CYCLIC AMP PROTECTS STEM CELL (CD34+) APOPTOSIS.

NEGROTTO, SOLEDAD; D´ATRI, LINA P; POZNER, ROBERTO G.; LAZZARI, MARIA A.; GOMEZ, RICARDO M.; SCHATTNER, MIRTA

Sydney, Australia.

Congreso; XXth Congress of the International Society on Thrombosis and Haemostasis.; 2005

International Society on 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 (PGI2) 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 >=95%). Apoptosis was triggered by two different stimuli: serum deprivation (SD) or exposing cells to the NO donor, PAPA/NO (100 µM). 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 PGI2 (3–30 µM) 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 PGI2 appeared to be due to the absence of its surface receptor in undifferentiated cells since the cAMP permeable analog, Dibutiril-cAMP (100 µM) 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.