Date of Submission

3-2020

Document Type

Thesis

Degree Name

Master of Science in Cellular and Molecular Biology

Department

Biology and Environmental Sciences

Advisor

Dr. Alireza G. Senejani

Committee Member

Dr. Christina Zito

Committee Member

Dr. Eva Sapi

Keywords

Base excision repair, Oxidative stress menadione, Cell viability analysis, Protein folding prediction

MeSH

DNA polymerase beta, Protein Isoforms, Neuroblastoma, DNA repair, DNA damage, Vitamin K 3, Reverse transcription, Polymerase chain reaction, Flow cytometry, RNA Messenger

LCSH

DNA ligases, Neuroblastoma, Oxidative stress, DNA repair, DNA damage, Polymerase chain reaction, Flow cytometry, Messenger RNA

Abstract

The DNA Base Excision Repair (BER) pathway is involved in repairing many types of DNA damages such as oxidation. This study investigates the effect of the oxidative stress menadione on expression of a key BER enzyme called DNA polymerase beta (POLB) in neuroblastoma (BE(2)C), neuron-like (HEK-293), and human lung fibroblast (MRC-5) cell lines. This was accomplished through cell viability analysis, reverse transcription-PCR, sequencing, quantitative PCR, mRNA and protein folding prediction, and flow cytometry. Results demonstrated that BE(2)C and HEK-293 are sensitive to relatively low doses of oxidative stress, particularly 25 uM menadione, but are resistant to high doses of hydrogen peroxide. Regardless of exposure oxidative stress, several splicing variants of POLB including exons 2, 4, and 9 were detected across all cell lines, though the most common was the exon 2 deleted isoform. Despite lower expression of POLB as a whole, this exon 2 deleted POLB variant was expressed at higher levels in BE(2)C cancer cells in both the normal condition and after exposure to menadione, whereas it was expressed more in HEK-293 after exposure to menadione. mRNA and protein folding suggest that the POLB isoforms, particularly the exon 2 deleted variant, may have a role as mRNA molecules rather than proteins. This role could be involved in oxidative stress pathways or could be related to apoptosis resistance since the BE(2)C cells which express more of the exon 2 deleted variant were observed to decrease in apoptosis after exposure to menadione unlike HEK-293 and MRC-5. The results from this study suggest that the presence of upregulated exon 2 deleted POLB isoforms may be a potential biomarker for cancers that may become aggressive and resistant to therapies.

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