Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

全文データ差替え待ち / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20268号 / 医博第4227号 / 新制||医||1021(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 松田 道行, 教授 武藤 学, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DAM

BAP1 (BRCA-Associated Protein1) is a tumor suppressor gene encoding a deubiquitinating enzyme (DUB) that regulates many facets of cellular biology. Genetic studies have demonstrated that somatic BAP1 mutations occur in numerous cancer types and that germline BAP1 mutations lead to a cancer susceptibility disorder that predisposes individuals to various tumors, in particular malignant mesothelioma (MM) and both uveal melanoma (UM) and cutaneous melanoma (CM). The Testa laboratory has identified several families (including one in Louisiana, designated Lou) with germline BAP1 mutations, in which there were recurrent cases of MM, UM, and CM. We generated a Bap1 mutant mouse model with a knockin mutation identical to that observed in the Lou family (Bap1+/Lou ) to test whether this mutation alone confers susceptibility to ultraviolet (UV) light-induced melanomagenesis either alone or in combination with a mutation found in a well-established Hepatocyte Growth Factor (HGF)/Scatter Factor transgenic mouse model. Neither Bap1+/Lou, HGF, nor Bap1+/Lou;HGF mice showed a significantly higher incidence or shorter latency of UV light-induced melanoma than wild type (WT) mice. The study also suggests that germline mutation of Bap1 alone does not cause an increased incidence of UV-induced melanomas under the conditions used in this investigation. Recent evidence indicates that BAP1 participates in the DNA damage repair response, suggesting that BAP1’s role in tumorigenesis could be particularly important in cancers associated with environmental carcinogens such as ultraviolet irradiation (UVR). To further investigate the role of BAP1 (Bap1 in the mouse) in DNA damage, we first knocked down BAP1 in human melanocytes as well as Melan-A and Melan-C mouse melanocytes and then exposed the cells to UVR, followed by analysis of DNA damage repair. UVR-induced and steady state levels of DNA damage were higher in BAP1-knockdown cells compared to shGFP-control cells. Levels of UVR-related DNA damage markers such as p53, γH2AX and CPD (cyclobutane pyrimidine dimers) were increased following BAP1 loss and UVR treatment. Cell cycle analysis by flow cytometry demonstrated that cells with knockdown of BAP1 and post UVR treatment showed a higher proportion of cells in S/G2 phase. Such an effect could be due to BAP1 loss and consequent inability to repair DNA damage and/or cell cycle progression. These data are consistent with a role for BAP1 in UVR-induced DNA damage repair. In MM, it is unclear to what extent BAP1 mutations cooperate tumorigenically with mutations of other tumor suppressor genes (TSGs) implicated in MM, such as CDKN2A and NF2. While germline mutations of BAP1 clearly predispose to MM, whether somatic mutations of BAP1 drive a more aggressive, metastatic tumor phenotype may depend on the disease type. For such studies, we used conditional knockout (CKO) mice along with intrathoracic (IT) or intraperitoneal (IP) injection of adenovirus expressing Cre recombinase (Adeno-Cre) to excise critical homozygously floxed TSGs in the mesothelial lining. These labor-intensive experiments demonstrated that while homozygous deletion of Bap1, Cdkn2a, or Nf2 alone in the pleural cavity (IT) of genetically engineered mouse (GEM) models gave rise to few or no MMs, inactivation of Bap1 cooperated with loss of either Nf2 or Cdkn2a to drive development of MM in ~20% of double CKO mice, and a high incidence (22/26, 85%) of MMs with short latency (12 weeks) was observed in Bap1;Nf2;Cdkn2a (triple)-CKO mice. The same trend was confirmed when the same gene combinations were homozygously deleted IP in these same GEM models, except that a much higher incidence of MM was observed in homozygously floxed Bap1 (Bap1f/f) mice injected IP versus IT, which may be due to a larger cell surface area of the peritoneum. Adeno-Cre treatment of normal mesothelial cells from Bap1f/f;Nf2 f/f;Cdkn2 f/f mice, but not from mice with knockout of one or any two of these tumor suppressor genes, resulted in robust spheroid formation in vitro, suggesting that homozygous deletion of all three of these TSGs is sufficient to drive a cancer stem cell-like potential. RNA-seq analysis of pleural MMs from triple-CKO mice revealed enrichment of many genes transcriptionally regulated by the polycomb repressive complex 2 (PRC2). Other genes upregulated in MMs from triple-CKO mice included Vegfd and Pak3, which encode proteins involved in angiogenic and cell motility pathways. In conclusion, we hypothesize that inherited mutations of BAP1 may increase susceptibility to certain environmental factors that may induce DNA damage and contribute to cancer development. Our data also indicate that cooperative somatic inactivation of Bap1, Nf2, and Cdkn2a results in rapid, highly aggressive MMs, and that deletion of Bap1 contributes to tumorigenesis, in part, by loss of PRC2-mediated gene repression of tumorigenic target genes and by acquisition of stem-cell potential. Thus, our studies suggest a potential avenue for therapeutic intervention. / Biomedical Sciences

