[SUPPRESSION OF TUMOR GROWTH AFTER XENOGRAFTING OF HUMAN COLORECTAL CARCINOMA CELLS].

Previously we've described the obtainment of a subpopulation of cancer stem cells from a human colorec- tal carcinoma cell line MIP101. These cells possess elevated clonogenic and tumorigenic capacities. According to our data, depletion of stem compartment in a cancer cell population blocks its tumorigenicity. The current work is dedicated to the comparison of tumorigenic potential between cell populations with enriched or depleted stem compartment. We show that tumor growth following xenografting of enriched stem cell population can be suppressed by intramuscular injections of ganciclovir. Thus, we report a method to obtain a cell population with high Oct4 promoter expression within the MIP101 colorectal carcinoma cell line and to eliminate these cells from the population in vitro as well as in vivo.

[THE STUDY OF THE ORIGIN OF HUMAN GLIOMA BASED ON CELL LINES AND TUMOR SPECIMENS].

The present study is dedicated to the search of human glioma "cells of origin". Specimens of the tumor tissue have been analyzed via the RT-PCR method to potentially reveal the expression of molecular markers characteristic of various cells of nerve tissue (NeuN, MOG, MBP, NG2, Olig2, Vimentin, GFAP, AldhlL1), as well as markers of stem (Oct4, c-Kit) and cancerous stem (CD133) cells. We have shown that the expression profiles of these markers for different types of gliomas intersect, which does not allow to determine the type of "cells of origin". So, in order to study the origin of glioma using cell lines derived from primary cultures, we need a more sophisticated culture conditions, rather than the commonly used serum-based media.

[A comparative analysis of colorectal carcinoma cell lines that differ in metastatic potential].

In the current work we make an attempt to compare cancer cells of one origin, but differing in the expression of CEA protein, a clinical marker of metastatic carcinomas, presumably one of the key factors in metastatic activity. We have explored the morphology of cell colonies in vitro, expression patterns of epithelial markers, the ability of these cells to form tumors and metastases in vivo, and evaluated their stem compartment with the aid of a suicidal genetic construct sensitive to the embryonic stem cell marker, Oct4.

[In vitro derivation and characterization of a colorectal cancer stem cell subpopulation].

In present publication we describe for the first time the obtainment of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells' primary role in the development of malignant neoplasms.

[Epithelial intestine cells transdifferentiate into bladder urothelium in experiments in vivo].

The autoplastic surgery by intestine tissue has been used for reconstructive therapy of the urinary tract since the middle of the last century; however, cell mechanisms of the urothelium engraftment are still obscure. Intestine stem cells possess plasticity and presumably enable after the autoplastic surgery to transdifferentiate into mature cells of urinary tract. Using the preliminary developed in vivo model for evaluation of somatic cells transdifferentiation into urothelium, we have found that the epithelial intestine cells producing Gfp transdifferentiate into the cryoinjured bladder urothelium of the syngenetic C57BL mice. Gfp was detected in the bladder tissue of mice-recipients using reverted polymerase chain reaction, primary fluorescence and immunofluorescence, while colocalization of the Gfp and Her-4 revealing similar to urothelium staining pattern was demonstrated in a few urothelium cells by double immunohistochemical staining of the bladder tissue with specific antibodies. The results obtained suggest that epithelial intestine cells enable to transdifferentiate into bladder urothelium, however the transdifferentiation level is low and presumably can not provide full functional urothelium engraftment in the case of autoplastic bladder surgery by intestine tissue.

[The interaction between CDX2 transcription factor and DDX5 RNA helicase].

Previously, our results of a two-hybrid screening essay allowed us to recognize p68 (DDX5) as a possible partner of CDX2. The recent part of research was carried out to confirm this interaction. We show the co-localization of these proteins in the nuclei of colon carcinoma cell line and of epithelium of villi. By means of GST-pulldown we reveal DDX5 as a part of a complex with CDX2. During the investigation of the effect of DDX5-CDX2 interaction upon beta-catenin-mediated transcription regulation we note that in each of three investigated cell lines Cdx2 acts as an activator of luciferase expression. In T98G and U20S cell lines we observe a partial decline of beta-catenin transcription enhancing effect while interacting with CDX2. In the cell systems studied, DDX5 acts as a weak repressor both solely and together with CDX2 and beta-catenin. Concerning the influence upon D1 cyclin promoter, we find that, depending on environment, CDX2 may either decline its transcription (U20S line) or raise it (T98G). Besides, PDGF reduces CDX2 activity both in activation and repression. When DDX5 and CDX2 are transfected in T98G cells together, the repressing activity of DDX5 is leveled with activation by Cdx2. In both cell lines the native DDX5 acts as a weak repressor of D1 cycline; PDGF treatment does no significant effect on its activity.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

[Genetic manipulation and studying of differentiation properties of rat induced pluripotent stem cells].

