Effects of Hypoxia on HIF, Notch, Akt, and NF-κB Signaling in Leukemia Cell Lines
Authors: Itoh Mai , Takahashi Yusuke , Okuhashi Yuki , Tohda Shuji 
Published: October 2019

Abstract

Background
Leukemia stem cells reside in the bone marrow niche under hypoxic conditions. The activity of various signaling pathways in the hypoxic environment should be known to understand the pathophysiology of leukemia stem cells. Hypoxia is known to stabilize HIF1α, which transactivates various genes allowing the cell to adapt to hypoxic conditions. Two theories have been reported regarding crosstalk between HIF and Notch signaling; one suggests that HIF1α binds to cleaved Notch1, which results in stabilization of Notch signaling, and the second suggests that HIF1α represses a negative feedback loop of the Notch1-Hes1 system by inhibiting Hes1 binding to the HES1 promoter and thus enhancing Notch signaling. In this study, we examined the activity of various signaling pathways in cells cultured under normoxia and hypoxia. We discovered novel processes for HIF, Notch, and their down-stream signaling.

Methods
Two T-ALL cell lines (KOPT-K1 and DND-41), 3 AML cell lines (NB4, THP-1, and TMD7), and 1 CML cell line (K562) were used in this study. Cells were cultured under normoxia or hypoxia (1% O2). The effect of hypoxia on cell growth was examined using a colorimetric WST-1 assay. The effect of hypoxia on replication of clonogenic cells was studied by colony assay after suspension culture under normoxia or hypoxia. The expression and activation of signaling proteins were examined by immunoblotting using lysates from cells cultured under normoxia or hypoxia for 4, 8, and 24 hours. In some experiments, we used lysates from cells cultured with recombinant Notch ligand Delta1 protein.

Results
Hypoxic conditions reduced cell growth and amplification of clonogenic cells when compared to the normoxic condition. Hypoxia enhanced HIF1α levels in all 6 cell lines and increased HIF2α levels in 4 cell lines. HIF2α was not expressed in NB4 and THP-1 cells. Hypoxia reduced the levels of Notch1 and cleaved Notch1 in KOPT-K1, DND-41, and NB4 cells. Notch1 expression was not affected by hypoxia in THP-1, TMD7, and K562 cells. In KOPT-K1, DND-41, and NB4 cells, hypoxia reduced Hes1 expression. In KOPT-K1 cells, Hes1 levels decreased after 4–8 hours under hypoxic conditions; however, the levels increased after 24 hours. Hypoxia suppressed Myc expression in 4 cell lines. Hypoxia also promoted Akt phosphorylation in KOPT-K1, NB4, and K562 cells but suppressed Akt phosphorylation in DND-41 cells without affecting total Akt levels. Hypoxia suppressed NF-κB phosphorylation without affecting total NF-κB protein levels in all cell lines. Delta1 enhanced cleaved Notch1 fragment levels in 4 myeloid cell lines under normoxia and hypoxia. The enhanced Hes1 expression was not significantly different between normoxia and hypoxia.

Discussion
We discovered a novel relationship between hypoxia and Notch signaling. Specifically, hypoxia suppressed Notch1 expression and activation, while hypoxia increased HIF1α and HIF2α levels. We found that Hes1 levels decreased and subsequently increased in KOPT-K1 cells cultured under hypoxia. This could be due to the decrease of cleaved Notch1 as shown here, and subsequent enhancement of Notch signaling owing to increased HIF1α as mentioned in Background section. Hypoxia also suppressed Myc expression and NF-κB phosphorylation, which was likely due to Notch1 suppression. The effect of hypoxia on Akt phosphorylation varied depending on cell lines; however, determining the underlying cause requires further study. In conclusion, we determined that the activities in various signaling pathways under hypoxia differ from those under normoxia. These findings would contribute to the development of various molecular-targeted therapies against leukemic cells, especially leukemia stem cells under hypoxic conditions.

Disclosures:
No relevant conflicts of interest to declare.