APOPTOSIS,5 LIPOXYGENASE ACTIVATING PROTEIN AND BCL X

活性氧和细胞凋亡的几个效应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，但可能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，已知诱导细胞凋亡的氧化产物是合理的候选物。脂氧合酶是这些产物的可能来源，并且已经使用各种抑制剂来暗示这些酶参与细胞凋亡。 然而，这些抑制剂中的几种，包括阻断5-脂氧合酶激活蛋白（FLAP）的MK 886，即使在缺乏LOX的细胞中也诱导凋亡。 我们还表明，NM 886诱导细胞凋亡独立于FLAP。 尽管如此，我们的发现表明FLAP和抗凋亡蛋白的BCL家族之间的联系，即过表达bcl-xL的FL5.12细胞具有减少的FLAP水平，并且在撤出IL-3后，FLAP从这些细胞中迅速丧失。 在用mK 886处理的细胞中也存在bcl-xL和bcl-2蛋白的快速损失。 本项目的总体目标是通过确定MK 886的凋亡机制来增强我们对与凋亡相关的机制的理解。 一个相关的目标将是澄清FLAP和bcl-XL之间的关系，诱导细胞凋亡。 待检验的假设是：1）MK 886对过氧化物酶体增殖物激活受体（PPAR）的抑制在诱导的细胞凋亡中起作用，2）MK 886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传导途径，以及3）破坏脂肪酸信号传导调节bcl-xL的表达。 初步数据显示，MK 886是一种有效的PPARalpha抑制剂。 将更详细地研究这种独特的作用，并通过上调和下调PPAR和确定MK 886的凋亡效力来研究与凋亡的任何联系。 第二个假设将通过测定用MK 886处理后的脂肪酸氧化，比较MK 886与包括氧化物质在内的外源性脂肪酸的作用，并检查谷胱甘肽是否可以影响MK 886诱导的细胞凋亡来研究。 FLAP和bcl-XL之间的任何关系将通过测量bcl-XL过表达对其他细胞系中FLAP表达的影响、评估在IL-3撤除或用MK 886处理后过表达bcl-XL的细胞中FLAP和bcl-XL蛋白和mRNA水平、以及评估各种蛋白水解途径在bcl-XL和FLAP损失中的作用来确定。 这些研究的结果将提高我们对脂肪酸信号传导和凋亡死亡与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.