Biochemistry (Moscow) (v.73, #7)

Interaction of cells with extracellular matrix (ECM) largely defines migration capacity of cells and ways of their dissemination in normal tissue processes and during tumor progression. We review current knowledge about structure of cell adhesions with ECM and their alterations during carcinogenesis. We analyze how changes in structure of cell—matrix adhesions and ECM itself lead to acquisition of neoplastic properties by cells. Modern concepts of tumor cell motility and changes in the relationships of cells with ECM during tumor development are presented. Contemporary approaches for influencing the cell—ECM adhesion structures for inhibition of invasion and metastasis are briefly discussed.
Keywords: focal adhesions; actin cytoskeleton; extracellular matrix; cell migration; invasion

Cadherin-mediated cell—cell adhesion defines the integrity of most tissues. Cell—cell adherens junctions are dynamic structures whose functional state is regulated by interactions of cadherin with β-catenin, p120, and actin cytoskeleton structures. Small GTPases of the Rho family and GTPase Rap1 play the central role in the formation and maintenance of cell—cell adhesion. Aberrant activation of signaling pathways, transcriptional repression of the E-cadherin gene, ectopic expression of N-cadherin, and disturbances in regulation of adhesive and transcriptional functions of β-catenin stimulate tumor progression.
Keywords: cell—cell adherens junctions; cadherins; actin cytoskeleton; epithelial-mesenchymal transformation

Mechanisms of angiogenesis by A. F. Karamysheva (751-762).
Tissue activity of angiogenesis depends on the balance of many stimulating or inhibiting factors. The key signaling system that regulates proliferation and migration of endothelial cells forming the basis of any vessel are vascular endothelium growth factors (VEGF) and their receptors. The VEGF-dependent signaling system is necessary for formation of the embryonic vascular system. Neoangiogenesis during tumor growth is also associated with activation of this signaling system. The biological significance of the effect of such system on the cells depends on the content in tissue of various factors of the VEGF family and their receptors, while in the case of VEGFA it is defined by the ratio of different isoforms of this growth factor. A number of other signaling systems are also involved in regulation of the main steps of vessel formation. The signaling system D114/Notch regulates selection of endothelial cells for beginning of angiogenic expansion by endowing particular properties to endothelial cells leading in this process. An important step in vessel stabilization and maturation is vascular wall formation. Signaling system PDGFB/PDGFRß as well as angiopoietins Ang1, Ang2, and their receptor Tie2 are involved in recruiting mural cells (pericytes and smooth muscle cells). Identification of key molecules involved in the regulation of angiogenesis may provide new possibilities for development of drugs suitable for inhibition of angiogenesis or its stimulation in various pathologies.
Keywords: angiogenesis; vascular endothelium growth factors (VEGF); neuropilins; PDGFB; angiopoietins

Carcinogenesis induced by foreign bodies by T. G. Moizhess (763-775).
This review deals with the contemporary investigations of carcinogenesis induced by foreign bodies. The main attention is given to the interactions of macrophages with an implanted foreign body and their possible role in tumorigenesis.
Keywords: carcinogenesis; foreign body; inflammation; macrophages; cytokines; free radicals

Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium by Mohamed Helmy; Doa Baddar; Mohamed Hisham El’Masry (776-782).
Bacterial colonies were isolated from different Egyptian soil samples. From these isolates, one bacterial species was found to produce siderophore. Using classical and biochemical identification methods, the siderophore producing iso-late was identified as Pseudomonas fluorescens. Based on the affinity of siderophores for metal ions, an affinity chromatography system was designed for the purification of the siderophore in one step. It was possible to isolate 25 mg siderophore per liter of culture media. The purified siderophore was found to exist in two forms of approximately 30 and 90 kD. They are believed to be polymers of several siderophore molecules. Both forms were found to be active against the pathogen Erwinia carotovora var. carotovora, the causal bacteria of soft rot disease on potato tubers. The advantage of this method over other purification methods is that it uses metal ion so it can be applied for the purification of the known types of siderophores. Moreover, the purification is based on affinity chromatography, so the siderophore purity state permits several biotechnological applications without further treatments.
Keywords: affinity chromatography; Erwinia carotovora ; siderophore; soft rot bacterium

