Suppressor Genes in Breast Cancer
Abstract
Several tumor suppressor genes (TSGs) have been cloned and found to be mutated in a variety of cancers, including breast cancer. However, few breast cancer specific TSGs are known. The purposes of this proposal are to: (1) generate cDNA expression libraries from reduction mammoplasties, (2) use a novel functional assay to clone new TSGs specific to human breast cancer, and (3) identify their characteristics, regulation and function. We are utilizing the tetracycline (tet) regulable system. We have constructed a cDNA library from normal human breast epithelia and cloned this cDNA library into a vector that is negatively regulated by tat repressor (tetR) and simultaneously expresses the enhanced green fluorescent protein. These vectors were then co-transfected into LCC6, MDA231, and MCF-7 cells that have the capability to express tetR. Upon withdrawal of tet, the repressed expression of the cDNA of interest is released, and the cDNA is expressed. Using a novel dye retained in nonproliferating cells, we were able to identify growth inhibited clones which were then sorted by Flow Cytometry. This functional screen has provided the basis for identifying TSGs that are expressed in the growth inhibited cells. Using PCR, we have obtained and sequenced two insert sequences. One is a putative TSG on chromosome #9 (725 bp) but the other sequence is vector DNA.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2001
- Accession Number
- ADA405468
Entities
People
- Robert R. Clarke
Organizations
- Georgetown University