Yes / Malignant pleural mesothelioma (MPM) is associated with an extremely poor prognosis, and most patients initially are or rapidly become unresponsive to platinum-based chemotherapy. MicroRNA-31 (miR-31) is encoded on a genomic fragile site, 9p21.3, which is reportedly lost in many MPM tumors. Based on previous findings in a variety of other cancers, we hypothesized that miR-31 alters chemosensitivity and that miR-31 reconstitution may influence sensitivity to chemotherapeutics in MPM. Reintroduction of miR-31 into miR-31 null NCI-H2452 cells significantly enhanced clonogenic resistance to cisplatin and carboplatin. Although miR-31 re-expression increased chemoresistance, paradoxically, a higher relative intracellular accumulation of platinum was detected. This was coupled to a significantly decreased intranuclear concentration of platinum. Linked with a downregulation of OCT1, a bipotential transcriptional regulator with multiple miR-31 target binding sites, we subsequently identified an indirect miR-31-mediated upregulation of ABCB9, a transporter associated with drug accumulation in lysosomes, and increased uptake of platinum to lysosomes. However, when overexpressed directly, ABCB9 promoted cellular chemosensitivity, suggesting that miR-31 promotes chemoresistance largely via an ABCB9-independent mechanism. Overall, our data suggest that miR-31 loss from MPM tumors promotes chemosensitivity and may be prognostically beneficial in the context of therapeutic sensitivity.

Malignant pleural mesothelioma (MPM) is a form of aggressive tumour that is almost always associated with prior exposure to asbestos. Currently responsible for over 47,000 deaths worldwide each year and rising, it poses a serious threat to global public health. Many clinical studies of MPM, including its diagnosis, prognostic planning, and the evaluation of a treatment, necessitate the accurate quantification of tumours based on medical image scans, primarily computed tomography (CT). Currently, clinical best practice requires application of the MPM-adapted Response Evaluation Criteria in Solid Tumours (MPM-RECIST) scheme, which provides a uni-dimensional measure of the tumour's size. However, the low CT contrast between the tumour and surrounding tissues, the extensive elongated growth pattern characteristic of MPM, and, as a consequence, the pronounced partial volume effect, collectively contribute to the significant intra- and inter-observer variations in MPM-RECIST values seen in clinical practice, which in turn greatly affect clinical judgement and outcome. In this thesis, we present a novel computer-assisted approach to evaluate MPM patient response to treatments, based on the volumetric segmentation of tumours (VTA) on CT. We have developed a 3D segmentation routine based on the Random Walk (RW) segmentation framework by L. Grady, which is notable for its good performance in handling weak tissue boundaries and the ability to segment any arbitrary shapes with appropriately placed initialisation points. Results also show its benefit with regard to computation time, as compared to other candidate methods such as level sets. We have also added a boundary enhancement regulariser to RW, to improve its performance with smooth MPM boundaries. The regulariser is inspired by anisotropic diffusion. To reduce the required level of user supervision, we developed a registration-assisted segmentation option. Finally, we achieved effective and highly manoeuvrable partial volume correction by applying a reverse diffusion-based interpolation. To assess its clinical utility, we applied our method to a set of 48 CT studies from a group of 15 MPM patients and compared the findings to the MPM-RECIST observations made by a clinical specialist. Correlations confirm the utility of our algorithm for assessing MPM treatment response. Furthermore, our 3D algorithm found applications in monitoring the patient quality of life and palliative care planning. For example, segmented aerated lungs demonstrated very good correlation with the VTA-derived patient responses, suggesting their use in assessing the pulmonary function impairment caused by the disease. Likewise, segmented fluids highlight sites of pleural effusion and may potentially assist in intra-pleural fluid drainage planning. Throughout this thesis, to meet the demands of probabilistic analyses of data, we have used the Non-Parametric Windows (NPW) probability density estimator. NPW outperforms the histogram in terms of its smoothness and kernel density estimator in its parameter setting, and preserves signal properties such as the order of occurrence and band-limitedness of the sample, which are important for tissue reconstruction from discrete image data. We have also worked on extending this estimator to analysing vector-valued quantities; which are essential for multi-feature studies involving values such as image colour, texture, heterogeneity and entropy.

