PPARG activation promotes the proliferation of colorectal cancer cell lines and enhances the antiproliferative effect of 5-fluorouracil.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARG) is a member of the nuclear receptor family. It is involved in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, vascular homeostasis and inflammation. In addition, PPARG agonists, known as thiazolidinediones, are well established in the treatment of type 2 diabetes mellitus. PPARGs role in cancer is a matter of debate, as pro- and anti-tumour properties have been described in various tumour entities. Currently, the specific role of PPARG in patients with colorectal cancer (CRC) is not fully understood. MATERIAL AND METHODS: The prognostic impact of PPARG expression was investigated by immunohistochemistry in a case-control study using a matched pair selection of CRC tumours (n = 246) with either distant metastases to the liver (n = 82), lung (n = 82) or without distant metastases (n = 82). Its effect on proliferation as well as the sensitivity to the chemotherapeutic drug 5-fluorouracil (5-FU) was examined after activation, inhibition, and transient gene knockdown of PPARG in the CRC cell lines SW403 and HT29. RESULTS: High PPARG expression was significantly associated with pulmonary metastasis (p = 0.019). Patients without distant metastases had a significantly longer overall survival with low PPARG expression in their tumours compared to patients with high PPARG expression (p = 0.045). In the pulmonary metastasis cohort instead, a trend towards longer survival was observed for patients with high PPARG expression in their tumour (p = 0.059). Activation of PPARG by pioglitazone and rosiglitazone resulted in a significant dose-dependent increase in proliferation of CRC cell lines. Inhibition of PPARG by its specific inhibitor GW9662 and siRNA-mediated knockdown of PPARG significantly decreased proliferation. Activating PPARG significantly increased the CRC cell lines sensitivity to 5-FU while its inhibition decreased it. CONCLUSION: The prognostic effect of PPARG expression depends on the metastasis localization in advanced CRC patients. Activation of PPARG increased malignancy associated traits such as proliferation in CRC cell lines but also increases sensitivity towards the chemotherapeutic agent 5-FU. Based on this finding, a combination therapy of PPARG agonists and 5-FU-based chemotherapy constitutes a promising strategy which should be further investigated.

Case of a Patient With Pancreatic Cancer With Sporadic Microsatellite Instability Associated With a BRAF Fusion Achieving Excellent Response to Immunotherapy.

In this case report, we discuss a case of pancreatic cancer bearing a BRAF fusion, leading to MAPK activation, MLHph, and finally MSI.

Clinical Impact of Structured Post-Operative Surveillance in Resected Pancreatic Adenocarcinoma: Results from a Retrospective Cohort Study.

INTRODUCTION: To this date, surgery remains the only potentially curative approach in the treatment of pancreatic cancer. To analyse the clinical impact of a structured post-operative follow-up programme, we retrospectively analysed a cohort of resected pancreatic adenocarcinoma patients treated at LMU Munich. METHODS: Pancreatic adenocarcinoma patients who underwent resection and presented for regular follow-up visits at our centre between 2002 and 2017 were identified from two existing study cohorts. Diagnosis of recurrences was categorised by timing (within or outside a scheduled follow-up visit) and detection modality (imaging, CA 19-9 increase, or clinical deterioration) and correlated with disease-free survival and overall survival (OS). RESULTS: One hundred and twenty-five patients with resected pancreatic adenocarcinoma were included in this analysis. Median OS in the whole cohort was 21.1 months. Of these 125 patients, 103 (82.4%) patients had a documented relapse. Tumour recurrences detected within a scheduled follow-up visit (n = 86, 83.5%) compared to recurrences becoming apparent at an unplanned visit (n = 17, 16.5%) were associated with a significantly improved OS (median 25.5 vs. 20.2 months, p = 0.019). Compared to patients with recurrence detected by clinical deterioration (n = 4, 3.9%), patients with recurrences detected by imaging or laboratory abnormalities (n = 99, 96.0%) had a longer median OS (24.8 vs. 15.1 months, p = 0.007). DISCUSSION: A structured follow-up after pancreatic ductal adenocarcinoma resection may have an impact on patient outcome. Prospective trials are needed to evaluate the clinical impact of post-operative follow-up programmes.

