The role of polyamines in supporting growth of mammalian cells is mediated through their requirement for translation initiation and elongation

Abstract

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps. L-alpha-difluoromethylornithine treatment resulting in polyamine depletion reduces protein synthesis via inhibition of translation initiation. N1-guanyl-diaminoheptane (GC7), a spermidine analogue that inhibits eukaryotic initiation factor 5A (eIF5A) hypusination, also caused inhibition of translation initiation. In contrast, depletion of eIF5A by short hairpin RNA inhibits translation elongation as was recently demonstrated in yeast and Drosophila. These results suggest that in addition to competing with spermidine in the hypusination reaction, GC7 also competes with spermidine at yet undefined sites required for translation initiation. Finally, we show that either polyamine depletion or GC7 treatment induced eIF2alpha phosphorylation and reduced phosphorylation of 4E-BP, thus setting the molecular basis for the observed inhibition of translation initiation.

FIGURE 1.

Polyamine depletion arrests growth of mammalian cells and inhibits eIF5A hypusination. A, NIH3T3 cells were treated with DFMO (1 mm) and with GC7 (100 μm). Cells were counted at the indicated times (the presented results represent triplicate experiments). Error bars indicate S.D. B, NIH3T3 were treated with GC7 and DFMO for the indicated times, and cellular extracts were prepared and subjected to polyamine analysis as described under “Materials and Methods.” The position of the polyamine markers is indicated. Put, putrescine; Spd, spermidine; Spm, spermine. C, NIH3T3 cells were treated with GC7. At the indicated time points, cellular extracts were prepared and subjected to Western blot analysis using anti-Az antibodies. D, NIH3T3 cells were treated with GC7 for 3 days and with DFMO for 4 days. Cellular extracts were resolved in a slab isoelectric focusing gel (pH 3–7), electroblotted to nitrocellulose, and probed with anti-eIF5A antibodies.

FIGURE 2.

DFMO and GC7 inhibit translation at the initiation step. A, NIH3T3 cells were treated with DFMO or GC7. At the indicated time points, the cells were labeled with [³⁵S]methionine ([³⁵]S-Met), and radioactivity incorporated into proteins was determined by trichloroacetic acid precipitation (the presented results represent two experiments). Error bars indicate S.D.; PSL, photostimulus luminescence. B, NIH3T3 cells were treated with DFMO (for 4 days) or GC7 (for 3 days). Cycloheximide was added to the growth medium shortly before harvesting. Whole cell extracts were prepared and resolved on sucrose gradients to visualize the indicated ribosomal species. The ratio between polysomes and monosomes (P/M) was calculated by dividing the area under the polysomes by that under the 40 S, 60 S, and 80 S peaks. C, NIH3T3 cells were treated with arsenite (250 μm) for 40 min. Whole cell extracts were prepared and fractionated as in A. D, cells were either untreated or treated with cycloheximide (5 μg/ml) 30 min prior to their harvest. Whole cell extracts were prepared and fractionated as in A. E, control NIH3T3 cells and cells treated with GC7 (100 μm for 3 days) were labeled with 100 μCi/ml [³⁵S]methionine for 10 min before harvesting. Whole cell extracts were prepared and fractionated to obtain the polysomal profiles. Aliquots from the individual fractions were spotted on a filter paper, and the radioactivity incorporated into proteins was determined using a PhosphorImager. F, control and GC7-treated cells were harvested as described and fractionated in sucrose gradients containing 45 mm or 200 mm KCl.

FIGURE 3.

GC7 inhibits translation initiation without depleting cellular polyamines. A, control NIH3T3 cells stably transfected with empty vector (E) and NIH3T3 cells stably expressing trypanosome ODC (Tryp.ODC) were treated with GC7 (100 μm) for 3 days. Cellular extracts were prepared and fractionated in a slab isoelectric focusing gel as described in the legend for Fig. 1. B, control NIH3T3 and trypanosome ODC-expressing NIH3T3 cells were treated with GC7 (100 μm) for 3 days and subjected to polyamine analysis as in Fig. 1B. Put, putrescine; Spd, spermidine; Spm, spermine. C, control NIH3T3 and trypanosome ODC-expressing NIH3T3 cells were treated with GC7, and their growth rate was determined as in Fig. 1A (the presented results represent triplicate experiments). Error bars indicate S.D. D, wild type and trypanosome ODC-expressing NIH3T3 cells were treated with GC7 (100 μm for 3 days) and polysomal profiles were prepared as in Fig. 2A. P/M, the ratio between polysomes and monosomes.

FIGURE 4.

Depletion of eIF5A results in inhibition of translation elongation. A, NIH3T3 cells were infected with a lentivirus encoding shRNA to eIF5A. Cellular extracts were prepared at the indicated times, and the level of eIF5A was determined by Western blot analysis. B and C, cells were harvested 6 days after infection either with (B, +CHX) or without (C, −CHX) cycloheximide and fractionated on a sucrose gradient as in Fig. 2.

FIGURE 5.

Treatment with either GC7 or DFMO affects the phosphorylation state of 4E-BP and eIF2α. Control NIH3T3 cells, stably transfected with empty vector or trypanosome ODC-expressing (Tryp.ODC) NIH3T3 cells, were treated with GC7 (3 days) or DFMO (4 days). Cellular extracts were prepared and subjected to Western blot analysis using anti-eIF2α, anti-eIF2α-P (recognizing only its phosphorylated form), and anti-4E-BP. P indicates phosphorylation.

FIGURE 6.

The translation initiation block induced by DFMO or GC7 is developed from early time points. NIH3T3 cells were treated with DFMO (A and C) or GC7 (B and D). Cellular extracts were prepared at the indicated time points and used for determination of the phosphorylation (P) state of eIF2α and 4E-BP and the hypusination state of eIF5A (A and B) or for the preparation of polysomal profiles (C and D). Ctrl, control. P/M, the ratio between polysomes and monosomes.