[Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells]. / Aktivnost' telomerazy, ékspressiia gena mTert i dlina telomer v otdalennyi period posle γ- i γ,n-oblucheniia v mezenkhimal'nykh stvolovykh kletkakh myshi i v opukholiakh, obrazovavshikhsia iz étikh kletok.

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

[Current trends in bladder cancer diagnosis].

In the Russian Federation, a proportion of bladder cancer in the overall structure of malignant tumors is about 2.8% and among oncological diseases of genitourinary system its incidence is second only to prostate cancer. Bladder cancer ranks as ninth most prevalent in males and as eighteens in females. The most important issue is to determine a recurrence rate of non-muscle invasive bladder cancer, which can reach 80%. In this regard, currently, all over the world much more attention is paid to studying and creation of early detection, including non-invasive, which will be reliable in the early stages. It can possibly lead to a reduction the number of cystoscopy and become as "golden" standard of non-invasive diagnosis bladder cancer.

CHARACTERISTICS OF TUMORS THAT DEVELOPED IN MICE AFTER TREATMENT WITH IRRADIATED SYNGENEIC MESENCHYMAL STEM CELLS OF BONE MARROW.

Mesenchymal stem cells (MSCs) are present in almost all organs and tissues of the organism. It is believed, that MSCs could be transformed into cancer stem cells spontaneously or under influence of genotoxic factors and trigger the growth of tumors. The aim of this work was to study the possibility of malignant transformation of cultured MSCs from murine bone marrow (MSCs-BM) after g-irradiation in vitro and characterize of biochemical and histological features of the tumors that developed after transplantation of MSCs-BM into syngeneic mice. Tumors were observed in 3­4 months after MSCs-BM transplantation. After administration of MSCs-BM irradiated at a dose of 1 Gy, tumors were seen in 2 of 5 mice. After transplantation of MSCs-BM irradiated at a dose of 6 Gy, tumors were found in all 5 of 5 mice. In the case of control MSCs-BM, only one tumor appeared in 6 months after transplantation. The telomerase activity was two times higher in the tumor developed from 6 Gy irradiated MSCs-BM than from 1 Gy irradiated MSCs-BM. The tumors developed from control and irradiated MSCs-BM were classified as multicomponent mesenchymomas («mixture of sarcomas¼). Histological examination showed that tumors contained tissue areas of different histogenesis. Thus, MSCs-BM g-irradiated at doses of 1 and 6 Gy and, much less frequently, control MSCs-BM can transform into tumor cells and induce development of multicomponent mesenchymomas.

[The role of telomerase activity in non-invasive diagnostics of bladder cancer].

PURPOSE: To evaluate the potentials of determining the telomerase activity (TA) in the cellular material of the urine for noninvasive diagnosis of bladder cancer (BC). MATERIALS AND METHODS: Evaluation of TA was performed in the urine of 48 patients with bladder cancer (study group) before and after transurethral resection of the bladder wall (n=38), an open resection of the bladder (n=4), and cystectomy (n=6). TA was also evaluated in 48 tumor tissue samples obtained from these patients during removal of the bladder tumor. Each sample of the tumor tissue was separated into two parts, one of which was subjected to histological examination, and the latter was used to determine the telomerase activity. In all cases, the diagnosis of bladder cancer was confirmed morphologically. Determination of TA in the samples was performed by the modified TRAP-method (telomerase repeat amplification protocol), RT-PCR, PCR, and electrophoresis. As a control, cell material of the urine and tissue in 12 patients with chronic cystitis was investigated. RESULTS: TA before surgery was found in 45 (93.75%) of 48 samples of cellular material of the urine from patients with suspected bladder cancer. BC was histologically verified in all patients in this group. In the postoperative period, TA was not observed in the 48 samples of cellular material of the urine from patients with BC. In the control group of patients with histologically verified cystitis, weak TA was determined only in one sample of cellular material of the urine. The analysis indicates statistically significant predominance of patients with bladder cancer in case of TA in the urine (P=0.001). TA was detected in all samples of tumor tissue. We also analyzed the dependence of TA levels in urine and tissue on the degree of BC differentiation. In patients with highly differentiated BC, mean AT in the cellular materials of the urine was 0,61% (n=15), in patients with moderately differentiated BC - 0.95% (n=23), in patients with low-grade bladder cancer - 1.33% (n=10); in other words, increase in the TA levels with decreasing the degree of differentiation was observed. This finding can be used in the prognosis of the course of disease based on determining the TA level in these patients. CONCLUSIONS: Preliminary data indicate the possibility of use of determining the TA in cellular material of the urine for the diagnosis and monitoring of bladder cancer recurrence.

