ATP7A-dependent copper sequestration contributes to termination of ß-CATENIN signaling during early adipogenesis.

OBJECTIVES: Adipocyte fate determination is tightly regulated by extrinsic signaling pathways and intrinsic metabolic and morphologic changes that maintain adipose tissue function. Copper (Cu) homeostasis is required for the normal metabolism of mature adipocytes, whereas the role of Cu in adipogenesis is unclear. METHODS: To determine the role of Cu is adipocytes differentiation, we used 3T3-L1 adipocytes, immunocytochemistry, X-ray fluorescence, mass-spectrometry, pharmacological treatments, and manipulations of copper levels. RESULTS: In differentiating 3T3-L1 cells, adipogenic stimuli trigger the upregulation and trafficking of the Cu transporter Atp7a, thus causing Cu redistribution from the cytosol to vesicles. Disrupting Cu homeostasis by the deletion of Atp7a results in Cu elevation and inhibition of adipogenesis. The upregulation of C/EBPß, an initial step of adipogenesis, is not affected in Atp7a-/- cells, whereas the subsequent upregulation of PPARγ is inhibited. Comparison of changes in the Atp7a-/- and wild type cells proteomes during early adipogenesis revealed stabilization of ß-catenin, a negative regulator of adipogenesis. Cu chelation, or overexpression of the Cu transporter ATP7B in Atp7a-/- cells, restored ß-catenin down-regulation and intracellular targeting. CONCLUSIONS: Cu buffering during early adipogenesis contributes to termination of ß-catenin signaling. Abnormal upregulation of ß-catenin was also observed in vivo in the livers of Atp7b-/- mice, which accumulate Cu, suggesting a tissue-independent crosstalk between Cu homeostasis and the Wnt/ß-catenin pathway. These results point to a new regulatory role of Cu in adipocytes and contribute to better understanding of human disorders of Cu misbalance.

Faraday effect in magnetoplasmonic nanostructures with spatial modulation of magnetization.

For the first time, to the best of our knowledge, the properties of the Faraday effect are addressed in a magnetoplasmonic nanostructure with nonuniform spatial distribution of the magnetization. It is shown that the coincidence in period and phase between magnetization modulation and the field of the optical mode provides the resonant enhancement of the Faraday effect. This effect is observed for both the surface plasmon polariton and waveguide modes.

Biological Activity of Agaricinic Acid Nanoparticles against Human Hepatoma HepG2 Cells.

A stable preparation of agaricinic acid nanoparticles was obtained. The mean hydrodynamic size of nanoparticles according to photon correlation spectroscopy was 200 nm and zeta potential was -57 mV. Cytotoxic activity of agaricinic acid nanoparticles against human HepG2 hepatoma cells was evaluated. Nanoparticles with a low concentration of agaricinic acid stimulated and with high concentration - suppressed metabolic activity and viability of hepatoma cells. The EC50 for the stimulating effect was 32.8 µg/ml, and the IC50=602.1 mg/ml. The preparation of agaricinic acid nanoparticles can be used in medicine as a potential antitumor agent.

ROS and RNS signalling: adaptive redox switches through oxidative/nitrosative protein modifications.

Over the last decade, a dual character of cell response to oxidative stress, eustress versus distress, has become increasingly recognized. A growing body of evidence indicates that under physiological conditions, low concentrations of reactive oxygen and nitrogen species (RONS) maintained by the activity of endogenous antioxidant system (AOS) allow reversible oxidative/nitrosative modifications of key redox-sensitive residues in regulatory proteins. The reversibility of redox modifications such as Cys S-sulphenylation/S-glutathionylation/S-nitrosylation/S-persulphidation and disulphide bond formation, or Tyr nitration, which occur through electrophilic attack of RONS to nucleophilic groups in amino acid residues provides redox switches in the activities of signalling proteins. Key requirement for the involvement of the redox modifications in RONS signalling including ROS-MAPK, ROS-PI3K/Akt, and RNS-TNF-α/NF-kB signalling is their specificity provided by a residue microenvironment and reaction kinetics. Glutathione, glutathione peroxidases, peroxiredoxins, thioredoxin, glutathione reductases, and glutaredoxins modulate RONS level and cell signalling, while some of the modulators (glutathione, glutathione peroxidases and peroxiredoxins) are themselves targets for redox modifications. Additionally, gene expression, activities of transcription factors, and epigenetic pathways are also under redox regulation. The present review focuses on RONS sources (NADPH-oxidases, mitochondrial electron-transportation chain (ETC), nitric oxide synthase (NOS), etc.), and their cross-talks, which influence reversible redox modifications of proteins as physiological phenomenon attained by living cells during the evolution to control cell signalling in the oxygen-enriched environment. We discussed recent advances in investigation of mechanisms of protein redox modifications and adaptive redox switches such as MAPK/PI3K/PTEN, Nrf2/Keap1, and NF-κB/IκB, powerful regulators of numerous physiological processes, also implicated in various diseases.

