Abstract Loss of SNF5, a core subunit of the SWI/SNF ATP-dependent chromatin-remodeling complex, has been linked to development of malignant rhabdoid tumors (MRT). MRTs, a deadly form of pediatric cancer, can occur in almost any soft tissue of the body, including the brain. We have previously shown that introduction of SNF5, a known tumor suppressor gene, into deficient cell lines results in a G1 growth arrest through p21(CIP1/WAF1) mRNA induction. However, additional gene expression changes that may contribute to MRT development or may be involved in SNF5 cell cycle arrest remain uncharacterized. To discover novel binding targets of SNF5, we used a Biosciences Human Cell Cycle RT2Profiler PCR expression array to identify genes whose expression significantly changed upon reexpression of SNF5-GFP in the MRT cell line A204.1. The genes identified include p16(INK4A), p21(CIP1/WAF1), CCNG2, CCNH, CDK8, and HERC5. p16(INK4A) and p21(CIP1/WAF1) have been previously validated as direct binding targets of SNF5. Cyclin G2 (CCNG2) is a noncanonical cyclin that appears to negatively control cell cycle progression. HERC5 is involved in interferon signaling and ISGylation of proteins. CDK8 is a member of the mediator complex that plays a role in transcription. Cyclin H (CCNH) has been linked to development of some neural cancers. We developed an adenoviral vector system containing a novel HA tagged SNF5 construct. We used RT-PCR to validate the gene expression changes seen in the array in A204.1 and other MRT cell lines using this novel construct. An empty adenoviral vector was used as a control. While all genes showed increases in A204.1 after SNF5 reexpression, CCNG2 and CCNH mRNA levels increased in only a few other MRT cell lines. CDK8 expression levels were unchanged or decreased in all other MRT cell lines examined. HERC5 mRNA expression was greatly increased in all MRT cell lines at significantly higher levels than any of the other genes identified in this study. Chromatin immunoprecipitation (ChIP) analysis of the HERC5 and CCNG2 promoter regions in A204.1 confirmed direct SNF5 binding. Maximal enrichment was observed upstream of the transcriptional start site of both promoters. RNA Polymerase II also increased across the promoter regions in a pattern similar to SNF5 binding. Western blotting was also performed, confirming the upregulation of HERC5. CCNG2 protein levels did not follow mRNA expression, possibly because of tight regulation due to involvement in the cell cycle. Overall, we have identified the HERC5 and CCNG2 promoters as direct binding targets of SNF5 that may play roles in the development of this cancer. Interestingly, previous studies have shown that interferon signaling is activated upon SNF5 introduction into MRTs, indicating the potential biological relevance of HERC5. Future studies are needed to fully characterize their relationship to MRT oncogenesis and the mechanism of SWI/SNF regulation at their proximal promoter region. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2011-4028

In earlier work application of carbon column chromatography to the products resulting from hydrolysis of umbilical cord hyaluronic acid by testicular hyaluronidase yielded fractions differing in average molecular weight (2). Monosaccharides were not detected in any of the digests, equivalence of hexosamine and uranic acid content was demonstrated in all fractions, and the constancy of certain end-group values was established. It was concluded that purified preparations of the enzyme hydrolyzed the polymer to oligosaccharides, in agreement with earlier workers, and that only the glucosaminidic linkages were affected, the uronidic linkages remaining intact. The present report is concerned with the application of ion exchange and paper chromatography to the isolation of the individual oligosaccharides, and with the determination of their molecular sizes, as well as with the development of analytical methods suitable for their estimation in mixtures. The oligosaccharides isolated are designated oligosaccharides I to VII, in order of increasing size.

Activity of the interferon-induced enzyme 2'-5' oligoadenylate synthetase (2-5 OAS) was measured in peripheral blood mononuclear cells (PBMCs) and serum of patients with chronic phase Ph'-positive chronic myelogenous leukemia (CML) treated with interferon-alpha (IFN-alpha) (4 x 10(6) IU/m2) alone or in combination with 50 micrograms IFN-gamma. At the beginning of IFN therapy, marked elevation of 2-5 OAS titers was detected in PBMCs (pretreatment 0.03-1.62, median 0.2; during treatment 0.8-13.14, median 4.31; 22 patients studied) and in serum (pretreatment 21-156 pmol/dl, median 62; during treatment 532-1740 pmol/dl, median 800; eight patients studied). However, 2-5 OAS titers were not related to clinical outcome or IFN therapy and also during IFN resistance elevated 2-5 OAS activity in PBMCs (median 3.45; range 1.05-13.14; 11 patients studied) were detected. These data argue against direct involvement of the 2-5 OAS system in the therapeutic effect of IFN in CML. However, 2-5 OAS titers in PBMCs or serum appear to be a reliable control of biologically active IFN therapy.

