Expression of CASC8 RNA in Human Pancreatic Cancer Cell Lines.

A lot of long non-coding RNAs (lncRNAs) are expressed in human cells in a number of transcripts of different lengths and composition of exons. In case of cancer-associated lncRNAs, an actual task is to determine their specific isoforms, since each transcript can perform its own function in carcinogenesis and might have a unique expression profile in various types of tumors. For the first time, we analyzed the expression of CASC8 lncRNA in human pancreatic ductal adenocarcinoma cell lines and found an abundant isoform that was previously considered as the minor one in this type of cancer. We also revealed extremely high expression levels of all CASC8 transcripts in MIA PaCa-2 cells and, conversely, the lack of this lncRNA in PANC-1. This allows to use them as convenient models for further in vitro studies.

Deregulation of signaling pathways involved in sorafenib resistance of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide. Hepatocarcinogenesis is associated with deregulation of the cell signaling thus targeted therapy can decelerate HCC progression by specific inhibition of alternated signaling cascades. Sorafenib is the only multitarget drug approved for HCC treatment that blocks several crucial oncogenic signaling pathways thus suppressing tumor growth, metastasis and providing survival benefit for subset of patients sensitive to sorafenib. Compensatory activation of other tumorigenic mechanisms may lead to decrease of HCC sensitivity to sorafenib. HCC are heterogenic tumors of epithelial origin, and presence of low-differentiated subpopulations of cancer stem cells or dedifferentiated fibroblastoid cells, that are less sensitive to sorafenib due to resistance to growth-inhibitory action of the drug, promotes HCC resistance to sorafenib. Analysis of the expression profile of genes encoding tissue-specific proteins, components of cell junctions, stem cell and mesenchymal markers can reveal sorafenib-resistant populations in HCC and identify signaling pathways that reduce response to sorafenib. Identification of individual sorafenib resistance mechanisms may be useful for rational choice of an appropriate combination of targeted drugs for retardation of HCC progression and improving the efficacy of therapy

[Partial reversion of the phenotype of a poorly differentiated hepatocellular carcinoma in a three-dimensional culture].

In vivo, normal tissues and organs have a three-dimensional structure and function in a three-dimensional environment. The standard two-dimensional cell culture conditions drastically differ from those in vivo. For this reason, three-dimensional cultures based on different variants of the extracellular matrix are more adequate for analyzing normal and tumor cell growth. Culturing a poorly differentiated hepatocellular carcinoma in a collagen gel yielded spheroids whose growth pattern shifted towards the epithelial phenotype. The shift was expressed in changes in the cytoskeleton, enhanced formation of extracellular matrix fibrils between cells, and formation of fibronectin fibrils on the outer surface of spheroids. Analysis of 25 genes reflecting the level of morphological and functional hepatocyte differentiation showed that the expression of the gene encoding the transforming growth factor TGFbeta2 was suppressed the most significantly.

[Hepatocyte nuclear factor 4 (HNF4) in epithelial development and carcinogenesis].

Hepatocyte nuclear factors play the key role in the establishing and maintenance of hepatocyte differentiation. They not only control the expression of functional hepatic genes but are also involved in the regulation of proliferation, morphogenesis and detoxication in the liver. In this review we consider the main biological properties of the central regulator of hepatic differentiation HNF4alpha, patterns of its expression in embryogenesis and different adult tissues, mechanisms of regulation of its activity and transcriptional properties, and the essential target genes. Based on the studies of gene expression on the experimental models of rodent carcinogenesis and clinical samples of human liver tumors, the clear association of HNF4alpha transcriptional repression with progression and dedifferentiation of this type of tumors was shown. The possibility of the reversion of dedifferentiated hepatocarcinoma malignant phenotype by means of HNF4alpha exogenous expression confirms the important role of this factor in the coordination of proliferation, differentiation and the maintenance of epithelial morphology in several types of epithelial cells.

Electrochromatography: a method for automatic immunoaffinity chromatography on porous membranes.

Electrochromatography (ECHR) exploits a very high electro-osmotic counterflow developed in porous membranes at discontinuous electrophoresis. This counterflow exceeds considerably the anodic migration of any negatively charged protein and is used as a 'conveyer belt' for sequential transfer of immunoreagents to the specific adsorbents (antigens or antibodies) fixed on the nitrocellulose membrane. This approach was applied for simultaneous detection of two antigens (alpha-fetoprotein and carcino-embryonic antigen) in one sample, for determination of subfractions of alpha-fetoprotein, different in their epitope specificity, and for detection of L chains with certain idiotype on the background of heterogeneous L fraction. ECHR was used also for the partition of different antibodies to DNA adducts, demonstrating the possibility of applying this method to the study of DNA-binding proteins.

