The broad objective of this proposal is to continue to investigate cellular, molecular, and electrophysiologic mechanisms by which retinoic acid (RA) induces the differentiation of human neuroblastoma cells. Based upon results obtained during the previous funding period, the present proposal emphasizes an understanding of synergistic interactions between RA and interferon-gamma (IFN-gamma) as a means of ultimately developing an optimal protocol of """"""""differentiation therapy"""""""" for this disease. One hypothesis is that IFN-gamma impacts on the RA differentiation program and that this interaction leads to enhanced retinoid activity. The first specific aim to address this hypothesis is to define which steps in the RA pathway are modified by IFN-gamma treatment. This goal will be accomplished by assessing IFN-gamma effects on 1) RA uptake and metabolism; 2) cytosolic RA binding protein content/affinity; and 3) RA nuclear receptor (RAR) expression. These experiments will define the conditions and level(s) of regulation by which IFN-gamma can influence RA-induced differentiation. Due to its clinical significance, special emphasis is given to understanding the interaction of IFN-gamma and RA in the regulation of N-myc expression as an endpoint for evaluating the efficacy of differentiation treatment. To this end, a series of experiments will precisely determine the level of regulation (transcriptional, post-tran- scriptional) by which these agents decrease N-myc expression; then unidirectional deletion mutants of N-myc promoter-CAT constructs will be utilized in transient transfection experiments to identify and compare RA and IFN-gamma response elements in the N-myc promoter. In addition to gaining new insight into interactions between RA and IFN-gamma, these investigations should increase our general understanding of N-myc promoter function and gene regulation. The last part of this proposal continues our studies into the direct action of RA on transmembrane Ca2+ fluxes and Ca2+ channels as a potential membrane mechanism which may combine with the classical genomic (RAR-dependent) pathway in mediating retinoid activity. These investigations will employ a new generation of long-wavelength Ca2+- sensitive probes (fluo-3) and the recently described """"""""perforated-patch"""""""" variation of the whole-cell patch clamp technique to delineate RA effects on voltage-gated Ca2+ channels in human neuroblastoma cells. By showing a relationship between channel-function and biological responses, this work will shed new light on the relevance of certain types of ion channels during neuronal development.
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