PubMed Health⌕ Search

Biomedical subjects

Tesshi Yamada

Publications and source records attributed to Tesshi Yamada.

At least 19 recordsLinked to original sources
PubMed2006

Mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells.

Actinin-4 was originally identified as an actin-binding protein associated with cell motility and cancer invasion and metastasis. However, actinin-4 forms complexes with a large number of different partner proteins and is speculated to have several distinct functions depending on its partner. The level of actinin-4 expression was found to be significantly lower in prostate cancer cells than in non-cancerous basal cells, and restoration of actinin-4 expression inhibited cell proliferation by prostate cancer cell line 22RV1. Immunoprecipitation and mass spectrometry analysis revealed that actinin-4 forms native complexes with several partner proteins in 22RV1 cells, including with beta/gamma-actin, calmodulin, the clathrin heavy chain, non-muscular myosin heavy chain, heterogeneous nuclear ribonucleoprotein A1, and Ras-GTPase-activating protein SH3 domain-binding protein. Clathrin is a coat protein that covers the internalized membrane pit that forms during early endocytosis. We found that other clathrin-related and unrelated cargo proteins, including dynamin, adaptin-delta, beta subunit of neuronal adaptin-like protein, and p47A, also interact with actinin-4. Immunofluorescence microscopy revealed that dynamin and clathrin co-localized with actinin-4 at the sites of membrane ruffling, and transfection of actinin-4 cDNA facilitated the transport of transferrin into perinuclear endosomes. Endocytosis terminates signaling evoked by cell surface receptors and regulates the recycling of receptors and ligands. We identified a panel of proteins whose expression and/or subcellular localization was regulated by actinin-4 by performing organelle fractionation and ICAT-LC-MS/MS. The decreased expression of actinin-4 protein in prostate cancer cells may cause aberrations in the intracellular trafficking of various cell surface molecules and contribute to carcinogenesis.

Actinin↗
PubMed2006

Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry.

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.

Actinin↗
PubMed2006

Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye.

CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.

Cell Line, Tumor↗
PubMed2006

[Early detection of pancreatic cancer by novel proteomic technique].

Pancreatic cancer is the fifth leading cause of cancer-related mortality in Japan. Early detection in pancreatic cancer is one of the most feasible strategies to improve outcome. We compared plasma proteome between pancreatic cancer patients and healthy controls using surface-enhanced laser desorption/ionization coupled with hybrid quadrupole time-of-flight mass spectrometry. Proteomic spectra were generated from a total of 245 plasma samples obtained from two institutes. A discriminating proteomic pattern was built from training cohort using machine learning algorithm and was applied two validation cohorts. This set discriminating cancer patients in the first validation cohort with a sensitivity of 90.9% and a specificity of 91.9%, and was further validated in an independent cohort at a second institution. When combined with CA19-9, 100% tumor of pancreatic cancers, including early stage tumors, were detected. In this report, we describe a possible detection of early pancreatic cancer using novel proteomic technique.

Biomarkers, Tumor↗
PubMed2005

Possible detection of pancreatic cancer by plasma protein profiling.

The survival rate of pancreatic cancer patients is the lowest among those with common solid tumors, and early detection is one of the most feasible means of improving outcomes. We compared plasma proteomes between pancreatic cancer patients and sex- and age-matched healthy controls using surface-enhanced laser desorption/ionization coupled with hybrid quadrupole time-of-flight mass spectrometry. Proteomic spectra were generated from a total of 245 plasma samples obtained from two institutes. A discriminating proteomic pattern was extracted from a training cohort (71 pancreatic cancer patients and 71 healthy controls) using a support vector machine learning algorithm and was applied to two validation cohorts. We recognized a set of four mass peaks at 8,766, 17,272, 28,080, and 14,779 m/z, whose mean intensities differed significantly (Mann-Whitney U test, P < 0.01), as most accurately discriminating cancer patients from healthy controls in the training cohort [sensitivity of 97.2% (69 of 71), specificity of 94.4% (67 of 71), and area under the curve value of 0.978]. This set discriminated cancer patients in the first validation cohort with a sensitivity of 90.9% (30 of 33) and a specificity of 91.1% (41 of 45), and its discriminating capacity was further validated in an independent cohort at a second institution. When combined with CA19-9, 100% (29 of 29 patients) of pancreatic cancers, including early-stage (stages I and II) tumors, were detected. Although a multi-institutional large-scale study will be necessary to confirm clinical significance, the biomarker set identified in this study may be applicable to using plasma samples to diagnose pancreatic cancer.

