Effects of geldanamycin, a ligand of heat shock protein 90, on cell cycle progression and induction of apoptosis in human lymphocytes and jurkat cells

thesis
dc.abstract.enHeat shock protein 90 (HSP90), which is implicated in post-translational folding, stability, and maturation of proteins, controls several key cell cycle regulators. Thus, the hypothesis was raised that geldanamycin, a specific and potent inhibitor of HSP90 function, may have pronounced effects on cell cycle progression. The objective of this study was to test this hypothesis in normal and cancer cells of human origin. The experiments performed on human lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) indicated that 100 nM or 150 nM geldanamycin induces transition of cells to the G0 state of cell cycle. This was documented utilizing acridine orange, a metachromatic dye which differentially stains DNA versus RNA. The same experimental protocol allowed demonstration that geldanamycin is a potent inducer of apoptosis in PHA-activated cells. Importantly, both the block in G0 and induction of apoptosis were reversible and returned to control values upon removal of geldanamycin. Similar conclusions were reached when cell number in cultures was analyzed, excluding the possibility that a relevant fraction of cells was disintegrated during the incubation period. Experiments on Jurkat line of acute T-cell leukemia were performed next. Jurkat cells were used here as a model system in which the cytostatic and cytotoxic properties of geldanamycin on cancer cells can be tested. Initial experiments determined the time course and concentration-dependence of geldanamycin-induced alterations in cell cycle distribution and apoptosis. In contrast to human lymphocytes, geldanamycin did not induce G0 arrest in Jurkat cells, but inhibited them initially in the G2 phase, and at later time points in the G1 phase. The G2 was distinguished from mitosis by the absence of phosphorylation of histone H3, a specific marker of mitotic cells. The inhibition of Jurkat cells in G1 was linked to a decrease in phosphorylation of retinoblastoma protein. Finally, the exposure of Jurkat cells to geldanamycin resulted in induction of apoptosis, predominantly in cells being arrested in G1 and G2/M phases of the cell cycle. Finally, to address the possibility that stimulation of nuclear factor kappa-B (NF-kB), downstream of HSP90, modulates the effects of geldanamycin on cancer cells, Jurkat IkBaM line was employed. These cells cannot activate their NF-kB-mediated responses because of the mutation in its inhibitory protein, IkB. In the absence of functional NF-kB, geldanamycin-mediated induction of apoptosis and loss of cycling cells were markedly higher than when NF-kB was functional. These results were further corroborated by experiments in which parthenolide, a plant-derived inhibitor of NF-kB was employed. Geldanamycin-treated Jurkat cells responded to the parthenolide challenge by partial arrest in S phase and increased cell death by apoptosis. In conclusion, inhibition of HSP90 by geldanamycin blocks cell cycle progression and induces apoptosis of Jurkat cells, and NF-kB mediates these effects. The newly identified network of interactions may facilitate understanding of the mechanism of cytostatic and cytotoxic action of geldanamycin derivatives used currently in clinical trials.pl
dc.affiliationWydział Biochemii, Biofizyki i Biotechnologii : Zakład Biofizyki Komórkipl
dc.contributor.advisorDobrucki, Jerzy - 127740 pl
dc.contributor.authorKajstura, Małgorzatapl
dc.contributor.institutionJagiellonian University. Faculty of Biochemistry, Biophysics and Biotechnologypl
dc.contributor.reviewerKmieć, Zbigniewpl
dc.contributor.reviewerPiwocka, Katarzynapl
dc.date.accessioned2019-03-05T12:04:48Z
dc.date.available2019-03-05T12:04:48Z
dc.date.openaccess0
dc.date.submitted2019-01-18pl
dc.description.accesstimew momencie opublikowania
dc.description.additionalBibliogr. s. 59-75pl
dc.description.physical75pl
dc.description.versionostateczna wersja autorska (postprint)
dc.identifier.callnumberDokt. 2019/011pl
dc.identifier.projectROD UJ / OPpl
dc.identifier.urihttps://ruj.uj.edu.pl/xmlui/handle/item/69784
dc.languageengpl
dc.placeKrakówpl
dc.rightsCopyright*
dc.rights.licenceInna otwarta licencja
dc.rights.simpleviewWolny dostęp
dc.rights.urihttp://ruj.uj.edu.pl/4dspace/License/copyright/licencja_copyright.pdf*
dc.share.typeotwarte repozytorium
dc.subject.engedladamycinpl
dc.subject.enheat shock protein 90pl
dc.subject.encell cyclepl
dc.subject.enapoptosispl
dc.subject.enflow cytometrypl
dc.subject.plgeldanamycynapl
dc.subject.plbiałko szoku cieplnego 90pl
dc.subject.plcykl komórkowypl
dc.subject.plapoptozapl
dc.subject.plcytometria przepływowapl
dc.titleEffects of geldanamycin, a ligand of heat shock protein 90, on cell cycle progression and induction of apoptosis in human lymphocytes and jurkat cellspl
dc.title.alternativeWpływ geldanamycyny, inhibitora białka szoku cieplnego 90, na przebieg cyklu komórkowego i indikcję apoptozy w ludzkich limfocytach i komórkach Jurkatpl
dc.typeThesispl
