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. 2015 Jun 15;468(3):435-47.
doi: 10.1042/BJ20150168. Epub 2015 Apr 7.

Depletion of the Polyamines Spermidine and Spermine by Overexpression of Spermidine/Spermine N¹-acetyltransferase 1 (SAT1) Leads to Mitochondria-Mediated Apoptosis in Mammalian Cells

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Depletion of the Polyamines Spermidine and Spermine by Overexpression of Spermidine/Spermine N¹-acetyltransferase 1 (SAT1) Leads to Mitochondria-Mediated Apoptosis in Mammalian Cells

Swati Mandal et al. Biochem J. .
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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.

Keywords: acetyltransferase; electron microscopy (EM); mitochondrial apoptosis; polyamine; spermidine; spermine.


FIGURE 1. Effects of AdSAT1 transduction, cycloheximide, DFMO, putrescine, N1-acetylspermidine, α-MeSpd, Me2Spm and BENSpm on cell viability
Viability of 293T cells was examined using a Live/dead cell imaging kit at 24, 48 and 72 h after seeding with no treatment (control), or AdSAT1 transduction, or treatment with cycloheximide (1 μg/ml), or with DFMO (5 mM), putrescine (5 mM) or N1-acetylspermidine (5 mM) or AdSAT1 transduction with added BENSpm (10 μM), α-MeSpd (100 μM), Me2Spm (100 μM). The concentrations of BENSpm, α-MeSpd and Me2Spm were chosen based on previous reports [9, 42]. Cells were stained with calcein and dead-red to visualize live and dead cells, respectively. Live cells fluoresce a bright green, whereas dead cells with damaged membranes fluoresce a red color. Representative images from three independent experiments are shown.
FIGURE 2. Effects of various compounds on the growth and the levels of SAT1 and ODC in untransduced, AdGFP-transduced and AdSAT1-transduced 293T cells
The compounds were added to the culture medium at the time of seeding and cell growth was monitored using Cell Counting Kit-8 (CCK-8) as described under Experimental Procedures. Average values from two independent experiments carried out in triplicate were plotted. (A) effects of AdGFP transduction, AdSAT1 transduction, or DFMO (5 mM) treatment on 293T cell growth. (B) effects of MDL 72527 (100 μM and 300 μM) and DFMO(5 mM) on the growth of untransduced or AdSAT1-transduced 293T cells. (C) effects of N-Acetyl-cysteine (NAC) on growth of untransduced and AdSAT1-transduced 293T cells. D, α-MeSpd (100 μM), Me2Spm (100 μM), or BENSpm (10 μM) on the growth of untransduced or AdSAT1-transduced cells. E, The time course of SAT1 and ODC protein levels were examined by western blotting.
FIGURE 3. Specificity of the hypusine-specific eIF5A antibody and effect of SAT1 overexpression on the hypusinated eIF5A levels
(A) Purified eIF5A proteins of various forms were tested using the hypusine-specific eIF5A antibody (top panel) [27] or eIF5A antibody (BD Biosciences) that recognizes all eIF5A forms (bottom panel). Lanes 1–3, recombinant human eIF5A containing lysine, deoxyhypusine or hypusine at residue 50. Lane 4, purified, acetylated eIF5A obtained by acetylation of recombinant human eIF5A(Hpu) using SAT1, lane 5, recombinant human eIF5A K50R mutant protein, lane 6, endogenous hypusine- containing eIF5A purified from CHO cells. Lane 7, unmodified eIF5A precursor isolated from DFMO-treated CHO cells. (B) Cell lysates from untransduced, AdGFP-transduced and AdSAT1-transduced 293T cells harvested at indicated times were analyzed using hypusine-specific eIF5A antibody verified in A. Abbreviations are: h5A(Lys), human eIF5A containing Lys at 50th residue; h5A(Dhp), human eIF5A containing deoxyhypusine; h5A(Hpu), human eIF5A containing hypusine; h5A(AcHpu), human eIF5A containing acetylhypusine; h5A(K50R), human eIF5A containing Arg at 50th residue.
FIGURE 4. Annexin V/PI FACS analyses, TUNEL assay and caspase 3 assay of AdSAT1-transduced and control 293T cells
(A) Apoptosis was measured at 24, 48 and 72 h of treatment by FACS analyses using Annexin V-FITC/PI double staining. 100 μM of MDL72527 or 1 μg/ml of cycloheximide was added to the sample as indicated. The percentage of apoptotic cells was calculated from fluorescence dot plots using quadrant statistics (10,000 cells/experiment). One representative experiment is shown. (B) Cells transduced with AdSAT1 or AdGFP were fixed and stained for the TUNEL assay at 48 h as described in Experimental Procedures. After the TUNEL assay, SAT1 immunocytochemistry (SAT1-ICC) was performed. The arrows indicate TUNEL positive cells which coincide with those with high expression of SAT1 and those with nuclear fragmentation upon staining with DAPI. The photomicrographs are representative images from three independent experiments. (C) Caspase 3 activity was measured as described under “Methods” with cell lysates prepared at 24 h, 48 h and 72 h of transduction. Bar graphs show data (mean ± SD) from three independent experiments done in duplicate.
FIGURE 5. Analyses of mitochondrial membrane potential, western blotting of apoptosis markers and cytochrome c release
(A) Untransduced and AdSAT1-transduced cells were stained with the mitoprobe JC-1 dye as described under Experimental Procedures. Red fluorescence represent cells with normal mitochondria membrane potential and green fluorescence represent those with depolarized mitochondrial membrane. Experiments were carried out twice in duplicates with 30,000 cells per analysis. One representative data is shown. (B) changes in the levels of apoptotic marker proteins in untransduced, AdGFP- or AdSAT1-transduced cells. (C) Western blot analyses of cytochrome c in the cytosol and mitochondrial fractions at different time points after adenoviral transduction. β-actin and Cox-IV were used as markers for cytoplasm and mitochondria, respectively.
FIGURE 6. Transmission electron microscopy of untransduced, AdGFP-transduced and AdSAT1-transduced 293T cells
TEM was performed on thin sections of 293T cells, untransduced at 48 h (panel set I), AdGFP-transduced at 48 h (Panel set II) or AdSAT1-transduced at 24, 48 and 72 h (panel sets III, IV and V, respectively). The images on the right side are at higher magnification. Mitochondria are indicated by arrows, nucleus by N, vacuole by V and lipid body by LB. Representative images are shown.

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