Christopher Switzer

Targeting SET/I2PP2A Oncoprotein Functions as a Multi-pathway Strategy for Cancer Therapy

The SET oncoprotein participates in cancer progression by affecting multiple cellular processes, inhibiting the tumor suppressor protein phosphatase 2A (PP2A), and inhibiting the metastasis suppressor nm23-H1. On the basis of these multiple activities, we hypothesized that targeted inhibition of SET would have multiple discrete and measurable effects on cancer cells. Here, the effects of inhibiting SET oncoprotein function on intracellular signaling and proliferation of human cancer cell lines was investigated. We observed the effects of COG112, a novel SET interacting peptide, on PP2A activity, Akt signaling, nm23-H1 activity and cellular migration/invasion in human U87 glioblastoma and MDA-MB-231 breast adenocarcinoma cancer cell lines. We found that COG112 interacted with SET protein and inhibited the association between SET and PP2A catalytic subunit (PP2A-c) and nm23-H1. The interaction between COG112 and SET caused PP2A phosphatase and nm23-H1 exonuclease activities to increase. COG112-mediated increases in PP2A activity resulted in the inhibition of Akt signaling and cellular proliferation. Additionally, COG112 inhibited SET association with Ras-related C3 botulinum toxin substrate 1 (Rac1), leading to decreased cellular migration and invasion. COG112 treatment releases the SET-mediated inhibition of the tumor suppressor PP2A, as well as the metastasis suppressor nm23-H1. These results establish SET as a novel molecular target and that the inhibition of SET may have beneficial effects in cancer chemotherapy.

Thrombospondin-1 Inhibits Nitric Oxide Signaling via CD36 by Inhibiting Myristic Acid Uptake

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.

Two Watson−Crick-Like Metallo Base-Pairs

Two Watson-Crick-like metallo base-pairs are described with mutually independent geometries that have similar dimensions and stabilities to their natural, hydrogen-bonded counterparts.

DNA bending by a phantom protein

BACKGROUND Despite its stiffness, duplex DNA is extensively bent and folded during packaging and gene expression in biological systems. Modulation of the electrostatic repulsion between phosphates in the DNA backbone may be important in the bending of DNA by proteins. Here, we analyze the shape of DNA molecules that have been modified chemically to mimic the electrostatic consequences of a bound protein. RESULTS We have simulated salt bridges between DNA phosphates and cationic amino acid sidechains of a phantom protein by tethering ammonium cations to one face of the DNA helix. Tethered ammonium cations, but not neutral acetylated controls, induce DNA to bend toward its neutralized surface. CONCLUSIONS The shape of DNA molecules bearing a laterally-asymmetric distribution of tethered cations agrees qualitatively with theoretical predictions and with results previously obtained using neutral phosphate analogs. These data suggest principles that might be applied to the design of artificial DNA-bending proteins.

Combinatorial Synthesis of Thrombin-Binding Aptamers Containing iso-Guanine

A library of all possible substitutions of guanine by iso-guanine (iG) in the thrombin aptamer was prepared by split and mix synthesis. A colorimetric assay was used to screen for functional oligomers in the library. Colorimetrically active oligonucleotides were selected and sequenced by the Maxam-Gilbert method. The sequenced oligonucleotides were individually resynthesized, and their affinities for thrombin were assayed by isothermal titration calorimetry. Three aptamer sequences containing iG were found to have enhanced binding activity to human alpha-thrombin compared to the parent aptamer.

An alternative nucleobase code: characterization of purine-purine DNA double helices bearing guanine-isoguanine and diaminopurine 7-deaza-xanthine base pairs.