Palmitate-induced uncoupling, which involves ADP/ATP and aspartate/glutamate antiporters, has been studied in liver mitochondria of old rats (22–26 months) under conditions of lipid peroxidation and inhibition of oxidative stress by antioxidants—thiourea, Trolox, and ionol. It has been shown that in liver mitochondria of old rats in the absence of antioxidants and under conditions of overproduction of conjugated dienes, the protonophoric uncoupling activity of palmitate is not suppressed by either carboxyatractylate or aspartate used separately. However, the combination of carboxyatractylate and aspartate decreased uncoupling activity of palmitate by 81%. In this case, palmitate-induced uncoupling is limited by a stage insensitive to both carboxyatractylate and aspartate. In the presence of antioxidants, the palmitate-induced protonophoric uncoupling activity is suppressed by either carboxyatractylate or aspartate used separately. Under these conditions, palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter). In the absence of antioxidants, the uncoupling activity of palmitate is not suppressed by ADP either in the absence or in the presence of aspartate. However, in the presence of thiourea, Trolox, or ionol ADP decreased the uncoupling activity of palmitate by 38%. It is concluded that in liver mitochondria of old rats the development of oxidative stress in the presence of physiological substrates of ADP/ATP and aspartate/glutamate antiporters (ADP and aspartate) results in an increase of the protonophoric uncoupling activity of palmitate.
Keywords: liver mitochondria; aging; oxidative stress; reactive oxygen species; fatty acids; uncoupling; ADP/ATP antiporter; aspartate/glutamate antiporter

Implication of α5β1 integrin in invasion of drug-resistant MCF-7/ADR breast carcinoma cells: a role for MMP-2 collagenase by G. E. Morozevich; N. I. Kozlova; I. B. Cheglakov; N. A. Ushakova; M. E. Preobrazhenskaya; A. E. Berman (791-796).
Expression of α5β1 integrin in the drug-resistant MCF-7/ADR breast carcinoma cells was inhibited by treatment of these cells with α5-specific siRNA. The decrease of α5β1 expression resulted in a sharp decrease of expression of MMP-2 collagenase and inhibition of invasion activity of these cells in vitro. Similar decrease of invasion was also observed during inhibition of MMP-2 expression by treatment of these cells with MMP-2-specific siRNA. Inhibition of α5β1 expression was also accompanied by significant decrease in cell content of active (phosphorylated) forms of signal protein kinases Akt and Erk1/2. Inhibition of activity of these kinases by treatment of cells with PI-3K/Akt-specific inhibitor LY294002 or Erk-specific inhibitor PD98059 resulted in inhibition of MMP-2 expression and the decrease of invasion in vitro. These data suggest that α5β1 controls invasion ability of these cells by regulating expression of MMP-2, which involves PI-3K and Erk1/2 protein kinase signaling.
Keywords: integrins; signal protein kinases; matrix proteases; metastasis; invasion

Localization of Mullerian inhibiting substance receptors in various human cancer cell lines by A. V. Rodina; N. V. Gukasova; V. A. Makarov; I. G. Kondrasheva; A. V. Khomyakova; G. A. Posypanova; O. N. Popova; E. Yu. Moskaleva; S. E. Severin (797-805).
Recombinant human MIS (rhMIS) produced in transfected Chinese hamster ovary cells has been purified by immunoaffinity chromatography. In the absence of reducing agents, 140 kD homodimer and several oligomers with molecular masses from 280 to 1000 kD are present. Homodimer, tetramer, and higher-molecular-weight rhMIS fractions reduced survival of tumor cells. For these experiments, FITC-labeled rhMIS was used for binding and endocytosis studies by flow cytometry. Flow cytometry performed on MIS-sensitive cancer cell lines demonstrated specific binding of rhMIS. The majority of rhMIS receptors have cytosolic localization. Thus, the level of MIS receptors on the cell membrane was pro-portional to the content of MIS-binding proteins in the whole cell and defines a level of receptor-mediated endocytosis. The immunopurified rhMIS caused significant growth inhibition of ovarian and prostate adenocarcinoma and melanoma human cell lines in inhibition assays.
Keywords: human recombinant MIS; receptor localization; biological activity