Biomarkers, Tumor↗
PubMed2005

Possible prediction of chemoradiosensitivity of esophageal cancer by serum protein profiling.

PURPOSE: Establishment of a reliable method of predicting the efficacy of chemotherapy and radiotherapy is necessary to provide the most suitable treatment for each cancer patient. We investigated whether proteomic profiles of serum samples obtained from untreated patients were capable of being used to predict the efficacy of combined preoperative chemoradiotherapy against esophageal cancer. EXPERIMENTAL DESIGN: Proteomic spectra were obtained from a training set of 27 serum samples (15 pathologically diagnosed responders to preoperative chemoradiotherapy and 12 nonresponders) by surface-enhanced laser desorption and ionization coupled with hybrid quadrupole time-of-flight mass spectrometry. A proteomic pattern prediction model was constructed from the training set by machine learning algorithms, and it was then tested with an independent validation set consisting of serum samples from 15 esophageal cancer patients in a blinded manner. RESULTS: We selected a set of four mass peaks, at 7,420, 9,112, 17,123, and 12,867 m/z, from a total of 859 protein peaks, as perfectly distinguishing responders from nonresponders in the training set with a support vector machine algorithm. This set of peaks (i.e., the classifier) correctly diagnosed chemoradiosensitivity in 93.3% (14 of 15) of the cases in the validation set. CONCLUSIONS: Recent mass spectrometric approaches have revealed that serum contains a large volume of information that reflects the microenvironment of diseased organs. Although a multi-institutional large-scale study will be necessary to confirm each component of the classifier, there is a subtle but definite difference in serum proteomic profile between responders and nonresponders to chemoradiotherapy.

Aged↗
PubMed2005

E-cadherin regulates the association between beta-catenin and actinin-4.

The E-cadherin/catenin system acts as an invasion suppressor of epithelial malignancies. This invasion suppressive activity seems be mediated not only by the cell adhesive activity of E-cadherin but by other undetermined signaling pathways elicited by beta-catenin. In fact, cancer cells that have infiltrated the stroma reduce the expression of E-cadherin and accumulate beta-catenin. We attempted to identify the alternative partner proteins that make complexes with beta-catenin in the absence of E-cadherin. An approximately 100-kDa protein was constantly coimmunoprecipitated with beta-catenin from SW480 colorectal cancer cells, which lack the expression of E-cadherin, and was identified as actinin-4 by mass spectrometry. Transfection of E-cadherin cDNA suppressed the association between beta-catenin and actinin-4. Inhibition of E-cadherin by RNA interference transferred the beta-catenin and actinin-4 proteins into the membrane protrusions of DLD-1 cells. Immunofluorescence histochemistry of clinical colorectal cancer specimens showed that the beta-catenin and actinin-4 proteins were colocalized in colorectal cancer cells infiltrating the stroma. We reported previously that overexpression of actinin-4 induces cell motility and specifically promotes lymph node metastasis by colorectal cancer. The association between beta-catenin and actinin-4 and its regulation by E-cadherin may represent a novel molecular link connecting cell adhesion and motility. Shutting down the signals mediating this association may be worth considering as a therapeutic approach to cancer invasion and metastasis.

Actinin↗
PubMed2005

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells.

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.

Amino Acid Sequence↗
PubMed2005

Morphological and transcriptional responses of untransformed intestinal epithelial cells to an oncogenic beta-catenin protein.

Aberrant transactivation of a certain set of target genes by the beta-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor complexes has been implicated in the process of intestinal epithelial cells entering early colorectal carcinogenesis. A rat intestinal epithelial cell line IEC6 became elongated, extended protrusions at cell periphery, and increased stress fibers and focal contacts upon the induction of beta-catenin protein stabilized by deletion of the N-terminal glycogen synthase kinase-3beta (GSKbeta) phosphorylation sites (beta-catenin DeltaN89). We used the GeneChiptrade mark oligonucleotide microarray system to examine approximately 24 000 genes and identified 13 genes whose expression was altered during the course of this morphological transformation. Those genes included known negative regulators of the Wnt signaling pathway, Sfrp4 and Axin2; extracellular matrix and related molecule, Hxb and Crtl1; cell adhesion and cytoskeletal proteins, Podxl, Igaf4, and Itab6; and molecules involved in the insulin and insulin-like growth factor (IGF) signaling pathways, Enpp1, Igfbp2, and Sgk. We report the finding that insulin-like growth factor-binding protein-2 (IGFBP2) is a direct target gene of the beta-catenin and TCF/LEF complexes. The IGFBP2 protein interacts with integrins. Disruption of the multigene network system regulating cell adhesion and cytoskeleton may be crucial in the initiation of colorectal carcinogenesis.

