Targeted treatment of chondrosarcoma with a bacteriophage-based particle delivering a secreted tumor necrosis factor-related apoptosis-inducing ligand.

Chondrosarcoma (CS) is a malignant cartilage-forming bone tumor that is inherently resistant to chemotherapy and radiotherapy, leaving surgery as the only treatment option. We have designed a tumor-targeted bacteriophage (phage)-derived particle (PDP), for targeted systemic delivery of cytokine-encoding transgenes to solid tumors. Phage has no intrinsic tropism for mammalian cells; therefore, it was engineered to display a double cyclic RGD4C ligand on the capsid to target tumors. To induce cancer cell death, we constructed a transgene cassette expressing a secreted form of the cytokine tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). We detected high expression of αvß3 and αvß5 integrin receptors of the RGD4C ligand, and of the TRAIL receptor-2 in human CS cells (SW1353), but not in primary normal chondrocytes. The RGD4C.PDP-Luc particle carrying a luciferase reporter gene, Luc, effectively and selectively mediated gene delivery to SW1353 cells, but not primary chondrocytes. Transduction of SW1353 cells with RGD4C.PDP-sTRAIL encoding a human sTRAIL, resulted in the expression of TRAIL and subsequent cell death without harming the normal chondrocytes. Intravenous administration of RGD4C.PDP-sTRAIL to mice with established human CS resulted in a decrease in tumor size and tumor viability. Altogether, RGD4C.PDP-sTRAIL can be used to target systemic treatment of CS with the sTRAIL.

ASCs derived from burn patients are more prone to increased oxidative metabolism and reactive oxygen species upon passaging.

BACKGROUND: Patients with severe burn injury (over 20% of the total body surface area) experience profound hypermetabolism which significantly prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds, including the potential for autologous ASC expansion. While subcutaneous adipocytes display an altered metabolic profile post-burn, it is not known if this is the case with the stem cells associated with the adipose tissue. METHODS: ASCs were isolated from discarded burn skin of severely injured human subjects (BH, n = 6) and unburned subcutaneous adipose tissue of patients undergoing elective abdominoplasty (UH, n = 6) and were analyzed at passages 2, 4, and 6. Flow cytometry was used to quantify ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively, while JC-10 Mitochondrial Membrane Potential Assays were also performed. Mitochondrial respiration and glycolysis were analyzed with a high-resolution respirometer (Seahorse XFe24 Analyzer). RESULTS: There was no difference in age between BH and UH (34 ± 6 and 41 ± 4 years, respectively, P = 0.49). While passage 2 ASCs had lower ASC marker expression than subsequent passages, there were no significant differences in the expression between BH and UH ASCs. Similarly, no differences in mitochondrial abundance or membrane potential were found amongst passages or groups. Two-way ANOVA showed a significant effect (P < 0.01) of passaging on mitochondrial ROS production, with increased ROS in BH ASCs at later passages. Oxidative phosphorylation capacities (leak and maximal respiration) increased significantly in BH ASCs (P = 0.035) but not UH ASCs. On the contrary, basal glycolysis significantly decreased in BH ASCs (P = 0.011) with subsequent passaging, but not UH ASCs. CONCLUSIONS: In conclusion, ASCs from burned individuals become increasingly oxidative and less glycolytic upon passaging when compared to ASCs from unburned patients. This increase in oxidative capacities was associated with ROS production in later passages. While the autologous expansion of ASCs holds great promise for treating burned patients with limited donor sites, the potential negative consequences of using them require further investigation.

Efficacy of systemic temozolomide-activated phage-targeted gene therapy in human glioblastoma.

