Biomedical subjects

D K Han

Publications and source records attributed to D K Han.

At least 19 recordsLinked to original sources

PubMed2006

Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues.

Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.

Amino Acid Sequence↗

PubMed2001

Effects of protein kinase CK2, extracellular signal-regulated kinase 2, and protein phosphatase 2A on a phosphatidic acid-preferring phospholipase A1.

A soluble, phosphatidic acid-preferring phospholipase A1, expressed in mature bovine testes but not in newborn calf testes, may contribute to the formation or function of sperm. Here we incubated a recombinant preparation of the phospholipase in vitro with several enzymes including protein kinase CK2 (CK2), extracellular signal-regulated kinase 2 (ERK2), and protein phosphatase 2A (PP2A) to identify effects that might be of regulatory importance in vivo. Major findings were that 1) CK2 phosphorylated the phospholipase on serines 93, 105, and 716; 2) ERK2 phosphorylated the enzyme on serine 730; 3) there was cross-antagonism between the reactions that phosphorylated serines 716 and 730; 4) PP2A selectively hydrolyzed phosphate groups that were esterified to serines 716 and 730; 5) CK2alpha formed a stable, MgATP/MgGTP-dependent complex with the phospholipase by a novel mechanism; and 6) the complex showed reduced phospholipase activity and resembled a complex identified in homogenates of macaque testis. These results provide the first available information about the effects of reactions of phosphorylation and dephosphorylation on the behavior of the phospholipase, shed light on properties of CK2alpha that may be required for the formation of complexes with its substrates, and raise the possibility that a complex containing CK2alpha and the phospholipase may play a special biological role in the testis.

Amino Acid Sequence↗

PubMed2001

Toward a high-throughput approach to quantitative proteomic analysis: expression-dependent protein identification by mass spectrometry.

The isotope-coded affinity tag (ICAT) technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.

Autoanalysis↗

PubMed2001

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naïve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.

Affinity Labels↗

PubMed2001

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from endothelial cells.

Although Fas-mediated cell death may play a role in atherogenesis, causal data in support of this hypothesis are lacking. The present study investigated the possibility that endothelial cells are involved in vascular smooth muscle cell (VSMC) apoptosis via the Fas-FasL pathway, and hence in atherogenesis. FACS analysis detected FasL on the surface of human umbilical vein endothelial cells (HUVECs) and immunofluorescence staining of the HUVECs demonstrated high levels of FasL in the intracellular compartment. FasL was down-regulated 4 h after tumor necrosis factor (TNFalpha) treatment, coinciding with maximal surface expression of the adhesion molecules vascular cell adhesion molecule-1 and E-selectin. However, the down-regulation of FasL expression was transient, as surface expression returned within 24 h of TNFalpha treatment. When cocultured with VSMCs, the FasL-expressing EC could kill the VSMCs in a manner that could be blocked by recombinant Fas-Fc, deployed as a soluble receptor for Fas. Moreover, when human coronary arteries were studied with immunohistochemistry using G247-4 monoclonal antibody for the detection of FasL, few FasL positive EC were observed in diffuse intimal thickening. In contrast, endothelium overlying the plaque showed prominent and uniform expression of FasL. These findings suggest that the Fas/FasL pathway can be used by EC to induce VSMC apoptosis in the atherosclerotic lesion.

Adult↗

PubMed2000

Caspase-3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces cerevisiae and mammalian cells.

Using a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of XIAP>c-IAP2>c-IAP1>survivin. The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto-activating caspase-3. The relative anti-apoptotic activities of each IAP depended on the particular death stimulus. For IAP-expressing cells treated with camptothecin, survival correlated with their intrinsic anti-caspase-3 activity. However, c-IAP1-transfected cells were disproportionately resistant to tumor necrosis factor-alpha, suggesting that its anti-apoptotic activities extend beyond caspase-3 or -7 inhibition. Yeast-based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Expression of cellular FLICE-inhibitory protein in human coronary arteries and in a rat vascular injury model.

