Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online June 7, 2005
Stem Cells Vol. 23 No. 7 August 2005, pp. 983 -991
doi:10.1634/stemcells.2004-0240; www.StemCells.com
© 2005 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0240v1
23/7/983    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muscari, C.
Right arrow Articles by Caldarera, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Muscari, C.
Right arrow Articles by Caldarera, C. M.

Polyamine Depletion Reduces TNF{alpha}/MG132-Induced Apoptosis in Bone Marrow Stromal Cells

Claudio Muscari, Francesca Bonafé, Ivana Stanic, Flavio Flamigni, Claudio Stefanelli, Giovanna Farruggia, Carlo Guarnieri, Claudio Marcello Caldarera

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{at}unibo.it


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polyamines are powerful modulators of both growth and survival in mammalian cells. In this study, we investigated the possibility of attenuating the process of apoptosis in bone marrow stromal cells (BMSCs), which comprise mesenchymal stem cells, by reducing the intracellular levels of polyamines. BMSCs were isolated from rat femurs and expanded for 12 days. At this time, BMSCs were CD34neg, CD45neg, and mostly CD90pos. BMSCs were grown for an additional 2 days in the presence of 1 mM {alpha}-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which reduced the content of both putrescine and spermidine by nearly 90%. DFMO treatment progressively slowed down BMSC proliferation, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, without arresting their growth completely. The effect of polyamine depletion on caspase-3 activity was evaluated in BMSCs after treatment with 500 U/ml tumor necrosis factor-{alpha} (TNF{alpha}) and 5 µM MG132, an inhibitor of proteasome. Caspase-3 activity increased linearly over a period of 24-hour stimulation (p < .01), but this augmentation was blunted by 50% after DFMO administration (p < .05). The effect of DFMO on TNF{alpha}/MG132-induced upregulation of caspase-3 activity was reversed by the addition of 100 µM putrescine, confirming that polyamines were really involved in the apoptotic process. Also, the number of apoptotic BMSCs after TNF{alpha}/MG132 treatment, as determined by terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay, were threefold reduced after polyamine depletion (p < .05). On the contrary, DFMO did not affect the MG132-mediated increase in p53 abundance, nor its translocation to the nucleus. Thus, polyamine depletion can be considered a useful tool for counteracting programmed cell death in BMSCs without involving the p53 proapoptotic protein.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mesenchymal stem cells represent a self-renewing stem-cell population that can be isolated from various tissues [1] and differentiated into several cell lineages, especially after homing at damaged tissues [2]. However, a constant outcome that dramatically impairs the efficacy of their engraftment is the limited number of stem cells surviving after transplantation [3]. 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 [4]. They also demonstrated that in mesenchymal stem cells engineered with Akt, a survival gene [5], 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 [6].

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 [7], just as polyamine depletion can protect cells exposed to death signals under different experimental conditions [8]. Apoptosis could be partially mediated by polyamines according to several proposed mechanisms, such as the production of hydrogen peroxide during their catabolism [9] or the stimulation of cytochrome-c release from mitochondria [10]. 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 [11] or that depleting cells of polyamines leads to cell death [12]. 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-{alpha} (TNF{alpha}) and a proteasome inhibitor. Cells were pretreated with {alpha}-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), which is the rate-limiting enzyme in polyamine biosynthesis [13]. 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 [14]. 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 [15].


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DFMO was a kind gift of Dr. Patrick M. Woster, of Wayne State University, Detroit. Polyamines, acetyl-Asp-Glu-Val-Asp- amino-4-methylcoumarin (Ac-DEVD-AMC), 5-azacytidine, Alizarin red, and all biochemical reagents were Sigma products (St. Louis, http://www.sigmaaldrich.com).

