Polyamine Depletion Reduces TNF/MG132-Induced Apoptosis in Bone Marrow Stromal Cells
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《干细胞学杂志》
Department of Biochemistry "G. Moruzzi," University of Bologna, Bologna, Italy
Key Words. Mesenchymal stem cells ? Apoptosis ? Caspase ? p53 ? Polyamines
Correspondence: Claudio Muscari, M.D., Department of Biochemistry "G. Moruzzi," University of Bologna, Via Irnerio 48, 40126 Bologna, Italy. Telephone: 0039-0512091245; Fax: 0039-0512091245; e-mail: claudio.muscari@unibo.it
ABSTRACT
Mesenchymal stem cells represent a self-renewing stem-cell population that can be isolated from various tissues and differentiated into several cell lineages, especially after homing at damaged tissues . However, a constant outcome that dramatically impairs the efficacy of their engraftment is the limited number of stem cells surviving after transplantation . Mangi et al. have recently shown that approximately 70% of mesenchymal stem cells injected into the border zone of the ischemic left ventricle of rat hearts died of apoptosis within 24 hours . They also demonstrated that in mesenchymal stem cells engineered with Akt, a survival gene , apoptosis was markedly reduced within the first day of their injection in the infarcted area, and both systolic and diastolic cardiac functions were normalized. Therefore, protecting mesenchymal stem cells from apoptosis should consistently improve their survival during the early phase of graft, thereby ameliorating the overall process of tissue regeneration. However, because stimulation of Akt is often paralleled by increased cell proliferation, it is not known whether Akt-gene transfection could lead mesenchymal stem cells to uncontrolled growth or malignant transformation .
To investigate novel conditions useful for protecting mesenchymal stem cells against apoptosis, we attempted to confer increased resistance against cell death by depleting them of polyamines. Excessive polyamine levels trigger apoptosis , just as polyamine depletion can protect cells exposed to death signals under different experimental conditions . Apoptosis could be partially mediated by polyamines according to several proposed mechanisms, such as the production of hydrogen peroxide during their catabolism or the stimulation of cytochrome-c release from mitochondria . However, the actual role of polyamines in apoptosis is more complex and not yet completely defined. Indeed, several studies have shown that polyamines can also protect cells from apoptosis or that depleting cells of polyamines leads to cell death . In the latter, the activity of polyamines appears to be consistent with their growth-stimulatory effects and promotion of cell cycle.
In this study, we show that polyamine depletion protected bone marrow stromal cells (BMSCs), which comprise mesenchymal stem cells, against apoptosis induced by simultaneous treatment with tumor necrosis factor- (TNF) and a proteasome inhibitor. Cells were pretreated with -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), which is the rate-limiting enzyme in polyamine biosynthesis . We chose DFMO from among various compounds able to decrease intracellular polyamine concentrations because its pharmacological properties have already been widely investigated in several cells and in vivo systems, showing a very low toxicity . Moreover, DFMO is extremely effective in reducing the contents of putrescine and spermidine in cultured cells, and the intracellular levels of these polyamines remain low for few days even after DFMO removal .
MATERIALS AND METHODS
Adherent Bone Marrow Cells Show Phenotypic Features of BMSCs and Can Be Committed to Mesenchymal Lineages
After 10–12 days of culture expansion on polystyrene surface, adherent bone marrow cells did not express the hematopoietic markers CD34 and CD45, nor a specific mononuclear phagocyte marker (Fig. 1). On the contrary, a very low positivity to CD59, an Sca-1 homologue, was observed, whereas 53% of total BMSCs were positive to CD90, a superficial antigen expressed by rat BMSCs .
Figure 1. Antigenic characterization of BMSCs. CD90 and CD59 superficial antigens were first determined by immunofluorescence and then quantified by FACS analysis. CD34, CD45, and MP marker were determined only by immunofluorescence because BMSCs resulted negative to these antigens. After 11 days from seeding, adherent bone marrow cells were mostly positive to CD90, dimly positive to CD59, and completely negative to the other tested hematopoietic cell markers. Abbreviations: BMSC, bone marrow stromal cell; FACS, fluorescent-activated cell sorting; MP, mononuclear phagocyte.
