Kinetics of metallo-enzymatic paramagnetic centers and free radicals in the rat liver in lung carcinogenesis

Kinetics of the content of nonheme iron-sulphur-containing (iron-sulphur) proteins, free radicals of electron-transport mitochondrial system, as well as of microsome terminal oxidase cytochrome P-450 is studied in the liver of rats at early stages of carcinogenesis and in the process of tumour growth induced by intratracheal administration of various benz(a)pyrene doses. It is found that the content of iron-sulphur proteins increases after the first administration, then it falls against a background of higher concentration of free radicals. A degree of pronounced changes in the content of the studied iron-sulphur proteins correlates with carcinogen dose. The cytochrome P-450 content is lowered for almost the whole period of carcinogen administration. In later periods animals with morphologically determinable pretumour changes exhibit a much higher content of iron-sulphur proteins, somewhat increased concentration of free radicals and a tendency to an increased level of cytochrome P-450. The appearance and growth of malignant tumours is followed by a considerable decrease in the content of iron-sulphur proteins and cytochrome P-450. On the basis of the results obtained it is supposed that the changes in the content of iron-sulphur proteins in the rat liver is the earliest and most pronounced reaction which depends on the benz(a)pyrene dose and may be of prognostic significance.     

Nitric oxide-induced bacteriostasis and modification of iron-sulphur proteins in Escherichia coli

The nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids (BCAA) anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (IlvD), an iron-sulphur enzyme essential for the BCAA biosynthesis, is completely inactivated in cells by NO with the concomitant formation of the IlvD-bound dinitrosyl iron complex (DNIC). Fractionation of the cell extracts prepared from the NO-exposed cells reveals that a large number of different protein-bound DNICs are formed by NO. While the IlvD-bound DNIC and other protein-bound DNICs are stable in cells under anaerobic growth conditions, they are efficiently repaired under aerobic growth conditions even without new protein synthesis. Additional studies indicate that L-cysteine may have an important role in repairing the NO-modified iron-sulphur proteins in aerobically growing E. coli cells. The results suggest that cellular deficiency to repair the NO-modified iron-sulphur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions.     

Location and nucleotide sequence of frdB, the gene coding for the iron-sulphur protein subunit of the fumarate reductase of Escherichia coli

The frdB gene, encoding the iron-sulphur protein subunit of fumarate reductase, has been located and its complete nucleotide sequence determined. The identity of the gene was confirmed by protein chemical studies and determination of the NH2-terminal sequence of the FrdB protein. The frdB gene is situated distal to and partially overlapped by frdA which codes for the flavoprotein subunit of the reductase. Its reading frame contains 244 codons and predicts a protein of Mr 27092. In composition, the FrdB protein is strikingly similar to the corresponding subunit of the related flavoenzyme, succinate dehydrogenase. Analysis of the protein's primary structure revealed several features characteristic of iron-sulphur proteins.     

Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation.

The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.     

Protein myristoylation in health and disease

N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.     

人肺癌变早期阻断及基分子机制研究

以人胚肺为实验材料,进行了ｏｌｔｉｐｒａｚ对人肺癌变早期阻断及其分子机制的研究,结果显示：ｏｌｔｉｐｒａｚ阻断人肺癌变在香烟凝聚物（ＣＳＣ）运用前或同时运行效果较好;ｏｌｔｉｐｒａｚ阻断人肺癌变时能使谷胱甘肽－Ｓ－转移酶（ＧＳＴｓ）活性和ＧＳＴ－π蛋白含量降低;ｏｌｔｉｐｒａｚ能使ｍｐ５３蛋白表达减弱;ｏｌｔｉｐｒａｚ仅能阻止而不能逆转ｒａｓ基因突变;ｏｌｔｉｐｒａｚ能诱导人肺腺癌细胞凋亡,在使用     

Expression of heat shock protein 70 in renal cell carcinoma and its relation to tumor progression and prognosis

