William J. Faught

Multiple Measurements of Gene Expression in Single, Living Cells Enable Molecular Analysis of Endocrine Cell Heterogeneitya

The last decade has witnessed an unprecedented emergence of new techniques in the field of molecular biology accompanied by the parallel development of more sophisticated and sensitive analytical tools. This combination has allowed investigators to answer questions at levels heretofore unattainable due to technical limitations. Taking advantage of such progress, our group has devised a strategy that enables the dynamic monitoring and quantification of hormonal gene expression in single, living pituitary cells.1,2 This has been feasible through the combined use of luciferase reporter gene methodologies and ultrasensitive low-light detector devices. Owing to its configuration, our approach can be applied concurrently with other “single-cell” techniques. This provides a more complete understanding of the relationship between gene expression and other parameters involved in the process of hormone secretion within individual endocrine cells.3,4 Here, we review the most relevant aspects of this technique as well as some of the results obtained by its application to the study of prolactin (PRL) gene expression in rat lactotropes.

4-Hydroxytamoxifen differentially exerts estrogenic and antiestrogenic effects on discrete subpopulations of human breast cancer cells.

Functional heterogeneity within populations of breast cancer cells contribute to the seemingly paradoxical effects of antiestrogens and the development of antiestrogen “resistance.” Our objectives were to determine the degree to which T-47D cells may respond inappropriately (positively) to the antiestrogen 4-hydroxytamoxifen (HOT) alone, and whether all cells that respond to the stimulatory effects of estradiol-17β (E2) are inhibited by the addition of HOT. Single, living T-47D cells were transfected by microinjection with an estrogen response element (ERE)-driven luciferase reporter plasmid. Transfected cells were then treated with medium alone, HOT, E2 or a combination thereof on consecutive days, exposed to the substrate luciferin and subjected to quantification of photonic emissions reflective of ERE-stimulated activity. This analysis revealed a subpopulation of cells that exhibited increased ERE-driven photonic activity in response to HOT. In companion studies, E2-stimulated ERE activity was reversed (on average) with HOT addition. However, analysis of individual cells revealed that although HOT reduced photonic activity in the majority (89.2%) of E2-responsive cells, there was a small subset (10.8% of the population) that was stimulated by E2 + HOT co-treatment. Our data support the hypothesis that these cells possess an intrinsic “resistance” to antiestrogenic agents, and that this could contribute to the remodeling of tumor cell populations toward a “resistant’ phenotype.