[Development of noninvasive bladder cancer diagnosis on basis of telomerase and it's subunits hTERT and hTR detection].

Telomerase activity (TA) and expression of genes coding it's subunits (hTERT and hTR) have been examined in tumor tissue and urine sediment samples taken from patients with bladder cancer (BC) using the modified TRAP assay (in the case of telomerase detection) and RT-PCR (in the case of hTERT and hTR expression). Results obtained in this study demonstrate possibility of noninvasive diagnosis of BC with sensitivity of 96% and specificity of 100% in the case of telomerase detection and with sensitivity of 80% and specificity of 100% in the case of hTERT detection in urine sediment samples.

Telomerase as a tumor marker in diagnosis of prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis. METHODS: Complex urological patient evaluation and assessment of telomerase activity. RESULTS: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients. CONCLUSIONS: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients.

[Development of a set of diagnostic test systems for analysis of expression of C-myc, mad1, max, p53, and E2F1 gene oncomarkers by the reverse transcription reaction-polymerase chain reaction alignment technique].

Based on the performed studies, the authors developed test systems to analyze the expression of mRNA of the p53, C-myc, mad1, max, and E2F1 genes. These test systems could reveal a statistically significant difference between follicular adenoma and carcinoma of the thyroid in their expression of p53 mRNA. It should be noted that the use of our developed test systems is promising when searching for the diagnostic and prognostic markers of cancer, analyzing, and creating the genetic networks characterizing this or that cancer.

[Detection of telomerase activity in gastric cancer].

Telomerase activity (TA) was examined in gastric adenocarcinomas and gastric lymphoma using a modified TRAP assay. TA was present in 16 of 18 (89%) gastric adenocarcinomas and in gastric lymphoma, whereas no TA was detected in normal tissue. Almost all samples had "high" and "very high" TA levels. Telomerase is undoubtedly associated with the process of malignant transformation and therefore can be an important marker for diagnostics of gastric cancer.

[Puncture hystobiopsy with tissue telomerase activity measurement in preoperative thyroid nodes diagnostics].

Demonstrated, that puncture thyroid hystobiopsy is safe and informative method of preoperative thyroid nodes diagnostics. Telomerase activity in tissue samples was significantly higher in case of malignant thyroid disease, although a positive correlation of telomerase activity was also with the amount of lymphocytes in bioptates. Combination of thyroid hystobiopsy with tissue telomerase activity measurement proved to be an effective means of preoperative diagnostic of patients with nodular goiter.

[Prostate-specific antigen and telomerase in prostatic cancers].

A correlation between the blood level of prostate-specific antigen (PSA) and the prostatic tissue activity of telomerase was analyzed in prostate cancer (PC), low- and high-grade prostatic intraepithelial neoplasia (LG PIN, HG PIN) and in benign prostatic hyperplasia (BPH). The study was based on the results of a comprehensive examination of 92 patients from the Clinic of Urology, I. M. Sechenov Moscow Medical Academy. 44% of the patients were diagnosed as having PC; 49% had LG PIN and BPH; 7% had HG PIN and BPH. Active telomerase in the prostate biopsy specimens was found in 98% of the patients with PC, in 33% of those with HG PIN, and in 20% of those with LG PIN. When active telomerase was detected in the prostate biopsy specimens of patients with urological cancer (PC, PIN, BPH, the blood content of total PSA) was statistically significantly higher than that in the absence of this enzyme.

[PCR system for the detection of the RNA of hepatitis A virus]. / Razrabotka i ispytanie PTSR test-sistemy dlia detektsii RNK virusa gepatita A.