Estimating thyroid masses for children, infants, and fetuses in Ukraine exposed to (131)I from the Chernobyl accident.

For the purpose of improving retrospective internal thyroid dose estimations for children and adolescents following the Chernobyl accident, age- and gender-dependent thyroid masses have been estimated for the children of Kiev and Zhytomyr oblasts, which are two of the most contaminated regions of Northern Ukraine. For children ages 6-16 y, the thyroid masses were based on the measurements by ultrasound of the thyroid volumes of about 60,000 children performed by the Sasakawa Memorial Health Foundation in the 1990s. For children aged 0 to 36 mo, because thyroid mass values for Ukrainian children were not found in the literature, autopsies were performed for the specific purpose of this paper. Thyroid mass values for children aged 3-5 and 17-18 y were either interpolated or extrapolated from the measured data sets. The results for children aged 6-16 y indicate that the thyroid masses of rural children are, on average, slightly higher (by about 8%) than the thyroid masses of urban children. The geometric means of the thyroid masses were estimated as 5.2 g, 9.0 g, and 15.8 g for boys and 5.2 g, 9.4 g, and 16.0 g for girls aged 5, 10, and 15 y, respectively. Those values are greater than the reference values that ICRP recommends for iodine-sufficient populations, thus reflecting the fact that the northern part of Ukraine is iodine-deficient.

Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson's disease.

The ideal gene therapy for metabolical liver disorders would target hepatocytes before the onset of disease and be durable, non-toxic and non-immunogenic. Early gestational gene transfer can achieve such goals. Here, we demonstrate that prenatal gene transfer of human Atp7b reduces liver pathology and improves biochemical markers in Atp7b(-/-) mice, a murine model of Wilson's disease (WD). Following prenatal injection of lentivirus vector containing the human Atp7b gene under the transcriptional control of a liver-specific promoter, the full-length ATP7B was detectable in mouse livers for the entire duration of experiments (20 weeks after birth). In contrast to a marked pathology in non-injected animals, livers from age-matched treated mice consistently demonstrated normal gross and histological morphology. Hepatic copper content was decreased in the majority of treated mice, although remaining copper levels varied. Improvement of hepatic copper metabolism was further apparent from the presence of copper-bound ceruloplasmin in the sera and normalization of the mRNA levels for HMG CoA-reductase. With this approach, the complete loss of copper transport function can be ameliorated, as evident from phenotypical improvement in treated Atp7b(-/-) mice. This study provides proof of principle for in utero gene therapy in WD and other liver-based enzyme deficiencies.

Endostatin: current concepts about its biological role and mechanisms of action.

Endogenous inhibitors of angiogenesis are proved to be a major factor preventing the emergence of clinically manifested stages of human cancer. The protein endostatin, a 20-kD proteolytic fragment of type XVIII collagen, is one of the most active natural inhibitors of angiogenesis. Endostatin specifically inhibits the in vitro and in vivo proliferation of endothelial cells, inducing their apoptosis through inhibition of cyclin D1. On the surface of endothelial cells, endostatin binds with the integrin alpha(5)beta(1) that activates the Src-kinase pathway. The binding of endostatin with integrins also down-regulates the activity of RhoA GTPase and inhibits signaling pathways mediated by small kinases of the Ras and Raf families. All these events promote disassembly of the actin cytoskeleton, disorders in cell-matrix interactions, and decrease in endotheliocyte mobility, i.e., promote the suppression of angiogenesis. Endostatin displays a high antitumor activity in vivo: it inhibits the progression of more than 60 types of tumors. This review summarizes results of numerous studies concerning the biological activity and action mechanism of endostatin.

Tumor angiogenesis inhibitors.

