The role of 5-methylcytosine in the expression of O(6)-methylguanine DNA-methyltransferase

Persistent Link:
http://hdl.handle.net/10150/282599
Title:
The role of 5-methylcytosine in the expression of O(6)-methylguanine DNA-methyltransferase
Author:
Watts, George Somerset, 1971-
Issue Date:
1998
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Regulation of the gene which encodes the human DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is of clinical interest and provides a gene model for the study of CpG methylation's role in transcriptional regulation. Expression of MGMT is important to the maintenance of genomic integrity because it repairs O6-alkylguanine DNA lesions, which, if left unrepaired, can lead to mutation. In the clinical setting of cancer chemotherapy, however, MGMT provides cancer cells with resistance to the nitrosoureas, a class of cancer chemotherapeutics whose cytotoxic activity is via formation of O6-alkylguanine adducts, and includes N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU) and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU). Expression of MGMT is regulated at the level of transcription in a cell-cycle-independent manner. It has been found that up to 25% of cultured cell lines do not express MGMT activity due to a lack of MGMT transcription. This dissertation describes the use of the human multiple myeloma cell line 8226/S and its MGMT negative (MGMT- variant 8226/V to study the role of CpG methylation in repression of MGMT transcription. Bisulfite sequencing showed that 5-methylation of cytosines within the CpG island of the 8226/V MGMT promoter correlated with loss of MGMT expression. Furthermore, the increase in CpG methylation is associated with the formation of nuclease-resistant chromatin structure around the MGMT transcription start site as determined by chromatin accessibility assays. Additional evidence is presented which shows that CpG methylation increased in the 8226/V genome as a result of selection with verapamil, and that the expression of genes which increase the clonigenic capacity of 8226/V were also affected by the selection. Southern analysis of cytosine methylation across the mdr1, c-myc, H19, K-ras, and thymidine kinase genes is presented, along with development of a sensitive control for the technique which utilizes the mitochondrial genome. This work has established the 8226/S and 8226/V cell lines as a model to study the mechanisms and effects of cytosine methylation. The results presented here support the importance of 5-methylcytosine as an epigenetic mechanism of MGMT gene regulation, and suggests epigenotoxicity as a cause for the increased cancer risk seen in patients receiving verapamil and other calcium channel blockers.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Health Sciences, Pharmacology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Pharmacology and Toxicology
Degree Grantor:
University of Arizona
Advisor:
French, Edward; Futscher, Bernard W.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleThe role of 5-methylcytosine in the expression of O(6)-methylguanine DNA-methyltransferaseen_US
dc.creatorWatts, George Somerset, 1971-en_US
dc.contributor.authorWatts, George Somerset, 1971-en_US
dc.date.issued1998en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractRegulation of the gene which encodes the human DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is of clinical interest and provides a gene model for the study of CpG methylation's role in transcriptional regulation. Expression of MGMT is important to the maintenance of genomic integrity because it repairs O6-alkylguanine DNA lesions, which, if left unrepaired, can lead to mutation. In the clinical setting of cancer chemotherapy, however, MGMT provides cancer cells with resistance to the nitrosoureas, a class of cancer chemotherapeutics whose cytotoxic activity is via formation of O6-alkylguanine adducts, and includes N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU) and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU). Expression of MGMT is regulated at the level of transcription in a cell-cycle-independent manner. It has been found that up to 25% of cultured cell lines do not express MGMT activity due to a lack of MGMT transcription. This dissertation describes the use of the human multiple myeloma cell line 8226/S and its MGMT negative (MGMT- variant 8226/V to study the role of CpG methylation in repression of MGMT transcription. Bisulfite sequencing showed that 5-methylation of cytosines within the CpG island of the 8226/V MGMT promoter correlated with loss of MGMT expression. Furthermore, the increase in CpG methylation is associated with the formation of nuclease-resistant chromatin structure around the MGMT transcription start site as determined by chromatin accessibility assays. Additional evidence is presented which shows that CpG methylation increased in the 8226/V genome as a result of selection with verapamil, and that the expression of genes which increase the clonigenic capacity of 8226/V were also affected by the selection. Southern analysis of cytosine methylation across the mdr1, c-myc, H19, K-ras, and thymidine kinase genes is presented, along with development of a sensitive control for the technique which utilizes the mitochondrial genome. This work has established the 8226/S and 8226/V cell lines as a model to study the mechanisms and effects of cytosine methylation. The results presented here support the importance of 5-methylcytosine as an epigenetic mechanism of MGMT gene regulation, and suggests epigenotoxicity as a cause for the increased cancer risk seen in patients receiving verapamil and other calcium channel blockers.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectHealth Sciences, Pharmacology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePharmacology and Toxicologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorFrench, Edwarden_US
dc.contributor.advisorFutscher, Bernard W.en_US
dc.identifier.proquest9829326en_US
dc.identifier.bibrecord.b38551913en_US
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