Human SAT1 adenoviral particles

The polyamines putrescine, spermidine, and spermine are closely associated with the regulation of cell growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with adenovirus encoding the key polyamine catabolic enzyme spermidine N1-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1) resulted in rapid depletion of spermidine and spermine, cell growth arrest, and decreased cell viability. Annexin V/propidium iodide FACS analysis, terminal uridine nucleotide end labeling (TUNEL), and caspase 3 assays showed clear signs of apoptosis in AdSAT1-transduced cells (24-72 hours), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in polyamine-depleted cells occurred via 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 mitochondrial release of cytochrome c following transduction with AdSAT1. In addition, TEM images of AdSAT1-transduced cells showed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial changes, vacuolization, and membrane blebbing. Inhibition of polyamine oxidase did not restore growth or prevent apoptosis in AdSAT1-transduced cells, indicating that growth arrest and apoptosis were not caused 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.

Annexin V/PI FACS analysis showed that early and late apoptotic cells were significantly increased 24-72 hours after AdSAT1 transduction compared with non-transduced cells (Figure 1A). Therefore, apoptosis in AdSAT1-transduced cells appears to be mainly due to the depletion of spermidine and spermine rather than oxidative stress. The increase in early and late apoptotic cells caused by cycloheximide treatment was relatively small compared with AdSAT1 transduction. TUNEL assay also showed that DNA fragmentation was increased in SAT1-overexpressing cells 48 hours after AdSAT1 transduction (Figure 1B, white arrows). Increased nuclear fragmentation was also detected in these cells by DAPI staining (Figure 1B, white arrows). Caspase 3 activity was significantly increased in AdSAT1-transduced cells, compared to untransduced and AdGFP-transduced cells at 24-72 h of transduction (Figure 1C). In contrast to AdSAT1-transduced cells, no or little DNA or nuclear fragmentation was observed in AdGFP-transduced cells (Figure 1B), and there were only small increases in caspase 3 over untransduced cells (Figure 1C), suggesting that the apoptosis is primarily due to SAT1 overexpression but not due to adenoviral infection.

Figure 1. Annexin V/PI FACS analyses, TUNEL assay and caspase 3 assay of AdSAT1-transduced and control 293T cells. (Mandal S, Mandal A, Park M H., 2015)