IMIDODIPEPTIDES AND AMINO ACID METABOLITES IN CELL REGULATION AND CARCINOGENESIS

脯氨酸氧化酶的诱导伴随p53依赖性诱导细胞凋亡表明脯氨酸代谢途径在程序性细胞死亡中起作用。我们正在以下水平研究该代谢途径：a）脯氨酸氧化酶-其诱导及其在细胞凋亡中的作用，B）吡咯啉5-羧酸（P5 C）作为信号传导和调节分子，以及c）亚胺二肽和脯氨酰二肽酶。a）脯氨酸氧化酶-其诱导及其在细胞凋亡中的作用。在过去的一年里，我们集中在脯氨酸氧化酶，催化脯氨酸转化为吡咯啉5-羧酸的酶。该酶与线粒体内膜结合，并向复合物II提供电子。最近，其他人已经表明，脯氨酸氧化酶（POX）是只有11个基因（超过7000基因监测SAGE）高度诱导伴随p53依赖性诱导大肠癌细胞凋亡。为了研究POX的作用，我们首先使用RT-PCR检测几种细胞类型和导致POX诱导的刺激。在人大肠癌细胞（LoVo）、温度敏感性SV 40转化的小鼠结肠上皮细胞（YAMC）以及多种癌细胞系中，我们发现血清饥饿和细胞毒药物都能诱导脯氨酸氧化酶的产生。我们提出脯氨酸氧化酶可能通过产生活性氧（ROS）参与伴随细胞凋亡的代谢事件。在YAMC细胞中检查了这种可能性，其中用荧光探针和激光细胞术监测ROS。有趣的是，加入脯氨酸以浓度依赖性方式显著增加ROS的产生，但仅伴随阿霉素诱导的细胞凋亡。另一方面，谷氨酸盐没有效果。我们目前正在确定其他脯氨酸调节的事件下游的细胞凋亡途径。B）吡咯啉5-羧酸酯（P5 C）作为信号传导和调节分子。吡咯啉5-羧酸（P5 C），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）的表达已被证明是由氨基酸代谢物刺激，我们测试了P5 C对小鼠巨噬细胞中NOS II表达的影响，发现P5 C增加了NOS II蛋白和mRNA的水平。NOS Ⅱ mRNA表达的增加依赖于P5 C浓度和暴露时间。然而，只有当细胞在缺氧条件下培养时，P5 C的这种作用才明显。虽然NOSII的表达响应脂多糖和干扰素γ在常氧条件下，P5 C是没有效果，除了在缺氧条件下。我们目前正在确定P5 C这种明显的转录作用的分子机制。c）亚胺二肽和脯氨酰二肽酶。含有脯氨酸或羟脯氨酸的亚氨基二肽来源于组织基质降解或膳食蛋白。它们不是一般肽酶的底物，而是循环到组织中，在组织中被脯氨酰二肽酶水解释放脯氨酸。由于脯氨酸在细胞凋亡中可能是重要的，因此脯氨酸二肽酶对脯氨酸释放的调节是令人感兴趣的。我们发现，细胞内脯氨酰二肽酶的水平是由细胞外胶原通过整合素受体发挥作用。因此，亚氨基二肽的水解响应于细胞与细胞外基质的相互作用。d）由前列腺特异性抗原刺激的ROS产生。前列腺特异性抗原（PSA）是前列腺癌的临床标志物，也可能是一种信号分子。PSA是LNCaP细胞中ROS产生的有效刺激剂，并且可能是雄激素对ROS产生的影响的介导剂。PSA对ROS的影响不依赖于雄激素机制。PSA对PC-3、DU 145细胞及LNCaP均有影响。我们正在研究相关机制。- 代谢，一氧化氮，营养，氧化应激，信号传导，

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,