Heat shock proteins (HSPs) play an important role in the cellular response to environmental stress and exert a cytoprotective effect. Especially HSP70 is an effective inhibitor of apoptosis, suggesting a role of HSP70 in carcinogenesis and tumor progression. To explore the relevance of HSP70 in renal cell carcinomas (RCCs), we analyzed nuclear and cytoplasmic HSP70 protein expression in formalin-fixed tissue from 145 clear cell RCCs by immunohistochemistry as well as Western blot analysis. Nuclear HSP70 expression was found in all RCCs and 75% of the tumors also exhibited a cytoplasmic HSP70 staining. Importantly, RCCs showed significantly reduced cytoplasmic (p=0.001) and combined nuclear/cytoplasmic (p=0.0022) HSP70 expression when compared with their cells of origin. A significant (p=0.0176) decrease of nuclear HSP70 expression became evident from well to poorly differentiated clear cell RCCs. Quite similarly, a trend (p=0.0558) for reduced combined nuclear/cytoplasmic HSP70 expression was shown from early (pT1) to advanced (pT3) tumor stages. Nevertheless, no correlation between HSP70 expression and patients survival became evident. In conclusion, our investigation demonstrates a significant decrease of antiapoptotic HSP70 protein expression during carcinogenesis and during progression from well (G1) to poorly (G3) differentiated clear cell RCCs. Our results suggest that HSP70-mediated inhibition of apoptosis seems to be of minor importance for carcinogenesis and tumor progression in RCCs.     

Hormone-independent polyamine metabolism of squamous cell carcinoma of the prostate

The levels of polyamines and their synthesizing enzymes in squamous cell carcinoma of prostate implanted in intact as well as castrated male rats were determined after certain hormonal manipulations. The tumour was found to grow with an identical rate in non-castrated and castrated rats. Polyamine content and activities of polyamine synthesizing enzymes in the tumour were found to be much lower compared to their values in ventral prostate. Moreover, the levels of these parameters were comparable in tumours whether implanted in non-castrated or gonadectomized animals. The sequential analyses of putrescine and spermidine and activities of L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase of tumours at different time intervals showed a significant reduction in their levels at 30 days compared to 10 days post implantation in non-castrated as well as castrated rats. Daily intramuscular administration of tumour-bearing intact or castrated animals with testosterone (50 micrograms/g), beta-estradiol (2 micrograms/g) or cyproterone (12.5 micrograms/g) for 10 days did not influence polyamine metabolism in tumour tissue. However, either beta-estradiol and cyproterone treatments or castration were found to decrease polyamine synthesis in ventral prostate. At the same time, the testosterone replacement therapy did not allow polyamine levels or activities of polyamine synthesizing enzymes to decline in the ventral prostate of castrated rats. Our results demonstrated that contrary to ventral prostate, the polyamine metabolism in squamous cell carcinoma of prostate is independent of hormonal control. The loss of hormonal sensitivity of polyamine metabolism in the prostatic tumour could be the result of qualitative changes that occurred during transformation.     

Fourth derivative UV-spectroscopy of proteins under high pressure II. Application to reversible structural changes

The structural basis and the thermodynamics of pressure induced reversible spectral transitions in the fourth derivative ultraviolet absorbance spectra of proteins were analysed as described in the preceding paper. Three proteins were studied: adrenodoxin (a small iron-sulphur protein that serves as an electron donor for cytochrome P450scc), ribonuclease A, and methanol dehydrogenase (a tetrameric protein). Fourth derivative spectroscopy is used to probe important mechanistic aspects of these proteins. For adrenodoxin, the results suggest that one or two phenylalanines interact with the iron-sulphur redox centre. High pressure denaturation of ribonuclease leads to a molten globule like structure that also occurs as an intermediate in the high temperature induced denaturation process. This state is characterised by the local dielectric constant in the vicinity of tyrosines. Methanol dehydrogenase was found to be very stable towards pressure. High pressure appears to strengthen the interaction between the two -subunits possibly through the increased interaction of four tryptophans with other aromatic amino acids.     

Abundance and localization of skeletal muscle-related erbB2 may stimulate tumour growth during initial stages of oral oncogenesis

ErbB2 and erbB3 transmembrane receptors, known to be associated with neuronal and skeletal muscle developmental function, seem to play an important role in human oral oncogenesis. This study was designed to determine gradual erbB2 and erbB3 expression in an experimental animal system of induced oral carcinogenesis in Syrian golden hamsters. Thirty-seven animals were divided into one control group (N=7) and three experimental groups (N=10 each one), which were treated with carcinogen 9,10-dimethyl-1,2-benzanthracene and sacrificed at 10, 14 and 19 weeks after treatment. The histological status of observed lesions in the three experimental groups corresponded well with tumour advancement (from oral mucosal dysplasia to moderately differentiated squamous cell carcinoma). Tissue sections ranging from normal mucosa to squamous cell carcinoma were studied using monoclonal antibodies against erbB2 and erbB3 proteins. Cytoplasmic erbB2 expression was gradually increased in pre-cancerous stages, remained stable in initial tumour stages and substantially decreased in moderately-differentiated carcinomas, suggesting that it may be useful as an early prognostic factor. On the contrary, erbB3 was not expressed at all either in normal or tumour tissue.     

