Live cell imaging is a unique tool for cellular research with a wide variety of applications. By streaming digital microscopic images an investigator can observe the dynamic morphology of a cell, track cell movement on a surface, and measure quantities or localization patterns of fluorescently labeled proteins or molecules. Digital image sequences contain a vast amount of information in the form of visually detectable morphological changes in the cell. We designed computer programs that allow the manual identification of visible events in live cell digital image sequences [Davis et al. 2007]. Once identified, the data are analyzed using algorithms to calculate the yield of individual events per cell over the time course of image acquisition. The sequence of event data is also constructed into directed acyclic graphs and through the use of a subgraph isomorphism algorithm we are able to detect specified patterns of events originating from a single cell. Two projects in the field of cancer research are here discussed that describe and validate the application of the event analysis programs. In the first project, mitotic catastrophe (MC) research [Ianzini and Mackey, 1997; Ianzini and Mackey, 1998; reviewed by Ianzini and Mackey, 2007] is enhanced with the addition of live cell imaging to traditional laboratory experiments. The event analysis program is used to describe the yield of normal or abnormal divisions, fusions, and cell death, and to detect patterns of reductive division and depolyploidization in cells undergoing radiation-induced MC. Additionally, the biochemical and molecular data used in conjunction with live cell imaging data are presented to illustrate the usefulness of combining biology and engineering techniques to elucidate pathways involved in cell survival under different detrimental cell conditions. The results show that the timing of depolyploidization in MC cells correlates with increased multipolar divisions, up-regulation of meiosis-specific genes, and the production of mononucleated cell progeny. It was confirmed that mononucleated cells are produced from multipolar divisions and these cells are capable of resuming normal divisions [Ianzini et al., 2009]. The implications for the induction of meiosis as a mechanism of survival after radiation treatment are discussed. In the second project, the effects of long-term fluorescence excitation light exposure are examined through measurements of cell division and cell death. In the field of live cell imaging, probably the most modern and most widely utilized technique is fluorescence detection for intracellular organelles, proteins, and molecules. While the technologies required to label and detect fluorescent molecules in a cell are well developed, they are not idealized for long term measurements as both the probes and excitation light are toxic to the cells [Wang and Nixon, 1978; Bradley and Sharkey, 1977]. From the event analysis data it was determined that fluorescence excitation light is toxic to multiple cell lines observed as the reduction of normal cell division, induction of cell death, and apparent morphological aberrations.

Radioimmunotherapy is becoming important as a new therapeutic strategy for treatment of tumour diseases. Lately monoclonal antibodies tagged with radionuclides have demonstrated encouraging results in treatment of hematological malignancies. The progress in treatment of solid tumours using radioimmunotherapy, however, has been slow. New strategies to improve the treatment response need to be evaluated. Such new strategies include the combination of radioimmunotherapy with other treatment modalities but also elucidation and exploration of the death effector mechanisms involved in tumour eradication. As the combination of radioimmunotherapy and radiotherapy provides several potential synergistic effects, we started out by optimising a treatment schedule to detect benefits combining these treatment modalities. An anti-cytokeratin antibody labelled with 125I administered before, after, or simultaneously with radiotherapy, indicated that the highest dose to the tumour was delivered when radiotherapy was given prior to the antibody administration. The optimised treatment schedule was then applied therapeutically in an experimental study on HeLa Hep2 tumour bearing nude mice given radiotherapy prior to administration of 131I-labelled monoclonal antibodies. Combining these treatment regimes enhanced the effect of either of the treatment modalities given alone, and a significant reduction in tumour volumes could be demonstrated. This treatment caused a dramatic change in tumour morphology, with increased amounts of connective tissue, giant cells and cysts. Furthermore cellular alterations like heterogeneity of nuclear and cytoplasmic size and shape were observed, and at least a fraction of the tumour cells presented some characteristics of apoptosis. The induced sequential events in Hela Hep2 cells exposed to 2.5-10 Gy of ionizing radiation were studied further, with special emphasis on cell cycle arrest, mitotic aberrations and finally cell death. Following radiation HeLa Hep2 cells initiated a transient G2/M arrest trying to repair cellular damage. This arrest was followed by a sequence of disturbed mitoses with anaphase bridges, lagging chromosomal material, hyperamplification of centrosomes and multipolar mitotic spindles. These mitotic disturbances produced multinuclear polyploid cells and cells with multiple micronuclei, cells that were destined to die via mitotic catastrophes and delayed apoptosis. Induction of apoptosis in HeLa Hep2 cells following radiation doses and dose-rates equivalent to those delivered at radioimmunotherapy was concurrently studied in vitro. Significant induction of apoptosis was obtained and found to be induced relatively slowly, peaking 72-168 hours post irradiation. Caspases from the intrinsic pathway as well as the extrinsic pathway were found to be activated in response to ionizing radiation. Furthermore caspase-2, which has recently been acknowledged for its role as an initiator caspase was found to be activated following radiation and seems to play an important role in this delayed apoptosis.

