Interakcija mitogenom aktiviranih protein kinaza i autofagije u diferencijaciji HL-60 leukemijskih ćelija u makrofage forbol-12-miristat-13-acetatom
Interaction of mitogen-activated protein kinases and autophagy in macrophage differentiation of HL-60 leukemia cells by phorbol-12-myristate-13-acetate
Author
Mandić, MilošMentor
Trajković, VladimirCommittee members
Misirkić Marjanović, MajaPerović, Vladimir
Ćirić, Darko
Ristić, Biljana
Metadata
Show full item recordAbstract
Forbol-12-miristat-13-acetat (PMA), aktivator protein kinaze C (PKC), indukuje diferencijaciju
ćelija akutne mijeloidne leukemije (AML) u makrofage. Pokazano je da autofagija učestvuje u
diferencijaciji ćelija hematopoeze i leukemijskih ćelija. Mitogenom aktivirane protein kinaze
(MAPK) mogu da aktiviraju autofagiju i diferencijaciju ćelija. Cilj ove disertacije je da se ispita
uloga autofagije i MAPK u diferencijaciji HL-60 leukemijskih ćelija u makrofage pod dejstvom
PMA, kao i uloga MAPK u indukciji autofagije. PMA je indukovala diferencijaciju HL-60 ćelija u
makrofage, što je pokazano povećanom ekspresijom makrofagnih markera CD11b, CD13, CD14,
CD45, EGR1, CSF1R i IL-8. Prisustvo autofagije u diferencijaciji HL-60 ćelija pokazano је
detekcijom zakišeljavanja citoplazme, povećanjem autofagnog fluksa, povećanjem ekspresije ATG
gena i oslobađanjem beklina-1 iz kompleksa sa Bcl-2. Autofagija je aktivirana izlaskom autofagnog
represora ZKSCAN3 iz jedra i ulaskom aktivatora autofagije FO...XO1/3 i TFEB u jedro HL-60
ćelija. Aktivacija autofagije je nezavisna od AMPK i Akt/mTORC1. Međutim, PMA je aktivacijom
PKC dovela do stimulacije ERK i JNK, što je povećalo konverziju LC3-I u LC3-II. Farmakološka i
genska inhibicija ERK ili JNK je smanjila konverziju LC3, ekspresiju ATG gena, premeštanje
FOXO1/3 i TFEB u jedro, disocijaciju beklina-1 iz kompleksa sa Bcl-2 i ekspresiju makrofagnih
markera CD11b, CD45, CSF1R, EGR1 i IL-8. Farmakološka i genska inhibicija autofagije je
sprečila diferencijaciju HL-60 ćelija u makrofage. Ovi rezultati ukazuju da ERK i JNK učestvuju u
diferencijaciji HL-60 ćelija u makrofage pod dejstvom PMA, aktivacijom autofagije, što predstavlja
potencijalnu metu za poboljšanje diferencijacione terapije AML.
Protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) induces macrophage
differentiation of acute myeloid leukemia (AML) cells. Autophagy has been shown to participate in
the differentiation of hematopoietic and leukemic cells. It is well known that mitogen-activated
protein kinases (MAPK) can activate autophagy and cell differentiation. The aim of this thesis is to
examine the role of autophagy and MAPK activation in PMA-induced macrophage differentiation
of HL-60 leukemia cells as well as the role of MAPK in the induction of autophagy. PMA induced
macrophage differentiation of HL-60 cells, as shown by increased expression of macrophage
markers CD11b, CD13, CD14, CD45, CSF1R, EGR1 and IL-8. The activation of autophagy during
the differentiation of HL-60 cells has been demonstrated by the increase of cytoplasmic
acidification, augmented autophagic flux, increased ATG gene expression, and the release of beclin-
1 from the complex with Bcl-2. Autophagy in HL-60 cells has... been activated by the cytoplasmic
translocation of the autophagy repressor ZKSCAN3 and nuclear translocation of autophagy
activators FOXO1/3 and TFEB. Stimulation of autophagy was independent of AMPK and
Akt/mTORC1 activation. However, PMA activation of PKC stimulated ERK and JNK and
increased the LC3 conversion. Pharmacological and genetic inhibition of ERK or JNK reduced LC3
conversion, ATG gene expression, FOXO1/3 and TFEB nuclear translocation and expression of
macrophage markers CD11b, CD45, CSF1R, EGR1 and IL-8. Inhibition of autophagy suppressed
differentiation of HL-60 leukemia cells induced by PMA. These results indicate that ERK and JNK-
dependent autophagy participates in PMA-induced differentiation of HL-60 cells into macrophages,
which represents a potential target for improving AML differentiation therapy.