Engineering, expression, and purification of “soluble” human cytochrome P45017α and its functional characterization by T. A. Pechurskaya; O. P. Lukashevich; A. A. Gilep; S. A. Usanov (806-811).
To engineer a “soluble” form of membrane-bound cytochrome P45017α (CYP17)—a key enzyme in steroid hormone biosynthesis—in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (δ23)) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a “soluble” form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is shown that replacement of hydrophobic amino acid residues in the FG-loop region does not change the metabolic profile during hydroxylation of steroids that is characteristic for wild type CYP17. Besides, the modification of the hemeprotein does not affect the affinity of CYP17 to steroid substrates. The engineered “soluble” form of human CYP17 is used as a subject for crystallization of the hemeprotein.
Keywords: cytochrome P45017α; heterologous expression; CYP17 in bacteria; “soluble” form of cytochrome P45017α

Lectin concentration (activity) increases in plant tissues upon infection by pathogens, in response to abiotic stress, as well as during growth and development of tissues. Such a broad range of events accompanied by accumulation of lectins is indicative of their involvement in regulation of integral processes in plant cells. Data concerning the role of lectins in regulation of oxidative stress and stress-induced cytoskeleton rearrangements are presented.
Keywords: lectins; stress; pathogenesis; phytohormones; reactive oxygen species; cytoskeleton

Induction of β-1,3-glucanase in callus cultures in vitro by E. A. Gunter; O. M. Kapustina; O. V. Popeyko; T. I. Chelpanova; E. A. Efimtseva; Yu. S. Ovodov (826-832).
Sodium salicylate (NaSA) increased induction of both intracellular and extracellular β-1,3-glucanases in calluses of campion and duckweed. NaSA concentrations from 30 to 100 mM were optimal for induction of intracellular glucanase in the campion callus, and for induction of extracellular glucanase the optimal concentration varied from 5 to 100 mM. The glucanase activity in the duckweed callus was lower than in the campion callus, and co-cultivation of the campion callus with Trichoderma harzianum mycelium increased the production of intracellular and extracellular β-1,3-glucanases and polygalacturonase in the callus. Biosynthesis by T. harzianum of glucanases, extracellular polygalacturonase and xylanase, and of intracellular galactosidase was increased. The cocultivation was accompanied by increased activity of intracellular acidic isoform of glucanase Glu-3 secreted by the callus cells into the medium, whereas NaSA activated in the callus culture the extracellular acidic isoform Glu-1 and extracellular basic isoform Glu-5. These data indicate the induction of these isoforms and the specificity of protective response of plant cells to different factors.
Keywords: Silene vulgaris (M.) G.; Lemna minor L.; Trichoderma harzianum ; callus; β-1,3-glucanase; polygalacturonase; sodium salicylate

Tolerance to antimicrobial agents and persistence of Escherichia coli and cyanobacteria by V. D. Samuilov; A. V. Bulakhov; D. B. Kiselevsky; Yu. E. Kuznetsova; D. V. Molchanova; S. V. Sinitsyn; A. A. Shestak (833-838).
Bacterial persistence is the tolerance of a small part of a cell population to bactericidal agents, which is attained by a suppression of important cell functions and subsequent deceleration or cessation of cell division. The growth rate is the decisive factor in the transition of the cells to the persister state. A comparative study of quickly growing Escherichia coli K-12 strain MC 4100 and cyanobacteria Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 growing slowly was performed. The cyanobacterial cells, like E. coli cells, differed in sensitivity to antimicrobial substances depending on the growth phase. Carbenicillin inhibiting the synthesis of peptidoglycan, a component of the bacterial cell wall, and lincomycin inhibiting the protein synthesis gave rise to nucleoid decay in cells from exponential cultures of Synechocystis 6803 and did not influence the nucleoids in cells from stationary cultures. Carbenicillin suppressed the growth of exponential cultures and had no effect on cyanobacterial stationary cultures. A suppression of Synechocystis 6803 growth in the exponential phase by lincomycin was stronger than in the stationary phase. Similar data were obtained with cyanobacterial cells under the action of H2O2 or menadione, an inducer of reactive oxygen species production. Slowly growing cyanobacteria were similar to quickly growing E. coli in their characteristics. Persistence is a characteristic feature of cyanobacteria.
Keywords: persistence; tolerance; antibiotics; carbenicillin; lincomycin; reactive oxygen species; E. coli ; cyanobacteria