Protein expression profiling in chemical carcinogenesis: a proteomic-based approach

The simultaneous analysis of a wide array of proteins may provide valuable information on the activation and suppression of cellular systems at different stages of the exposure-disease continuum. In this review, results of proteomic studies in the field of toxicology are covered, focusing on the effects of chemical carcinogens. So far, alterations of highly abundant proteins have been identified which, irrespective of the wide differences in study design and technologies used, can be grossly assigned to three functional classes: proteins related to cellular stress response, inflammation, and stimulation of the immune system. It is obvious that the observed protein alterations are not causal factors in the development of chemically induced cancer but rather reflect common reactions to cellular perturbations. In order to gain deeper insights into the process of chemical carcinogenesis, the previously applied "shotgun" analyses have to be abandoned in favour of targeted proteomic approaches focusing on the accurate identification and quantification of selected proteins. Advanced analytical techniques such as selective reaction monitoring (SRM) and multiple reaction monitoring (MRM) offer this opportunity. If toxicoproteomic research moves into that direction and takes advantage of such techniques it will have the potential to contribute to the elucidation of chemical carcinogenesis.     

Depletion of mitochondrial DNA and enzyme in estrogen-induced hamster kidney tumors: a rodent model of hormonal carcinogenesis

Mitochondrial DNA (mtDNA) encodes for 13 polypeptides critical for normal functioning of the electron transport chain and damage to mtDNA has been associated with aging, and implicated in several disease processes. Although damage to mtDNA is being implicated in mutagenesis and carcinogenesis, there are limited studies demonstrating the role and extent of mtDNA damage in human or rodent cancers. Using serial dilution and competitive polymerase chain reaction analysis, we have quantitated the amount of total mtDNA and analyzed the extent of mtDNA damage in estrogen-induced and estrogen-dependent hamster kidney tumors. The hamster kidney tumor model is a useful and widely investigated rodent model of hormonal carcinogenesis, which shares several characteristics with human breast and uterine cancers, and point to a common mechanistic pathway. Our data indicate a significant decrease in the copy number of total mtDNA and the activity of a nuclear-encoded mitochondrial enzyme citrate synthase in hamster kidney tumors compared to age-matched controls. Since there are several hundred mitochondria in a cell and each mitochondrion has multiple copies of mtDNA, a very small percentage of somatic deletion mutation may not be enough to result in a decreased capacity of the mitochondrial genome. However, a significant increase in deletion mutations or a decrease in the mtDNA copy number can result in a decreased oxidative phosphorylation capacity of the mitochondria and decreased energetics, and thus increased susceptibility to the disease process. Therefore, estrogen-induced hamster kidney tumor model can be a useful rodent model of carcinogenesis to understand the role of mtDNA damage in cancer progression and development.     

Evolution and cellular function of monothiol glutaredoxins: involvement in iron-sulphur cluster assembly

A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron-sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron-sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell.     

Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2

Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: -3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4-7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of alpha-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase alpha (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.     

The cooperative role of OsCnfU-1A domain I and domain II in the iron sulphur cluster transfer process as revealed by NMR

OsCnfU-1A is a chloroplast-type Nfu-like protein that consists of tandem repeats sharing high sequence homology. Domain I of this protein, but not domain II, has a C-X-X-C motif that is thought to assemble an iron-sulphur cluster. Herein we report the solution structure of OsCnfU-1A domain I (73-153). Although OsCnfU-1A domain I is structurally similar to OsCnfU-1A domain II (154-226), the electrostatic surface potential of the 2 domains differs. Domain I has an acidic surface, whereas that of domain II is predominantly basic. Chemical shift perturbation studies on OsCnfU-1A domain I and domain II with ferredoxin revealed negligible chemical shift changes in domain I, whereas much larger chemical shift changes were observed in domain II. The residues with larger chemical shift changes were located on the basic surface of domain II. Considering that ferredoxin is predominantly negatively charged, we propose the following hypothesis: First, an iron-sulphur cluster is assembled on domain I. Next, domain II interacts with the ferredoxin, thus tethering domain I close to the ferredoxin. Finally, domain I transfers the iron-sulphur cluster to the ferredoxin. Thus, domain II facilitates the efficient transfer of the iron-sulphur cluster from domain I to the ferredoxin.     

