PO-17 - Identification of IgG bound to plasminogen in oncologic diseases.

INTRODUCTION: The binding of plasminogen (Pg) to cell receptors and extracellular ligands facilitates its activation to plasmin, which stimulates the extracellular matrix degradation, neoangiogenesis and tumor invasion. Plasmin can also degrade IgG thereby exposing C-terminal lysine residues. Previously, we have found IgG specifically bounded to Pg in the plasma of patients with malignant tumors. AIM: To identify IgG degraded by plasmin in the plasma of cancer patients. MATERIALS AND METHODS: Methods of ELISA were used for comparative research of levels of IgG bound to Pg in plasma of patients with the prostate cancer (PC, n=25) and lung cancer (LC, n=17). Plasma of healthy donors (n=29) was used as control. All patients signed informed consent for participation in this study. Affinity chromatography on Pg-sepharose was used for the quantification of IgG. Carboxypeptidase was used for remove of C-terminal lysine residues of the IgG. The program ATTESTAT was used for nonparametric analysis. RESULTS: The frequency of occurence of elevated levels of IgG to Pg in plasma was detected in 68% of patients with PC, 59% of patients with LC and only 12% of healthy women and 10% of healthy men. The quantification of antibodies in plasma samples showed that the quantity of IgG to Pg in patients with PC was 27% from the total amount of IgG and in healthy men - 9%. Treatment of diluted plasma samples with carboxypeptidase B abolished the elevated levels of IgG to Pg, as well as the specific activity of the purified IgG to Pg-sepharose. CONCLUSIONS: C-terminal lysine residues which are formed as a result of degradation of native IgG with plasmin can bind to lysine binding sites on the kringle domains of Pg. Increased levels of these degraded IgG can be marker at cancer.

Autoantibodies to plasminogen and their role in tumor diseases.

Plasma level of IgG autoantibodies to plasminogen was measured by ELISA in patients with benign prostatic hyperplasia (n=25), prostatic cancer (n=17), lung cancer (n=15), and healthy volunteers (n=44). High levels of IgG to plasminogen were found in 2 (12%) of 17 healthy women, in 1 (3.6%) of 27 specimens in a healthy man, in 17 (68%) of 25 specimens in prostatic cancer, in 10 (59%) of 17 specimens in lung cancer, and in 5 (30%) of 15 specimens in benign prostatic hyperplasia. Comparison of plasma levels of anti-plasminogen IgG by affinity chromatography showed 3-fold higher levels in patients with prostatic cancer vs. healthy men.

Two-dimensional electrophoretic proteome study of serum thermostable fraction from patients with various tumor conditions.

One of the problems of plasma proteomics is a presence of large major components. In this work, we use the thermostable fraction as a way to deplete these major proteins. The thermostable fraction of serum samples from patients with ovarian, uterus, and breast cancers and benign ovarian tumor was analyzed using two-dimensional electrophoresis combined with MALDI-TOF(-TOF)-mass spectrometry. Of them, alpha-1-acid glycoprotein and clusterin are expressly down-regulated in breast cancer, whereas transthyretin is decreased specifically in ovarian cancer. Apolipoprotein A-I forms have decreased spot volumes, while haptoglobin alpha1, in contrast, is elevated in several tumors. These data are partly consistent with previous art studies on cancer proteomics, which involve mass-spectrometry-based serum profiling techniques. Serum thermostable fraction may be recommended as a good tool for medium and small protein proteome investigation, in particular, by 2D-electrophoresis.

Comparative analysis of different typing methods for Helicobacter pylori clinical isolates.

The goal of the present work was to compare different techniques of molecular typing using as an example clinical isolates of Helicobacter pylori obtained from patients in different regions of Russia. DNA-macroarray genome scanning using individual genes was employed to set up our basic classification of isolates that did or did not contain pathogenicity islands. In parallel, DNA of the same isolates was used in the conventional random amplified polymorphic DNA (RAPD) PCR analysis, and the isolates were also genotyped (cagA, vacA, iceA, and babA status) and their proteomic maps were obtained by means of unidimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with identification using peptide mass fingerprinting by MALDI-TOF mass spectrometry. A statistically significant correlation (coefficient of correlation r = 0.25, p = 0.005) was observed between the results of genome scanning and 1D-SDS-PAGE. No correlation was found between RAPD-PCR typing and genome scanning.

