APC-dependent regulation of polyamine metabolism and apoptosis in human colon tumor cells

Persistent Link:
http://hdl.handle.net/10150/280176
Title:
APC-dependent regulation of polyamine metabolism and apoptosis in human colon tumor cells
Author:
Fultz, Kimberly Elizabeth
Issue Date:
2002
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:
Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, the human colon tumor cell line HT29 (wtAPC -/-) was stably transfected with a zinc-inducible wild-type APC gene. Addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein, and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the beta-galactosidase (betaGal) gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. To examine the role of APC-dependent regulation of ODC, the two sets of E-boxes were analyzed. When the E-box domain in the 5' flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results indicate that APC expression can inhibit ODC via the 5' E-box. Using the cell model previously described, APC selectively represses the ODC A allele, apparently through selective binding of Mad1. These results demonstrate that wild-type APC suppresses c-myc and activates Mad1 expression in HT29 colon-derived cells. Treatment of Min mice with the ODC inhibitor, difluoromethylornithine (DFMO), suppresses intestinal polyamine contents and intestinal tumorigenesis. The data presented in this dissertation indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. Apoptosis is significantly reduced in both the small intestines and colons of Min (multiple intestinal neoplasia) mice when compared to normal littermates. Apoptotic indices can be restored by treating the mice with alpha-difluoromethylornithine (DFMO). DFMO is a specific, irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. These results indicate that APC induces apoptosis via the mitochondrial pathway rather than through the death receptor pathway. APC also affects a variety of other proteins involved in the regulation of apoptosis including transcription factors (i.e., ets2, FKHR, JunB, etc.) and bcl-2 (i.e., Bcl-xL) family members. The multiple levels at which APC functions suggest a variety of possible targets for the prevention and treatment of colon cancer. (Abstract shortened by UMI.)
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Biology, Cell.; Health Sciences, Oncology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Molecular and Cellular Biology
Degree Grantor:
University of Arizona
Advisor:
Gerner, Eugene W.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleAPC-dependent regulation of polyamine metabolism and apoptosis in human colon tumor cellsen_US
dc.creatorFultz, Kimberly Elizabethen_US
dc.contributor.authorFultz, Kimberly Elizabethen_US
dc.date.issued2002en_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.abstractMutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, the human colon tumor cell line HT29 (wtAPC -/-) was stably transfected with a zinc-inducible wild-type APC gene. Addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein, and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the beta-galactosidase (betaGal) gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. To examine the role of APC-dependent regulation of ODC, the two sets of E-boxes were analyzed. When the E-box domain in the 5' flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results indicate that APC expression can inhibit ODC via the 5' E-box. Using the cell model previously described, APC selectively represses the ODC A allele, apparently through selective binding of Mad1. These results demonstrate that wild-type APC suppresses c-myc and activates Mad1 expression in HT29 colon-derived cells. Treatment of Min mice with the ODC inhibitor, difluoromethylornithine (DFMO), suppresses intestinal polyamine contents and intestinal tumorigenesis. The data presented in this dissertation indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. Apoptosis is significantly reduced in both the small intestines and colons of Min (multiple intestinal neoplasia) mice when compared to normal littermates. Apoptotic indices can be restored by treating the mice with alpha-difluoromethylornithine (DFMO). DFMO is a specific, irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. These results indicate that APC induces apoptosis via the mitochondrial pathway rather than through the death receptor pathway. APC also affects a variety of other proteins involved in the regulation of apoptosis including transcription factors (i.e., ets2, FKHR, JunB, etc.) and bcl-2 (i.e., Bcl-xL) family members. The multiple levels at which APC functions suggest a variety of possible targets for the prevention and treatment of colon cancer. (Abstract shortened by UMI.)en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectBiology, Cell.en_US
dc.subjectHealth Sciences, Oncology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMolecular and Cellular Biologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGerner, Eugene W.en_US
dc.identifier.proquest3073220en_US
dc.identifier.bibrecord.b43468159en_US
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