Interaction with AEG-1 and MDM2 is associated with glioma development and progression and correlates with poor prognosis

Glioma is the most common central nervous system tumor with poor prognosis. The AEG-1 (Astrocyte Elevated Gene 1) gene displays oncogenic characteristics, including proliferation, metastasis, chemoresistance, invasion, and evasion of apoptosis, and is strongly linked to the occurrence of glioma. Here, we elucidated the potential contribution of AEG-1 in human glioma pathogenesis. In glioma cells, AEG-1 could directly interact with Murine Double Minute-2 (MDM2) protein resulting in MDM2-p53-mediated cell proliferation and apoptosis. MDM2 is being revealed as an oncoprotein, which is involved in many human cancers progression. By immunohistochemical and a multivariate analysis, expressions of AEG-1 and MDM2 were elevated in glioma and high AEG-1 and MDM2 expressions were showed to be correlated with poor prognosis. AEG-1-MDM2 interaction prolonged stabilization of MDM2 where AEG-1 inhibited ubiquitination and subsequent proteasome-mediated degradation of MDM2 protein. Moreover, slicing AEG-1 blocked MDM2 expression and then impacted MDM2-p53 pathway that influenced cell proliferation and apoptosis. These findings uncover a novel AEG-1-MDM2 interplay by which AEG-1 augments glioma progression and reveal a viable potential therapy for the treatment of glioma patients.     

Crystal and molecular structures of cyclomaltoheptaose inclusion complexes with HI and with methanol

β-Cyclodextrin (β-CD; cyclomaltoheptaose; cyclohepta-amylose; C₄₂H₇₀O₃₅) crystallises from aqueous solutions of HI and of MeOH in the form of stout prisms, which are isomorphous to each other with monoclinic space-group P2₁; cell constants for C₄₂H₇₀O₃₅ · 2HI · 8 H₂O: a = 21.25(3), b = 10.28(2), c = 15.30(2) Å, β = 113.25(9)°, and Z = 2; and for C₄₂H₇₀O₃₅ · MeOH · 6.5 H₂O: a = 21.03(3), b = 10.11(2), c = 15.33(2) Å, β = 111.02(9)°, and Z = 2. X-ray counter data were used to determine the structures of both crystals, which belong to the cage type, with β-CD molecules in nearly identical, “round” shapes. In the HI complex, one I^- is located inside, and one outside, the β-CD cavity; in the MeOH complex, the MeOH is within the cavity. The cavity is closed at the O-2,O-3 side by adjacent β-CD molecules, and at the O-6 side by water molecules hydrogen-bonded to the guest and to surrounding β-CD molecules. Interstices between β-CD molecules are filled by water of hydration molecules in distorted co-ordination.     

Zinc complexes with tricarballylic acid and Lewis bases with zero- and two-dimensional structures

Three ternary zinc complexes of the open chain polycarboxylic acid, tricarballylic (1,2,3-propane-tricarboxylic) acid (PTCH₃) have been isolated and characterized with crystallographic and physicochemical techniques. [Zn(PTCH)(phen)(H₂O)]₂ · 4H₂O (1) (where phen = 1,10-phenanthroline) has a unique dinuclear structure, while [Zn(PTCH)(bpy)]_n · 3nH₂O (2) and [Zn(PTCH)(epy)]_n · 4nH₂O (3) (where bpy = 4,4′-bipyridine and epy = 1,2-bis(4-pyridine)ethane) have 2D polymeric structures. The bis-deprotonated ligand, in all three complexes, uses for coordination only two oxygen atoms, which belong to the same carboxylate in 1, and to two different carboxylates in 2 and 3.     

Manganese-porphyrin reactions with lipids and lipoproteins

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.     

In vitro reactions of isopropyl methanesulfonate with DNA and with 2'-deoxyribonucleosides

Isopropyl methanesulfonate (IPMS), an SN1 alkylating agent, is a direct-acting mutagen in bacteria. We recently reported that s.c. and topical administration of IPMS to mice resulted in the rapid induction of thymic lymphomas. Thymic lymphoma induction was not observed following administration of the SN2 alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). We have studied the reactions of IPMS with dAdo, dCyd, dGuo and dThd at pH 6.5 to 7.5 and 37 degrees C for 3 h. IPMS formed the following isopropyl (IP) adducts: 7-IP-Gua (4% yield), O6-IP-Gua (8%), O2-IP-Cyt (1%), O2-IP-dThd (2%), 3-IP-dThd (1%), and O4-IP-dThd (0.4%). Adducts were characterized from UV and mass spectra. IPMS was reacted in vitro with calf thymus DNA (pH 6.5 to 7.5, 37 degrees C, 3 h) and yielded (nmol/mg DNA): 7-IP-Gua (22) O6-IP-dGuo (11), O2-IP-Cyt (9), O2-IP-dThd (2), O4-IP-dThd (2), 3-IP-Ade (0.2) and 3-IP-dThd (0.2). The relatively greater alkylation of exocyclic oxygen atoms in DNA by IPMS compared to values for MMS and EMS reported by others, may play a role in the induction of thymic lymphomas in mice by IPMS and the lack of such activity by MMS and EMS.     

