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Iodine-131 induces mitotic catastrophes and activates apoptotic pathways in HeLa Hep2 cells
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. (Stigbrand)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. (Stigbrand)
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
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2008 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 23, no 5, 541-549 p.Article in journal (Refereed) Published
Abstract [en]

Iodine-131 (131I) has been used both in unconjugated form and conjugated to antibody derivates (i.e., radioimmunotherapy; RIT) to treat malignant diseases. The mechanisms by which 131I-irradiation causes growth retardation are, however, inadequately understood. The aim of this study was to elucidate the sequential molecular and cellular events that initiate cell death in HeLa Hep2 cells exposed to 131I. In this paper, HeLa Hep2 cells were found to display a transient G2-M arrest following irradiation, but then reentered the cell cycle still containing unrepaired cellular damage. An increase of multipolar mitotic spindles, as well as a significant increase in centrosome numbers from 8.8% +/- 1.9% in controls to 54.7% +/- 2.2% in irradiated cells, was observed (p < 0.0001). A subsequent failure of cytokinesis caused the cells to progress into mitotic catastrophe. This was accompanied by the formation of giant cells with multiple nuclei, multilobulated nuclei, and an increased frequency of polyploidy cells. A fraction of the cells also displayed apoptotic features, including the activation of initiator caspases-2, -8, -9, and effector caspase-3, as well as cleavage of poly(ADP-ribose) polymerase, a cell-death substrate for active caspase-3. These findings demonstrate that mitotic catastrophes and the activation of a delayed type of apoptosis might be important mechanisms involved in cell death following the RIT of solid tumors with -emitting radionuclides, such as 131I.

Place, publisher, year, edition, pages
2008. Vol. 23, no 5, 541-549 p.
Keyword [en]
iodine-131, mitotic catastrophe, apoptosis, caspases, centrosomes, radioimmunotherapy
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-21104DOI: 10.1089/cbr.2007.0471PubMedID: 18986216OAI: oai:DiVA.org:umu-21104DiVA: diva2:210798
Available from: 2009-04-06 Created: 2009-04-02 Last updated: 2013-09-13Bibliographically approved
In thesis
1. Regulation of apoptosis during treatment and resistance development in tumour cells
Open this publication in new window or tab >>Regulation of apoptosis during treatment and resistance development in tumour cells
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Induction of apoptosis is the most studied cell death process and it is a tightly regulated physiological event that enables elimination of damaged and unwanted cells. Apoptosis can be induced via activation of either the intrinsic or the extrinsic signalling pathway. The intrinsic pathway involves activation of the mitochondria by stress stimuli, whereas the extrinsic pathway is triggered by ligand induced activation of death receptors such as Fas. Apoptosis induction via Fas activation plays an important role in the function of cytotoxic T lymphocytes and in the control of immune cell homeostasis.

Several studies have shown that anticancer therapies require functional cell death signalling pathways. Irradiation based therapy has been successful in treatment of several malignancies but the usage of high doses has been associated with side effects. Therefore, low dose therapies, that either is optimized for specific delivery or administrated in combination with other treatments, are promising modalities. However, in order to achieve high-quality effects of such treatments, the death effector mechanisms involved in tumour eradication needs to be further explored. Importantly, tumour cells frequently acquire resistance to apoptosis, which consequently allows tumour cells to escape from elimination by the immune system and/or treatment.

Interferons constitute a large family of pleotrophic cytokines that are important for the immune response against viruses and other microorganisms. The interferon signalling pathway mediates transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. Interferon has successfully been used in therapy against some tumours. However, several drawbacks have been reported, such as reduced sensitivity to interferon during treatment.

The aim of this thesis was to elucidate mechanisms that mediate resistance to death receptor or interferon induced apoptosis in human tumour cell models, as well as investigate what molecular events that underlie cell death following radiation therapy of tumour cells.

In order to elucidate mechanisms involved in acquired resistance to Fas- or interferon-induced apoptosis, a Fas- and interferon-sensitive human cell line, U937, was subjected to conditions where resistance to either Fas- or interferon induced apoptosis was acquired. Characterization of the Fas resistant cells showed that multiple resistant mechanisms had been acquired. Reduced Fas expression and increased cFLIP expression, which is an inhibitor of death receptor signalling, were two important changes found. To further examine the importance of these two alterations, clones from the Fas resistant population were established. The reduced Fas expression was determined to account for the resistant phenotype in approximately 70% of the clones. In the Fas resistant clones with normal Fas expression, the importance of an increased amount of the cFLIP protein was confirmed with shRNA interference. A cross-resistance to death receptor induced apoptosis was detected in the interferon resistant variant, which illustrates that a connection between death receptor and interferon induced apoptosis exists. Notably, interferon resistant cells also contained increased cFLIP expression, which were determined to mediate resistance to both interferon and death receptor mediated apoptosis. Finally, when cell death induced by irradiation treatment was investigated in HeLa Hep2 cells we could demonstrate that cell death was mediated by centrosome hyperamplification and mitotic aberrations, which forced the cells into mitotic catastrophes and delayed apoptosis.