Isolation and Culture of BMSCs
BMSCs were isolated from rat bone marrow according to the method of Javazon et al. [16]. Femurs of 3-month-old Wistar rats were flushed with 30 ml of {alpha}-modified Eagle’s medium ({alpha}-MEM) containing 20% fetal bovine serum (FBS), 0.1 µg/ml L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 250 µg/ml amphotericin B. Cells were filtered through a 70-µm nylon filter and plated into a 75-cm2 flask. The cells were grown in complete {alpha}-MEM at 37°C and 5% CO2 for 3 days, the medium was replaced with fresh medium, and the adherent cells were grown for 10–12 days to become subconfluent. Then, adherent cells (BMSCs) were washed with phosphate-buffered saline (PBS) and detached by incubation with 0.25% trypsin and 0.1% EDTA for 3 minutes at 37°C. Complete medium was added to inactivate the trypsin. The cells were centrifuged at 450g for 10 minutes, the supernatant was removed, and the cells were resuspended in complete medium and counted in duplicate using a Burker hemocytometer (Brand GmbH, Wertheim, Germany, http://www.brand.de).

All animal work was performed under guidelines on animal care and welfare determined by the Italian Bioethics Committee.

Osteogenic Differentiation of BMSCs
BMSCs were induced to differentiate into osteoblast lineage according to the method of Javazon et al. [16]. Briefly, BMSCs were grown to approximately 80% confluency, and then the medium was substituted with the osteogenic medium containing {alpha}–MEM, 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomicin, 250 µg/ml amphotericin B, 10–8 M dexamethasone, 0.2 mM ascorbic acid 2-phosphate, and 10 mM ß-glycerol phosphate. The osteogenic medium was replaced every 3–4 days. Mineralization was assessed after 2–3 weeks by staining with 40 mM Alizarin red, pH 4.1 [17].

Myogenic Differentiation of BMSCs
BMSCs were induced to differentiate into myogenic lineage according to the method of Makino et al. [18]. Cells were treated with 5 µM 5-azacytidine for 24 hours. The expression of desmin, a marker of myogenic differentiation, was assessed by Western blotting after 3 weeks from 5-azacytidine treatment.

Immunofluorescence
Cells grown on glass coverslip were fixed for 15 minutes at room temperature with 3% paraformaldehyde in PBS, rinsed twice with PBS, and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. After blocking with 1% bovine serum albumin and 0.1% Tween 20 in PBS for 1 hour at room temperature, the cells were incubated with primary antibodies at room temperature for 1 hour and washed with PBS. Cy3-conjugated antimouse antibody (Sigma) was used as secondary antibody (1:2,000 diluted in blocking buffer).

The primary antibodies were mouse antirat, obtained from the following source: p53 (Biosource Int., Camarillo, CA, http://www.biosource.com), CD34, CD45, CD59 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), CD90, mononuclear phagocyte marker (Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). All antibodies were used 1:100 diluted in blocking buffer.

After immunofluorescent staining, cells were mounted with Moviol on standard glass slides and observed with an IX50 inverted microscope (Olympus, Tokyo, http://www.olympus.com).

Fluorescent-Activated Cell Sorting Analysis
Flow cytometry analysis was performed using an Epics Elite (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com) flow cytometer equipped with a 15-mW argon ion laser tuned to 488 nm. Green and red fluorescences were collected at 525 nm and 605 nm, respectively.

Superficial Antigen Determination   Cells were detached with a solution containing 500 mg/ml trypsin and 200 mg/ml EDTA (BioWhittaker, Walkersville, MD, http://www.clonetics.com), washed in PBS, and centrifuged at 500g for 5 minutes. The pellets were resuspended in 3% paraformaldehyde for 15 minutes at room temperature and then washed in PBS.

Nonspecific antigens were blocked by incubating the cells at room temperature for 1 hour in a solution containing 1% BSA and 0.1% FBS in PBS. Aliquots (2 to 3 x 105 cells) were incubated with mouse antiCD90 or rabbit antiCD59 primary antibodies for 45 minutes at room temperature. The cells were washed by centrifugation in PBS, and the pellet was resuspended in antimouse fluorescein isothiocyanate (FITC)–conjugated (Sigma) or antirabbit rhodamine-conjugated (Sigma) secondary antibodies for 20 minutes at room temperature in the dark. Primary antibodies were used 1:100 for CD90 and 2 µg/ml for CD59 diluted in blocking buffer. Secondary antibodies were used 1:500 diluted in blocking buffer.

The cells were then washed in PBS and resuspended in 1 ml PBS for fluorescent-activated cell sorting (FACS) analysis. Non-specific fluorescence was determined using equal aliquots of the cell preparation that were incubated with antimouse FITC-conjugated or antirabbit rhodamine-conjugated antibodies.

