+/+ and Mrad1+/- keratinocytes. Western blotting analysis of p21 and p53 protein levels in keratinocytes incubated for four days after isolation. The first two lanes are the protein levels in cells without DMBA treatment, and the last two lanes are proteins from cells treated with 0.15 μg/ml DMBA for 24 hours. The data in the figure represent results from three independent western blotting experiments. +/+ and +/- indicate Mrad1+/+ and Mrad1+/- genotypes, respectively. B. Mrad9 and Mhus1 expression levels in Mrad1+/+ and Mrad1+/- keratinocytes. The gene expression levels were analyzed with real-time quantitative RT-PCR. Each result is the average ratio of the PCR results of an indicated gene relative to β-actin level for three independent samples, and each PCR result is the mean of triplicate PCR of the same sample. The difference of the Mrad1 expression levels between Mrad1+/- and Mrad1+/- cells is statistically significant (n = 3, P = 0.022). Either Mrad9 or Mhus1 expression levels were similar in both Mrad1+/+ and Mrad1+/- keratinocytes. C. Mrad1 expression levels in Mrad1+/+ and Mrad1+/- tumors. The expression levels of Mrad1 were analyzed by PCR as in B in this figure legend, and no statistical significance (n = 3, P = 0.34).

Mentions: In a previous study, we reported that deletion of either one or two alleles of Mrad9 induced expression of cell cycle checkpoint genes and dramatically reduced cell proliferation of keratinocytes in culture. The results herein show that Mrad1+/- keratinocytes have only a slightly lower proliferation rate than the Mrad1+/+ controlcells (Fig. 4A). We therefore examined whether expression levels of p53 and p21 in Mrad1+/- keratinocytes are higher than in the related Mrad1+/+ cells. Indeed, heterozygous deletion of Mrad1 did not by itself alter expression of p21or p53 in skin keratinocytes in culture (Fig. 5A). These results can explain why Mrad1+/- keratinocytes do not have significantly higher levels of apoptosis and only a slightly lower proliferation rate than those of the wild type keratinocyte control. We further investigated p21 and p53 expression levels in keratinocytes treated with DMBA (0.15 μg/ml, 24 h) and found that the treatment induced expression most dramatically of p53 but also somewhat p21 in both Mrad1+/+ and Mrad1+/- keratinocytes (Fig. 5A). The expression levels of Mrad9 and Mhus1 in keratinocytes withMrad1+/+ and Mrad1+/- genotypes were also examined by real-time PCR. The results show that Mrad9 and Mhus1 expression levels are not altered by one allele Mrad1 deletion (Fig. 5B), suggesting that there is no regulatory effect of Mrad1 on either Mrad9 or Mhus1 expression in mouse keratinocytes. Interestingly, Mrad1 expression levels in Mrad1+/- mouse skin tumors were higher than those in Mrad1+/+mouse skin tumors, but not statistically significantly (Fig. 5C).


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)

Expression of cell cycle checkpoint genes. A. Expression of p21 and p53 in Mrad1+/+ and Mrad1+/- keratinocytes. Western blotting analysis of p21 and p53 protein levels in keratinocytes incubated for four days after isolation. The first two lanes are the protein levels in cells without DMBA treatment, and the last two lanes are proteins from cells treated with 0.15 μg/ml DMBA for 24 hours. The data in the figure represent results from three independent western blotting experiments. +/+ and +/- indicate Mrad1+/+ and Mrad1+/- genotypes, respectively. B. Mrad9 and Mhus1 expression levels in Mrad1+/+ and Mrad1+/- keratinocytes. The gene expression levels were analyzed with real-time quantitative RT-PCR. Each result is the average ratio of the PCR results of an indicated gene relative to β-actin level for three independent samples, and each PCR result is the mean of triplicate PCR of the same sample. The difference of the Mrad1 expression levels between Mrad1+/- and Mrad1+/- cells is statistically significant (n = 3, P = 0.022). Either Mrad9 or Mhus1 expression levels were similar in both Mrad1+/+ and Mrad1+/- keratinocytes. C. Mrad1 expression levels in Mrad1+/+ and Mrad1+/- tumors. The expression levels of Mrad1 were analyzed by PCR as in B in this figure legend, and no statistical significance (n = 3, P = 0.34).
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Figure 5: Expression of cell cycle checkpoint genes. A. Expression of p21 and p53 in Mrad1+/+ and Mrad1+/- keratinocytes. Western blotting analysis of p21 and p53 protein levels in keratinocytes incubated for four days after isolation. The first two lanes are the protein levels in cells without DMBA treatment, and the last two lanes are proteins from cells treated with 0.15 μg/ml DMBA for 24 hours. The data in the figure represent results from three independent western blotting experiments. +/+ and +/- indicate Mrad1+/+ and Mrad1+/- genotypes, respectively. B. Mrad9 and Mhus1 expression levels in Mrad1+/+ and Mrad1+/- keratinocytes. The gene expression levels were analyzed with real-time quantitative RT-PCR. Each result is the average ratio of the PCR results of an indicated gene relative to β-actin level for three independent samples, and each PCR result is the mean of triplicate PCR of the same sample. The difference of the Mrad1 expression levels between Mrad1+/- and Mrad1+/- cells is statistically significant (n = 3, P = 0.022). Either Mrad9 or Mhus1 expression levels were similar in both Mrad1+/+ and Mrad1+/- keratinocytes. C. Mrad1 expression levels in Mrad1+/+ and Mrad1+/- tumors. The expression levels of Mrad1 were analyzed by PCR as in B in this figure legend, and no statistical significance (n = 3, P = 0.34).
Mentions: In a previous study, we reported that deletion of either one or two alleles of Mrad9 induced expression of cell cycle checkpoint genes and dramatically reduced cell proliferation of keratinocytes in culture. The results herein show that Mrad1+/- keratinocytes have only a slightly lower proliferation rate than the Mrad1+/+ controlcells (Fig. 4A). We therefore examined whether expression levels of p53 and p21 in Mrad1+/- keratinocytes are higher than in the related Mrad1+/+ cells. Indeed, heterozygous deletion of Mrad1 did not by itself alter expression of p21or p53 in skin keratinocytes in culture (Fig. 5A). These results can explain why Mrad1+/- keratinocytes do not have significantly higher levels of apoptosis and only a slightly lower proliferation rate than those of the wild type keratinocyte control. We further investigated p21 and p53 expression levels in keratinocytes treated with DMBA (0.15 μg/ml, 24 h) and found that the treatment induced expression most dramatically of p53 but also somewhat p21 in both Mrad1+/+ and Mrad1+/- keratinocytes (Fig. 5A). The expression levels of Mrad9 and Mhus1 in keratinocytes withMrad1+/+ and Mrad1+/- genotypes were also examined by real-time PCR. The results show that Mrad9 and Mhus1 expression levels are not altered by one allele Mrad1 deletion (Fig. 5B), suggesting that there is no regulatory effect of Mrad1 on either Mrad9 or Mhus1 expression in mouse keratinocytes. Interestingly, Mrad1 expression levels in Mrad1+/- mouse skin tumors were higher than those in Mrad1+/+mouse skin tumors, but not statistically significantly (Fig. 5C).

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