dspace.entity.typePublication
dc.abstract.enpl
Heat shock protein 90 (HSP90), which is implicated in post-translational folding, stability, and maturation of proteins, controls several key cell cycle regulators. Thus, the hypothesis was raised that geldanamycin, a specific and potent inhibitor of HSP90 function, may have pronounced effects on cell cycle progression. The objective of this study was to test this hypothesis in normal and cancer cells of human origin. The experiments performed on human lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) indicated that 100 nM or 150 nM geldanamycin induces transition of cells to the G0 state of cell cycle. This was documented utilizing acridine orange, a metachromatic dye which differentially stains DNA versus RNA. The same experimental protocol allowed demonstration that geldanamycin is a potent inducer of apoptosis in PHA-activated cells. Importantly, both the block in G0 and induction of apoptosis were reversible and returned to control values upon removal of geldanamycin. Similar conclusions were reached when cell number in cultures was analyzed, excluding the possibility that a relevant fraction of cells was disintegrated during the incubation period. Experiments on Jurkat line of acute T-cell leukemia were performed next. Jurkat cells were used here as a model system in which the cytostatic and cytotoxic properties of geldanamycin on cancer cells can be tested. Initial experiments determined the time course and concentration-dependence of geldanamycin-induced alterations in cell cycle distribution and apoptosis. In contrast to human lymphocytes, geldanamycin did not induce G0 arrest in Jurkat cells, but inhibited them initially in the G2 phase, and at later time points in the G1 phase. The G2 was distinguished from mitosis by the absence of phosphorylation of histone H3, a specific marker of mitotic cells. The inhibition of Jurkat cells in G1 was linked to a decrease in phosphorylation of retinoblastoma protein. Finally, the exposure of Jurkat cells to geldanamycin resulted in induction of apoptosis, predominantly in cells being arrested in G1 and G2/M phases of the cell cycle. Finally, to address the possibility that stimulation of nuclear factor kappa-B (NF-kB), downstream of HSP90, modulates the effects of geldanamycin on cancer cells, Jurkat IkBaM line was employed. These cells cannot activate their NF-kB-mediated responses because of the mutation in its inhibitory protein, IkB. In the absence of functional NF-kB, geldanamycin-mediated induction of apoptosis and loss of cycling cells were markedly higher than when NF-kB was functional. These results were further corroborated by experiments in which parthenolide, a plant-derived inhibitor of NF-kB was employed. Geldanamycin-treated Jurkat cells responded to the parthenolide challenge by partial arrest in S phase and increased cell death by apoptosis. In conclusion, inhibition of HSP90 by geldanamycin blocks cell cycle progression and induces apoptosis of Jurkat cells, and NF-kB mediates these effects. The newly identified network of interactions may facilitate understanding of the mechanism of cytostatic and cytotoxic action of geldanamycin derivatives used currently in clinical trials.
dc.affiliationpl
Wydział Biochemii, Biofizyki i Biotechnologii : Zakład Biofizyki Komórki
dc.contributor.advisorpl
Dobrucki, Jerzy - 127740
dc.contributor.authorpl
Kajstura, Małgorzata
dc.contributor.institutionpl
Jagiellonian University. Faculty of Biochemistry, Biophysics and Biotechnology
dc.contributor.reviewerpl
Kmieć, Zbigniew
dc.contributor.reviewerpl
Piwocka, Katarzyna
dc.date.accessioned
2019-03-05T12:04:48Z
dc.date.available
2019-03-05T12:04:48Z
dc.date.openaccess
0
dc.date.submittedpl
2019-01-18
dc.description.accesstime
w momencie opublikowania
dc.description.additionalpl
Bibliogr. s. 59-75
dc.description.physicalpl
75
dc.description.version
ostateczna wersja autorska (postprint)
dc.identifier.callnumberpl
Dokt. 2019/011
dc.identifier.projectpl
ROD UJ / OP
dc.identifier.uri
https://ruj.uj.edu.pl/xmlui/handle/item/69784
dc.languagepl
eng
dc.placepl
Kraków
dc.rights*
Copyright
dc.rights.licence
Inna otwarta licencja
dc.rights.simpleview
Wolny dostęp
dc.rights.uri*
http://ruj.uj.edu.pl/4dspace/License/copyright/licencja_copyright.pdf
dc.share.type
otwarte repozytorium
dc.subject.enpl
gedladamycin
dc.subject.enpl
heat shock protein 90
dc.subject.enpl
cell cycle
dc.subject.enpl
apoptosis
dc.subject.enpl
flow cytometry
dc.subject.plpl
geldanamycyna
dc.subject.plpl
białko szoku cieplnego 90
dc.subject.plpl
cykl komórkowy
dc.subject.plpl
apoptoza
dc.subject.plpl
cytometria przepływowa
dc.titlepl
Effects of geldanamycin, a ligand of heat shock protein 90, on cell cycle progression and induction of apoptosis in human lymphocytes and jurkat cells
dc.title.alternativepl
Wpływ geldanamycyny, inhibitora białka szoku cieplnego 90, na przebieg cyklu komórkowego i indikcję apoptozy w ludzkich limfocytach i komórkach Jurkat
dc.typepl
Thesis
dspace.entity.type
Publication
Affiliations

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