Chizuko Tsurumi

Proteasomes and Antigen Processing

Publisher Summary This chapter discusses the recent findings related to the roles of proteasomes in the major histocompatibility complex (MHC) class I-restricted antigen-processing pathway. One of the most important responses in this antigen-specific immune system is to distinguish correctly non-self-antigens from self-antigens for selective elimination because deviation from this recognition would lead to a variety of opportunistic infections or autoimmune diseases. The chapter focuses on the roles of proteasomes and their regulators in antigen processing with special reference to the influence of γ-interferon (γ-IFN) on both the structure and the functions of the proteasome. Proteasomes play a central role in various biological processes, one of which is the generation of the peptides presented by MHC class I molecules to the circulating T lymphocytes. Proteasomes have been implicated as the processing enzyme for the generation of the ligand peptides for MHC class I receptors. Future studies should address how proteasome genes have evolved. Acquisition of the γ-IFN-responsive proteasomal and PA28 family genes may be related to that of multiple MHC and TAP genes during evolution. Studies on molecular evolution may provide new insight into the alternative roles of proteasome genes in immunity.

The 26S proteasome: subunits and functions

The 26S proteasome is an eukaryotic ATP-dependent, dumbbell-shaped protease complex with a molecular mass of approximately 2000 kDa. It consists of a central 20S proteasome, functioning as a catalytic machine, and two large V-shaped terminal modules, having possible regulatory roles, composed of multiple subunits of 25–110 kDa attached to the central portion in opposite orientations. The primary structures of all the subunits of mammalian and yeast 20S proteasomes have been determined by recombinant DNA techniques, but structural analyses of the regulatory subunits of the 26S proteasome are still in progress. The regulatory subunits are classified into two subgroups, a subgroup of at least 6 ATPases that constitute a unique multi-gene family encoding homologous polypeptides conserved during evolution and a subgroup of approximately 15 non-ATPase subunits, most of which are structurally unrelated to each other.

Non-Invasive In Vivo Imaging of Tumor-Associated CD133/Prominin

Background Cancer stem cells are thought to play a pivotal role in tumor maintenance, metastasis, tumor therapy resistance and relapse. Hence, the development of methods for non-invasive in vivo detection of cancer stem cells is of great importance. Methodology/Principal Findings Here, we describe successful in vivo detection of CD133/prominin, a cancer stem cell surface marker for a variety of tumor entities. The CD133-specific monoclonal antibody AC133.1 was used for quantitative fluorescence-based optical imaging of mouse xenograft models based on isogenic pairs of CD133 positive and negative cell lines. A first set consisted of wild-type U251 glioblastoma cells, which do not express CD133, and lentivirally transduced CD133-overexpressing U251 cells. A second set made use of HCT116 colon carcinoma cells, which uniformly express CD133 at levels comparable to primary glioblastoma stem cells, and a CD133-negative HCT116 derivative. Not surprisingly, visualization and quantification of CD133 in overexpressing U251 xenografts was successful; more importantly, however, significant differences were also found in matched HCT116 xenograft pairs, despite the lower CD133 expression levels. The binding of i.v.-injected AC133.1 antibodies to CD133 positive, but not negative, tumor cells isolated from xenografts was confirmed by flow cytometry. Conclusions/Significance Taken together, our results show that non-invasive antibody-based in vivo imaging of tumor-associated CD133 is feasible and that CD133 antibody-based tumor targeting is efficient. This should facilitate developing clinically applicable cancer stem cell imaging methods and CD133 antibody-based therapeutics.

Delayed cell death associated with mitotic catastrophe in γ-irradiated stem-like glioma cells

Background and PurposeStem-like tumor cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumor cells. Here, early and late radioresponse of patient-derived stem-like glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors.Materials and methodsPrimary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by serum-containing medium without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots.ResultsSLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10 Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10 Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce γH2AX levels. Nonetheless, in two p53-deficient SLGC lines examined γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe. In a line containing CD133-positive and -negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe.ConclusionsOur results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of p53-deficient SLGCs. This may have therapeutic implications. We further show that the stem-cell culture cytokines EGF plus FGF-2 activate DNA repair and thus confound in vitro comparisons of DNA damage repair between stem-like and more differentiated tumor cells.

cDNA cloning of bovine thrombospondin 1 and its expression in odontoblasts and predentin

The extracellular matrix protein thrombospondin 1 (TSP1) was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. The 5289 bp cDNA contains a complete open reading frame of 1170 amino acids. Bovine TSP1 has high homologies to its human and mouse counterparts. In immunohistochemical analyses of bovine anterior teeth with anti-TSP1 monoclonal antibody, TSP1 was only detectable at the position of predentin, located between dentin and unmineralized dental pulp. Northern blot analysis showed high levels of two sizes of TSP1 mRNAs in odontoblasts but not dental pulp and gingiva. Previously we found that osteotropic factors such as calcitriol and TGF-beta induce TSP1 at the transcriptional level in clonal rat dental pulp cells. These results suggest a role of TSP1 in dentinogenesis and/or maintenance of dentin and dental pulp.