Bcl-xL as prognostic marker and potential therapeutic target in cholangiocarcinoma.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-xL and Mcl-1. Expression of Bcl-xL , Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-xL and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl-xL (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-xL and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-xL as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Exhaustion of Skeletal Muscle Fibers Within Seconds: Incorporating Phosphate Kinetics Into a Hill-Type Model.

Initiated by neural impulses and subsequent calcium release, skeletal muscle fibers contract (actively generate force) as a result of repetitive power strokes of acto-myosin cross-bridges. The energy required for performing these cross-bridge cycles is provided by the hydrolysis of adenosine triphosphate (ATP). The reaction products, adenosine diphosphate (ADP) and inorganic phosphate (P i ), are then used-among other reactants, such as creatine phosphate-to refuel the ATP energy storage. However, similar to yeasts that perish at the hands of their own waste, the hydrolysis reaction products diminish the chemical potential of ATP and thus inhibit the muscle's force generation as their concentration rises. We suggest to use the term "exhaustion" for force reduction (fatigue) that is caused by combined P i and ADP accumulation along with a possible reduction in ATP concentration. On the basis of bio-chemical kinetics, we present a model of muscle fiber exhaustion based on hydrolytic ATP-ADP-P i dynamics, which are assumed to be length- and calcium activity-dependent. Written in terms of differential-algebraic equations, the new sub-model allows to enhance existing Hill-type excitation-contraction models in a straightforward way. Measured time courses of force decay during isometric contractions of rabbit M. gastrocnemius and M. plantaris were employed for model verification, with the finding that our suggested model enhancement proved eminently promising. We discuss implications of our model approach for enhancing muscle models in general, as well as a few aspects regarding the significance of phosphate kinetics as one contributor to muscle fatigue.

An enhanced model of cross-bridge operation with internal elasticity.

A recent study has shown (as reported by Rosenfeld, Eur Biophys J 41:733-753, 2012) that an apparatus consisting of a cycling pump, a lever, and charged beads is able to generate force in accordance with Hill's force-velocity relation. Here, we show that a spring integrated into this microscopic model of a myosin motor allows reproducing, in general terms, the muscle fiber responses to sudden changes in fiber length. The time course of relaxation is governed by the same hindering force that determines the maximal value of muscle contraction velocity. Any single one of the exceptionally simple parts of the proposed model device corresponds to some element of the real myosin head and interacts with any other part in accordance with the laws of Newton, Coulomb, and Hooke. In essence, the model demonstrates that Coulomb repulsion should be understood as the physical source of muscle force. Accordingly, some fictitious master equation with ad hoc postulated rate constants is not needed to explain the essential mechanical characteristics of a muscle. The current model still contains no mechanism that could account for superfast relaxations within periods of about 0.1 ms.

A polyphosphoester conjugate of melphalan as antitumoral agent.

The low molecular weight of many chemotherapeutics causes their untargeted distribution in the body and fast renal clearance, which leads to a loss of therapeutic activity and to unspecific toxic side effects. Therefore, there is a growing interest in conjugating anticancer drugs to water soluble polymers and thus, take advantage of the 'enhanced permeability and retention' (EPR) effect in tumors. In this study, water soluble polyphosphoesters were used as polymer carriers of melphalan hydrochloride (hydrochloride of p-bis(2-chloroethyl)amino-L-phenylalanine), which is a multifunctional alkylating agent. Melphalan was chemically immobilized by covalent bonding to poly(oxyethylene H-phosphonate) under Atherton-Todd reaction conditions. Novel polymer-melphalan complexes with ionic and hydrogen bonds were designed as controls, basing on two other biodegradable polyphosphoesters: poly(hydroxyoxyethylene phosphate) and poly(methyloxyethylene phosphate). The structure of the formed products was elucidated by (1)H, (13)C, (31)P NMR and FT-IR spectroscopy. The cytotoxic effect of the melphalan formulations was evaluated on different tumor cell lines. The novel polymer formulations showed a concentration dependent antitumoral activity, comparable to the effect of unmodified melphalan. The polymer-melphalan conjugate was also evaluated in vivo in the human hepatocellular carcinoma HuH7 xenograft mouse model. It improved the therapeutic efficacy of pure melphalan without causing side effects.