Adenoma↗
PubMed2005

Prognostic significance of tissue factor in pancreatic ductal adenocarcinoma.

Tissue factor (TF) is a transmembrane glycoprotein that plays roles in the blood coagulation and intracellular signaling pathways, and has also been suggested to modulate the biological behavior of cancer cells. In order to examine the clinicopathologic significance of TF expression in pancreatic ductal adenocarcinoma, TF expression was determined by immunohistochemistry using a newly raised anti-TF monoclonal antibody in 113 patients who had undergone surgical resection of pancreatic ductal adenocarcinoma. According to the incidence of tumor cell immunopositivity, patients were divided into "negative TF" (0%), "weak TF" (<25%), or "high TF" (25% or more) groups, which accounted for 11.6% (n = 13), 44.2% (n = 50), and 44.2% (n = 50) of the total, respectively. Increased TF expression was correlated with the extent of the primary tumor (P = 0.0043), lymph node metastasis (P = 0.0043), lymphatic distant metastasis (P = 0.0039), advanced tumor-node-metastasis stage (P = 0.0002), and high tumor grade (P = 0.0164). Multivariate analysis using the Cox proportional hazards model showed that high TF expression was an independent negative predictor for survival (hazard ratio, 2.014; P = 0.0076). Moreover, patients with TF-negative tumors had a significantly better prognosis even if lymph node metastasis was present (P < 0.0001). We also showed that TF knockdown by RNA interference suppressed the invasiveness of a pancreatic adenocarcinoma cell line in vitro. These results indicate that TF expression may contribute to the aggressiveness of pancreatic ductal adenocarcinoma by stimulating tumor invasiveness, and that evaluation of the primary tumor for TF expression may identify patients with a poor prognosis.

Aged↗
PubMed2005

Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye.

To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.

Adult↗
PubMed2005

Proteomic signatures for histological types of lung cancer.

We performed proteomic studies on lung cancer cells to elucidate the mechanisms that determine histological phenotype. Thirty lung cancer cell lines with three different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma and adenocarcinoma) were subjected to two-dimensional difference gel electrophoresis (2-D DIGE) and grouped by multivariate analyses on the basis of their protein expression profiles. 2-D DIGE achieves more accurate quantification of protein expression by using highly sensitive fluorescence dyes to label the cysteine residues of proteins prior to two-dimensional polyacrylamide gel electrophoresis. We found that hierarchical clustering analysis and principal component analysis divided the cell lines according to their original histology. Spot ranking analysis using a support vector machine algorithm and unsupervised classification methods identified 32 protein spots essential for the classification. The proteins corresponding to the spots were identified by mass spectrometry. Next, lung cancer cells isolated from tumor tissue by laser microdissection were classified on the basis of the expression pattern of these 32 protein spots. Based on the expression profile of the 32 spots, the isolated cancer cells were categorized into three histological groups: the squamous cell carcinoma group, the adenocarcinoma group, and a group of carcinomas with other histological types. In conclusion, our results demonstrate the utility of quantitative proteomic analysis for molecular diagnosis and classification of lung cancer cells.

Adenocarcinoma↗
PubMed2005

Protein expression pattern distinguishes different lymphoid neoplasms.

To identify proteins associated with the histological subtypes of lymphoid neoplasms, we studied the proteomes of 42 cell lines from human lymphoid neoplasms including Hodgkin's lymphoma (HL; four cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell lymphoma (three cell lines). The protein spots were sequentially selected by (i) Wilcoxon or Kruskal-Wallis tests to find the spots whose intensity was significantly (p <0.05) different among the cell line groups, (ii) by statistical-learning methods to prioritize the spots according to their contribution to the classification, and (iii) by unsupervised classification methods to validate the classification robustness by the selected spots. The selected spots discriminated (i) between HL cells and other cells, (ii) between the cells from B cell malignancies, T cell malignancies, and NK cell lymphoma cells, and (iii) between HL cells and anaplastic large cell lymphoma cells. Among the 31 informative protein spots, MS identified 24 proteins corresponding to 23 spots. Previous reports did not correlate these proteins to lymphocyte differentiation, suggesting that a proteomic study would identify the novel mechanisms responsible for the histogenesis of lymphoid neoplasms. These proteins may have potential as differential diagnostic markers for lymphoid neoplasms.

