Mehanizam diferencijacije stanica akutne mijeloične leukemije potaknute citarabinom

doctoral thesis

• Title (english): The mechanism of cytarabine-induced differentiation of acute myeloid leukemia cells
• Author: Tomislav Smoljo
• Mentor: Dora Višnjić (mentor)
• Committee member: Rajko Kušec (predsjednik povjerenstva)
• Committee member: Danka Grčević (član povjerenstva)
• Committee member: Radovan Vrhovac (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2024-04-26, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Cytology, Histology and Embryology
• Universal decimal classification (UDC): 61 - Medical sciences

Abstract

Citarabin u malim koncentracijama potiče diferencijaciju stanica akutne mijeloične leukemije (AML), ali mehanizam nije u potpunosti objašnjen. Naša prethodna istraživanja pokazala su da inhibitori sinteze pirimidina potiču diferencijaciju leukemijskih stanica aktivacijom kinaze Chk1 (prema engl. checkpoint kinase 1). Cilj ovog istraživanja je ispitati ulogu Chk1 u diferencijaciji leukemijskih stanica potaknutoj citarabinom te ispitati učinke stromalnih stanica na diferencijaciju leukemijskih stanica. Leukemijske stanične linije U937, THP-1 i MOLM-13, te stromalnu staničnu liniju MS-5 smo tretirali citarabinom i inhibitorima sinteze pirimidina. Koristeći Western blot, protočnu citometriju, farmakološke inhibitore i siRNA transfekciju pokazali smo da niska doza citarabina uzrokuje diferencijaciju aktivacijom Chk1. Prisutnost stromalnih stanica smanjuje učinke niskih doza citarabina na zastoj u staničnom ciklusu, signalizaciju oštećenja DNA i diferencijaciju stanica AML-a. Iako je sekvenciranje RNA pokazalo da stroma smanjuje izražaj gena uključenih u signalizaciju citokinima i oksidativni stres, rezultati dobiveni pomoću farmakoloških inhibitora i neutralizirajućih protutijela nisu podržali ulogu CXCL12, TGF- i slobodnih radikala kisika. Ovi rezultati pokazuju da niska doza citarabina potiče diferencijaciju leukemijskih stanica aktivacijom kinaze Chk1 te da stromalne stanice smanjuju diferencijaciju stanica AML-a potaknutu niskim dozama citarabina in vitro.

Abstract (english)

Low-dose cytarabine promotes differentiation of acute myeloid leukemia (AML) cells, but the mechanism is not fully elucidated. Our previous research showed that pyrimidine synthesis inhibitors stimulate the differentiation of AML cells by activating checkpoint kinase 1 (Chk1). This research aims to investigate the role of Chk1 in the cytarabine-induced differentiation of AML cells and to examine the effects of stromal cells on AML differentiation. Leukemia cell lines U937, THP-1, and MOLM-13, and stromal cell line MS-5 were treated with cytarabine and pyrimidine synthesis inhibitors. Using immunoblotting, flow cytometry analyses, pharmacologic inhibitors, and genetic inactivation of Chk1 we show that low-dose cytarabine induces differentiation by activating Chk1. The presence of stromal cells prevented cell cycle arrest, DNA damage signaling, and AML cells differentiation induced by low-dose cytarabine. Although transcriptomic analysis revealed that the stroma reduced the expression of genes involved in cytokine signaling, and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role of CXCL12, TGF-β, or reactive oxygen species. This study reveals that that low-dose cytarabine promotes leukemic cell differentiation by activating Chk1 kinase and that stromal cells reduce low-dose cytarabine-induced AML differentiation in vitro.

• Keywords: Chk1; Protočna citometrija; Leukemija; mijeloidna; akutna; Citarabin; Stromalne stanice
• Keywords (english): Chk1; Flow Cytometry; Leukemia; Myeloid; Acute; Cytarabine; Stromal Cells
• Language: croatian
• URN:NBN: urn:nbn:hr:105:023718
• Promotion: 2024
• Project: Number: IP-2016-06-4581; Title: Signalni mehanizmi i metaboličke promjene u diferencijaciji stanica akutne mijeloične leukemije; Title: Signaling mechanisms and metabolic changes in differentiation of acute leukemia cells; Acronym: SignalmetabAML; Leader: Dora Višnjić; Jurisdiction: Croatia; Funder: Hrvatska zaklada za znanost; Funding stream: Research Projects
• Project: Number: IP 2022-10-9146; Title: Oštećenje DNA i diferencijacija akutne mijeloične leukemije”; Leader: Dora Višnjić; Jurisdiction: Croatia; Funder: Hrvatska zaklada za znanost; Funding stream: Research Projects
• Project: Number: KK.01.1.1.01.0007; Title: Znanstveni centar izvrsnosti - eksperimentalna i klinička istraživanja hipoksijsko-ishemijskog oštećenja mozga u perinatalnoj i odrasloj dobi; Title: Scientific center of excellence - Experimental and clinical research of hypoxic-ischemic brain damage in perinatal and adulthood; Acronym: ZCI-NEURO; Leader: Miloš Judaš; Jurisdiction: eu; Funder: Europska komisija; Funding stream: European Regional Development Fund
• Study programme: Title: Biomedicine and Health Sciences; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
• Type of resource: Text
• Extent: 91 str.
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-05-10 11:24:38