To assess whether adherent cells were really prone to differentiate into mesenchymal lineages, we grew them in the osteogenic medium, inducing a marked production of calcium minerals after 2 weeks, as shown by the positivity to Alizarin red staining (Fig. 2A). Figure 2B shows that BMSCs can also differentiate into myocyte phenotype when exposed to 5 μM 5-azacytidine. The protein expression of desmin, a specific marker for both striate and smooth muscle cells, was observed after 3 weeks of the differentiating treatment.
Figure 2. Differentiation of BMSCs into mesenchymal lineages. (A): Osteogenic differentiation was demonstrated by intense Alizarin red staining revealing mineral deposition only in the cultures treated with the osteogenic medium for 2 weeks. (B): Myogenic differentiation was shown by desmin expression after 3 weeks from 5 μM 5-azacytidine treatment of 24 hours. Desmin was determined at the end of the treatment by Western blotting, using a gut polyclonal primary antibody. Abbreviation: BMSC, bone marrow stromal cell.
ODC Inhibition Results in Depletion of Intracellular Polyamines
Figure 3 shows that 1 mM DFMO treatment of BMSCs for 24 hours resulted in a decrease of the intracellular levels of putrescine and spermidine by approximately 70% and 40%, respectively. The intracellular levels of both putrescine and spermidine continued to decline after 48 hours, reaching almost undetectable levels after 48 hours. Higher concentrations of DFMO (up to 5 mM) did not accentuate this effect, where as lower concentrations (0.1–0.5 mM) were slightly less effective in decreasing polyamine contents (data not shown). The treatment with DFMO for 72 hours did not further modify the levels of these polyamines. On the contrary, the ODC inhibitor did not substantially affect spermine concentration in BMSCs, in accordance with similar results obtained under various conditions of DFMO treatment .
Figure 3. Time-dependent changes in polyamine concentrations after treatment of BMSCs with DFMO. The treatment of BMSCs with 1 mM DFMO almost completely depleted the cells of putrescine and spermidine after 48 hours, whereas spermine concentration was not reduced with respect to control. One mM DFMO treatment of 72 hours did not further decrease intracellular polyamine levels. The polyamine contents of BMSCs were determined by high-performance liquid chromatography after derivatization with dansyl chloride. Values represent the mean ± SEM of duplicate experiments and are expressed as a percentage of the corresponding value obtained at each selected time from untreated BMSCs (100%). Abbreviations: BMSC, bone marrow stromal cell; DFMO, -difluoromethylornithine.
TNF Treatment, Together with Proteasome Inhibition, Increases Caspase-3 Activity
The presence of 500 U/ml TNF alone did not induce any increase in caspase-3 activity in BMSCs over a period of 24 hours (Fig. 4). On the contrary, the combined effect of 500 U/ml TNF with 5 μM MG132, an inhibitor of proteasome, stimulated caspase-3 activity in a time-dependent manner and increased the basal value approximately fourfold after 24 hours of treatment. MG132 by itself also increased caspase-3 activity, but to a lesser extent, suggesting that TNF together with MG132 exerted synergistic effects.
Figure 4. Stimulation of caspase-3 activity by treatment of BMSCs with TNF and MG132. BMSCs were treated with 500 U/ml TNF, or 5 μM MG132, or both, over a period of 24 hours. Caspase-3 activity was then measured using Ac-DEVD-AMC as a fluorigenic substrate. The treatment with only TNF (500 U/ml) was not able to increase caspase-3 activity in BMSCs, but a synergistic effect was observed when, in addition to this cytokine, 5 μM MG132 was also present in the culture medium. MG132 by itself could also stimulate caspase-3 activity, but to a lesser extent with respect to the effect obtained together with TNF. Values represent the mean ± SEM of duplicate experiments. The one-way analysis of variance, followed by Bonferroni’s post hoc test, was applied to compare the values obtained at each selected time. * p < .05, ** p < .01 versus the corresponding control value. # p < .05 versus the corresponding MG132 value. Abbreviations: BMSC, bone marrow stromal cell; TNF, tumor necrosis factor-.