Characterization of in vivo chemoresistant human hepatocellular carcinoma cells with transendothelial differentiation capacities.

BACKGROUND: Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model. METHODS: SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel. RESULTS: HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression. CONCLUSIONS: Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

A Comprehensive Gene Expression Analysis of Resistance Formation upon Metronomic Cyclophosphamide Therapy.

Resistance formation is one of the major hurdles in cancer therapy. Metronomic anti-angiogenic treatment of xenografted prostate cancer tumors in severe combined-immunodeficiency (SCID) mice with cyclophosphamide (CPA) results in the appearance of resistant tumors. To investigate the complex molecular changes occurring during resistance formation, we performed a comprehensive gene expression analysis of the resistant tumors in vivo. We observed a multitude of differentially expressed genes, e.g., PAS domain containing protein 1, annexin A3 (ANXA3), neurotensin, or plasminogen activator tissue (PLAT), when comparing resistant to in vivo passaged tumor samples. Furthermore, tumor cells from in vivo and in vitro conditions showed a significant difference in target gene expression. We assigned the differentially expressed genes to functional pathways like axon guidance, steroid biosynthesis, and complement and coagulation cascades. Most of these genes were involved in anti-coagulation. Up-regulation of anticoagulatory ANXA3 and PLAT and down-regulation of PLAT inhibitor serpin peptidase inhibitor clade A were validated by quantitative real-time polymerase chain reaction. In contrast, coagulation factor F3 was upregulated, accompanied by the expression of an altered gene product. These findings give insights into the resistance mechanisms of metronomic CPA treatment, suggesting an important role of anti-coagulation in resistance formation.

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)ß(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.

Climbing in hexapods: a plain model for heavy slopes.

Usually, a climbing cockroach attaches with three legs to a substrate. According to a recent model study, pulling forces underneath the front leg are required at some critical slope angle in upward locomotion. This critical angle depends on the animal's anatomy and leg positioning. In this study, we asked especially how this critical angle can be biased by one parameter that may be controlled during climbing: the body height above the substrate. We found that the typical ratio between body height and length (0.2) adopted by cockroaches is slightly higher than the very ratio (0.15) at which the critical slope angle can be increased most strongly for a given decrease in body height. In other words, it is likely that a geometrical body design of cockroaches evolved, which enables a delicate reduction in body height perfectly suitable for preventing the danger of slipping or even falling over rearwards at steepening slopes (approaching the vertical). In that sense, our model predicts, not just for hexapods but rather for any three-point climber, that taking up a low ratio of body height to the distance between the foremost and the hindmost attachment point (very crouched posture) makes body height a good parameter for climbing control.

Dual-targeted polyplexes: one step towards a synthetic virus for cancer gene therapy.

Incorporating ligands into nano-scale carriers for specific delivery of therapeutic nucleic acids to tumor sites is a promising approach in anti-cancer strategies. Current artificial vector systems however still suffer from efficient and specific delivery, compared to their natural counterparts and addressed receptor types rarely are exclusively expressed on target cells. In this study synthetic dual receptor targeted polyplexes were developed, mimicking biphasic cell entry characteristics of natural viruses to increase efficiency and specificity by a dual-receptor internalization mechanism. For engineering the synthetic dual targeted vector system, the transferrin targeting peptide B6 was evaluated for the first time in the context of PEGylated PEI based polyplexes. As a second ligand, arginine-glycine-aspartic acid (RGD) containing peptide was incorporated for simultaneous integrin targeting. Cellular association, cellular uptake, transfection efficiency and accordant competition experiments displayed specificity of both ligands for each targeted receptor in two prostate cancer cell lines. A clear synergy of dual targeting over the combination of single-targeted polyplexes was found, suggesting that the dual targeting strategy is one step towards safe vectors for therapeutic approaches.

Pyridylhydrazone-based PEGylation for pH-reversible lipopolyplex shielding.