Carbon nanotubes (CNT) are a new form of industrially relevant nano-scale particle and are seen as the cutting edge of the burgeoning nanotechnology revolution which promises to impact on all our lives. Due to high length to diameter ratio, CNT are perhaps the most well known of a growing collection of high aspect ratio nanoparticles (HARN). However the production and use of carbon nanotubes has presented an interesting toxicological question based on its structure and raised the question ‘are carbon nanotubes like asbestos?’. Few people are unaware of the devastating global pandemic of diseases caused by asbestos and similarities in needle-like shape between asbestos and nanotubes have raised fears that nanotubes may mimic asbestos-type disease. The purpose of this study was to investigate this link, based on the wealth of information known about the toxic effects of certain forms of fibre on the respiratory system. From this we hope to identify those carbon nanotubes which are hazardous whilst not prejudicing the use of those industrially relevant materials which can be used safely. Within fibre toxicology there exists a central paradigm which outlines the main properties a fibrous particle must possess if it is to exert pathogenic effects in the body. This paradigm outlines the importance of length, thinness and biopersistence to a fibre and an absence of one or more of these attributes results in a loss of pathogenicity. We took this paradigm and, using suitable asbestos and non-asbestos controls, applied it various morphological forms of carbon nanotubes using an in vivo model. The resultant data demonstrates for the first time that asbestos-like pathogenic behaviour associated with carbon nanotubes is closely linked to the morphology of the nanotubes and their aggregates. Specifically our results showed that CNT which possessed a long, straight length were highly inflammogenic and fibrogenic within the peritoneal cavity of mice; a model sensitive to the pathogenic effects of fibres. As well as length, the importance of biopersistence in the pathogenesis of fibrous particles has been known for many years and is a central attribute affecting the pathogenicity of fibres. Amphibole asbestos is known to be durable, a commercially exploited attribute and as such is biopersistent in the lung which is a key feature of its pathogenicity. Glass fibre on the other hand is bio-soluble, and whilst long and inhalable, does not cause significant disease due to its lack of biopersistence. Based on the grapheme structure of CNT which impart exceptional strength and rigidity and the chemical inertness of carbon we would hypothesis that CNT would be biopersistent and therefore fulfil another of the criteria of the fibre pathogenicity paradigm (FPP). Our aim therefore has been to establish the durability of CNT against fibrous particles of known durability using a synthetic solution maintained at a pH to simulate the lung environment. Using a range of 4 CNT and using both durable and non-durable fibres such as amphibole asbestos and glass fibre to bench mark our result; we demonstrated that 3 of the 4 CNT tested displayed exceptional durability whilst the fourth lost approximately 30% of its mass during the experiment with concomitant reduction in pathogenicity. As well as length and biopersistence, the surface of a particle has been shown to contribute to the overall toxicity of a particle and in certain circumstances, such as that of quartz, the surface of the particle can be the biologically active component. In the case of carbon nanotubes, surface functionalisation is commonly used for various endpoints including the addition of various tags and labels for tracking. As such our further aim was to investigate the relationship between the length-dependent pathogenicity of a fibre sample and the surface of the fibre. By using different forms of functional groups attached to the surface of a pathogenic carbon nanotube we aim to critically test if the level of inflammation and fibrosis triggered in vivo can be altered by simple alteration of the surface. Our results showed that surface modification of CNT could alter the inflammogenic and fibrogenic effects of CNT which may have important implications when considering the hazard assessment of functionalised HARN. As CNT are not the only form of fibrous nanomaterial and within this project we also attempted to determine the applicability of the FPP to further high aspect ratio nanomaterials. In order to do this we set out to determine the generality of this hypothesis by asking whether nickel nanowires, a radically different form of HARN to CNT, show length-dependent pathogenicity. Nickel oxide nanowires synthesised to be predominantly long (>20 μm) act similarly to amphibole asbestos in showing the ability to elicit strong inflammation in the mouse peritoneal model in a dose dependent manner; inflammation was not seen with the short (<5 μm) nanowires. In summation, the results from this study are the first to show that long HARN can indeed behave like asbestos when in contact with the sensitive mesothelium. This study suggests a potential link between inhalation exposure to long nanotubes and asbestos-related disease, especially mesothelioma and as such this may have immediate implications across many disciplines if care is to be taken to avoid a long term legacy of harm.

