付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23090号 / 医博第4717号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 小川 修, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24521号 / 医博第4963号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 萩原 正敏, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A large number of anti-cancer chemotherapeutics target DNA topoisomerases. Etoposide is a specific topoisomerase II poison which causes reversible double strand DNA breaks. The focus of this project is to analyze the repair of DNA damage induced by etoposide.. Double strand DNA break repair is mediated by through either non-homologous end joining (NHEJ) or homologous recombination. NHEJ repairs through direct ligation of a double stranded break while homologous recombination utilizes a homologous template to recover the wild type sequence. A reporter cassette, RYDR-GFP, has been stably integrated into HeLa cells. This reporter contains an ultra-high affinity topoisomerase II cleavage site (RY) placed in the middle of a mutant GFP sequence. Flanking this sequence is a corresponding stretch of wild type GFP that is used as template to repair the break and restore gene function yielding GFP positive cells. Titrations with etoposide have shown that a logarithmic increase in drug concentration yields a corresponding increase in repair through homologous recombination (HR). This result demonstrates that topoisomerase II mediated damage is efficiently repaired by the process of HR. To examine NHEJ repair, a doxycycline inducible, stably integrated NHEJ HeLa cell reporter cassette was also evaluated. The data indicates that repair of topoisomerase II mediated DNA damage occurs more efficiently through the HR pathway. Collectively, the data suggests that tumor cells proficient in HR repair may effectively elude treatment by topoisomerase II targeting drugs.

T cells play an important role in adaptive immune system against viral infections, while premature aging and dysfunction of T cells induced by unrepaired DNA damages are always non-negligible snags during the long-term of fighting with chronic viral infections, such as Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Human Immunodeficiency Virus (HIV) infection. In this dissertation, we investigated the role of topological DNA damage in reprogramming telomeric DNA damage responses (DDR), mitochondrial metabolisms, and T cell functions using CD4+ T cells derived from individuals with chronic viral infections or healthy subjects treated with topoisomerase inhibitors. The healthy human T cells were treated with camptothecin (CPT) for mitochondrial topoisomerases I (Top1mt) or ICRF-193 or etoposide (ETP) for topoisomerases IIα (Top2α) as models. We found a significant suppression of Top2α and Top1mt protein levels and enzymatic activity in CD4+ T cells in chronically HCV/HIV-infected patients compared to age and gender-matched healthy subjects, along with an accumulation of the topoisomerase cleavage complex (Topcc) in genomic DNA as well as mitochondrial DNA (mtDNA). Mechanistically, topoisomerase inhibition in healthy CD4+ T cells caused topological DNA damage, telomere attrition, mitochondrial metabolic disorder and T cell apoptosis or dysfunction via inducing Topcc accumulation, PARP1 cleavage and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. In addition, T cells from virally infected subjects with lower topoisomerase levels were vulnerable to the inhibitor-induced cell apoptosis, indicating an important role for Top2α and Top1mt in preventing DNA topological disruption and cell death. These results demonstrate that accumulation of Topcc and topoisomerase deficiency lead to unrepaired DNA damage and render virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to mitochondrial metabolic disturbance or dysfunction in CD4+ T cell during chronic HCV or HIV infection. This study reveals a novel mechanism by which topoisomerase deficiency promotes telomeric DNA or mtDNA damage and premature T cell aging, and provides a new therapeutic target for restoring the DNA topologic machinery protecting T cells from unwanted DNA damage and to maintain immune competence.

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No / PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.

Yes / Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional setting. / RMP and MS funded by Yorkshire Cancer Research (pump priming grant BPP 046). IJS and AL funded by NIHR Research & Innovation Division, Strategic Project Funding 2013 and Manchester Pharmacy School Fellowship.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.