Non-natural nucleic acid frameworks have exhibited functional behavior as independent, expanded, and potentially prebiotic genetic systems. While success has been achieved in these areas by replacing the native chemical functionality that is found in the sugar-phosphate backbone or bases with nonnative groups, less-divergent structural alternatives to nucleic acids still remain to be fully explored. To date, nonstandard base pairs rely on alternate hydrogen-bonded motifs, complementary hydrophobic surfaces, dimensional homologues, and metal coordination. One minimally divergent alternative that is yet to be fully explored is an all-purine genetic system. Crick proposed that a genetic system that incorporates only adenine and hypoxanthine might have preceded the modern genetic code. In this connection, a wide array of helical structures have been reported for all-purine nucleic acids, including those that contain three, four, and five strands. Doublestranded purine–purine structures are known in the context of both nonstandard 11] and natural 13, 14] nucleic acids; however, only two reports describe all-purine duplexes of DNA that display Watson–Crick or reverse Watson–Crick pairing for association. Whereas model systems have so far failed to demonstrate nonenzymatic oligomerization by purine–purine pairs, insufficient information exists to date concerning the fitness of an all-purine double helix to assess its suitability as a precursor or independent genetic material. In an extension of our earlier work on iG self-pairing, 10] we report the characterization of the G·iG and D·X base-pairing system in DNA (Scheme 1). Several considerations guided purine nucleobase selection. To minimize potential mispairs (e.g. , A·G 17] or H·iG), diaminopurine and xanthine motifs were utilized in place of adenine and hypoxanthine. Further, 7-deazaxanthine was deemed a more-suitable complement to diaminopurine than xanthine because the lack of a 7-nitrogen atom leads to reduced susceptibility to depurination and an increased pKa. [18] Additionally, the nitrogen substitution in 7-deazaxanthine removes a HoogACHTUNGTRENNUNGsteen-face hydrogen-bond acceptor, thereby inhibiting alternative modes of strand association. Isoguanine, via its N3-H tautomer, was chosen to complement guanine in analogy with homo-DNA pairing. It is notable that both D·X and iG·G base pairs benefit from the potential to form three hydrogen bonds via their Watson–Crick faces. The oligonucleotides displayed in Scheme 1 were synthesized from commercially available phosphoramidites on an Expedite 8909 DNA synthesizer. To assess the association of complementary strands, UV-monitored thermal denaturation experiments were performed on each of two strands individually, and then combined. Because the optimal wavelength at which to observe hyperchromicity is expected to be unique for a given nucleic acid complex, multiple wavelengths were monitored simultaneously during initial experiments. A clear melting transition was observed with maximal hyperchromicity at 250 nm for 1·2 (Figure 1 A). It can also be seen from Figure 1 that profiles for 1 and 2 alone do not sum to the denaturation profile of 1 and 2 combined. These results are consistent with duplex formation between 1 and 2. Similarly, clear melting transitions are seen for 5·6 and 9·10 (Figure 1 C, E). Thermodynamic values for strand association were obtained by nonlinear regression of the melting curve traces and a van’t Hoff plot (Table 1). Whereas clear evidence is seen for association between oligomers 1 and 2, thermal denaturation of the individual strands revealed that oligomer 2 self associates in the absence of its Scheme 1. A) Purine–purine and purine–pyrimidine base-pair structures ; B) oligonucleotide sequences.

SYNTHESIS OF OLIGONUCLEOTIDES BEARING THE NON-STANDARD BASES ISO-C AND ISO-G. COMPARISON OF ISO-C-ISO-G, C-G AND U-A BASE-PAIR STABILITIES IN RNA/DNA DUPLEXES

Abstract The synthesis of iso -cytidine and 2′-deoxy- iso -guanosine phosphoramidites in protected form is described. The synthesis of complementary dodecanucleotides containing a central iso -C- iso -G base-pair was achieved. It is found that the iso -C- iso -G base-pair has comparable stability to a C-G base-pair.

Polymerase Recognition of a Watson–Crick‐Like Metal‐Mediated Base Pair: Purine‐2,6‐Dicarboxylate⋅Copper(II)⋅ Pyridine

