Retroviral transfer of antisense sequences results in reduction of c-Abl and induction of apoptosis in hemopoietic cells

The c-abl proto-oncogene is ubiquitously expressed during mammalian development. Activated forms of c-Abl proteins are oncogenic and have been shown to suppress apoptosis. The biological role of normal c-Abl protein is unknown. In this study, we have introduced c-abl antisense sequences into various hemopoietic cells by retroviral gene transfer. Introduction and expression of the antisense sequence effectively reduced the amount of c-Abl protein in a number of transduced hemopoietic cells, that consequently underwent apoptosis. When factor-dependent cell lines were examined, we observed that the addition of sufficient amounts of growth factors could suppress apoptosis in myeloid but not in lymphoid lines. The ability of myeloid cells to be rescued by growth factors correlated with upregulation of mRNA level of IL-3 receptor subunits. Our data suggest that c-Abl provides an anti-apoptotic signal during mammalian cell growth, and that myeloid and lymphoid cells are different in their resistance to apoptosis.     

Expression of c-src and c-abl in embryonal carcinoma cells and adult mouse tissues

We have studied the expression of c-src and c-abl proto-oncogenes in early mouse development using embryonal carcinoma (EC) cells as a model system, and compared this to the expression pattern in adult tissues. In all three EC lines tested (F9, PC13, and P19), c-src and c-abl mRNA can be detected. When F9 and PC13 are induced to differentiate they form endodermal cells characteristic of the early embryo, and we found no change in c-src or c-abl expression. In contrast, P19 cells showed increased levels of both mRNAs both mRNAs when induced to differentiate along the neural pathway by retinoic acid, whereas differentiation along the muscle pathway by dimethyl sulfoxide resulted in decreased levels of c-abl expression. These results are consistent with the idea that c-src and c-abl have important functions in the differentiation of the cell types of the later embryo, but not in those of the early embryo.     

Suitability of stratagene reference RNA for analysis of lymphoid tissues

We evaluated a lymphoid RNA standard prepared in our laboratory for spotted microarrays against the Universal Human Reference standard from Stratagene. Our goal was to determine if the Stratagene standard, which contains only two lymphoid cell lines out of a pool of 10 human cancer cell lines, had acceptable gene coverage to serve as a comprehensive standard for gene expression profiling of lymphoid tissues. Our lymphoid standard was prepared from thymus, spleen, tonsil, and cell lines representing immature B cells, plasma cells, and natural killer (NK) cells, thus covering the entire spectrum of lymphoid cells and most stromal elements present in specialized lymphoid tissues. The two standards were co-hybridized on oligonucleotide microarrays containing 17,260 genes, and both had fluorescence intensities above background for approximately 85% of the genes. Despite the limited representation of lymphoid cells in the Stratagene standard, only 4.2% genes exhibited expression differences greater than 2-fold including only 0.35% with differences greater than 4-fold. Although the lymphoid standard reflected a more comprehensive representation of immune system-associated genes, the Stratagene standard has the advantage of being commercially available, enabling easier comparison across laboratories and allowing comparative studies across a long period of time.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

Protein kinases predominately expressed in human ES cell lines during differentiation

Capacity of human embryonic stem cells (ESC) for unlimited proliferation and differentiation make them an attractive object in fundamental science and medicine. Little is known about the mechanisms that direct cells to particular differentiation or sustain them in an undifferentiated state. Activation of these mechanisms is determined by gene expression mediated by cascades of signal transduction. Protein kinases are essential components of signal pathways. The study of protein kinases expression in ESC and embryoid bodies facilitates a better understanding of the processes underlying the differentiation stages. We isolated cDNA libraries with fragments of catalytic domains of protein kinases expressed in human ESC and embryoid bodies (EB) of hESM01 and hESM02 cell lines. Using Northern hybridization, we revealed a high level of protein kinases MAK-V in human ESC. Expressions of MAK-V, A-RAF-1, MAPK3, IGF1R, NEK3, and NEK7 in ESC and EB in hESM01 and hESM02 cell lines were compared by the semiquantitative method RT-PCR.     

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.     

Lineage and stage specific expression of HOX 1 genes in the human hematopoietic system.

Recent observations have demonstrated the expression of several members of the homeobox-containing (HOX) gene complexes within the hematopoietic compartment. We have analyzed the expression pattern of the entire HOX 1 locus in a panel of leukemia-derived human cell lines representing various blood phenotypes. The expression of the eleven HOX 1 genes is lineage-restricted and these genes are predominantly detected within cells of myelomonocytic origin. This is in strong contrast with the erythro-megakaryocytic specific expression of HOX 2 genes. Furthermore, we have observed that the expression of three HOX 1 genes within B lymphoid lineages is stage-related and that the expression of several of them is switched off during TPA-induced differentiation of Kg1 and U937. These observations suggest that HOX 1 homeoproteins could be regulators of lineage determination during hematopoiesis.     

Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.     