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. / 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.

No description available.

Apoptoseresistenz ist einer der Gründe für ein Versagen von Chemotherapie bei vielen Krebserkrankungen, darunter das Burkitt Lymphom. Um die molekularen Mechanismen der Apoptoseresistenz aufzuklären, wurde die Apoptoseinduktion in 15 Burkitt-Lymphom-Zelllinien nach Behandlung mit den Spindelgiften Taxol (Paclitaxel), Nocodazol und Vincristin untersucht. Interessanterweise entwickelten Zellen, die sich als resistent gegenüber Taxol- und Nocodazol-induzierter Apoptose erwiesen, nach Behandlung eine Polyploidie (>4N DNA), was eine inverse Relation von Apoptose und Polyploidie aufzeigt. In den sensitiven Zelllinien war die Taxol- und Nocodazol-induzierte Apoptose von Caspase-Aktivierung, Bid-Spaltung und Herunterregulation von Mcl-1 begleitet. Im Gegensatz zu den sensitiven Zelllinien wiesen die meisten apoptoseresistenten Zellen einen Verlust von Bax und Bak auf und waren durch einen anhaltenden mitotischen Arrest mit Auftreten eines >4N DNA-Gehalts nach Behandlung charakterisiert. Um weitere Einblicke in den Mechanismus der Spindelgift-induzierten Apoptose zu erhalten, wurde die Rolle der mitotische Kinase PLK1 (polo-like kinase) näher untersucht. Eine dominant-negative PLK1-Mutante induziert Apoptose. Allerdings zeigte eine zusätzliche Behandlung mit Spindelgiften keinen synergistischen Effekt, was darauf schließen lässt, dass sowohl Inhibierung von PLK1 als auch Mikrotubuli-destabilisierende Agenzien den gleichen Stress-Signalweg aktivieren. Andererseits unterstützte Überexpression von Wildtyp-PLK1 in Taxol behandelten Zellen die Zellzyklus-Progression. Dies deutet auf eine Verbindung zwischen Zelltodresistenz und genetischer Instabilität (Aneuplodie) hin. Inhibition von Apoptose in sensitiven Zelllinien durch Caspase-Inhibierung förderte Polypoidie, welche die inverse Relation bestätigte. Medikamente, welche die Caspase-Aktivierung unabhängig von Bax und Bak induzieren, könnten eine weitere Möglichkeit zur Behandlung von resistenten Burkitt-Lymphomen darstellen. / Apoptosis resistance is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphomas (BL). To elucidate molecular mechanisms regulating the development of apoptosis resistance, a panel of 15 BL cell lines was investigated for apoptosis induction upon treatment with microtubule inhibitors taxol, nocodazole and vincristine. Significant differences were observed in the extent of apoptosis induction among BL cell lines examined. Interestingly, cell lines exhibiting resistance to taxol- or nocodazole-induced apoptosis, showed development of polyploidy (>4N) and vice versa, displaying an inverse relationship between apoptosis and polyploidy induction. Further, in sensitive cell lines taxol-induced apoptosis was accompanied by caspase activation, Bid cleavage and Mcl-1 down-regulation. In contrast, most apoptosis resistant cell lines exhibited a loss of Bax and Bak expression and showed prolonged mitotic arrest with >4N DNA content upon treatment. To gain mechanistic insights into microtubule inhibitor-induced cell death, the role of the mitotic kinase PLK1 was addressed. Dominant negative PLK1 mutant induced apoptosis, however, failed to show synergism in induction of apoptosis in combination with microtubule inhibitors. This indicates that PLK1 inhibition and spindle toxins might trigger a similar mitotic stress pathway. Conversely, overexpression of wildtype PLK1 promoted cell cycle progression in cells treated with taxol. Remarkably, inhibition of apoptosis in sensitive cell lines by caspase inhibition promoted polyploidy confirming the inverse relationship between apoptosis and polyploidization. Considering targets to induce Bax/Bak independent caspase activation would be of great importance to avoid undesirable events leading to chromosomal imbalances in treating resistant cancers.

