Pegg A E, Madhubala R, Kameji T, Bergeron R J
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
J Biol Chem. 1988 Aug 5;263(22):11008-14.
The regulation of ornithine decarboxylase (ODC) activity by the polyamine derivatives N1,N8-bis(ethyl)-spermidine and N1,N12-bis(ethyl)spermine was studied using a line of L1210 cells resistant to alpha-difluoromethylornithine (D-R cells), which contain very high levels of ODC, and a synthetic mRNA prepared from a plasmid containing an insert corresponding to ODC mRNA adjacent to an SP6 RNA polymerase promoter. Studies in which ODC protein was labeled in the D-R cells by exposure to [35S]methionine indicated that the polyamine derivatives and their physiological counterparts led to an increased rate of degradation of ODC and to a rapid reduction in ODC synthesis without affecting the content of ODC mRNA. Direct evidence that the polyamine derivatives act by inhibiting the translation of the ODC mRNA was obtained by studying their effects on the translation of ODC mRNA in reticulocyte lysates. This translation was strongly inhibited by the addition of N1,N8-bis(ethyl)spermidine, spermidine, N1,N12-bis(ethyl)spermine, or spermine but was not affected much by putrescine. The inhibition of the translation of ODC mRNA by either of the bis(ethyl) polyamine derivatives occurred at concentrations which stimulated total protein synthesis showing the selectivity of the reduction in ODC. The effects of polyamine derivatives and polyamines on translation of the plasmid-derived ODC mRNA were identical with those found with the D-R L1210 cell mRNA. This synthetic ODC mRNA lacks 261 bases of the 5'-leader sequences and 200 bases plus the poly(A) section from the 3'-nontranslated sequence. Therefore, these regions appear not to influence sensitivity of the ODC mRNA to inhibition of translation by polyamine derivatives.
利用对α-二氟甲基鸟氨酸具有抗性的L1210细胞系（D-R细胞）研究了多胺衍生物N1,N8-双（乙基）-亚精胺和N1,N12-双（乙基）-精胺对鸟氨酸脱羧酶（ODC）活性的调节作用，D-R细胞含有非常高水平的ODC，还研究了由含有与ODC mRNA对应的插入片段且该片段紧邻SP6 RNA聚合酶启动子的质粒制备的合成mRNA。在D-R细胞中通过暴露于[35S]甲硫氨酸对ODC蛋白进行标记的研究表明，多胺衍生物及其生理对应物导致ODC降解速率增加以及ODC合成迅速减少，而不影响ODC mRNA的含量。通过研究它们对网织红细胞裂解物中ODC mRNA翻译的影响，获得了多胺衍生物通过抑制ODC mRNA翻译起作用的直接证据。添加N1,N8-双（乙基）-亚精胺、亚精胺、N1,N12-双（乙基）-精胺或精胺会强烈抑制这种翻译，但腐胺对其影响不大。两种双（乙基）多胺衍生物对ODC mRNA翻译的抑制发生在刺激总蛋白合成的浓度下，这表明ODC减少具有选择性。多胺衍生物和多胺对质粒衍生的ODC mRNA翻译的影响与D-R L1210细胞mRNA的情况相同。这种合成的ODC mRNA缺乏5'-前导序列的261个碱基以及3'-非翻译序列的200个碱基加上聚（A）区段。因此，这些区域似乎不影响ODC mRNA对多胺衍生物抑制翻译的敏感性。
