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Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.

We developed a software program (titled Precursor Ion Calibration software for LTQ or, in short, PICsL) that increases the reliability of precursor ion assignations from LC-MS analysis using ultra zoom scanning of LTQ linear ion trap MS and automatically corrects the assignations. Although existing software calculates the theoretical isotopic distribution according to m/z with a computational algorithm, our method simply searches for ions close to the theoretical mass value using both MS/MS raw data and Mascot search result files, followed by a second database search that identifies the proteins using the regenerated peak list files. Our software program mimics the manual inspection of the spectral data of precursor ions and is expected to be applicable not only for low resolution MS, such as LTQ, but also for a wide variety of MS instruments.

The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.

A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups. In addition to many housekeeping proteins found in common with other cell types, the subset contained a group of regulatory proteins that may determine unique ES cell functions. We identified 39 transcription factors including Oct-3/4, Sox-2, and undifferentiated embryonic cell transcription factor I, which are characteristic of ES cells, 88 plasma membrane proteins including cell surface markers such as CD9 and CD81, 44 potential proteinaceous ligands for cell surface receptors including growth factors, cytokines, and hormones, and 100 cell signaling molecules. The subset also contained the products of 60 ES-specific and 41 stemness genes defined previously by the DNA microarray analysis of Ramalho-Santos et al. (Ramalho-Santos et al., Science 2002, 298, 597-600), as well as a number of components characteristic of differentiated cell types such as hematopoietic and neural cells. We also identified potential post-translational modifications in a number of ES cell proteins including five Lys acetylation sites and a single phosphorylation site. To our knowledge, this study provides the largest proteomic dataset characterized to date for a single mammalian cell species, and serves as a basic catalogue of a major proteomic subset that is expressed in mouse ES cells.

Protein expression and de novo synthesis in normal and prostate cancer cell lines derived from the same patient were compared by proteomic analysis, and the effects of INFalpha and INFgamma (INF=interferon) determined. The expressions of several INF-inducible proteins, including MxA, Nmi, PA28a and IFP53, were downregulated in the cancer cells. INFgamma induced a more than twofold increase or decrease in the synthesis rates of almost twice as many proteins in the cancer cell line. The positive regulator of INF-induced transcription ISGF3gamma was upregulated in the cancer cells and inversely regulated by INFalpha and INFgamma in the normal and cancer cells. Moreover, ISGF3gamma's induction by INFgamma in the cancer cells was more enhanced by simultaneous stimulation with EGF, than its induction in the normal cells. In all, 31 differentially regulated proteins were identified by mass spectrometry analysis, several of which are involved in chaperone-assisted protein folding in the endoplasmic reticulum (ER) or in regulated protein degradation. Our results suggest that the exclusion of proteins by the ER quality control system, crosstalk between the EGF- and INF-induced signalling pathways and the regulation of INF-inducible genes are all altered in the prostate cancer cells. The combination of upregulated activity in the growth-promoting PI3K/Akt pathway, suppression of Nmi and overexpression of hnRNP-K and c-myc proteins may explain why the prostate cancer cells were found to be more resistant to the growth inhibitory effects of INFgamma.

Phospholipase C delta4 (PLCdelta4) is one of the delta-type PLC isozymes, the expression of which is induced in nuclei by treatment with serum and also in some cancer cells. We isolated and analyzed a promoter region of the murine PLCdelta4 gene. DNA sequence analysis showed that this region is GC-rich and has no TATA box, and the region from -143 to -127 was found, by luciferase activity and gel mobility-shift assay, to be essential for transcription of PLCdelta4. We also found that the promoter activity of PLCdelta4 was stimulated by treatment with growth factors such as bradykinin, lysophosphatidic acid, and Ca2+ ionophore in addition to serum. In parallel, we detected PLCdelta4 mRNA induction and an increase in complex formation of the promoter region and nuclear protein from HeLa cells on stimulation with these growth factors. Finally, we found that trapping the growth factor-induced cytoplasmic Ca2+-inhibited activation of the promoter activity and protein induction in nuclei. These results show that PLCdelta4 may have an important role in nuclei in response to growth factors, and its expression may be partially regulated by an increase in cytoplasmic Ca2+.

