Farnesol-induced cell death and stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in tobacco cv bright yellow-2 cells

Abstract

Growth inhibition of tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells by mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) could be partially overcome by the addition of farnesol. However, farnesol alone inhibited cell division and growth as measured by determination of fresh weight increase. When 7-d-old tobacco cv Bright Yellow-2 cells were diluted 40-fold into fresh culture, the cells exhibited a dose-dependent sensitivity to farnesol, with 25 microM sufficient to cause 100% cell death, as measured by different staining techniques, cytometry, and monitoring of fragmentation of genomic DNA. Cells were less sensitive to the effects of farnesol when diluted only 4-fold. Farnesol was absorbed by the cells, as examined by [1-(3)H]farnesol uptake, with a greater relative enrichment by the more diluted cells. Both mevinolin and farnesol treatments stimulated apparent HMGR activity. The stimulation by farnesol was also reflected in corresponding changes in the steady-state levels of HMGR mRNA and enzyme protein with respect to HMGR gene expression and enzyme protein accumulation.

Figure 1

Growth inhibition of TBY-2 suspension cells by farnesol treatment. A, Seven-day-old cells were subcultured (2–80 mL of modified Murashige and Skoog medium; dilution 1:41) and were grown for 1 week in the absence (control) or in the presence of increasing concentrations of farnesol. The 100% level corresponds to 32 g. B, Seven-day-old cells were subcultured (20–80 mL of modified Murashige and Skoog medium; dilution 1:5) and were grown for 48 h in the absence (control) or in the presence of increasing concentrations of farnesol. The 100% level corresponds to 8 g. Fresh weight was determined, compared with controls (100%), and normalized to percentage of growth. Mean of two to five replicates.

Figure 2

Dependence of farnesol-induced inhibition on initial cell density. A, Seven-day-old cells were subcultured (2, 5, 10, or 20 mL, respectively, was added to 80 mL of modified Murashige and Skoog medium, yielding dilution factors of 41-, 17-, 9-, and 5-fold, respectively) and were further cultivated for 4 d in the presence (+) or in the absence (−) of 25 μm farnesol.

Figure 3

Radiolabeling of 7-d-old cells for 48 h following subculture with 122 nCi/mL of [1-³H]farnesol. Data are expressed as percentage ratio between incorporation observed with highly diluted cells (2 mL added to 80 mL of modified Murashige and Skoog medium, 41-fold dilution) and with less diluted ones (20–80 mL, 5-fold dilution). Radioactivity was determined in aliquots of the cell juice and of the medium after centrifugation of cells.

Figure 4

Influence of farnesol treatment on apparent activity of microsome-bound HMGR. TBY-2 cells were treated for 48 h with different concentrations of farnesol before the isolation of membranes. A, Enzyme activity in TBY-2 cells treated by farnesol. Activity was always measured in the presence of 30 μm R,S-[3-¹⁴C]HMG-CoA (10-fold K_m) and of 30 μg of microsomal protein. Control, Untreated cells. 100% activity corresponds to 4.8 nmol mg⁻¹ h⁻¹ (80 pmol mg⁻¹ min⁻¹). Mean of three independent experiments. B, Western-blot analysis of HMGR protein. All lanes contained 15 μg of protein; an identical gel was run in parallel and used for silver staining to be sure that the intensity of individual protein bands was identical for each lane (not shown). C, Northern-blot analysis of HMGR RNA. All lanes contained 20 μg of total RNA. Intensity of major ribosomal RNA bands on the agarose gel was identical for each lane. *, Not determined.

Figure 5

Determination of HMGR activity in mevinolin- plus farnesol-treated cells. Microsomes were isolated from TBY-2 cells described in Table I and were used directly or buffer-washed before corresponding HMGR activity was measured. 100% activity (243.3 pmol mg⁻¹ min⁻¹) corresponds to that found with unwashed microsomes isolated from TBY-2 cells, which were treated with 5 μm mevinolin. Microsomes isolated from TBY-2 cells that were cultivated for 72 h in the absence of both mevinolin and farnesol showed an apparent HMGR activity of 126 pmol mg⁻¹ min⁻¹). The experiments were repeated three times, and sds are indicated.

Figure 6

Farnesol-induced cell death in TBY-2 suspension cells. A, Seven-day-old cells were subcultured (2–80 mL, dilution 41-fold) in the presence of different farnesol concentrations. After 5 d of culture the proportion of dead cells was determined. Cells were stained with fluorescein diacetate (specific for living cells) and propidium iodide (penetrates only into dead cells and stains nuclei orange-red). The percentage of dead cells corresponds to the proportion of red nuclei. Asterisks indicate values below 3% of dead cells, as was found in control cultures. B, Seven-day-old cells were subcultured by addition of 2, 5, 10, or 20 mL, respectively, to 80 mL of modified Murashige and Skoog medium (dilution factors of 41-, 17-, 9-, and 5-fold, respectively) containing 25 μm farnesol (final concentration). The percentage of dead cells was determined as in A.

Figure 7

Photographs of cells that were treated with 25 μm farnesol for 5 d. The culture was started by adding 10 mL of 7-d-old TBY-2 cells to 80 mL of new modified Murashige and Skoog medium. A through C, Same conditions showing differential effects on the induction of cell death and on morphological features. D, Control cells. Cells were doubly stained as described in Figure 6. The white bar in A corresponds to 100 μm in A and D, and to 50 μm in B and C. Under the conditions presented in A through C, 14% of total cells were dead and the total fresh weight of suspension cells was reduced from 23.5 (control) to 4.6 g. At the dilution of 2 and 5 mL of 7-d-old TBY-2 cells into 80 mL, 100% of cells were dead (only red nuclei were visible, data not shown).

Figure 8

Farnesol-induced degradation of genomic DNA in TBY-2 cells. Cells (20 mL) from a 7-d-old culture were added to 80 mL of modified Murashige and Skoog medium (5-fold dilution). A, Genomic DNA fragmentation induced by farnesol treatment (1, 100 μm, 48 h; 2, 1-kb ladder, Gibco-BRL, Cleveland). For comparison mevinolin-induced DNA degradation is included (3, untreated cells with intact genomic DNA; 4, 5 μm mevinolin, 24 h; 5, same, but 48 h; 6, same, but 72 h; 7, same, but 96 h; and 8, same, but 168 h). DNA was stained with ethidium bromide. B, Cytometric analysis of the nuclei from TBY-2 cells treated with farnesol. Percentage of cells in different phases of the cell cycle was calculated by automatic microscopic scanning of Feulgen-stained cells. Fol, Farnesol, 100 μm; Co, control cells without treatment. 2c and 4c, The values are presented as relative nDNA content per cell. Intermediate values are characteristic of cells in S phase (DNA synthesis), values below the window 2c (G0, G1) indicate DNA degradation. Analyses are based on 300 nuclei each.