[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.

[Gene expression of vascular endothelial growth factors and their receptors in different variants of the course of multiple myeloma].

AIM: To determine the significance of the angiogenic activity estimated from the gene expression of the vascular endothelial growth factors (VEGFs) VEGF-A, VEGF-C, and VEGF-D and their receptors VEGFR1, VEGFRls, VEGFR2, and VEGFR3 in the mononuclear cell fraction of bone marrow (BM) aspirates with tumor plasma cells predominating in different variants of the course of multiple myeloma (MM). MATERIALS AND METHODS: The gene expression of VEGF-A, VEGF-C, and VEGF-D and their receptors VEGFRI, VEGFRls, VEGFR2, and VEGFR3 was determined by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: VEGF-A, VEGF-C, VEGF-D, as well as VEGFR1, VEGFRls, VEGFR2, and VEGFR3 were expressed showing different intensities in the mononuclear cell fraction of BM aspirates with a predominance of tumor plasma cells in the patients with MM, which allowed patient groups to be identified. In the group of high gene expression of VEGFs and their receptors, the number of clusters of plasma cells and vascular endothelium in the BM aspirates and the degree of osteolysis in the skeletal bones of patients with MM were significantly higher than those in the group of low or absent gene expression. The survival in the latter group was significantly higher. CONCLUSION: The investigation could provide an estimate of angiogenic processes in MM and establish their association with clinical manifestations and cytological characteristics.

[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.

[Relationship between the induction of MDR1, a multidrug resistance gene in tumor cells, and apoptosis]. / Sviaz' mezhdu inductsiei aktivnosti gena mnozhestvennoi lekarstvennoi ustoichivosti opukholevykh kletok MDR1 i apoptozom.

Gene MDR1 coding for P-glycoprotein belongs to a group of genes responsible for cell defense. Overexpression of this gene determines the resistance of tumor cells to a series of chemotherapeutic drugs known as multidrug resistance. Many chemotherapeuticals induce both apoptosis and transcriptional activity of the MDR1 gene in tumor cells. It is not known, however, how these two processes are associated with each other. In order to elucidate a possible link between them, we have studied the sphyngomyelinic pathway of signal transduction. This pathway is activated in response to various stress factors and includes the hydrolysis of sphyngomyelin of cytoplasmic membrane resulting in an accumulation of intracellular ceramide, which activates cascades of enzymatic reactions leading to various cell responses, including apoptosis. C2 ceramide (N-acetyl-D-sphyngosine) and cytosar (1 beta-D-arabinosylcytosine, or ara C) were used to induce the sphyngomyelinic pathway. Their effects on human hemoblastosis cell lines (K562 and H9 cell lines) were examined. C2 ceramide and ara C induced apoptosis in both cell lines over an 18-h incubation. C2 ceramide also induced an increase in the expression of the gene MDR1 in both cell lines, while ara C increased the activity of the gene MDR1 only in H9 cells. The results obtained provide evidence for the contribution of ceramide-mediated signal pathway to the control of MDR1 activity.

[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.

[The clinical significance of the expression of the multiple-drug resistance protein P-glycoprotein in chronic myeloleukemia]. / Klinicheskoe znachenie ékspressii belka mnozhestvennoi lekarstvennoi ustoichivosti P-glikoproteina pri khronicheskom mieloleikoze.

Cell resistance to pharmaceutical agents arises among other causes because of multiple drug resistance induced by P-glycoprotein (P-gp). The analysis of expression of P-gp and differentiation antigens of hemopoietic cells has been made on myeloid cells from 14 patients in CML chronic phase and 25 with CML acceleration and in blast crisis. Surface antigen expression was evaluated at flow cytofluorimetry (FACScan unit). Fluorescent dye rodomin (Rh123) helped examine P-gp functional activity. A close relationship is shown between P-gp expression and CD34 (r = 0.69. p = 0.0004), this giving evidence of these antigens expression on the same cells. In chronic phase P-gp is expressed on a few cells in some patients, its activity being low or absent. The appearance of UIC-2+ cells was unrelated to previous chemotherapy and brought no resistance to treatment. In terminal stage P-gp is expressed in 50% of cases. Functional tests identified the active protein in blast populations with a large number of UIC-2+ cells and in some patients with a small number of cells expressing P-gp. Therefore, comprehensive clinical investigations are needed of multiple drug resistance, though in half of the resistant patients in AML blast crisis P-gp+ cells were not identified suggesting the existence of other mechanisms responsible for resistance to treatment.

[The normalization of tumor cell morphology related to an increase in their size: research on giant cells produced by mitomycin C treatment]. / Normalizatsiia morfologii opukholevykh kletok, sviazannaia s uvelicheniem ikh razmerov: issledovanie gigantskikh kletok, poluchennykh obrabotkoi mitomitsinom C.

