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DNA strand break associated bystander effect (DSB-ABE) is linked to gene mutations in naïve cells and indicates the involvement of a MAPK pathway

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dc.contributor.authorJalal, Nasir, author
dc.contributor.authorLiber, Howard L., advisor
dc.contributor.authorBailey, Susan M., advisor
dc.contributor.authorNickoloff, Jac, committee member
dc.contributor.authorBamburg, James, committee member
dc.date.accessioned2007-01-03T08:11:23Z
dc.date.available2007-01-03T08:11:23Z
dc.date.issued2012
dc.description.abstractThe goal of this project was to investigate whether radiation independent DNA damage, specifically a single induced DNA double strand break (DSB) can produce bystander signal, which is capable of inducing genomic instability in non-targeted cells. Previously uncharacterized E18 (modified TK6 cells) with a unique I-Sce1 insert in intron 2 of the thymidine kinase (TK1) gene were allowed to generate a bystander signal following electroporation of the rare cutting restriction enzyme I-Sce1 carried by a plasmid to induce DNA damage at the I-Sce1 site. Mutation assays were carried out to measure mutation fraction (MF) in directly targeted cells and using medium transfer, in non-targeted cells as a measure of bystander signal production. Transfection of the plasmid carrying I-Sce1 gene resulted in production of sufficient bystander signal into the medium to increase the bystander MF, when conditioned medium was applied from directly targeted to naïve E18 cells. The DSB-ABE exhibited temporal kinetics over a 10 hour duration. The relative direct and bystander MF increase due to the electroporation of three rare cutting restriction enzymes namely Not1 an 8 base cutter, Sfi1 a 13 base cutter (technically 8 because of the 5 non-specific bases in sequence) and I-Sce1 an 18 base cutter, showed that DSB-ABE does not show a dose-response. The bystander signal inhibition using superoxide dismutase (an enzyme that degrades reactive oxygen species) and PD 98059 (MEK1/2 inhibitor) indicated that the bystander signal activated the MAPK pathway in naïve cells. Higher levels of bystander mutation fraction were attempted through chemical inhibition of the three known enzymes of DNA repair (ATM, ATR and DNA PK) in directly targeted cells. Results reveal that inhibition of repair of I-Sce1 induced damage was not linked to bystander response. Further, sufficient bystander signal was produced by a presumably single I-Sce1 induced DNA break. The bystander signal produced, exhibited a dose-response in naïve cells and there was suggestion for the involvement of MAPK pathway.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierJalal_colostate_0053A_11327.pdf
dc.identifierETDF2012400342CMBO
dc.identifier.urihttp://hdl.handle.net/10217/68182
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectbystander effect
dc.subjectDNA damage and repair
dc.subjectDNA repair capacity
dc.subjectintercellular signaling
dc.subjectMAPK pathway
dc.subjectradiation independent DNA damage
dc.titleDNA strand break associated bystander effect (DSB-ABE) is linked to gene mutations in naïve cells and indicates the involvement of a MAPK pathway
dc.typeText
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineCell and Molecular Biology
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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