Estradiol enhances liposome-mediated uptake, preferential nuclear accumulation and functional expression of exogenous genes in MDA-MB231 breast tumor cells.
Exposure of p53 mutated estrogen-receptor-negative MDA-MB231 human breast tumor cells to a pharmacological concentration of estradiol enhances liposome-mediated uptake and expression of SV-40 luciferase. Unexpectedly, the effect of estradiol on SV-40 expression is evident even when estradiol exposure occurs after the initial uptake phase; this suggests that estradiol may influence gene expression by mechanisms other than increasing gene uptake alone, such as altering the intracellular distribution of the gene. We determined that while uptake of SV-40 luciferase is increased only three-fold by estradiol, there is a 30-fold increase in the nuclear/cytoplasmic ratio of the gene. In order to demonstrate that the influence of estradiol on gene uptake and expression is translated into a functional response, the effects of estradiol on the function of an exogenous gene, in this case the apoptotic function of p53, were assessed in the p53 mutated MDA-MB231 breast tumor cell. While liposome-mediated delivery of CMV-p53 alone was ineffective in promoting cell death, incubation with estradiol and the liposomal p53 complex resulted in a two-fold increase in cell killing over that observed in cells transfected with the corresponding mock vector (empty vector for p53). Evidence that cell killing was occurring through apoptosis included apoptotic body formation, cell shrinkage and an increase in fluorescence after terminal transferase end-labeling. The capacity of estradiol to promote apoptosis in MDA-MB231 cells by a p53-liposome complex is likely to be related to the preferential redistribution of the gene from the cytoplasm to the nucleus which could occur during both the uptake and post-uptake phases. Consequently, although direct effects on gene expression, and the stability of message and protein cannot be ruled out, the predominant effect of estradiol in this experimental system appears to be to influence DNA translocation from the cytoplasm to the cell nucleus.
Jain
P T
PT
Department of Pharmacology, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980230, Richmond, VA 23298, USA.
Seth
P
P
Gewirtz
D A
DA
D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms; D002467:Cell Nucleus / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005814:Genes, Viral; D016158:Genes, p53; D020287:In Situ Nick-End Labeling; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D008858:Microscopy, Phase-Contrast; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D008081:Liposomes; D004958:Estradiol; D008156:Luciferases