This study shows that artificial increase in cell site leads to morphological normalization of transformed fibroblasts. Mouse L cells (clone 171/5) were used. As most transformed cells, they were poorly spread on the substratum, made only dot-like focal contacts with it, rounded quickly at room temperature and did not contain prominent actin cables. Giant cells were obtained by incubation of these cells in the medium supplemented with mitomycin C (0.15-0.20 mcg/ml). DNA synthesis and mitosis were blocked by this treatment, while protein synthesis was changing very slightly. As a consequence, the cell size increased dramatically from 3 to 11 days of the cell incubation in the mitomycin containing medium. The degree of cell spreading per mcg of protein increased significantly in the giant cells. These cells do not round after moderate cooling, and well developed system of actin cables and matured streak-like focal contacts associated with these cables are formed in them. These results, along with our previous data on the restoration of cell spreading and cytoskeleton structure in giant multinucleated cells, provide strong evidences that the increase in cell size per se can induce qualitative changes in cell morphology. It can be suggested that there are some scaling-dependent factors regulating the processes of cytoskeleton assembly and formation of cell-substrate contacts.

[Multiple drug resistance and differentiation in the population of transformed dog kidney cells MDCK]. / Mnozhestvennaia lekarstvennaia ustoichivost' i differentsirovka v populiatsii transformirovannykh kletok pochki sobaki MSCK.

Sublines of the canine kidney epithelium cells (MDCK) resistant to various colchicine doses (0.3-5.0 mcg.ml-1) were obtained by multistep selection. The new lines were shown to possess multidrug-resistance (MDR) by means of Rhodamine 123 staining. The initial steps of resistance (2 mcg.ml-1, 300-fold increase in resistance) are evidently due to enhancement of mdr gene transcription. Approximately 4-fold gene amplification was revealed in the cells resistant to 3 mcg.ml-1 of colchicine. All the drug-resistant lines tested were found to be more prone to differentiation than the wild type cells (both spontaneous and induced by several drugs such as DMSO, cAMP and others). The data suggest that the selection for overexpression of mdr gene results in selection of variants that are more capable of differentiation than the rest of the population.

[The cells of murine sarcoma PC-103 induced by a polymeric plate secret transforming growth factor alpha]. / Kletki sarkomy PS-103 myshei, indutsirovannoi polimernoi plastinkoi, sekretiruiut transformiruiushchii faktor rosta al'fa.

It was shown previously that cells of sarcoma PS-103 induced in mouse by subcutaneous transplantation of plastic film produce growth-stimulating activity. In this communication clone 3 sb, isolated from sarcoma PS-103, was studied. Growth factor produced by the cells of this clone stimulated proliferation of quasi-normal (3T3, NRK) cells and transformed cells both in monolayer and in semi-solid medium. It was shown by gel filtration chromatography that the main growth-stimulating activity migrated with the protein fraction m.m. 10-15 kDa. The addition of this growth factor to the culture medium significantly (90%) inhibited 125I-EGF (epidermal growth factor) binding by A-431 cells. These data suggest that the growth factor produced by the studied tumor cells is transforming growth factor alpha.

[Effects of monoclonal antibodies to bovine nerve growth factor on NGF-induced cell differentiation in culture]. / Vliianie monoklonal'nykh antitel k faktoru rosta nervov byka na FRN-indutsirovannuiu differentsirovku kletok v kul'ture.

Five Hybridoma clones producing monoclonal antibodies (MAT) to bovine nerve growth factor (NGF) were developed. The biological effects of antibodies were studied: the influence of MAT on neurit outgrowth induced by NGF in rat pheochromocytoma PC12 or spinal chicken ganglia was investigated. MAT fell into two groups. Two of them inhibited neurit induction by NGF, three others stimulated this process. The stimulation of the neurit outgrowth by MAT was observed at low concentration of NGF (3 ng/ml of culture medium). Mechanisms of antibodies effects are discussed.

[Characteristics of crossed resistance of neoplastic cells with high level of colchicine resistance]. / Osobennosti perekrestnoi rezistentnosti opukholevykh kletok s vysokim urovnem ustoichivosti k kolkhitsinu.

Using a long-term selection the authors obtained the cells of a hamster with a rather high level of stability to colchicine (800-16,600 times). The nature of resistance of these cells to actinomycin differed from those having general origin with the newly obtained but having a lower level of stability to colchicine. This level of stability did not correlate with that to a selective agent.

[Relation of the signs of malignant degeneration and tumor cell reaction to a growth-stimulating factor]. / Sviaz' priznakov malignizatsii i reaktsii opukholevykh kletok na roststimuliruiushchii faktor.

