J Vet Sci.  2010 Dec;11(4):291-297. 10.4142/jvs.2010.11.4.291.

Conditional knockout of brca1/2 and p53 in mouse ovarian surface epithelium: Do they play a role in ovarian carcinogenesis?

• Kim KY
• Park DW
• Jeung EB
• Choi KC

• KMID: 1072177
• DOI: http://doi.org/10.4142/jvs.2010.11.4.291

Abstract

Alterations of genes are known to be critical for the induction of tumorigenesis, but the mechanism of ovarian carcinogenesis is little understood and remains to be elucidated. In this study, we investigated the roles of brca1, brca2 and p53 genes in the development of ovarian cancer using conditional knockout mice generated by a Cre-loxP recombinant system. Following the application of recombinant adenovirus expressing Cre in vitro, the proliferation of ovarian surface epithelium (OSE) was increased. For instance, a significant increase in cell growth was observed in OSE cells in vitro by conditional knockout isolated from the mice bearing concurrent floxed copies of brca1 and brca2/p53. However, the proliferative effect of the ovarian cells was not observed in concurrent brca1/brca2 or p53 knockout mice in vivo, indicating that we could not observe the direct evidence of the involvement of brca1, brca2, and p53 in ovarian carcinogenesis. Since morphological changes including tumor formation were not observed in mice bearing floxed copies of concurrent brca1/brca2 or p53, the inactivation of brca1/2 or p53 is not sufficient for the induction of tumor formation. Taken together, these results suggest that the deficiency of these genes may not be involved directly in the mechanism of ovarian carcinogenesis.

Keyword

BRCA; p53; conditional knockout; ovarian cancer; tumor suppressor genes

MeSH Terms

Animals
BRCA1 Protein/*genetics/metabolism
BRCA2 Protein/*genetics/metabolism
Cell Proliferation
Cell Transformation, Neoplastic/*genetics
Epithelium/*pathology
Extracellular Matrix Proteins/genetics
Female
Gene Silencing
Mice
Mice, Knockout
Ovarian Neoplasms/*genetics
Protein-Lysine 6-Oxidase/genetics
Tumor Cells, Cultured
Tumor Suppressor Protein p53/*genetics/metabolism

Figure

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  Fig. 1 Genotyping of floxed mice for brca1 and brca2. (A) The predicted 461-bp (5' floxed) and 562-bp (3' floxed) PCR products of were observed in brca1loxP/loxP mice (Lanes 1-6). (B) The predicted 376-bp (5' floxed) and 529-bp (3' floxed) PCR products of brca2 were confirmed in brca2loxP/loxP mice (Lanes 1-6). (C) Confirmation of PCR products of homozygous p53loxP/loxP mice (Lanes 3 and 5). Lanes 6 and 7, heterozygous p53loxP/loxP mice. Lane M: Molecular weight marker, Lane W: wild-type.

• 

  Fig. 2 Evaluation of multiplicity of infection (MOI) by adeno-Cre virus (AdCMVCre) infection for deleting the genes in ovarian surface epithelium. (A) The expression of brca1 and brca2 following treatment with serial dilutions of AdCMVCre. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to confirm the same amount of loading. (B) Schematic explanation of Cre-loxP recombination.

• 

  Fig. 3 Effect of inactivation of brca1, brca2, and p53 on ovarian surface epithelium (OSE) cell proliferation. OSE cells from diverse genotypes were infected with AdCMVCre at MOI 200 for 1.5 h. Then, cell proliferation was measured by thymidine incorporation assay in the OSE cells with brca1loxP/loxP, brca1/2loxP/loxP, brca2/p53loxP/loxP and p53loxP/loxP after infection. Adeno-EGFP (AdCMVEGFP) was used as a control. Proliferation levels are expressed as a relative fold change. Values are the mean ± SD. *p < 0.05 vs. AdCMVEGFP infection.

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