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.

Fig. 1.

Lack of effect of SAT1 overexpression on mRNA levels of GFP and two endogenous genes. (A) Fold change in GFP and SAT1 mRNA levels compared with untransfected control shown by real-time PCR. Total RNA was isolated from HeLa cells using the RNeasy kit and reverse transcription was performed using ThermoScript reverse transcription-PCR kit (Invitrogen) in a 20-µL reaction mixture containing 500 ng purified RNA, according to the manufacturer’s instructions. PCR was performed in triplicates using the IQ SYBR Green Super mix (Bio-Rad) and the Bio-Rad Q-cycler machine, as follows: 50 °C for 2 min and 95 °C for 10 s followed by 40 cycles at 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 20 s. The generation of specific PCR products was confirmed by melting curve analysis. Error bars refer to SD for three independent experiments done in triplicate. GFP and SAT1 mRNA levels were normalized using GAPDH as an internal control. (B) RNA FISH image showing GFP mRNA (Q570, yellow) and SAT1 mRNA (Q670, red) in high and low magnifications. HeLa cells cultured on glass coverslips were washed with PBS, fixed in 3.7% (vol/vol) formaldehyde for 10 min at room temperature, washed twice with PBS, and permeabilized at 4 °C in 70% (vol/vol) EtOH for 1 h. A total of 1 μL of respective probe stock (12.5 μM) in 100 μL of hybridization solution was added and cells were incubated in a dark chamber at 37 °C overnight. After washing with the wash buffer [2× SSC with 10% (vol/vol) formamide], DAPI nuclear stain in PBS (5 ng/mL) was added to counterstain the nuclei. (C) RNA FISH of endogenous GAPDH and eIF5A-1 mRNAs (Q570, yellow) and SAT1 mRNA (Q670, red) in cotransfected HeLa cells. Images were obtained using a Zeiss LSM510 META inverted confocal system. pGFP, pCEFL/GFP; pEV, pCMV7.1. 3xFLAG empty vector; and pSAT1, pCMV7.1.3xFLAG/SAT1.

Fig. 2.

Fluorescence imaging of nascent synthesis of protein, RNA, and DNA in HeLa cells. HeLa cells were transfected with the empty vector (pEV) (Upper) or the FLAG-SAT1 vector (Lower). After 24 h of transfection, new syntheses of protein (A), RNA (B), and DNA (C) were imaged using three respective Click-IT assay kits (Invitrogen) by incubation with L-azidohomoalanine, 5-ethylene uridine, and 5-ethynyl-2′-deoxyuridine, respectively, for 30 min at 37 °C. They were fixed in 3.7% (vol/vol) formaldehyde for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and fluorescent labeling was performed by addition of Click-IT reaction mixtures. SAT1 overexpressing cells were visualized by counterimmunostaining with anti-FLAG antibody and are indicated by white arrows.

Fig. 3.

Effect of AdSAT1 and AdGFP cotransduction and the time course of overexpression of SAT1 upon AdSAT1 transduction. (A) GFP fluorescence and RNA FISH of GFP and SAT1 mRNAs. HEK293 cells were transduced with AdGFP alone or cotransduced with AdSAT1 and RNA FISH was performed at 24 h. (B) Time course of induction of SAT1 activity upon AdSAT1 transduction. SAT1 activity assays were performed as described under SI Materials and Methods using different amounts of cell lysate proteins (0.3–3 μg), 0.5 μCi [³H]ΑcCoA (2.53 Ci/mmol, 8 μM), and 1 mM spermidine. (C) Western blotting of lysates (50 μg proteins) of AdSAT1-transduced cells using SAT1 antibody (Santa Cruz) at the indicated time after transduction. β-Actin was used as a loading control. AdGFP-transduced and untransduced cell lysates showed no SAT1 signals at any time points under the same condition.

Fig. 4.

The temporal effects of AdGFP or AdSAT1 transduction on cell growth, protein synthesis, and polyamine content. HEK293 cells were transduced with AdGFP or AdSAT1 viruses. (A) Cell growth was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in triplicates. (B) Protein synthesis was measured in cells in 24-well dishes at indicated time points by measurement of radioactivity incorporated into TCA precipitable material after 2 h incubation in 0.5 mL medium containing 5 μCi of [³H]leucine (Left graph). Newly synthesized proteins of cells radiolabeled by incubation in leucine-free DMEM containing 10% (vol/vol) FBS and 20 μCi/mL of [³H]leucine for 1 h at 24 h of transduction was visualized by fluorography (Left) after SDS/PAGE. Immunoblotting of the same samples with antiadnovirus type 5 antibody is shown on the Right. (C) Cellular content of putrescine, spermidine, spermine, and N¹-acetylspermidine was measured as described in SI Materials and Methods. The experiments were carried out in duplicate and repeated two or three times with similar results. Representative data are shown.

Fig. 5.

The effects of polyamine analogs and APAO inhibitors on GFP expression or protein synthesis. (A) GFP expression in HeLa cells transfected with GFP vector alone or cotransfected with SAT1 vector. The APAO inhibitors, MDL72527 and MDL72521 (200 μM each), or polyamine analogs (10 μM) were added at the time of transfection and images were taken at 24 h. (B) Polyamines and their analogs (10 and 100 μM) were tested as substrates of SAT1 in a reaction mixture (25 μL containing 50 mM Tris⋅HCl, pH 8.0, 1 mM DTT, 10% (vol/vol) glycerol, 0.1 mM EDTA, 0.5 μCi [³H]AcCoA (3.62 Ci/mmol, 5.5 μM), and 20 ng of recombinant human SAT1). After incubation for 10 min at 30 °C, 10 μL of reaction mixture was spotted in duplicates on phosphocellulose P81 filter disk (Whatman; 2.5 cm diameter), and the filters were washed three times in water and the radioactivity on filters was measured. (C) Protein synthesis was measured as in Fig. 4 in HEK293 cells after 24 h of adenovirus transduction and incubation with or without 10 μM BENSpm. Spd, spermidine; Spm, spermine; Ptc, putrescine; MeSpd, methylspermidine; hSpd, symhomospermidine; Me₂Spm, 1,12-dimethyl spermine; cis, Spd, N-(3-aminopropyl)-1,4-diamino-cis-but-2-ene; GC7, N¹-monoguanyl-1,7-diaminoheptane; and BENSpm, N¹, N¹¹-bis(ethyl)norspermine.

Fig. 6.

Polysome profiles of HEK293 cells untransduced or transduced with AdGFP or AdSAT1 viruses. HEK293 cell lysates were prepared at 12 and 24 h after transduction and polysome profiles were obtained as described under SI Materials and Methods using 0.1 mm/s speed and Bio-Rad EM-1 flow cell at 254 nm. Representative set of profiles from three independent experiments is shown.