We previously isolated MACH-related inducer of toxicity (MRIT), a homolog of caspase 8. MRIT, also known as c-FLICE-inhibitory protein (c-FLIP), is an enzymatically inactive homolog of caspase 8 with homology to viral FLIP (v-FLIP). Because of this homology and resemblance to dominant negative proteins, c-FLIP is widely believed to be an antagonist to the death receptor-initiated apoptotic pathways that use caspase 8. We generated a polyclonal antibody, MAG1, and show that this antibody specifically recognizes two splice forms, long form (c-FLIPL) and short form (c-FLIPS). By in situ hybridization and immunohistochemistry, we demonstrate that c-FLIP is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs) both in human coronary arteries and in cultured cells. In an uninjured rat carotid arteries, c-FLIP protein is abundant in the vascular media. After balloon angioplasty, c-FLIP protein is rapidly down-regulated in medial SMCs for 2 weeks and regains expression by 4 weeks. In contrast, the neointima is strongly immunoreactive to c-FLIP from day 7 after the initial injury and remains strongly immunoreactive until 4 to 6 weeks. Similarly there is strong c-FLIP immunoreactivity in SMCs from nonatherosclerotic diffuse intimal thickening and in the overlying endothelial cells. In contrast, c-FLIP immunoreactivity is uneven and often absent in SMCs within the atherosclerotic plaque. Double labeling with c-FLIP antibody and terminal deoxynucleotidyltransferase-mediated UDP end labeling (TUNEL) in the injured rat common carotid artery show that TUNEL-positive cells in the first 2 days after injury lack detectable c-FLIP, suggested a role for caspase 8 in this form of death. In contrast, there is no correlation of c-FLIP with the spontaneous elevation in death of intima seen at 7 days after injury. For human atherosclerotic plaques, the majority of TUNEL-positive cells lack detectable c-FLIP. The expression pattern of c-FLIP and the relation between c-FLIP and TUNEL suggest a role for c-FLIP- and caspase 8-driven death in control of viability of the cells of the atherosclerotic intima.

Adult↗

PubMed2000

Cellular FLIP is expressed in cardiomyocytes and down-regulated in TUNEL-positive grafted cardiac tissues.

OBJECTIVE: c-FLIP is a natural homologue of caspase 8, and may antagonize activation of death pathways mediated by FADD. c-FLIP is highly expressed in the heart, and a recent report suggests that c-FLIP may protect against certain types of myocyte death. The present study was designed to define the expression patterns of c-FLIP in the heart. METHODS: The expression pattern of c-FLIP in end-stage human hearts, and rat cardiomyocyte grafting models was analyzed by in situ hybridization, immunohistochemistry and TUNEL assay. In addition, to determine whether Fas-dependent pathway is active in cardiomyocytes in vitro, we examined whether activated monocytes can kill neonatal cardiomyocytes in a co-culture system. RESULTS: c-FLIP mRNA and protein were abundantly expressed in normal cardiomyocytes from failing human heart. In animal models, c-FLIP protein was absent in TUNEL-positive grafted cardiomyocytes. Double staining demonstrated that c-FLIP-positive cells rarely had fragmented DNA, while TUNEL-positive cells rarely contained c-FLIP. Finally, activated monocytes induced death of neonatal rat cardiomyocytes via the Fas/FasL system. CONCLUSIONS: Loss of c-FLIP expression correlates with cardiomyocyte cell death. We hypothesize that diminished c-FLIP expression may predispose cardiomyocytes to apoptotic death.

Adult↗

PubMed2000

Heparinized bovine pericardium as a novel cardiovascular bioprosthesis.

A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.

Animals↗

PubMed2000

Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells.

Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.

Adaptor Proteins, Signal Transducing↗

PubMed2000

CD91: a receptor for heat shock protein gp96.

Antigen presenting cells (APCs) can take up exogenous antigenic peptides chaperoned by heat shock protein gp96 and re-present them through the endogenous pathway on their major histocompatibility class I molecules. The high efficiency of this process has been attributed previously to a receptor for gp96 on APCs. The CD91 molecule (also called alpha 2-macroglobulin receptor or the low density lipoprotein-related protein) is shown here to be a cell surface receptor for the heat shock protein gp96. CD91 binds gp96 directly, rather than through another ligand for CD91. The previously known CD91 ligand, alpha 2-macroglobulin, inhibits re-presentation of gp96-chaperoned antigenic peptides by macrophages, as do antibodies to CD91. As gp96 is exclusively intracellular and is released as a result of necrotic but not apoptotic cell death, we propose that CD91 acts as a sensor for necrotic cell death.

Amino Acid Sequence↗

PubMed1999

Caspase-3 inhibits growth in Saccharomyces cerevisiae without causing cell death.

Caspase-3, a member of the caspase family of cell death proteases, cleaves cytoplasmic and nuclear substrates and promotes apoptotic cell death in mammalian cells. Although yeast homologs of apoptotic genes have not been identified, some components of apoptotic pathways retain function in yeast. Here we show that the expression of caspase-3 delays cell growth in Saccharomyces cerevesiae without causing cell death. Mutation of the caspase-3 QACRG active site abolished effects on yeast growth. Co-expression of caspase inhibitors alleviated growth inhibition in yeast as did the tripeptide caspase inhibitor ZVAD-fmk. These results suggest that substrates for caspase-3 are present in S. cerevesiae and may participate in the normal cell growth and division processes.

Apoptosis↗

PubMed1999

Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era.

The post-genomic era is characterized by the deposition of sequence information for entire genomes in databases. Currently, besides the protein sequences for known human proteins, there are partial sequences from thousands more human proteins for which no biological function has been assigned. A powerful new tool for the unambiguous identification and characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching. This combination provides the cancer biologist with the ability to (i) identify the potential protein:protein associations and (ii) fully characterize function-critical post-translational modifications, both directly from silver-stained polyacrylamide gels. In this report we describe the application of tandem mass spectrometry and database searching to two problems which are prototypical for cancer research and indeed for biomedical research in general. The first is the identification of gel-separated, low abundance proteins based on amino acid sequence composition following coimmunoprecipitation with the human apoptosis inhibitor protein BclX(L). The second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E-BP1, which controls mRNA translation.

