Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells

Abstract

The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromolecular synthesis in vitro, their specific functions in vivo are poorly understood. We investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N 1 -acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with GFP and SAT1 vectors suppressed GFP protein expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect is most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggests a direct role for polyamines in translation initiation. Our data provide strong evidence for a primary function of polyamines, spermidine and spermine, in translation in mammalian cells.[NH 2 (CH 2 ) 4 NH(CH 2 ) 3 NH 2 ], and spermine [NH 2 (CH 2 ) 3 NH (CH 2 ) 4 NH(CH 2 ) 3 NH 2 ], are ubiquitous in living organisms and are essential for eukaryotic cell proliferation (1-3). Because their primary and secondary amino groups are protonated at physiological pH in cells, these polycations interact with negatively charged molecules such as DNA, RNA, proteins, and phospholipids (4). Polyamines have been implicated in diverse biological processes, including replication, transcription, translation, posttranslational modification, ion channel gating, and membrane stability (4), and they regulate cellular proliferation, transformation, differentiation, apoptosis, and tumorigenesis (3, 5). Dysregulation of cellular polyamines is associated with various pathological conditions, including cancer, and polyamine pathways have been explored as targets for cancer chemotherapy and chemoprevention (5, 6). However, the precise physiological functions of polycationic polyamines in vivo and the mechanism of their actions in mammalian cell proliferation have remained largely obscure.One known critical function of polyamines in eukaryotes is the role of spermidine for the covalent modification of one cellular protein, eukaryotic initiation factor 5A (eIF5A), resulting in an unusual amino acid, hypusine [N e -(4-amino-2-hydroxybutyl) lysine] (7, 8). eIF5A and hypusine modification are absolutely required for the viability and growth of mammalian cells (9-12).The functions of polyamines in mammalian cells have b...

Activation of SAT1 engages polyamine metabolism with p53-mediated ferroptotic responses

“…Although other groups using transient transfection (27,43) previously reported that SAT1 overexpression causes rapid cell-growth arrest in HeLa cells and apoptosis in glioblastoma cells, we observed neither of these phenomena in our inducible expression system. These differing results may be attributable to the levels of SAT1 being expressed in cells, because the lower level of SAT1 expression induced by tetracycline may mimic the physiological condition better than transient transfection.…”

“…1A, SAT1 catalyzes the acetylation of spermidine and spermine to form N 1 -acetylspermidine and N 1 -acetylspermine, which then are either exported from the cells or converted back to putrescine or spermidine by N 1 -acetylpolyamine oxidase (26). Therefore, overexpression of SAT1 leads to an overall depletion of spermidine and spermine while increasing the levels of putrescine, N 1 -acetylspermidine, and N 1 -acetylspermine (27). In fact, N 1 , N 11 -di(ethyl)norspermine (DENSpm), a polyamine analog that is known to induce SAT1, has been applied in several clinical trials in patients with advanced malignancies (28)(29)(30)(31).…”

“…SAT1 is a key polyamine catabolism enzyme that mediates the acetylation of spermidine and spermine. Overexpression of SAT1 has been shown to cause a rapid depletion of the polyamine pool (27). To investigate the effect of SAT1 on cell proliferation and survival in a physiological setting, we generated a SAT1 Tet-on cell line using p53-null H1299 cells.…”

The effects of putrescine are partly overlapping with osmotic stress processes in wheat

“…Involvement of PAs in plant responses to environmental stimuli has been demonstrated by several studies. The protective effects of elevated endogenous level (either due to exogenous PA treatment or to transgenic modification) have been reported in several plant species under various stress conditions [1][2][3][4][5][6][7][8][9][10][11]. Besides their direct protective role PAs regulate fundamental cellular processes as signalling molecules, suggesting that abiotic stress tolerance is mainly influenced via signalling processes rather than by their accumulation [12].…”

Tailoring recombinant protein quality by rational media design

“…The literature has reported a link between spermine and growth. Depletion of spermine and spermidine led to an entire arrest in translation and growth in HEK293 cells, and a direct role for polyamines in the initiation of translation was suggested 240 . Other scientists observed that polyamines were needed at the initiation and at the elongation steps of translation: they showed that spermine is a precursor in the hypusination of the eukaryotic initiation factor 5A (eIF5A) that acts as a translation elongation factor 241 .…”