PEGylation, which is reversed after the therapeutic agent reaches the target cell, presents an attractive feature for drug, protein or nucleic acid delivery. For bioreversible surface shielding of DNA lipopolyplexes, the bifunctional, endosomal pH-cleavable reagent OPSS-PEG-HZN-Chol [ω-2-pyridyldithio poly (ethylene) glycol α-(butyraldehyde) (N(1)-cholesteryloxycarbonyl-1, 2-diaminoethane amidocarboxy) pyridyl hydrazone] was synthesized. Both OPSS-PEG-HZN-Chol and its corresponding pH stable analog OPSS-PEG-Chol form micelles which remain stable and shielded at pH 7.4 and 37 °C as demonstrated by size exclusion chromatography. At endosomal pH 5.4, OPSS-PEG-HZN-Chol micelles are destroyed within 30 min at 37 °C, while OPSS-PEG-Chol micelles remain stable. Lipopolyplexes composed of DNA condensed with polyethylenimine (PEI), phospholipids including dioleoyl phosphatidylethanolamine (DOPE) and pH-labile OPSS-PEG-HZN-Chol were prepared by the ethanol injection technique. The optimal formulation yielded particles of 160 nm size and a zeta potential of +7 mV. At physiological neutral conditions pyridylhydrazone-based PEGylated lipopolyplexes were as stable as standard PEGylated lipopolyplexes. At pH 5.4, an increase in size was observed only for pH-reversible HZN lipopolyplexes. Transfection with plasmids encoding for luciferase or EGFP resulted in an upto 40-fold enhancement in gene expression with the reversibly PEGylated lipopolyplexes compared to the stably PEGylated ones. Incorporation of a synthetic peptidic ligand for transferrin receptor targeting further improved transfection efficiency in transferrin overexpressing tumor cells. Cellular association and uptake studies by flow cytometry and confocal laser scanning microscopy with an Alexa-labeled PEG shield revealed intracellular deshielding in case of the HZN lipopolyplexes.

A macroscopic ansatz to deduce the Hill relation.

In this study, we derive the hyperbolic force-velocity relation of concentric muscular contraction, first formulated empirically by A.V. Hill in 1938, from three essential model assumptions: (1) the structural assembly of three well-known elements - i.e. active, parallel damping, and serial - fulfilling a force equilibrium, (2) the parallel damping coefficient explicitly depending on muscle force output and three parameters, and (3) the kinematic gearing ratio between active and serial element being assigned to a parameter. The energy source within the muscle represented by the force of the active element is an additional fifth parameter. As a result we find the Hill "constants" A and B as functions of our five model parameters. Using A and B values from literature on experimental data, we predict heat power release of our model. By calculating enthalpy rate and mechanical efficiency, we compare the model heat power to predictions from another Hill-type model, to Hill's original findings, and to findings from modern muscle heat measurements. We reconsider why the biggest share of heat rate during isometric contractions (maintenance heat) and the velocity-dependent heat rate during concentric contractions in addition to maintenance heat rate (shortening heat rate) may be traced back to the same mechanism represented by the kinematic gearing ratio. Namely, we suggest that the serial element transfers attachment-detachment fluctuations of actin-myosin crossbridges within one sarcomere to others in the same sarcomere and to those in parallel and in series. Numerically, in case of negligible passive muscular damping, we find the ratio between A and isometric force (relative A) to depend exclusively on the kinematic gearing ratio, whereas the maintenance heat rate scales with the square of relative A. Moreover, this mechanical coupling internal to the muscle fibres may also be behind the macroscopic force dependency of the overall parallel damping coefficient.

Novel approaches in anti-angiogenic treatment targeting endothelial F-actin: a new anti-angiogenic strategy?

As a functional blood supply is crucial for growth of solid tumors, the development of anticancer agents to inhibit the formation of new tumor blood vessels is an area of extensive research. Endothelial cell motility driven by the dynamics of the cytoskeleton is a key feature of angiogenesis. Agents that preferentially target endothelial tubulin are well established, and inhibition of the endothelial actin dynamics appears to be another promising anti-angiogenic strategy. Remodeling of the actin cytoskeleton is regulated by several pathways involving a large number of signaling proteins. Therefore, therapeutic strategies for the modulation of actin dynamics include agents that target the actin cytoskeleton directly, as well as inhibitors of actin binding proteins and regulators in upstream pathways. This review provides an overview of the regulation of the actin cytoskeleton and proteins that could potentially be targeted by therapeutic agents. In addition, an outline of promising agents, which includes recombinant proteins, endogenous effectors and treatment regimes that exert anti-angiogenic effects partly mediated by affecting endothelial actin dynamics is provided.

