A. S. Mikaelyan

A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells

The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. Here we address whether the transcriptional characteristics of HCC can provide insight into the cellular origin of the tumor. We integrated gene expression data from rat fetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors in this newly identified HCC subtype might have key roles in tumor development.

Application of comparative functional genomics to identify best-fit mouse models to study human cancer

Genetically modified mice have been extensively used for analyzing the molecular events that occur during tumor development. In many, if not all, cases, however, it is uncertain to what extent the mouse models reproduce features observed in the corresponding human conditions. This is due largely to lack of precise methods for direct and comprehensive comparison at the molecular level of the mouse and human tumors. Here we use global gene expression patterns of 68 hepatocellular carcinomas (HCCs) from seven different mouse models and 91 human HCCs from predefined subclasses to obtain direct comparison of the molecular features of mouse and human HCCs. Gene expression patterns in HCCs from Myc, E2f1 and Myc E2f1 transgenic mice were most similar to those of the better survival group of human HCCs, whereas the expression patterns in HCCs from Myc Tgfa transgenic mice and in diethylnitrosamine-induced mouse HCCs were most similar to those of the poorer survival group of human HCCs. Gene expression patterns in HCCs from Acox1−/− mice and in ciprofibrate-induced HCCs were least similar to those observed in human HCCs. We conclude that our approach can effectively identify appropriate mouse models to study human cancers.

HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway

Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier‐protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac‐GTPase and Rac‐specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin‐induced hy‐perpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin‐induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115‐RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac‐dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF‐induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against throm‐bin‐induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac‐dependent mechanism of HGF‐induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeabilityvia dynamic interactions between Rho‐ and Tiam1/Rac‐medi‐ated pathways.—Birukova, A., Alekseeva, E., Mikaelyan, A., and Birukov, K. G. HGF attenuates thrombin‐induced endothelial permeability by Tiam1‐mediated activation of the Rac pathway and by Tiam1/Rac‐dependent inhibition of the Rho pathway. FASEB J. 21, 2776–2786 (2007)

Tiam1 and βPIX mediate Rac-dependent endothelial barrier protective response to oxidized phospholipids

Oxidized 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphorylcholine (OxPAPC) exhibits potent barrier protective effects on pulmonary endothelium, which are mediated by small GTPases Rac and Cdc42. However, upstream mechanisms of OxPAPC‐induced small GTPase activation are not known. We studied involvement of Rac/Cdc42‐specific guanine nucleotide exchange factors (GEFs) Tiam1 and βPIX in OxPAPC‐induced Rac activation, cytoskeletal remodeling, and barrier protective responses in the human pulmonary endothelial cells (EC). OxPAPC induced membrane translocation of Tiam1, βPIX, Cdc42, and Rac, but did not affect intracellular distribution of Rho and Rho‐specific GEF p115‐RhoGEF. Protein depletion of Tiam1 and βPIX using siRNA approach abolished OxPAPC‐induced activation of Rac and its effector PAK1. EC transfection with Tiam1‐, βPIX‐, or PAK1‐specific siRNA dramatically attenuated OxPAPC‐induced barrier enhancement, peripheral actin cytoskeletal enhancement, and translocation of actin‐binding proteins cortactin and Arp3. These results show for the first time that Tiam1 and βPIX mediate OxPAPC‐induced Rac activation, cytoskeletal remodeling, and barrier protective response in pulmonary endothelium. J. Cell. Physiol. 211: 608–617, 2007.

Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification

Single-stranded long oligonucleotide-based (50- to 70-mer) microarrays offer several advantages over conventional cDNA microarrays. These include the easy preparation of the probes, low cost of array production, and low cross-contamination during probe handling. However, the application of oligonucleotide microarrays for the analysis of global gene expression with small amounts of total RNA using the conventional oligo(dT)-T7 promoter-based amplification is hampered by the single-stranded nature (sense strand) of oligonucleotide probes in microarrays. In this report, we describe modified RNA amplification methods generating antisense-labeled cDNA targets and a successful application for oligonucleotide microarray gene expression analysis. In the first round, mRNA was amplified linearly with oligo(dT)24T7-primed reverse transcription and in vitro transcription by T7 RNA polymerase. In the second round, random 9-mer T3 primers and T3 RNA polymerase were used to generate sense-strand amplified RNA (aRNA). Fluorescently labeled cDNA targets were generated from the aRNA and hybridized to the oligonucleotide microarrays. Our data show that the amplification provides highly reproducible results, as evidenced by a significant correlation between the amplified and nonamplified samples. We also demonstrate that amplification of RNA derived from laser-microdissected tumor samples reproduced the gene expression profiles that were obtained from total RNA isolated from the same samples.

Molecular and Genetic Regulation of Osteogenic Differentiation of Mesenchymal Stromal Cells

The review summarizes current concepts concerning the molecular genetic mechanisms of the osteogenic differentiation of mesenchymal stromal cells, which is controlled by a complex of signaling proteins and transcription factors. The interaction of regulatory factors involved in the most important signaling pathways at different stages of this differentiation is discussed.

Comparative characterization of mesenchymal bone marrow stromal cells at early and late stages of culturing

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1–4) but disappear at later stages (passages 9–11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6).

Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro

Purpose: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing β-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [1, 2]. Recently, we have shown that beneficial effects on β-cells require cadherin contacts between bNCSCs and β-cells [3, 4]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on β-cells. Materials and Methods: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of β-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. Results: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced β-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and β-cells. Even in direct contact between β-cells and HF-NCSCs, no cadherin expression was detected. Conclusions: These observations indicate that HF-NCSCs do not confer the same positive effect on β-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support β-cells in coculture and after transplantation.

Interaction of the ss and CG5017 genes in the regulation of morphogensis of limbs in Drosophila melanogaster

The influence of the P-element built into the area of the CG5017 gene on the mutation of the spineless (ss) gene was studied. It was shown that the insertion of the P-element decreased the level of transcription of CG5017 approximately twofold. Modulation of the level of transcription of the CG5017 gene helped demonstrate, for the first time, its influence on the phenotypic manifestation of the mutation of the ss gene, which shows their interaction in the process of regulation of morphogenesis of limbs in Drosophila melanogaster.

Molecular-genetic and immunophenotypic analysis of antigen profile and osteogenic and adipogenic potentials of mesenchymal stromal cells from fetal liver and adult bone marrow in rats

Comparative characteristics of mesenchymal stromal cells (MSCs) from adult bone marrow and fetal liver are of great interest due to the similar functions performed by these organs on the organization of a hemopoietic microenvironment at various developmental periods. It is known that MSCs play a pivotal role in the formation of niches for hemopoietic stem cells. The histogenetic relation of MSCs from these two hemopoietic organs cannot be ruled out. An analysis of antigen profile using immunocytochemistry and RT-PCR has confirmed that the studied cell populations fit the MSC criteria and have no contaminations of hemopoietic, lymphoid, and endothelial cells beginning at the second passage. Comparative analysis of osteogenic and adipogenic marker expression revealed MSC from fetal liver to have a weaker potential for adipogenesis and the extremely low capability for terminal osteogenic differentiation, in contrast to pronounced osteo- and adipogenic potentials of adult bone marrow MSC. The similar cell phenotype but different differentiation potentials under identical conditions of cultivation in vitro seem to be due to different developmental programs of the pre- and postnatal histogenesis of these MSC.