Titanium and vanadium complexes with tetraphenylimidodiphosphinate

Treatment of [Ti(OPrⁱ)₂Cl₂] with K(tpip) (tpip⁻ = [N(PPh₂O)₂]⁻) followed by chlorination with HCl afforded cis-[Ti(tpip)₂Cl]₂ (1). Reduction of 1 with Na/Hg in THF gave [Ti(tpip)₃] (2), which could also be prepared from [TiCl₃(THF)₃] and K(tpip). Recrystallization of [V(O)(tpip)₂] (3) from CH₂Cl₂-Et₂O in air afforded trinuclear [{V(O)}₃(μ-tpip)₃(μ-O)₃] (4). Treatment of [Cr(NBu^t)₂Cl₂] and [Cr(NBu^t)Cl₃(dme)] (dme = 1,2-dimethoxyethane) with [Ag(tpip)]₄ led to isolation of [Cr(tpip)₃] (6) and [Cr(NBu^t)(tpip)₂Cl] (7), respectively. The Ti- and V-tpip complexes are capable of catalyzing oxidation of sulfides with tert-butyl hydroperoxide and H₂O₂. The crystal structures of 1, 2, and 4 have been determined.     

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.     

Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2

Disabled-2 (Dab2) is a widely expressed relative of Disabled-1, a neuron-specific signal-transduction protein that binds to and receives signals from members of the low-density lipoprotein receptor (LDLR) family. Members of the LDLR family internalize through clathrin-coated pits and vesicles to endosomes, from where they return to the cell surface through the secretory pathway. In this study, we show that the Dab2 phosphotyrosine-binding domain binds peptides containing the sequence FXNPXY. This core sequence is found in the intracellular domains of LDLR family members and is important for receptor internalization. Dab2 transiently colocalizes with the LDLR in clathrin-coated pits, but is absent from endosomes and lysosomes. Dab2 is alternatively spliced and its localization depends on a region of the protein that contains two DPF motifs that are present in the p96 Dab2 protein and absent in the p67 splice variant. This region is sufficient to confer Dab2 binding to the α-adaptin subunit of the clathrin adaptor protein, AP-2. Overexpression of p96 but not of p67 Dab2 disrupts the localization of AP-2. These findings suggest that in addition to previously reported signal-transduction functions, Dab2 could also act as an adaptor protein that may regulate protein trafficking.     

Immunotherapy with intralesional and systemic interleukin-2 of patients with non-small-cell lung cancer

Eight patients affected by non-small-cell lung cancer were treated with intralesional and systemic recombinant IL-2(rIL-2) injection with the aim of activating both tumour-infiltrating lymphocytes and circulating cytotoxic or killer cells. The schedule of treatment was as follows: a daily fine-needle transparietal intralesional rIL-2 injection (1×10⁵ Cetus units) from day 1 to day 5 and systemic rIL-2 infusion (1×10⁵ Cetus units kg^–1 day^–1) from day 6 to day 10. One to four cycles of treatment were received by each patient. Clinical and immunological evaluations were performed (a) before treatment, (b) following the intralesional rIL-2 administration, (c) 1 h after the beginning of rIL-2 infusion and (d) at the end of the systemic rIL-2 infusion. No complete remission was achieved, two patients showed a partial remission, three resulted in stable disease and three patients progressed. Natural killer and lymphokine-activated killer cell activity dramatically decreased 1 h after the beginning of rIL-2 infusion and increased at the end of treatment. A progressive increase of circulating CD8⁺ and HLA class II⁺ T cells as well as of CD8⁺ T cell clones, most of which displayed NK activity, was recorded following rIL-2 infusion. Present data indicate that (a) the local administration of rIL-2 coupled with systemic rIL-2 infusion may be suggested as an alternative approach for the immunotherapy of lung cancer, (b) rIL-2 induces different immunological modifications according to the route and the time of its administration and (c) rIL-2 administration increases the amount of circulating immune cells with potential antitumour activity.     

