[Modern molecular approaches to diagnosis and treatment of high-grade brain gliomas].

The review analyzes the current state of the problem of diagnosis and therapy of high-grade gliomas on the basis of the most promising present-day approaches. The diagnostic and treatment perspectives of the molecular genetic analysis of glioblastoma markers located on the tumor cell surface are considered. Gene therapy and the use of dendritic cells and oncolytic viruses are considered as the most interesting approaches to therapy of high-grade gliomas.

[Connection of intracellular protein YB-1 localization in cell cultures of human tumors with multidrug resistance].

In this study, we investigated how the protein YB-1 influenced on the expression of genes coding ABC transporters and on drug resistance in several cell lines, in which originally gene MDR1, coding P-glycoprotein, was not expressed. These populations were significantly different in the presence of mRNA YB-1 and the nature of the intracellular localization of the protein YB-1. However incubation of cells in all studied populations in the culture medium with serum after starvation led to translocation of YB-1 in the cell nucleus. The increase of the number of cells with nuclear localization of YB-1 correlated with increased amount of mRNA YB-1. Processing of cells with drug LY-294,002 by PI3K/Akt inhibitor prevented the translocation of the protein YB-1 into the nuclei of cells, and the cells became more sensitive to the toxic action. Thus, we observed that the signaling pathways involved in control of cell proliferation, in particular a signaling cascade PI3K/Akt were involved in the control of the intracellular localization of YB-1 in cell populations of ovarian cancer, melanoma and human prostate cancer. In these cells the nuclear localization of YB-1 correlated with an expression of MDR and MRP1 DCRP genes and with a sensitivity of cells to a number of drugs.

[Hydrophilic regulator hexapeptides as inhibitors of tumor cell multiple drug resistance].

Multiple drug resistance (MDR) of tumor cells to cytostatics is one of the most often and severe complications of chemotherapy in oncological patients. The phenomenon of MDR could be due to a sharp increase of the activity of the ATP-dependent transport proteins of the ABC system, that provides pumping of the drug from the cells to the extracellular space. Up to now, all the attempts to design agents preventing MDR were of no success. One of the prospective trends is the use of hydrophilic regulator hexapeptides. Three regulator hydrophilic hexapeptides of the linear and cyclic structure were used as the MDR modulators. The sensitivity of the tumor cells to various cytostatics in the presence of the peptides was determined by the MTT-test and the direct counting of the survived cells. The effect of the hexapeptides on MCF7, KB8-5 and PC3 cells was investigated. It was concluded that the hydrophilic hexapeptides of the linear and cyclic structure increased the sensitivity to doxorubicin (a cytostatic). The tumor cell MDR inhibition was mediated by the ATP-dependent transport protein MRP. Such a characteristic of the hexapeptides is of interest for their use as agents preventing MDR.

[Determination of the amount of YB-1 gene mRNA in the breast tumor tissues to predict the course of disease].

The purpose of the study was to develop a test of the multifunctional protein YB-1 in the intraoperative biopsy specimen to predict the course of breast cancer (BC). Its tasks were to use of real-time reverse-transcription polymerase chain reaction (RT-RCR) to substantiate the data previously obtained by semiquantitative RT-PCR and to clarify whether there was a correlation between the amount of YB-1 mRNA in the BC tissue and the status of steroid hormone receptors of these tumors. The determination of the tumor amount of YB-1 mRNA was shown to predict the course of BC: a statistically significant correlation was found between the higher content of YB-1 mRNA and the aggressive course of BC--the emergence of distant metastases. Comparing the content of YB-1 mRNA and the hormonal status of a tumor (the number of estrogen and progesterone receptors) revealed no correlations. The findings indicate that the determination of YB-1 mRNA by both real-time RT-PCR and semiquantitative RT-PCR may be used to predict BC metastases in distant organs.

[Studies of YB-1 protein in breast tumors].