Amino Acid Sequence↗

PubMed1999

Effect of cross-link density and hydrophilicity of PU on blood compatibility of hydrophobic PS/hydrophilic PU IPNs.

To investigate the effect of the hydrophilic and hydrophobic microdomain structure on blood compatibility, a series of interpenetrating polymer networks (IPNs) composed of hydrophilic polyurethane (PU) and hydrophobic polystyrene (PS) was prepared. One series was prepared with varying cross-link densities of each network, the other with varying hydrophilicity of the PU component. All PU/PS IPNs exhibited microphase-separated structures that had dispersed PS domains in the continuous PU matrix. The domain size decreased with decreasing the hydrophilicity of the PU component and increasing the cross-link density of each network. As the cross-link density and hydrophobicity of the PU component was increased, an inward shift of Tgs was observed, which was due to the decrease in phase separation between the hydrophobic PS component and hydrophilic PU component. In the in vitro platelet adhesion test, as the microdomain size of PU/PS IPN surface decreased, the number of adhered platelets on the PU/PS IPN surface was reduced and deformation of the adhered platelets decreased. It could be concluded that blood compatibility of PU/PS IPN was mainly affected by the degree of mixing between PU and PS component, which was reflected by the domain size of PS rich phase.

Cross-Linking Reagents↗

PubMed1998

Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1.

Mst1 is a ubiquitously expressed serine-threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. In this paper we show that Mst1 is specifically cleaved by a caspase 3-like activity during apoptosis induced by either cross-linking CD95/Fas or by staurosporine treatment. CD95/Fas-induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD-fmk, the more selective caspase inhibitor DEVD-CHO and by the viral serpin CrmA. Caspase-mediated cleavage of Mst1 removes the C-terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase-mediated cleavage activating Mst1. Overexpression of either wild-type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase-dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co-transfection assays, suggesting that Mst1 may activate these pathways. Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Surface characteristics and biocompatibility of lactide-based poly(ethylene glycol) scaffolds for tissue engineering.

Novel lactide-based poly(ethylene glycol) (PEG) polymer networks (GL9-PEGs) were prepared by UV copolymerization of a glycerol-lactide triacrylate (GL9-Ac) with PEG monoacrylate (PEG-Ac) to use as scaffolds in tissue engineering, and the surface properties and biocompatibility of these networks were investigated as a function of PEG molecular weight and content. Analysis by ATR-FTIR and ESCA revealed that PEG was incorporated well within the GL9-PEG polymer networks and was enriched at the surfaces. From the results of SEM, AFM, and contact angle analyses, GL9-PEG networks showed relatively rough and irregular surfaces compared to GL9 network, but the mobile PEG chains coupled at their termini were readily exposed toward the aqueous environment when contacting water such that the surfaces became smoother and more hydrophilic. This reorientation and increase in hydrophilicity were more extensive with increasing PEG molecular weight and content. As compared to GL9 network lacking PEG, protein adsorption as well as platelet and S. epidermidis adhesion to GL9-PEG networks were significantly reduced as the molecular weight and content of PEG was increased, indicating that GL9-PEG networks are more biocompatible than the GL9 network due to PEG's passivity. Based on the physical and biological characterization reported, the GL9-PEG materials would appear to be interesting candidates as matrices for tissue engineering.

Bacterial Adhesion↗

PubMed1998

Sulfonated poly(ethylene oxide)-grafted polyurethane copolymer for biomedical applications.

Sulfonated poly(ethylene oxide) (PEO-SO3)-grafted polyurethane (PU) copolymer (PU-PEO-SO3) was developed for utilization as a material for biomedical applications. Its bulk properties and biocompatibility were investigated and the results were compared to those of PU and PU-PEO (not sulfonated). PU-PEO-SO3 copolymer showed higher water uptake than PU and PU-PEO, indicative of increased hydrophilicity due to the grafting of PEO-SO3. Compared with PU, PU-PEO-SO3 displayed lower glass transition temperature (Tg) and melting endotherm from DSC analysis, suggesting the increase of microphase separation and the suppression of hard-segment packing. PU-PEO-SO3 had practically similar mechanical properties to PU, indicating that the chemical modification of PU was accomplished without reducing its bulk properties. From the results of biocompatibility tests, PU-PEO-SO3 copolymer showed significantly lower adhesion of platelets and S. epidermidis than PU control and PU-PEO. In addition, the platelet factor 4 (PF4) released from platelets was the lowest in PU-PEO-SO3 among the test materials. PU-PEO-SO3 copolymer, which has excellent mechanical properties and biocompatibility, is expected to be useful as coating, molding, and blending materials for artificial organs and medical devices.

Alkanesulfonates↗

PubMed1997

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death.

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

Adult↗