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The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.

Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.

The cleavage and isomerization of 3‘,5‘-uridyluridine catalyzed by morpholine buffers and by imidazole buffers has been reinvestigated. The key evidence for a previously proposed partitioning mechanismin which the buffer acid BH+ converts the substrate to a phosphorane monoanion intermediate which then partitions either to the 2‘,5‘ isomer of the substrate or (with buffer base catalysis) to the cleavage productis confirmed. The negative catalytic effect of the buffer base on the isomerization reaction is not due to a medium effect. Indeed the medium effect on this reaction is in the opposite direction, strengthening the catalytic evidence. However, this branching mechanism with sequential bifunctional catalysis of the cleavage reaction is accompanied by an additional cleavage path using the buffer base only. This additional path, for which several alternative mechanisms are possible, is required by the results of improved studies on the imidazole catalysis. These show that the previously reported decrease...

Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m -carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro . Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.