Evaluation of two xenobiotic reductases from Pseudomonas putida for their suitability for magnetic nanoparticle-directed enzyme prodrug therapy as a novel approach to cancer treatment.

Directed enzyme prodrug therapy (DEPT) is a cancer chemotherapy strategy in which bacterial enzymes are delivered to a cancer site before prodrug administration, resulting in prodrug activation at the cancer site and more localized treatment. A major limitation to DEPT is the poor effectiveness of the most studied enzyme for the CB1954 prodrug, NfnB from Escherichia coli, at concentrations suitable for human use. Much research into finding alternative enzymes to NfnB has resulted in the identification of the Xenobiotic reductases, XenA and XenB, which have been shown in the literature to reduce environmentally polluting nitro-compounds. In this study, they were assessed for their potential use in cancer prodrug therapy strategies. Both proteins were cloned into the pET28a+ expression vector to give the genetically modified proteins XenA-his and XenB-his, of which only XenB-his was active when tested with CB1954. XenB-his was further modified to include a cysteine-tag to facilitate direct immobilization on to a gold surface for future magnetic nanoparticle DEPT (MNDEPT) treatments and was named XenB-cys. When tested using high-performance liquid chromatography (HPLC), XenB-his and XenB-cys both demonstrated a preference for reducing CB1954 at the 4-nitro position. Furthermore, XenB-his and XenB-cys successfully induced cell death in SK-OV-3 cells when combined with CB1954. This led to XenB-cys being identified as a promising candidate for use in future MNDEPT treatments.

The Pseudoalteromonas luteoviolacea L-amino Acid Oxidase with Antimicrobial Activity Is a Flavoenzyme.

The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing.

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio-temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore-assisted light inactivation (CALI) of proteins, as well as for photo-induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini-antibodies or selective scaffold proteins and photo-induced cytotoxicity of miniSOG, which is particularly promising for selective non-invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven inflammation.

SEE PLUCHINO AND PERUZZOTTI-JAMETTI DOI101093/AWW266 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Myelin regeneration (remyelination) is a spontaneous process that occurs following central nervous system demyelination. However, for reasons that remain poorly understood, remyelination fails in the progressive phase of multiple sclerosis. Emerging evidence indicates that alternatively activated macrophages in central nervous system lesions are required for oligodendrocyte progenitor differentiation into remyelinating oligodendrocytes. Here, we show that an alternatively activated macrophage secreted enzyme, interleukin-four induced one (IL4I1), is upregulated at the onset of inflammation resolution and remyelination in mouse central nervous system lesions after lysolecithin-induced focal demyelination. Focal demyelination in mice lacking IL4I1 or interleukin 4 receptor alpha (IL4Rα) results in increased proinflammatory macrophage density, remyelination impairment, and axonal injury in central nervous system lesions. Conversely, recombinant IL4I1 administration into central nervous system lesions reduces proinflammatory macrophage density, enhances remyelination, and rescues remyelination impairment in IL4Rα deficient mice. We find that IL4I1 does not directly affect oligodendrocyte differentiation, but modulates inflammation by reducing interferon gamma and IL17 expression in lesioned central nervous system tissues, and in activated T cells from splenocyte cultures. Remarkably, intravenous injection of IL4I1 into mice with experimental autoimmune encephalomyelitis at disease onset significantly reversed disease severity, resulting in recovery from hindlimb paralysis. Analysis of post-mortem tissues reveals reduced axonal dystrophy in spinal cord, and decreased CD4+ T cell populations in spinal cord and spleen tissues. These results indicate that IL4I1 modulates inflammation by regulating T cell expansion, thereby permitting the formation of a favourable environment in the central nervous system tissue for remyelination. Therefore, IL4I1 is a potentially novel therapeutic for promoting central nervous system repair in multiple sclerosis.

A new anticancer toxin based on HER2/neu-specific DARPin and photoactive flavoprotein miniSOG.

Cytotoxic effects of a new targeted phototoxin DARPin-miniSOG and mechanism of its action were investigated in vitro. It was determined that DARPin-miniSOG causes light-induced death of HER2/neu-positive cancer cells (IC50 0.8 µM). Treatment of the cells with DARPin-miniSOG in the presence of ascorbic acid eliminated the light-induced cytotoxic action of the protein. This observation suggests the involvement of oxidative stress in the mechanism of the phototoxin action. DNA fragmentation analysis, caspase-3 activity assay and PI-staining of HER2/neu-positive cancer cells treated with DARPin-miniSOG indicated that phototoxin induces necrotic cell death under blue light illumination. Co-localization analysis showed that DARPin-miniSOG accumulates mostly in endosomes and lysosomes.