Induced pluripotent stem (iPS) cells are derived from somatic cells reprogrammed to the pluripotent state by the induced expression of defined transcription factors, achieved for the first time by the seminal work of Takahashi and Yamanaka. This new type of pluripotent cells has offered new exciting options in regenerative medicine allowing the replacement of cells and organs with the patient's own cells thereby avoiding immunological complications. In order to develop such technologies in approved animal models, iPS cells were also generated from rodents. Of course, the most important model for studying of different diseases is rat. In this study, we present a method suitable for rat iPS cells genetic modification by stable transfection and show necessary conditions for the first stages of direct differentiation.

[Chromosomal instability of in vitro cultured mouse embryonic stem cells and induced pluripotent stem cells].

A perspective of using embryonic stem (ES) and induced pluripotent stem (iPS) cells in clinical medicine makes karyological analysis of these cells an important issue. Using methods of classical and molecular cytogenetics chromosomal analysis, we have carried out karyological study of two mouse ES and two iPS cell lines derived de novo. We have found monosomy of X chromosome in all studied ES and iPS cell lines, thus making a modal number of chromosomes in these cell lines 39. A chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of iPS cell line. Our findings underline the importance of careful cytogenetic evaluation of pluripotent cell lines, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.

[Model in vivo to study the transdifferentiation of the somatic cell into urothelium].

Development of reconstructive therapy of the urinary tract using pluripotent and somatic stem cells, for example mesenchymal stem cell (MSCs), recently goes through the stage of experimental studies. These studies include investigation of the main functions of MSCs and urothelium lining from inside the organs of the urinary tract. An important role in the regulation of proliferation and differentiation of urothelium belongs to EGF and Wnt-beta-catenin signaling pathways which activity may be accessed by the level of Her-4 and Tcf3,4, accordingly. We found here that MSCs labeled by transgenic green fluorescence protein (GFP) did not produce in vitro Her-4 and Tcf3,4 but activated their production after transfer into cryoinjured bladder of the syngenic mouse. After MSCs transplantation, GFP was detected in the bladder by RT-PCR and was colocalized with Her-4 or Tcf3,4 in a few urothelium cells detected by immunohistichemical staining with specific antibodies. These results suggest that MSCs labeled by GFP may be used as a good model to study transdifferentiation of somatic cells into urothelium.

[E-cadherin as a novel surface marker of spermatogonial stem cells].

Spermatogenesis is a fundamental biological process that ensures gee transmission from one generation to another trough gametes. This process relies on a rare population of testicular cells, called spermatogonial stem cells (SSCs), that self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding research of SSCs, their biological properties remain largely unknown which is partly due to very limited availability of these cells. Here we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs, which can be successfully used for their enrichment.

[The application of lentiviral vectors for tissue-specific gene manipulations].

The trophectoderm (TE) ofblastocysts, the first epithelium established in mammalian development, 1) plays signaling, supportive, and patterning functions during pre-implantation development, 2) ensures embryo implantation into the uterine wall, and 3) gives rise to extra-embryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust non-permeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass (ICM) from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.

Two distinct types of repression domain in engrailed: one interacts with the groucho corepressor and is preferentially active on integrated target genes.

Active transcriptional repression has been characterized as a function of many regulatory factors. It facilitates combinatorial regulation of gene expression by allowing repressors to be dominant over activators under certain conditions. Here, we show that the Engrailed protein uses two distinct mechanisms to repress transcription. One activity is predominant under normal transient transfection assay conditions in cultured cells. A second activity is predominant in an in vivo active repression assay. The domain mediating the in vivo activity (eh1) is highly conserved throughout several classes of homeoproteins and interacts specifically with the Groucho corepressor. While eh1 shows only weak activity in transient transfections, much stronger activity is seen in culture when an integrated target gene is used. In this assay, the relative activities of different repression domains closely parallel those seen in vivo, with eh1 showing the predominant activity. Reducing the amounts of repressor and target gene in a transient transfection assay also increases the sensitivity of the assay to the Groucho interaction domain, albeit to a lesser extent. This suggests that it utilizes rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepressor complex only on integrated target genes.

[The search for the DNA sequences characterizing the granulocytic lineage of cell differentiation in the HL-60 line]. / Poisk DNA-posledovatel'nostei, kharakterizuiushchikh granulotsitarnoe napravlenie differentsirovki kletok linii HL-60.

cDNA of HL-60 cells induced to differentiation into granulocytes was hybridized with the excess of mRNA of K 562 cells induced to differentiation into erythrocytes. Cloning of cDNA sequences characterizing the induced differentiation of HL-60 cells into granulocytes was performed. The clones obtained were shown to contain sequences encoding a zinc finger motif, which is specific for DNA-binding domain of protein activators of transcription.