Cell cycle analysis of tumor cells using adenoviral vectors

Pranjić, Blanka (2008) Cell cycle analysis of tumor cells using adenoviral vectors. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

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.

Item Type: Thesis (Diploma thesis)
Keywords: cell cycle, p21WAF1/CIPgene, adenovirus, cancer
Supervisor: Kralj, Marijeta
Co-supervisor: Matulić, Maja
Date: 2008
Number of Pages: 63
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 15 Sep 2014 09:39
Last Modified: 15 Sep 2014 09:39
URI: http://digre.pmf.unizg.hr/id/eprint/2756

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