Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4 : 11) abnormality.

Rearrangements of the human ALL-1 gene are frequently encountered in acute lymphocytic leukemias (ALL) and acute myeloid leukemias (AML). These rearrangements are mostly due to chromosome translocations and result in production of chimeric proteins composed of the N-terminal fragment of ALL-1 and the C-terminal segments of the partner proteins. The most common chromosome translocation involving ALL-1 is the t(4 : 11) associated with ALL. ALL-1 is the human homologue of Drosophila trithorax and directly activates transcription of multiple Hox genes. A preliminary DNA microarray screen indicated that the Meis1, HoxA9 and AC133 genes were overexpressed in ALLs with t(4 : 11), compared to ALLs with very similar phenotype but without the chromosomal abnormality. These genes, as well as additional five Hox genes, were subjected to comprehensive semi-quantitative or quantitative RT-PCR analysis in 57 primary ALL and AML tumors. Meis1 and HoxA9 were found expressed in 13/14 of ALLs with the t(4 : 11) and in 8/8 of AMLs with ALL-1 rearrangements. The two genes were not consistently transcribed in other types of ALL. AC133 was transcribed in 13/14 of ALLs with t(4 : 11), but in only 4/8 of AMLs with ALL-1 rearrangements. HoxA10 was expressed in most leukemias with ALL-1 alterations, but was also transcribed in PrePreB CD10(-) ALLs lacking the t(4 : 11). Expression of HoxA5, HoxA7, HoxC8 and HoxC10 did not correlate with ALL-1 rearrangements. Coexpression of Meis1 and HoxA9, overexpression of HoxA10, and overexpression or fusion of HoxA9 were previously implicated in certain acute myeloid leukemias in mice and humans. The present work suggests that upregulation of Meis1, HoxA9, and possibly HoxA10 might also play a role in pathogenesis of acute lymphocytic and acute myeloid leukemias associated with ALL-1 fusions.

huASH1 protein, a putative transcription factor encoded by a human homologue of the Drosophila ash1 gene, localizes to both nuclei and cell-cell tight junctions.

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an approximately 10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.

Estrogen reduces cholecystokinin-induced c-Fos expression in the rat brain.

Recent reports have shown that estradiol increases the hypophagic effect of exogenous cholecystokinin-octapeptide (CCK). CCK is known to increase the expression of Fos, a marker of neuronal activation, in specific medullary and hypothalamic nuclei. The present experiment tested the hypothesis that as estradiol enhances that behavioral effects of CCK, there is a parallel amplification of CCK-induced Fos expression. Instead, estradiol pretreatment reduced the level of CCK-induced Fos expression in the nucleus of the solitary tract (NTS) and in the medial parvocellular region of the hypothalamic paraventricular nucleus (PVN). The number of Fos-containing cells in the supraoptic nucleus and other regions of the PVN were not affected by estradiol pretreatment. The results are consistent with the hypothesis that the NTS and/or the PVN may mediate the estrogen-induced increased sensitivity to peripheral inhibitory signals in the control of food intake.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex.

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene.

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

Nuclear punctate distribution of ALL-1 is conferred by distinct elements at the N terminus of the protein.

The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.

The fourth immunoglobulin domain of the stem cell factor receptor couples ligand binding to signal transduction.

Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.

Convergence of signaling by interleukin-3, granulocyte-macrophage colony-stimulating factor, and mast cell growth factor on JAK2 tyrosine kinase.

Mast cell growth factor (MGF) (also called stem cell factor) synergizes with several lymphokines, including interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), to promote proliferation and differentiation of certain hemopoietic progenitor cells. Although similar patterns of tyrosine-phosphorylated proteins characterize cells stimulated by MGF, IL-3, and GM-CSF, only the MGF receptor is a tyrosine kinase, and the heterodimeric receptors for IL-3 and GM-CSF share a common beta subunit that is devoid of enzymatic activity. Here we show that signaling pathways utilized by all three cytokines include the cytoplasmic tyrosine kinase JAK2. Analysis of several factor-dependent myeloid cell lines indicated that JAK2 is physically associated with the common beta subunit and with MGF receptor (c-Kit) even prior to ligand binding. However, each of the ligands induced elevated tyrosine phosphorylation of JAK2 and a consequent increase in its catalytic activity. These results demonstrate for the first time the convergence within the same myeloid cells of signaling pathways originating in two distinct lymphokine receptors and a tyrosine kinase receptor on activation of a cytoplasmic tyrosine kinase.

Protein kinase C-dependent release of a functional whole extracellular domain of the mast cell growth factor (MGF) receptor by MGF-dependent human myeloid cells.