Genetically encoded immunophotosensitizer 4D5scFv-miniSOG is a highly selective agent for targeted photokilling of tumor cells in vitro.

Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.

The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver.

UNLABELLED: Cytochrome P450 2E1 (CYP2E1) is clinically and toxicologically important and exhibits 24-hour periodicity in its activity. In the present study, we investigated whether hepatic nuclear factor-1alpha (HNF-1alpha) and clock genes with a striking 24-hour rhythm in mouse liver contributed to the 24-hour regulation of CYP2E1 expression. The results demonstrated that the expression of CYP2E1 messenger RNA (mRNA) in the liver was affected by HNF-1alpha and the circadian organization of molecular clocks. The mRNA levels of CYP2E1 in the liver increased from the late light phase to the early dark phase. Luciferase reporter gene analysis revealed that HNF-1alpha activated CYP2E1 promoter activity, which was restricted by CRY1, a member of the circadian organization of molecular clocks. Repressor activity of CRY1 was observed on the HNF-1alpha binding site of the CYP2E1 promoter region with mutated E-box. Serum shock induced approximately 24-hour oscillation in CYP2E1 mRNA in HepG2. Transfection of HNF-1alpha and CRY1 small interfering RNA dampened the oscillation of CYP2E1 mRNA in HepG2. Chromatin immunoprecipitation assay in the CYP2E1 promoter indicated that HNF-1alpha binding to the CYP2E1 promoter increased from the late light phase to the early dark phase. Using the chromatin immunoprecipitation reimmunoprecipitation assay, time-dependent differences were demonstrated for CRY1 protein interaction with HNF-1alpha transcriptional complexes, including coactivator p300 on the HNF-1alpha binding site in the CYP2E1 promoter. CONCLUSION: Our results suggest that the transcription activator of HNF-1alpha acts periodically and the negative limbs of molecular clocks periodically inhibit CYP2E1 transcription, resulting in the 24-hour rhythm of its mRNA expression.

Circadian- and light-dependent regulation of resting membrane potential and spontaneous action potential firing of Drosophila circadian pacemaker neurons.

The ventral lateral neurons (LNvs) of adult Drosophila brain express oscillating clock proteins and regulate circadian behavior. Whole cell current-clamp recordings of large LNvs in freshly dissected Drosophila whole brain preparations reveal two spontaneous activity patterns that correlate with two underlying patterns of oscillating membrane potential: tonic and burst firing of sodium-dependent action potentials. Resting membrane potential and spontaneous action potential firing are rapidly and reversibly regulated by acute changes in light intensity. The LNv electrophysiological light response is attenuated, but not abolished, in cry(b) mutant flies hypomorphic for the cell-autonomous light-sensing protein CRYPTOCHROME. The electrical activity of the large LNv is circadian regulated, as shown by significantly higher resting membrane potential and frequency of spontaneous action potential firing rate and burst firing pattern during circadian subjective day relative to subjective night. The circadian regulation of membrane potential, spontaneous action potential firing frequency, and pattern of Drosophila large LNvs closely resemble mammalian circadian neuron electrical characteristics, suggesting a general evolutionary conservation of both physiological and molecular oscillator mechanisms in pacemaker neurons.

The mitochondrial death pathway and cardiac myocyte apoptosis.

Apoptosis has been causally linked to the pathogenesis of myocardial infarction and heart failure in rodent models. This death process is mediated by two central pathways, an extrinsic pathway involving cell surface receptors and an intrinsic pathway using mitochondria and the endoplasmic reticulum. Each of these pathways has been implicated in myocardial pathology. In this review, we summarize recent advances in the understanding of the intrinsic pathway and how it relates to cardiac myocyte death and heart disease.

HFR1, a putative bHLH transcription factor, mediates both phytochrome A and cryptochrome signalling.

Plants are very sensitive to their light environment. They use cryptochromes and phytochromes to scan the light spectrum. Those two families of photoreceptors mediate a number of similar physiological responses. The putative bHLH (basic Helix Loop Helix) transcription factor long hypocotyl in far-red (HFR1) is important for a subset of phytochrome A (phyA)-mediated light responses. Interestingly, hfr1 alleles also have reduced de-etiolation responses, including hypocotyl growth, cotyledon opening and anthocyanin accumulation, when grown in blue light. This phenotype is particularly apparent under high fluence rates. The analysis of double mutants between hfr1 and different blue light photoreceptor mutants demonstrates that, in addition to its role in phyA signalling, HFR1 is a component of cryptochrome 1 (cry1)-mediated light signalling. Moreover, HFR1 mRNA levels are high both in blue and in far-red light but low in red light. These results identify HFR1 as a positively acting component of cry1 signalling and indicate that HFR1 integrates light signals from both phyA and cry1.

