Pulsatile exocytosis is functionally associated with GnRH gene expression in immortalized GnRH-expressing cells.

Pulsatile release of GnRH is essential for proper reproductive function, but little information is available on the molecular processes underlying this intermittent activity. Recently, GnRH gene expression (GnRH-GE) episodes and exocytotic pulses have been identified separately in individual GnRH-expressing cells, raising the exciting possibility that both activities are linked functionally and are fundamental to the pulsatile process. To explore this, we monitored GnRH-GE (using a GnRH promoter-driven luciferase reporter) and exocytosis (by FM1-43 fluorescence) in the same, living GT1-7 cells. Our results revealed a strong temporal association between exocytotic pulses and GnRH-GE episodes. To determine whether a functional link existed, we blocked one process and evaluated the other. Transcriptional inhibition with actinomycin D had only a modest influence on exocytosis, suggesting that exocytotic pulse activity was not dictated acutely by episodes of gene expression. In contrast, blockage of exocytosis with anti-SNAP-25 (which obstructs secretory granule fusion) abolished GnRH-GE pulse activity, indicating that part of the exocytotic process is responsible for triggering episodes of GnRH-GE. When taken together, our findings suggest that a careful balance is maintained between release and biosynthesis in GT1-7 cells. Such a property may be important in the hypothalamus to ensure that GnRH neurons are in a constant state of readiness to respond to changes in reproductive function.

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-17beta (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 cotreatment. 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.

Simultaneous indirect activity measurements of GH and PRL genes in the same, living mammosomatotrope.

Dynamic intracellular processes in endocrine cells are usually controlled by the coordinated modulation of two or more functionally related genes. Attempts to gain a more complete understanding of these processes would be facilitated greatly by a method enabling activity measurements of two genes at the same time. Here we describe how we developed such a system and used it to determine indirectly whether individual, living pituitary cells could concurrently express both the growth hormone (GH) and prolactin (PRL) genes. Our results demonstrate that coexpression of these genes is indeed possible. Moreover, our findings provide a general paradigm for future "real-time" analysis of other interrelated genes involved in the regulation of endocrine processes.