Cell-Cycle Investigation   The investigation of cell-cycle phases in BMSCs was performed according to the protocol of Darzynkiewicz [19]. Cells were plated in 10 cm–diameter dishes at a density of 2,000 cells per cm2. At the end of the incubation period, cells were detached by 0.25% trypsin, the pellet was washed twice in PBS, and then fixed in 70% ethanol and starved at –20°C. Just before the analysis, cells were centrifuged at 200g for 5 minutes, and the pellet was resuspended in 0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8, to obtain a final density of approximately 1 x 106 cells/ml. Cells were then incubated at 37°C for 30 minutes and centrifuged at 1,500g for 10 minutes, and the pellet was resuspended in 1 ml of the staining solution containing propidium iodide (0.1% Triton X-100 in PBS, 2 µg RNAase, 1 mg/ml propidium iodide).

Western Blotting
Protein expression of both desmin and cyclin D2 was determined by western blotting. BMSCs were homogenized in a glass tissue grinder in 5 mM dithiothreitol, 2 mM EDTA, 0.1% CHAPS, 0.1% Triton X-100, and protease inhibitors in 20 mM HEPES, pH 7.5. The homogenate was centrifuged at 15,000g for 15 minutes, and the supernatant (diluted in loading buffer [2% SDS, 5% glycerol, 0.002% bromophenol blue, 4% ß-mercaptoethanol in 0.25 M Tris-HCl, pH 6.8]) was denatured by boiling for 3 minutes. Aliquots corresponding to 80 µg protein were analyzed by SDS-PAGE (7.5% gel). Proteins were transferred onto a nitrocellulose membrane for 1 hour. The nitrocellulose membrane was then saturated with 5% nonfat dry milk for 1 hour, washed with Tris-buffered saline, and probed overnight at 4°C with the specific primary antibodies (1:1,000 gut polyclonal antidesmin and 1:1,000 rabbit polyclonal anticyclin D2; Santa Cruz Biotechnology). The membrane was then incubated for 60 minutes with the secondary antibody (1:2,500 horseradish peroxidase–conjugated species-specific anti-IgG; Santa Cruz Biotechnology).

Determination of Polyamine Concentrations
Cells were washed twice with PBS, harvested in 0.4 ml of chilled 0.3 M perchloric acid, and subjected to two cycles of freeze-thawing. After centrifugation at 12,000g for 5 minutes at 4°C, 0.3 ml of the clear supernatant was used for polyamine analysis, whereas the pellet was dissolved in 0.4 ml of 0.3 M NaOH for protein determination. Polyamines were separated and quantified by high-performance liquid chromatography after derivatization with dansyl chloride [20].

TUNEL Assay and Trypan Blue staining
To label apoptotic nuclei, the 3'-OH end of DNA fragments were visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), using the Apoptosis Detection System fluorescence kit (Promega, WI, http://www.promega.com) according to the manufacturer’s instructions. The nuclei of apoptotic and nonapoptotic cells were counterstained with 4,6-diamindino-2-phenylindole (DAPI) (0.1 g/ml). The labeled cells were analyzed by fluorescence microscopy. The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL-positive cells to the total number of DAPI-stained cells, counted in six different random fields. The morphological features of apoptosis (chromatin condensation and nuclear fragmentation) were monitored by fluorescence microscopy.

The number of dead cells was determined by counting trypan blue–stained cells with a Burker hemochromocytometer and calculating the ratio of dead cells to total number of cells.

Determination of Caspase-3 Activity
The activity of the mainly effector caspases 3, 6, and 7 was measured by the cleavage of the fluorogenic peptide substrates Ac-DEVD-AMC during a 15-minute incubation at 37°C, as detailed elsewhere [21]. Because caspase-3 is the most represented isoform into cell among the three executioner caspases [22], we conventionally referred to the obtained values as a measure of caspase-3 activity. Enzyme activity is expressed as U/mg protein, 1 U being defined as the amount of cleaved 1.0 pmol substrate/minute, in the described standard conditions.

