[Circulating microRNAs in lung cancer: prospects for diagnostics, prognosis and prediction of antitumor treatment efficiency].

The major methods of microRNA extraction from different biological fluids (particularly, serum and plasma), approaches to the analysis of microRNA concentration and composition, normalization methods used in data analysis are outlined in the review. The advantages and disadvantages of the described methodological approaches are being highlighted. Special attention is given to microRNAs, circulating in blood, which could be used as the markers for minimally invasive lung cancer diagnostics, prediction of antitumor treatment efficiency and disease prognosis. Prospects and limitations arising from the evaluation of clinical significance of microRNAs as the potential tumor markers, and emerging as roles of various microRNAs in the pathogenesis of lung cancer become known, are discussed.

[Assay of methylated gene RARbeta2 in circulating DNA of blood from patients with lung cancer as a potential prognostic marker].

Blood-based methylated DNA gene RARbeta2 in circulating plasma (cir DNA) and one associated with blood cell surface were assayed in patients with non small cell lung cancer before and after combined treatment. The levels in both appeared to be significantly higher than in healthy subjects. Enhanced levels prior to treatment were associated with greater advancement of the disease and unfavorable prognosis (overall survival). After two courses of neoadjuvant therapy plus surgery methylation indices fell down to match those in healthy subjects. Our data may be instrumental in working out additional criteria to be used in diagnosis, prognosis and follow-up of patients with non small cell lung cancer.

[Molecular-genetic markers in lung cancer diagnostics].

The major approaches to different lung cancer marker development are outlined in the review, including genetic, epigenetic, protein, transcryptomic, proteomic, metabolic, and miRNA markers. As far as epigenetic changes are among the earliest events in malignant transformation, methylated markers are thoroughly discussed. Special attention is given to minimally invasive tumor markers, which could be detected in easily accessible biological fluids, because they can be useful for screening and early diagnostics of cancer (before its clinical manifestation) as well as for verification of standard methods of diagnostics. Extracellular nucleic acids, circulating in blood (cirNA), are highlighted as the potential source of material for the early lung cancer diagnostics, prediction of antitumor treatment efficiency, post-treatment monitoring and disease prognosis.

[Immunochemical and molecular-genetic markers in gastric cancer diagnostics].

Studies of molecular mechanisms of neoplastic transfromation are being under the thorough investigation, reveal the new genes involved into the tumor development. These genes and their products are potential tumor markers and levels of their expression can be detected using modem assays characterized by high specificity and sensitivity. The review highlights the current status of the molecular diagnostics of gastric cancer, including protein and nucleic acids biomarkers. The genetic and epigenetic changes, which are detected in the malignant and nonmalignant tumor tissue DNA and in the blood plasma DNA from tumor bearing patients, are summarized.

[Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood].

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of patients with breast tumors and healthy controls. Frequency of RASSF1A, Cyclin D2 and RARbeta2 methylation was detected using methylation-specific PCR in the extracellular DNA, extracted from plasma and cell-surface bound fractions of patient blood. Methylation of at least one of these genes was found in plasma of 13% patients with benign breast fibroadenoma and in 60% of breast cancer patients. Using cell-surface bound DNA as a substrate for PCR have lead to increase of gene methylation detection frequency up to 87% in fibroadenoma and 95% in breast cancer patients without false positive controls. GAPDH, RASSF8, Ki-67 RNA and 18S RNA were quantified using RT-qPCR of the extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The main part of the extracellular RNA was shown to be cell-surface bound. Results show a higher amount of RASSF8, Ki-67 RNA and 18S RNA in plasma and cell-bound fraction of patients with breast cancer compared with patients with benign tumors and healthy controls. The data indicate that the specific RNA quantification in blood plasma is valuable for discrimination between cancer and benign tumors, which can be detected with high sensitivity using analysis of methylated RASSF1A, Cyclin D2 and RARbeta2 genes in extracellular circulating DNA.

[Circulating nucleic acids in blood of patients with stomach and colon neoplasms].

Concentrations of extracellular deoxy- and ribonucleic acids in blood plasma and cell-surface-bound of blood cells were investigated in healthy donors and patients with malignant gastrointestinal tumors. Our results indicate that high concentration of extracellular DNA in blood plasma along with decreased level of extracellular RNA on the surface of blood cells correlate with development of gastrointestinal cancer. Ratio of nucleic acids in plasma to total amount of nucleic acids circulated in blood is a characteristic parameter distinguishing cancer patients from healthy persons.

[Pharmacokinetics and stability in the blood in vivo of phosphodiester and modified oligonucleotide derivatives]. / Issledovanie farmakokinetiki i stabil'nosti v krovi in vivo fosfodiéfirnykh i modifitsirovannykh proizvodnykh oligonukleotidov.

In vivo stability and distribution of deoxyribooligonucleotides and 3'-end modified oligodeoxyribooligonucleotides were studied in blood serum and cells. Substitution of two 3'-end internucleotide phosphodiester bonds increased the stability of modified oligonucleotides. Modified oligonucleotides were shown to be stable in blood serum for up to 24 hours. Leukocytes did not bind oligonucleotides significantly, whereas erythrocytes accumulate mono-, di-, three-, tetranucleotides appeared in the blood stream as degradation products of the parent oligonucleotide.