Polyamine Depletion Inhibits Caspase-3 Activity in Both Untreated and TNF/MG132-Stimulated BMSCs
Direct addition of 1 mM DFMO to BMSC suspension did not affect caspase-3 activity, whereas 48 hours of 1 mM DFMO treatment decreased its basal enzymatic activity (63.3% ± 5.93% of control). The inhibiting effect of DFMO on caspase-3 activity was also observed after 8, 16, and 24 hours of stimulation with TNF/MG132 (Fig. 5A), suggesting that polyamine depletion elicited a marked and sustained antiapoptotic effect. The addition of 100 μM putrescine to BMSCs, pretreated with DFMO and stimulated for an additional 24 hours with TNF/MG132, restored the activity of caspase-3 over the value obtained in the presence of TNF/MG132 alone (839.5% ± 64.5% and 459.0% ± 60.4% of control, respectively). These results confirmed that the protective effect of DFMO was actually related to its ability to deplete BMSCs of polyamines.
Figure 5. Effect of intracellular polyamine depletion on caspase-3 activity. (A): One mM DFMO significantly reduced caspase-3 activity in BMSCs stimulated with 500 U/ml TNF and 5 μM MG132 for 8, 16, and 24 hours. DFMO was preadministered for 48 hours and remained in the culture medium for the whole period of TNF/MG132 treatment. Values represent the mean ± SEM of 8 to 12 separate experiments and are expressed as a percentage of the corresponding untreated control values obtained at each selected time (100%). * p < .05, ** p < .01 versus TNF/MG132. (B): One mM DFMO pretreatment for 48 hours also reduced TNF/MG132-induced apoptosis as determined by TUNEL assay. Values are means ± SEM of duplicate experiments. * p < .05 versus control, #p < .05 versus TNF/MG132. Abbreviations: BMSC, bone marrow stromal cell; DFMO, -difluoromethylornithine; TNF, tumor necrosis factor-; TUNEL, terminal transferase-mediated dUTP nick end-labeling.
Polyamine-Depleted BMSCs Are Protected Against Death Stimuli
Quantification of apoptotic cells by TUNEL assay supported the hypothesis that polyamine depletion significantly protected BMSCs against programmed cell death during TNF/MG132 stimulation. Figure 5B shows that 24 hours of TNF/MG132 treatment made positive to TUNEL 16.9% ± 4.4% of total BMSCs, but only 6.45% ± 1.05% of these cells became apoptotic after polyamine depletion by DFMO. DFMO treatment also prevented general cell death in TNF/MG132-stimulated BMSCs, because the number of BMSCs stained with trypan blue, a dye excluded by viable cells, was approximately 50% lower after polyamine depletion (data not shown).
MG132 Increases the Level of p53 in BMSCs
The proteasome inhibition induced by MG132 alone led to stimulation of caspase-3 activity. We tested the hypothesis that this effect was related to apoptosis that usually follows inhibition of p53 protein degradation. It is known that the turnover of p53 is highly regulated by its presentation to proteasome after the formation of murine double minute-2 (MDM2)/p53 complex, which can be subsequently ubiquitinated and then degraded . We showed that control BMSCs were negative to p53 immunostaining, especially at the nuclear level, whereas just after 8 hours of stimulation with 5 μM MG132, they became markedly positive to this proapoptotic factor (Fig. 6). No further increase in p53 abundance was observed when TNF was added together with MG132 for 8 hours; TNF alone gave results similar to control. Moreover, DFMO did not prevent the increase in p53 concentration either in MG132- or TNF/MG132-treated BMSCs, suggesting that the protective effect of polyamine depletion against apoptosis was not due to a reduction in the level of p53.
