APOPTOSIS,5 LIPOXYGENASE ACTIVATING PROTEIN AND BCL X

据报道，活性氧物种与几种细胞凋亡效应因子之间的关系已被报道。虽然氧化剂诱导细胞凋亡的机制尚不清楚，但它很可能是产生了一些信号因子。由于多不饱和脂肪酸对氧化高度敏感，已知可诱导细胞凋亡的氧化产物是合理的候选者。脂氧合酶是这类产物的一个可能来源，各种抑制剂已被用于将这些酶与细胞凋亡联系起来。然而，其中几种抑制剂，包括阻断5-脂氧合酶激活蛋白的MK886，即使在缺乏LOX的细胞中也能诱导细胞凋亡。我们还表明，NM886诱导的细胞凋亡不依赖于皮瓣。然而，我们的研究结果表明，FLAP与抗凋亡蛋白BCL家族之间存在联系，过量表达bCL-XL的FL5.12细胞的FL5·12水平降低，当IL-3退出时，这些细胞中的FLAP迅速丢失。在mK886处理的细胞中，bclxl和bcl2蛋白也迅速丢失。本项目的总体目标是通过确定MK886的凋亡机制来加深我们对与细胞凋亡相关的机制的理解。一个相关的目标将是阐明Flat和bclxl之间的关系，以及诱导细胞凋亡。需要检验的假设是：1)MK886对PPAR的抑制在诱导细胞凋亡中起作用；2)MK886阻断不饱和脂肪酸的结合，增加其细胞含量。然后这些脂肪酸直接或在转化为其他物种后激活凋亡信号通路，3)扰乱脂肪酸信号调节bclxl的表达。初步数据显示，MK886是一种有效的PPARα抑制剂。这一独特的作用将被更详细地研究，并通过上调和下调PPAR和确定MK886的凋亡效力来研究与细胞凋亡的任何联系。第二个假设将通过测定MK886处理后的脂肪酸氧化，比较MK886与包括氧化物种在内的外源脂肪酸的作用，以及检查谷胱甘肽是否会影响MK886诱导的细胞凋亡来研究第二个假设。通过测量BCL-XL在其他细胞系中过表达对FLOW表达的影响，评估停用IL-3或MK886处理后BCL-XL过表达细胞中FIFP和BCL-XL蛋白和mRNA的水平，以及评估各种蛋白降解途径在BCL-XL和FLAP丢失中的作用，将确定FLOW和BCL-XL之间的任何关系。这些研究的结果将提高我们对脂肪酸信号和凋亡死亡与FLAP和BCL蛋白的理解。

A relationship between reactive oxygen species and several effectors of apoptosis has been reported. Although the mechanism by which oxidants induce apoptosis is unknown, it is likely some signaling factor is generated. Since polyunsaturated fatty acids are highly susceptible to oxidation, oxidized products that are known to induce apoptosis are reasonable candidates. Lipoxygenase enzymes are a possible source of such products and various inhibitors have been used to implicate these enzymes in apoptosis. However, several of these inhibitors, including MK886 which blocks 5-lipoxygenase activating protein (FLAP), induce apoptosis even in cells lacking LOX. We have also shown that NM886 induces apoptosis independent of FLAP. Nevertheless, a link between FLAP and the BCL family of anti-apoptotic proteins is suggested by our findings that FL5.12 cells overexpressing bcl-xL have diminished levels of FLAP and there is a rapid loss of FLAP from such cells upon withdrawal of IL-3. There is also a rapid loss of bcl-xL and bcl-2 protein in cells treated with mK886. The overall goal of this project is to enhance our understanding of the mechanisms associated with apoptosis by determining the apoptotic mechanism of MK886. A related goal will be to clarify the relationship between FLAP and bcl-XL, and the induction of apoptosis. The hypotheses to be tested are that: 1) the inhibition of peroxisome proliferator activated receptors (PPAR) by MK886 plays a role in the induced apoptosis, 2) MK886 blocks the binding of unsaturated fatty acids increasing their cellular content. These fatty acids then activate apoptotic signaling pathways either directly or following conversion to other species, and 3) disrupting fatty acid signaling modulates the expression of bcl-xL. Preliminary data show that MK886 is a potent inhibitor of PPARalpha. This unique effect will be studied in more detail and any link to apoptosis investigated by up- and down-regulating PPAR and determining the apoptotic potency of MK886. The 2nd hypothesis will be studied by determining fatty acid oxidation after treatment with MK886, comparing the effects of MK886 to those of exogenous fatty acids including oxidized species, and examining whether glutathione can affect MK886-induced apoptosis. Any relationship between FLAP and bcl-XL will be determined by measuring the effect of bcl-XL overexpression on FLAP expression in other cell lines, assessing FLAP and bcl-XL protein and mRNA levels in bcl-XL overexpressing cells following withdrawal of IL-3 or treatment with MK886, and assessing the role of various proteolytic pathways in the loss of bcl-XL and FLAP. The results of these studies will improve our understanding of fatty acid signaling and apoptotic death in conjunction with FLAP and bcl proteins.