Synthesis of nucleic acids and proteins in synchronized Ehrlich tumour cells

The activation of RNA synthesis in Ehrlich tumour cells occurs during the transition: G1 leads to S simultaneously with the onset of DNA replication and is intermittent. A high rate of synthesis is maintained at a constant level for some period of time and is decreased only by the end of the mitotic cycle. Actinomycin D (0.05 mkg/ml) inhibits the label incorporation into RNA in the S- and G2 phases, but has no inhibiting effect at earlier stages. These findings and the data from polyacrylamide gel electrophoresis suggest that all types of rRNA and tRNA are synthesized in the course of the S- and G2 phases. The rate of protein synthesis is correlated with that of protein synthesis in tumour cells at all stages of the cycle. Electrophoresis in polyacrylamide gel shows that the spectra of nuclear proteins and Ehrlich tumour cell cytoplasm are not significantly changed throughout the mitotic cycle. The amount of histones in the nuclei is increased simultaneously with the increase in the level of DNA, so that the histone/DNA ratio remains constant throughout the cycle and is equal to 0,96 +/- 0,03.     

Factors reducing the superoxide dismutase activity of the liver of rats with Pliss lymphosarcoma

The procedures for isolation and purification of Cu/Zn superoxide dismutase (SOD) from small amounts of rat liver at different stages of Pliss lymphosarcoma growth were developed. Two stages of tumour growth were distinguished. At the first stage (4-5th day after reinoculation) the intensity of optical (680 nm) and EPR spectra of SOD was decreased, on the average, to 30%, while at the second stage--by 40% at 680 nm and by 32%, at 260 nm. The intensity of the EPR spectra was also diminished by 40% as compared to the control. It was assumed that at initial stages of tumour growth the decrease of the SOD activity is mainly due to the reduction of enzyme Cu, while at the second stage--to the decrease of the protein content. In all probability, the observed changes are induced by the activation of lipid peroxidation involving active O2 species. The second stage is associated with the superoxide-induced impairment of the function of nucleic acids involved in protein biosynthesis.     

The source of the iron binding with nitric oxide in activated macrophages]

No decrease in iron-sulphur centers was found in cultured macrophage cells (J774) after the treatment with nitric oxide (10(-7) M NO/10(7) cells) during 5 min. The center content was controlled by the electron spin resonance (ESR) method. The macrophages pretreated with dithionite + methyl viologen showed the formation of dinitrosyl iron complexes (DNIC) with a characteristic ESR signal at g approximately 2.03. The data suggest that loosely bound nonheme iron (free iron) mostly contributes to the formation of these complexes. Iron from iron-containing proteins does not release from these centers under the direct action of nitric oxide. The iron-sulphur centers can be destroyed by the products of nitric oxide oxidation (NO2, N2O3, etc.) as oxidizing and acid agents.     

Endocrine cells in gastric carcinoma and adjacent mucosa. An immunohistochemical and ultrastructural study

Endocrine cells are often found in human gastric carcinoma and may be recognized by the immunoreactivity of their chromogranin A, peptides and biogenic amines content. Anti-chromogranin A was used to investigate the morphology of endocrine cells using light and electron microscope immunohistochemical techniques. The hormone content of endocrine cells was examined in both tumour tissue and tumour-adjacent mucosa. It was found that the endocrine cells in tumour tissue were malignant, often had amphocrine differentiation and did not resemble a normal cell type. The hormone content of endocrine cells in tumour tissue seldom corresponded to the hormonal content of endocrine cells in tumour-adjacent mucosa. In intestinal-type carcinoma and in some parts of diffuse-type gastric carcinomas, endocrine cell hyperplasia and an alteration of the differentiation in the tumour-adjacent mucosa were discovered. The distribution of endocrine cells in the tumour tissue was different in both types of gastric carcinoma. The results reported here suggest that endocrine cell differentiation of malignant endocrine cells in human gastric carcinoma develops in a different way from that of endocrine cells in tumour-adjacent mucosa, and as a result, diverse hormonal products may appear in tumour tissue.