BIOLOGICAL CHEMISTRY OF FOLATE RECEPTORS

Project: Research project

Description

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.
StatusFinished
Effective start/end date5/16/944/30/04

Funding

  • National Institutes of Health: $201,263.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $204,734.00
  • National Institutes of Health: $201,150.00
  • National Institutes of Health: $160,875.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $201,150.00

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Folic Acid
HeLa Cells
Therapeutics
Ganciclovir
Complementary DNA
Folate Receptor 1
Up-Regulation
Cisplatin
Messenger RNA
KB Cells
Thymidine Kinase
Carcinoma
Neoplasms

ASJC

  • Medicine(all)