Persistent Link:
http://hdl.handle.net/10150/289132
Title:
Mechanisms of imexon action in human myeloma cells
Author:
Dvorakova, Katerina
Issue Date:
2000
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:
Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule dependent and long exposures ( ≥ 48 hours) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also shown by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione (GSH). Oxidative stress in myeloma cells exposed to imexon was detected by staining with dihydroethidium (HE) and flow cytometry, and by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG) followed by confocal microscopy. This antibody can also recognize 8-hydroxyguanine and 8-hydroxyguanosine. The images showed increased levels of oxidized nucleotides in the cytoplasm of cells treated with imexon. Interestingly, 8-OHdG staining was not observed in the nucleus of imexon treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis on electron microscopy and phosphatidylserine exposure detected as Annexin-V binding on the cell surface. Mitochondrial enlargement was observed by electron microscopy in the cells treated with 90 μM imexon at as early as 4 hours. Morphometric studies indicated that mitochondrial volume significantly increased from 14.1% in control cells to 18.7% in cells treated with 45 μM imexon for 24 hours. In flow cytometric experiments it was shown that the increase in levels of ROS (measured by staining with dihydroethidium) in myeloma cells exposed to imexon precedes the loss of Δψ(m) (detected by MitoTracker Red labeling). Damage of mitochondrial DNA was detected by semiquantitative PCR assay in myeloma cells treated with imexon; however nuclear DNA remained unaffected by imexon treatment. Moreover, release of cytochrome c from mitochondria to cytosol was observed in imexon treated cells. Partial protection of myeloma cells against imexon cytotoxicity was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex 11. Myeloma cell line was developed that is stably resistant to imexon. This cell line shows an increase in the expression of mitochondrial proteins (e.g. Bcl-2, thioredoxin 2) with antioxidant properties. These changes are consistent with drug-induced mitochondrial oxidation and apoptotic signaling.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Cell.; Health Sciences, Pharmacology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Pharmacology and Toxicology
Degree Grantor:
University of Arizona
Advisor:
Dorr, Robert T.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleMechanisms of imexon action in human myeloma cellsen_US
dc.creatorDvorakova, Katerinaen_US
dc.contributor.authorDvorakova, Katerinaen_US
dc.date.issued2000en_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.abstractImexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule dependent and long exposures ( ≥ 48 hours) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also shown by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione (GSH). Oxidative stress in myeloma cells exposed to imexon was detected by staining with dihydroethidium (HE) and flow cytometry, and by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG) followed by confocal microscopy. This antibody can also recognize 8-hydroxyguanine and 8-hydroxyguanosine. The images showed increased levels of oxidized nucleotides in the cytoplasm of cells treated with imexon. Interestingly, 8-OHdG staining was not observed in the nucleus of imexon treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis on electron microscopy and phosphatidylserine exposure detected as Annexin-V binding on the cell surface. Mitochondrial enlargement was observed by electron microscopy in the cells treated with 90 μM imexon at as early as 4 hours. Morphometric studies indicated that mitochondrial volume significantly increased from 14.1% in control cells to 18.7% in cells treated with 45 μM imexon for 24 hours. In flow cytometric experiments it was shown that the increase in levels of ROS (measured by staining with dihydroethidium) in myeloma cells exposed to imexon precedes the loss of Δψ(m) (detected by MitoTracker Red labeling). Damage of mitochondrial DNA was detected by semiquantitative PCR assay in myeloma cells treated with imexon; however nuclear DNA remained unaffected by imexon treatment. Moreover, release of cytochrome c from mitochondria to cytosol was observed in imexon treated cells. Partial protection of myeloma cells against imexon cytotoxicity was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex 11. Myeloma cell line was developed that is stably resistant to imexon. This cell line shows an increase in the expression of mitochondrial proteins (e.g. Bcl-2, thioredoxin 2) with antioxidant properties. These changes are consistent with drug-induced mitochondrial oxidation and apoptotic signaling.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Cell.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.advisorDorr, Robert T.en_US
dc.identifier.proquest9972072en_US
dc.identifier.bibrecord.b40637682en_US
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