APOPTOSIS,5 LIPOXYGENASE ACTIVATING PROTEIN AND BCL X

细胞凋亡、5 脂氧合酶激活蛋白和 BCL X

• 批准号: 6514212
• 负责人: James P Kehrer
• 金额: $ 25.43万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514212
• 关键词: antisense nucleic acid; apoptosis; autosomal dominant trait; complementary DNA; enzyme activity; enzyme linked immunosorbent assay; fatty acid metabolism; gel electrophoresis; glutathione; interleukin 3; lipoxygenase; oncoproteins; oxidoreductase inhibitor; peroxides; peroxisome proliferator activated receptor; protooncogene; tissue /cell culture; transfection

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.

活性氧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，但可能是产生了某种信号因子。由于多不饱和脂肪酸极易氧化，因此已知可诱导细胞凋亡的氧化产物是合理的候选者。脂氧合酶是这种产物的可能来源，各种抑制剂已被用于使这些酶参与细胞凋亡。然而，其中一些抑制剂，包括阻断5-脂氧合酶激活蛋白（FLAP）的MK886，即使在缺乏LOX的细胞中也会诱导细胞凋亡。我们还发现NM886诱导细胞凋亡不依赖于FLAP。然而，我们的研究结果表明，FLAP与BCL家族抗凋亡蛋白之间存在联系，过表达BCL - xl的FL5.12细胞的FLAP水平降低，并且在退出IL-3后，这些细胞的FLAP迅速丧失。在用mK886处理的细胞中，bcl-xL和bcl-2蛋白也会迅速丧失。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凋亡相关机制的理解。一个相关的目标将是阐明皮瓣和bcl-XL之间的关系，以及诱导细胞凋亡。有待验证的假设为：1)MK886对过氧化物酶体增殖物激活受体（PPAR）的抑制作用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，这些脂肪酸直接激活凋亡信号通路或随后转化为其他物种，3)破坏脂肪酸信号通路调节bcl-xL的表达。初步数据显示MK886是一种有效的pparα抑制剂。通过上调和下调PPAR以及确定MK886的凋亡效力，我们将对这种独特的作用进行更详细的研究，并研究其与细胞凋亡的关系。第二种假说将通过测定MK886处理后的脂肪酸氧化，比较MK886与外源脂肪酸（包括氧化种）的作用，以及检测谷胱甘肽是否会影响MK886诱导的细胞凋亡来研究。通过测量bcl-XL过表达对其他细胞系FLAP表达的影响，评估IL-3停药或MK886治疗后bcl-XL过表达细胞中FLAP和bcl-XL蛋白及mRNA水平，评估各种蛋白水解途径在bcl-XL和FLAP缺失中的作用，来确定FLAP与bcl-XL之间的关系。这些研究结果将提高我们对脂肪酸信号和凋亡死亡与FLAP和bcl蛋白相关的理解。