Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

Design and Synthesis of 4-N-Alkanoyl and 4-N-Alkyl Gemcitabine Analogues Suitable for Positron Emission Tomography

Gemcitabine is a highly potent chemotherapeutic nucleoside agent used in the treatment of several cancers and solid tumors. However, it is therapeutically limitated because of toxicity to normal cells and its rapid intracellular deamination by cytidine deaminase into the inactive uracil derivative. Modification at the 4-(N) position of gemcitabine's exocyclic amine to an -amide functionality is a well reported prodrug strategy which has been that confers a resistance to intracellular deamination while also altering pharmacokinetics of the parent drug. Coupling of gemcitabine to carboxylic acids with varying terminal moieties afforded the 4-N-alkanoylgemcitabines whereas reaction of 4-N-tosylgemcitabine with the corresponding alkyl amines gave the 4-N-alkylgemcitabines. The 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues with a terminal hydroxyl group on the 4-N-alkanoyl or 4-N-alkyl chain were efficiently fluorinated either with diethylaminosulfur trifluoride or under conditions that are compatible with the synthetic protocols for 18F labeling, such as displacement of the corresponding mesylate with KF/Kryptofix 2.2.2. The 4-N-alkanoylgemcitabine analogues displayed potent cytostatic activities against murine and human tumor cell lines with 50% inhibitory concentration (IC50) values in the range of low nM, whereas cytotoxicity of the 4-N-alkylgemcitabine derivatives were in the low to modest µM range. The cytostatic activity of the 4-N-alkanoylgemcitabines was reduced by several orders of magnitude in the 2'-deoxycytidine kinase (dCK)-deficient CEM/dCK- cell line while the 4-N-alkylgemcitabines were only lowered by 2-5 times. None of the 4-N-modified gemcitabines were found to be substrates for cytosolic dCK, however all were found to inhibit DNA synthesis. As such, the 4-N-alkanoyl gemcitabine derivatives likely need to be converted to gemcitabine prior to achieving their significant cytostatic potential, whereas the 4-N-alkylgemcitabines reach their modest activity without "measurable" conversion to gemcitabine. Thus, the 4-N-alkylgemcitabines provide valuable insight on the metabolism of 4-N-modified gemcitabine prodrugs.