Glioblastoma multiforme (GBM) is the most lethal primary intracranial malignant neoplasm in adults and most resistant to treatment. Integration of gene therapy and chemotherapy, chemovirotherapy, has the potential to improve treatment. We have introduced an intravenous bacteriophage (phage) vector for dual targeting of therapeutic genes to glioblastoma. It is a hybrid AAV/phage, AAVP, designed to deliver a recombinant adeno-associated virus genome (rAAV) by the capsid of M13 phage. In this vector, dual tumor targeting is first achieved by phage capsid display of the RGD4C ligand that binds the αvß3 integrin receptor. Second, genes are expressed from a tumor-activated and temozolomide (TMZ)-induced promoter of the glucose-regulated protein, Grp78 Here, we investigated systemic combination therapy using TMZ and targeted suicide gene therapy by the RGD4C/AAVP-Grp78 Firstly, in vitro we showed that TMZ increases endogenous Grp78 gene expression and boosts transgene expression from the RGD4C/AAVP-Grp78 in human GBM cells. Next, RGD4C/AAVP-Grp78 targets intracranial tumors in mice following intravenous administration. Finally, combination of TMZ and RGD4C/AAVP-Grp78 targeted gene therapy exerts a synergistic effect to suppress growth of orthotopic glioblastoma.

Characterization of Mesenchymal-Fibroblast Cells Using the Col1a2 Promoter/Enhancer.

Excessive deposition of extracellular matrix (ECM) is a common hallmark of fibrotic diseases in various organs. Chiefly among this ECM are collagen types I and III, secreted by local fibroblasts, and other mesenchymal cells recruited for repair purposes. In the last two decades, the search for a fibroblast-specific promoter/enhancer has intensified in order to control the regulation of ECM in these cells and limit the scarring of the fibrotic process. In our previous work, we characterized an enhancer region 17 kb upstream of the Col1a2 gene transcription start site. This enhancer in transgenic mice is expressed mainly in mesenchymal cells during development and in adults upon injury. When driving transgenes such as beta-galactosidase or luciferase, this construct acts as an informative reporter of collagen transcription and is predictive of collagen type I deposition. In this chapter, we provide detailed protocols for identifying similar enhancers and using the sequence to generate a construct for transfection and producing transgenic animals. We also provided information on the use of luminescence in transgenic mice, tissue processing, as well as using cre/lox system to obtain conditional gain and loss of function in mice.

Generation of a mouse line harboring a Bi-transgene expressing luciferase and tamoxifen-activatable creER(T2) recombinase in cartilage.

We have used an aggrecan gene enhancer to generate a transgenic murine line (Acan-CreER-Ires-Luc) expressing firefly luciferase and tamoxifen activatable Cre recombinase (Cre-ER(T2) ). The expression and efficiency of the inducible Cre recombinase activity were tested in double transgenic mice created by crossing the Acan-CreER-Ires-Luc line with a Rosa26-lacZ reporter mouse. The expression pattern of the transgene of our line was restricted to cartilage from embryonic to adult stages. ß-galactosidase staining was observed in growth plate, articular cartilage, as well as fibrocartilage of meniscus, trachea, and intervertebral discs. Similar staining was observed in a previously described Agc1 (tm(IRES-creERT2)) murine line. The presence of luciferase in our transgene allows the visualization of the transgene expression in live animals. Weekly measurements from 2 to 8 weeks of age showed a reduction in luminescence in knee joints between 2 and 4 weeks of age, but stabilization thereafter. Following the surgical induction of osteoarthritis at 12 weeks of age, the level of luminescence remained the same in the knee joints for 8 weeks. This Acan-CreER-Ires-Luc murine line allows indirect monitoring of the transcriptional activity of the Acan gene via expression of luciferase, while the inducible Cre recombinase activity facilitates studies involving gain or loss of gene expression in cartilage.

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes.

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.

Tungsten-induced protein aggregation: solution behavior.

Tungsten has been associated with protein aggregation in prefilled syringes (PFSs). This study probed the relationship between PFSs, tungsten, visible particles, and protein aggregates. Experiments were carried out spiking solutions of two different model proteins with tungsten species obtained from the extraction of tungsten pins typically used in syringe manufacturing processes. These results were compared to those obtained with various soluble tungsten species from commercial sources. Although visible protein particles and aggregates were induced by tungsten from both sources, the extract from tungsten pins was more effective at inducing the formation of the soluble protein aggregates than the tungsten from other sources. Furthermore, our studies showed that the effect of tungsten on protein aggregation is dependent on the pH of the buffer used, the tungsten species, and the tungsten concentration present. The lower pH and increased tungsten concentration induced more protein aggregation. The protein molecules in the tungsten-induced aggregates had mostly nativelike structure, and aggregation was at least partly reversible. The aggregation was dependent on tungsten and protein concentration, and the ratio of these two and appears to arise through electrostatic interaction between protein and tungsten molecules. The level of tungsten required from the various sources was different, but in all cases it was at least an order of magnitude greater than the typical soluble tungsten levels measured in commercial PFS.