In conclusion, we have described model systems where selection for resistance to Fas or interferon induced apoptosis generated a heterogeneous population, where several signalling molecules were altered. Furthermore, we have shown that a complex cell death network was activated by irradiation based therapy.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Medicinska fakulteten), 2008. 65 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1199
Keyword
Tumörbiologi
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1851 (URN)978-917264-627-8 (ISBN)
Public defence
2008-10-03, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2010-04-06Bibliographically approved
2. Optimizing experimental radioimmunotherapy: investigating the different mechanisms behind radiation induced cell deaths
Open this publication in new window or tab >>Optimizing experimental radioimmunotherapy: investigating the different mechanisms behind radiation induced cell deaths
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Optimering av experimentell radioimunoterapi : utredning av de olika mekanismerna bakom strålningsinducerade celldöder
Abstract [en]

Background. Radiation therapy is an important treatment regimen for malignant disease. Radiation therapy uses ionizing radiation to induce DNA damage in tumor cells in order to kill them. Tumor cells are more sensitive than normal cells, since they have an increased proliferation rate and often lack the ability to properly repair the induced damage. Radiation can be delivered by an external source outside the body, by brachytherapy delivered inside the patient near the tumor, or systemically by injection into the blood stream. When delivered systemically, the radiation is administered as radioisotope alone or conjugated to antibodies targeting tumor antigens (radioimmunotherapy). Radiotherapy (RT) usually is administered using high doses, causing necrotic cell death. Low doses of radiation (by RT or RIT) have been observed to induce different types of cell deaths, like apoptosis, mitotic catastrophe or senescence.Aims. We wanted to elucidate the molecular and cellular events responsible for the induction of cell death in cells of different origin and p53 status. We also wanted to identify the kinetics behind gene expression alterations induced in response to irradiation and correlate these to cell death specific molecular and cellular events. In the end this research aims to identify key regulators of the main radiation induced cell death modalities in order to improve our understanding and potentially use this knowledge to increase treatment efficacy of radiation therapy.

Methods. Four different cell lines were used in these studies to elucidate the role of p53 status cell origin in radiation induced cell death. HeLa Hep2 tumor cells have been used previously in our group in several RIT and RT studies. During these studies we observed morphological alterations in shrinking tumors that were typical for mitotic catastrophe. This led to studies on the underlying mechanisms causing these aberrations. Isogenic solid tumor cell lines HCT116 p53 +/+ and HCT116 p53 -/- were included to further elucidate the role of p53, and also to study senescence, one of the main outcomes in irradiated tumor cells. MOLT-4 was finally included to compare these finding to classical apoptosis. Gene expression analysis was done using Illumina bead chip arrays, and pathway analysis was performed using MetaCore (Thomson Reuters).

Results. In paper I, II, and III, transient G2/M arrests were observed in HeLa Hep2 and HCT116 p53 -/- cells following irradiation. The lack of p53 in these cells caused checkpoint adaptation due to an unscheduled accumulation of genes promoting mitosis. Anaphase bridges were observedivin HeLa Hep2 cells, as a consequence of premature mitotic entry with unrepaired DNA damage. Centrosome amplification, as well as deregulation of genes involved in centrosome amplification and clustering was observed in both cell lines. We observed changes in expression of several genes responsible for maintaining the spindle assembly checkpoint (SAC) arrest. A prolonged SAC arrest has been shown to be important for execution of mitotic catastrophe. SAC activation was followed by mitotic slippage and a subsequent failure of cytokinesis. We observed multipolar mitoses (both cell lines), multiple- and micronuclei (HeLa Hep2, paper I), and an increased frequency of tetraploid cells (HeLa Hep2 and HCT116 p53 -/- cells). A fraction of HeLa Hep2 cells also displayed apoptotic features, including caspase activation and DNA fragmentation (paper I). These findings indicate that mitotic catastrophe and the activation of a delayed type of apoptosis are involved in cell death following RIT.HCT116 p53 +/+ cells induced both G1 and G2 arrest following irradiation (paper III). Gene expression analysis revealed significantly decreased expression of genes responsible for cell cycle progression (pronounced decrease compared to HeLa Hep2 and HCT116 p53 -/-), especially mitotic genes. The prolonged arrest transitioned into senescence starting 3 days following irradiation and peaked after 7 days. Several genes associated with SASP were upregulated in the same time frame as senescence was induced, further supporting the fact that senescence is the main radiation induced response in HCT116 p53 +/+ cells.MOLT-4 cells, similar to HCT116 p53 +/+ cells, induced both G1 and G2 arrests in response to irradiation (paper IV). Morphological studies revealed apoptotic features like shrunken cells with condensed DNA. Caspase assays showed increased activity of caspases -3, -8, and -9. Gene expression analysis confirmed an increased expression of genes important for both extrinsic (FAS and TRAIL) and intrinsic (BAX) apoptosis. Furthermore, changed expression also included genes involved in cell cycle checkpoints and their regulation and genes important for T-cell activation/proliferation.

