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Keywords:

  • Mesenchymal stem cells;
  • Apoptosis;
  • Caspase;
  • p53;
  • Polyamines

Abstract

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 α-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-α (TNFα) 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 TNFα/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 TNFα/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.