Meis2 as a critical player in MN1-induced leukemia.

Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia (AML), with high expression linked to all-trans retinoic acid resistance and poor survival. MN1 is also a potent and sufficient oncogene in murine leukemia models, strongly dependent on the MEIS1/AbdB-like HOX protein complex to transform common myeloid progenitors, block myeloid differentiation, and promote leukemic stem cell self-renewal. To identify key genes and pathways underlying leukemic activity, we functionally assessed MN1 cell phenotypic heterogeneity, revealing leukemic and non-leukemic subsets. Using gene expression profiling of these subsets combined with previously published comparisons of full-length MN1 and mutants with varying leukemogenic activity, we identified candidate genes critical to leukemia. Functional analysis identified Hlf and Hoxa9 as critical to MN1 in vitro proliferation, self-renewal and impaired myeloid differentiation. Although critical to transformation, Meis1 knockdown had little impact on these properties in vitro. However, we identified Meis2 as critical to MN1-induced leukemia, with essential roles in proliferation, self-renewal, impairment of differentiation and disease progression in vitro and in vivo. Here, we provide evidence of phenotypic and functional hierarchy in MN1-induced leukemic cells, characterise contributions of Hlf, Hoxa9 and Meis1 to in vitro leukemic properties, and reveal Meis2 as a novel player in MN1-induced leukemogenesis.

Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and in vivo.

Several studies point to multiple members of the Hox transcription factor family as playing key roles in normal hematopoietic development, and they link the imbalanced expression of these transcription factors, in particular of the Abd-like A cluster HOX genes HOXA9 and HOXA10, to leukemogenesis. To test directly the hypothesis that HOXA10 is involved in human hematopoietic development, the gene was retrovirally overexpressed in human highly purified CD34(+)/GFP(+) hematopoietic progenitor cells derived from cord blood or fetal liver sources, and the impact of aberrant gene expression was analyzed on differentiation and proliferation in vitro and in vivo. HOXA10 misexpression profoundly impaired myeloid differentiation with a higher yield of blast cells in liquid culture and a greater than 100-fold increased generation of blast colonies after in vitro expansion or after replating of primary colonies first plated in methylcellulose directly after transduction (P < .01). Furthermore, aberrant HOXA10 expression almost completely blocked erythroid differentiation in methylcellulose (P < .02). HOXA10 deregulation also severely perturbed the differentiation of human progenitors in vivo, reducing B-cell development by 70% in repopulated NOD/SCID mice and enhancing myelopoiesis in the transduced compartment. The data provide evidence that the balanced expression of HOXA10 is pivotal for normal human hematopoietic development and that aberrant expression of the gene contributes to impaired differentiation and increased proliferation of human hematopoietic progenitor cells. These results also provide a framework to initiate more detailed analyses of HOX regulatory domains and HOX cofactors in the human system in vitro and in vivo.

SHIPs ahoy.

In 1996 three groups independently cloned a hemopoietic specific, src homology 2-containing inositol 5'-phosphatase which, based on its structure, was called SHIP. More recently, a second more widely expressed SHIP-like protein has been cloned and called SHIP2. Both specifically hydrolyze phosphatidylinositol-3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vitro. Moreover, SHIP has been shown in vivo to be the primary enzyme responsible for breaking down phosphatidylinositol-3,4,5-trisphosphate to phosphatidylinositol-3,4-bisphosphate in normal mast cells and, as a result, limits normal and prevents inappropriate mast cell degranulation. Because of their ability to break down phosphatidylinositol-3,4,5-trisphosphate, the SHIPs have the potential to regulate many, if not all, phosphatidylinositol-3-kinase induced events including, proliferation, differentiation, apoptosis, end cell activation, cell movement and adhesion and will thus likely be the subject of intensive research over the next few years.

Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span.

SHIP is a 145-kD SH2-containing inositol-5-phosphatase widely expressed in hemopoietic cells. It was first identified as a tyrosine phosphoprotein associated with Shc in response to numerous cytokines. SHIP has been implicated in FcgammaRIIB receptor-mediated negative signaling in B cells and mast cells and is postulated to down-regulate cytokine signal transduction in myeloid cells. To define further its role in the proliferation and differentiation of hemopoietic progenitors, as well as its function in mature cells, we have generated embryonic stem cells and mice bearing a targeted disruption of both SHIP alleles. Here we show that although SHIP null mice are viable and fertile, they fail to thrive and survival is only 40% by 14 weeks of age. Mortality is associated with extensive consolidation of the lungs resulting from infiltration by myeloid cells. Increased numbers of granulocyte-macrophage progenitors are observed in both the bone marrow and spleen of SHIP-/- mice, perhaps as a consequence of hyper-responsiveness to stimulation by macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor, interleukin-3, or Steel factor as observed in vitro. In contrast, numbers of bone marrow lymphoid and late erythroid progenitors (CFU-E) are reduced. Thus, homozygous disruption of SHIP establishes the crucial role of this molecule in modulating cytokine signaling within the hemopoietic system and provides a powerful model for further delineating its function.

The role of erythropoietin receptor tyrosine phosphorylation in erythropoietin-induced proliferation.

