Title Uloga tumorskog supresora p53 u fenotipu Rpl24-heterozigotnih miševa : doktorski rad Title (english) The role of tumor suppressor p53 in phenotype of RPL24-heterozygous mice Author Martina Barkić Mentor Siniša Volarević (mentor)Committee member Bojan Polić (predsjednik povjerenstva)Committee member Srećko Gajović (član povjerenstva)Committee member Nives Jonjić (član povjerenstva)Committee member Siniša Volarević (član povjerenstva)Granter University of Rijeka Defense date and country 2010-11-30, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract Cilj istraživanja: Testirati izražaj p53 u Rpl24-heterozigotnim miševima. Ukoliko je tumor supresor p53 izražen, testirati njegovu ulogu u fenotipu Rpl24-heterozigotnih miševa te odrediti molekularne mehanizme putem kojih p53 uzrokuje patološki fenotip Rpl24-heterozigotnih miševa.
Materijali i metode: U istraživanju je korišten miš s heterozigotnom mutacijom u genu za ribosomalni protein l24 (Rpl24) koja je nastala spontano, a koja rezultira specifičnim fenotipom ovih miševa: sljepoćom, abnormalnosti skeleta, smanjenom masom i zavijenim repom (Bst, engl. Belly Spot and Tail). Izražaj p53 utvrđen je imunohistokemijski i westernskom analizom u različitim razvojnim stadijima miševa Rpl24Bst/+. Korištenjem imunohistokemije analizirani su stanični ciklus nakon ugradnje 5-BrdU i apoptoza (prisutnost aktivirane kaspaze 3). Genska inaktivacija p53 ili p21 korištena je u analizi utjecaja ovih molekula na patološki fenotip miševa Rpl24Bst/+. Izražaj Rpl24 utvrđen je nakon izolacije RNA ili proteina northernskom ili westernskom analizom. Ciljne mRNA i miRNA koje su transkripcijski ovisne o p53 identificirane su korištenjem kvantitativne reakcije lančanom polimerazom (PCR, engl. polymerase chain reaction) i DNA nizova nakon izolacije RNA i sinteze komplementarne DNA (cDNA, engl. complementary DNA).
Rezultati: Predloženo je da je patološki fenotip Rpl24-heterozigotnih miševa posljedica pogreške u sintezi ribosoma. U ovom radu sam pokazala da je p53 izražen u specifičnim stadijima embrionalnog razvoja miševa Rpl24Bst/+ . Za razliku od toga, mutacija Rpl24Bst/+ ne aktivira p53-ovisni kontrolni mehanizam u odraslih miševa što bi moglo biti posljedica normalne razine Rpl24 u stanicama, iako je razina mRNA za Rpl24 snižena slično kao u embrijima. Genska inaktivacija p53 gotovo u potpunosti sprječava razvoj patološkog fenotipa Bst. Nasuprot tome, preživljavanje Rpl24Bst/+;p53-/- je značajno smanjeno. Analizom p53-ovisnih mRNA i miRNA utvrđeno da su većina mRNA i miRNA čiji izražaj je poremećen u embrijima Rpl24Bst/+ uključene u regulaciju apoptoze. Analiza apoptoze u neuralnim tkivima embrija ukazala je na njenu ovisnost o statusu p53. Nadalje, p53- i Bax-ovisna apoptoza u pretečama melanocita vjerojatno dovodi do smanjenja broja tih stanica te pojave bijele pjege na trbuhu miševa Rpl24Bst/+. Normalan pupilarni refleks na svjetlo nakon inaktivacije p53 ukazuje da je i ovaj fenotip ovisan o p53 unatoč tome što ni p53 ni apoptoza nisu bili aktivirani u retinama embrija Rpl24Bst/+ . Prisutnost apoptoze u nedostaku poremećaja diobe stanica u repovima embrija Rpl24Bst/+ ukazuje da zavijen rep u Rpl24-heterozigotnih miševa može biti posljedica aktivacije apoptoze.
