Neoplastic Consequences of a Mutator Phenotype in Human Breast Epithelial Cells: A Prospective Analysis
Abstract
We have established an ex vivo culture system using MCF-10A cells. The doubling time for this cell line in our laboratory is ^3-5 days. We have determined the cytotoxicity of three antibiotics towards MCF-10A cells. The cells were found to be more sensitive to antibiotics than other cell lines we have used experimentally. The effective concentrations for selection of MCF-10A cells are: zeocin, 300 micrograms/ml; puromycin, 0.5 micrograms/ml; and hygromycin, 40 micrograms/ml. We have standardized our method for transfection (lipofection) of the MCF-10A cells. Two expression vectors for DNA polymerase beta (polbeta) have been constructed: pCDNA4-based and pIRES-based. Stable zeocin-resistant MCF-10A clones were isolated following transfection of pCDNA-polbeta. While endogenous polbeta protein was detected by Western analyses, no exogenous protein was detectable in these clones. Stable selection using the pIRES-beta/pIRESpuro vector pair was only partially successful, as the MCF-10A, puromycin-resistant clones became senescent. However, Western analyses of polbeta-pIRES transfected MCF-10A cells demonstrated the exogenous expression of His-polbeta in the clones. These results demonstrate that our proposed experiments are feasible. However, the time frame needed to complete the project may be substantially longer than anticipated, due to the slow growth rate of the MCF-10A cell line and sensitivity to antibiotics.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2001
- Accession Number
- ADA396659
Entities
People
- Danny R. Welch
- Kristin A. Eckert
Organizations
- Penn State College of Medicine