Matsumoto H, Hayashi S, Hatashita M, Ohnishi K, Ohtsubo T, Kitai R, Shioura H, Ohnishi T, Kano E
Department of Experimental Radiology and Health Physics, Fukui Medical University, Matsuoka, Japan.
Cancer Res. 1999 Jul 1;59(13):3239-44.
Nitric oxide is known to be a multifunctional physiological substance. Recently, it was suggested that nitric oxide is involved in p53-dependent response to many kinds of stress, such as heat shock and changes in cellular metabolism. To verify this hypothesis, we examined the effect of nitric oxide produced endogenously by heat-shocked cells on nonstressed cells using a human glioblastoma cell line, A-172, and its mutant p53 (mp53) transfectant (A-172/mp53). The accumulation of inducible nitric oxide synthase was caused by heat treatment of the mtp53 cells but not of the wild-type p53 (wtp53) cells. The accumulation of heat shock protein 72 (hsp72) and p53 was observed in nontreated mtp53 cells cocultivated with heated mp53 cells, and the accumulation of these proteins was suppressed by the addition of a specific inducible nitric oxide synthase inhibitor, aminoguanidine, to the medium. Furthermore, the accumulation of these proteins was observed in the wtp53 cells after exposure to the conditioned medium by preculture of the heated mp53 cells, and the accumulation was completely blocked by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. In addition, the accumulation of hsp72 and p53 in the wtp53 cells was induced by the administration of an nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, to the medium. Finally, the thermosensitivity of the wtp53 cells was reduced in the conditioned medium by preculture of the heated mp53 cells as compared with conventional fresh growth medium. Our finding of the accumulation of hsp72 and p53 in nitric oxide-recipient cells cocultivated with heated nitric oxide-donor cells provides the first evidence for an intercellular signal transduction pathway via nitric oxide as intermediate without cell-to-cell interactions such as gap junctions.
一氧化氮是一种多功能的生理物质。最近，有人提出一氧化氮参与了p53依赖的对多种应激的反应，如热休克和细胞代谢变化。为了验证这一假设，我们使用人胶质母细胞瘤细胞系A-172及其突变型p53（mp53）转染细胞（A-172/mp53），研究了热休克细胞内源性产生的一氧化氮对未受应激细胞的影响。mtp53细胞经热处理可导致诱导型一氧化氮合酶的积累，而野生型p53（wtp53）细胞则不会。在与加热的mp53细胞共培养的未处理mtp53细胞中观察到热休克蛋白72（hsp72）和p53的积累，向培养基中添加特异性诱导型一氧化氮合酶抑制剂氨基胍可抑制这些蛋白的积累。此外，在加热的mp53细胞预培养后的条件培养基处理wtp53细胞后，观察到这些蛋白的积累，向培养基中添加特异性一氧化氮清除剂2-(4-羧基苯基)-4,4,5,5-四甲基-咪唑啉-1-氧基-3-氧化物可完全阻断这种积累。此外，向培养基中添加一氧化氮生成剂S-亚硝基-N-乙酰青霉胺可诱导wtp53细胞中hsp72和p53的积累。最后，与传统的新鲜生长培养基相比，加热的mp53细胞预培养后的条件培养基降低了wtp53细胞的热敏感性。我们发现，在与加热的一氧化氮供体细胞共培养的一氧化氮受体细胞中积累hsp72和p53，这首次证明了存在一条通过一氧化氮作为中间体的细胞间信号转导途径，而无需细胞间相互作用，如间隙连接。