Figure 6. p53 protein expression in BMSCs treated with DFMO, TNF, and MG132. This is a representative figure of triplicate experiments showing that both control BMSCs and BMSCs treated for 8 hours with TNF did not present elevated concentrations of p53, especially at the nuclear level. On the contrary, 5 μM MG132 treatment of 8 hours remarkably increased p53 abundance, whereas the simultaneous addition of 500 U/ml TNF did not cause any further modification of p53 concentration. Also, the amount of p53 in BMSCs stimulated for 8 hours with MG132 alone or TNF/MG132 was not affected by pretreatment with 1 mM DFMO. Immunostaining of p53 was performed using a mouse monoclonal antibody together with an anti-immunoglobulin G secondary antibody conjugated with Cy3 (magnification x20). Abbreviations: BMSC, bone marrow stromal cell; CTR, control; DFMO, -difluoromethylornithine; TNF, tumor necrosis factor-
Polyamine Depletion Attenuates the Rate of BMSC Proliferation but Does Not Reduce Their Differentiation Potential
In most cell types, polyamine depletion leads to inhibition of proliferation, and we observed this effect also in BMSCs (Fig. 7A). The rate of BMSC proliferation was significantly reduced with respect to control just after 1 mM DFMO treatment of 48 hours, corresponding to the maximal depletion of both putrescine and spermidine (Fig. 3). Anyway, the inhibition of growth was not complete, and this may be due to the inability to also reduce the intracellular level of spermine.
Figure 7. Effect of polyamine depletion on BMSC proliferation and differentiation. (A): One mM DFMO was added to the culture medium over a period of 72 hours. Cell proliferation was estimated by quantifying cell number through the MTT assay described in Materials and Methods. DFMO decreased the rate of proliferation of BMSCs. The positivity to MTT became significantly lower in DFMO-treated BMSCs than control after 48 hours of treatment. Values are expressed as means ± SEM of quadruplicate experiments. *p < .05 versus control evaluated at the same time. (B): Cyclin D2 protein expression was determined by Western blotting using a rabbit polyclonal primary antibody. The analysis was performed in BMSCs after 1 mM DFMO treatment of 72 hours. (C): Cell cycle was investigated by cytofluorimetry after 72 hours of 1 mM DFMO treatment. X-axis indicates fluorescence intensity of DNA, and y-axis the absolute cell number. Inside are expressed the percentage of BMSCs in the following cell cycle phases: G = G0/G1, H = S, I = G2/M. (D): In the first dish, BMSCs were depleted of polyamines by 48 hours of treatment with 1 mM DFMO, washed, and then expanded in normal medium. The second culture of BMSCs grew for 3 weeks in the osteogenic medium described in Materials and Methods. The third culture of BMSCs was treated with 1 mM DFMO for 48 hours, washed, and then expanded in the same osteogenic medium for 3 weeks. Abbreviations: BMSC, bone marrow stromal cell; DFMO, -difluoromethylornithine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide.
Cyclin D2 expression (Fig. 7B) was partially reduced after DFMO treatment, showing that polyamine depletion could affect the cell cycle, at least in part, by inhibiting progression through G1. Cell-cycle analysis by FACS (Fig. 7C) showed that the percentage of BMSCs in G0/G1 increased only slightly after DFMO treatment with respect to control cells. Besides, it is well known that the effect of DFMO on cell proliferation is reversible upon removal of the inhibitor . Therefore, these data suggest that there was only a small amount of cells blocked in G0/G1 after DFMO administration with respect to control and, therefore, that BMSCs were still able to ensure a substantial self-renewing activity. Furthermore, a transient DFMO treatment neither reduced the ability of BMSCs to differentiate, nor induced spontaneous differentiation. Figure 7D shows that polyamine depletion did not attenuate the osteogenic differentiation of BMSCs after 3 weeks from 1 mM DFMO treatment of 48 hours. Moreover, DFMO administration alone was not sufficient to commit BMSCs into osteoblastic phenotype.
DISCUSSION
This research was supported with grants from MIUR (FIRB 2001), Rome, and Compagnia di San Paolo, Turin, Italy. We are very grateful to Prof. J. Prockop (Tulane University Health Sciences Center, New Orleans) for helpful suggestions, especially about BMSC isolation and characterization. We also thank Mr. Massimo Sgarbi for his technical assistance.