17 Beta-estradiol interaction with haptoglobin and its complex with hemoglobin

The estradiol-binding capacity of sheep haptoglobin and its complex with hemoglobin was investigated. It was shown that in the presence of H2O2 the haptoglobin-hemoglobin complex tightly binds 17 beta-estradiol. No dissociation of the estradiol-protein complex occurs after its precipitation with trichloroacetic acid and steroid extraction with the ester. It is supposed that the 17 beta-estradiol is bound to the protein by covalent bonds; this binding is characteristic only of the haptoglobin-hemoglobin complex but not of haptoglobin alone.     

Combined radioimmunotherapy and chemotherapy of breast tumors with Y-90-labeled anti-Her2 and anti-CEA antibodies with taxol

Because breast cancer cells often express either Her2/neu or carcinoembryonic antigen (CEA) or both, these tumor markers are good targets for radioimmunotherapy using Y-90-labeled antibodies. We performed studies on nude mice bearing xenografts from MCF7, a cell line that has low Her2 and CEA expression, to more accurately reflect the more usual situation in breast cancer. Although uptake of In-111 anti-CEA into tumors was lower than that for In-111-labeled anti-Her2, radioimmunotherapy (RIT) with Y-90 anti-CEA was equivalent to that of Y-90 anti-Her2. When either Y-90 antibody was combined with a split-dose treatment with Taxol, the antitumor effect was greater than with either agent alone. When Y-90 anti-CEA was combined with a single dose of Taxol, the results were equivalent to the split-dose regimen. RIT plus cold Herceptin had no additional effects on tumor size reduction over RIT alone. When animals were first treated with Y-90 anti-Her2 and imaged 1-2 weeks later with In-111 anti-CEA or anti-Her2, tumor uptake was higher for anti-CEA and improved over tumor uptake with no prior RIT. These results suggest that a split dose of RIT with anti-Her2 antibody followed by anti-CEA antibody would be more effective than a single dose of either. This prediction was partially confirmed in a controlled study comparing single- vs split-dose anti-Her2 RIT followed by either anti-Her2 or anti-CEA RIT. These studies suggest that combined RIT and Taxol therapy are suitable in breast cancers expressing either low amounts of Her2 or CEA, thus expanding the number of eligible patients for combined therapies. They further suggest that split-dose RIT using different combinations of Y-90-labeled antibodies is effective in antitumor therapy.     

Heparanase 2 Interacts with Heparan Sulfate with High Affinity and Inhibits Heparanase Activity

Heparanase activity is highly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis. Heparanase homolog, termed heparanase 2 (Hpa2), was cloned based on sequence homology. Detailed characterization of Hpa2 at the biochemical, cellular, and clinical levels has not been so far reported, and its role in normal physiology and pathological disorders is obscure. We provide evidence that unlike heparanase, Hpa2 is not subjected to proteolytic processing and exhibits no enzymatic activity typical of heparanase. Notably, the full-length Hpa2c protein inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and heparan sulfate and its ability to associate physically with heparanase. Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence (follow-up to failure; p = 0.006) and inversely correlating with tumor cell dissemination to regional lymph nodes (N-stage; p = 0.03). Hpa2 appears to restrain tumor metastasis, likely by attenuating heparanase enzymatic activity, conferring a favorable outcome of head and neck cancer patients.     

Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.     

Preparation and properties of prostaglandin D2 labelled with tritium with high molar radioactivity

Kinetic parameters of enzymatic and non-enzymatic transformations of [3H]prostaglandin H2 (PGH2) were determined; the maximum yield of [3H]PGD2 being obtained at the keobs/koobs ratio equal to 10. The two-stage enzymatic synthesis of [3H]PGD2 with high molar radioactivity (3.15 TBq/mmol) from [3H]arachidonic acid carried out. Its identity in properties to the natural PGD2 was shown in experiments on the inhibition of ADP-induced aggregation of thrombocytes and on enzymatic oxidation with 15-hydroxyprostaglandin dehydrogenase.     

