Cyclic AMP Protects Stem Cell (CD34+) Apoptosis.

NEGROTTO, S; D’ATRI, LP; POZNER, RG; LAZZARI, MA; GOMEZ, RM; SCHATTNER, M

Sydney, Australia

Congreso; International Society of Thrombosis and Haemostasis; 2005

International Society of Thrombosis and Haemostasis

Several studies have shown that cAMP promotes or inhibits apoptosis according cell type. We have previously demonstrated that a cAMP-elevating agent such as prostacyclin inhibits nitric oxide (NO)-induced megakaryocyte apoptosis. We have now investigated cAMP effect on stem cells (CD34+) survival. CD34+ cells were isolated from human cord blood by positive magnetic immunoselection (% of purity ³ 95%). Apoptosis was triggered by two different stimuli: serum deprivation (SD) or exposing cells to the NO donor, PAPA/NO (100 mM). Cell death was evaluated 24 h later by detection of apoptosis/necrosis using fluorescence microscopy and identification of hypodiploid cells by flow cytometry. Results showed that prostacyclin (3-30 mM) failed to protect CD34+ apoptosis (C: 4±1, SD: 26±3, PGI2+SD: 27±1, ON: 17±2, PGI2+ON: 18±1 % of apoptosis, n=5). The lack of effect by prostacyclin appeared to be due to the absence of its surface receptor in undifferentiated cells since the cAMP permeable analog, Dibutiril-cAMP (100 mM) significantly prevented both SD- (12±3# %, n=4) and NO-induced apoptosis (10±2& %, n=4). In order to identify whether cAMP effect on cell survival was mediated by regulation of BCL-xL and caspase-3 levels, the expression of both molecules were measured by flow cytometry. While SD decreased BCL-xL levels (C: 95±5, SD: 66±3* % of positive cells, n=3) and increased caspase-3 expression (C: 12±1, SD: 36±3 % of positive cells, n=3), pretreatment with Dibutiril-cAMP inhibited both effects (96±2# %, n=3 and 20±2# %, n=3 respectively). These results suggest that cAMP promotes CD34+ cell survival through BCL-xL and caspase-3 regulation.*p<0.05 vs control, #p<0.05 vs SD, &p<0.05 vs ON.