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Key Words. Mesenchymal stem cells ? Apoptosis ? Caspase ? p53 ? Polyamines
Correspondence: Claudio Muscari, M.D., Department of Biochemistry "G. Moruzzi," University of Bologna, Via Irnerio 48, 40126 Bologna, Italy. Telephone: 0039-0512091245; Fax: 0039-0512091245; e-mail: claudio.muscari@unibo.it
ABSTRACT
Mesenchymal stem cells represent a self-renewing stem-cell population that can be isolated from various tissues and differentiated into several cell lineages, especially after homing at damaged tissues . However, a constant outcome that dramatically impairs the efficacy of their engraftment is the limited number of stem cells surviving after transplantation . Mangi et al. have recently shown that approximately 70% of mesenchymal stem cells injected into the border zone of the ischemic left ventricle of rat hearts died of apoptosis within 24 hours . They also demonstrated that in mesenchymal stem cells engineered with Akt, a survival gene , apoptosis was markedly reduced within the first day of their injection in the infarcted area, and both systolic and diastolic cardiac functions were normalized. Therefore, protecting mesenchymal stem cells from apoptosis should consistently improve their survival during the early phase of graft, thereby ameliorating the overall process of tissue regeneration. However, because stimulation of Akt is often paralleled by increased cell proliferation, it is not known whether Akt-gene transfection could lead mesenchymal stem cells to uncontrolled growth or malignant transformation .
To investigate novel conditions useful for protecting mesenchymal stem cells against apoptosis, we attempted to confer increased resistance against cell death by depleting them of polyamines. Excessive polyamine levels trigger apoptosis , just as polyamine depletion can protect cells exposed to death signals under different experimental conditions . Apoptosis could be partially mediated by polyamines according to several proposed mechanisms, such as the production of hydrogen peroxide during their catabolism or the stimulation of cytochrome-c release from mitochondria . However, the actual role of polyamines in apoptosis is more complex and not yet completely defined. Indeed, several studies have shown that polyamines can also protect cells from apoptosis or that depleting cells of polyamines leads to cell death . In the latter, the activity of polyamines appears to be consistent with their growth-stimulatory effects and promotion of cell cycle.
In this study, we show that polyamine depletion protected bone marrow stromal cells (BMSCs), which comprise mesenchymal stem cells, against apoptosis induced by simultaneous treatment with tumor necrosis factor- (TNF) and a proteasome inhibitor. Cells were pretreated with -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), which is the rate-limiting enzyme in polyamine biosynthesis . We chose DFMO from among various compounds able to decrease intracellular polyamine concentrations because its pharmacological properties have already been widely investigated in several cells and in vivo systems, showing a very low toxicity . Moreover, DFMO is extremely effective in reducing the contents of putrescine and spermidine in cultured cells, and the intracellular levels of these polyamines remain low for few days even after DFMO removal .
MATERIALS AND METHODS
Adherent Bone Marrow Cells Show Phenotypic Features of BMSCs and Can Be Committed to Mesenchymal Lineages
After 10–12 days of culture expansion on polystyrene surface, adherent bone marrow cells did not express the hematopoietic markers CD34 and CD45, nor a specific mononuclear phagocyte marker (Fig. 1). On the contrary, a very low positivity to CD59, an Sca-1 homologue, was observed, whereas 53% of total BMSCs were positive to CD90, a superficial antigen expressed by rat BMSCs .
Figure 1. Antigenic characterization of BMSCs. CD90 and CD59 superficial antigens were first determined by immunofluorescence and then quantified by FACS analysis. CD34, CD45, and MP marker were determined only by immunofluorescence because BMSCs resulted negative to these antigens. After 11 days from seeding, adherent bone marrow cells were mostly positive to CD90, dimly positive to CD59, and completely negative to the other tested hematopoietic cell markers. Abbreviations: BMSC, bone marrow stromal cell; FACS, fluorescent-activated cell sorting; MP, mononuclear phagocyte.