Cadherin conformations associated with dimerization and adhesion

To investigate conformations of C-cadherin associated with functional activity and physiological regulation, we generated monoclonal antibodies (mAbs) that bind differentially to monomeric or dimeric forms. These mAbs recognize conformational epitopes at multiple sites along the C-cadherin ectodomain aside from the well known Trp-2-mediated dimer interface in the N-terminal EC1 domain. Group 1 mAbs, which bind monomer better than dimer and the Trp-2-mutated protein (W2A) better than wild type, recognize epitopes in EC4 or EC5. Dimerization of the W2A mutant protein via a C-terminal immunoglobulin Fc domain restored the dimeric mAb-binding properties to EC4-5 and partial homophilic binding activity but did not restore full cell adhesion activity. Group 2 and Group 3 mAbs, which bind dimer better than monomer and wild type better than W2A, recognize epitopes in EC1 and the interface between EC1 and EC2, respectively. None of the mAbs could distinguish between different physiological states of C-cadherin at the cell surface of either Xenopus embryonic cells or Colo 205 cultured cells, demonstrating that changes in dimerization do not underlie regulation of adhesion activity. On the cell surface the EC3-EC5 domains are much less accessible to mAb binding than EC1-EC2, suggesting that they are masked by the state of cadherin organization or by other molecules. Thus, the EC2-EC5 domains either reflect, or are involved in, cadherin dimerization and organization at the cell surface.     

Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.     

Caveolin-1 Interacts with Derlin-1 and Promotes Ubiquitination and Degradation of Cyclooxygenase-2 via Collaboration with p97 Complex

Caveolin-1 (Cav-1) interacts with and mediates protein trafficking and various cellular functions. Derlin-1 is a candidate for the retrotranslocation channel of endoplasmic reticulum proteins. However, little is known about how Derlin-1 mediates glycosylated protein degradation. Here, we identified Cav-1 as a key player in Derlin-1- and p97-mediated cyclooxygenase 2 (COX-2) ubiquitination and degradation. Derlin-1 augmented the interaction of Cav-1 and COX-2 and mediated the degradation of COX-2 in a COX-2 C terminus-dependent manner. Suppression of Cav-1 decreased the ubiquitination of COX-2, and mutation of Asn-594 to Ala to disrupt N-glycosylation at the C terminus of COX-2 reduced the interaction of COX-2 with Cav-1 but not Derlin-1. Moreover, suppression of p97 increased the ubiquitination of COX-2 and up-regulated COX-2 but not COX-1. Cav-1 enhanced the interaction of p97 with Ufd1 and Derlin-1 and collaborated with p97 to interact with COX-2. Cav-1 may be a cofactor in the interaction of Derlin-1 and N-glycosylated COX-2 and may facilitate Derlin-1- and p97 complex-mediated COX-2 ubiquitination, retrotranslocation, and degradation.     

Sterically hindered cisplatin derivatives with multiple carboxylate auxiliary arms: synthesis and reactions with guanosine-5'-monophosphate and plasmid DNA.

Two novel sterically hindered cisplatin derivatives with the ligand L=NH(2)C(CH(2)CH(2)COOH)(3) were prepared: cis-PtCl(2)L(2) and cis-PtCl(2)L(NH(3)). The starting compound for the syntheses was NH(2)C(CH(2)CH(2)COOtBu)(3), also known as a building block for dendrimers. cis-PtCl(2)L(2) was prepared from K(2)PtCl(4) in an unusual two-phase reaction in water-chloroform, followed by deprotection of the tert-butyl protective groups with formic acid to yield a water-soluble complex. The mixed-ligand compound cis-PtCl(2)L(NH(3)) was prepared from [PPh(4)][PtCl(3)(NH(3))] in methanol, with subsequent deprotection in formic acid. DNA-binding properties of the two compounds were investigated using the model base guanosine-5'-monophosphate (5'-GMP) and pBR322 plasmid DNA. While cisplatin [cis-PtCl(2)(NH(3))(2)] induced an unwinding of 12 degrees in pBR322 plasmid DNA, cis-PtCl(2)L(NH(3)) induced only 3 degrees unwinding, which is indicative of a monofunctional binding mode. Remarkably, cis-PtCl(2)L(2) did not induce any distortion in plasmid DNA, which strongly suggests that the compound does not bind to DNA. Test reactions with 5'-GMP, monitored by 1H and 195Pt NMR, confirmed that cis-PtCl(2)L(2) is unable to bind to DNA, whereas cis-PtCl(2)L(NH(3)) binds only one nucleotide. Apparently, binding of platinum to nucleotides at the coordination site cis with respect to the ligand L is prevented by steric crowding. Thus, cis-PtCl(2)L(NH(3)) must bind DNA monofunctionally at the trans position. Besides, both compounds have a chloride replaced by one of the carboxylate arms, forming a a seven-membered chelate ring. In theory, cis-PtCl(2)L(2) could also form a second chelate ring, but this was not observed.     

CRIT peptide interacts with factor B and interferes with alternative pathway activation

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4beta chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.