The detection of the causative agent of hepatitis A in the patient's body is the necessary element of the diagnostics of this disease. In this work the PCR system for the analysis of the RNA of hepatitis A virus in the patient's blood is presented and characterized. The method of the detection of the RNA of hepatitis A virus is based on the "nest" principle and consists of two consecutive reactions. In the first reaction the reverse transcription and amplification with the external pair of primers are carried out. The product thus obtained is used as material for the second reaction with the internal pair of primers. This method was used for the study of 44 blood samples from hepatitis A patients and 23 blood samples from healthy donors. The detection rate of the RNA of hepatitis A virus in blood samples from the patients was 82%. Viral RNA could be detected in the serum in 72% of cases, both in the serum and in mononuclear blood cells in 20% of cases, in mononuclear blood cells only in 8% of cases.

[Analysis of virulent and avirulent strains of Vibrio cholerae by the nest polymerase chain reaction]. / [Aanaliz virulentnykh i avirulentnykh shtammov Vibrio cholerae metodom gnezdovoi polimeraznoi tsepnoi reaktsii.

The detection of cholera enterotoxin in environmental objects and isolation of patients are considered to be the most reliable indices of that the cholera agent is present. Described in the case study is a method of nested polymerase chain reaction (PCR) based on amplifying the ctxA gene fragment coding subunit A of enterotoxin. A possibility was shown to use the above method to confirm the virulence of strains Vibrio cholerae isolated from different sources. The method was tested with 18 virulent and avirulent strains V. cholerae as well as (for the sake of verifying the analysis specificity) with DNAs of other human-pathogenic microorganisms and with the human genome DNA. The results showed a high efficiency of nested PCR in detecting the pathogenicity of cholera-agent strains.

[Study of the effectiveness of the method of the nest polymerase chain reaction and the field of its use in hepatitis A diagnosis]. / Issledovanie éffektivnosti i oblasti primeneniia metoda gnezdovoi PTSR pri diagnostike gepatita A.

To determine the causative agent of hepatitis A in the blood of patients, we developed the method of the nest polymerase chain reaction (PCR) with reverse transcription. For the comparative evaluation of the diagnostic efficacy of the nest PCR and the enzyme immunoassay (EIA) serum samples and mononuclear blood cells obtained from 15 patients with diagnosed hepatitis A and from 33 patients having signs and carrying markers of other form of hepatitis were analyzed. On the whole, the method of PCR confirmed the results of EIA and in some cases exceeded it in sensitivity. In addition, hepatitis A virus RNA was detected in the blood of a clinically healthy person having had a contact with a hepatitis A patient. The PCR data were shown to correlate with the activity of liver amino transferases. The results obtained in this study indicate that the field of the use of PCR in the analysis of hepatitis A virus may include cases of mixed infection and virus carriership.

[Use of nested PCR in detection of the plague pathogen]. / Primenenie gnezdovoi PTsR v detektsii vozbuditelia chumy.

Causative agents of plague, i.e. bacterium Yersina pestis (in the subcutaneous tissues of rodents) and their cutaneous parasites need to be isolated to enable plague prevention. A comparatively new method of polymerase chain reaction (PCR) opens up new possibilities of determining Y. pestis just within several hours and without any cultivation. The article contains a description of the PCR-method, which makes it possible to distinguish the culture of Y. pestis from cultures of other microorganism, including speci of Yersina. The method is of the cluster-type, i.e. it is made up of subsequent PC reactions with the substrate for the second reaction being the product of the first one. The cluster nature of the method preconditions a higher sensitivity and specificity versus the ordinary PCR.

[Use of the nested polymerase chain reaction in the differential diagnosis of human herpes simplex virus]. / Primenenie metoda gnezdovoi polimeraznoi tsepnoi reaktsii dlia differentsial'noi diagnostiki virusa prostogo gerpesa cheloveka.

