MECHANISMS OF ACROLEIN ON PROLIFERATION AND APOPTOSIS

丙烯醛是一种高度亲电的α-β-不饱和醛，人类在各种环境条件下都会接触到它。在低剂量时，丙烯醛抑制细胞增殖而不导致细胞死亡，并可以促进次级毒素的凋亡。本项目的总体目标是确定丙烯醛促进细胞凋亡和抑制细胞增殖的分子机制(S)。要研究的假设是，丙烯醛促进了包括细胞周期非特异性抗癌药物在内的次要应激源的细胞凋亡，这是由于调节这一过程的基因或转录因子的变化。由于丙烯醛是环磷酰胺的代谢物，这一假说得出的结论是，这种抗癌药物的独特有效性是丙烯醛和磷酰胺芥末代谢物的特征。一个相关的假设是，丙烯醛介导的细胞增殖下降是由于一个或多个生长或应激相关基因或转录因子的表达发生变化，继而导致谷胱甘肽的减少，而谷胱甘肽在丙烯醛处理后迅速耗尽。本项目针对这些假说的具体目的是：1)详细评估丙烯醛在二次促凋亡治疗中增强细胞对凋亡的敏感性的能力；以及2)确定丙烯醛治疗后促进细胞凋亡和抑制细胞增殖的机制。由于丙烯醛会瞬间耗尽GSH，因此将进行实验，以区分与抗氧化能力丧失直接相关的影响和与转录因子或基因表达变化有关的影响(即使在那些继发于GSH丢失的影响中，GSH的丢失可能会使辅助试剂更好地接触分子靶点)。初步数据表明，核因子-kappaB是丙烯醛的靶标。这将通过测定马来酸二乙酯(DEM)(在不耗尽GSH的剂量下阻断NF-kappaB的激活)影响细胞凋亡的能力，并通过检测丙烯醛在不耗尽GSH的剂量下对NF-kappaB的激活的影响(在不耗尽GSH的剂量下)来影响细胞凋亡，以及通过检测丙烯醛对第二细胞系中的NF-kappaB和细胞凋亡的影响来进一步研究这一点。将确定其他选定基因在丙烯醛影响中的作用。具体地说，将测量AP-1的激活以及在丙烯醛损伤后不同时间p53、c-jun、c-fos、c-myc、bcl2、bclx和bax的mRNA水平和蛋白产物的变化。这些研究的结果将为调控细胞增殖和凋亡的途径提供重要的新信息，并提供与低水平接触活性物质(如丙烯醛)相关的风险数据。

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde to which humans are exposed in a variety of environmental situations. At low doses, acrolein inhibits cell proliferation without causing cell death and can enhance apoptosis from secondary toxins. The overall goals of this project are to determine the molecular mechanism(s) by which acrolein enhances apoptosis and inhibits proliferation. The hypothesis to be studied is that acrolein enhances apoptosis from secondary stressors including cell-cycle-non-specific anti-cancer drugs and that this is due to changes in genes or transcription factors regulating this process. Since acrolein is a metabolite of cyclophosphamide, this hypothesis leads to the conclusion that the unique effectiveness of this anti-cancer drug is a feature of both acrolein and the phosphoramide mustard metabolite. A related hypothesis is that the acrolein-mediated decrease in cell proliferation is caused by effecting changes in the expression of one or more growth- or stress-related genes or transcription factors secondary to a reduction in GSH which is rapidly depleted following acrolein treatment. The specific aims of this project to address these hypotheses are: 1) to assess in detail the ability of acrolein to enhance the susceptibility of cells to apoptosis form a secondary pro-apoptotic treatment; and 2) to determine the mechanism of the enhanced apoptosis and decreased proliferation following acrolein treatment. Since acrolein transiently depletes GSH, experiments will be run to differentiate effects directly related to a loss of antioxidant capacity to those related to changes in transcription factors or gene expression (even in those are secondary to the loss of GSH which may allow secondary agents better access to molecular targets). Preliminary data have implicated NF-kappaB as a target for acrolein. This will be further studied by determining the ability of diethyl maleate (DEM) (which blocks NF-kappaB activation at doses that do not deplete GSH) to affect apoptosis and by examining the effects of acrolein on NF-kappaB activation at doses that do not deplete GSH) to affect apoptosis and by examining the effects of acrolein on NF-kappaB and apoptosis in a second cell line. A role for other selected genes in the effects of acrolein will be determined. Specifically, the activation of AP-1 will be measured as well as any changes in mRNA levels and protein products of p53, c-jun, c-fos, c-myc, bcl-2, bcl-x, and bax at various times following acrolein insult. The results of these studies will provide important new information on pathways regulating cell proliferation and apoptosis, as well as provide data on the risk associated with low-level exposure to reactive species such as acrolein.