Regulacija diferencijacije stanica akutne promijelocitne leukemije koštanim morfogenetskim proteinima [Effect of bone morphogenetic proteins on differentiation of human
promyelocytic leukemia cells]
Acute promyelocytic leukemia (APL) is malignant hematologic disease characterized by
translocation t(15;17). This translocation results in the formation of PML/RARα oncogene and
accumulation of malignant promyelocytes that do not differentiate into mature granulocytes.
Treatment of this type of leukemia is based on differentiation agents all-trans retinoic acid
(ATRA) and arsenic-trioxide, which induce degradation and conformational changes of the
oncogene PML/RARα. However, prolonged treatment with ATRA often results in relapse, due to
development of ATRA resistance by leukemic cells, and serious side-effects.
In this research we aimed to investigate possible mechanisms of BMP-mediated suppression of
ATRA-induced differentiation using myeloid leukemia cell lines NB4 and HL60. We used two
myeloid leukemia cell lines: NB4, which carries the specific t(15;17) translocation, and HL60, a
myeloblastic leukemia cell line that lacks t(15;17) translocation, to test if the BMP effect is
PML/RARα specific. Cell lines were treated with ATRA and BMPs, and, in some experiments,
BMP-antagonists NOGGIN.
ATRA induced differentiation of malignant promyelocytes into neutrophils in both tested cell
lines. Addition of BMPs in ATRA-treated samples significantly reduced the percentage of
differentiated cells. This anti-differentiation effect of BMPs was reversed by BMP-antagonist
NOGGIN.
To investigate the mechanism of anti-differentiation effect of BMPs on ATRA-treated myeloid
leukemia cell lines NB4 and HL60, we analyzed the expression pattern of several genes involved
in proliferation, differentiation and apoptosis, which are, at the same time, involved in both
BMP- and ATRA-signaling pathway. Those in vitro results were confirmed in bone marrow and
peripheral blood samples of APL patients.
Gene expression analysis revealed induction of ID expression in both NB4 cell line and bone
marrow APL samples. Addition of BMPs further enhanced ID gene expression in NB4 cells.
Moreover, ATRA induced expression of differentiation genes PU.1 and C/EBPε. This effect was
suppressed by combined treatment with ATRA and BMP. Also, we confirmed significant
positive correlation between expression of total receptor RARα, important for differentiation
effect of retinoic acid, and transcription factor PU.1 in bone marrow samples of APL patients. In
the same samples, cKit expression negatively correlated with PML/RARα oncogene, but
positively with total RARα, indicationg the role of cKit in differentiation of APL cells. Finally,
the expression of total RARα was in negative correlation with BMP-6 and in positive correlation
with BMP-antagonist BAMBI.
Based on the obtained results, we concluded that ID genes and proteins could mediate antidifferentation
effect of BMPs. Also, our findings suggest that PU.1 and C/EBPε could be
responsible for the interference of BMP-signal and ATRA-differentiation effect. Results of our
investigation confirmed the important role of BMPs in the pathogenesis of APL, and indicate
that BMP molecules may be involved in the development of ATRA-resistance in APL patients