Tripeptidyl Peptidase II Plays a Role in the Radiation Response of Selected Primary Cell Types but not Based on Nuclear Translocation and p53 Stabilization

The giant cytosolic protease tripeptidyl peptidase II (TPPII) was recently proposed to play a role in the DNA damage response. Shown were nuclear translocation of TPPII after gamma-irradiation, lack of radiation-induced p53 stabilization in TPPII-siRNA-treated cells, and complete tumor regression in mice after gamma-irradiation when combined with TPPII-siRNA silencing or a protease inhibitor reported to inhibit TPPII. This suggested that TPPII could be a novel target for tumor radiosensitization and prompted us to study radiation responses using TPPII-knockout mice. Neither the sensitivity to total body irradiation nor the radiosensitivity of resting lymphoid cells, which both strongly depend on p53, was altered in the absence of TPPII. Functional integrity of p53 in TPPII-knockout cells is further shown by a proper G(1) arrest and by the accumulation of p53 and its transcriptional targets, p21, Bax, and Fas, on gamma-irradiation. Furthermore, we could not confirm radiation-induced nuclear translocation of TPPII. Nevertheless, after gamma-irradiation, we found slightly increased mitotic catastrophe of TPPII-deficient primary fibroblasts and increased apoptosis of TPPII-deficient activated CD8(+) T cells. The latter was accompanied by delayed resolution of the DNA double-strand break marker gammaH2AX. This could, however, be due to increased apoptotic DNA damage rather than reduced DNA damage repair. Our data do not confirm a role for TPPII in the DNA damage response based on nuclear TPPII translocation and p53 stabilization but nevertheless do show increased radiation-induced cell death of selected nontransformed cell types in the absence of the TPPII protease.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

Protein and gene structures of 20S and 26S proteasomes.

The two types of proteasomes with apparent sedimentation coefficients of 20S and 26S consist of a number of heterogeneous polypeptides and are unusually large protein complexes of approximately 750 kDa and 2000 kDa, respectively. The 26S proteasome is a cylindrical caterpillar-shaped complex with a symmetrical assembly of a four-layered central 20S proteasome and two terminal 22S regulators each with a V-like structure. The central core and the terminal structures are formed by multiple polypeptides with molecular masses of 21-31 kDa and 28-112 kDa, respectively. We have been studying their detailed structures by protein-chemical and molecular biological techniques. In this review, we summarize the structural features of eukaryotic 20S and 26S proteasomes. We also discuss the possible function(s) of the terminal multi-protein regulator complex based on current information.

Abstract 5322: Bioluminescence, fluorescence, and biomarker mediated monitoring of an orthotopic model of colon cancer treated with VX-680

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL With 10% of all newly diagnosed cases per year, colon cancer ranks within the top 5 cancer types within the USA, and, with a similar figure of people succumbing to the disease, is a major cause of tumor-related deaths. Primary tumors may often be removed via surgery, but metastatic spread poses considerable difficulties and chemotherapeutic treatments are rarely curative. Novel drugs, and predictive pre-clinical model systems to better mimic human disease are therefore urgently needed. Although subcutaneously implanted tumor cells have been used for pre-clinical drug testing for decades now, the predictive value of these models has recently become the subject of controversy. Orthotopically implanted cells reflect the human disease much better in that organo-typical tumor stromal interactions are enabled, and tumor cells spread to other sites with distribution patterns reflecting the human disease. Orthotopical implantation requires sensitive imaging modalities, similar to the patient situation. Bioluminescence imaging (BLI) is a well established modality for pre-clinical models, but is not translatable to the clinic. Recently, fluorescence molecular tomography (FTM) imaging has been shown to offer good sensitivity and tissue penetration, and sensitive probes – that may be used in the clinic – have been developed. A pre-clinical experiment was performed to compare the growth behaviour of orthotopically implanted, luciferase marked HCT116 wild-type and p53 KO human colon cancer cells and test their sensitivity towards the aurora-inhibitor VX-680. Growth of the tumors, and treatment effects were followed in vivo using BLI, and a final imaging analysis with integrisense, using FMT. The in vivo imaging data were validated with necropsy data, including sensitive luciferase assays to detect metastases in organs. In vivo BLI suggested that p53 KO tumors grew much more aggressive, giving rise to larger, more fragmented signals than their wild-type counterparts. Imaging using integrisense confirmed the presence of much larger tumor masses in the p53 KO than in the wild-type group. VX-680 slightly reduced the BLI signals from both groups. Necropsy results confirmed the tumor size differences between wild-type and p53 KO tumors, also the VX-680 effect against wild-type tumors (−50%, SD 70%). However, the drug appeared ineffective against p53 KO tumors (+120%, SD 70%). Luciferase assays from potential target organs detected an increased metastasation in p53 KO tumor bearing animals compared to those with wild-type tumors. Interestingly, metastasation of the p53 KO tumors to the spleen was sensitive to VX-680 treatment (−90%, SD 20%). The effect of the drug could also be demonstrated in skin biopsies taken from the mice as a surrogate tissue. This study represents a ‘high content’, partially translatable mouse model applicable also for the testing of other targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5322. doi:10.1158/1538-7445.AM2011-5322