Accumulation of hypoxia-inducible factor-1 alpha protein and its role in the differentiation of myeloid leukemic cells induced by all-trans retinoic acid.
BACKGROUND
The clinical activities of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia, have triggered extensive studies aimed at defining the mechanisms by which this compound induces differentiation of leukemic cells. Recent studies show that hypoxia-inducible factor-1 alpha (HIF-1 alpha) contributes to the differentiation of acute myeloid leukemia cells via transcriptional activity-independent mechanisms. We investigated whether all-trans retinoic acid affects HIF-1 alpha protein and whether this has a role in all-trans retinoic acid-induced differentiation.


METHODS
The acute myeloid leukemia cell lines NB4 and U937 were treated with all-trans retinoic acid, and HIF-1 alpha/HIF-1 beta mRNA and proteins were measured respectively by real-time quantitative reverse transcriptase polymerase chain reaction and western blotting. To investigate the role of HIF-1 alpha in all-trans retinoic acid-induced differentiation, NB4 cells, U937 cells, U937 cells in which HIF-1 alpha was induced by the withdrawal of tetracycline and U937 cells with stable expression of specific short hairpin RNA against HIF-1 alpha, Runx1, C/EBP alpha and PU.1, were treated with all-trans retinoic acid and/or the hypoxiamimetic agent cobalt chloride (CoCl(2)). Cellular differentiation was evaluated by morphological criteria and myeloid differentiation antigens.


RESULTS
all-trans retinoic acid rapidly increased endogenous and inducible expressed or CoCl(2)-stabilized HIF-1 alpha protein in leukemic cells under normoxia. Importantly, suppression of HIF-1 alpha expression by specific short hairpin RNA partially but significantly inhibited all-trans retinoic acid-induced differentiation of the U937 cell line. Reciprocally, the differentiation induced by all-trans retinoic acid was significantly enhanced by conditional HIF-1 alpha induction and HIF-1 alpha-stabilizing CoCl(2) treatment. Furthermore, knock-down of PU.1, Runx1 and C/EBP alpha, three transcriptional factors crucial for normal hematopoiesis, greatly inhibited the differentiation cooperation of all-trans retinoic acid and HIF-1 alpha induction.


CONCLUSIONS
This work provides the first demonstration that HIF-1 alpha, a protein rapidly responsive to all-trans retinoic acid, plays a role in all-trans retinoic acid-induced differentiation of leukemic cells. These observations shed new light on the molecular mechanisms underlying all-trans retinoic acid-induced differentiation of acute myeloid leukemia cells.
Zhang
Jing
J
Institute of Health Science, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences (SIBS)-Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai, China.
Song
Li-Ping
LP
Huang
Ying
Y
Zhao
Qian
Q
Zhao
Ke-Wen
KW
Chen
Guo-Qiang
GQ
D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D003300:Copper / Q000494:pharmacology; D050676:Core Binding Factor Alpha 2 Subunit / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D000099232:Proto-Oncogene Protein Spi-1
D022763:CCAAT-Enhancer-Binding Protein-alpha; D003300:Copper; D050676:Core Binding Factor Alpha 2 Subunit; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014212:Tretinoin; D000099232:Proto-Oncogene Protein Spi-1; C029892:cupric chloride