Polyamine depletion reduces TNFalpha/MG132-induced apoptosis in bone marrow stromal cells.
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 (TNFalpha) and 5 microM 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 TNFalpha/MG132-induced upregulation of caspase-3 activity was reversed by the addition of 100 microM putrescine, confirming that polyamines were really involved in the apoptotic process. Also, the number of apoptotic BMSCs after TNFalpha/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.
Muscari
Claudio
C
Department of Biochemistry G. Moruzzi, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy. claudio.muscari@unibo.it
Bonafé
Francesca
F
Stanic
Ivana
I
Flamigni
Flavio
F
Stefanelli
Claudio
C
Farruggia
Giovanna
G
Guarnieri
Carlo
C
Caldarera
Claudio Marcello
CM
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D017209:Apoptosis; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D004396:Coloring Agents / Q000494:pharmacology; D000518:Eflornithine / Q000494:pharmacology; D005269:Femur / Q000378:metabolism; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D007976:Leupeptins / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009955:Ornithine Decarboxylase / Q000378:metabolism; D010011:Osteocytes / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011700:Putrescine / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013095:Spermidine / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018952:Antigens, CD34; D004396:Coloring Agents; D007976:Leupeptins; D011073:Polyamines; D061988:Proteasome Inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D017493:Leukocyte Common Antigens; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D009955:Ornithine Decarboxylase; C022616:thiazolyl blue; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D013095:Spermidine; D011700:Putrescine; D000518:Eflornithine