TRAIL IS INVOLVED IN HYPOXIA-INDUCED APOPTOSIS OF ACUTE MYELOID LEUKEMIA T(8;21)-POSITIVE CELLS

Introduction. The acute myeloid leukaemia (AML) is a clonal neoplasia of the hematopoietic system characterized by a block of proliferation control and hematopoietic differentiation. 15-20% of AML, defined corebinding factor AML (CBF-AML), is caused by two chromosomic aberration, t(8;21) or inv(16). In particular, t(8;21) AML is defined by the expression of AML1/ETO fusion protein, responsible for HDAC recruitment, determining transcriptional repression of target genes involved in myeloid maturation. We investigated the effects of severe hypoxia on inhibition of proliferation, induction of apoptosis and histone acetylation, and modulation of AML1/ETO expression. Moreover, we deepened the role of TRAIL in the apoptosis induced by hypoxia. Methods. We used Kasumi-1 t(8;21)-positive cell line and U937-A/E cell line, characterized by AML1/ETO-inducible expression. Cells were incubated in 0.3% O2 or normoxia for several days, and treated or not with blocking antibody for TRAIL or control antibody of the same isotype. Cells were harvested at different time points and analysed as for their number, proliferation by propidium iodide staining and viability by the annexin-V test, or lysed to undergo western blotting or real-time PCR. Results. Hypoxia reduced cell viability of Kasumi-1, that is correlated to a reduction of proliferation, as indicated from the increase of cells percentage in phase G0/G1 and the decrease of that in phases S and G2/M, and an increase of apoptosis. Hypoxia-induced apoptosis is confirmed by caspase-9, -8 and -3 activation. Hypoxia additionally induced AML1/ETO disappearance and acetylation of histone H4. Histone acetylation is induced before the disappearance of fusion protein to indicate that hypoxia function as an epigenetic modulator. When we used U937-A/E cell line, we showed that hypoxia induced a high amount of apoptosis only in AML1/ETOexpressing cells, underlining the relationship between hypoxia, as apoptotic agent, and AML1/ETO as sensitizing factor to a pro-apoptotic condition. Then we showed that the presence of AML1/ETO induced the expression of TRAIL and DR5 mRNA. In AML1/ETO-expressing cells, hypoxia initially determined a further increase of TRAIL expression. Moreover Kasumi-1 cells physiologically expressed TRAIL and DR5 mRNA levels similar to those of U937-A/E AML1/ETO-expressing cells. Although hypoxia maintained stable TRAIL mRNA, TRAIL protein is markedly induced by hypoxia. Finally we pointed out that the blocking antibody for TRAIL significantly reduced hypoxia-induced apoptosis (about 20%) of Kasumi-1 cells and completely impaired that of U937- A/E AML1/ETO-expressing respect to not expressing cells. Conclusion. Hypoxia seems to play a relevant anti-leukemic effect, inhibiting cell proliferation, inducing apoptosis and working as an epigenetic modulator. The high sensitivity of AML1/ETO-positive cells to the apoptotic effect of hypoxia involves TRAIL extrinsic pathways of apoptosis.