MTT Assay
BMSCs were seeded in a 96-multiwell plate (1,500 cells/well) containing {alpha}-MEM, 10% FBS. BMSC proliferation was then determined over a 72-hour period by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay [23], using a multiwell spectrophotometer (Victor 2, PerkinElmer, Wellesley, MA, http://www.perkinelmer.com). A standard curve of BMSCs was previously plotted to correlate the number of living cells with the absorbance values.

Statistical Analysis
Results were expressed as mean ± SEM. Statistical analysis was performed using the one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test for comparison of multiple mean values. The unpaired Student’s t-test was performed to compare two mean values. Probability of null hypothesis <5% (p < .05) was considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go). 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 [16].



View larger version (37K):
[in this window]
[in a new window]
  		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. 2AGo). Figure 2BGo 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.



View larger version (58K):
[in this window]
[in a new window]
  		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 3Go 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 [24].



View larger version (16K):
[in this window]
[in a new window]
  		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, {alpha}-difluoromethylornithine.

 
TNF{alpha} Treatment, Together with Proteasome Inhibition, Increases Caspase-3 Activity
The presence of 500 U/ml TNF{alpha} alone did not induce any increase in caspase-3 activity in BMSCs over a period of 24 hours (Fig. 4Go). On the contrary, the combined effect of 500 U/ml TNF{alpha} 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{alpha} together with MG132 exerted synergistic effects.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Stimulation of caspase-3 activity by treatment of BMSCs with TNF{alpha} and MG132. BMSCs were treated with 500 U/ml TNF{alpha}, 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{alpha} (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{alpha}. 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{alpha}, tumor necrosis factor-{alpha}.

 
Polyamine Depletion Inhibits Caspase-3 Activity in Both Untreated and TNF{alpha}/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{alpha}/MG132 (Fig. 5AGo), 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{alpha}/MG132, restored the activity of caspase-3 over the value obtained in the presence of TNF{alpha}/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.



View larger version (34K):
[in this window]
[in a new window]
  		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{alpha} 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{alpha}/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{alpha}/MG132. (B): One mM DFMO pretreatment for 48 hours also reduced TNF{alpha}/MG132-induced apoptosis as determined by TUNEL assay. Values are means ± SEM of duplicate experiments. * p < .05 versus control, #p < .05 versus TNF{alpha}/MG132. Abbreviations: BMSC, bone marrow stromal cell; DFMO, {alpha}-difluoromethylornithine; TNF{alpha}, tumor necrosis factor-{alpha}; 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{alpha}/MG132 stimulation. Figure 5BGo shows that 24 hours of TNF{alpha}/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{alpha}/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 [25]. 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. 6Go). No further increase in p53 abundance was observed when TNF{alpha} was added together with MG132 for 8 hours; TNF{alpha} alone gave results similar to control. Moreover, DFMO did not prevent the increase in p53 concentration either in MG132- or TNF{alpha}/MG132-treated BMSCs, suggesting that the protective effect of polyamine depletion against apoptosis was not due to a reduction in the level of p53.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 6. p53 protein expression in BMSCs treated with DFMO, TNF{alpha}, and MG132. This is a representative figure of triplicate experiments showing that both control BMSCs and BMSCs treated for 8 hours with TNF{alpha} 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{alpha} 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{alpha}/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, {alpha}-difluoromethylornithine; TNF{alpha}, tumor necrosis factor-{alpha}

 
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. 7AGo). 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. 3Go). Anyway, the inhibition of growth was not complete, and this may be due to the inability to also reduce the intracellular level of spermine.



View larger version (59K):
[in this window]
[in a new window]
  		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, {alpha}-difluoromethylornithine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide.

