Folate receptors (FR) which mediate cellular folate uptake in cervical carcinoma (HeLa-IU1) cells are inversely regulated by extracellular folate concentrations. However, there is a dearth of information on underlying mechanisms. The applicants have recently identified that post-transcriptional modulation of FR-alpha mRNA synthetic rates is the dominant mechanism underlying up- and down-regulation of FR in these cells. Furthermore, the interaction of an 18-base cis-element in the 5'-UTR of FR-alpha mRNA and a cytosolic 46-kDa trans-factor (46 kDa TF*) is critical for translation of FR mRNA in vitro because an 18-mer antisense DNA to the cis-element abolishes translation. A model to explain the inverse translational regulation of FR by folates is that the absence of folates promotes maximal interaction of the 18-base cis-element and 46 kDa TF*, and this leads to optimal translation of FR mRNA into FR and consequent up-regulation of FR. Conversely, in folate replete states, down-regulation of FR is accomplished by folate-mediated reduction in the affinity of interaction of the cis-element and trans-factor, and this in turn leads to a reduction in FR mRNA translation. The applicant proposes four inter-related hypothesis-driven specific aims to comprehensively test this model. The applicant will first isolate and characterize the 46 kDA TF* from HeLa-IU1 cells and determine the role of folates in modulating the interaction of the cis-element and 46-kDA-TF* within the context of translation of FR mRNA. Second, he will determine if transient or stable (long-term) expression of the 18-mer antisense DNA in HeLa-IU1 cells has effects on the synthesis of FR under conditions of limiting and excess extracellular folate, an whether this leads to megaloblastosis and premature cell death in vitro and in vivo. Third, he will test the hypothesis that down-regulation of FR can be experimentally induced in cervical carcinoma cells by stable adeno-associated virus 2 (AAV)-mediated transduction and over-expression of the antisense cDNA of the 46 kDA TF*; this will necessitate molecular cloning of the 46 kDa TF* cDNA. Finally, because there is also an interaction of the FR-alpha mRNA cis-element with nuclear trans-factors (nucTF) in HeLa-IU1 cells, the applicant will test the hypothesis that this protein is structurally and functionally distinct from the cytoplasmic 46 kDa TF* in these cells. Together, these studies will help to develop a comprehensive model for the translational regulation of FR. This knowledge will likely lead to new methods of enhancing cytotoxicity toward cervical carcinoma through the application of molecular medicine to exploit the FR metabolic pathway.
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