Utjecaj ekspresije coxsackie -adenovirusnih receptora na ekspresiju integrina u stanicama rabdomiosarkoma embrija čovjeka

master's thesis

• Title (english): The Influence of Coxsackie Adenovirus Receptor Expression on Integrin Expression in Human Embryonal Rhabdomyosarcoma Cell Line
• Author: Tea Špeljko
• Mentor: Andreja Ambriović Ristov (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Andreja Ambriović Ristov (član povjerenstva)
• Committee member: Dubravka Hranilović (član povjerenstva)
• Committee member: Dijana Škorić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2010-09-23, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Coxsackie-adenovirusni receptor (CAR) komponenta je međustaničnih veza, primarni receptor adenovirusa tip 5 (Ad5) i tumor-supresor. Oskudni podaci iz literature o tome kako povećanje ekspresije CAR utječe na ekspresiju integrina uključenih u transdukciju Ad5-vektorima, su kontradiktorni. Ovim radom je pokazano da u klonovima stanica rabdomiosarkoma embrija čovjeka (RD) s povećanom ekspresijom CAR dolazi do smanjene ekspresije integrinske podjedinice αv i heterodimera αvβ3 i αvβ5. Navedeno smanjenje zabilježeno je na proteinskoj razini metodom protočne citometrije, dok se razine mRNA, mjerene semikvantitativnim RT-PCR-om, u stabilnim CAR-transfektantima u odnosu na RD-stanice nisu mijenjale. Ispitivanjem djelotvornosti transdukcije RD stanica i dva CAR stabilna transfektanta, rekombinatnim Ad5 sa skraćenim vlaknom (Ad5FbΔ639 i Ad5FbΔ639RGD4C), potvrđen je negativan utjecaj smanjenja razine integrina αvβ3 i αvβ5 na transdukciju Ad5FbΔ639RGD4C. Metodom Western blot utvrđeno je povećanje aktivacije kinaze regulirane izvanstaničnim signalom, ERK1/2, u stabilnim transfektantima RD-CAR u odnosu na RD-stanice. Ovi rezultati su važni za razumijevanje biološke funkcije CAR i otkrivaju da povećana ekspresija CAR smanjuje ekspresiju αvβ3 i αvβ5 integrina, što može imati važne posljedice na adheziju stanica, njihovu pokretljivost i/ili metastatski potencijal, ali i na uspješnost transdukcije adenovirusnim vektorima.

Abstract (english)

Coxsackie adenovirus receptor (CAR) is a component of intercellular junctions, a primary receptor for adenovirus type 5 (Ad5) and a tumor suppressor. Little is known about the influence of increased CAR expression on expression of integrins involved in Ad5 vector transduction, and the information gathered so far is contradictory. The present study demonstrates that augmented CAR expression in clones of human embryonal rhabdomyosarcoma (RD) cell line leads to reduced expression of integrin subunit αv and integrin heterodimers αvβ3 and αvβ5. The aforementioned reduction was recorded on a protein level, using flow cytometry, whereas the mRNA level, measured by semiquantitative RT-PCR, did not vary in RD clones overexpressing CAR as compared to RD cells. By testing the transduction efficacy of RD cells and two RD-CAR transfectants by two recombinant Ad5s with shortened fibers, Ad5FbΔ639 and Ad5FbΔ639RGD4C the negative effect of reduced levels of αvβ3 and αvβ5 on Ad5FbΔ639RGD4C transduction was confirmed. Western blot analysis demonstrates an increase in activation of extracellular signal-regulated kinase (ERK1/2) in RD clones overexpressing CAR as compared to RD cells. These findings are important for understanding the biological function of CAR and reveal that increased expression of CAR reduces integrin αvβ3 and αvβ5 expression, which may have serious effects on adhesion, mobility and/or metastatic potential of cells, but also on the transduction efficacy of adenoviral vectors.

• Keywords: coxsackie adenovirus receptor; integrin; pERK1/2; adenovirus; genska terapija tumora
• Keywords (english): coxsackie adenovirus receptor; integrin; pERK1/2; adenovirus; tumor gene therapy
• Language: croatian
• URN:NBN: urn:nbn:hr:217:988531
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-05-04 08:03:16