 
Cyclin D2 expression (Fig. 7BGo) 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. 7CGo) 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 [24]. 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 7DGo 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several superficial antigens have been proposed for distinguishing human BMSCs from hematopoietic cells [26]. However, only a few works describe the pattern of markers distributed on BMSC plasma membrane in rats [4, 16]. In the present study, the main hematopoietic cell markers, namely CD34 and CD45, were not present in BMSCs, except for CD90 and CD59. The former, also named Thy-1, although it is typically expressed by T cells [27], has been described as a marker of BMSCs [26]. According to Javazon et al. [16], rat BMSCs are widely positive to CD90, and we also observed its abundance in bone marrow-derived adherent cells. Moreover, the absence of monocyte-phagocyte antigen in cultured bone marrow cells excludes the possibility of a contamination with macrophages. Mesenchymal stem cells, a sub-population of BMSCs, are multipotent cells that can differentiate into several cell lineages in either in vitro or in vivo conditions. The possibility of these cells to be committed into mesenchymal lineages was also assessed in this study by the development of either osteoblast or myocyte phenotype during their cultivation in appropriate differentiating media. However, the ability of mesenchymal stem cells to regenerate in vivo damaged tissues is largely compromised by the high degree of cell death that occurs after their transplantation [3]. With the present study, we demonstrate that BMSCs are strongly protected from cell death and, in particular, apoptosis, after polyamine depletion. The choice of TNF{alpha} as a cytokine stimulating apoptosis was mainly due to the fact that it is largely produced in the microenvironment of the ischemic/infarcted area of the myocardium [28], one of the main targets for mesenchymal stem cell transplantation [29]. However, the pretreatment of BMSCs with TNF{alpha} by itself was not able to provoke apoptosis. Indeed, it has been reported that TNF{alpha} can simultaneously stimulate both caspase activity and the transcription factor nuclear factor {kappa}B (NF-{kappa}B), which may prevent cell death by increasing the expression of anti-apoptotic proteins [30]. Under nonstress conditions, NF-{kappa}B is sequestered in the cytoplasm by I{kappa}B, which blocks its translocation to the nucleus [31]. In response to TNF{alpha} stimulation, I{kappa}B is phosphorylated and then degraded by the ubiquitin-proteasome pathway [32], allowing free NF-{kappa}B to activate protein transcription. Therefore, to induce apoptosis, we treated BMSCs not only with TNF{alpha} but with MG132, a proteasome inhibitor that blocks I{kappa}B degradation and, therefore, NF-{kappa}B-dependent gene expression [33]. Moreover, we observed that MG132 alone was able to increase the amount of p53, a protein naturally sequestrated by MDM2 and subsequently degraded by the ubiquitin-proteasome pathway [25]. It is well known that p53 can alternatively inhibit cell growth or promote apoptosis, depending on the type and duration of the stimulation [34]. TNF{alpha} activates the cascade of caspase enzymes, especially by means of the receptor-mediated pathway of apoptosis [35], whereas p53 promotes cell death mainly by increasing the biosynthesis of some mitochondrial proteins exerting proapoptotic effects [36].

The treatment of BMSCs with DFMO markedly attenuated both the TNF{alpha}/MG132-mediated caspase-3 activation and the process of apoptosis. These findings confirm similar results obtained in different cell types [8]. Many studies using various cell systems show a fast and sustained increase in ODC activity just after the early induction of apoptotis, suggesting for polyamines a role of mediators in programmed cell death [37]. TNF{alpha}, in particular, has been described as stimulating ODC activity in human fibroblasts [38], and selective proteasome inhibitors can increase polyamine biosynthesis by reducing ODC degradation [39]. However, because in our experimental model the inhibiting effect of DFMO on caspase-3 activity was observed also in the absence of TNF{alpha}/MG132, the DFMO-dependent protection against cell death seems to occur independently of higher polyamine concentrations in the process of apoptosis.

How polyamine depletion leads to caspase-3 inhibition is not known at present. We can speculate that this effect is not mediated by NF-{kappa}B, as described in other conditions of polyamine depletion [40], because we used MG132, which blocked I{kappa}B degradation and NF-{kappa}B activity. Also, the effect of DFMO was downstream or independent of p53, because it did not prevent the early enhancement of p53 concentration induced by MG132. Some possible mechanisms have been explored in other cell types depleted of polyamines. DFMO treatment in rat intestinal epithelial cells (IEC-6) prevented the translocation of Bax to mitochondria, thus inhibiting the release of cytochrome c into cytoplasm [41]. In the same cells, both caspase-8 activity and cleavage of Bid were decreased, whereas the expression of antiapoptotic proteins Bcl-x(L) and Bcl-2 was increased. Moreover, a link between polyamine depletion and increased protein expression has been suggested by Veress et al., who demonstrated that several transcripts accumulated in DFMO-treated Rat-2 cells because of increased stabilization of mRNAs [42].

