ROLE OF ERK5 IN THE SURVIVAL OF MYELOID LEUKEMIA CELLS IN HYPOXIA

Introduction. Bone marrow is a physiologically hypoxic tissue, where oxygen levels are likely decreased further in leukemic patients as a result of cell overcrowding as well as anemia, which are frequent consequences of leukemia cell growth. The effects of severe hypoxia on myeloid leukemia cells have been investigated in murine erythroleukemia (MEL) cells to characterize the interference of hypoxia with intracellular signalling pathways, MAPK in particular, relevant to leukemia cell survival and growth. Methods. Cells were incubated in 0.3% O2 or normoxia for several days. Cells were harvested at different time points and analysed as for their number and viability by the annexin V test, or lysed to undergo SDS-PAGE and western blotting.Results. Hypoxia prevented the cell number increase which occurred in normoxia and determined early and massive apoptosis, as well as cell cycle arrest of surviving cells. Consistently, the AKT protein, an important pro-survival signal, was cleaved in hypoxia. Hypoxia decreased the intensity and duration of ERK1/2, p38 and JNK phosphorylation/activation occurring in normoxia, without altering the expression of these proteins. On the other hand, hypoxia suppressed p120ERK5 constitutive activation and protein expression, unchanged in normoxia. ERK5 mRNA was not decreased in hypoxia. Phosphorylation of a p82 ERK5 form was also abrogated in hypoxia, but not normoxia, although the protein level massively increased. This down-modulation was also found in the HL60 and K562 human leukemia cells undergoing hypoxia-induced apoptosis. Disappearance of p120ERK5 and dephosphorylation of p82ERK5 were prevented by treatment with the pan-caspase inhibitor z-VAD. Accordingly, when a dominant/negative form of ERK5 was overexpressed in K562 cells, cell number was significantly decreased after 48- 72 hours in hypoxia, but not normoxia, apparently due to cell cycle arrest of cells in G0/G1 and modest apoptosis. Moreover, silencing of ERK5 with shRNA determined massive apoptosis after 72 hours of incubation in hypoxia, but not normoxia. Conclusions. These findings are consistent with a role of ERK5 as a pro-survival signal which is suppressed in leukemia cells undergoing hypoxia-induced apoptosis.