The multifunctional mammalian protein YB-1 is involved in multiple DNA- and mRNA-dependent events in the cell and regulates gene expression at different levels. The intracellular localization and relative mRNA content of YB-1 in the breast tumors were studied. The presence of cells with nuclear YB-1 localization in the tumor cell population is a poor predictor that correlates with larger tumors (more than 5 cm). The high YB-1 mRNA content in the breast tumors promotes metastasis of small neoplasms and patients with breast cancer who have high tumor tissue YB-1 mRNA levels may referred to as an early distant metastasis risk group. The findings suggest that combined determination of YB-1 intercellular localization and mRNA levels can ensure a more reliable prognosis of breast cancer.

[Role of PTEN protein in multidrug resistance of prostate cancer cells].

In a past decade became evident that phosphatidylinositol-3-kinase controlled signal transduction cascade (PI3K/Akt/PTEN/mTOR) is implicated in resistance of tumor cells to anticancer drugs. Another well studied mechanism of multidrug resistance is associated with the activity of drug transporters of ABC superfamily (first of all P-glycoprotein (Pgp), MRP1, BCRP). Several mechanisms of cell defense can be turned on in one cell. The interconnections between different mechanisms involved in drug resistance are poorly studied. In the present study we used PC3 and DU145 human prostate cell lines to show that PTEN functional status determines level of cell resistance to some drugs, it correlates with expression level of MRP1 and BCRP proteins. We showed that Pgp is not involved in development of drug resistance in these cells. Transfection of PTEN into PTEN-deficient PC3 as well as rapamycin treatment caused the inhibition of PI3K/Akt/mTOR signaling and resulted in cell sensitization to the action of doxorubicin and vinblastine. We showed that PTEN transfection leads to the change in expression of MRP1 and BCRP. Our results show that in prostate cancer cells at least two mechanisms of drug resistance are interconnected. PTEN and mTOR signaling were shown: to be involved into regulation of MRP1 and BCRP.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

[Functional activity and expression of P-glycoprotein in chronic myeloid leukemia]. / Funktsional'naia aktivnost' i ékspressiia P-glikoproteina pri khronicheskom mieloleikoze.

AIM: To evaluate the prognostic significance of P-glycoprotein (Pgp) in chronic myeloid leukemia (CML). MATERIALS AND METHODS: Functional activity (rhodamine 123 test) and expression of Pgp (binding of UIC2 monoclonal antibodies by cells) were evaluated by flow cytofluorometry. A total of 141 samples of peripheral blood from 121 patients with various stages of CML were examined. RESULTS: The number of patients whose cells express functionally active Pgp increases during the blast crisis (BC) in comparison with the chronic phase (CP). Repeated testing of patients with BC and CP showed that Pgp-expressing cells can disappear from the peripheral blood of patients despite the treatment by Pgp preparations and substrates. However the number of cases with expression and functional activity of Pgp increases in the course of BC. Several patients in whom functionally active Pgp was not detected during diagnosis of BC had longer BC phase than patients with the active protein. CONCLUSION: These data suggest that active Pgp contributes to CML BC (presumably to patient's response to therapy) but this contribution is not decisive.

[Adaptation of reverse transcriptase polymerase chain reaction for clinical diagnosis of expression of MDR1 gene]. / Adaptatsiia metoda obratnotranskriptaznoi tsepnoi reaktsii dlia klinicheskoi diagnostiki ékspressii gena MDR1.

Chemotherapy of malignant tumors is ineffective usually because of tumor cell resistance to it. Two types of resistance are known: cell resistance to a certain drug and multiple drug resistance (MDR). MDR covers a wide spectrum of drugs with different chemical structure and mechanisms of action. The most frequent cause of MDR is hyperexpression in the plasma membrane of P glycoprotein cells, which is coded for by MDR1 gene realizing active release of many cytotoxic substances from cells (Pgp-MDR). Acquisition of MDR phenotype by patient's cells impedes therapy and is often a poor prognostic sign, and therefore testing of material from cancer patients for MDR phenotype is important for selecting tumor therapy. We adapted the reverse transcriptase polymerase chain reaction (RT-PCR) to evaluating the MDR1 gene expression in peripheral blood cells of patients with hemoblastosis, assessed its sensitivity and specificity, and carried out clinical trials with blood samples from patients with MDR. Comparison of the results of RT-PCR with the findings of other methods used for detection of Pg-MDR showed their good correlation in the majority of cases. These results recommend these method for clinical practice in patients with hemoblastosis.