Oligoethylenimine-grafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery.

Several grafted polypropylenimine dendrimers were synthesized by modifying either polypropylenimine (PPI) dendrimer generation 2 (G2) or generation 3 (G3) via 1.6-hexandioldiacrylate with branched oligoethylenimine 800Da (OEI) or PPI dendrimer G2. The resulting derivatives were characterized ((1)H NMR, GPC) and their biophysical properties such as DNA condensing ability, colloidal stability and hydrodynamic diameters were determined. All grafted dendrimers were able to efficiently compact DNA to nanosized polyplexes (100-200 nm) and exhibited an increased colloidal stability as compared to their unmodified counterparts. In vitro, grafted dendrimers resulted in much higher transfection levels as compared to the unmodified ones displaying alongside a clear structure-activity relationship regarding their transfection/toxicity profile. Transfection levels of OEI-grafted dendrimers were the highest, being similar or even higher as compared to standard polyethylenimines (linear and branched), demonstrating that the incorporation of ethylenimine moieties is the key factor contributing to this boosted transfection efficiency. None of the compounds resulted in polymer-induced erythrocyte aggregation. Upon i.v. injection of OEI-grafted dendrimer polyplexes into tumor-bearing mice transgene expression was predominantly found in the (subcutaneous) tumors. Importantly, the tumor gene expression levels significantly increased with the higher dendrimer core generation.

Electrophoretic purification of tumor-targeted polyethylenimine-based polyplexes reduces toxic side effects in vivo.

Non-viral vectors based on polyethylenimine (PEI) are usually generated with an excess of PEI. However, the amount of unbound polymer correlates with toxicity limiting the in vivo use of these gene carriers. Purification based on size exclusion chromatography of PEI/DNA polyplexes smaller than 200 nm has been shown to efficiently remove unbound PEI polymer. A novel purification method based on electrophoresis can purify PEI polyplexes independent of their size resulting in polyplexes with final PEI nitrogen/DNA phosphate ratios between 2.6 and 3.1. Also unbound PEI conjugates like PEGylated PEI and transferrin-conjugated PEI can be separated from the polyplexes, providing formulations with clearly defined compositions. Purified polyplexes can mediate in vitro gene transfer with high transfection efficiencies while demonstrating lower cellular toxicity. Purified polyplexes were well-tolerated when systemically delivered into tumor-bearing mice at 100 microg/20 g body weight, with tumor gene expression levels up to 5-fold higher than the non-purified polyplexes. Mice receiving non-purified gene carriers exhibited severe toxicity leading to high mortality and unfavourable gene expression patterns.

Specific targets in tumor tissue for the delivery of therapeutic genes.

Gene therapy is part of a growing field in molecular medicine, which will gain importance in the treatment of human diseases. Until now, almost two thirds of all clinical trials performed in gene therapy are directed against Cancer As solid tumors exceeding a certain size rely on blood supply, the administration of particulate gene delivery vectors via the bloodstream is a promising concept. Tumor cells and the tumor vasculature both offer specific molecular targets, which can be utilized for the site directed delivery of therapeutic genes. Passive targeting of macromolecular drugs including gene delivery vectors to tumors can be achieved by the so called enhanced permeability and retention (EPR) effect. The specificity can be markedly enhanced when tumor targeting ligands are used. Viral vectors, which usually do not have a natural tropism for tumor tissue, were generated to carry tumor targeting molecules on their surface. Synthetic gene delivery vectors, based on cationic lipids or cationic polymers were biochemically modified to incorporate ligands specific for tumor cells or tumor vasculature. For systemic application, these delivery systems have to fulfill certain conditions. The delivery vector should not induce any immunogenic and inflammatory responses. Several studies were conducted to reduce the immunogenicity of viral vectors; surface modification of non-viral gene delivery systems reduced their non-specific interaction with blood components. On the genetic level, tumor specific promoters add additional layers of specificity restricting the transgene expression to the tumor tissue. This review will cover the systemic application of particulate gene transfer vectors targeted to tumors and will give an overview of therapeutic concepts for cancer gene therapy.

Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli.

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.