Our long-term goal is to target folate receptor (FR)-expressing cervical carcinoma cells for gene therapy to further up-regulate FR expression. This will assist tumor growth Control in 3 ways: first, because of an inverse relationship between FR overexpression and cell proliferation in vivo, tumor doubling will be dramatically delayed from 4.5 to 9 days; second, transduced cells will be more receptive to the uptake of cisplatin that is encapsulated in folate-tethered liposomes (F ) which enter cells via FR, thereby predicting a lower IC50 with less systemic toxicity; and third, the increase in thymidine kinase (TK) activity which accompanies FR overexpression will render these cells hypersensitive to otherwise nontoxic concentrations of azidothymidine (or ganciclovir). While pursuing this goal, we propose to continue our basic studies to elucidate the (potential clinical) significance of the interaction of the 18-base cis-element in the 5'-UTR of FR mRNA and 46-kDa-trans-factor in the translation of FR in cells. So we will test the hypothesis that this cis-element and trans-factor interaction is a critical determinant for the synthesis of FR in cultured HeLa-IU1 cells by introducing antisera to the purified trans-factor and antisense oligonucleotides to the cis-element into cells via F and determining if quenching FR (under conditions when FR expression is essential for the viability of the cell) will trigger a folate-deficient cell death. We will also test the hypothesis that transduction of the sense 46-kDa-trans-factor cDNA (or higher-affinity mutant) can up-regulate FR in HeLa-IU1 cells, decrease cell proliferation, and increase susceptibility to TK-induced cytotoxicity by ganciclovir. This could identify new leads to modulate FR expression by means other than through direct transduction of FR genes. During these studies, we will also initiate a program to systematically test a series of hypotheses to confirm the superiority of F to deliver either cisplatin or retroviral constructs to cervical carcinoma cells, and to verify that ganciclovir augments the cytotoxicity of transduced Hela-IU1 cells that overexpress FR in vitro and in vivo in tumor-bearing mice. This will place us in a position to eventually test the hypothesis that amplification of FR on HeLa-IU1 cell-derived subcutaneous flank tumors by gene therapy with F containing retroviral constructs bearing HeLa-specific promoter-driven sense FR cDNA will (a) reduce tumor doubling; (b) render cells more receptive to subsequent uptake of F +cisplatin resulting in greater cytotoxicity. And because of the parallel increase in TK activity, (c) the addition of ganciclovir will lead to synergistic cytotoxic effects to tumor cells with F +cisplatin thereby reducing its IC50 and toxicity to normal tissues. This approach is innovative because of its inherent 'translational' nature wherein we are attempting to bridge exciting results that have arisen from bench research to the preclinical setting as a prelude to using this approach for women with advanced cervical cancer.
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