To assess whether adherent cells were really prone to differentiate into mesenchymal lineages, we grew them in the osteogenic medium, inducing a marked production of calcium minerals after 2 weeks, as shown by the positivity to Alizarin red staining (Fig. 2A). Figure 2B shows that BMSCs can also differentiate into myocyte phenotype when exposed to 5 μM 5-azacytidine. The protein expression of desmin, a specific marker for both striate and smooth muscle cells, was observed after 3 weeks of the differentiating treatment.
Figure 2. Differentiation of BMSCs into mesenchymal lineages. (A): Osteogenic differentiation was demonstrated by intense Alizarin red staining revealing mineral deposition only in the cultures treated with the osteogenic medium for 2 weeks. (B): Myogenic differentiation was shown by desmin expression after 3 weeks from 5 μM 5-azacytidine treatment of 24 hours. Desmin was determined at the end of the treatment by Western blotting, using a gut polyclonal primary antibody. Abbreviation: BMSC, bone marrow stromal cell.
ODC Inhibition Results in Depletion of Intracellular Polyamines
Figure 3 shows that 1 mM DFMO treatment of BMSCs for 24 hours resulted in a decrease of the intracellular levels of putrescine and spermidine by approximately 70% and 40%, respectively. The intracellular levels of both putrescine and spermidine continued to decline after 48 hours, reaching almost undetectable levels after 48 hours. Higher concentrations of DFMO (up to 5 mM) did not accentuate this effect, where as lower concentrations (0.1–0.5 mM) were slightly less effective in decreasing polyamine contents (data not shown). The treatment with DFMO for 72 hours did not further modify the levels of these polyamines. On the contrary, the ODC inhibitor did not substantially affect spermine concentration in BMSCs, in accordance with similar results obtained under various conditions of DFMO treatment .
Figure 3. Time-dependent changes in polyamine concentrations after treatment of BMSCs with DFMO. The treatment of BMSCs with 1 mM DFMO almost completely depleted the cells of putrescine and spermidine after 48 hours, whereas spermine concentration was not reduced with respect to control. One mM DFMO treatment of 72 hours did not further decrease intracellular polyamine levels. The polyamine contents of BMSCs were determined by high-performance liquid chromatography after derivatization with dansyl chloride. Values represent the mean ± SEM of duplicate experiments and are expressed as a percentage of the corresponding value obtained at each selected time from untreated BMSCs (100%). Abbreviations: BMSC, bone marrow stromal cell; DFMO, -difluoromethylornithine.
TNF Treatment, Together with Proteasome Inhibition, Increases Caspase-3 Activity
The presence of 500 U/ml TNF alone did not induce any increase in caspase-3 activity in BMSCs over a period of 24 hours (Fig. 4). On the contrary, the combined effect of 500 U/ml TNF with 5 μM MG132, an inhibitor of proteasome, stimulated caspase-3 activity in a time-dependent manner and increased the basal value approximately fourfold after 24 hours of treatment. MG132 by itself also increased caspase-3 activity, but to a lesser extent, suggesting that TNF together with MG132 exerted synergistic effects.
Figure 4. Stimulation of caspase-3 activity by treatment of BMSCs with TNF and MG132. BMSCs were treated with 500 U/ml TNF, or 5 μM MG132, or both, over a period of 24 hours. Caspase-3 activity was then measured using Ac-DEVD-AMC as a fluorigenic substrate. The treatment with only TNF (500 U/ml) was not able to increase caspase-3 activity in BMSCs, but a synergistic effect was observed when, in addition to this cytokine, 5 μM MG132 was also present in the culture medium. MG132 by itself could also stimulate caspase-3 activity, but to a lesser extent with respect to the effect obtained together with TNF. Values represent the mean ± SEM of duplicate experiments. The one-way analysis of variance, followed by Bonferroni’s post hoc test, was applied to compare the values obtained at each selected time. * p < .05, ** p < .01 versus the corresponding control value. # p < .05 versus the corresponding MG132 value. Abbreviations: BMSC, bone marrow stromal cell; TNF, tumor necrosis factor-.
