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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23761号 / 医博第4807号 / 新制||医||1056(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 YOUSSEFIAN Shohab, 教授 羽賀 博典, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p> Reduced oxygen tension (i.e. hypoxia) regulates gene expression in various chromaffin cell types that synthesize catecholamines. In this study, the effect of chronic hypoxia on tyrosine hydroxylase (TH) mRNA and protein expression was investigated in the adrenomedullary chromaffin MAH cell line. RT-PCR results indicated that TH mRNA was expressed in MAH cells both during normoxia (20% 0 2) and hypoxia (5% 02). However, TH mRNA expression during chronic hypoxia was significantly higher than that during normoxia, increasing by approximately 2- fold after 16 hour exposure to chronic hypoxia. Western Blot analysis of the regulation of TH gene expression by chronic hypoxia indicated that TH protein initially decreased during 10 hr exposure to hypoxia and this was followed by a rapid increase in expression over the next 10 hr, and then by a slower increase (up to 1.3x initial control) after 72 hr exposure. Therefore, TH mRNA and protein levels were changed in MAH cells by hypoxia in a time-dependent manner. Surprisingly, cobalt treatment of MAH cells, expected to mimic the effects of chronic hypoxia, had little effect on TH gene expression. Interestingly, the decrease in TH expression protein after 10 hr exposure to hypoxia was prevented by nifedipine, an L-type calcium channel blocker. These results suggest that MAH cells represent a useful model system for examining hypoxia-induced gene regulation in an 02-sensitive cell line. Additionally, preliminary studies on HIF-1a expression in MAH cells showed that HIF-la mRNA was expressed and remained stable under both hypoxic and normoxic conditions. </p> / Thesis / Master of Science (MSc)

No / Background: Previous studies have shown extensive vascularisation surrounding subcutaneously implanted fibres when the duration of the US National Cancer Institute (NCI) hollow fibre assay was prolonged. Materials and Methods: The feasibility of adapting the NCI assay for evaluating agents targeting the tumour vasculature was investigated in vitro and in vivo. Finally, in the optimised assay, changes in neovasculature formation around the fibres following treatment with the anti-vascular agent paclitaxel were quantified by immunohistochemistry. Results: Correlations between cell number seeded, time in culture and vascular endothelial growth factor (VEGF) secretion were seen. In vivo studies showed that transplanting single rather than 3 fibres at a site reduced inflammation, reducing the length of the fibre transplanted, as did without any significant loss in cell growth over 21 days. A statistically significant reduction in neovascularisation surrounding the fibres was seen accompanying paclitaxel treatment. Conclusion: Modifications made here to the NCI hollow fibre assay demonstrate its potential for analysing anti-tumour vasculature agents.

No / The in vitro measurement of drug-induced apoptosis provides a mechanism-based test for the chemosensitivity of tumor cells isolated from a patient or from a specific cell line. The goal of this study was to investigate the effects of p-glycoprotein reversal agents on apoptosis and necrosis in Burkitt lymphoma cells. These effects were determined by microscopic observation and by electrophoretic separation of DNA fragments. We demonstrated induction of apoptosis in Burkitt lymphoma Raji Thymidine Kinase+ (TK+)and TK- cells using different subclasses of p-glycoprotein reversal agents. A low dose of doxorubicin was also used. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of TK-deficient cells. The first phase of the present study involved morphological analyses and DNA degradation on agarose gel electrophoresis. The second phase analyzed DNA damage using the Comet assay and tail moments calculated with Komet imaging software. Electrophoretic separation resulted in a ladder pattern, indicating that the p-glycoprotein reversal agents were able to induce apoptosis and necrosis. The morphological frequency of apoptosis and necrosis in the cells was significantly increased. Most p-glycoprotein reversal agents showed an increase in tail moments in the Comet assay. The results indicate that indomethacin and quercetin may help to overcome chemoresistance in Burkitt’s lymphoma.

No / Dystroglycan is a ubiquitously expressed cell adhesion molecule frequently found to be altered or reduced in adenocarcinomas, however the mechanisms or consequences of dystroglycan loss have not been studied extensively. We examined the consequence of overexpression or RNAi depletion of dystroglycan on properties of in vitro growth migration and invasion of LNCaP, PC3, and DU145 prostate cancer cell lines. RESULTS: Using LNCaP cells we observed cell density-dependent changes in beta-dystroglycan with the appearance of several lower molecular weight species ranging in size from 43 to 26 kDa. The bands of 31 and 26 kDa were attributed to proteolysis, whereas bands between 43 and 38 kDa were a consequence of mis-glycosylation. The localization of beta-dystroglycan in LNCaP colonies in culture also varied, cells with a mesenchymal appearance at the periphery of the colony had more pronounced membrane localization of dystroglycan. Whereas some cells demonstrated nuclear dystroglycan. Increased dystroglycan levels were inhibitory to growth in soft agar but promoted Matrigel invasion, whereas reduced dystroglycan levels promoted growth in soft agar but inhibited invasion. Similar results were also obtained for PC3 and DU145 cells. This study suggests that changes in beta-dystroglycan distribution within the cell and/or the loss of dystroglycan during tumorigenesis, through a combination of proteolysis and altered glycosylation, leads to an increased ability to grow in an anchorage independent manner, however dystroglycan may need to be re-expressed for cell invasion and metastasis to occur.

Yes / Background: Platelet rich plasma (PRP), processed from autologous peripheral blood, is used to treat androgenetic alopecia (AGA). Objective: To determine the efficacy of PRP for hair growth promotion in AGA patients in a randomized, blinded, placebo controlled, pilot clinical trial (NCT02074943). Methods: The efficacy of an 8 week, 5 session, PRP treatment course was determined by measuring hair density and hair caliber changes in 10 AGA affected patients. For each PRP sample, the concentrations of selected growth factors were determined using a multiplex assay system. The clinical results were then correlated to the growth factor concentrations in PRP. Results: At 16 weeks, 8 weeks after the last PRP injection, treated areas exhibited increased mean hair density (+12.76%) over baseline compared to placebo (+0.99%). Mean hair caliber decreased in both treated and placebo regions (-16.22% and -19.46% respectively). Serial analysis of PRP significant variability in concentrations between patients. Overall, there was a positive correlation between GDNF concentration and hair density (p= 0.004). Trends, though not statistically significant, were also observed for FGF2 and VEGF. Limitations: Small sample size and lack of comparative cohorts receiving protocol variations limit confidence in the study data. Conclusions: This small pilot clinical trial suggests PRP treatment may be beneficial for AGA. However, the variable hair growth responses between patients indicate there is a significant opportunity to improve PRP therapy protocols for hair growth promotion. The variability in growth factor concentration in PRP suggests standardization of growth factors post-processing might improve hair growth responses. / RepliCel Life Sciences Inc. (Canada)

The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events. / Master of Science / The CRISPR/Cas9 system is the newest generation of genetic engineering tool for genome editing. Genetic modifications can be introduced while repairing double strand breaks (DSBs) on DNA, induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing pig fibroblast under low amount of nutrient could enrich the cells in G0/G1 phase of cell cycle, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.