Sequence and expression of murine cDNAs encoding Xlr3a and Xlr3b, defining a new X-linked lymphocyte-regulated Xlr gene subfamily

Using a subtractive cDNA approach we have identified two nearly identical genes, Xlr3a and Xlr3b (X-linked lymphocyte regulated), expressed at a consistently high level in 14 out of 14 murine plasmacytoma cell lines, at a high level in 1 out of 8 B-lymphoma cell lines, and at a very low level in 2 out of the 8 B-lymphoma cell lines. The messages are not detected in 10 pre-B-lymphoma cell lines. These genes express 2.0-kb mRNAs that encode 226-amino-acid proteins that are extremely basic, with an estimated pI of 8.1 and 9.0, respectively. By sequence comparison they are homologous to Xlr1, an acidic nuclear protein that is produced in lymphoid cell lines corresponding to the late stages of lymphocyte differentiation. Xlr2 is a highly homologous gene that is expressed in differentiating male germ cells. Xlr3a and Xlr3b are members of a new subfamily in the Xlr multigene family. Like Xlrl, they are up-regulated during B-cell terminal differentiation in normal and neoplastic B-cells, and cross-hybridize with a message in testis RNA. Also, like Xlrl, they do not cross-hybridize with human genomic DNA.     

Ethanol Alters Proliferation and Differentiation of Normal and Chromosomally Abnormal Human Embryonic Stem Cell‐Derived Neurospheres

Ethanol is a powerful substance and, when consumed during pregnancy, has significant psychoactive and developmental effects on the developing fetus. These abnormalities include growth retardation, neurological deficits, and behavioral and cognitive deficiencies, commonly referred to as fetal alcohol spectrum disorder. The effect of ethanol has been reported to affect cellular development on the embryonic level, however, not much is known about mutations contributing to the influence of ethanol. The purpose of our study was to determine if mutation contribute to changes in differentiation patterning, cell‐cycle regulatory gene expression, and DNA methylation in human embryonic stem cells after ethanol exposure. We exposed human embryonic stem cells (with and without know DNA mutations) to a low concentration (20 mM) of ethanol and measured neurosphere proliferation and differentiation, glial protein levels, expression of various cell‐cycle genes, and DNA methylation. Ethanol altered cell‐cycle gene expression between the two cell lines; however, gene methylation was not affected in ether lines.. Birth Defects Res (Part B) 98:283–295, 2013. © 2013 Wiley Periodicals, Inc.     

Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency‐associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by real‐time gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct‐4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct‐4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines.     

Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties.

The v-abl protein of Abelson murine leukemia virus is a tyrosine-specific kinase. Its normal cellular homolog, murine c-abl, does not possess detectable tyrosine kinase activity in vitro. Previously, we have detected tyrosine kinase activity in vitro for an altered c-abl gene product (c-abl P210) in the K562 human chronic myelogenous leukemia cell line. The expression of this variant c-abl gene product correlates with chromosomal translocation and amplification of the c-abl gene in K562 cells. Like v-abl, c-abl P210 is a fusion protein containing non-abl sequences near the amino terminus of c-abl. We compared the in vitro tyrosine kinase activity of c-abl P210 with that of wild-type murine v-abl. The remarkable similarities of these two proteins with respect to cis-acting autophosphorylation, trans-acting phosphorylation of exogenous substrates, and kinase inhibition, using site-directed abl-specific antisera, suggested that c-abl P210 could function similarly to v-abl in vivo. In addition, c-abl P210 possessed an associated serine kinase activity in immunoprecipitates. The serine kinase activity was not inhibited by site-directed, abl-specific antisera that inhibit the tyrosine kinase activity, suggesting that the serine kinase activity is not an intrinsic property of c-abl P210. Thus, the activation of the c-abl gene in a human leukemia cell line may have functional consequences analogous to activation of the c-abl gene in Abelson murine leukemia virus.     

Isolation and characterization of a novel human gene expressed specifically in the cells of hematopoietic lineage.

A novel cDNA clone designated as HS1, which show an expression pattern limited to human hematopoietic cells, was isolated. About 2kb mRNA of the clone was accumulated in all the mature and immature lymphoid and myeloid cell lines tested, and two of three erythroblastoid cell lines, but not in any cell lines of non-hematopoietic tissues. The same mRNA was also detected in normal lymphoid and myeloid tissues and peripheral blood lymphocytes, granulocytes and macrophages, but again not in non-hematopoietic tissues. Nucleotide sequence of the HS1 predicts a protein of 486 amino acids (Mr 53,931). N-terminal half of the protein retains unique repeating motifs, each of which shows a significant homology with the helix-turn-helix DNA-binding motif of several proteins reported previously. C-terminal half of the protein retains a region conserved between non-receptor tyrosine kinases (src family), phospholipase C(PLC)-148 and the crk oncogene product. A unique feature of HS1 suggests that the protein may be involved in signal transduction and regulation of gene expression.