Accumulation of succinimide in a recombinant monoclonal antibody in mildly acidic buffers under elevated temperatures.

PURPOSE: The purpose of this paper was to identify the location of a succinimide and determine the rate of its formation and hydrolysis in a recombinant human monoclonal IgG2 antibody aged in mildly acidic buffers at elevated temperatures. MATERIALS AND METHODS: Cation exchange (CEX) HPLC separated multiple Main Peaks and high levels (up to 50%) of basic variants, the identification of which was an analytical challenge and required several complementary techniques. The relative abundance of the CEX basic variants was used to quantify the percentage of succinimide and to study the rates of its formation and hydrolysis. RESULTS: Mass decrease by approximately 18 Da for intact antibodies from the CEX basic fractions suggested succinimide formation from aspartic acid as the major modification. Reversed-phase HPLC/MS of the reduced and trypsin-digested samples detected an isoaspartate 30 (isoD30) in the light chain peptide A25-R37. Direct evidence that isoD30 was from succinimide was obtained by performing succinimide hydrolysis in H2(18)O followed by tryptic digestion in H2(16)O. CONCLUSIONS: Succinimide formation increased as pH became more acidic, whereas its hydrolysis was faster as pH became neutral and alkaline. Succinimide hydrolysis in a denatured sample was estimated to have completed in less than 2 h, but approximately three days for a similar pH but without denaturant. These observations suggest that protein conformation affects succinimide hydrolysis.

18O labeling method for identification and quantification of succinimide in proteins.

We have developed a new method for identification and quantification of succinimide in proteins. The method utilizes 18O water to monitor succinimide hydrolysis. 18O-labeled isoaspartic acid and aspartic acid peptides were produced by hydrolysis of a succinimide-containing protein in 18O water (H218O) followed by tryptic digestion in regular water (H216O). The peptides that had 18O incorporated were 2 Da heavier than their 16O native counterparts. The mass difference was detected and quantified by electrospray time-of-flight mass spectrometry. The amount of 18O incorporation into the isoaspartic acid- and aspartic acid-containing peptides was used to quantify the amount of succinimide present in the native sample. The method was applied to analyze a degraded recombinant monoclonal antibody, which exhibited the accumulation of succinimide after storage in mildly acidic buffers at elevated temperatures for a few weeks. We unambiguously identified amino acid residue 30 located in the antibody light chain as the site of aspartic acid isomerization. At this site, there were 20% isoaspartic acid and 80% aspartic acid detected by peptide mapping in the degraded sample (8 weeks, 45 degrees C, pH 5.0). Hydrolysis in 18O water showed that 80% of the isoaspartic acid and 6% of the aspartic acid had 18O incorporated. The only explanation of 18O incorporation was the presence of succinimide in the sample. Together, a total of 21% (0.8x20% isoaspartic acid+0.06x80% aspartic acid) of aspartic acid residue 30 was found to be present in the form of succinimide in this degraded sample. As a control, the same sample, analyzed using regular 16O water did not show any incorporation of 18O water. By monitoring the amount of 18O-labeled isoaspartic acid and aspartic acid over time under both denaturing and native conditions at pH 8.2, we found that, at denaturing conditions, succinimide at light chain residue 30 hydrolyzed very rapidly (in less than 5 s), but slower (succinimide half-life of approximately 6 h) under native conditions. We also found that, under denaturing conditions, succinimide hydrolyzed at an isoaspartic acid/aspartic acid ratio of 3.5:1, but hydrolyzed almost exclusively to aspartic acid under native conditions. This finding indicates that protein structure plays an important role in the kinetics of succinimide hydrolysis as well as in the generation of the hydrolysis products isoaspartic acid and aspartic acid.

Functional genetic screen for genes involved in senescence: role of Tid1, a homologue of the Drosophila tumor suppressor l(2)tid, in senescence and cell survival.

