Next-generation biomarkers based on 100-parameter functional super-resolution microscopy TIS

Functional super-resolution (fSR) microscopy is based on the automated toponome imaging system (TIS). fSR-TIS provides insight into the myriad of different cellular functionalities by direct imaging of large subcellular protein networks in morphologically intact cells and tissues, referred to as the toponome. By cyclical fluorescence imaging of at least 100 molecular cell components, fSR-TIS overcomes the spectral limitations of fluorescence microscopy, which is the essential condition for the detection of protein network structures in situ/in vivo. The resulting data sets precisely discriminate between cell types, subcellular structures, cell states and diseases (fSR). With up to 16 bits per protein, the power of combinatorial molecular discrimination (PCMD) is at least 2(100) per subcellular data point. It provides the dimensionality necessary to uncover thousands of distinct protein clusters including their subcellular hierarchies controlling protein network topology and function in the one cell or tissue section. Here we review the technology and findings showing that functional protein networks of the cell surface in different cancers encompass the same hierarchical and spatial coding principle, but express cancer-specific toponome codes within that scheme (referred to as TIS codes). Findings suggest that TIS codes, extracted from large-scale toponome data, have the potential to be next-generation biomarkers because of their cell type and disease specificity. This is functionally substantiated by the observation that blocking toponome-specific lead proteins results in disassembly of molecular networks and loss of function.     

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.     

Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures

This protocol details sample preparation and measurement procedures for a fluorescence technology capable of colocalizing hundreds of different proteins in a cell or tissue section. The procedure relies on fixation of samples and on the use of dye-conjugated tag libraries. To colocalize proteins, a sample is placed on the microscope stage of an imaging system (toponome imaging system (TIS)) performing sequential cycles of tag-dye incubation, imaging and bleaching to generate images for each localization cycle. TIS overcomes the spectral limitations of traditional fluorescence microscopy. Image processing reveals toponome maps, uncovering the coexistence of proteins at a location (protein clusters). The approach provides direct insight into the topological organization of proteins on a proteomic scale for the first time. If, for example, two dyes are used per cycle, 18 proteins in 4 visual fields can be colocalized in 21 h. Parallel TIS procedures using more than two dyes per cycle enhance the throughput.     

A three-symbol code for organized proteomes based on cyclical imaging of protein locations.

BACKGROUND: A major challenge in the post genomic era is to map and decipher the functional molecular networks of proteins directly in a cell or a tissue. This task requires technologies for the colocalization of random numbers of different molecular components (e.g. proteins) in one sample in one experiment. METHODS: Multi-epitope-ligand-"kartographie" (MELK) was developed as a microscopic imaging technology running cycles of iterative fluorescence tagging, imaging, and bleaching, to colocalize a large number of proteins in one sample (morphologically intact routinely fixed cells or tissue). RESULTS: In the present study, 18 different cell surface proteins were colocalized by MELK in cells and tissue sections in different compartments of the human immune system. From the resulting sets of multidimensional binary vectors the most prominent groups of protein-epitope arrangements were extracted and imaged as protein "toponome" maps providing direct insight in the higher order topological organization of immune compartments uncovering new tissue domains. The data sets suggest that protein networks, topologically organized in proteomes in situ, obey a unique protein-colocation and -anticolocation code describable by three symbols. CONCLUSION: The technology has the potential to colocalize hundreds of proteins and other molecular components in one sample and may offer many applications in biology and medicine.     

Toponomics and neurotoponomics: a new way to medical systems biology

The fluorescence robot imaging technology multi-epitope-ligand-cartography/toponome imaging system has revolutionized the field of proteomics/functional genomics, because it enables the investigator to locate and decipher functional protein networks, the toponome, consisting of hundreds of different proteins in a single cell or tissue section. The technology has been proven to solve key problems in biology and therapy research. It has uncovered a new cellular transdifferentiation mechanism of vascular cells giving rise to myogenic cells in situ and in vivo; a finding that has led to efficient cell therapy models of muscle disorders, and discovered a new target protein in sporadic amyotrophic lateral sclerosis by hierarchical protein network analysis, a finding that has been confirmed by a mouse knockout model. A lead target protein in tumor cells that controls cell polarization as a mechanism that is fundamental for migration and metastasis formation has also been uncovered, and new functional territories in the CNS defined by high-dimensional synaptic protein clusters have been unveiled. The technology can be effectively interlocked with genomics and proteomics to optimize time-to-market and the overall attrition rate of new drugs. This review outlines major proofs of principle with an emphasis on neurotoponomics.     