わが国では,使用済燃料はエネルギー資源の備蓄として,再処理されるまでの間,適切に貯蔵・管理される。欧米では,直接処分または再処理までの間の貯蔵技術として,金属・コンクリートキャスク貯蔵,ボールト貯蔵横型サイロ貯蔵,等が開発されている。わが国では,金属キャスク貯蔵の許認可が得られている。今後は,使用済みの高燃焼度燃料やMOX燃料も貯蔵の対象となり,貯蔵コスト低減のための研究等が行われている。

We performed here MS-based cell surface proteome profiling of HCT-116 cells by two distinct methods based on biotin labeling and glycoprotein capturing. In total, 742 biotinylated and 219 glycosylated proteins were identified by the biotin labeling and glycoprotein capturing, of which 224 and 138 proteins known to be located on plasma membrane were included, respectively, according to ingenuity pathway analysis. Although 104 plasma membrane proteins were identified by both methods, the rest of 154 were identified only by one. Almost all the identified plasma membrane proteins possessed consensus N-glycosylation sites, and proteins having various numbers of glycosylation sites were identified by both methods. Thus, the discrepancies of the identified proteins obtained from those two methods might not be only due to the number of glycosylation sites, but also to the expression and/or glycosylation level of the cell surface proteins. We also identified 312 N-glycosylated proteins from xenograft samples by glycoprotein capturing of which 135 were known as plasma membrane proteins. Although a number of highly-expressed plasma membrane proteins were common between culture and xenograft cells, some proteins showed culture- or xenograft-specific expression, suggesting that those proteins might contribute to grow in different environment.

We performed here MS-based phosphoproteomics using both metal oxide affinity chromatography (pSTY proteomics) and anti-phosphotyrosine antibody (pY proteomics). The former method identified mainly phospho-serine and -threonine of nuclear or cytoplasmic proteins, whereas the latter did phosphotyrosine including more plasma membrane proteins and kinases. The overlap between these two methods was limited (24 tyrosine phosphorylation sites out of 325) and, by combining the two, coverage of the signaling molecules was enhanced as exemplified by Erk signaling. We also performed whole cell proteomics using an off-gel fractionator, and found 68.9% of the proteins identified by phosphoproteomics. Thus, the expression levels of phosphoproteins were roughly estimated. In addition to many uncharacterized phosphorylation sites, the dataset includes 136 sites that were experimentally verified elsewhere to be phosphorylated by a total of 83 kinases and kinase groups out of the 256 registered in the Phospho.ELM database. With the integration of various proteomic analyses and information from database, the responsible kinases of the identified phosphorylation sites and possibly their activity status were predicted by phosphorylation status and expression levels of their substrates, and thus our method may be able to monitor the activity status of phosphorylation signaling.

For 15 patients with refractory gastric cancer and peritoneal metastasis, intraperitoneal hyperthermic perfusion (IPHP) using mitomycin C combined with extensive surgery was prescribed. The antitumor effects were assessed cytohistologically in pre-IPHP and post-IPHP specimens of the abdominal effusion and peritoneal tissue. Gastric cancer cells in the abdominal effusion and/or lavage vanished from post-IPHP peritoneal exudate obtained from the pouch of Douglas. Peritoneal tissues from nine patients were harvested just after the IPHP treatment. All the nuclei of cancer cells were pyknotic in three of nine patients, and two of these three patients are alive with no local recurrence; one patient died of hepatic metastasis. In the remaining six patients, four with preoperative ascitic effusion and positive post-IPHP histologic findings died of peritoneal, intraabdominal, and pericardial metastases. The other two had some residual microscopic foci in the subperitoneal deep layer; one patient died of pleural recurrence, and the other is alive with no evidence of recurrence 42 months after the IPHP. Among the other six patients, whose post-IPHP peritoneal tissues were not available because of disappearance of disseminating foci as a result of the IPHP, two are living with no recurrence and, of the remaining four patients, three died of hepatic and intraabdominal metastases and the other one died of other causes. The histologic findings are suggestive of the following: (1) uniform heat and drug distribution in the abdominal cavity with IPHP treatment, except for an area adjacent to the inflow point of the perfusate; and (2) limited penetration of heat and drug through the subperitoneal layer. Thus, IPHP treatment results in complete destruction of cancer cells in the abdominal effusion and on and just beneath the peritoneum.