We performed a genetic suppressor element screen to identify genes whose inhibition bypasses cellular senescence. A normalized library of fragmented cDNAs was used to select for elements that promote immortalization of rat embryo fibroblasts. Fragments isolated by the screen include those with homology to genes that function in intracellular signaling, cellular adhesion and contact, protein degradation, and apoptosis. They include mouse Tid1, a homologue of the Drosophila tumor suppressor gene l(2)tid, recently implicated in modulation of apoptosis as well as gamma interferon and NF-kappaB signaling. We show that GSE-Tid1 enhances immortalization by human papillomavirus E7 and simian virus 40 T antigen and cooperates with activated ras for transformation. Expression of Tid1 is upregulated upon cellular senescence in rat and mouse embryo fibroblasts and premature senescence of REF52 cells triggered by activated ras. In accordance with this, spontaneous immortalization of rat embryo fibroblasts is suppressed upon ectopic expression of Tid1. Modulation of endogenous Tid1 activity by GSE-Tid1 or Tid1-specific RNA interference alleviates the suppression of tumor necrosis factor alpha-induced NF-kappaB activity by Tid1. We also show that NF-kappaB sequence-specific binding is strongly downregulated upon senescence in rat embryo fibroblasts. We therefore propose that Tid1 contributes to senescence by acting as a repressor of NF-kappaB signaling.

Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function.

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 microm(-1) s(-1) and 0.22 s(-1), respectively, yielding an O(2) affinity of 21 microm(-1), which is approximately 20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O(2) binding. Resonance Raman spectra show that the Fe-O(2) vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe-O-O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe-O-O angle is 110 degrees, the O-O bond is pointed toward the heme alpha-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O(2) and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O-O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin alpha-meso-carbon by preventing premature O-O bond cleavage.

The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae.

Crystal structures of the ferric and ferrous heme complexes of HmuO, a 24-kDa heme oxygenase of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 A resolution, respectively. The HmuO structures show that the heme group is closely sandwiched between the proximal and distal helices. The imidazole group of His-20 is the proximal heme ligand, which closely eclipses the beta- and delta-meso axis of the porphyrin ring. A long range hydrogen bonding network is present, connecting the iron-bound water ligand to the solvent water molecule. This enables proton transfer from the solvent to the catalytic site, where the oxygen activation occurs. In comparison to the ferric complex, the proximal and distal helices move closer to the heme plane in the ferrous complex. Together with the kinked distal helix, this movement leaves only the alpha-meso carbon atom accessible to the iron-bound dioxygen. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediate by preventing premature heterolytic O-O bond cleavage. This allows the enzyme to oxygenate selectively at the alpha-meso carbon in HmuO catalysis.

Solution 1H NMR investigation of the active site molecular and electronic structures of substrate-bound, cyanide-inhibited HmuO, a bacterial heme oxygenase from Corynebacterium diphtheriae.

The molecular structure and dynamic properties of the active site environment of HmuO, a heme oxygenase (HO) from the pathogenic bacterium Corynebacterium diphtheriae, have been investigated by (1)H NMR spectroscopy using the human HO (hHO) complex as a homology model. It is demonstrated that not only the spatial contacts among residues and between residues and heme, but the magnetic axes that can be related to the direction and magnitude of the steric tilt of the FeCN unit are strongly conserved in the two HO complexes. The results indicate that very similar contributions of steric blockage of several meso positions and steric tilt of the attacking ligand are operative. A distal H-bond network that involves numerous very strong H-bonds and immobilized water molecules is identified in HmuO that is analogous to that previously identified in hHO (Li, Y., Syvitski, R. T., Auclair, K., Wilks, A., Ortiz de Montellano, P. R., and La Mar, G. N. (2002) J. Biol. Chem. 277, 33018-33031). The NMR results are completely consistent with the very recent crystal structure of the HmuO.substrate complex. The H-bond network/ordered water molecules are proposed to orient the distal water molecule near the catalytically key Asp(136) (Asp(140) in hHO) that stabilizes the hydroperoxy intermediate. The dynamic stability of this H-bond network in HmuO is significantly greater than in hHO and may account for the slower catalytic rate in bacterial HO compared with mammalian HO.