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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.

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O objetivo do estudo foi avaliar a importância da expressão das proteínas topoisomerase II alfa (topo II) e HER-2 como fatores preditvos da resposta à quimioterapia neoadjuvante e prognóstico em pacientes com câncer de mama nos estádio clínico II e III. Pacientes e métodos: 99 pacientes receberam quimioterapia neoadjuvante com docetaxel (75mg /m²) e epirrubicina (50 mg/m²) em infusão endovenosa no dia 1 a cada 3 semanas após terem sido submetidas a biópsia incisional. Foi complementado tratamento sistêmico com quimioterapia adjuvante com CMF ou FEC de acordo com o estado axilar avaliada após a cirurgia definitiva e/ou hormonioterapia de acordo com a avaliacãodos receptores hormonais. Avaliamos a taxa de resposta ao tratamento neoadjuvante e a influência da topo II alfa e do HER-2 na taxa de resposta à quimioterapia neoadjuvante bem comona sobrevida livre de doença e sobrevida global. Também foram avaliadas a expressão dos receptores hormonais. Resultados: a taxa de resposta clínica objetiva foi de 80,8 % com 9,1 % de resposta patológica completa. A expressão da topo II alfa nao apresentou significância nas taxas de resposta ou na sobrevida das pacietnes e nao houve correlação entre a expressão desta proteína e de HER-2. A superexpressão da proteína HER-2 foi associada com uma redução significante nas taxas de sobrevida livre de doença e sobrevida global (p= 0,04 e p= 0,004, respectivamente). Conclusão: a expressão da topo II alfa não demonstrou, em nosso estudo, ser fator preditivo ou prognóstico nas pácientes submetidas a quimioterapia neoadjuvante com docetaxel e epirrubicina. / The objective of this study is to evaluate the importance of the expression of the proteins topoisomerase II alpha (topo II) and HER-2 as predictive factors to response to neoadjuvant chemotherapy and the prognosis of patients diagnosed with clinical stage II and stage III breast cancer. Patients and methods: 99 patients have received neoadjuvant chemotherapy with docetaxel (75mg /m²) and epirrubicine (50 mg/m²) through intravenous infusion on D1 q3 weeks, after submitted to pathologic specimen harvest. Systemic treatment was then complemented with CMF or FEC according to the status of axilla involvement after surgical staging and/or hormone therapy according tohormone receptor status. We evaluated the response rate to neoadjuvant treatment and the influence of topo II alpha and HER-2 expression on the response rate and disease free survival and overall survival. The expression of hormone receptors was also evaluated. Results: Objective clinical response was 78,8%, with 8,2% of complete pathological response.Topo II alpha expression did not correlate to response to chemotherapy or survival and there was no correlation between topo II alpha expression and HER-2 expression. Superexpression of HER-2 protein was associated to a significant reduction in disease free survival and overall survival (p=0,04 and p=0,004, respectively). Conclusion: topo II alpha expression did not demonstrate, in our study, to be a predictive nor prognostic factor to the patientssubmitted to neoadjuvant with docetaxel and epirrubicin.

