Promoter DNA Methylation of Oncostatin M receptor-β as a Novel Diagnostic and Therapeutic Marker in Colon Cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.     

Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state.Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.     

Exploiting the Natural Diversity of Microviridin Gene Clusters for Discovery of Novel Tricyclic Depsipeptides

Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.Bloom-forming freshwater cyanobacteria are a rich source of natural products (30). They flourish in lakes and ponds of different temperate zones, where they can eventually form thick scums at the surface. Microcystis is one of the predominant genera composing these blooms, particularly in lakes suffering from eutrophication (17). Like other bloom-forming species, it is infamous for the production of the hepatotoxin microcystin, a nonribosomal peptide toxin that inhibits protein phosphatases in a broad range of eukaryotes from zooplankton to humans (1, 9). Moreover, Microcystis is known to produce a multitude of other peptides that are considered to be potential drug leads (14, 30).Microviridins form one of the most intriguing classes of peptides, since they feature a cage-like structure (e.g., microviridin B) (Fig. ​(Fig.1A).1A). The highly unusual tricyclic architecture results from ω-ester and ω-amide bonds between amino acid side chains. The past few years have afforded 13 variants of the peptide from both field samples and laboratory strains (5, 8, 13, 15, 21, 22). Most of the variants show inhibitory activities against serine-type proteases, most notably against elastase, which is a target enzyme in the treatment of lung emphysema (28). One of the peptide isoforms, microviridin J, has been shown to inhibit the molting process of Daphnia, leading to death of the animals (23).Open in a separate windowFIG. 1.(A) Representative structure of microviridin B and three-dimensional model. (B) Schematic representation of the microviridin gene cluster of M. aeruginosa NIES298. The positions of primers for the amplification of mdnA, -B, and -C are indicated. GNAT, GCN5-related N-acetyltransferase. (C) PCR gel picture showing the amplification of mdnB from a selection of laboratory strains.Recent studies have revealed a unique biosynthetic mechanism for microviridins in Microcystis and the filamentous cyanobacterial genus Planktothrix (18, 31). The 14-amino-acid (aa) peptide sequence is encoded at the C terminus of the ribosomal precursor peptide MdnA (31). Macrocyclization of the peptide depends on the activities of two ATP grasp-type ligases, MdnB and -C, that are encoded downstream of the precursor gene. Further enzymes encoded by the cluster include the N-acetyltransferase MdnD and the putative transporter-peptidase MdnE (31) (Fig. ​(Fig.1B).1B). The activities of the enzymes MdnB, -C, and -D were confirmed by heterologous production of microviridins in Escherichia coli (31). The MdnB and -C orthologs of the filamentous cyanobacterial strain Planktothrix agardhii CYA126, MvdC and MvdD, were characterized using synthetic precursor peptides in vitro (18). Furthermore, Philmus et al. have shown limited flexibility of MvdD, catalyzing two rounds of microviridin lactonization to accept substrates with altered amino acid compositions and a stringent ring size requirement (19).Recent genome-sequencing projects with cyanobacteria have revealed widespread occurrence of microviridin-related gene clusters in various cyanobacterial genera, although the production of microviridins by these strains remains to be shown (18, 24, 31). Here, we present data showing an unprecedented natural diversity of microviridin precursor genes in a set of closely related Microcystis laboratory strains and field samples. The insights gained from our survey provide lessons about the flexibility of the microviridin ligases in a small group of cyanobacteria and an estimate of the size of the natural microviridin library that still awaits discovery. The detection of novel natural microviridin prepeptides from Microcystis can ultimately guide the heterologous production of novel microviridins in E. coli. As proof of principle, we have identified and characterized the new variant microviridin L from the strain Microcystis aeruginosa NIES843.     

Neurofilament Heavy Polypeptide Regulates the Akt-β-Catenin Pathway in Human Esophageal Squamous Cell Carcinoma

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.     

Effects of platelet-activating factor and thromboxane A2 on isolated perfused guinea pig liver

Lipid mediators, thromboxane A2 (TxA2) and platelet-activating factor (PAF), are potent vasoconstrictors, and have been implicated as mediators of liver diseases, such as ischemic-reperfusion injury. We determined the effects of a TxA2 analogue (U-46619) and PAF on the vascular resistance distribution and liver weight (wt) in isolated guinea pig livers perfused with blood via the portal vein. The sinusoidal pressure was measured by the double occlusion pressure (P(do)), and was used to determine the pre- (R(pre)) and post-sinusoidal (R(post)) resistances. U-46619 and PAF concentration-dependently increased the hepatic total vascular resistance (R(t)). The minimum concentration at which significant vasoconstriction occurs was 0.001 microM for PAF and 0.1 microM for U-46619. Moreover, the concentration of U-46619 required to increase R(t) to the same magnitude is 100 times higher than PAF. Thus, the responsiveness to PAF was greater than that to U-46619. Both agents increased predominantly R(pre) over R(post). U-46619 caused a sustained liver weight loss. In contrast, PAF also caused liver weight loss at lower concentrations, but it produced liver weight gain at higher concentrations (2.5 +/- 0.3 per 10g liver weight at 1 microM PAF), which was caused by substantial post-sinusoidal constriction and increased P(do). In conclusion, both TxA2 and PAF contract predominantly the pre-sinusoidal veins. TxA2 causes liver weight loss, while PAF at high concentrations increases liver weight due to substantial post-sinusoidal constriction in isolated guinea pig livers.     

Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants

Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

Contribution of the T cell receptor BJ gene to recognition of the P91A tumor antigen in DBA/2 mice

 To understand specific immune responses against a tumor, it is important to characterize T cells that recognize the tumor antigen. The mouse P91A antigen is one of the well-defined tumor antigens that is expressed on the P911 cell line, and T cells responding to the antigen in DBA/2 mice were reported to be restricted to BV8S2/S3 families in their T cell receptor (TCR) BV gene usage. We have further characterized the P91A-responding T cells in DBA/2 mice, focusing on TCR BJ gene usage and using the polymerase chain reaction/enzyme-linked immunosorbent assay and DNA sequencing studies of their third complementarity-determining (CDR3) regions. As a result, T cells with cytotoxic activity to the P91A antigen, induced from murine spleen cells both in vivo and in vitro, showed predominant use of the BJ2S1 gene segment in both BV8S2 and BV8S3 T cells compared to unmanipulated murine spleen cells. Sequencing studies of the CDR3 regions in the BV8S3 T cells revealed clonal expansion of T cells with the BV8S3-BJ2S1 combination in two of three DBA/2 mice tested. In the remaining mouse, clonal expansion was not detected despite predominant use of the BJ2S1 segment by these T cells. These data suggest that P91A-recognizing T cells would predominantly use the BV8S2/S3-BJ2S1 combination. Analysis of T cells with these TCR BV-BJ gene combinations may contribute to the evaluation, monitoring and development of a T-cell-mediated immunotherapeutic strategy. Received: 3 July 1997 / Accepted: 17 November 1997