Heparin trisaccharides having the sequence O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-alpha-L- idopyranosyluronic acid-(1----4)-2,5-anhydro-D-[1-3H]mannitol have been prepared, as substrate models for studying sulfatases of heparan sulfate catabolism, by alpha-L-iduronidase cleavage of previously reported heparin tetrasaccharides, with additional chemical and enzymic modification as required. Three series are described, including isomeric sulfate esters of that trisaccharide with no N-substituent, with N-acetyl substitution, and with N-sulfate substitution. New features of the substrate specificity of the hydrolases used, including iduronate sulfatase, alpha-L-iduronidase, glucosamine 6-sulfate sulfatase, and heparin sulfamidase, were observed, and simple procedures for partial purification of these hydrolases are reported. The structures assigned to the trisaccharides are supported by the mode of preparation, reactions, regularities in electrophoretic behavior, and identities of the products of deamination.

A study is reported of the reactivities of the disaccharides isolated after deamination of beef-lung heparin and reduction of the products by sodium borotritide: 2,5-anhydro- O -(α- l -idopyranosyluronic acid sulfate)- d -mannitol sulfate, SIMS; 2,5-anhydro- O -(α- l -idopyranosyluronic acid)- d -mannitol sulfate, IMS; 2,5-anhydro- O -(α- l -idopyranosyluronic acid sulfate)- d -mannitol, SIM; and 2,5-anhydro- O -(β- d -glucopyranosyluronic acid)- d -mannitol sulfate, GMS. Results for the non-sulfated disaccharides IM and GM, prepared by desulfation of SIMS and GMS, are also reported. SIMS and SIM were inert to purified α- l -iduronidase, showed unexpected resistance to periodate oxidation, and lost sulfate rapidly in 50m m hydrochloric acid at 100°. Hydrolysis of IM and of IMS was catalyzed by α- l -iduronidase, and of GM and GMS by β- d -glucuronidase; the radioactive products were identified as 2,5-anhydro- d -mannitol (aM) and its sulfate (aMS). The products SIM and IMS obtained by deamination of heparin and desulfation of SIMS (the major deamination product) are apparently identical. In heparin partially desulfated by methanolic hydrogen chloride, residual sulfate groups were mostly linked to l -iduronic acid residues. Chemical, chromatographic, and electrophoretic methods that are valuable for separation and characterization of the disaccharides are described.

Partial N -desulfation of beef-lung heparin prior to degradative deamination with butyl nitrite and reduction with sodium borotritide yielded many large fragments. From these, a tetrasaccharide tetra- O -sulfate (II-4NH; 8% yield from heparin) and a mixture of tetrasaccharide tri- O -sulfates (II-3NHh; 6% yield) were isolated by sequential chromatography on Sephadex G-25 and DEAE-Sephadex. For these and the other tetrasaccharide preparations, the radioactive disaccharides produced by deamination, with and without subsequent relabelling with sodium borotritide, have been quantitatively determined by the methodology described in the preceding paper. In most cases, the results permit a unique reconstruction of the relative proportions of monosaccharide components and of their sequences in the compounds present. Tetrasaccharide II-4NH appeared homogeneous and has the structure (IdoA-SO 4 )(GN-O-SO 4 )(IdoA-SO 4 )(anhMan-SO 4 ). In tetrasaccharide preparation II-3NHh, the preponderant species (57%) lacks ester sulfate at the terminal l -iduronic residue in the structure just mentioned, and five other species are present. By treatment of the tetra- O -sulfate with mild acid, tetrasaccharide preparations with 3, 2, 1, and no ester sulfate were produced and could be isolated. The isomeric tetrasaccharide tri- O -sulfate species have been partially resolved. Composition and sequence data are given for all of the preparations. The resolution of numerous small fractions suggests minor irregularities in the fine structure of heparin. Ion-exchange electrophoresis was applied to the acidic oligosaccharides and was found to be a useful technique.

Condensation of dimeric 3,4,6-tri-O-acetyl-2-deoxy-2-nitroso-α-d-glucopyranosyl chloride with 4-methylumbelliferone gave crystalline 4-methylumbelliferyl 3,4,6-tri-O-acetyl-2-deoxy-2-oximino-α-d-arabino-hexopyranoside. Acetylation of this adduct, reduction of the resulting crude O-acetyloxime with borane in oxolane, and acetylation gave the 3,4,6-tri-O-acetyl derivative of 4-methylumbelliferyl 2-acetamido-2-deoxy-α-d-glucopyranoside (1). A new sensitive assay of N-acetyl-α-d-glucos-aminidase (EC 3.2.1.50) is made possible by fluorometric measurement of 4-methyl-umbelliferone liberated by enzymic hydrolysis of glycoside 1. Such assays are illustrated by results obtained with enzyme preparations from pig liver and human-blood serum.