Formation of the blood supply system is a critical step in malignant transformation of neoplasms which results in the penetration of tumor cells into neighboring tissues and metastatic growth. Significant progress in the elucidation of mechanisms underlying tumor angiogenesis and the discovery of a great diversity of biomolecules involved in its regulation have culminated in the development of a radically new approach to antitumor therapy based on the search for efficient inhibitors of tumor angiogenesis. This review is devoted to the analysis of action mechanisms and expression of the major endogenous inhibitors involved in regulation of tumor and physiological angiogenesis. The antiangiogenic effects of the majority of currently known synthetic inhibitors are considered in the context of their roles in the main steps of tumor angiogenesis. Possible applications of antiangiogenic therapy in the chemotherapy of cancer diseases are discussed.

Molecular mechanisms of tumor angiogenesis.

The maintenance of growth of malignant tumors is closely related with the development of the vascular network supplying the tumor with blood. The vascularization of tumor tissue is similar to physiological angiogenesis, but in tumors it has some specific features. During the last 25 years a vast number of biomolecules have been found and described which are involved in the regulation of tumor angiogenesis. This review considers the action mechanisms and specific features of expression of the main angiogenic growth factors, such as the vascular endothelium growth factor (VEGF), angiopoietins (Ang-1, Ang-2), and the basic fibroblast growth factor (bFGF). The roles of cytokines, growth factors, proteolytic enzymes, and cell adhesion molecules in the regulation of the key steps of blood vessel generation in the tumor are considered. The significance of angiogenesis in the treatment of oncological diseases and possible approaches for inhibition of the regulatory signals of angiogenic factors are discussed.

Function and regulation of the mammalian copper-transporting ATPases: insights from biochemical and cell biological approaches.

Copper is an essential trace element that plays a very important role in cell physiology. In humans, disruption of normal copper homeostasis leads to severe disorders, such as Menkes disease and Wilson's disease. Recent genetic, cell biological, and biochemical studies have begun to dissect the molecular mechanisms involved in transmembrane transport and intracellular distribution of copper in mammalian cells. In this review, we summarize the advances that have been made in understanding of structure, function, and regulation of the key human copper transporters, the Menkes disease and Wilson's disease proteins.

The Wilson's disease protein expressed in Sf9 cells is phosphorylated.

The Wilson's disease protein (WNDP), a copper transporter, is a crucial mediator of copper homoeostasis in mammalian cells. We recently found that changes in copper concentration regulate the phosphorylation level of WNDP. WNDP phosphorylation was observed in several mammalian cell lines, suggesting that a common phosphorylation pathway exists in these cells. Here we demonstrate that WNDP expressed in Sf9 insect cells is also phosphorylated, as evidenced by metabolic labelling of these cells with [(32)P]P(i). Because the baculovirus system allows us to generate large amounts of protein, we are using this expression method to isolate WNDP and map the sites of WNDP phosphorylation. The identification of phosphorylation sites is the first step towards understanding the physiological role of WNDP phosphorylation.

Cytotoxic and antitumor activities of doxorubicin conjugates with the epidermal growth factor and its receptor-binding fragment.

The epidermal growth factor (EGF) receptor is expressed at high levels on many types of tumor cells, such as squamous carcinoma, breast cancer and endothelial cells. We studied targeted delivery of the anticancer drug doxorubicin (DOX) using EGF and its receptor-binding fragment (EGFfr) to cells able to overexpress EGF receptors. EGF-DOX and EGFfr-DOX conjugates were synthesized via a glutaraldehyde bridge. The cytotoxic activities (CTA) of the conjugates were studied in vitro in different tumor cell lines (MCF-7Wt, MCF-7AdrR, B16) and endothelial cells using MTT-test. The antitumor effects of the conjugates were examined in vivo in mice with a subcutaneous B16 model. In the case of MCF-7Wt cells, CTA of EGF-DOX and EGFfr-DOX conjugates exceeded 7.7- and 68-fold that of free DOX. Besides, the conjugates effectively decreased the drug resistance of MCF-7AdrR cells. CTA of the conjugates against endothelial cell cultures markedly exceeded that of free DOX. It is of note that proliferating endothelial cells were much more sensitive to the effects of the conjugates than confluent endothelial cells. Administration of EGF-DOX and EGFfr-DOX conjugates significantly inhibited tumor growth and increased the mean life span of experimental animals by 46 and 48.5%, respectively.

Copper specifically regulates intracellular phosphorylation of the Wilson's disease protein, a human copper-transporting ATPase.