Zaključci: Pogreška u sintezi ribosoma uzrokovana smanjenim izražajem Rpl24 u Rpl24-heterozigotnim miševima dovodi do aktivacije tumorskog supresora p53 koji putem regulacije svojih transkripcijskih ciljeva dovodi do razvoja fenotipa Bst. Ovi rezultati ukazuju da p53 regulira morfološki fenotip Rpl24-heterozigotnih miševa putem p21-neovisnih mehanizama kao što je npr. apoptoza. Suprotno tome, p53 regulira preživljavanje ovih miševa barem djelomično putem p21-ovisnog mehanizma.
Abstract (english) Objectives: To investigate if tumor suppressor p53 is upregulated during the development of Rpl24-heterozygous mice. If so, to determinate the biological role of p53-dependent ckeckpoint in the development of Rpl24-heterozygous mice pathological phenotype.
Material and Methods: Spontaneous heterozygous mutation in one allele of ribosomal protein l24 gene (Rpl24) in mice results in defects of the eye, skeleton, mass and coat pigmentation (Bst, Belly Spot and Tail mice). Rpl24- heterozygous mice were tested during different developmental stages for the expression of p53 by using immunohistochemistry and Western blot. p53-dependent cell cycle block (5-BrdU incorporation) and apoptosis (cleaved caspase 3 expression) were also tested by using immunohistochemistry. Genetic inactivation of p53 or p21 in Rpl24Bst/+ mice was used for further evaluation of p53 and p21 role in phenotype of Rpl24Bst/+ mice. Rpl24 expression level was determinated by Northern blot and Western blot after RNA or protein isolation. p53 transcriptional target mRNAs an miRNAs were identified in Rpl24Bst/+ embryos using quantitative polymerase chain reaction (PCR) array after RNA isolation and complementary DNA (cDNA) synthesis.
Results: It has been hypothesized that pathological manifestations of Rpl24-heterozygous mice result exclusively from faulty protein synthesis. We showed here that p53 is upregulated during the restricted period of embryonic development in Rpl24Bst/+ mice. Rpl24Bst/+ mutation does not elicit a p53-dependent ckeckpoint response in adult mice. This difference could be explained by the normal amount of Rpl24 in adult Rpl24Bst/+ cells, although the defect in Rpl24 mRNA expression was comparable to the one in embryos. Genetic inactivation of p53 led to almost complete rescue of Bst phenotype but the survival of Rpl24Bst/+;p53-/- mice was greatly aflicted. Analysis of p53-target mRNAs and miRNAs in Rpl24Bst/+ embryos identified several molecules involved in apoptosis. We showed that apoptosis in neural tissue is p53-dependent and that p53 and Bax dependent apoptosis in melanocyte precursors could result in decreased number of melanocyte precursors and be responsible for the white ventral middle spot in Rpl24-heterozygous mice. Rescue of pupillar defect after p53-inactivation cleary showed that this phenotype is p53-regulated, although p53 or apopotosis could not be detected in retinas of Rpl24Bst/+ mice. Proliferative defect in tails of Rpl24Bst/+ embryos was not detectable but apoptosis was activated suggesting that kinked tail phenotype in Rpl24Bst/+ mice could also be apotosis dependent.
Conclusions: p53 upregulation during restricted period of embryonic development of Rpl24-heterozygous mice as a consequene of impaired ribosome synthesis significantly contributes to the Bst phenotype possibly by an apoptosis-dependent mechanism but promotes their survival at least partially via p21-dependent mechanism.
Keywords
apoptoza
dioba stanice
kontrolni mehanizam ovisan o p53
nedostatak ribosomalnih proteina
rast stanice
ribosom
ribosomalni protein l24
Keywords (english)
apoptosis
cell division
cell growth
ribosme
ribosomal protein l24
ribosomal protein deficiency
p53 checkpoint control
Language croatian URN:NBN urn:nbn:hr:188:649444 Project Number: 062-1081347-0370 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL https://libraries.uniri.hr/cgi-bin/unilib.cgi?form=D1120820052 Type of resource Text Extent 148 str. File origin Born digital Access conditions Closed access Terms of use Created on 2017-01-19 17:08:38