Bibliography: p. 159-193. / x, 200 p. : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis compares the rates of mitotic activity of the ovarian surface epithelium (OSE) and the peritoneal mesothelium (PM) and the effects of ovarian hyperstimulation using a rodent model. The study provides also information about cytokine expression and production in benign and malignant ovarian tissue, both in humans and animals. / Thesis (M.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1997?

Apoptosis is a special form of cell death that if non-functional may lead to diseases such as cancer. A reduction of the intracellular potassium ion (K+) content is necessary for activating enzymes important for the execution of apoptosis. Pharmacological modulation of K+ fluxes to reduce intracellular K+ in cancer cells might therefore force the cells into apoptosis and decrease tumour cell mass. Human malignant pleural mesothelioma (MPM) is a form of cancer often caused by asbestos exposure. Although asbestos has been banned in the Western World, the incidence of MPM is expected to increase. Cisplatin is the first-line chemotherapy for MPM, but acquired resistance to the drug is a clinical problem. This thesis is mainly based on work with the human malignant pleural mesothelioma cell line (P31 wt) and a cisplatin-resistant sub-line (P31 res). The aim was to first characterize K+ fluxes in P31 wt and P31 res cells, and then manipulate them in order to reduce intracellular K+ and induce apoptosis with K+ manipulation alone or in combination with cisplatin. Characterization of K+ fluxes in P31 wt cells showed that: 1) ouabain, a digitalis-like drug, and specific blocker of the Na+, K+, ATPase pump, effectively inhibited K+ uptake, 2) bumetanide, a diuretic, and an inhibitor of the Na+, K+, 2Cl-¬-cotransporter, had a transient effect on K+ uptake, and 3) the antifungal drug amphotericin B stimulated K+ efflux. In order to determine intracellular K+ content, the potassium-binding fluorescent probe PBFI-AM was used in a 96-well plate assay. After a 3-h incubation with ouabain, with or without bumetanide, combined with amphotericin B, the intracellular K+ content was reduced in P31 wt cells but not in P31 res cells. Ouabain induced apoptosis in both P31 wt and P31 res cells. P31 res cells were sensitized to cisplatin by ouabain, since 10 mg/L cisplatin in combination with ouabain induced about the same percentage of apoptotic cells as 40 mg/L cisplatin. Apoptosis was executed via caspase-3 activation in both P31 wt and P31 res cells. Amphotericin B enhanced ouabain-induced apoptosis in P31 wt cells via caspase-9 activation, with increased caspase-3 activation and DNA fragmentation as consequences. Ouabain-induced apoptosis in P31 res cells was executed via increased expression of pro-apoptotic Bak. The combination of cisplatin with ouabain and amphotericin B was stressful to both P31 wt and P31 res cells, since SAPK/JNK a known factor in stress-induced apoptosis was activated. In conclusion, K+ flux manipulation with clinical used drugs can induce apoptosis per se and also enhance cisplatin-induced apoptosis in P31 wt and P31 res cells.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.