The mast cell growth factor (MGF) affects migration, proliferation and differentiation of erythroid and myeloid progenitor cells by binding to a transmembrane receptor tyrosine kinase encoded by the c-Kit proto-oncogene. By using MGF-dependent human myeloid cell lines (M-07e and TF-1), here we show that a Kit-related 100 kDa protein is associated with the cell but it undergoes release into the medium upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. Immunological analysis with a series of antibodies to Kit indicated that the released protein (p100Kit) contains the whole glycosylated extracellular portion of the transmembrane Kit protein (p145Kit). The secreted protein retained the ability to specifically bind MGF. Moreover, p100Kit was able to block the mitogenic effect of MGF on cultured M-07e cells, suggesting that the soluble protein may function as a physiological antagonist of MGF.

Steel factor and c-kit protooncogene: genetic lessons in signal transduction.

Despite extensive research on the molecular mechanisms of signal transduction by growth factors and their oncogenic receptor tyrosine kinases, the physiological relevance of these pathways, especially in mammals, remains largely unknown. A unique exception is the Steel factor (SLF) and its c-kit-encoded receptor, because many natural germ line mutations of both the ligand and the receptor exist in mice. The protooncogene c-kit encodes a cell surface receptor that belongs to the immunoglobulin gene family and carries an intrinsic tyrosine kinase activity in its cytoplasmic portion. The precursor of the Kit ligand, SLF, is also a transmembrane protein that exists as a soluble factor as well as a cell surface protein. The interaction of Kit with SLF leads to receptor dimerization, kinase activation, and tyrosine phosphorylation of cytoplasmic proteins that contain Src homology 2 motifs. Various mutations in Kit and SLF result in a defective signaling pathway and underly the complex phenotypes of W and Sl mice, respectively. The early development of at least four cell lineages is affected. These are erythrocytes, melanocytes, germ cells, and mast cells. Correlation between the behavior of these lineages and specific mutations uncovered interesting physiological aspects of the mechanism of signal transduction by a polypeptide growth factor. These include the different degrees of severity of affected lineages, indications for distinct functions during early embryonic development and at late phases, the significance of synergy between a growth factor and lymphokines, the interaction between mutant and wild-type proteins in heterozygous animals, and the possibility that a surface-anchored ligand may act differently than a soluble factor. Predictably, the lessons learned with Kit and Sl mice will be widely relevant to other pairs of ligands and receptors that control the function of different cell lineages and physiological processes.

Structure-function analyses of the kit receptor for the steel factor.

Binding of the Steel factor (SLF) to the product of the c-kit proto-oncogene stimulates the receptor's intrinsic tyrosine kinase that phosphorylates a set of cytoplasmic signaling molecules. Germ-line mutations in the genes that encode the receptor or the ligand result in remarkably similar phenotypes that affect melanogenesis, erythropoiesis and gametogenesis in mice. We concentrated on the initial events of the signal transduction pathway that underlies these processes. The extracellular portion of Kit is comprised of five immunoglobulin-(Ig)-like domains. Ligand binding to this domain induces rapid and extensive dimerization of the receptor molecules in a mechanism that involves monovalent binding of the dimeric ligand, followed by an increase in receptors' affinity and gradual stabilization of the dimers. It thus appears that Kit has at least two functions: ligand binding and ligand-induced receptor dimerization, in addition to the kinase activity. Both functions are independent of the transmembrane and cytoplasmic domains, as a recombinant soluble ectodomain retained high affinity to SLF and ligand-dependent dimerization. In order to correlate these functions with specific structures, we employed ligand-competitive monoclonal antibodies, soluble deletion mutants of the ectodomain and chimeric human-mouse Kit proteins. These approaches indicated that the N-terminal three Ig-like domains constitute the binding site, whose core is the second domain. Further experiments suggested that a putative dimerization site is distinct from the binding cleft and may be located on the fourth Ig-like domain.

Interspecies molecular chimeras of kit help define the binding site of the stem cell factor.

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.

Soluble c-kit proteins and antireceptor monoclonal antibodies confine the binding site of the stem cell factor.

The binding of the stem cell factor (SCF) to the c-kit-encoded receptor tyrosine kinase stimulates a variety of biochemical responses that culminate in cellular proliferation, migration, or survival. The extracellular domain of p145kit consists of five immunoglobulin-like domains. To confine the ligand binding site to this portion of the receptor we generated a panel of murine monoclonal antibodies (mAbs) to the Kit protein and identified two mAbs that efficiently displaced receptor-bound SCF and also inhibited proliferation of SCF-dependent human megakaryocytes. To map the epitopes of these mAbs we constructed and expressed soluble portions of the extracellular domain of Kit, which included either the two amino-terminal Ig-like domains (denoted Kit 1-2), three Ig-like domains (Kit 1-2-3), or the entire extracellular portion (Kit-X). All three recombinant proteins were recognized by the ligand inhibitory mAbs, suggesting that the SCF binding site resides in the amino-terminal half of the ecto-domain. Consistent with this conclusion, all of the soluble proteins inhibited SCF binding to Kit-expressing cells, and they also underwent specific covalent cross-linking to the radiolabeled ligand. However, whereas Kit 1-2-3 and Kit-X displayed comparable ligand affinities, deletion of the third Ig-like domain, in Kit 1-2, involved significant reduction in SCF binding. Hence, the binding site of SCF probably includes Ig-like domains 1 and 2, but structural determinants distal to this portion may also participate in ligand recognition.

