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In vitro repression of Brca1-associated RING domain gene, Bard1, induces phenotypic changes in mammary epithelial
I Irminger-Finger1, J V Soriano, G Vaudan
1Laboratory of Biology of Aging and Department of Geriatrics, University of Geneva, Switzerland. iirminger@cmu.unige.ch
Abstract:
BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1-BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.
Insights
The breast cancer suppressor gene BRCA1 interacts with BARD1. Reducing BARD1 expression in mammary cells caused changes indicative of a premalignant phenotype, suggesting BARD1
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- BRCA1-associated RING domain (BARD1) interacts with BRCA1, a key breast cancer suppressor gene.
- Mutations in BRCA1 and BARD1 are linked to breast cancer, suggesting BARD1's role in tumor suppression.
- BARD1 gene expression in murine tissues mirrors that of Brca1.
Purpose of the Study:
- To investigate the functional role of BARD1 in mammary epithelial cells.
- To determine the consequences of reduced Bard1 gene expression on cell phenotype and behavior.
Main Methods:
- Cloning of the murine Bard1 gene.
- Partial repression of Bard1 gene expression in TAC-2 murine mammary epithelial cells using ribozymes and antisense RNAs.
- Analysis of phenotypic changes, cell cycle progression, and growth characteristics in vitro.
Main Results:
- Reduced Bard1 expression led to significant alterations in cell morphology, including changes in shape, increased size, and multinucleation.
- Bard1 repression resulted in aberrant cell cycle progression.
- Bard1-repressed cells exhibited loss of contact inhibition and impaired luminal structure formation in 3D cultures, indicative of a premalignant phenotype.
Conclusions:
- Bard1 plays a crucial role in maintaining normal mammary epithelial cell properties.
- The repression of Bard1 induces complex cellular changes strongly suggesting a role in BRCA1-mediated tumor suppression.
- These findings highlight BARD1 as a potential target in breast cancer research.