The mitochondrial apoptosis-inducing factor plays a role in E2F-1-induced apoptosis in human colon cancer cells.

BACKGROUND: Overexpression of the transcription factor E2F-1 provokes apoptosis in cancer cells; the mechanism, however, is not completely understood. We sought to evaluate E2F-1 gene therapy in human colon cancer and to investigate the apoptotic pathway involved. METHODS: Adenoviral vectors were used to transfect the E2F-1 gene (Ad5E2F-1) or the control gene luciferase (Ad5Luc) into four human colon carcinoma cell lines. Apoptosis was confirmed by flow cytometry and poly (ADP-ribose) polymerase cleavage. Expression of apoptotic factors was determined with Western blot analysis. Inhibitory assays were used to determine the involvement of caspases in the apoptotic pathway. RESULTS: Overexpression of E2F-1 was evident in all cells treated with Ad5E2F-1; upregulation of Bcl-2, and activation of caspases were noted. The apoptosis-inducing factor in the cytosolic fraction was markedly upregulated after Ad5E2F-1 treatment. E2F-1 overexpression inhibited proliferation and induced significant apoptosis in all cell lines (P <.005). This apoptotic response could be only partially blocked by caspase inhibitors. CONCLUSIONS: These findings demonstrate that E2F-1 induces apoptosis and inhibits proliferation in human colon cancer cell lines. The marked upregulation of apoptosis-inducing factor and the fact that E2F-1-induced apoptosis is incompletely blocked by caspase inhibitors suggest a caspase-independent pathway of E2F-1-mediated apoptosis, reported here for the first time.

Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes.

The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70 degrees ) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-alpha, anti-Fas ligand, or anti-transforming growth factor-beta antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68,000-70,000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.

Alternative splicing yields novel BMAL2 variants: tissue distribution and functional characterization.

The BMAL2 gene encodes a member of the basic helix-loop-helix PER-ARNT-SIM family of transcription factors, which control diverse physiological processes including circadian rhythms. We identified four novel human BMAL2 transcripts that differ by alternative splicing within their NH2-terminal regions. Divergent expression of these and previously reported transcripts was observed among human tissues. The functional consequences of alternative splicing for transcriptional activation by CLOCK:BMAL2 heterodimers were assessed using luciferase reporter gene constructs that contained one of three diurnally regulated promoters, namely, those of the mouse period1, mouse vasopressin, and human plasminogen activator inhibitor-1 genes. These studies revealed that alternative splicing generates BMAL2 isoforms possessing high, medium, low, or no transcriptional activity. Similar results were obtained with each promoter, suggesting that alternative splicing may influence the amplitudes of both central and peripheral oscillators. Indeed, alternative splicing of BMAL2 may provide tissues with a rheostat capable of regulating CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities to accommodate varied metabolic demands and physiological roles.

Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta.

The goal of this study was to determine whether the intracellular distribution of the proapoptotic enzyme glycogen synthase kinase-3 beta (GSK-3 beta) is dynamically regulated by conditions that activate apoptotic signaling cascades. In untreated human neuroblastoma SH-SY5Y cells, GSK-3 beta was predominantly cytosolic, although a low level was also detected in the nucleus. The nuclear level of GSK-3 beta was rapidly increased after exposure of cells to serum-free media, heat shock, or staurosporine. Although each of these conditions caused changes in the serine 9 and/or tyrosine phosphorylation of GSK-3 beta, neither of these modifications was correlated with nuclear accumulation of GSK-3 beta. Heat shock and staurosporine treatments increased nuclear GSK-3 beta prior to activation of caspase-9 and caspase-3, and this nuclear accumulation of GSK-3 beta was unaltered by pretreatment with a general caspase inhibitor. The GSK-3 beta inhibitor lithium did not alter heat shock-induced nuclear accumulation of GSK-3 beta but increased the nuclear level of cyclin D1, indicating that cyclin D1 is a substrate of nuclear GSK-3 beta. Thus, the intracellular distribution of GSK-3 beta is dynamically regulated by signaling cascades, and apoptotic stimuli cause increased nuclear levels of GSK-3 beta, which facilitates interactions with nuclear substrates.

Active roles of caspase-3 in human gastric carcinoma cell death by apoptosis inducing nucleosides from CD57+HLA-DRbright natural suppressor cell line.

The six apoptosis inducing nucleosides (AINs), which were isolated by high performance liquid chromatography from CD57+HLA-DRbright natural suppressor (57.DR-NS) cell cultures, induced apoptosis in human gastric carcinoma (GCIY) cells demonstrating the accumulation of sub-G1 DNA content and morphological changes. By means of DNA unwinding assay, it has been revealed that DNA strand breaks were initially involved in the apoptotic cell death of GCIY cells treated with AINs followed by activation of caspase cascades, especially caspase-3. Actually, the cleavage of fluorogenic substrate for caspase-3 was identified in the reaction. Whereas, the addition of caspase-3 inhibitor into the reaction prevented the cleavage of fluorogenic substrate for caspase-3 and resulted in the blockage of the sub-G1 DNA accumulation and DNA fragmentation as apoptotic signals. Thus, it was definitely elucidated that the activation of caspase-3 displayed a key feature during AIN-induced apoptosis in GCIY cells.

