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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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Targeted deletion of Mrad1. A, Top panel: Mrad1 genomic DNA; Bottom panel: targeting construct. White boxes: exons; Gray box: promotorless neo gene; black thin lines: introns; Black thick lines: DNA sequences outside of Mrad1 genomic DNA; Numbers above: lengths of introns in bp; Numbers below: lengths of exons in bp; White arrows: restriction enzyme cutting sites (targeting construct) and locations around the DNA sequence to be removed (genomic DNA); B, Southern blot analysis of the Mrad1 gene in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. C, PCR to assess genotypes in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. D, Southern blot analysis of the Mrad1 gene in mice. E, Mrad1 genotyping in mice using PCR.
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Figure 1: Targeted deletion of Mrad1. A, Top panel: Mrad1 genomic DNA; Bottom panel: targeting construct. White boxes: exons; Gray box: promotorless neo gene; black thin lines: introns; Black thick lines: DNA sequences outside of Mrad1 genomic DNA; Numbers above: lengths of introns in bp; Numbers below: lengths of exons in bp; White arrows: restriction enzyme cutting sites (targeting construct) and locations around the DNA sequence to be removed (genomic DNA); B, Southern blot analysis of the Mrad1 gene in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. C, PCR to assess genotypes in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. D, Southern blot analysis of the Mrad1 gene in mice. E, Mrad1 genotyping in mice using PCR.

Mentions: To obtain Mrad1-disrupted ES cells, we used a promoterless gene targeting strategy to delete the gene [20]. Mrad1 was disrupted by homologous recombination in ES cells using the targeting vector illustrated in Fig. 1A. This targeting construct contains a selectable neo gene. This gene, which lacked the start codon (ATG), was inserted between the third exon and third intron of Mrad1, deleting parts of the third exon and third intron. Homologous recombination of the targeting construct into mouse genomic Mrad1 was predicted to generate mutant cells that express a fusion protein containing the 77AA of the Mrad1 N-terminus and a full length neo protein, but lacking the rest of the Mrad1 protein (part of the third exon and the complete 4, 5 and 6 exons). We then targeted a 129Sv/Ev ES cell line obtained from Dr. Victor Lin's laboratory (Columbia University) and Mrad1+/- ES cell clones were obtained after transfection and challenge with 300 μg/ml G418. Multiple heterozygous Mrad1-deleted clones were identified by Southern blot analysis (Fig. 1B). A few were selected for PCR genotyping and confirmed to be Mrad1 heterozygous (Fig. 1C).


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)

Targeted deletion of Mrad1. A, Top panel: Mrad1 genomic DNA; Bottom panel: targeting construct. White boxes: exons; Gray box: promotorless neo gene; black thin lines: introns; Black thick lines: DNA sequences outside of Mrad1 genomic DNA; Numbers above: lengths of introns in bp; Numbers below: lengths of exons in bp; White arrows: restriction enzyme cutting sites (targeting construct) and locations around the DNA sequence to be removed (genomic DNA); B, Southern blot analysis of the Mrad1 gene in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. C, PCR to assess genotypes in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. D, Southern blot analysis of the Mrad1 gene in mice. E, Mrad1 genotyping in mice using PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864214&req=5

Figure 1: Targeted deletion of Mrad1. A, Top panel: Mrad1 genomic DNA; Bottom panel: targeting construct. White boxes: exons; Gray box: promotorless neo gene; black thin lines: introns; Black thick lines: DNA sequences outside of Mrad1 genomic DNA; Numbers above: lengths of introns in bp; Numbers below: lengths of exons in bp; White arrows: restriction enzyme cutting sites (targeting construct) and locations around the DNA sequence to be removed (genomic DNA); B, Southern blot analysis of the Mrad1 gene in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. C, PCR to assess genotypes in mouse ES cells. Bands indicate wild-type and deleted Mrad1 allele. D, Southern blot analysis of the Mrad1 gene in mice. E, Mrad1 genotyping in mice using PCR.
Mentions: To obtain Mrad1-disrupted ES cells, we used a promoterless gene targeting strategy to delete the gene [20]. Mrad1 was disrupted by homologous recombination in ES cells using the targeting vector illustrated in Fig. 1A. This targeting construct contains a selectable neo gene. This gene, which lacked the start codon (ATG), was inserted between the third exon and third intron of Mrad1, deleting parts of the third exon and third intron. Homologous recombination of the targeting construct into mouse genomic Mrad1 was predicted to generate mutant cells that express a fusion protein containing the 77AA of the Mrad1 N-terminus and a full length neo protein, but lacking the rest of the Mrad1 protein (part of the third exon and the complete 4, 5 and 6 exons). We then targeted a 129Sv/Ev ES cell line obtained from Dr. Victor Lin's laboratory (Columbia University) and Mrad1+/- ES cell clones were obtained after transfection and challenge with 300 μg/ml G418. Multiple heterozygous Mrad1-deleted clones were identified by Southern blot analysis (Fig. 1B). A few were selected for PCR genotyping and confirmed to be Mrad1 heterozygous (Fig. 1C).

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