Authors:	Su, Zhanhai
Title:	Modulation of apoptosis by endogenously produced nitric oxide
Online publication date:	9-Aug-2006
Year of first publication:	2006
Language:	english
Abstract:	Untersuchungen zur Expression der induzierbaren NO-Synthetase (NOS2) belegen eine häufige Expression dieses Enzyms in Tumoren unterschiedlicher Gewebe. Bislang ist jedoch ungeklärt, ob die Expression der NOS2 in Tumorzellen die apoptotische Eliminierung durch zytotoxische T-Zellen beeinflussen kann. In der vorliegenden Arbeit wurden die Folgen einer endogenen NO-Synthese auf die Apoptosesensitivität von HEK293-Zellen untersucht. Um primäre NO-Wirkungen von NO-induzierten, sekundären (kompensatorischen) Veränderungen zu trennen, wurde mit einem induzierbaren Vektorsystem gearbeitet. Die NOS2 wurde zunächst unter der Kontrolle eines Ecdyson-sensitiven Promoters in HEK293-Zellen kloniert. Es konnten regulierbare NOS2-Klone selektiert werden, die nach Ponasteronbehandlung dosisabhängig die NOS2 exprimieren und NO synthetisieren. Die NOS2-Expression wurde durch Western Blot Analyse und Immunfluoreszenzfärbung dargestellt und die NO-Produktion mit Hilfe der Griess-Reaktion gemessen. An den NOS2-induzierten Zellen wurde dann der Einfluss von NO auf die CD95-vermittelte Apoptose analysiert. Es zeigte sich nach Stimulation des CD95-Rezeptors eine deutliche Korrelation der Apoptoserate mit der NOS2-Expression. In Kokulturexperimenten mit Peptid-spezifischen zytotoxischen T-Zellen zeigte sich, dass NO-produzierende Zielzellen effektiver eliminiert werden konnten. Auch nach Behandlung der Zellen mit TRAIL ergab sich eine höhere Apoptoserate in NO-produzierenden Zellen. Die weitere Analyse der durch NO beeinflussten Signalwege ergab eine Beteiligung von ER-Stress-vermittelten Apoptosewegen. Dies zeigte sich an der Hochregulation des ER-Stress-Proteins Grp78 (BiP) nach NOS2-Expression und der Spaltung der am ER-lokalisierten Caspase-4. Darüber hinaus konnte der schnellere Verlust des mitochondrialen Membranpotentials in Abhängigkeit von der NOS2-Expression nachgewiesen werden. Weiterhin wurde die Wirkung einer dauerhaften NO-Exposition auf die Apoptosesensitivität der Zellen untersucht. Auch ohne zusätzliche CD95-Stimulation induzierte eine kontinuierliche NOS2-Expression nach wenigen Tagen in den EcR293-NOS2-Zellen Apoptose. Diese Dauerbehandlung führte zum nahezu vollständigen Absterben der Kulturen. Einige Zellen überlebten jedoch diese Behandlung und wuchsen zu Zellklonen. Diese NO-resistenten Klone konnten isoliert werden. Sie zeigten eine zusätzliche Resistenz für CD95-vermittelte Apoptosesignale und waren besser vor dem Angriff Peptid-spezifischer CTLs geschützt. Die Apoptoseresistenz blieb auch nach längerer Kultur erhalten und scheint auf NO-induzierter Genotoxizität zu beruhen. Anhand dieser Arbeit konnte gezeigt werden, dass allein durch chronische NO-Behandlung eine Selektion apoptoseresistenter Zellen stattfinden kann. Nitric oxide (NO) has many important roles in the human nervous, immune and vascular system, and it also plays crucial roles in tumorgenesis. Many human tumors express inducible NO synthetase (NOS2/iNOS); however the roles of NO in tumor development and in the immune system are not fully understood. The role of nitric oxide (NO) in cells is controversial. Both cytotoxic and cyto-protective roles have been reported. The effects of endogenous inducible NO synthesis on apoptosis sensitivity have been attempted to be elucidated in this study, and apoptosis mediated by the CD95 or TRAIL system was given particular attention. The hypothesis tested was whether persistent exposure to NO induces apoptosis and can select for cells with reduced sensitivity to NO and apoptosis. A NOS2 expression vector driven by an ecdysone-inducible promoter was constructed; the vector was then introduced into EcR293 cells and EcR293-NOS2 stable cells were established. Ponasterone A, an ecdysone analog, was used to induce NOS2 expression. The advantage of this system is that cell lines could be established without constant exposure to NO. The expression of NOS2 and NO products were analyzed by Western blot, immunofluoresence staining, and Griess assays. The influences of NO on CD95- and TRAIL-mediated apoptosis were determined through FACS analysis, cytotoxicity assays, Western blots, and caspase activity. The expression of NOS2 and NO product (nitrite) were detectable in a number of cell clones after induction by ponasterone A and NOS2 expression was dependent on the dose of ponasterone A. As the dose of ponasterone A increased, apoptosis induced by treatment with agonistic CD95 antibody (anti-APO-1) or TRAIL, proportionally increased compared to controls. In CD95-mediated apoptosis, loss of mitochondria membrane potential, the activation of caspase-8, -9, and -3 were observed. The same results also were obtained from TRAIL-mediated apoptosis pathway. In EcR293-NOS2 cells, it was found that NO can reduce cell proliferation compared with uninduced cells. The cytotoxicity of NO may involve mitochondria depolarization and endoplasmic reticulum (ER) stress signalling pathway, because ER stress-associated proteins, such CHOP, BiP and ER-associated caspase-4 activation, were enhanced under the expression of NOS2. The effects of long-term exposure to NO were further examined. NO induction increased the apoptosis substantially over uninduced controls after nine days of culture. To test the hypothesis that persistent expression of NO selects for cells with reduced sensitivity to apoptosis, EcR293-NOS2 cells were cultured under long term NO induction. Significant cell death was observed in these cultures but cell colonies grew which were selected for further cultivation. After approximately three months of culturing in the presence of ponasterone A, cell clones were analyzed for NO production. All the selected colonies, which survived cultivation in the presence of high concentration of ponasterone A, also produced NO. These results demonstrated that the survival of the colonies was due to NO resistance and not merely to loss of ponasterone inducible NO production. To assess the effects on apoptosis, cells were treated with agonistic CD95 antibodies and analyzed by FACS. Interestingly, it was found that the NO resistant cell lines were cross resistant to CD95-mediated apoptosis and NO resistant cell lines showed a reduced sensitivity to CTL killing. These results support the idea that chronic exposure to NO selects cells with reduced sensitivity to apoptosis. On the basis of these results, it is suggested that the apoptosis-enhancing effect of NO results in the elimination of cells with a fully functional apoptotic response and the retention of a subpopulation of cells with an aberrant or attenuated response to death inducing signals. Chronic exposure to NO thus facilitates the clonal evolution of a population of cells that can circumvent normal death inducing signals, including those derived from CTLs during immune surveillance. These results support a role for NO in accelerating tumor formation and progression.
DDC:	570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 10 Biologie
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-4127
URN:	urn:nbn:de:hebis:77-11209
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Appears in collections:	JGU-Publikationen