Immunoglobulin-specific T-B cell interaction. IV. B cell presentation of idiotypic determinant(s) of monoclonal anti-surface immunoglobulin antibody to idiotope-recognizing helper T clones.

Previously, we have demonstrated T-B cell interactions mediated by T cell recognition of immunoglobulin (Ig) peptide/major histocompatibility complex (MHC) class II complexes derived by the B cell processing of endogenously synthesized Ig molecules. In this report Ig-specific T-B cell interaction mediated by B cell presentation of idiotopes (Id) of anti-sIg antibodies to Id-specific T cell clones has been studied in Ig kappa-1-congenic rat strains. A panel of August (RT-1c; Ig kappa-1a) rat T helper clones specific for Id of syngeneic anti-Ig kappa-1b C3B9 monoclonal antibody (mAb) has been developed to study IdC3B9 presentation by Ig kappa-1b-bearing B cells from congenic August.1b (RT-1c; Ig kappa-1b) rats. Five of seven IdC3B9-specific T clones responded even at very low concentrations (100-200 pg/ml) of C3B9 mAb presented by Ig kappa-1b+ B cells. In contrast, the presentation of intact C3B9 mAb by nonspecific antigen-presenting cells (macrophages, Ig kappa-1a+ B cells, etc.) to IdC3B9-specific T cells was of low efficiency. The IdC3B9-specific T cell response to idiotopes of anti-Ig kappa-1b C3B9 mAb was found to be restricted by RT-1B molecule and required the processing of intact C3B9 molecule. IdC3B9 epitope recognized by C31 and C5 clones was mapped to the heavy chain of C3B9 mAb. Thus, B cells are able to present peptides related to the V region of anti-sIg Ab, i.e. Id peptide/MHC class II complexes, to Id-recognizing T cells. IdC3B9-presenting B cells are specifically activated both to proliferation and Ig production upon interaction with IdC3B9-specific T clones. Based on the results of our studies on B cell presentation of Ig epitopes to T cells a hypothetical model of Ig peptide-driven T-B cell interaction has been proposed.

Immunoglobulin-specific T-B cell interaction. II. T cell clones recognize the processed form of B cells' own surface immunoglobulin in the context of the major histocompatibility complex class II molecule.

In the preceding report (Eur. J. Immunol. 1989. 19: 1677) we have demonstrated that normal B cells, including small B cells, are capable of presenting Ig kappa-1b allotypic determinants of their endogeneously synthesized Ig+ to Ig kappa-1b-immune major histocompatibility complex (MHC) class II-restricted T cells. A panel of Ig kappa-1b allotype-specific T cell clones from August rats has been developed to study further the presentation of self surface Ig by B cells from Ig kappa-1-congeneic August.1b rats. All the clones studied were of the T helper/inducer phenotype (W3/25+,OX8-) and restricted by the RT-1Bc molecule. These clones responded both to the serum IgG(Ig kappa-1b) in the presence of irradiated spleen cells (SC) from August rats and to the Ig kappa-1b-bearing irradiated B cells from August.1b rats. SC presentation of secreted IgG was much less effective than B cell presentation of membrane Ig. Using CNBr cleavage of isolated C kappa (Ig kappa-1b) domain followed by high-performance liquid chromatography fractionation of the derived antigenic peptides, the kappa chain sequence between amino acids 176 and 214 has been identified as the T cell epitope recognized by all T cell clones in association with RT-1Bc. The fragment 176-214 of the Ig kappa-1b allotype differs from that of Ig kappa-1a allotype by three amino acid substitutions at positions 184, 185, 188. T cell recognition of pL kappa-1b(176-214) required no additional processing by the antigen-presenting cell: the efficient presentation of the peptide but not of intact IgG(Ig kappa-1b) by the paraformaldehyde-fixed SC was observed. These data provide clear-cut evidence for an absolute requirement of the processing of Ig molecules for T cell recognition to occur in our experimental system. Although the fixation of B cells from August.1b rats diminished their Ig kappa-1b-presenting ability, fixed Ig kappa-1b+ B cells were still able to induce Ig kappa-1b-specific T cell clone responses. Our results suggest that B cells can express the processed form of self-synthesized surface Ig in addition to intact surface Ig molecules. The former can be recognized by MHC-restricted T cell.