Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC50 16¿640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a ¿gene-like sequence¿ of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC50 = 6.25 ¿M in a susceptible cell line, compared with > 100 ¿M for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes. / Yorkshire Cancer Research, BMSS, School of Life Sciences and the Frank Hudson Memorial Fund

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management. / AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC₅₀ 16-640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a 'gene-like sequence' of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC₅₀ = 6.25 μM in a susceptible cell line, compared with > 100 μM for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes.

Preclinical testing is an important part of cancer drug development. The aim of this thesis was to establish and evaluate preclinical in vitro methods useful in the development of new anticancer drugs. In paper I, the development of non-clonogenic assays (FMCA-GM) using CD34+ stem cells for assessment of haematological toxicity was described. A high correlation was seen when comparing the 50% inhibitory concentrations (IC50) from FMCA-GM with the IC50 from the established clonogenic assay (CFU-GM). In paper II, FMCA-GM was complemented with additional cell models, establishing a normal cell panel. In vitro toxicity towards the five normal cell types was compared with known clinical adverse event profiles. The normal cell panel roughly reflected the tissue specific toxicities but was most useful in the prediction of therapeutic index. In paper III the use of peripheral blood lymphocytes from human, dog, rat and mouse to detect species differences in cellular drug sensitivity was described. Good agreement between our method and the established CFU-GM assay was observed. In paper II the benefit of using primary tumour cells from patients to predict cancer diagnosis-specific activity was studied. The in vitro activity of fourteen anticancer drugs was tested in tumour samples of both haematological and solid tumour origin. In general, clinical activity was well reflected. In paper IV, the efficacy and toxicity models were applied for experimental follow-up of a novel inhibitor of the ubiquitin-proteasome system, CB3 (Phosphoric acid, 2,3-dihydro-1,1-dioxido-3-thienyl diphenyl ester). In the preliminary characterization of CB3, antitumour activity and a favourable toxicity profile were displayed, although the exact mechanism of action remains to be elucidated. CB3 will therefore be further investigated. In conclusion, the work presented here contributes to different parts of the preclinical drug development and the methods may aid in the characterization of anticancer compounds

Los efectos biológicos producidos por dosis bajas de radiación continúan siendo objeto de controversia. Cada cierto tiempo aparecen nuevos estudios a escala molecular y celular que arrojan luz sobre los mecanismos moleculares básicos que subyacen en las respuestas de los sistemas vivos a las radiaciones ionizantes. En esta tesis se muestran tanto la metodología empleada como los resultados obtenidos tras la irradiación in vitro de diferentes líneas celulares tumorales y no tumorales. Para irradiar los cultivos con dosis bajas (del orden del mGy) de radiación alfa se ha puesto a punto un dispositivo para disolver el gas radiactivo 222Rn en el medio de cultivo celular.Los resultados más interesantes han sido los obtenidos tras la irradiación de la línea MCF-7 de cáncer de mama humano metastásico. Junto con la observación de cambios en el crecimiento (no relacionadas linealmente con la dosis), los análisis genéticos revelaron modificaciones en la expresión de genes de la familia Bcl-2, reguladores de la muerte celular por apoptosis. En concreto, el gen bcl-xS (relacionado con quimiorresistencia en esta línea celular) sólo se expresó en los cultivos irradiados. Tras este resultado se analizó la influencia de la radiación sobre la sensibilidad de las MCF-7 a los fármacos Taxol y VP-16, observándose que los cultivos previamente irradiados respondían mejor al tratamiento con ambos quimioterápicos. / Biological effects due to low doses of radiation still remain as a controversial issue. Every certain time new studies on a cellular and molecular basis give out light on the basic mechanisms underlying the response of living systems to ionising radiation. In this thesis the methodology and results obtained after in vitro irradiation of tumour and normal cell lines are described. In order to expose cells to low doses of alpha particles, a new device for dissolving radioactive gas 222Rn in the culture medium was set up.The most remarkable results are those obtained after the irradiation of human breast metastasic cells of the line MCF 7. Together with influences on the growth rate (non linearly related with the dose of radiation), also genetic changes were observed in the expression of some Bcl 2 gene family members related with apoptotic cell death. In short, bcl xs (related with multidrug resistance phenomena in this cellular line) was only expressed in irradiated cell cultures. After this result, the influence of alpha irradiation on the sensibility of MCF 7 to the chemotherapeutic drugs Taxol and VP 16 was analysed, obtaining that those cell cultures previously irradiated showed an improved response to the action of the drugs.

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 2002</p> / digitalisering@umu

The nucleotide excision repair (NER) pathway is essential for repair of UV-induced bulky DNA lesions. NER is divided into two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). UVC radiation has been shown to result in the formation of bulky DNA lesions, which are removed by NER. Previous published reports have shown a role for the p53 tumour suppressor protein in GGR and TCR, but the involvement of p53 in TCR has been controversial. In addition, it has also been suggested that hypoxia affects NER and expression of p53. In the present work, the role of p53, hypoxia and HIF-lα in NER was investigated. It was determined that p53 overexpression in primary human fibroblasts resulted in up-regulation of both the GGR and TCR subpathways of a UV -damaged reporter gene. Pre-treatment of cells with low UVC-fluence and p53 overexpression also induced an upregulation of GGR and TCR. These results are consistent with a p53-dependent upregulation of TCR and GGR of the UVC-damaged reporter gene, as well with a UV-inducible TCR and GGR that is dependent on p53 expression prior to UV treatment. Hypoxia coupled to low pH induced a transient up-regulation of p53 expression and NER in human primary normal fibroblasts and a concomitant decrease in UVC sensitivity. In contrast, in tumour cells hypoxia coupled to low pH resulted in a delayed, but not absent up-regulation of NER, which was p53-independent and did not result in a decrease in UVC sensitivity. We report here that it is the early transient p53-dependent up-regulation induced by hypoxia coupled to acidosis in human primary normal fibroblasts that may play a significant role in cellular UVC sensitivity. These data suggest a different cellular NER response to hypoxia compared to hypoxia coupled to low pH. The NER response to hypoxia and hypoxia coupled with acidosis was also different in primary cells when compared to tumour-derived cells. It was demonstrated that expression of dominant-negative HIF-lα in rat prostate tumour cells results in a reduction in host cell reactivation (HCR) of a UV-damaged reporter gene when compared to that in wild-type HIF-lα cells under normoxic conditions suggesting that basal HIF-lα expression may play an important role in NER. In addition we showed that hypoxia induced an up-regulation of NER in human primary normal fibroblasts that was delayed, but not absent in TCR-deficient CSB cells, suggesting a role for hypoxia in up-regulation of the GGR pathway of NER of a UVdamaged reporter gene. In contrast, HIF-lα-overexpression under conditions of hypoxia resulted in a down-regulation of NER in normal fibroblasts, which was delayed, but not absent in CSB fibroblasts. These results suggest that HIF-1α and CSB are involved in a hypoxia-induced NER response. This work provides further evidence that both GGR and TCR are p53-dependent. In addition, this study provides evidence that hypoxia and hypoxia coupled to acidosis can up-regulate NER in both primary and tumour cells, and that HIF-lα and the CSB protein play an important role in a hypoxia-induced NER response. / Thesis / Doctor of Philosophy (PhD)

Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs. / Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.