The effects of DFMO on BMSCs included not only an increase of cell survival, but also, as expected, a reduction of cell proliferation. However, the growth rate of BMSCs was not completely arrested in the presence of DFMO, since it slowed down by approximately 50%. Indeed, BMSCs were depleted of putrescine and spermidine, but not spermine, after DFMO treatment, and this condition likely allowed BMSCs to grow, albeit slowly. Moreover, the effect of DFMO on cell proliferation was opposite to that expected for Akt-gene transfection, suggesting that polyamine depletion not only counteracts cell death, but also does not expose BMSCs to the risk of uncontrolled proliferation. The possibility that DFMO might compromise self-renewing and differentiation potential of BMSCs is also excluded by its inability to markedly and irreversibly modify cell-cycle progression and to change the course of the osteogenic differentiation.

In conclusion, DFMO treatment prevents excessive apoptosis in BMSCs stimulated with TNF{alpha} and MG132. This protective effect is related to the intracellular depletion of both putrescine and spermidine and is paralleled by a marked inhibition of caspase-3 activity.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pittinger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.[Abstract/Free Full Text]

  2. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568–584.[CrossRef][Medline]

  3. Toma C, Pittenger MF, Cahill KS et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002;105:93–98.[Abstract/Free Full Text]

  4. Mangi AA, Noiseux N, Kong D et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003;9:1195–1201.[CrossRef][Medline]

  5. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three AKTs. Genes Dev 1999;13:2905–2927.[Free Full Text]

  6. Koc ON, Gerson SL. Akt helps stem cells heal the heart. Nat Med 2003;9:1109–1110.[CrossRef][Medline]

  7. Poulin R, Pelletier G, Pegg AE. Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells. Biochem J 1995;311:723–727.

  8. Thomas T, Thomas TJ. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 2001;58:244–258.[CrossRef][Medline]

  9. Bonneau MJ, Poulin R. Spermine oxidation leads to necrosis with plasma membrane phosphatidylserine redistribution in mouse leukemia cells. Exp Cell Res 2000;259:23–34.[CrossRef][Medline]

  10. Stefanelli C, Stanic I, Zini M et al. Polyamines directly induce release of cytochrome C from heart mitochondria. Biochem J 2000;347 Pt3:875–880.

  11. Schipper RG, Penning LC, Verhofstad AJ. Involvement of polyamines in apoptosis. Facts and controversies: effectors or protectors? Semin Cancer Biol 2000;10:55–68.[CrossRef][Medline]

  12. Penning LC, Schipper RG, Vercammen D et al. Sensitization of TNF-induced apoptosis with polyamine synthesis inhibitors in different human and murine tumor cell lines. Cytokine 1998;10:423–431.[CrossRef][Medline]

  13. Wallace HM, Fraser AV, Hughes A. A perspective of polyamine metabolism. Biochem J 2003;376:1–4.[CrossRef][Medline]

  14. Marton LJ, Pegg AE. Polyamines as target for therapeutic intervention. Annu Rev Pharmacol Toxicol 1995;35:55–91.[CrossRef][Medline]

  15. Gerner EW, Mamont PS. Restoration of the polyamine contents in rat hepatoma tissue-culture cells after inhibition of polyamine biosynthesis. Relationship with cell proliferation. Eur J Biochem 1986;156:31–35.[Medline]

  16. Javazon EH, Colter DC, Schwarz EJ et al. Rat bone marrow cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. STEM CELLS 2001;19:219–225.[Abstract/Free Full Text]

  17. DiGirolamo CM, Stokes D, Colter D et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275–281.[CrossRef][Medline]

  18. Makino S, Fukuda K, Miyoshi S et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697–705.[Medline]

  19. Darzynkiewicz Z. Flow cytometry in cytopathology. Overview and perspectives. Anal Quant Cytol Histol 1988;10:459–461.[Medline]

  20. Stefanelli C, Carati D, Rossoni C. Separation of N1- and N8-acetylspermidine isomers by reversed phase chromatography after derivatization with dansyl chloride. J Chromatogr 1986;375:49–55.[Medline]

  21. Stefanelli C, Bonavita F, Stanic I et al. Inhibition of etoposide-induced apoptosis with peptide aldehyde inhibitors of proteasome. Biochem J 1998;332:661–665.