[Determination of DNA-binding proteins by countercurrent isotachopheresis on nitrocellulose membranes. I. Antibodies to DNA and its adducts]. / Opredelenie DNK-sviazyvaiushchikh belkov metodom protivotochnogo izotakhoforeza na nitrotselliuloznykh membranakh. I. Antitela k DNK i ee adduktam.

A very strong electroosmotic counterflow was produced on nitrocellulose membranes during isotachophoresis in a system of 0.06 M Tris-HCl (pH 6.7) as the leading electrolyte and 0.012 M Tris-beta-alanine (pII 8.6) as the terminating one. This counterflow was equal in rate and opposite in direction to the migration of the Cl-/beta-alanine boundary. The rate of counterflow was much higher than the rate of migration of any organic anions, including different proteins. Double-stranded and single-stranded DNA or its adducts were fixed on the nitrocellulose membrane, and the membrane was blocked with unrelated proteins. DNA-binding proteins, namely antibodies to DNA, followed by peroxidase-conjugated anti-IgG, were introduced into the counterflow, which transferred them one after another to the DNA spots. Thus, sequential binding and washing was performed automatically. In this way, antibodies were detected to ds- and ss-DNA, to BrdU-DNA, to Z-DNA, to biotinylated DNA and DNA modified with trans-Pt, as well as development of biotinylated DNA dots by steptavidin-peroxidase.

[Suppression of alpha-fetoprotein gene expression in somatic hybrids of AFP-positive and AFP-negative clones of rat hepatoma]. / Podavlenie ékspressii gena al'fa-fetoproteina v somaticheskikh gibridakh AFP-polozhitel'nogo i AFP-otritsatel'nogo klonov krysinoi gepatomy.

The presented study is devoted to investigation of molecular mechanisms regulating alpha-fetoprotein (AFP) gene expression at transcriptional level. The study was carried out on AFP-positive and AFP-negative clones of rat hepatoma McA-RH 7777 that also differ in hepatocyte nuclear factors (HNF) 1 and 4 transcription levels. To examine a hypothesis of existence in AFP-non-producing clones a transcriptional factor that downregulates this gene expression, we have obtained somatic hybrids of AFP-positive and AFP-negative clones. In the obtained hybrids AFP gene expression is decreased while expression of HNF1, one of the main AFP promoter activators, is maintained. These data indicate an existence of a repressor in AFP-negative clones that determines AFP gene downregulation regardless of the HNF1 expression level.

[Expression of tissue-specific genes with progression of mouse hepatocellular carcinoma]. / Ekspressiia tkanespetsificheskikh genov pri progressii gepatokartsinom myshi.

Expression of hepatocyte-specific genes in slow- and fast-growing hepatocellular mouse carcinomas was studied. The fast-growing poorly differentiated passaged hepatocarcinoma (fHC) originated from the well-differentiated slow-growing variant (sHC). In contrast to the parental hepatocarcinoma, in fHC the expression of the hepatocyte nuclear factor 4 (HNF4), in fHC a key factor responsible for hepatocyte differentiation, and several HNF-4-responsive genes, such as those for transferrin, transthyretin, hepatocyte nuclear factor 1 (HNF1), and serum albumin, was significantly suppressed. The expression of exogenous HNF4 in the fHC cell culture partially restored the expression of hepatocyte marker genes and the appearance of epithelial cell islands in the culture. The described system may serve as a convenient model for further analysis of mechanisms underlying hepatocarcinogenesis and liver tumor progression.

Deregulation of hepatocyte nuclear factor 4 (HNF4)as a marker of epithelial tumors progression.

Tissue-specific transcription factors forming the regulatory cascades which determine the specification and differentiation of epithelial cells during embryogenesis, play the central role in the control of functional and morphological properties of different cell types. Hepatocyte nuclear factors (HNFs) network is one of the most investigated tissue-specific regulatory systems which controls the specification and maintenance of differentiation of several epithelial cell types. Nuclear receptor HNF4α is one of the central elements of this regulatory network in the liver. We have found that deregulation of this gene is associated with rodent and human hepatocellular carcinoma (HCC) progression and induces the increase of proliferation rate, loss of epithelial morphology, dedifferentiation and metastasis. Restoration of HNF4α expression in dedifferentiated cells induced partial reversion of highly malignant phenotype both in vitro and in vivo. In human HCC samples HNF4α transcription was completely lost or significantly decreased in about 70% of HCCs, not associated with hepatitis B virus infection. Decrease of HNF4α isoforms expression correlated with poor prognosis. Thus we propose HNF4α is a candidate tumor suppressor for hepatic cells. Dysfunction of different HNFs was also reported in other epithelial tumors. We suppose that tissue-specific transcription factors which control the key steps of definite differentiation programs and are capable to receive and modulate extracellular signals can be considered as promising tumor suppressor candidates for their corresponding tissues.

