In previous studies, we have obtained evidence for inactivation of eIF5A by selective acetylation of eIF5A at hypusine residue by a polyamine catabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). Investigation of the molecular interaction between eIF5A and SSAT1 by coexpression in mammalian cells led us to study the functions of cellular polyamines as SSAT1 overexpression leads to total depletion of spermidine and spermine. Under this circumstance, we observed total inhibition of expression of GFP-eIF5A, GFP or any other cotransfected gene. The inhibition was at the level of translation but not at transcription, since GFP mRNA or GFP-eIF5A mRNA did not decrease upon cotransfection with SSAT1. Overexpression of SSAT1 in 293T cells by use of adenovirus resulted in a rapid depletion of spermidine and spermine, arrest in protein synthesis, inhibition of cell growth and apoptotic cell death. Addition of polyamine analogs, such as alpha-methylspermidine and dimethylspermine that are not substrates for SSAT1, restored translation and cell growth, providing evidence that depletion of polyamines caused growth arrest and cell death. Inhibition of polyamine oxidases by MDL72527 did not restore cell growth suggesting that growth arrest was not induced by oxidative stress resulting from accelerated polyamine catabolism. Annexin V staining and propidium iodide Fluorescent Activated Cell Sorter (FACS) analyses showed a clear increase in apoptosis at 48 h after AdSAT1 transduction. Nuclear fragmentation and an increase in caspase 3 activity, Poly ADP ribose polymerase (PARP) cleavage and release of cytochrome c from mitochondria into cytoplasm further suggest apoptosis by the intrinsic mitochondrial pathway in these polyamine-depleted cells. Moreover, electron microscopy images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including nuclear fragmentation, cytoplasm shrinkage, mitochondrial alteration, vacuolization and membrane blebbing. The effects of control GFP adenovirus (AdGFP) transduction were minor compared to those of AdSAT1 transduction, suggesting that the apoptosis was mainly caused by an over-expression of SAT1 rather than adenoviral transduction per se. These data provide strong evidence that the polyamines spermidine and spermine are vital for cell viability.
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