Toponomics and neurotoponomics: a new way to medical systems biology

The fluorescence robot imaging technology multi-epitope-ligand-cartography/toponome imaging system has revolutionized the field of proteomics/functional genomics, because it enables the investigator to locate and decipher functional protein networks, the toponome, consisting of hundreds of different proteins in a single cell or tissue section. The technology has been proven to solve key problems in biology and therapy research. It has uncovered a new cellular transdifferentiation mechanism of vascular cells giving rise to myogenic cells in situ and in vivo; a finding that has led to efficient cell therapy models of muscle disorders, and discovered a new target protein in sporadic amyotrophic lateral sclerosis by hierarchical protein network analysis, a finding that has been confirmed by a mouse knockout model. A lead target protein in tumor cells that controls cell polarization as a mechanism that is fundamental for migration and metastasis formation has also been uncovered, and new functional territories in the CNS defined by high-dimensional synaptic protein clusters have been unveiled. The technology can be effectively interlocked with genomics and proteomics to optimize time-to-market and the overall attrition rate of new drugs. This review outlines major proofs of principle with an emphasis on neurotoponomics.     

TIS11D is a Candidate Pro-Apoptotic p53 Target Gene

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes by differential display, we have identified TIS11D as a p53-inducible gene. Induction of TIS11D mRNA was confirmed by Northern Blot in response to p53 expression. Inducible expression of TIS11D resulted in inhibition of cell proliferation and apoptosis. These data suggest TIS11D as a candidate p53 target gene that may be part of the network of genes responsible for p53-dependent apoptosis.     

Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms

The immediate early gene tristetraprolin (TTP) is induced transiently in many cell types by numerous extracellular stimuli. TTP encodes a zinc finger protein that can bind and destabilize mRNAs that encode tumor necrosis factor-alpha (TNFalpha) and other cytokines. We hypothesize that TTP also has a broader role in growth factor-responsive pathways. In support of this model, we have previously determined that TTP induces apoptosis through the mitochondrial pathway, analogously to certain oncogenes and other immediate-early genes, and that TTP sensitizes cells to the pro-apoptotic signals of TNFalpha. In this study, we show that TTP and the related proteins TIS11b and TIS11d bind specifically to 14-3-3 proteins and that individual 14-3-3 isoforms preferentially bind to different phosphorylated TTP species. 14-3-3 binding does not appear to inhibit or promote induction of apoptosis by TTP but is one of multiple mechanisms that localize TTP to the cytoplasm. Our results provide the first example of 14-3-3 interacting functionally with an RNA binding protein and binding in vivo to a Type II 14-3-3 binding site. They also suggest that 14-3-3 binding is part of a complex network of stimuli and interactions that regulate TTP function.     

B cell translocation gene 2 enhances susceptibility of HeLa cells to doxorubicin-induced oxidative damage

BTG2/TIS21/PC3 (B cell translocation gene 2) has been known as a p53 target gene and functions as a tumor suppressor in carcinogenesis of thymus, prostate, kidney, and liver. Although it has been known that the expression of BTG2/TIS21/PC3 is induced during chemotherapy-mediated apoptosis in cancer cells, a role of BTG2/TIS21/PC3 in cell death remains to be elucidated. In this study, the mechanism and role of BTG2 involved in the enhancement of doxorubicin (DOXO)-induced cell death were examined. Treatment of HeLa cells with DOXO revealed apoptotic phenomena, such as chromatin condensation and cleavage of poly(ADP-ribose) polymerase and lamin A/C with concomitant increase of BTG2/TIS21/PC3 expression. Employing infections of Ad-TIS21 virus and lentivirus with short hairpin RNA to BTG2, the effect of BTG2/TIS21/PC3 on the DOXO-induced apoptosis of HeLa cells and liver cancer cells was evaluated. Not only short hairpin RNA-BTG2 but also N-acetyl-L-cysteine significantly reduced the DOXO-induced HeLa cell death and generation of H2O2. Moreover, forced expression of BTG2/TIS21/PC3 using adenoviral vector augmented DOXO-induced cancer cell death concomitantly with increase of manganese-superoxide dismutase but not catalase, CuZnSOD, and glutathione peroxidase 1. The increased apoptosis by forced expression of BTG2/TIS21/PC3 could be inhibited by N-acetyl-L-cysteine and polyethylene glycol-catalase. These results therefore suggest that BTG2/TIS21/PC3 works as an enhancer of DOXO-induced cell death via accumulation of H2O2 by up-regulating manganese-superoxide dismutase without any other antioxidant enzymes. In summary, BTG2/TIS21/PC3 enhances cancer cell death by accumulating H2O2 via imbalance of the antioxidant enzymes in response to chemotherapy.     

Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.     

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.     

Molecular properties of a galaptin and its activity in normal and leukemic erythroid tissue