O objetivo do estudo foi avaliar a importância da expressão das proteínas topoisomerase II alfa (topo II) e HER-2 como fatores preditvos da resposta à quimioterapia neoadjuvante e prognóstico em pacientes com câncer de mama nos estádio clínico II e III. Pacientes e métodos: 99 pacientes receberam quimioterapia neoadjuvante com docetaxel (75mg /m²) e epirrubicina (50 mg/m²) em infusão endovenosa no dia 1 a cada 3 semanas após terem sido submetidas a biópsia incisional. Foi complementado tratamento sistêmico com quimioterapia adjuvante com CMF ou FEC de acordo com o estado axilar avaliada após a cirurgia definitiva e/ou hormonioterapia de acordo com a avaliacãodos receptores hormonais. Avaliamos a taxa de resposta ao tratamento neoadjuvante e a influência da topo II alfa e do HER-2 na taxa de resposta à quimioterapia neoadjuvante bem comona sobrevida livre de doença e sobrevida global. Também foram avaliadas a expressão dos receptores hormonais. Resultados: a taxa de resposta clínica objetiva foi de 80,8 % com 9,1 % de resposta patológica completa. A expressão da topo II alfa nao apresentou significância nas taxas de resposta ou na sobrevida das pacietnes e nao houve correlação entre a expressão desta proteína e de HER-2. A superexpressão da proteína HER-2 foi associada com uma redução significante nas taxas de sobrevida livre de doença e sobrevida global (p= 0,04 e p= 0,004, respectivamente). Conclusão: a expressão da topo II alfa não demonstrou, em nosso estudo, ser fator preditivo ou prognóstico nas pácientes submetidas a quimioterapia neoadjuvante com docetaxel e epirrubicina. / The objective of this study is to evaluate the importance of the expression of the proteins topoisomerase II alpha (topo II) and HER-2 as predictive factors to response to neoadjuvant chemotherapy and the prognosis of patients diagnosed with clinical stage II and stage III breast cancer. Patients and methods: 99 patients have received neoadjuvant chemotherapy with docetaxel (75mg /m²) and epirrubicine (50 mg/m²) through intravenous infusion on D1 q3 weeks, after submitted to pathologic specimen harvest. Systemic treatment was then complemented with CMF or FEC according to the status of axilla involvement after surgical staging and/or hormone therapy according tohormone receptor status. We evaluated the response rate to neoadjuvant treatment and the influence of topo II alpha and HER-2 expression on the response rate and disease free survival and overall survival. The expression of hormone receptors was also evaluated. Results: Objective clinical response was 78,8%, with 8,2% of complete pathological response.Topo II alpha expression did not correlate to response to chemotherapy or survival and there was no correlation between topo II alpha expression and HER-2 expression. Superexpression of HER-2 protein was associated to a significant reduction in disease free survival and overall survival (p=0,04 and p=0,004, respectively). Conclusion: topo II alpha expression did not demonstrate, in our study, to be a predictive nor prognostic factor to the patientssubmitted to neoadjuvant with docetaxel and epirrubicin.

The clearance of viruses is largely dependent upon the activation of T cells to generate a robust immune response. However, host responses are suppressed during chronic viral infections. In this thesis, we explored the role of Top2α in DNA topology in individuals with chronic HBV, HCV, and HIV infections. We found that Top2α protein expression and activity were low in T cells derived from chronically virus-infected individuals compared to healthy subjects. Using CD4+ T cells treated with Top2α inhibitor or poisoner as a model, we demonstrated that Top2α inhibition disrupts the DNA topology, suppresses DNA repair kinase (ATM), and telomere protein (TRF2) expression, and induces T cell dysfunction. These findings reveal that Top2α inhibition is a mechanism by which viruses evade the host responses and establish persistent infection, and thus, restoring Top2α levels could be a way of boosting immune responses during chronic viral infections.

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24520号 / 医博第4962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 生田 宏一, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Nikola Jaščevská Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effects of topoisomerase II beta on the sensitivity of the cancer cells to the antineoplastics Topoisomerase II (TOP II) is a cellular enzyme responsible for solving topological problems of double-stranded DNA. Alpha and beta isoforms of TOP II are different gene products having similar catalytic activities. The expression of TOP IIα is cell-cycle dependent, peaking in G2/M phase, while TOP II isoform is expressed constitutively throughout the cell cycle. It is therefore present also in non-proliferating differentiated cells. Anthracycline antibiotics are an old class of anticancer drugs, belonging to TOP II poisons. Although their clinical usefulness is high, the incidence of side effects (especially myelotoxicity and cardiotoxicity) may limit the therapy. The key role of TOP II inhibition, which is present also in cardiomyocytes, has been increasingly discussed. Dexrazoxane, the only clinically used cardioprotective, leads to depletion of TOP II in cardiomyocytes, which may explain its cardioprotection. Although TOP II was previously shown to be dispensable for cellular proliferation, its possible...

Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIβ isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIβ is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.

Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.