Molecular Determinants of Antiestrogen and Drug Sensitivity in Breast Carcinoma Cells.

摘要

We have found that genetic suppressor elements (OSEs) conferring drug resistance may function in combination rather than individually. To determine optimal conditions for the selection and analysis of OSE combinations, we have investigated parameters affecting the integration and gene expression from retroviral vector LNCX, used in the prior GSE studies. We have found that multicopy integration, which would allow combinatorial effects, is common even at intermediate infection rates, and that 0418 selection for the neo gene of LNCX may result in inactivation of the unselected gene. We have generated a series of retroviral vectors carrying green fluorescent protein (GFP) rather than neo as a selectable marker and identified vectors that provide high GFP expression in MCF7 breast carcinoma cells. These vectors will be tested for optimal expression of the unselected reporter gene in MCF7. The most efficient vector will be used to clone a normalized population of cDNA fragments, which we have generated from the mRNA of MCF7 cells. To optimize phenotypic analysis of individual GSEs, we have introduced the lacI repressor into a subline of MCF7 cells and demonstrated regulated expression of a reporter gene from a p- galactoside regulated retroviral vector. We have also continued the analysis of OSE-mediated inhibition of the BCL2 gene in MCF7 cells.