Copper is a trace element essential for normal cell homeostasis. The major physiological role of copper is to serve as a cofactor to a number of key metabolic enzymes. In humans, genetic defects of copper distribution, such as Wilson's disease, lead to severe pathologies, including neurodegeneration, liver lesions, and behavior abnormalities. Here, we demonstrate that, in addition to its role as a cofactor, copper can regulate important post-translational events such as protein phosphorylation. Specifically, in human cells copper modulates phosphorylation of a key copper transporter, the Wilson's disease protein (WNDP). Copper-induced phosphorylation of WNDP is rapid, specific, and reversible and correlates with the intracellular location of this copper transporter. WNDP is found to have at least two phosphorylation sites, a basal phosphorylation site and a site modified in response to increased copper concentration. Comparative analysis of WNDP, the WNDP pineal isoform, and WNDP C-terminal truncation mutants revealed that the basal phosphorylation site is located in the C-terminal Ser(796)-Tyr(1384) region of WNDP. The copper-induced phosphorylation appears to require the presence of the functional N-terminal domain of this protein. The novel physiological role of copper as a modulator of protein phosphorylation could be central to understanding how copper transport is regulated in mammalian cells.

The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner.

Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.

Cytotoxic activity, accumulation, and intracellular distribution of anthracycline antibiotics and their conjugates with the epidermal growth factor in sensitive and resistant MCF-7 cells.

Cytotoxic activities, accumulation levels and dynamics, and intracellular distribution of the anthracycline antibiotics doxorubicin (DR) and carminomycin (CM) in the free forms or within conjugates with the epidermal growth factor (EGF) were for the first time compared in human breast carcinoma cell lines MCF-7Wt and MCF-7AdrR. The cytotoxic activities of DR and CM conjugates with EGF were higher than the cytotoxic activities of the free antibiotics in both cell lines. The accumulation levels of the free anthracyclines in both cell lines were lower than those of the conjugates and significantly depended on the cell sensitivities to the antibiotics. On receptor-mediated endocytosis of the anthracycline-EGF conjugates, the accumulation levels did not significantly depend on the cell sensitivities to the antibiotics. Both DR and CM, either free or conjugated with EGF, were mainly accumulated in nuclei. The free drugs were accumulated more rapidly, and the accumulation rates of both free and EGF-conjugated CM were higher than those of DR preparations. The intracellular distribution of the free antibiotics significantly depended on the cell sensitivities to the anthracyclines, whereas the cell sensitivities had no effect on the distribution of the conjugates between the nucleus and cytoplasm. The rate of intracellular degradation of DR and CM delivered to target cells within conjugates with EGF was twice lower than that of the free antibiotics. The difference in the accumulation levels and dynamics and in the intracellular distribution of the free and conjugated DR and CM is likely to underlie the higher cytotoxic activities of the anthracycline conjugates with EGF compared to the free drugs.

Antitumor activity of alpha-fetoprotein conjugate with doxorubicin in vitro and in vivo.

alpha-Fetoprotein (AFP) was conjugated with doxorubicin (DR) using glutaraldehyde as a cross-linking agent. The protein/DR molar ratio in the conjugate is 1 : 2. Cytotoxic activities (CTA) of the AFP-DR conjugate and of the free DR were compared using human mammary gland carcinoma cells, both DR-sensitive (MCF-7Wt) and DR-resistant (MCF-7AdrR). The CTA of the AFP-DR conjugate was fivefold higher than the CTA of the free DR for sensitive cells of the MCF-7Wt line and sevenfold higher for resistant cells of the MCF-7AdrR line. The CTA of the AFP-DR conjugate was also studied in vitro using the proliferating endothelium taken for a model of endothelial cell lining of blood vessels that supply the tumor. The AFP-DR conjugate was shown to have a high CTA for the endothelial cells (IC50 = 2.5 nM); thus, the conjugate is suggested to manifest an anti-angiogenic effect in vivo. The antitumor activity of the AFP-DR conjugate was studied using mice with inoculated melanoma B16 tumors. The treatment of animals significantly inhibited the tumor growth (>97%) and increased by 60% the mean life span of the animals compared to the control. The high antitumor efficiency of the AFP-DR conjugate and the possibility to significantly decrease the tumor cell resistance to DR make this conjugate a promising chemotherapeutic agent.

