Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica

master's thesis

• Title (english): Cell cycle analysis of tumor cells using adenoviral vectors
• Author: Blanka Pranjić
• Mentor: Marijeta Kralj (mentor)
• Mentor: Maja Matulić (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Marijeta Kralj (član povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Committee member: Željka Vidaković Cifrek (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2008, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Poremećaj regulacije staničnog ciklusa uzrokuje nekontroliranu proliferaciju stanica te je jedan od glavnih uzroka pojave raka. Postoji niz gena koji reguliraju stanični ciklus i čuvaju stanice od zloćudne preobrazbe, među kojima je gen p21WAF1/CIP1 čiji proteinski produkt je inhibitor kompleksa kinaze ovisne o ciklinu (CDK) i ciklina. Njegova povećana ekspresija u tumorskim stanicama predstavlja potencijalno oružje u genskom liječenju raka, međutim istraživanje njegove uloge ukazuje na niz kontradiktornih rezultata zbog kojih je nužno dodatno rasvijetliti njegovu ulogu u tumorskim stanicama. U ovom je radu praćen utjecaj povećane ekspresije gena p21 unošenjem njegove cDNA replikacijski nesposobnim adenovirusnim vektorima (Ad5-p21) u tumorske stanice karcinoma debelog crijeva SW480 i SW620, koje potječu od istog pacijenta, ali imaju različit stupanj zloćudne preobrazbe. Kao kontrolni vektor je korišten adenovirus s ugrađenim genom dojavljivačem (Ad5-β-gal). Oba vektora umnožena su u visokoj koncentraciji u komplementirajućim stanicama HEK 293, izolirana i pročišćena. Virusni titar je izmjeren određivanjem koncentracije ukupnih i infektivnih virusnih čestica. Djelotvornost transdukcije stanica karcinoma debelog crijeva SW620, SW480, a i HCT116 adenovirusnim vektorom Ad5-β- gal određena je praćenjem pojave plavog obojenja aktivnošću enzima β-galaktozidaze. Uočena je slabija djelotvornost transdukcije SW620 stanica putem Ad5-β-gal u odnosu na SW480 i HCT116 stanice. Također je primijećeno toksično djelovanje samog vektora na tumorske stanice, poglavito na stanice SW620. Utjecaj Ad5-p21 i Ad5-β-gal na stanični ciklus SW620 i SW480 stanica praćen je metodom protočne citometrije. Povećana ekspresija gena p21 izaziva zastoj u G1 fazi staničnog ciklusa stanica SW480. Njegova je uloga u stanicama SW620 upitna zbog snažnog utjecaja samog adenovirusnog vektora na stanični ciklus, budući da je uočen zastoj staničnog ciklusa u G2 fazi nakon zaražavanja stanica s kontrolnim vektorom Ad5-β-gal. Kako i sami adenovirusni vektori mogu utjecati na stanični ciklus te uzrokovati lažne rezultate, potreban je izuzetan oprez kod njihovog korištenja u istraživanju gena koji reguliraju stanični ciklus.

Abstract (english)

Deregulation of the cell cycle induces uncontrolled cell proliferation and is one of the main causes of cancer development. A variety of genes has cell cycle regulating properties and can therefore protect cells from malignant transformation. An example is p21WAF1/CIP1 which is an inhibitor of cyclin-dependent kinases (CDK)/cyclin complexes. Overexpression of p21WAF1/CIP1 in tumor cells might represent a potential tool for cancer gene therapy. However, the study of p21WAF1/CIP1 functions in tumor cells revealed contradictory findings and therefore it is necessary to clarify its role in tumor cells with different genetic background. The effect of p21WAF1/CIP1 overexpression was monitored by introducing p21 cDNA into colon carcinoma cells SW480 and SW620, using the replication-deficient recombinant adenoviral vectors. These cell lines were derived from the same patient, but from cells with different level of malignancy. Adenoviral vector containing the reporter gene for β-galactosidase was used as a control. Both vectors were propagated in HEK 293 helper cells in high concentrations, isolated and purified. Viral titer was determined measuring the concentration of total and infectious viral particles. The efficiency of transduction of colon carcinoma cells SW620, SW480, as well as HCT116, with adenoviral vectors was determined by β-galactosidase staining of infected cells. Transduction of SW620 with Ad5-β-gal had lower efficiency, as compared to SW480 and HCT116. In addition, a vector related toxic effect on tumor cells was detected, in particular on SW620 cells. The effect of Ad5- p21 and Ad5-β-gal on the cell cycle of SW620 and SW480 cells was monitored by flow cytometry. p21WAF1/CIP1 overexpression in SW480 cells induced cell cycle arrest in G1 phase. In SW620, a strong effect of the vector itself, Ad5-βgal, was detected, resulting in G2/M arrest. Since the adenoviral vector itself can effect the cell cycle leading to false results, their use in the investigation of the cell cycle regulating genes should be handled with care.

• Keywords: stanični ciklus; gen p21WAF1/CIP1; adenovirus; rak
• Keywords (english): cell cycle; p21WAF1/CIPgene; adenovirus; cancer
• Language: croatian
• URN:NBN: urn:nbn:hr:217:645812
• 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: Open access
• Created on: 2018-03-05 12:24:25