Conclusions. RIT is successfully used to treat lymphoma, but treatment of solid tumors with RIT is still difficult. This thesis elucidates cellular alterations characteristic for the 3 main radiation death modalities, i.e. mitotic catastrophe, senescence and apoptosis. Furthermore, cell death specific traits are correlated to alterations in gene expression. Treatment efficacy can potentially be improved by finding key cell death mediators to inhibit in combination with radiation.

Abstract [sv]

Bakgrund. Strålbehandling används för att bota eller lindra symptomen av cancer och består av joniserande strålning vars syfte är att skada DNAt i cellerna vilket leder till att de dör. Tumörceller är känsligare för strålning än normala celler eftersom de delar sig i snabbare takt och ofta saknar förmågan att reparera skadorna som uppstår. Det finns flera typer av strålbehandling: extern strålbehandling, d.v.s. när strålkällan är placerad utanför kroppen, brachyterapi, när strålkällan placeras i en kapsel inuti kroppen, eller systemisk strålning, där en radioisotop injiceras, antingen själv eller kopplad till en antikropp, då kallad radioimmunoterapi (RIT). Vid extern strålbehandling använder man sig ofta av relativt höga doser av strålning under ett kortare tidsintervall. Dessa celler dör ofta en nekrosliknande död. Med RIT kan man behandla patienterna med lägre doser under en längre tid och strålningen kan riktas specifikt till tumören, vilket minskar risken för bieffekter. Dessa celler dör av andra former av celldöd, apoptos, senescence eller mitotisk katastrof. Apoptos är för många synonymt med programmerad celldöd, och sker till exempel i respons till DNA skada. En apoptotisk cell känns igen på sitt utseende med fragmenterat DNA, nedbrutet cytoskelett och apoptotiska kroppar. Senescence är associerat med cellens åldrande men kan även orsakas av DNA-skador, och är en vanlig form av celldöd hos solida tumörceller med funktionell p53-signalering. Bestrålade solida tumörceller som saknar p53-signalering, antingen på grund av mutationer eller på grund av virusinducerad inaktivering, dör oftast i en helt annan celldöd, kallad mitotisk katastrof. Avsaknad av p53 leder till att en cell som erhållit skador på DNAt inte klarar av att uppehålla cellcykeln länge nog för att reparera skadorna. Inte heller apoptos induceras, eftersom p53 saknas. Detta leder till att cellen kommer att gå in i mitos med skador i sitt DNA som ej hunnit repareras. Celler i mitotisk katastrof har ett väldigt typiskt utseende med multipla kärnor, mikrokärnor (kromosomrester), multipla centrosomer och multipolära mitotiska spindlar. En del celler dör i mitosen medan andra försöker dela sig och kan överleva i flera generationer till, dock med skador på DNA. Målet med denna avhandling var att utreda de molekylära och transkriptionella mekanismerna bakom strålningsinducerad celldöd, och p53s roll i detta. Dessa studier kan så småningom leda till att viktiga regulatoriska proteiner av de strålnigsinducerade celldödsmekanismerna kan identifieras. Specifika inhibitorer riktade mot dessa proteiner kan med ökad kunskap strategiskt användas i kombination med strålning och potentiellt leda till förbättrade behandlingseffekter.

