Fogel-Petrovic M, Shappell N W, Bergeron R J, Porter C W
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263.
J Biol Chem. 1993 Sep 5;268(25):19118-25.
In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 microM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA. By Northern blot analysis three RNA transcripts were found to hybridize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximately 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.
在MALME - 3M人黑色素瘤细胞中，多胺类似物N1,N12 - 双（乙基）精胺（BESPM）可抑制关键的多胺生物合成酶——鸟氨酸脱羧酶和S - 腺苷甲硫氨酸脱羧酶，并使多胺分解代谢酶——亚精胺/精胺N1 - 乙酰转移酶（SSAT）增加200倍以上。在本研究中，发现用10 microM BESPM处理的MALME - 3M细胞中SSAT活性的增加伴随着SSAT mRNA的大量积累（高达45倍）。通过Northern印迹分析，发现三种RNA转录本与人类SSAT cDNA的编码区杂交：一种较小的高分子量（约3.5千碱基）的物种命名为A形式，以及两种较低分子量的物种命名为B形式和C形式（分别约为1.5和1.3千碱基）。在BESPM处理期间，A形式均匀增加，在核RNA制剂中最为明显。基于与基因转录区域的大小相似性、与SSAT cDNA编码区的杂交以及其在核mRNA制剂中的普遍性，A形式被认为代表前体SSAT RNA。C形式存在于对照细胞中，并在处理过程中稳定增加，而B形式在早期处理（1 - 3小时）期间短暂增加。通过RNase H消化试验，发现B形式具有长200个碱基对的poly(A)尾，因此可能代表C形式的前体。发现SSAT mRNA的积累是基因转录增加和SSAT mRNA稳定的结果。核延伸实验表明SSAT基因的转录速率增加了2 - 4倍。如放线菌素D实验所示，BESPM处理使SSAT mRNA半衰期从17小时增加到64小时。天然多胺精胺也增加了SSAT mRNA（24小时时增加5.5倍），并且在诱导相同三种转录本形式出现方面的表现与BESPM相似。在增加酶活性方面，该多胺的效果远不如类似物。用生物合成抑制剂降低细胞内多胺池可使基础SSAT mRNA水平降低至少70%，这表明该基因可被多胺下调以及上调。这些发现表明，SSAT代表了一个独特的例子，即多胺及其类似物在RNA水平上对基因表达产生正向影响。