Amino Acid Sequence↗
PubMed2005

Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer.

BACKGROUND & AIMS: Enhanced motility of cancer cells by remodeling of the actin cytoskeleton seems crucial in the process of cancer invasion and metastasis. We previously identified an actin-binding protein, actinin-4, as a new biomarker of cancer invasion and an indicator of prognosis for patients with breast cancer. However, its involvement in the mechanisms of cancer invasion and metastasis remains undetermined. The current study tested the role of actinin-4 in the motility and metastatic potential of colorectal cancer cells. METHODS & RESULTS: Quantitative immunofluorescence histochemistry showed that the expression level of the actinin-4 protein was increased in 73.1% (19/26) of the cases of colorectal cancer over the corresponding normal intestinal epithelium. The increased expression of actinin-4 was most significant in dedifferentiated cancer cells at the invasive front. A colorectal cancer cell clone capable of inducing actinin-4 using the tetracycline-regulatory system (designated DLD1 Tet-off ACTN-4) was established. Upon the induction of actinin-4, DLD1 Tet-off ACTN-4 cells spread filopodia and significantly increased their motility ( P = .00027); actinin-4 protein was concentrated at the leading edges of these actin-rich podia. When injected into the mesocecum of severe combined immunodeficient mice, DLD1 Tet-off ACTN4 cells, but not the control cells, metastasized into regional mesenteric lymph nodes, resembling the behavior of clinical cancers. The expression of actinin-4 in focally dedifferentiated cancer cells at the invasive front was significantly correlated with the frequency of lymph node metastasis of colorectal cancer ( P = .038). CONCLUSIONS: Actinin-4 actively increases cell motility and promotes lymph node metastasis of colorectal cancer.

Actinin↗
PubMed2005

Poly(ADP-ribose) polymerase-1 is a component of the oncogenic T-cell factor-4/beta-catenin complex.

BACKGROUND & AIMS: T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. METHODS & RESULTS: A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. CONCLUSIONS: In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Cell Transformation, Neoplastic↗
PubMed2005

Beta-catenin interacts with the FUS proto-oncogene product and regulates pre-mRNA splicing.

BACKGROUND & AIMS: beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin. METHODS: The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry. RESULTS: beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity. CONCLUSIONS: Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

Alternative Splicing↗
PubMed2005

Identification of 2 serum biomarkers of renal cell carcinoma by surface enhanced laser desorption/ionization mass spectrometry.

PURPOSE: Surface enhanced laser desorption/ionization mass spectrometry can generate robust information from a small amount of clinical samples such as serum and plasma. In this study we identified novel diagnostic biomarkers of renal cell carcinoma (RCC) by large-scale serum protein profiling using surface enhanced laser desorption/ionization mass spectrometry. MATERIALS AND METHODS: Proteomic spectra were generated by a time of flight mass spectrometer from a set of training samples (21 patients with RCC and 24 healthy volunteers) and another set of validation samples (19 patients with RCC, 20 healthy volunteers and 5 patients with pyelonephritis). Information on the peaks (intensity and m/z) was extracted from the mass spectra using newly developed algorithms, and the Mann-Whitney's U test and linear support vector machine were used to identify the peaks distinguishing RCC samples from the controls. RESULTS: Two peaks with molecular masses of 4,151 and 8,968 m/z were selected as significantly more prominent in RCC samples (p <0.01) among the 3,539 peaks in the range of 3,000 to 30,000 m/z obtained from the training samples. Simultaneous recognition of these 2 biomarkers was shown to have a sensitivity of 89.5% for the diagnosis of RCC and an overall specificity of 80.0% (95% [19 of 20] of healthy volunteers and 20% [1 of 5] of patients with pyelonephritis) in the blinded validation samples, and to allow detection of RCC in stage I (UICC) in 88.9% (16 of 18) of the cases. CONCLUSIONS: We identified 2 serum biomarkers potentially useful for the early diagnosis of RCC. This finding warrants a further large-scale multi-institutional analysis for clinical evaluation of the diagnostic significance of these biomarkers.

Adult↗