Year: 2018
Type: dissertation
Abstract: High-throughput proteomics approaches have been successfully employed in the inclusive and unbiased \ncharacterization of cellular proteome. In this thesis, we are aiming to comprehensively compare changes \nin proteome expression in mammalian cell lines under two different conditions. The first is an analysis of \nmAb producing CHO cells with a focus on comparing the productivity of inducible cells, and the second \ninvestigates human colorectal cancer cells with a focus on chemo-resistant cells. To this end, the \nquantitative proteomics technique iTRAQ was performed on the samples. \nThe production of monoclonal antibodies as therapeutic medicines has gained increasing attention and \napplications in the last decades. Improving production levels is an important issue that can economically \nimpact the industry. Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell lines \nfor the production of therapeutic mAbs and have been genetically modified for this purpose. Monitoring \nthe effects of genetic modification systems on the cells is very important to maintain the quality and \nconsistency of the cellular products. The possible changes or stresses forced on the cells after these \nmodifications can be observed by analyzing the cells’ proteome and can enable the development of \noptimized expression systems. Also, the biological characteristics of high-producing CHO cells could be \nobtained by proteomic comparison of these cells. \nWe quantified the expression levels of 1,616 unique proteins from an iTRAQ MS analysis and identified \n81 proteins with p-values lower than 0.05 where the highest fold-change belong to the mAb sequence. \nOverall, proteins did not show any large changes based on the fold-change and it is likely that the induction \nsystem with cumate gene switch did not cause large metabolic changes or stress responses in the CHO cells. \nWith these results we can suggest that the system has generated a stable cell line with a higher capacity for \nmAb production without inducing any major stress responses in the cells, and the cell line could have the \npotential for high mAb production using a stronger promoter system. \nThe second topic of interest in this thesis is therapy resistance colorectal cancer, which is either caused \nby a selection or induction of drug resistance in a subset of colorectal cancer cells. Drug resistance is the \nfirst reason for treatment failure or metastasis, therefore understanding the biological features of resistant \ncells can be helpful. To this end, the cancer cell line, HT-29, and a SN-38 drug-resistant HT-29 cell line \nwere explored by iTRAQ quantitative proteomic analysis. SN-38 is the active metabolite of the \nchemotherapy drug, irinotecan, a common mediation for treating colon cancer. \nThe expression levels of 1,665 proteins were quantified and 21 proteins had significant changes with \nmore than 1 log-fold-change. PAGE4 is the most up-regulated protein in the resistant HT-29 cells; it shows \nto have a role in cell viability and survival. Among the down-regulated proteins, cytokeratin (KRT20) had \na significant low expression in resistant HT-29 cells that can show the change in cell migration because it \nis known for circulating CRC cells. Biosynthesis of antibiotics is identified as the most enriched KEGG \npathway within the up-regulated genes; also, ribosome pathway is observed as the most enriched pathway \namong the down-regulated gene list. Overall, a large number of the differentially regulated genes were \nknown in the stem cancer cells, EMT processes and metastasis. The main functions among the differentially \nregulated proteins is related to cell adhesion and migration, cell proliferation, and the ribosome.
Source: UWSpace (University of Waterloo)
Author: Vida Talebian
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Funder: University of Waterloo
OpenAlex ID: W2804919590