Six clones of mouse sarcoma induced by subcutaneous implantation of a plastic film were studied. They differed in the degree of production of the transforming growth factor (TGF) and in stimulation of proliferation in semi-solid medium. The degree of cancerogenicity and cloning efficiency in semi-solid medium correlated with the reactivity of cells to TGF.

[Clonal structure of tumors: difference between clones based on the ability to secrete growth-regulating factors and to react to them]. / Klonal'naia struktura opukholi: razlichiia mezhdu klonami po sposobnosti vydeliat' faktory, reguliruiushchie razmnozhenie, i reagirovat' na nikh.

Conditioned medium isolated from different clones of tumour cell culture PS-103 (sarcoma induced in CBA mouse by plastic film implantation) varied in the ability to stimulate cell proliferation in semi-solid medium. The clones differed also in their response to the medium conditioned by culture PS-103: some clones responded by increased proliferation in semi-solid medium, some were inhibited and some failed to respond.

[Independence from the substrate of multiplication in nonclonal and clonal tumor cell populations]. / Nezavisimost' razmnozheniia ot substrata v neklonirovannoi i klonovoi populiatsiiakh opukholevykh kletok.

Experiments were made with sarcoma PS-103 cells cultured in vitro. The sarcoma was induced by implantation of a plastic plate into CBA mice. Clonal analysis of the cell culture demonstrated that 1) all 3 clones isolated from substrate (SB) grew in 1.2% methyl cellulose (MC) at the same efficiency as parental cells; 2) all 5 clones isolated from MC formed in a semi-solid medium 10-100-fold more colonies than PS-103. During the subcloning of one of PS-103 clones in solid substrate and in MC, it turned out that the majority of MC and SB subclones had the plating efficiency in MC similar to that in PS-103. Apparently, the PS-103 population contains clones with different degrees of anchorage independence.

[Relation between the attachment of cells to a substrate and their proliferative characteristics]. / Sviaz' mezhdu prikrepleniem kletok k substratu i ikh proliferativnymi kharakteristikami.

17 cloned cell lines of transformed mouse fibroblasts were used for the evaluation of a correlation between three traits of malignancy: cloning efficiency in semisolid medium (CE); cell dose inducing tumours in 50% of inoculated animals (TD50); degree of cell attachment to a substrate (RT50--rounding time for 50% of the cells under moderate cooling, 18 degrees C). It is shown that an increase in RT50 (better attachment of the cells to the substrate) is accompanied by a decrease in CE and an increase in TD50 (coefficients of rank correlation p = -0.76 and p = 0.58, respectively). A negative correlation between CE and TD50 was also revealed (p = -0.68). A certain increase in the percentage of cells containing actin filament bundles was found in cell clones better attached to the substrate. Mechanisms of disturbances in proliferation and attachment of malignant cells are discussed.

[Effect of polyploidization on the anchorage-independent multiplication of transformed cells]. / Izuchenie vliianiia poliploidizatsii na nezavisimost' razmnozheniia transformirovannykh kletok ot prikrepleniia k substratu.

Three nearhexaploid sublines were obtained from hypotriploid mouse L cells by means of colcemid treatment. When cultivated on solid substratum, all of them did not differ from the parental line either in doubling time or in cloning efficiency. The ability of polyploid cell variants to be initiated for proliferation in a semi-solid medium was equal to that of hypotriploid cells, while the average diameter of colonies formed by hexaploid cells in methyl cellulose turned out to be significantly smaller than the size of colonies of parental cells. The inhibition of growth in the semi-solid medium may reflect partial normalization of the transformed phenotype of polyploid L cells.

[Effect of ethylmethane sulfonate on the expression of one of the traits of malignancy by cultured mouse cells]. / Vliianie étilmetansul'fonata na ékspressiiu odnogo iz priznakov malignizatsii kul'tiviruemymi kletkami myshi.

The influence of ethyl methane sulfonate (methane sulfonic acid ethyl esther, EMS) on anchorage independence of tumor cells was studied. Mouse near-diploid spontaneously transformed clonal fibroblasts (CAK-25Agr were used. They were characterized by a stable low cloning efficiency in 1,2% methyl cellulose ((3-5) . 10(-5) per cell seeded into a semi-solid medium). EMS enhanced the quantity of CAK-25Agr colonies grown in methyl cellulose. However, this enhancement was only obtained when correction on the cloning efficiency of the cells in a liquid medium was introduced. Subclones of CAK-25Agr isolated from methyl cellulose were studied for their ability to form colonies in the semi-solid medium. The number of subclones with elevated anchorage independence in cultures treated by a mutagen and in untreated cultures did not differ.