POLYAMINE DEPLETION REDUCES TNF/MG₁₃₂ -INDUCED APOPTOSIS IN BONE MARROW STROMAL CELLS

¹ Department of Biochemistry "G. Moruzzi" - University of Bologna - Italy
² National Institute of Cardiovascular Research - Bologna - Italy

^* To whom correspondence should be addressed. E-mail: claudio.muscari{at}unibo.it.

	Abstract

Polyamines are powerful modulators of both growth and survival in mammalian cells. Here we investigated the possibility to attenuate the process of apoptosis in bone marrow stromal cells(BMSCc), 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 CD34^neg, CD45^neg and mostly CD90^pos. BMSCs were grown for further 2 days in the presence of 1 mM -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 TNF and 5 µM MG₁₃₂, an inhibitor of proteasome. Caspase-3 activity increased linearly over a period of 24 h stimulation (p<0.01), but this augmentation was blunted by 50 % after DFMO administration (p<0.05). The effect of DFMO on TNF/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 following TNF/MG₁₃₂ treatment, as determined by TUNEL assay, were three-fold reduced after polyamine depletion (p<0.05). 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 an useful tool to counteract programmed cell death in BMSCs, without involving the p53 proapoptotic protein.