Nasopharyngeal carcinoma (KB) cells contain undetectable amounts of reduced-folate transporters (RFT), but constitutively overexpress high affinity folate receptors (FR) which primarily mediate the entry of folates/antifolates. While FR expression is up- and down-regulated in response to low and high extracellular folate concentrations (EFC), respectively, over-activity of a membrane-associated FR-specific Mn2+dependent metalloprotease (FR-specific MP*) can also lead to reduction in FR expression. Thus, it is of significant importance to quantitate the kinetics of basal synthesis and degradation of FR and the mechanism(s) involved in steady-state alterations in these parameters following up- and down-regulation of FR in response to regulatory influences (EFC and FR- specific MP*). Identification of the influence of these variables which influence FR expression can provide a better rationale for scheduling antifolate therapy in human head and neck malignancies which are responsive to antifolates. To define kinetics of basal synthesis and degradation of FR, we will quantitate the rate of incorporation of heavy isotope labeled amino acids into newly-synthesized FR which can be separated from """"""""light"""""""" (""""""""old"""""""") FR by isopycnic density sedimentation on cesium chloride gradients; the rate of loss of old FR is a measure of rates of degradation. Then we will define the mechanism(s) accounting for steady-state switches in up-and down- regulation of FR (i) under low versus high EFC, respectively, and (ii) in KB cells containing transduced sense/antisense FR cDNA to analyze changes in kinetic parameters independent of the EFC. During these studies, we will also evaluate the influence of these variables on FR-specific MP* activity. To further analyze the independent role of FR-specific MP* on FR, we propose to isolate and characterize KB cell FR-specific MP* with a view to molecular cloning of its cDNA. Then following transduction of sense/antisense FR-specific MP* cDNA into KB cells, we can define the influence of primary over-and under expression of FR-specific MP* on FR expression, and the possibility that this will lead to short- and long-term adaptive changes in rates of steady-state synthesis and degradation of FR. These studies can therefore add significant new knowledge on the role of regulatory influences like the EFC and FR-specific MP* on FR regulation which will translate into better knowledge on the role of FR in antifolate sensitivity and resistance in cultured KB cells which are prototypical for human head and neck cancer.
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