INCUBATION OF MURINE ERYTHROLEUKEMIA CELLS IN SEVERE HYPOXIA INDUCES MASSIVE APOPTOSIS PARALLELED BY AKT AND ERK5 CLEAVAGE

We previously showed that severe hypoxia (0.1% O2) favours the self renewal of murine and human normal haematopoietic stem cell. The importance of hypoxia in the regulation of neoplastic stem cells also recently emerged. This study was undertaken to characterize the effects of hypoxia on a murine erythroleukemia cell line. To this purpose, Friend erythroleukemia cells were incubated in severe hypoxia (0,1% O2) or normoxia (20% O2) for 7 days; cells incubated in hypoxia (LC1) were then transferred to normoxia (LC2), to determine their potential for overall cell number expansion. The colony-formation efficiency of day-7 hypoxic cultures was unreduced when compared to that of normoxic cultures; however, the incubation in hypoxia during LC1 reduced cell proliferation rate after transfer to normoxia (LC2). The effects of hypoxia at different incubation times were determined with respect to cell cycle and viability. Total cell number was found strongly reduced after 3 days of incubation in hypoxia when compared to normoxia. The Annexin-V test showed that hypoxia doubled the percentage of cells in early as well as late apoptosis. At the end of LC1 (day- 5/6) almost all cells were in late apoptosis, while surviving cells (2%) were in a quiescent state (G0- G1 phase of cell cycle), as demonstrated by flow cytometry. Several molecular parameters were investigated in hypoxic cultures. Hypoxia was found to interfere with the AKT and ERK5 signalling systems. AKT cleavage, as determined by AKT disappearance and appearance of 40-44 kDa AKT fragments, was marked at day 3 of incubation in hypoxia, to increase significantly thereafter. Active (threonine/tyrosine phosphorylated) ERK5 was markedly reduced at day 3 in hypoxia, to disappear at day 6.On the other hand, the expression itself of ERK5 was significantly reduced already after a 1-day incubation in hypoxia; the downmodulation of ERK5 was paralleled by the appearance of a cleaved 30 kDa ERK5 fragment. Under the same conditions, the amount of ERK1/2 in hypoxia was unchanged. These results suggest that AKT and ERK5 are pro-survival signals in these cells and are specifically cleaved in hypoxia-induced apoptosis. The effects of hypoxia on histone acetylation were also determined. We observed that histone H4 was hypo-acetylated at day 3, suggesting that incubation in hypoxia interferes with transcriptional regulation.