Polyamine Depletion Inhibits Caspase-3 Activity in Both Untreated and TNF/MG132-Stimulated BMSCs
Direct addition of 1 mM DFMO to BMSC suspension did not affect caspase-3 activity, whereas 48 hours of 1 mM DFMO treatment decreased its basal enzymatic activity (63.3% ± 5.93% of control). The inhibiting effect of DFMO on caspase-3 activity was also observed after 8, 16, and 24 hours of stimulation with TNF/MG132 (Fig. 5A), suggesting that polyamine depletion elicited a marked and sustained antiapoptotic effect. The addition of 100 μM putrescine to BMSCs, pretreated with DFMO and stimulated for an additional 24 hours with TNF/MG132, restored the activity of caspase-3 over the value obtained in the presence of TNF/MG132 alone (839.5% ± 64.5% and 459.0% ± 60.4% of control, respectively). These results confirmed that the protective effect of DFMO was actually related to its ability to deplete BMSCs of polyamines.
Figure 5. Effect of intracellular polyamine depletion on caspase-3 activity. (A): One mM DFMO significantly reduced caspase-3 activity in BMSCs stimulated with 500 U/ml TNF and 5 μM MG132 for 8, 16, and 24 hours. DFMO was preadministered for 48 hours and remained in the culture medium for the whole period of TNF/MG132 treatment. Values represent the mean ± SEM of 8 to 12 separate experiments and are expressed as a percentage of the corresponding untreated control values obtained at each selected time (100%). * p < .05, ** p < .01 versus TNF/MG132. (B): One mM DFMO pretreatment for 48 hours also reduced TNF/MG132-induced apoptosis as determined by TUNEL assay. Values are means ± SEM of duplicate experiments. * p < .05 versus control, #p < .05 versus TNF/MG132. Abbreviations: BMSC, bone marrow stromal cell; DFMO, -difluoromethylornithine; TNF, tumor necrosis factor-; TUNEL, terminal transferase-mediated dUTP nick end-labeling.
Polyamine-Depleted BMSCs Are Protected Against Death Stimuli
Quantification of apoptotic cells by TUNEL assay supported the hypothesis that polyamine depletion significantly protected BMSCs against programmed cell death during TNF/MG132 stimulation. Figure 5B shows that 24 hours of TNF/MG132 treatment made positive to TUNEL 16.9% ± 4.4% of total BMSCs, but only 6.45% ± 1.05% of these cells became apoptotic after polyamine depletion by DFMO. DFMO treatment also prevented general cell death in TNF/MG132-stimulated BMSCs, because the number of BMSCs stained with trypan blue, a dye excluded by viable cells, was approximately 50% lower after polyamine depletion (data not shown).
MG132 Increases the Level of p53 in BMSCs
The proteasome inhibition induced by MG132 alone led to stimulation of caspase-3 activity. We tested the hypothesis that this effect was related to apoptosis that usually follows inhibition of p53 protein degradation. It is known that the turnover of p53 is highly regulated by its presentation to proteasome after the formation of murine double minute-2 (MDM2)/p53 complex, which can be subsequently ubiquitinated and then degraded . We showed that control BMSCs were negative to p53 immunostaining, especially at the nuclear level, whereas just after 8 hours of stimulation with 5 μM MG132, they became markedly positive to this proapoptotic factor (Fig. 6). No further increase in p53 abundance was observed when TNF was added together with MG132 for 8 hours; TNF alone gave results similar to control. Moreover, DFMO did not prevent the increase in p53 concentration either in MG132- or TNF/MG132-treated BMSCs, suggesting that the protective effect of polyamine depletion against apoptosis was not due to a reduction in the level of p53.
