Habibi Laleh, Shokrgozar Mohammad Ali, Motamedi Mahdieh, Akrami Seyed Mohammad
Dept. of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Iranian National Cell Bank, Pasteur Institute of Iran, Tehran, Iran.
Iran Biomed J. 2013;17(4):171-8. doi: 10.6091/ibj.1230.2013.
L1 retrotransposons are the most active mobile DNA elements in human genome. Unregulated L1 retrotransposition may have deleterious effect by disrupting vital genes and inducing genomic instabilities. Therefore, human cells control L1 elements by silencing their activities through epigenetic mechanisms. It has been shown that cell division and heavy metals stimulate the frequency of L1 activities. Removal of silencing by L1 motivators may restart L1 element functions. Here, we have proposed that weather neurotoxic environmental heavy metals (as L1 stimulating factors) have a role in removing L1 silencing and restating its activities in nondividing neuronal cells.
L1-RP green fluorescent protein (GFP)-tagged knock-in human neuroblastoma clones were prepared. Single-cell clone was treated with mitomycin-c combined with nontoxic and toxic concentrations of iron (Fe), copper (Cu), and mercury (Hg). Silencing status of engineered L1 elements in dividing and nondividing cells was determined through measuring the amount of GFP expressing cells with flow cytometry. The cytotoxic effect of mitomycin-c combined with metals was measured by MTT assay.
Hg in nondividing cells and Fe, Cu, and Hg in dividing neuroblastoma cells could significantly remove L1 silencing. Also, mitomycin-c treatment did not have any effect on metal toxicity status in neuroblastoma cells.
Totally, our findings have shown that cell division has a role in removing L1 silencing as well as L1 retrotransposition induced by environmental heavy metals. It has been also indicated that Hg at all concentrations could remove silencing of engineered L1 element regardless of cell cycle state.
L1逆转座子是人类基因组中最活跃的可移动DNA元件。不受调控的L1逆转座可能通过破坏重要基因和诱导基因组不稳定而产生有害影响。因此，人类细胞通过表观遗传机制沉默L1元件的活性来对其进行控制。研究表明，细胞分裂和重金属会刺激L1活性的频率。去除L1激活因子的沉默作用可能会重启L1元件的功能。在此，我们提出环境神经毒性重金属（作为L1刺激因子）是否在去除非分裂神经元细胞中L1的沉默并恢复其活性方面发挥作用。
制备了带有L1-RP绿色荧光蛋白（GFP）标签的人神经母细胞瘤敲入克隆。单细胞克隆用丝裂霉素-C与无毒和有毒浓度的铁（Fe）、铜（Cu）和汞（Hg）进行处理。通过流式细胞术测量表达GFP的细胞数量，以确定工程化L1元件在分裂和非分裂细胞中的沉默状态。用MTT法测定丝裂霉素-C与金属联合使用的细胞毒性作用。
非分裂细胞中的Hg以及分裂的神经母细胞瘤细胞中的Fe、Cu和Hg可显著去除L1的沉默。此外，丝裂霉素-C处理对神经母细胞瘤细胞中的金属毒性状态没有任何影响。
总的来说，我们的研究结果表明细胞分裂在去除L1沉默以及环境重金属诱导的L1逆转座方面发挥作用。还表明，无论细胞周期状态如何，所有浓度的Hg都能去除工程化L1元件的沉默。