A trisaccharide of sequence: (glucosamine O,N-disulfate)-(iduronic acid O-sulfate)-(3H-anhydromannitol O-sulfate) was prepared from degradation products of heparin and was used as a substrate to demonstrate, in rat and bovine tissues, a novel O,N-disulfoglucosamine O-sulfatase. The enzyme, purified 720-fold from extracts of beef kidney, has optimal activity at pH 4.1. It is distinct from arylsulfatases A or B, N-acetylglucosamine 6-sulfate sulfatase, and urinary 3,N-disulfoglucosamine 3-O-sulfatase. Data are given on the substrate specificities of the presently described O-sulfatase, of heparin sulfamidase, and of α-L-iduronidase.

Receptor-binding of "high-uptake" forms of lysosomal enzymes to human diploid skin fibroblasts had been predicted from the Michaelis--Menten kinetics of uptake of these enzymes [e.g., Sando, G.N. & Neufeld, E.F. (1977) Cell 12, 619--627]. We have now demonstrated such binding directly by using a sensitive assay for the bound enzyme. Cells deficient in alpha-L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to recover, and used in suspension. They were incubated with urinary alpha-L-iduronidase at 0 degrees C for 90 minutes and then washed by centrifugation through concentrated bovine serum albumin; the activity of the cell-associated enzyme was measured with 4-methylumbelliferyl alpha-L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kd of 1 x 10(-9) M for high-uptake alpha-L-iduronidase; binding of the low-uptake form was barely detectable. Mannose 6-phosphate, a known competitive inhibitor of uptake, inhibited the binding competitively, with Ki = 1 x 10(-4) M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of alpha-L-iduronidase at 35 degrees C, the receptors were regenerated every few minutes, even in the absence of protein synthesis.

This chapter explains the characterization of reference disaccharides from nitrous acid deamination of beef lung heparin. In a study described in the chapter, uronosyl (IdoA or GlcA) anhydromannitol (AM) disaccharides were prepared as markers and as model compounds for structural studies of heparin and, particularly, of its oligosaccharides, which were required as enzyme substrates. The chapter explains the sensitivity of disaccharide to sodium metaperiodate (8mM) and HCl (0.05M). It also explains the hydrolysis of disaccharides with β-D-glucuronidase and α-L-iduronidase, which showed that disaccharides not sulfated at the uronosyl residue were appropriately cleaved by the same. The chapter also illustrates the anion exchange electrophoresis patterns at pH 6 of crude tetrasaccharide mixture from lung heparin, tetrasaccharide A, tetrasaccharide B, and a digest of tetrasaccharide B with rat spleen homogenate.

The earlier preparation of cyclohexylammonium (phenyl α-l-idopyranosid)-uronate has been improved, and (4-methylumbelliferyl α-l-idopyranosid)uronic acid (14), a more sensitive substrate for α-l-iduronidase, has been synthesized by an analogous route. Zinc chloride-catalyzed condensation of 4-methylumbelliferone with 1,2,3,4,6-penta-O-acetyl-α-l-idopyranose (4) in 1,2-ethanediol diacetate gave crystalline 4-methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α-l-idopyranoside (7). O-Deacetylation and catalytic oxidation gave 14, characterized as a cyclohexylammonium salt. The starting material 4 was prepared, in 21 % yield from l-glucose, by conversion of the intermediate 1,2,3,4,6-penta-O-acetyl-β-l-glucopyranose to 2,3,4,6-tetra-O-acetyl-β-l-glucopyranosyl chloride and acetoxonium ion rearrangement, as described for the D-series.

Conditions studied earlier by Tracey [(1948) Biochem. J.43, 185] are used for acid decarboxylation in sealed tubes of uronide samples supplemented with 6-14C-labeled uronic acid. The specific activity of the CO2 evolved is measured as the ratio of radioactivity to area of the CO2 peak obtained in a gas chromatogram. By appropriate standardization, samples containing some 60 nmol of uronic acid can be analyzed with reproducibility and apparent accuracy of about ±2% (mean deviation). The techniques developed for uronic acid analysis should apply with minor modification to any problem requiring accurate measurement of CO2 in small amounts.

Use of phenyl α-L-iduronide as a test substrate now makes it possible to show occurrence in rat liver lysosomes of an α-L-iduronidase. The enzyme can be shown to be distinct from the well studied β-glucuronidase. With the phenyl glycoside, measurements can be made of this relatively weak mammalian activity, whose occurrence could previously be inferred only indirectly from the slow degradation of dermatan sulfate derivatives by tissue extracts. Trials with appropriate aryl glycosides indicate absence of detectible α-D-glucuronidase, α-D-mannuronidase, α-D-galacturonidase, or β-L-iduronidase activities from lysosomal extracts.