Among biomolecules, nucleic acids have unique physical properties that render them highly adapted to their role of information storage and transfer. Nevertheless, etiological deconstruction of nucleic acids has shown that considerable latitude exists for replacement of the component features, including in both the backbone and nucleobases. Natural nucleobases encode genetic information by presenting complementary arrays of hydrogen-bond donors and acceptors, leading to Watson–Crick base pairs incorporating (large) purine and (small) pyrimidine components. A goal of synthetic biology is to generalize the function of biomolecules in a way that extends their behaviors to new molecular systems. Efforts to extend the information-encoding capabilities of nucleobases have focused on altered hydrogen-bonding patterns, polarity/shape interactions, and metal coordination. 5] The first two strategies have successfully culminated in base pairs that can be replicated in vitro with high fidelity in the presence of the four natural nucleotides. Polymerases have been shown recently to incorporate metal-ion-mediated base pairs (MMBPs) of the canonical nucleobases T, C, and A, as T-Hg -T, C-Ag-C, and C-Ag-A. In one example, two of these base pairs were used as the basis for logic-gate operations. The presence of canonical nucleobases in these MMBPs, however, precludes their use for expanding the information content of the genetic code. Polymerase processing of a crosslinking salen-complex, dS-Cu-dS, has recently enabled the first in vitro copying and amplification of a MMBP that is orthogonal to the four natural bases. The requirement for an organic co-factor (ethylene diamine) in addition to the inorganic co-factor (Cu ) by the crosslinking salen complex can be expected to limit its applications relative to MMBPs relying on an inorganic co-factor alone. Additionally, the achievement of a metal-mediated hetero-base pair that is orthogonal to the four natural bases is desirable as it would make 152 “extra” codons possible, in comparison to the 61 extra codons that would be derived from adding a self pair (i.e. , 216 vs. 125 total codons, respectively, minus the standard 64 codons). As wobble base pairing at the third codon position is expected to reduce the number of unique codons actually realized in both cases, the additional codons owing to hetero-base pairing might not be inconsequential. Herein, we show that DNA polymerases can recognize a Cu -mediated Watson–Crick-like hetero-base pair. In past work, we have explored the properties of MMBP motifs with purine or pyrimidine cores to promote their possible use as substrates for natural enzymes. These efforts have included an examination of bipyridyland terpyridyl-like purine and pyrimidine nucleotides with affinities for Ni + or Ag in DNA double helices. Despite stabilities that are comparable to those of natural base pairs, these MMBPs have shortcomings as potential enzyme substrates, including their size, and, in the case of base pairs using Ag , a background affinity for natural nucleobases and acute toxicity. Accordingly, we sought to replace the pyridyl moieties with a smaller coordinating group. At the same time, we hoped that the new MMBP might recruit a metal ion with fewer undesirable effects. The culmination of our efforts to create an MMBP with a close likeness to the canonical base pairs, and thereby amenable to enzymatic processing, led to Pur·Cu + ·3-Py (Figure 1), an MMBP that is readily synthesized by a wide range of polymerases.

Dithiolethione modified valproate and diclofenac increase E-cadherin expression and decrease proliferation of non-small cell lung cancer cells

The effects of dithiolethione modified valproate, diclofenac and sulindac on non-small cell lung cancer (NSCLC) cells were investigated. Sulfur(S)-valproate and S-diclofenac at 1 microg/ml concentrations significantly reduced prostaglandin (PG)E(2) levels in NSCLC cell lines A549 and NCI-H1299 as did the COX-2 inhibitor DuP-697. In vitro, S-valproate, S-diclofenac and S-sulindac half-maximally inhibited the clonal growth of NCI-H1299 cells at 6, 6 and 15 microg/ml, respectively. Using the MTT assay, 10 microg/ml S-valproate, NO-aspirin and Cay10404, a selective COX-2 inhibitor, but not SC-560, a selective COX-1 inhibitor, inhibited the growth of A549 cells. In vivo, 18mg/kg i.p. of S-valproate and S-diclofenac, but not S-sulindac, significantly inhibited A549 or NCI-H1299 xenograft proliferation in nude mice, but had no effect on the nude mouse body weight. The mechanism by which S-valproate and S-diclofenac inhibited the growth of NSCLC cells was investigated. Nitric oxide-aspirin but not S-valproate caused apoptosis of NSCLC cells. By Western blot, S-valproate and S-diclofenac increased E-cadherin but reduced vimentin and ZEB1 (a transcriptional suppressor of E-cadherin) protein expression in NSCLC cells. Because S-valproate and S-diclofenac inhibit the growth of NSCLC cells and reduce PGE(2) levels, they may prove beneficial in the chemoprevention and/or therapy of NSCLC.

Bis(6-carboxypurine)-Cu2+: a possibly primitive metal-mediated nucleobase pair.

A metal-mediated self-pair is described that emulates Watson-Crick base pair properties in a DNA double helix.