[Early proteomics in ovarian cancer: myth or reality?]. / Ranniaia proteomika raka iaichnikov. Mif ili real'nost'?

Literature data summarizing new approaches and importance of early ovary cancer diagnostics have been reviewed. Alpha-feta-protein (AFP) and SA125 were the most reliable markers for determination of early ovary cancer stages. Nevertheless, these markers don't reflect the disease stage, malignance and they don't possess sufficient specificity. New methodical approaches have recently been introduced. They include combination of 2-D electrophoresis with mass-spectrometry. These methods allow to inventory and identify almost all proteins of various tissues. Using these methods for scanning proteins from biopsies of ovary cancer tissues new markers have been discovered.

Comparative analysis of proteome maps of Helicobacter pylori clinical isolates.

The gram-negative bacterium Helicobacter pylori is found in human gastric mucosa. A widely distributed H. pylori infection is associated with chronic gastritis, gastric and duodenal ulcers, and malignant neoplasms. In this study proteome maps of four H. pylori clinical isolates derived from patients of two Russian regions (Moscow/Moscow Region and Novosibirsk) were obtained using 2D-electrophoresis and MALDI-TOF-mass-spectrometry. Variability of some H. pylori proteins and the level of their expression have been evaluated. These four isolates could be easily subdivided into two equal groups characterized by the close proteome profiles and the isolate from Moscow Region and the isolate from Novosibirsk constituted one group. The present study demonstrates the potential of proteome technology, which can be employed together with genome and transcriptome studies for the multiparameter typing of clinical isolates of pathogenic microorganisms.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3.

The cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G(1) cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Sp1 and Sp3 activate p21 (WAF1/CIP1) gene transcription in the Caco-2 colon adenocarcinoma cell line.

The CDK inhibitor p21WAF1/CIP1 is a negative regulator of the cell cycle, and its expression is induced during terminal differentiation in vitro and in vivo. Expression of p21 is controlled at the transcriptional level by both p53-dependent and -independent mechanisms. Our previous studies established that p21 is expressed in the Caco-2 adenocarcinoma cell line, and its expression is induced by a p53-independent mechanism during differentiation of these cells. Here we have found that transcription of p21 in Caco-2 cells is controlled primarily by the transcription factors Sp1 and Sp3 through two Sp1 binding sites, Sp1-1 and Sp1-2, located between -119 and -114 bp and between -109 and -104 bp of the p21 promoter, respectively. Sp1 and Sp3 binding to the p21 promoter increased during Caco-2 cell differentiation, while the absolute level of Sp1 did not change and the absolute level of Sp3 increased approximately twofold. Transfection experiments in the SL2 Drosophila cell line that lacks endogenous Sp3 activity demonstrated that Sp1 transactivates the p21 promoter primarily through the Sp1-2 site, while Sp3 acts through the Sp1-1 site. In these cells Sp3 is a stronger transactivator of the p21 promoter than Sp1. Our data suggest that induction of p21 transcription during Caco-2 differentiation is modulated by Sp1/Sp3 interactions with the p21 promoter.

A role for E2F1 in Ras activation of p21(WAF1/CIP1) transcription.

We recently reported that E2F1 could transactivate the p21 promoter via cis-acting elements between -119 to +16 bp of the p21 gene. Here we show that activated V12-H-Ras can induce the p21 promoter through the same region of the p21 promoter by a p53-independent mechanism in NIH3T3 cells. In contrast, activated Ras was not able to induce the p21 promoter in E2F1-/- fibroblasts, suggesting that E2F1 is required for induction of the p21 promoter by activated Ras. Cotransfection of increasing concentrations of dominant negative E2F1 alone, or with dominant negative DP1 into NIH3T3 cells suppressed induction of the p21 promoter by activated Ras. These data suggest that p53-independent induction of the p21 promoter by activated Ras is mediated at least in part by E2F1. Oncogene (2000) 19, 961 - 964.

Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins.

The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo.

p53-independent induction of p21WAF1/CIP1 expression in pericentral hepatocytes following carbon tetrachloride intoxication.

The cyclin-dependent kinase, proliferating cell nuclear antigen, and stress-activated protein kinase/c-jun NH2 terminal kinase inhibitor p21WAF1/CIP1 can induce G1 arrest, and its expression coincides with the cessation of replication in many systems. We examined expression of p21 during the early stages of carbon tetrachloride intoxication in the mouse liver and observed a dramatic increase in p21 RNA levels between 4 and 8 h after administration. p21 expression, visualized by in situ hybridization, is induced in pericentral hepatocytes before carbon tetrachloride-induced necrosis. Examination of c-fos and c-myc expression patterns confirm that these immediate-early genes are induced in similar regions of the mouse liver. p21 induction is not dependent on p53; we observed similar levels and localization of p21 in wild-type and p53 null animals. Immunohistochemical localization of p21 and CCAAT/enhancer-binding protein expression shows that p21 protein accumulation is limited to a subset of CCAAT/enhancer-binding protein-positive hepatocytes. A second peak of periportal and intermediate zone-specific p21 gene expression, appearing 1-2 days after injection, is also p53 independent and may represent cell cycle checkpoints or postmitotic growth arrest. Sporadic p21 expression was also detected in pairs of hepatocytes distributed throughout the liver acini in healthy animals. Together, these data suggest several roles for p21 in the liver in response to toxicity, regeneration, and growth inhibition.

p21 (WAF1/CIP1) expression is induced in newly nondividing cells in diverse epithelia and during differentiation of the Caco-2 intestinal cell line.

We examined the relationship between expression of the p21 (WAF1/CIP1) inhibitor of cyclin-dependent kinases, cessation of proliferation, and terminal differentiation in the epithelia of the gastrointestinal tract. Using in situ hybridization, we performed a detailed study of patterns of p21 mRNA expression in different regions of the stomach, along the length of the intestine, and in tongue, cervix, and hair follicle. We detected strong hybridization only in cells that had ceased proliferation and begun the process of terminal differentiation. Induction of p21 transcription may serve as a useful marker for dissection of differentiation programs in these diverse epithelia. To determine the relative levels of p21 expressed in various regions of the gastrointestinal tract from the esophagus to the colon, we used quantitative RT-PCR with endogenous and exogenous sequences as internal standards. The highest levels of p21 expression were detected in the distal small intestine. To further investigate the role that cell cycle regulation may play during differentiation of intestinal epithelial cells, we examined the expression of p53, p21, cyclin D1, cyclin E, and E2F1 in the Caco-2 colon carcinoma cell line, which differentiates spontaneously after reaching confluence. p21 and p53 mRNA and protein levels increase as Caco-2 cells differentiate. In both undifferentiated and differentiated Caco-2 cells, p53 protein was not inducible by DNA damaging agents, suggesting the absence of functionally wildtype protein. Caco-2 cells should provide a useful model system for studying regulation of p21 and determining if it plays a role during intestinal epithelial cell differentiation.

[The previously unknown biological characteristics of the neurons of the paraventricular hypothalamic nucleus]. / O rannee neizvestnykh biologicheskikh osobennostiakh neironov paraventrikuliarnykh iader gipotalamusa.

Structural sex-depended differences were revealed in 7 out of 10 hypothalamic paraventricular nuclei under study in DB/DB C57 BL-K56 mice. Sex-dependent differences were also found in the responses to diabetes mellitus in 6 of these nuclei. The data obtained seem to corroborate the sex dependence of the paraventricular-vagal neuronal system involved in the control of carbohydrate homeostasis.

[Cellular organization of paraventricular hypothalamic nuclei of the rat]. / Kletochnaia organizatsiia paraventrikuliarnykh iader gipotalamusa krysy.

