IMIDODIPEPTIDES AND AMINO ACID METABOLITES IN CELL REGULATION AND CARCINOGENESIS

P53依赖的细胞凋亡诱导过程中伴随着Pro氧化酶的诱导，提示Pro代谢途径在细胞程序性死亡中起着重要作用。我们正在从以下水平研究这一代谢途径：a)Pro氧化酶-其诱导及其在细胞凋亡中的作用，b)吡咯烷5-羧酸(P5C)作为信号和调节分子，c)亚氨二肽和Prolidase。a)Pro氧化酶-其诱导及其在细胞凋亡中的作用。在过去的一年里，我们重点研究了Pro氧化酶，这是一种催化Pro转化为吡咯烷5-羧酸盐的酶。该酶结合到线粒体内膜并向复合体II提供电子。最近，其他研究表明，脯氨酸氧化酶(POX)是SAGE监测的仅有的11个基因(超过7000个基因)之一，它伴随着P53依赖的方式诱导结直肠癌细胞凋亡。为了研究POX的作用，我们首先使用RT-PCR检测了几种细胞类型和将导致POX诱导的刺激。在人结直肠癌(LoVo)细胞、温敏性SV40(YAMC)转化的小鼠结肠上皮细胞以及多种癌细胞系中，我们发现血清饥饿以及细胞毒性药物都能诱导Pro氧化酶的产生。我们认为，脯氨酸氧化酶可能通过产生活性氧(ROS)而参与伴随细胞凋亡的代谢事件。这种可能性在YAMC细胞中进行了检验，在YAMC细胞中，用荧光探针和激光细胞术监测ROS。有趣的是，添加Pro以浓度依赖的方式显著增加ROS的产生，但仅伴随着阿霉素诱导的细胞凋亡。另一方面，谷氨酸则没有效果。我们目前正在确定在细胞凋亡途径下游的其他受脯氨酸调节的事件。B)作为信号和调节分子的吡咯烷5-羧酸盐(P5C)。5-吡咯烷酸(P5C)是脯氨酸氧化酶的产物，已被证明具有调节活性。由于一氧化氮合酶II(NOS II)的表达已被证明受到氨基酸代谢产物的刺激，我们检测了P5C对小鼠巨噬细胞NOS II表达的影响，发现P5C增加了NOS II的蛋白和mRNA水平。一氧化氮合酶II基因表达的增加与P5C浓度和暴露时间有关。然而，只有当细胞在低氧条件下培养时，P5C的这种作用才明显。在常氧条件下，NOS II的表达对脂多糖和干扰素-γ有反应，而P5C在低氧条件下则不受影响。我们目前正在确定P5C.c)亚氨二肽和脯氨酸酶这种明显转录效应的分子机制。含有脯氨酸或羟脯氨酸的亚胺二肽来源于组织基质降解或来自饮食蛋白。它们不是普通多肽酶的底物，而是循环到组织中，在那里它们被脯氨酸酶水解以释放脯氨酸。由于脯氨酸可能在细胞凋亡中起重要作用，因此，在细胞凋亡过程中，由脯氨酸酶调节的脯氨酸释放是很有意义的。我们发现细胞内的脯氨酸酶水平是由细胞外的胶原通过整合素受体调节的。因此，亚胺二肽的水解反应是细胞与细胞外基质相互作用的结果。D)前列腺特异性抗原刺激的ROS生成。我们认为前列腺癌的临床标志物前列腺特异性抗原(PSA)也可能是一种信号分子。PSA是LNCaP细胞产生ROS的有力刺激物，可能是雄激素影响ROS产生的中介因子。PSA对ROS的影响与雄激素机制无关。PSA作用于PC-3、DU145细胞和LNCaP。我们正在调查涉及的机制。-新陈代谢、一氧化氮、营养、氧化应激、信号、

The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death. We are studying this metabolic pathway at the level of : a) Proline oxidase - its induction and its role in apoptosis, b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule and c) Imidodipeptides and prolidase.a) Proline oxidase - its induction and its role in apoptosis. During this last year, we have focused on proline oxidase, the enzyme which catalyzes the conversion of proline to pyrroline 5-carboxylate. The enzyme is bound to mitochondrial inner membranes and donates electrons to complex II. Recently others have shown that proline oxidase (POX) is one of only 11 genes (over 7000 genes monitored by SAGE) highly induced accompanying p53-dependent induction of apoptosis in colorectal tumor cells. To investigate the role of POX, we first used RT-PCR to examine several cell types and the stimuli which will result in POX induction. In human colorectal cancer (LoVo) cells, mouse colonic epithelial cells transformed by temperature sensitive SV40 (YAMC), as well as a variety of carcinoma cell lines, we found that serum starvation as well as cytotoxic drugs could induce proline oxidase. We proposed that proline oxidase may contribute to the metabolic events accompanying apoptosis by generating reactive oxygen species (ROS). This possibility was examined in YAMC cells in which ROS was monitored with a fluorescent probe and laser cytometry. Interestingly, the addition of proline markedly increased ROS generation in a concentration- dependent manner but only accompanying adriamycin-induced apoptosis. Glutamate, on the other hand, was without effect. We are currently identifying other proline-modulated events downstream in the apoptosis pathway. b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. Pyrroline 5-carboxylate (P5C), the product of proline oxidase, has been shown to have regulatory activities. Since the expression of Nitric Oxide Synthase II (NOS II) has been shown to be stimulated by amino acid metabolites, we tested the effect of P5C on NOS II expression in murine macrophages and found that P5C increased the level of NOS II protein and mRNA. The increase in NOS II mRNA was dependent on P5C concentration and duration of exposure. This effect of P5C, however, was evident only when cells were cultured under hypoxic conditions. Although NOS II expression responded to lipopolysaccharide and interferon gamma under normoxic conditions, P5C was without effect except under hypoxic conditions. We are currently defining the molecular mechanisms for this apparent transcriptional effect of P5C.c) Imidodipeptides and prolidase. Imidodipeptides containing proline or hydroxyproline originate from either tissue matrix degradation or from dietary protein. They are not substrate for generic peptidases but instead circulate to tissues where they are hydrolyzed by prolidase to release proline. Since proline may be important in apoptosis, the regulated release of proline by prolidase is of interest. We found that the level of cellular prolidase is regulated by extracellular collagen acting through integrin receptors. Thus, the hydrolysis of imidodipeptides is responsive to cellular interaction with extracellular matrix. d) ROS generation stimulated by prostate specific antigen. We are pursuing the hypothesis that prostate specific antigen (PSA), the well-known clinical marker for prostate cancer, also may be a signaling molecule. PSA is a potent stimiulator of ROS generation in LNCaP cells and may be the mediator of the effects of androgens on ROS generation. The effect of PSA on ROS is independent of androgen mechanisms. PSA affected PC-3, DU145 cells as well as LNCaP. We are investigating the involved mechanisms. - metabolism, Nitric oxide, nutrition, Oxidative stress, signaling,