  22. Slee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001;276:7320–7326.[Abstract/Free Full Text]

  23. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.[CrossRef][Medline]

  24. Pegg AE, McCann PP. Polyamine metabolism and function. Am J Physiol 1982;243:C212–C221.

  25. Meek DW, Knippschild U. Posttranslational modification of MDM2. Mol Cancer Res 2003;1:1017–1026.[Abstract/Free Full Text]

  26. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitors cells. J Cell Physiol 1999;181:67–73.[CrossRef][Medline]

  27. Killeen N. T-cell regulation: Thy-1 - hiding in full view. Curr Biol 1997;7: R774–R777.[CrossRef][Medline]

  28. Berthonneche C, Sulpice T, Boucher F et al. New insights into the pathological role of TNF-alpha in early cardiac dysfunction and subsequent heart failure after infarction in rats. Am J Physiol Heart Circ Physiol 2004;287:H340–H350.[Abstract/Free Full Text]

  29. Pittenger MF, Bradley JM. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:9–20.[Abstract/Free Full Text]

  30. Tamatani M, Che YH, Matsuzaki H et al. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF{kappa}B activation in primary hippocampal neurons. J Biol Chem 1999;274:8531–8538.[Abstract/Free Full Text]

  31. Krappmann D, Wulczyn FG, Scheidereit C. Different mechanisms control signal-induced degradation and basal turnover of the NF-{kappa}B inhibitor I{kappa}B alpha in vivo. EMBO J 1996;15:6716–6726.[Medline]

  32. Brown K, Gerstberger S, Carlson L et al. Control of IkB{alpha} proteolysis by site-specific, signal-induced phosphorylation. Science 1995;267:1485–1488.[Abstract/Free Full Text]

  33. Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 1998;8:397–403.[CrossRef][Medline]

  34. Vermeulen K, Berneman ZN, Van Bockstaele DR. Cell cycle and apoptosis. Cell Prolif 2003;36:165–175.[CrossRef][Medline]

  35. Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science 2002;296:1634–1635.[Abstract/Free Full Text]

  36. Shen Y, White E. p53-dependent apoptosis pathways. Adv Cancer Res 2001;82:55–84.[Medline]

  37. Sun Y. Identification and characterization of genes responsive to apoptosis: application of DNA chip technology and mRNA differential display. Histol Histopathol 2000;15:1271–1284.[Medline]

  38. Donato NJ, Rotbein J, Rosenblum MG. Tumor necrosis factor stimulates ornithine decarboxylase activity in human fibroblasts and tumor target cells. J Cell Biochem 1991;46:69–77.[CrossRef][Medline]

  39. Murakami Y, Tanahashi N, Tanaka K et al. Proteasome pathway for the degradation of ornithine decarboxylase in intact cells. Biochem J 1996;317:77–80.

  40. Pfeffer LM, Yang CH, Murti A et al. Polyamine depletion induces rapid NF-kB activation in IEC-6 cells. J Biol Chem 2001;276:45909–45913.[Abstract/Free Full Text]

  41. Yuan Q, Ray RM, Johnson LR. Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c. Am J Physiol Cell Physiol 2002;282:C1290–C1297.[Abstract/Free Full Text]

  42. Veress I, Haghighi S, Pulkka A et al. Changes in gene expression in response to polyamine depletion indicates selective stabilization of mRNAs. Biochem J 2000;346:185–191.

Received September 16, 2004; accepted for publication March 30, 2005.



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
S. Kaneko, E. Okuda-Ashitaka, A. Ando, K. Nishimura, K. Igarashi, M. Maeda, K. Furuta, M. Suzuki, M. Matsumura, and S. Ito
Polyamines upregulate the mRNA expression of cationic amino acid transporter-1 in human retinal pigment epithelial cells
Am J Physiol Cell Physiol, August 1, 2007; 293(2): C729 - C737.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0240v1
23/7/983    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Muscari, C.
Right arrow Articles by Caldarera, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Muscari, C.
Right arrow Articles by Caldarera, C. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.stemcellsportal.com/