Wahlfors J, Myöhänen S, Korhonen V P, Alhonen L, Jänne J
A.I. Virtanen Institute, University of Kuopio, Finland.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):299-303. doi: 10.1042/bj3100299.
(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
(1) 人骨髓瘤细胞系苏丹（Sultan）因鸟氨酸脱羧酶（ODC）基因扩增而对20 mM二氟甲基鸟氨酸（DFMO）具有抗性，在无DFMO的情况下培养10个月。撤去DFMO后监测ODC基因的基因拷贝数和甲基化状态。此外，在研究过程中反复记录ODC mRNA水平、免疫反应性ODC蛋白、ODC活性和多胺水平。(2) 结果显示，在无DFMO培养4周后，ODC基因拷贝数开始下降，最终水平低于原始基因剂量的30%。然而，ODC基因的甲基化状态几乎保持不变，在无DFMO培养10个月后仅显示甲基化有适度增加。ODC信使RNA的量迅速下降至原始值的75%，然后开始以基因拷贝数依赖的方式下降。在研究过程中，ODC蛋白的量紧密跟随mRNA水平，而ODC活性在第一周短暂增加后，4周后降至原始水平的一半。在6至16周之间，ODC活性稳定在原始值的五分之一，在随后的观察期内未检测到进一步变化。(3) 由于ODC酶活性大幅升高，腐胺和亚精胺水平在撤去DFMO后首先达到峰值，然后在6周时稳定下来。最终亚精胺水平与仅具有一个活性ODC基因拷贝的亲代苏丹细胞系相当。然而，腐胺含量显著升高，稳定在比亲代细胞高20倍的水平。在研究过程中精胺浓度基本不变。(4) 根据本研究获得的结果，人骨髓瘤细胞中ODC表达的异常水平在转录或转录后水平上部分受到抑制，但这不是由于基因甲基化增加所致。补偿ODC高度升高表达的主要调节机制是酶活性的调节。在无DFMO培养10个月后，这些细胞的ODC活性和腐胺浓度仍比具有单个ODC基因拷贝的骨髓瘤细胞系高约20倍。然而，它们没有显示出任何生长迟缓的迹象或与亲代细胞不同的其他特征。（摘要截短至400字）