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule-interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC-MS-based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer-Pro and pSer-Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit alpha (COPA) S173, suggesting that the events are not cell-type specific but events generally occurring in mitosis or induced by a microtubule-interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M-phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M-phase arrest.

The image diagnosis in early breast cancers in clinically studied.(1) Echography(1) After Hitachi EUB2D 7.5 MHZ being used, the accurate diagnosis rate and false negative rate in invasive breast cancers are 90.9% and 9.1%, in tumors less than 1.0 cm in diameter, and 92.0%, 8.0% in those from 1.0 to 2.0 cm respectively. All tumors more than 0.5 cm in diameter can be represented as tumor echo. (2) The accurate rate in non invasive breast cancers is 69.2%.(2) Mammography(1) Accurate and false negative rates in invasive breast cancers are 47.1% and 52.9%, in tumors less than 1.0 cm in diameter, and 80.7% and 19.3% in those from 1.0 to 2.0 cm respectively. Diagnostic rate in tumors less than 1.0 cm is bad. It is as not so good as 57.1% in non invasive breast cancers. (2) The 52.9% of infiltrating breast cancer tumors less than 1.0 cm and 83.2% of those from 1.0 to 2.0 cm can be represented as tumor shadow. The previous date was bad. Tumor shadow can be visualized in 57.1% of non invasive tumors. It is 64.7% in invasive ductal carcinomas with a predominant intraductal component. (3) The appearance rates of fine calcification (FC) are 23.5% in less than 1.0 cm and 43.2% in from 1.1 to 2.0 cm invasive cancers, while 50.0% in non invasive ones. (4) The cases having no tumor shadow but FC are found in 25.0% of invasive cancers less than 1.0 cm and 26.7% of those in from 1.1 to 2.0 cm. It is 33.3% in non invasive breast ones.

Embryonic stem cells (ESCs) can give rise to any adult cell type and thus offer enormous potential for regenerative medicine and drug discovery. Molecular biomarkers serve as valuable tools to classify and isolate ESCs and to monitor their differentiation state by antibody-based techniques. A number of biomarkers, such as certain cell surface antigens, are used to assign pluripotent ESCs; however, accumulating evidence suggests that ESCs are heterogeneous in morphology, phenotype and function, and are thereby classified into subpopulations characterized by multiple sets of molecular biomarkers. Biomarker discovery is also important for ESC biology to elucidate the molecular mechanisms that regulate pluripotency and differentiation. This review summarizes studies of ESC biomarker discovery. “Genome-wide” expression profiling of ESC mRNAs and proteins and direct analyses of the cell surface subproteome have demonstrated that ESCs express a diverse range of biomarkers, cell surface antigens, and signaling molecules found in different cell lineages, as well as a number of key molecules that assure “stemness”. Clearly, future quantitative proteomics approaches will enhance our knowledge of the stage- and lineage-specific expression of the proteome and its temporal changes upon differentiation, and provide a more detailed view of nascent and clonally amplified ESCs.

非機能性膵島腫瘍の1例を経験したので文献的考察を加え報告する.症例は, 35歳女性で, 心窩部不快感を主訴に精査を受け, 膵体部癌の診断のもと, 膵体尾部・脾合併切除術を施行した.病理組織検査で膵島癌と診断された.本症は比較的まれであり, 術前診断が難しい.一般に膵管癌に比べ予後は良く, 腫瘍の発育速度が遅いと考えられていることから積極的に腫瘍切除を行うべきと思われる.

The cold-sensitive fcsA29 mutation of Escherichia coli was found to be a new type of cold-sensitive allele of the polA gene encoding DNA polymerase I, caused by an Asp(116)--Asn change in the 5'--3' exonuclease domain. The fcsA29 mutant showed typical polA mutant phenotypes such as UV sensitivity and unacceptability of recA mutation. Cold-sensitive growth of the mutant was suppressed by introduction of a sulA mutation, indicating that cell filamentation was due to the SOS response.

Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology. From 965 unique peptides carrying biotin labels, we assigned 324 proteins including 235 proteins that have putative signal sequences and/or transmembrane segments. Receptors, transporters, and cell adhesion molecules were the major classes of proteins identified. Besides known cell surface markers of embryonic stem cells, such as alkaline phosphatase, the analysis identified 59 clusters of differentiation-related molecules and more than 80 components of multiple cell signaling pathways that are characteristic of a number of different cell lineages. We identified receptors for leukemia-inhibitory factor, interleukin 6, and bone morphogenetic protein, which play critical roles in the maintenance of undifferentiated mouse embryonic stem cells. We also identified receptors for growth factors/cytokines, such as fibroblast growth factor, platelet-derived growth factor, ephrin, Hedgehog, and Wnt, which transduce signals for cell differentiation and embryonic development. Finally we identified a variety of integrins, cell adhesion molecules, and matrix metalloproteases. These results suggest that D3 cells express diverse cell surface proteins that function to maintain pluripotency, enabling cells to respond to various external signals that initiate differentiation into a variety of cell types.

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E(3)-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin -ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

Leukemic cells from an 8-year-old girl with ANLL-M2 expressed precursor B-cell antigen CD19, but none of the myeloid antigens CD11b, CD13, CD14 and CD33. After culture, the cells expressed CD11b and CD13. The cells carried a high number of granulocyte colony-stimulating factor (G-CSF) receptors. In chromosome analysis, metaphase cells were obtained only in the case of culture with G-CSF. The karyotype was a variant of t(8;21)(q22;q22). Southern blot analysis revealed rearrangement of the AMLI gene located on chromosome 21. These observations may suggest that even without myeloid surface antigens and with precursor B-cell antigen, ANLL-M2 with t(8;21)(q22;q22) has apparent myeloid characteristics.

Tumor microenvironment is a major factor influencing treatment resistance to conventional anticancer therapies. Indeed, under “quasi in vivo” conditions, with low oxygen (1%), low pH (6.5) and low glucose concentration (0.01%) to mimic the environments in grafted tumors in mice or in clinical tumors, anti-proliferative activities of some conventional anticancer agents were diminished. Here, we describe the small molecule CH4938056, which has a novel chemical structure and was identified through cell-based screening under the “quasi in vivo” conditions followed by chemical modification. Our initial phenotypic profiling revealed that CH4938056 specifically arrests cells at the M phase and that it overcomes multiple resistance mechanisms to conventional anticancer agents including over-expression of MDR1 and BCRP. Then, after designing and identifying a water-soluble phosphate prodrug which successfully converts to CH4938056 after injection, we demonstrated antitumor efficacy of CH4938056 in a HCT116 xenograft model and a MDR1-overexpressing cancer model. Chemo-proteomic studies and consequent biochemical analysis revealed that CH4938056 binds to microtubule-associated protein 4 (MAP4). Moreover, CH4938056 inhibited MAP4-dependent microtubule assembly in a cell free system. siRNA-mediated knockdown of MAP4 induced chromosomal misalignment in metaphase cells, which closely resembles the primary phenotype of the CH4938056-treated cells. Under the “quasi in vivo” conditions, MAP4 expression turned out to be down-regulated (since MAP4 transcription is known to be negatively regulated by p53 which is up-regulated under these conditions) and when we knocked down MAP4 with siRNA, cancer cells became sensitive to CH4938056, which altogether is consistent with the fact that CH4935056 has antitumor activity even under the “quasi in vivo” conditions. From these observations, we conclude that CH4938056 inhibits proliferation of cancer cells by targeting MAP4. Targeting MAP4 with CH4938056 offers a novel approach for the treatment of cancer, especially for patients resistant to conventional anticancer 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 2524. doi:10.1158/1538-7445.AM2011-2524

1) Noninvasive breast cancer was found in 16 (5.1%) among 314 female patients with primary breast cancer and, these 16 patients were studied retrospectively. Careful, skilled palpation could detect a tumor-like mass or induration even in noninvasive cancer. Nipple discharge was observed in only 4/16 patients (25.0%). Neither class IV nor V was found in cytological examination for their smears. Mammography revealed a tumor shadow in 43.8%, malignant fine calcification in 50.0%, and no such findings in 37.5% of the patients. 2) Echography represented a tumor echo in 84.6% of the 16 patients. 3) Aspiration biopsy cytology revealed 30.0% of the patients to be class IV or V. 4) 68.8% of the patients could not be definitely diagnosed till an excisional biopsy was made. 5) Since a large number of the patients had a palpable tumor more than 2.1cm in diameter measured pathologically, standard radical mastectmy had been performed for 37.5% of the 16 patients.