La sénescence réplicative est le phénomène associé à un arrêt de croissance permanent causé par le raccourcissement progressif des télomères à chaque division. Lorsqu’ils atteignent une longueur critique, les télomères perdent leur structure terminale protectrice en t-loop, ce qui révèle l’extrémité du chromosome et déclenche une Réponse aux dommages à l’ADN (RDA) p53-dépendante. Le nombre de télomères ouverts nécessaire à la mise en place de la sénescence n’est pas connu, mais plusieurs évidences suggèrent que la cellule pourrait en tolérer un certain nombre avant de s’arrêter définitivement. Dans ce projet, nous utilisons un dominant négatif de Tin2 (Tin2DN), un membre du complexe nucléo-protéique nommé le télosome qui stabilise la t-loop, pour démontrer que la dysfonction chromatinienne télomérique seule ne suffit pas à déclencher un arrêt de croissance permanent. Lorsqu’il est exprimé, Tin2DN induit la formation de foyers de dommages de 53BP1, la RDA ainsi qu’un arrêt de croissance transitoire. De façon surprenante, nous observons que les cellules qui ont subi ce premier arrêt de croissance ré-entrent dans le cycle cellulaire et se divisent, et ce malgré la présence de foci télomériques. Cette réentrée cause l’apparition de cassures secondaires ainsi qu’une accumulation d’instabilités génomiques, telles que des ponts chromosomiques ou des micro-noyaux. Cet échappement des points de blocages du cycle cellulaire pourrait être expliqué par notre observation que la dysfonction télomérique induite par Tin2DN n’active que très faiblement p53 et p21, et pratiquement pas la kinase chkChk2. Néanmoins, en inhibant directement l’activité de p53, nous n’observons plus aucun arrêt de croissance mais une accumulation de foci et d’instabilités génomiques, avec une forte occurrence de catastrophes mitotiques. L’ensemble de ces résultats propose un nouveau modèle d’entrée en sénescence réplicative : l’ouverture des télomères induits une faible RDA menant à un premier arrêt de prolifération transitoire p53-dépendant. Les cellules échappent à cet arrêt et se divisent, mais l’ouverture des télomères ayant causé des fusions chromosomiques, la division crée alors de nouvelles cassures doubles brins dans le génome qui déclencheront une forte RDA et un nouvel arrêt de croissance permanent, la sénescence réplicative. / Replicative senescence is the physiological permanent growth arrest caused by telomeres shortening, at each round of replication. Once they have reach a critical length, the telomeres lose their t-loop structure, revealing the chromosome extremity that triggers a p53-dependant DNA damage response (DDR) and leads to proliferation arrest. The number of shortened telomeres that are necessary to onset senescence is not known, but accumulating evidences suggest that the cell is able to tolerate a certain level of telomere uncapping before stopping its divisions. Here, we used an inducible dominant negative form of Tin2 (Tin2DN), a member of the shelterin complex that stabilizes the t-loop, to show that telomeres uncapping alone is not sufficient to induce a stable growth arrest. When expressed, Tin2DN leads to the openingverture of the t-loop, creating a DDR with the formation of 53BP1 DNA damage foci (DDF) and a transient growth arrest. Indeed, we observed that the cells were re-entering the cell cycle and dividing, despite their uncapped DDF harbouring telomeres. As telomere uncapping creates chromosome fusions, such division leads to the apparition of secondary DNA breaks, with an accumulation of genomic instabilities, such as chromosomes bridges or micronuclei. We observed that Tin2 DN-induced telomere uncapping leads to a very weak activation of p53 and p21, with almost no phosphorylation of chkChk2. Nevertheless, when we infected our cells with a shp53, the primary growth arrest did not occur, leading to an amplification of the damages, with strong signs of instability and mitotic catastrophe. Altogether, these results propose a new model for replicative senescence: telomere uncapping induces a weak DDR that leads to a transitory growth arrest. The cells divide with fused chromosomes, creating new randomly distributed double strand breaks that trigger a stronger DDR and a permanent growth arrest. In that model, replicative senescence is not directly induced by telomere uncapping, but by an amplification of DNA damages through mitotic catastrophe.