Figure 6. p53 protein expression in BMSCs treated with DFMO, TNF, and MG132. This is a representative figure of triplicate experiments showing that both control BMSCs and BMSCs treated for 8 hours with TNF did not present elevated concentrations of p53, especially at the nuclear level. On the contrary, 5 μM MG132 treatment of 8 hours remarkably increased p53 abundance, whereas the simultaneous addition of 500 U/ml TNF did not cause any further modification of p53 concentration. Also, the amount of p53 in BMSCs stimulated for 8 hours with MG132 alone or TNF/MG132 was not affected by pretreatment with 1 mM DFMO. Immunostaining of p53 was performed using a mouse monoclonal antibody together with an anti-immunoglobulin G secondary antibody conjugated with Cy3 (magnification x20). Abbreviations: BMSC, bone marrow stromal cell; CTR, control; DFMO, -difluoromethylornithine; TNF, tumor necrosis factor-
Polyamine Depletion Attenuates the Rate of BMSC Proliferation but Does Not Reduce Their Differentiation Potential
In most cell types, polyamine depletion leads to inhibition of proliferation, and we observed this effect also in BMSCs (Fig. 7A). The rate of BMSC proliferation was significantly reduced with respect to control just after 1 mM DFMO treatment of 48 hours, corresponding to the maximal depletion of both putrescine and spermidine (Fig. 3). Anyway, the inhibition of growth was not complete, and this may be due to the inability to also reduce the intracellular level of spermine.
Figure 7. Effect of polyamine depletion on BMSC proliferation and differentiation. (A): One mM DFMO was added to the culture medium over a period of 72 hours. Cell proliferation was estimated by quantifying cell number through the MTT assay described in Materials and Methods. DFMO decreased the rate of proliferation of BMSCs. The positivity to MTT became significantly lower in DFMO-treated BMSCs than control after 48 hours of treatment. Values are expressed as means ± SEM of quadruplicate experiments. *p < .05 versus control evaluated at the same time. (B): Cyclin D2 protein expression was determined by Western blotting using a rabbit polyclonal primary antibody. The analysis was performed in BMSCs after 1 mM DFMO treatment of 72 hours. (C): Cell cycle was investigated by cytofluorimetry after 72 hours of 1 mM DFMO treatment. X-axis indicates fluorescence intensity of DNA, and y-axis the absolute cell number. Inside are expressed the percentage of BMSCs in the following cell cycle phases: G = G0/G1, H = S, I = G2/M. (D): In the first dish, BMSCs were depleted of polyamines by 48 hours of treatment with 1 mM DFMO, washed, and then expanded in normal medium. The second culture of BMSCs grew for 3 weeks in the osteogenic medium described in Materials and Methods. The third culture of BMSCs was treated with 1 mM DFMO for 48 hours, washed, and then expanded in the same osteogenic medium for 3 weeks. Abbreviations: BMSC, bone marrow stromal cell; DFMO, -difluoromethylornithine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide.
Cyclin D2 expression (Fig. 7B) was partially reduced after DFMO treatment, showing that polyamine depletion could affect the cell cycle, at least in part, by inhibiting progression through G1. Cell-cycle analysis by FACS (Fig. 7C) showed that the percentage of BMSCs in G0/G1 increased only slightly after DFMO treatment with respect to control cells. Besides, it is well known that the effect of DFMO on cell proliferation is reversible upon removal of the inhibitor . Therefore, these data suggest that there was only a small amount of cells blocked in G0/G1 after DFMO administration with respect to control and, therefore, that BMSCs were still able to ensure a substantial self-renewing activity. Furthermore, a transient DFMO treatment neither reduced the ability of BMSCs to differentiate, nor induced spontaneous differentiation. Figure 7D shows that polyamine depletion did not attenuate the osteogenic differentiation of BMSCs after 3 weeks from 1 mM DFMO treatment of 48 hours. Moreover, DFMO administration alone was not sufficient to commit BMSCs into osteoblastic phenotype.
DISCUSSION
This research was supported with grants from MIUR (FIRB 2001), Rome, and Compagnia di San Paolo, Turin, Italy. We are very grateful to Prof. J. Prockop (Tulane University Health Sciences Center, New Orleans) for helpful suggestions, especially about BMSC isolation and characterization. We also thank Mr. Massimo Sgarbi for his technical assistance.
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