Metoder. Vi använde fyra cellinjer med olika bakgrund och p53 status. Vi har tidigare studerat HeLa Hep2 (en solid tumörcellslinje infekterad medviHPV som slår ut funktionen av p53) och sett vid både RT och RIT studier, att cellernas morfologi avviker från klassiks apoptos (stora celler med stora mängder DNA, istället för små celler med lite DNA). Detta ledde till studier av mekanismerna bakom denna avvikande cellmorfologin, som är typisk för mitotisk katastrof. Vi utökade studien med HCT116 p53 +/+ och HCT116 p53 -/- som är identiska så när som på p53, där ena cellinjen saknar denna gen. Detta skulle ge ökad förståelse för p53s roll vid mitotisk katastrof och även visa mekanismerna bakom senescence, en annan vanlig celldödsmekanism i strålade solida tumörceller. Även MOLT-4 inkluderades i studien för att kunna jämföra våra resultat med en cellinje som genomgår klassisk apoptos och är mer känslig för strålning.

Resultat. I celler där mitotisk katastrof inducerades efter strålning (HeLa Hep2, HCT116 p53-/-) såg vi ett övergående G2 arrest. Eftersom cellerna inte klarade av att underhålla detta arrest, då de saknar p53, fortsatte de in i nästa fas av cellcykeln, mitos. Detta ledde till att DNA skador kvarstod och en ökad frekvens av anafasbryggor. Dessutom skedde en centrosomamplifiering i dessa celler vilket gav upphov till multipolära mitotiska spindlar och en efterföljande icke fungerande cytokines. Detta gav i sin tur celler med multipla kärnor eller mikrokärnor. En ökad frekvens av tetraploida och polyploidaEn förändrad expression av gener som kunde kopplas till flera av dessa för mitotisk katastrof specifika karaktäristika observerades också. Flera gener associerade med reglering av centrosomen och dess amplifiering, med kontrollen av cellens progression från G2 till M-fasen av cellcykeln, samt involverade i kontrollen av en rätt utförd mitos (SAC) hade en ändrad genexpression som korrelerade väl i tid med de ovan nämda fenotyperna. Caspaser som är viktiga för apoptos visade sig vara aktiva i HeLa Hep2, vilket indikerar att mitotisk katastrof kan leda till fördröjd apoptos. Men en del celler lyckas smita undan från apoptosinduktionen och fortsätter i en ny runda i cellcykeln, och detta kunde ses som en växande population viabla celler med ökad mängd DNA (tetraploida celler).HCT116 p53 +/+ celler som har funktionellt p53 kunde inducera både G1 and G2 arrest och genexpressionen visade att många gener som styr övergången till mitos var nedreglerade och förhindrade detta (till skillnad från HeLa Hep2 och HCT116 p53 -/-, där dessa nivåer var högre). Dessa arrester övergick till senescence 3 dagar efter strålning och många gener kopplade till senescence visade ett ökat uttryck. Vi såg ingen markant ökning av centrosomer eller polyploida celler vilket skiljde sig från HeLa Hep2 och HCT116 p53 -/-. Detta tyder på att senescence skiljer sig markant åt från mitotisk katastrof och att p53 är viktig för induktionen av denna form av celldöd.viiVi såg att MOLT-4, precis som HCT116 p53 +/+, inducerar både G1 and G2 arrest. Denna arrest resulterade dock i ökad expression av gener viktiga för cellcykelarrest och apoptosinduktion, och vi såg även en ökad aktivitet av caspaser. Morfologiska studier visade att strålade MOLT-4 celler ofta var små och hade kondenserat DNA, vilket är typiska kännetecken för apoptos. Strålning av MOLT-4 celler ledde till aktivering av klassisk apoptos, och tidsförloppet var mycket snabbare jämfört med de övriga cellinjerna.

Slutsats. RIT är en framgångsrik metod för att behandla hematologiska maligniteter, men solida tumörer svarar fortfarande dåligt på denna form av behandling. Denna avhandling visar på komplexiteten bakom strålningsinducerad celldöd och att det är viktigt att identifiera de reglerande mekanismerna för att kunna förbättra RIT av solida tumörer. Vi visar även på vikten av p53 vad gäller tumörens respons av strålbehandling. Genom att identifiera viktiga proteiner för mitotisk katastrof, senescence, och apoptos, kan man utveckla inhibitorer mot dessa och använda de i kobination med RT och RIT för att förbättra behandlingseffekten.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2013. 63 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1594
Keyword
mitotic catastrophe, senescence, cell cycle checkpoint, apoptosis, radiation, gene expression, p53
National Category
Cell and Molecular Biology
Research subject
Immunology; radiofysik
Identifiers
urn:nbn:se:umu:diva-80232 (URN)978-91-7459-717-2 (ISBN)
Public defence
2013-10-04, By 6E sal E04, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-09-13 Created: 2013-09-12 Last updated: 2013-09-13Bibliographically approved

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Eriksson, DavidBlomberg, JeanetteLindgren, TheresJohansson, LennartRiklund, KatrineStigbrand, Torgny

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