Herpes is one of the most widespread human viral pathologies. The article depicts a special modification of polymerized chain reaction--(PCR)--(referred to as "nested"), which has a higher sensitivity, specificity and reliability as compared to the ordinary PCR, and which is designed to detect the herpes viruses. The method was initially tested at purified preparation of viral DNA, and later--at clinical materials obtained from patients and healthy donors. Secretions from the urogenital tract (smears), scrapes from the urogenital tracts and urinal cellular samples were examined in patients. Herpes simplex was detected in all cases. As for the healthy people, the identical examinations produced in them mainly the negative findings. Therefore, the nested PCR is a simple, sensitive and effective instrument in the diagnostics and prevention of herpes; it can be recommended for a comprehensive usage in medical practice.

The tumor cell and telomerase.

Imbalanced activity of the mechanism that controls cell division is a prerequisite for malignant transformation of a normal cell. The present review considers this multi-step mechanism, which is usually called the G1-S checkpoint. Besides, tumor cells are characterized by the presence of telomerase, an enzyme responsible for restoration of chromosome ends after replication and thus providing for unlimited cell division. The main point of the present article is to find out whether the activation of telomerase is controlled by the G1-S checkpoint or does not depend on it. The principal components of the G1-S checkpoint, such as cyclin-dependent kinases, retinoblastoma and E2F proteins, control the activity of telomerase. In their turn, they accumulate and transmit signals from various sources inside and outside the cell. Thus, various changes in tumor cells can activate telomerase through the G1-S checkpoint. Such are the suggested effects on telomerase of Myc, p53, Waf1, protein kinases B and C, Wnt5A, TGFbeta, WT1, and estrogens. However, Myc, p53, WT1, estrogens, protein kinases B and C, and TGFbeta can also directly influence telomerase independently of the G1-S checkpoint mechanism. Moreover, in 30% of human tumors the gene of the key subunit of telomerase (hTERT) is amplified, possibly due to chromosomal rearrangements unassociated with the activity of the G1-S checkpoint. Thus, telomerase seems to be activated not by a single agent but due to combined action of various factors, both with involvement of the G1-S checkpoint mechanism and independently of it.

Inhibition of telomerase activity of melanoma cells in vitro by antisense oligonucleotides.

The effect of antisense oligonucleotides complementary to the RNA component of human telomerase on telomerase activity in cell extracts of the melanoma cell line SK-Mel-28 has been studied. It has been shown that the antisense oligonucleotide complementary to the hTR component in the region of the template synthesis of telomeric repeats is the most efficient inhibitor of telomerase activity in comparison with other antisense oligonucleotides. Pronounced inhibition of telomerase activity was observed at the oligonucleotide (Tel P5) concentration in the reaction mixture of about 5 nM. Complete inhibition of the enzyme occurs at the oligonucleotide concentration in the sample of about 20 nM.

[The determination of the content of the cholera toxin gene in the composition of the DNA from Vibrio cholerae strains by means of the nested polymerase chain reaction]. / Opredelenie soderzhaniia gena kholernogo toksina v sostave DNK iz shtammov Vibrio cholerae metodom "gnezdovoi" polimeraznoi tsepnoi reaktsii.

The possibility of detecting cholera toxin genes in V.cholerae enterotoxigenic strains by the method of "nested" polymerase chain reaction with the use of primers on the DNA area of operon ctx of AB genes. The possibility of the detection of several V.cholerae cells by this method was shown with the use of a series of bacterial lysate dilutions. The newly developed test system for the detection of cholera toxin gene on the basis of the analysis of bacterial lysates of V.cholerae nontoxigenic strains, as well as Escherichia coli toxigenic and nontoxigenic strains, was shown to be highly specific.

[A DNA analysis of Vibrio cholerae strains by the polymerase chain reaction]. / Analiz DNK shtammov Vibrio cholerae metodom polimeraznoi tsepnoi reaktsii.

The method for the analysis of cholera toxin gene in V. cholerae strains was developed on the basis of polymerase chain reaction (PCR). This specific and highly sensitive method using primers affecting the site of the DNA of the operon of cholera toxin gene made it possible to identify one copy of V. cholerae genome. For the first time the content of cholera toxin gene in 4 V. cholerae (eltor) strains, obtained from the clinical material of cholera patients in Tajikistan and Dagestan, was shown with the use of PCR.