Antitumor activity of alpha fetoprotein and epidermal growth factor conjugates in vitro and in vivo.

Conjugates of carminomycin (Cm) with alpha-fetoprotein (AFP) and epidermal growth factor (EGF) were prepared and their cytotoxic activities were studied in vitro. Both conjugates showed cytotoxic activity which exceeded that of free Cm in tumor cell cultures of MCF-7, SKOV3, QOS, P388 and B16 cells. The antitumor effects of the conjugates were studied in vivo in mice with subcutaneous tumors of B16 and P388 cells. The Cm-AFP and Cm-EGF conjugates inhibited tumor growth and noticeably increased the mean life span in experimental animals. Our results suggest that the therapeutic activity of Cm can be significantly enhanced by conjugation to AFP or EGF.

Expression of ZntA, a zinc-transporting P1-type ATPase, is specifically regulated by zinc and cadmium.

All cells have developed various mechanisms to regulate precisely the availability of important micronutrients such as zinc and copper; in many cells, this regulation is mediated by P1-type ATPases. Most of the P1-ATPases have been described very recently, and little is known about their molecular mechanism and regulation. Here, we demonstrate that the expression of ZntA, a Zn,Cd-transporting P-type ATPase of Escherichia coli, is specifically regulated by the transported cations, cadmium and zinc. Nickel, cobalt, and copper did not induce the expression of ZntA, even when present at concentrations as high as 0.6-1 mM. The effect of zinc and cadmium on the ZntA expression is concentration dependent, the apparent Km for Cd (19 microM) being markedly lower than that for Zn (100 microM). This metal selectivity is opposite to the known metal selectivity of transport by ZntA. Thus, we speculate that, to maintain zinc concentrations in the cell in the presence of cadmium, ZntA probably interacts with other proteins that modulate the ZntA selectivity towards transported cations.

Cytotoxic action of conjugates of alpha-fetoprotein and epidermal growth factor with photoheme, chlorines, and phthalocyanines.

Covalently bound conjugates of alpha-fetoprotein (AFP) and epidermal growth factor (EGF) with photoheme (PH), 3-desvinyl-3-formylchlorine p6 (Chl p6), chlorine e6 (Chl e6), aluminum disulfochloride phthalocyanine (PC(Al)), and cobalt octa-4,5-carboxyphthalocyanine (teraphthal, TP(Co)) were synthesized. Their molar ratios were 1:4 for AFP-cytotoxin conjugates (cf. 1:10 for AFP-TP(Co)) and 1:2 for EGF conjugates (cf. 1:1 for EGF-PC(Al)). Dark toxicity of both protein conjugates with PH, chlorines, and PC(Al) was much lower than their phototoxicity. Studies on phototoxicity demonstrated that PC(Al) conjugates with AFP and EGF and also EGF-Chl p6 were the most effective. The cytotoxic activity (CTA) of AFP-PC(Al) and EGF-Chl p6 was 80% and of EGF-PC(Al) 64% higher than the CTA of the free drugs. Conjugates with TP(Co) were much more toxic on their activation with ascorbic acid (AA): in the presence of AA the CTA of AFP-TP(Co) and of EGF-TP(Co) was 19 and 61.1% higher, respectively, than the CTA of the free TP(Co).

Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation.

The Atp7b protein is a copper-transporting ATPase expressed predominantly in the liver and to a lesser extent in most other tissues. Mutations in the ATP7B gene lead to Wilson disease, a copper toxicity disorder characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neuro-logical abnormalities. Using homologous recombination to disrupt the normal translation of ATP7B, we have generated a strain of mice that are homozygous mutants (null) for the Wilson disease gene. The ATP7B null mice display a gradual accumulation of hepatic copper that increases to a level 60-fold greater than normal by 5 months of age. An increase in copper concentration was also observed in the kidney, brain, placenta and lactating mammary glands of homo-zygous mutants, although milk from the mutant glands was copper deficient. Morphological abnormalities resembling cirrhosis developed in the majority of the livers from homozygous mutants older than 7 months of age. Progeny of the homozygous mutant females demonstrated neurological abnormalities and growth retardation characteristic of copper deficiency. Copper concentration in the livers of the newborn homozygous null mutants was decreased dramatically. In summary, inactivation of the murine ATP7B gene produces a form of cirrhotic liver disease that resembles Wilson disease in humans and the 'toxic milk' phenotype in the mouse.