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Functional characterization of genes involved in the development of breast cancer
Breast Cancer Research volume 7, Article number: P4.07 (2005)
Amplifications of large chromosomal regions have been detected by microarray-based comparative genomic hybridization in breast tumors and breast cancer cell lines. Many of the genes within these amplicons also show increased expression on microarrays, indicating that these genes are probably of importance in breast cancer development. A significant number of the genes showing both amplifications and increased expression are located in amplified regions of human chromosome 8q. Copy number abnormalities on chromosome 8q are further correlated with poor survival outcome and the presence of TP53 mutations. In our research we used cell-line-based functional assays to identify and characterize genes on human chromosome 8q to identify those that directly contribute to the evolution of breast tumors. Hierarchical clustering of tumor gene expression profiles has identified distinct subtypes of breast tumors. We have chosen specific breast cancer cell lines in our studies as models for two of these subtypes: basal-like and luminal breast tumors. In order to knock down gene expression in these cell lines, we constructed a self-inactivating murine stem cell virus (MSCV)-based shRNA expression vector. The vector, pSiRPG, contains a highly efficient selection marker (Puromycin) and an Enhanced Green Fluorescent Protein marker for the detection of cells harboring the constructs. We have demonstrated efficient expression of siRNAs in cell lines that are difficult to transfect using standard methods. We also constructed a Gateway-compatible expression vector, pRetroTRexD30, on the MSCV back bone. Full-length cDNAs from more than 30 chromosome 8q genes selected in our project have so far been transferred into this vector.
The siRNA constructs are currently being used to knock down gene expression in cell lines showing high endogenous expression of these 8q genes. In parallel experiments, the genes are overexpressed in cell lines showing low or undetectable endogenous expression. In these experiments, cell lines harboring overexpression or siRNA constructs are assayed for cancer-relevant phenotypes such as growth rate changes, the ability to grow in low serum concentrations and/or hypoxic conditions, sensitivity to chemotherapeutic drugs, apoptosis induction, and the ability to form colonies in agar.
This work is supported by the Research Council of Norway and the Norwegian Cancer Society, and by funds from the NCI Breast SPORE program to CMP (P50-CA58223-09A1).
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Gjernes, E., Størvold, G., Askautrud, H. et al. Functional characterization of genes involved in the development of breast cancer. Breast Cancer Res 7, P4.07 (2005) doi:10.1186/bcr1137
- Breast Cancer Cell Line
- Enhance Green Fluorescent Protein
- Poor Survival Outcome
- Specific Breast Cancer
- shRNA Expression Vector