According to the data on reconstruction of the serial sections of the cerebral hypothalamic area, using morphometric and electron microscopical methods, the structure of the paraventricular nuclei (PVN) has been studied in white rats (Wistar strain) hypothalamus. Ten subnuclei have been revealed. Each of them has individual characteristics. Owing to the analysis of the groups revealed, it is possible to divide the subnuclei into 3 main groups: magnocellular, parvicellular and subnuclei consisting of middle size neurons. The coordinated interaction of these subnuclei are supposed to ensure possible participation of PVN in regulation of the vegetative nervous and endocrine systems.

[Testosterone metabolism in discrete areas of the brain in rat fetuses]. / Metabolizm testosterona v diskretnykh oblastiakh mozga plodov krys.

Experiments in vitro with further thin-layer chromatography of steroids were staged to investigate changing of 3H-testosterone into 3H-estradiol-17 beta and 3H-5 alpha-reduced metabolites in a fraction of 1000 g of homogenates of the medial preoptic area (MPA), the paraventricular nuclei (PVN), the dorsal vagal complex (DVC) of the lower cerebral trunk, the rostral and caudal parts of the amygdaloid complex (ACr and ACc) of the brain of 21-day male and female rat fetuses. The activity of aromatase (AA) and 5 alpha-reductase (AR) was detected in all investigated areas. AA was maximum in PVN of females and minimum in ACc of males and females. The production of 5 alpha-dehydrotestosterone prevailed among 5 alpha-reduced products in PVN, DVC and ACr, 5 alpha- and rostane-3 alpha and 17 beta-diol--in MPA and ACr, AR in ACr was much higher than in ACc. AR in DVC of females was slightly higher than in males. Sex differences in the other cerebral areas were undetectable. The results of the investigation suggest that the above cerebral structures are dependent on sex hormones.

Experimental evidence for hypothalamic paraventricular nucleus involvement in the regulation of carbohydrate homeostasis.

An attempt was made to elucidate whether the hypothalamic paraventricular nucleus (PVN) is involved in carbohydrate homeostasis regulation in rats. The cellular responses to various disturbances of homeostasis were studied in the neuronal subdivisions (subnuclei) of the PVN. The responses were assessed in terms of changes in the sizes of the cell nuclei, nucleoli, and cytoplasm, and by the ultrastructural changes evident in cellular organelles. Significant cellular responses were observed in several subnuclei, with the nature of the responses being determined by the type of homeostatic alteration. The results indicate the possible involvement of the PVN subnuclei in the regulation of carbohydrate homeostasis.

[Reaction of specific cell populations of hypothalamic paraventricular nuclei of the rat to carbohydrate loading and fasting]. / Reaktsiia otdel'nykh kletochnykh populiatsii paraventrikuliarnykh iader gipotalamusa krys na uglevodnuiu nagruzku i golodanie.

The structural correlates of the cell responses in subnuclei of the hypothalamic paraventricular nuclei (PVN) in fasting and carbohydrate loaded adult male rats were studied using light microscopic morphometric methods. The data obtained revealed difference in responsiveness of the subnuclei studied to alternative shifts in the carbohydrate homeostasis: histophysiological features in five of ten cell-divisions, distinguished in the rat RVN, demonstrated their dependence on impaired carbohydrate homeostasis.

[Topography of the subnuclei of the paraventricular hypothalamic nucleus in rats and the sensitivity of their neurons to insulin deficiency]. / Topografiia sub"iader paraventrikuliarnogo iadra gipotalamusa u krys i chuvstvitel'nost' ikh neironov k defitsitu insulina.

Light microscopy was used to investigate the morphology and topography of hypothalamic paraventricular subnuclei in adult male rats. The subnuclear cell reaction to experimental alloxan diabetes was studied by karyometry. The paraventricular nucleus was established to contain 10 subnuclei, 8 of which responded to disorders in carbohydrate metabolism. The data obtained are consistent with the hypothesis of the involvement of the paraventricular nucleus in the control of carbohydrate homeostasis.