Characterization of factors inducing apoptosis in thymocytes of mice bearing a transplantable T-cell lymphoma of spontaneous origin.

It has been observed that the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin in murine host, designated as Dalton's lymphoma (DL), induces inhibition of various immune responses and is associated with an involution of thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes caused by induction of apoptosis with a decrease of CD4+CD8+, CD4+CD8- and CD4-CD8+ thymocytes. Here, we report that serum of DL-bearing mice contains soluble factors capable of inducing thymocyte apoptosis, the effectiveness of which increases with the progression of tumor growth. A decline of essential cytokines and hormones in the body due to their depletion by DL cells, which being a T-cell phenotype may have similar growth factor requirements, is ruled out by our results, suggesting additional apoptosis-inducing factors to be present in the tumor serum. Partial characterization of the serum to identify the biochemical nature of the putative serum-borne apoptosis inducing factor(s) showed that the same was proteinaceous. Further analysis of the sera of normal and DL-bearing mice by gel filtration using fast protein liquid chromatography (FPLC) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that protein profile in the two sera differed quantitatively as well as qualitatively. FPLC analysis could resolve six peaks in both the sera, out of which the peak containing protein(s) in the range of MW 35 kD showed a higher magnitude and apoptotic activity followed by peaks containing proteins of MW in the range of 67 and 116 kD respectively as compared to that of the corresponding peaks in the normal serum. These observations were also confirmed by SDS-PAGE, with the resolution of additional proteins in the range of 25-26 kD which were found to be absent in normal serum. Further, the paper discusses different possible factors that could be associated with the progression of DL growth.

Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis.

Apoptosis-inducing factor (AIF) is encoded by one single gene located on the X chromosome. AIF is ubiquitously expressed, both in normal tissues and in a variety of cancer cell lines. The AIF precursor is synthesized in the cytosol and is imported into mitochondria. The mature AIF protein, a flavoprotein (prosthetic group: flavine adenine dinucleotide) with significant homology to plant ascorbate reductases and bacterial NADH oxidases, is normally confined to the mitochondrial intermembrane space. In a variety of different apoptosis-inducing conditions, AIF translocates through the outer mitochondrial membrane to the cytosol and to the nucleus. Ectopic (extra-mitochondrial) AIF induces nuclear chromatin condensation, as well as large scale ( approximately 50 kb) DNA fragmentation. Thus, similar to cytochrome c, AIF is a phylogenetically old, bifunctional protein with an electron acceptor/donor (oxidoreductase) function and a second apoptogenic function. In contrast to cytochrome c, however, AIF acts in a caspase-independent fashion. The molecular mechanisms via which AIF induces apoptosis are discussed.

Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine.

Clozapine, an atypical antipsychotic used in the treatment of refractory schizophrenia, causes neutropenia and agranulocytosis in 3 and 0.8% of patients, respectively. Clozapine undergoes bioactivation to a chemically reactive nitrenium ion, which has been shown to cause neutrophil cytotoxicity. To define further the mechanism of cell death, we have investigated the toxicity of clozapine, its stable metabolites, and its chemically reactive nitrenium ion to neutrophils and lymphocytes. Clozapine was able to induce neutrophil apoptosis at therapeutic concentrations (1-3 microM) only when it was bioactivated to the nitrenium ion. The parent drug caused apoptosis at supratherapeutic concentrations (100-300 microM) only. Neutrophil apoptosis induced by the nitrenium ion, but not by the parent drug itself, was inhibited by antioxidants and genistein and was accompanied by cell surface haptenation (assessed by flow cytometry) and glutathione depletion. Dual-color flow cytometry showed that neutrophils that were haptenated were the same cells that underwent apoptosis. No apoptosis of lymphocytes was evident with the nitrenium ion or the parent drug, despite the fact that the former caused cell surface haptenation, glutathione depletion, and loss of membrane integrity. Demethylclozapine, the major stable metabolite in vivo, showed a profile that was similar to, although less marked than that observed with clozapine. N-oxidation of clozapine or replacement of the nitrogen (at position 5) by sulfur produced compounds that were entirely nontoxic to neutrophils. In conclusion, the findings of the study expand on potential mechanisms of clozapine-induced cytotoxicity, which may be of relevance to the major forms of toxicity encountered in patients taking this drug.