Tissue-specific transcription factors in progression of epithelial tumors.

Dedifferentiation and epithelial-mesenchymal transition are important steps in epithelial tumor progression. A central role in the control of functional and morphological properties of different cell types is attributed to tissue-specific transcription factors which form regulatory cascades that define specification and differentiation of epithelial cells during embryonic development. The main principles of the action of such regulatory systems are reviewed on an example of a network of hepatocyte nuclear factors (HNFs) which play a key role in establishment and maintenance of hepatocytes--the major functional type of liver cells. HNFs, described as proteins binding to promoters of most hepatospecific genes, not only control expression of functional liver genes, but are also involved in regulation of proliferation, morphogenesis, and detoxification processes. One of the central components of the hepatospecific regulatory network is nuclear receptor HNF4alpha. Derangement of the expression of this gene is associated with progression of rodent and human hepatocellular carcinomas (HCCs) and contributes to increase of proliferation, loss of epithelial morphology, and dedifferentiation. Dysfunction of HNF4alpha during HCC progression can be either caused by structural changes of this gene or occurs due to modification of up-stream regulatory signaling pathways. Investigations preformed on a model system of the mouse one-step HCC progression have shown that the restoration of HNF4alpha function in dedifferentiated cells causes partial reversion of malignant phenotype both in vitro and in vivo. Derangement of HNFs function was also described in other tumors of epithelial origin. We suppose that tissue-specific factors that underlie the key steps in differentiation programs of certain tissues and are able to receive or modulate signals from the cell environment might be considered as promising candidates for the role of tumor suppressors in the tissue types where they normally play the most significant role.

Molecular mechanisms of alpha-fetoprotein gene expression.

Alpha-fetoprotein (AFP) is the main component of mammalian fetal serum. It is synthesized by visceral endoderm of the yolk sac and by fetal liver. Immediately after birth AFP level in blood decreases dramatically. AFP synthesis is reactivated in liver tumors and germinogeneous teratoblastomas, in a lesser degree after chemical and mechanical liver injuries followed by regeneration (i.e., acute viral hepatitis). AFP blood level change is an important marker for liver tumors that is widely used in clinical practice. Therefore, the study of the molecular and cellular mechanisms participating in regulation of the oncoembryonal protein AFP is an important task. On various experimental models it has been shown that the expression is regulated mainly on the transcriptional level, the AFP gene having a 7 kb regulatory region upstream. Within this region a tissue-specific promoter, three independent enhancers, and a silencer that is at least partially responsible for AFP gene expression decrease in adult liver have been defined. Some ubiquitous and some tissue-specific transcription factors, including hepatocyte nuclear factors (HNFs), which mediate the transcription of most of the liver-specific genes, have been shown to bind to the promoter. However, the mechanisms determining drastic changes of AFP synthesis level in the course of ontogenesis and carcinogenesis remain incompletely clarified. Also, little is known about negative regulators of AFP gene expression in cells of non-hepatic origin and in adult liver.

Inducible protein in rat hepatomas with expression alternative to alpha-fetoprotein.

The rat hepatoma cell line McA RH7777 was cloned into alpha-fetoprotein-producing (AFP+) and non-producing (AFP-) sublines. A monoclonal antibody (MAb A2/3) reacting with an antigen (Ag A2/3) present only in AFP- clones or AFP- cells in mixed clones was obtained. Ag A2/3 was absent from the liver of embryonic, fetal, newborn and adult rats, but it was present in gastric and intestinal mucosa of adult rats. Ag A2/3 was found to be a heavy metal-inducible protein: Cd2+ and Pb2+ strongly induced the expression of Ag A2/3 in vivo in the liver of adult rats, while xenobiotics and CCl4 were not active in this respect. In vitro Cd2+ and Pb2+ induced Ag A2/3 expression in several AFP+ clones, leading to a simultaneous marked decrease of AFP+ cells from such clones. The effect of Cd2+ in the induction of Ag A2/3 and suppression of AFP was reversible. SDS PAGE revealed one protein band with an m.w. close to 45,000, which was not sensitive to mercaptoethanol. Despite its inducible properties, Ag A2/3 was shown not to belong to metallothioneins, cytochrome P-450, glutathion-transferase or heat shock proteins families, well-known as being inducible cell stress proteins. Expression of Ag A2/3 could be one of the factors determining the high amplitude of AFP production by individual liver tumors. The nature of Ag A2/3 and its alternative expression with respect to AFP remain to be studied.