Recently, the generic term “galaptins” was proposed for the group of low molecular weight, acidic, β-galactoside-specific protein lectins that have been isolated from a wide variety of animal tissues and are thought to have a role in cell-cell recognition and adhesion. A molecule of this type, called erythroid developmental agglutinin (EDA), has been isolated from rabbit bone marow where it seems to mediate the intererythroblast adhesion seen in erythroblastic islands during erythropoiesis in vivo. Here, we show that after purification, EDA shows 95%-100% Coomassie blue staining as a single component on electrophoresis in native, urea, and SDS polyacrylamide gels and electrofocuses as a single band at pH 5.6. EDA has a subunit molecular weight of 13,000 in SDS gels and, unlike the majority of other galaptins, which arc dimeric, native EDA is monomeric in solution. Another monomeric galaptin, chicken lactose lectin II, has been described recently, and it therefore seems that there may be two classes of galaptin distinguishable by their aggregation state in solution. We have previously reported that EDA agglutinates rabbit erythroblasts in vitro and that this reaction is inhibited by β-galactoside-containing sugars and by anti-EDA Fab fragments suggesting that EDA bridges directly between cell surface glycoproteins. The insensitivity of this reaction to cooling, or to the disruption of cellular metabolism or the cytoskeleton demonstrated here further supports this hypothesis. EDA-mediated erythroblast agglutination was also shown to be independent of divalent cations. Since galaptins are thought to be important in cohesion between normal cells, the possibility that EDA is not active in leukemic erythroid tissue was examined. The murine erythroleukemia cell line (MELC) provided an excellent system for this study since MELC are thought to be derived from an erythroid committed cell transformed at an early stage of development and can be induced by a number of chemical agents to differentiate terminally along the erythroid developmental pathway in culture. EDA of rabbit origin was found to agglutinate mouse erythroblasts in vitro and was used to investigate the response of MELC to EDA. It was found that the transformed cells were not readily agglutinated by EDA but on induction, and the concomitant loss of many of their transformed characteristics, MELC gained aggregation competence for EDA. The possible causes of these differences are discussed.     

Synthesis,degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs. PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, “pulse-chase” experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is' a non-nuclear, soluble intracellular protein. © 1994 Wiley-Liss, Inc.     

Syncytial apoptosis signaling network induced by the HIV-1 envelope glycoprotein complex: an overview

Infection by human immunodeficiency virus-1 (HIV-1) is associated with a progressive decrease in CD4 T-cell numbers and the consequent collapse of host immune defenses. The major pathogenic mechanism of AIDS is the massive apoptotic destruction of the immunocompetent cells, including uninfected cells. The latter process, also known as by-stander killing, operates by various mechanisms one of which involves the formation of syncytia which undergo cell death by following a complex pathway. We present here a detailed and curated map of the syncytial apoptosis signaling network, aimed at simplifying the whole mechanism that we have characterized at the molecular level in the last 15 years. The map was created using Systems Biology Graphical Notation language with the help of CellDesigner software and encompasses 36 components (proteins/genes) and 54 interactions. The simplification of this complex network paves the way for the development of novel therapeutic strategies to eradicate HIV-1 infection. Agents that induce the selective death of HIV-1-elicited syncytia might lead to the elimination of viral reservoirs and hence constitute an important complement to current antiretroviral therapies.     

Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.     

Changes in p34cdc2 kinase activity and cyclin A during induced differentiation of murine erythroleukemia cells.

Hexamethylene bisacetamide (HMBA)-induced murine erythroleukemia (MELC) differentiation is characterized by a prolongation of the initial G1 which follows passage through S phase in the presence of inducer. Commitment to terminal cell division is first detected in a portion of the cell population during this prolonged G1. HMBA-induced commitment is stochastic. This study has examined changes in two known cell cycle regulators, p34cdc2 and cyclin A, in cycle-synchronized MELC in the absence and presence of HMBA. Histone H1 kinase activity of p34cdc2, and the levels of CDC2Mm mRNA, 1.8-kilobase mRNA of cyclin A, and cyclin A protein changed during cell cycle progression in MELC, and all of them were suppressed during G1. The suppression of the H1 kinase activity and cyclin A expression continued through the prolonged G1 in MELC cultured with HMBA, whereas p34cdc2 protein level did not vary through the cell cycle in MELC cultured without or with inducer. Phosphorylation of p34cdc2 in uninduced MELC gradually increased as cells progressed from G1 to S. In induced MELC, an increase in phosphorylation of p34cdc2 occurred during the prolonged G1, and prior to the exit of the bulk of the cells from G1 to S. These results suggest that in HMBA-induced MELC, p34cdc2 phosphorylation per se is not a limiting factor in determining G1 to S progression. The persistent suppression of cyclin A expression and histone H1 kinase activity may play a role in HMBA-induced commitment to terminal differentiation.     

HBx参与肝细胞癌发生发展调控的分子机制

肝细胞癌 (hepatocellular carcinoma, HCC)是我国最常见的恶性肿瘤之一,而HBV慢性感染是肝癌发生的主要原因.乙型肝炎病毒(HBV)中X基因编码的一种多功能蛋白(HBx),参与众多重要生物学过程的调控,并促进肝细胞癌的发生. 早期研究表明,HBx在HCC发生过程中发挥重要的调控功能,但其确切分子机制尚未完全明确. 近几年,HBx参与生物学过程的分子机制研究有了较快的进展. 有趣的是,研究发现,HBx在不同的细胞系以及HBV感染的不同阶段发挥促抑凋亡的双重作用,HBx还参与细胞自噬的调控. 此外,在HBx参与细胞增殖及肿瘤侵袭和转移等方面,也产生了一些新的认识. 本文将从HBx对肝细胞凋亡、自噬和增殖的调控及其对肝癌细胞转移和侵袭的调控等方面,对HBx参与肝细胞癌发生发展调控机制做一综述.