The effect of chemical carcinogens on DNA bypass replication and the development of in vitro and in vivo models for chemical mutagenesis.

Persistent Link:
http://hdl.handle.net/10150/184689
Title:
The effect of chemical carcinogens on DNA bypass replication and the development of in vitro and in vivo models for chemical mutagenesis.
Author:
Yamanishi, Douglas Tadao.
Issue Date:
1989
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:
In the context of the somatic mutation theory of chemical carcinogenesis, mutations are thought to arise during the replication of DNA past carcinogen-DNA adducts. The work described in this thesis deals with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with the ultimate carcinogenic form of benzo (a) pyrene, 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE I). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. The data obtained were, therefore, supportive of the 'block-gap' model of DNA bypass replication in carcinogen damaged mammalian cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. It was determined that the integrated human c-Ha-ras gene was hypomethylated, and expressed at the messenger level. The human c-Ha-ras protein, p21, was also detected in these transfected cell lines. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-N-nitroso-N'-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines. Screening of the in vitro and in vivo treated, transformed cell lines by RNA:RNA duplex mismatch analysis led to the detection of no mutations within the first exon of the human c-Ha-ras oncogene.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Carcinogens -- Metabolism -- Genetic aspects.; Chemical mutagenesis.; DNA -- Synthesis -- Regulations.; DNA damage.; Oncogenes.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Molecular and Cellular Biology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Bowden, G. Tim

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleThe effect of chemical carcinogens on DNA bypass replication and the development of in vitro and in vivo models for chemical mutagenesis.en_US
dc.creatorYamanishi, Douglas Tadao.en_US
dc.contributor.authorYamanishi, Douglas Tadao.en_US
dc.date.issued1989en_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.abstractIn the context of the somatic mutation theory of chemical carcinogenesis, mutations are thought to arise during the replication of DNA past carcinogen-DNA adducts. The work described in this thesis deals with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with the ultimate carcinogenic form of benzo (a) pyrene, 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE I). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. The data obtained were, therefore, supportive of the 'block-gap' model of DNA bypass replication in carcinogen damaged mammalian cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. It was determined that the integrated human c-Ha-ras gene was hypomethylated, and expressed at the messenger level. The human c-Ha-ras protein, p21, was also detected in these transfected cell lines. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-N-nitroso-N'-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines. Screening of the in vitro and in vivo treated, transformed cell lines by RNA:RNA duplex mismatch analysis led to the detection of no mutations within the first exon of the human c-Ha-ras oncogene.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectCarcinogens -- Metabolism -- Genetic aspects.en_US
dc.subjectChemical mutagenesis.en_US
dc.subjectDNA -- Synthesis -- Regulations.en_US
dc.subjectDNA damage.en_US
dc.subjectOncogenes.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMolecular and Cellular Biologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorBowden, G. Timen_US
dc.contributor.committeememberDuffy, John J.en_US
dc.contributor.committeememberGerner, Eugene W.en_US
dc.contributor.committeememberMeyskens, Frank L.en_US
dc.contributor.committeememberLindell, Thomas J.en_US
dc.identifier.proquest8915995en_US
dc.identifier.oclc702372614en_US
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