David K. M. Han

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 α2-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, α2-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.

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 BclXL. The second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E‐BP1, which controls mRNA translation.

Toward a High-Throughput Approach to Quantitative Proteomic Analysis: Expression-Dependent Protein Identification by Mass Spectrometry

The isotope-coded affinity tag (ICAT) [1] 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.

A COMPARATIVE MOLECULAR ANALYSIS OF FOUR RAT SMOOTH MUSCLE CELL LINES

SummaryTranscriptional regulation of smooth muscle cell (SMC) differentiation is a rapidly growing area of interest that has relevance for understanding intimal disease. Despite the wealth of data accumulating in vitro, however, no study has compared the cell-specific marker profile, transfectability, promoter activity, and growth characteristics among several SMC culture systems. Accordingly, we performed a comprehensive analysis of the marker profile, growth properties, transfectability, and SMC promoter activity in four rat SMC lines (A7r5, adult and pup aortic, and PAC1). Despite alterations in chromosomal number and structure, A7r5, adult aortic, and PAC1 cells express all SMC markers studied including SM α-actin, SM calponin, SM22, tropoelastin, and to a lesser extent, SM myosin heavy chain (SMMHC). In contrast, pup aortic cells express very low or undetectable levels of all the above markers except tropoelastin. Adult aortic, pup, and PAC1 cells display similar growth curves and levels of proto-oncogene transcripts, whereas those in the A7r5 line are comparatively less. All cell lines studied except pup cells show expression of SMC differentiation genes during active growth, indicating that growth and differentiation are not mutually exclusive in cultured smooth muscle. Transfection studies reveal dramatic differences in DNA uptake and SMC-restricted promoter activity between cell lines. Collectively, these results provide detailed information relating to SMC molecular biology in culture that should facilitate the selection of a cell line for studying the transcriptional regulatory mechanisms underlying SMC differentiation.

Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate‐derived expressed sequence tags (ESTs). A total of 55 000 prostate ESTs were assembled into a set of 15 953 clusters putatively representing 15 953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen‐response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen‐regulated genes such as prostate‐specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen‐stimulated prostate cancer cells were generated by two‐dimensional electrophoresis (2‐DE). Mass spectrometric analysis of androgen‐regulated proteins in these cells identified the metastasis‐suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway.

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.

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.

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from monocytes/macrophage

Abstract Fas and its ligand (FasL), are a receptor-ligand pair identified as promoting cell death in several tissues. Apoptosis of vascular smooth muscle cells (VSMCs) in human atherosclerotic plaque may contribute to weakening of the fibrous cap, ultimately resulting in plaque rupture. We investigated the ability of monocytes to induce apoptosis of cultured VSMCs through Fas/FasL pathway. In addition, we examined the association of FasL with apoptosis in human coronary plaques. Both activated monocytes and the supernatant obtained from activated monocytes were able to kill cultured VSMCs. The apoptotic response of VSMCs was almost completely blocked by the caspase inhibitor z-VAD-fmk and was partially blocked by incubation with antagonistic anti-Fas IgG1 which suggests that Fas/FasL system was involved in the induction of cell death. An ≈30 kDa protein, which represents a cleaved, soluble form of FasL, was identified in culture medium from activated monocytes, but not in culture medium from control, unactivated monocytes. Immunohistochemical analysis of human atherosclerotic coronary lesions showed that FasL is expressed by macrophages, and microvessels in the adventitia as well as in the plaque. Finally, double-staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and FasL antibody showed that FasL enriched lesions always included a number of TUNEL-positive cells. These data suggest that Fas/FasL pathway can be employed by monocytes/macrophages to induce VSMC apoptosis in the atherosclerotic lesions.

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‐α, 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.

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.