Signalni mehanizmi i metaboličke promjene tijekom diferencijacije i proliferacije leukemijskih stanica

doctoral thesis

• Title (english): Signaling mechanisms and metabolic changes during differentiation and proliferation of leukemia cells
• Author: Barbara Tomić
• Mentor: Dora Višnjić (mentor)
• Committee member: Drago Batinić (predsjednik povjerenstva)
• Committee member: Danka Grčević (član povjerenstva)
• Committee member: Svjetlana Kalanj-Bognar (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2022-08-31, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Clinical Medical Sciences; Clinical Immunology
• Universal decimal classification (UDC): 616 - Pathology. Clinical medicine

Abstract

5-aminoimidazol-4-karboksamid ribonukleozid (AIKAr) prekursor je u biosintezi purina i poznati aktivator kinaze ovisne o AMP-u (AMPK). Naše je prethodno istraživanje pokazalo da AIKAr koči proliferaciju i potiče diferencijaciju stanica akutne mijeloične leukemije (AML) neovisno o AMPK-u. Cilj ovoga istraživanja bio je odrediti ulogu signalnoga puta ATR/Chk1 (prema engl. ataxia-telangiectasia-mutated-and-Rad3-related/checkpoint kinase 1) u diferencijaciji leukemijskih stanica potaknutoj AIKAr-om, koristeći metaboličke testove, tekućinsku kromatografiju s tandemskom spektrometrijom masa (LC/MS/MS), Western blot, protočnu citometriju, farmakološke inhibitore te RNA-interferenciju. Rezultati pokazuju kako učinci AIKAr-a na staničnu diferencijaciju ne ovise o potrošnji glukoze, već ovise o metabolizmu glutamina i sintezi nukleotida, odnosno aktivaciji kinaze Chk1 posljedično manjku pirimidina. LC/MS/MS analiza otkrila je da AIKAr povećava koncentraciju orotata i smanjuje koncentraciju uridin-monofosfata (UMP), u skladu s inhibicijom sinteze UMP-a u reakciji nakon dihidroorotat-dehidrogenaze (DHODH). AIKAr i inhibitor DHODH-a, brekvinar, povećali su izražaj biljega diferencijacije i zakočili stanice u S-fazi staničnog ciklusa. Inhibicija signalnoga puta Chk1 primjenom farmakoloških inhibitora i RNA-interferencije poništila je oba spomenuta učinka. Naši rezultati otkrivaju da je učinak AIKAr-a na diferencijaciju stanica AML uzrokovan poremećajem biosinteze pirimidina te posredovan aktivacijom signalnog puta u odgovoru na oštećenje DNA.

Abstract (english)

5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) is a precursor in purine biosynthesis and a well-known activator of AMP-dependent kinase (AMPK). Our previous research has shown that AICAr inhibits proliferation and promotes differentiation of acute myeloid leukemia (AML) cells in an AMPK-independent manner. The aim of this study was to determine the role of ataxia telangiectasia and RAD3-related (ATR)/checkpoint kinase 1 (Chk1) in AICAr-mediated differentiation of leukemia cells using metabolic tests, liquid chromatography with tandem mass spectrometry (LC/MS/MS), Western blot, flow cytometry, pharmacological inhibitors and RNA-interference. The results show that AICAr-mediated differentiation is independent of glucose consumption, but instead depends on glutamine metabolism, nucleotide synthesis and activation of Chk1 due to pyrimidine depletion. LC/MS/MS analysis revealed that AICAr increased orotate levels and decreased uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and DHODH inhibitor, brequinar, increased the expression of differentiation markers and arrested cells in the S-phase of the cell cycle. Pharmacologic and siRNA-mediated inhibition of Chk1 pathway diminished both effects. Our results reveal that the effect of AICAr on AML differentiation is caused by impaired pyrimidine biosynthesis and mediated by the activation of DNA damage response pathway.

• Keywords: AMP-aktivirane protein kinaze; Dihidroorotat-dehidrogenaza; Glutamin; RNA; mala interferencija; Protočna citometrija; 4-aminoimidazol; Ataksija telangiektazija; Leukemija; mijeloidna; akutna; Oštećenje DNK
• Keywords (english): AMP-Activated Protein Kinases; Dihydroorotate Dehydrogenase; Glutamine; RNA; Small Interfering; Flow Cytometry; 4-aminoimidazole; Ataxia Telangiectasia; Leukemia; Myeloid; Acute; DNA Damage
• Language: croatian
• URN:NBN: urn:nbn:hr:105:392291
• 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: 82 str.
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-10-28 11:39:14