Uloga proteina p21 WAF1/Cip1 i inhibicije šaperona u odgovoru na kemoterapeutike

master's thesis

• Title (english): The role of p21WAF1/Cip1 protein and chaperone inhbition in tumor cell response to therapy
• Author: Petra Jagušt
• Mentor: Marijeta Kralj (mentor)
• Mentor: Maja Matulić (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Marijeta Kralj (član povjerenstva)
• Committee member: Mladen Kučinić (član povjerenstva)
• Committee member: Inga Marijanović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2014-02-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Protein p21WAF1/Cip1 pripada porodici inhibitora kinaza ovisnih o ciklinima Cip/Kip, koji dovode do zaustavljanja staničnog ciklusa, potiče pojavu senescencije, ima dvojaku ulogu u regulaciji apoptoze, no njegova uloga u autofagiji, kao ni važnost u međudjelovanju različitih oblika staničnog odgovora nije jasna. Autofagija je proces lizosomalne razgradnje kojim se održava stanična homeostaza, ali se aktivira i kao odgovor na stres. Cilj ovog rada bio je istražiti ulogu proteina p21 u modulaciji apoptoze, autofagije i senescnecije te pokazati jesu li ovi procesi povezani s nastankom reaktivnih kisikovih vrsta (ROS). Stanice karcinoma debelog crijeva HCT 116 tretirali smo kemoterapeuticima cisplatinom i doksorubicinom, povećanu ekspresiju proteina p21 postigli smo unosom gena p21 adenovirusnim vektorom, dok smo njegovu eskpresiju utišali interferencijom RNA. Rezultati su pokazali da protein p21 podiže razinu ROS-a u, što dovodi do aktivacije autofagije i senescencije, posebno snažno u kombiniranim tretmanima kemoterepeuticima uz povišenu ekspresiju p21. S druge strane, p21 inhibira apoptozu aktiviranu kemoterapeuticima. Ove smo rezultate potvrdili inhibicijom bazalne ekspresije proteina p21. Dobiveni rezultati ukazuju na važnost reaktivnih kisikovih vrsta u pokretanju određenog tipa stanične smrti. Također smo ispitali utjecaj inhibitora šaperona HSP90 i HSP70 na preživljenje stanica nakon tretmana kemoterapeuticima. Pokazali smo da inhibicija HSP90 blago pojačava učinak doksorubicina, dok pifitrin-μ, nije imao učinak na stanice.

Abstract (english)

Protein p21WAF1/Cip1 belongs to the Cip/ Kip family of cyclin dependent kinases inhibitors that mediates cell cycle arrest, activates senescence, has dual role in apoptosis modulation, and still unknown role in autophagy and in the interplay of these cell responses to therapy. Autophagy is a process of lysosomal degradation which maintains cell homeostasis, but it is also a stress-response mechanism. The aim of this work was to investigate the role of p21 in the activation of apoptosis, autophagy and cellular senescence through regulation of reactive oxygen species (ROS). For this purpose, we treated HCT 116 colon cancer cells with chemotherapeutic agents, cisplatin and doxorubicin after transduction with adenoviral vectors bearing p21 sequence or silencing p21 expression with small interfering RNAs. The results showed that the p21 activated senescence and autophagy by mechanism involving ROS accumulation, while it inhibited apoptosis. The results indicate the importance of ROS in the initiation of certain types of cell death. To test the influence of HSP90 and HSP70 inhibitors on cell viability after treatment with chemotherapeutic agents, we used geldanamicin or pifitrin-μ. We showed that the inhibition of HSP90 with geldanamicin slightly enhanced the effect of doxorubicin, while pifitrin-μ had no effect on the cell viability.

• Keywords: protein p21; reaktivne vrste kisika; stanično starenje; apoptoza; autofagija; molekularni šaperoni
• Keywords (english): protein p21; reactive oxygen species; cellular senescence; apoptosis; autophagy; molecular chaperones
• Language: croatian
• URN:NBN: urn:nbn:hr:217:966521
• 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: 2017-04-26 09:32:02