[Development of a two-site immunoassay using polyclonal antibodies for determination of lactoferrin in human blood]. / Razrabotka dvukhsaitnogo immunoanaliza s ispol'zovaniem poliklonal'nykh antitel dlia opredeleniia laktoferrina v syvorotke krovi cheloveka.

The two-site enzyme-linked immunosorbent assay (ELISA) for lactoferrin using polyclonal antibodies to spatially distant epitopes has been developed. The assay sensitivity defined as minimal detectable lactoferrin concentration for p = 0.05 is 0.5 ng/ml. Accuracy of the assay (variance coefficient) is 7% within the clinical range of antigen concentrations. Human albumin, hemoglobin, and transferrin in concentrations up to 5 mg/ml practically do not interfere with the measurement. Sera of healthy donors and viral hepatitis patients were investigated using the two-site ELISA. The lactoferrin content in 44 donors' sera was 130 +/- 40 ng/ml (medium +/- standard deviation). A study of the serum specimens of 95 patients with hepatitis A, B, and C showed significant increase in serum lactoferrin concentration: 850 +/- 420, 780 +/- 580, and 680 +/- 500 ng/ml respectively. The assay showed good characteristics and may be recommended for lactoferrin measurement in patients' sera.

[Study of the oligonucleotide binding sites on an immunoglobulin molecule]. / Issledovanie uchastka sviazyvaniia oligonukleotidov na molekule immunoglobulina.

Interaction of IgG molecules with oligonucleotides using reactive derivatives of p(T)16 bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue at the 5'-terminal phosphate was investigated. The modified immunoglobulins were degraded with pepsin into Fab and Fc fragments and by incomplete CNBr hydrolysis into smaller peptides. It became obvious from the analysis of the peptides obtained that the oligonucleotides contacted with immunoglobulins at the antigen-binding Fab fragment of the molecule. The site of interaction is localized in the light-chain N-terminal fragment 178 amino acids long. These facts are in accordance with our previous data about the ability of a specific antigen to prevent the oligonucleotide-antibody interaction. On the contrary, an oligonucleotide covalently linked with immunoglobulin could not prevent the antigen-antibody reaction. Monoclonal Fab fragments modified with the alkylating p(T)16 derivative were found to interact with the specific antigen (human myoglobin) during affinity chromatography. This interaction deserves further investigation because of its importance in studying the fate of oligonucleotides in vivo.

[Interaction of oligonucleotides with blood serum proteins]. / Vzaimodeistvie oligonukleotidov s belkami syvorotki krovi.

The interaction of alkylating derivatives of deoxyribonucleotides whose 5'-terminal phosphate group contains a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue with serum proteins has been studied. Incubation of whole human sera with various concentrations of the alkylating derivative, p(T)16, resulted in affinity modification of several proteins, among which albumin as well as IgM and IgG were the most readily detectable ones. The type of dependence of the degree of modification of these proteins on oligonucleotide concentration suggests that the oligonucleotides display a higher affinity for IgM than for IgG and albumin. Binding of reactive oligomer derivatives to serum proteins was inhibited by polyanions of different oligomeric composition, two-chain DNA and heparin, the latter being the strongest inhibitor of the immunoglobulins. These data point to a role of nonspecific ion-to-ion interactions in the IgG-oligonucleotide complex formation. Oligonucleotide interaction with murine monoclonal antibodies GI was inhibited by a specific antigen which suggests that oligonucleotides may interact with immunoglobulin either at or near a site where the antigen is recognized by the antibody.

[Affinity modification of proteins, binding nucleic acids in mammalian cells, by alkylating derivatives of oligonucleotides]. / Affinnaia modifikatsiia belkov, sviazyvaiushchikh nukleinovye kisloty v kletkakh mlekopitaiushchikh, alkiliruiushchimi prozvodnymi oligonukleotidov.

Using the 32P-labelled alkylating oligonucleotide derivative (pT)16, the oligonucleotide interaction with mammalian cells has been studied. The majority of fibroblastoid cell lines tested in this study (COS-1, vero, L-671, Ag 17-1, CHO, B7) as well as mouse hepatocytes were found to contain proteins specifically interacting with oligonucleotides. Cells of Ag 17-1 and COS-1, apart from the 79 kDa protein specific for all fibroblast lines, contained also a protein with a molecular mass of 83 kDa. In BALB/c mouse hepatocytes the 83 kDa protein is the major oligonucleotide binding protein. Analysis of concentration dependencies of specific modification of receptor proteins in liver cells has made it possible to determine the values of constants for the oligonucleotide derivatives binding to the protein. The binding constant for the alkylating oligonucleotide derivative pT16 and the corresponding phosphothioate oligonucleotide has been found to be equal to 5 x 10(6) M-1.

[Rapid time-resolved fluoroimmunometric analysis of myoglobin]. / Bystryi vremiarazreshennyi fliuoroimmunometricheskii analiz na mioglobin.

High performance quantitative procedure for evaluation of myoglobin in blood serum is developed. The principle of two-site immunometry was used involving monoclonal antibodies against nonoverlapping epitopes of myoglobin molecule. The fluorescent compound containing europium served as a label of the antibody. Modification of the single-step assay enabled to increase the velocity of the assay to about 30 min and simplify it. The assay allowed estimation of myoglobin over a broad range of concentrations from 2 ng up to 1,000 ng/ml. The assay may be recommended for use in clinical practice for express diagnosis of myocardium infarction.