Mandal Swati, Mandal Ajeet, Park Myung Hee
Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Biochem J. 2015 Jun 15;468(3):435-47. doi: 10.1042/BJ20150168. Epub 2015 Apr 7.
The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis.
多胺腐胺、亚精胺和精胺密切参与细胞生长和活力的调节。用编码关键多胺分解代谢酶亚精胺N¹-乙酰基转移酶1（SSAT1）/SAT1的腺病毒转导人胚肾（HEK）293T细胞，会导致亚精胺和精胺迅速耗竭，细胞生长停滞，细胞活力下降。膜联蛋白V/碘化丙啶流式细胞术分析、末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）和半胱天冬酶3检测显示，在转导AdSAT1的细胞中（24 - 72小时）有明显的凋亡迹象，但在用编码绿色荧光蛋白的腺病毒（AdGFP）转导的细胞中则没有。多胺耗竭细胞中的凋亡通过线粒体内在途径发生，转导AdSAT1后，线粒体膜电位丧失、促凋亡蛋白Bax增加、抗凋亡蛋白Bcl-xl、Bcl2和Mcl-1减少以及细胞色素c从线粒体释放，均证明了这一点。此外，转导AdSAT1的细胞的透射电子显微镜图像显示出通常与凋亡相关的形态学变化，包括细胞皱缩、核碎裂、线粒体改变、空泡化和膜泡形成。凋亡似乎主要是由于多胺亚精胺和精胺的耗竭所致，因为不是SAT1底物的多胺类似物α-甲基亚精胺（α-MeSpd）和N¹,N¹²-二甲基精胺（Me₂Spm）可以部分恢复AdSAT1转导细胞的生长并防止其凋亡。抑制多胺氧化酶并不能恢复AdSAT1转导细胞的生长或阻止凋亡，这表明生长停滞和凋亡不是由多胺分解代谢加速导致的氧化应激所诱导。综上所述，这些数据提供了强有力的证据，表明多胺亚精胺和精胺的耗竭会导致线粒体介导的凋亡。