Although studies with truncated erythropoietin receptors (EpoRs) have suggested the tyrosine phosphorylation (Yphos) of the EpoR may not play a significant role in Epo-induced proliferation, we found, using a full length EpoR mutant designed Null, in which all 8 of the intracellular tyrosines (Ys) were substituted with phenylalanines (Fs), that Null cells required 5-10 fold more Epo than wild type (WT) EpoR containing cells in order to proliferate as well. Moreover, a comparison of Epo-induced proliferation with Epo-induced Yphos patterns, using DA-3 cells expressing WT, Null and various Y to F EpoR point mutants revealed that Stat5 Yphos and activation correlated directly with proliferation and was mediated primarily throuhg the most membrane proximal Y, i.e., Y343, although other tyrosines (most likely Y401 and Y431) within the EpoR could activate Stat5 in its absence. We also found that EpoR Yphos was essential for the Yphos of Shc and for the Yphos and association of a 145 kDa protein with Shc. We purified and cloned this Shc-associated 145 kDa protein and found that it was a unique SH2 containing inositol polyphosphate-5-phosphatase. This novel enzyme, which we have called SHIP for SH2-containing inositol-phosphatase, may modulate both Ras and inositol signaling pathways.

Cloning and characterization of human SHIP, the 145-kD inositol 5-phosphatase that associates with SHC after cytokine stimulation.

We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N-terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37.

The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Mapping of CD24 and homologous sequences to multiple chromosomal loci.

The human cell surface antigen, CD24, is a glycosyl phosphatidylinositol (GPI)-linked glycoprotein that has been implicated in the differentiation and activation of granulocytes and B lymphocytes. Changes in expression of the antigen occur at critical times during B lineage development. CD24 was cloned by its homology to mouse heat-stable antigen. Southern blot analysis suggested the presence of multiple CD24 related sequences in the human genome. We have now mapped CD24 homologous sequences to chromosomes 6q21, 15q21-q22, and Yq11 by screening a panel of somatic cell hybrid DNAs and by in situ hybridization. At least two additional homologues, one located on chromosome 1 at band p36 and one tentatively mapped to chromosome 20, that are distantly related to CD24 were identified. Southern analysis of male and female DNA samples confirmed the presence of CD24 homologous sequences on the human Y chromosome. Sequencing of DNA fragments amplified from monochromosomal somatic cell hybrids showed that the CD24 cDNA was derived from a transcript originating from a gene on chromosome 6. The CD24 gene on the Y chromosome had many base changes compared to the cDNA but had retained an open reading frame, leaving open the question of whether this gene is functional. Both ATGs in the translation initiation region of CD24 homologous sequences on chromosome 15 were converted to GTGs, making it unlikely that this gene, if functional, encodes a protein similar to CD24. CD24, therefore, is a member of a multigene family, but it remains to be determined whether any of the related genes are functional.

CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor.

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d, and the two Ag appear to have a common genetic ancestry, the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag, this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide, and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24.

Purification and purity assessment of toxic shock syndrome toxin 1.

Toxic shock syndrome toxin 1 (TSST-1) was partially purified from culture supernatants by SP-Sephadex C-25 ion-exchange chromatography and subsequent Sephadex G-75 gel filtration. This protein had an apparent molecular weight of 24,000 and an isoelectric point of 7.0. The NH2-amino acid sequence for the first 40 residues agreed completely with that predicted from the known TSST-1 genome. Ouchterlony immunodiffusion with monospecific rabbit antisera demonstrated a single line of identity with reference TSST-1 as well as with three preparations obtained from other investigators. When the purity of the different TSST-1 preparations was examined by Coomassie blue or silver staining after SDS-PAGE, only the major band at molecular weight 24,000 was apparent. However, multiple additional bands were seen in all preparations when visualized either by double staining with Coomassie blue stain followed by silver stain or by immunoblot using pooled human serum. Further purification of our preparation by reverse-phase high-performance liquid chromatography eliminated some, but not all, extraneous antigens. A final purification step by preparative SDS-PAGE resulted in an eluted protein that yielded only the 24-kDa TSST-1 band and a 48-kDa dimer by immunoblot. This material was endotoxin free (sensitivity limit, 10 pg/mL) and retained biologic activity for induction of cachectin and production of interleukin 1 by human monocytes. These data emphasize the need for stringent methods of assessment of purity in TSST-1 preparations.

Determination by western blot (immunoblot) of seroconversions to toxic shock syndrome (TSS) toxin 1 and enterotoxin A, B, or C during infection with TSS- and non-TSS-associated Staphylococcus aureus.

Serum antibody responses to toxic shock syndrome (TSS) toxin 1 (TSST-1) and staphylococcal enterotoxins A, B, and C were determined by western blot (immunoblot) analysis of acute- and convalescent-phase paired sera from 18 TSS- and 31 non-TSS-associated Staphylococcus aureus infections. Compared with non-TSS cases, seroconversion to TSST-1 was significantly more frequent among both menstrual (5 of 8 versus 1 of 31; P less than 0.001) and nonmenstrual (3 of 10; P less than 0.05) patients. Seroconversion to staphylococcal enterotoxin A was also more frequent among both menstrual (2 of 8 versus 0 of 31; P less than 0.05) and nonmenstrual (2 of 9; P less than 0.05) TSS patients. In general, patients with TSS associated with TSST-1-positive S. aureus were more likely to seroconvert exclusively to TSST-1 (4 of 12 versus 0 of 6; P = 0.16), whereas those associated with TSST-1-negative S. aureus were more likely to seroconvert exclusively to enterotoxins (3 of 6 versus 0 of 11; P less than 0.05). Concurrent seroconversions to multiple exoproteins were more frequent among both menstrual (3 of 8; P less than 0.05) and nonmenstrual (2 of 9; P less than 0.05) TSS patients compared with persons without TSS (0 of 31). These data suggest but do not prove that enterotoxins (especially staphylococcal enterotoxin A) in addition to TSST-1 may be involved in both menstrual and nonmenstrual TSS. Furthermore, since exposure to multiple exoproteins is more likely to occur during TSS-associated than non-TSS-associated S. aureus infections, the possibility of additive or synergistic effects of these putative toxins in the pathogenesis of TSS should be further explored.

Serologic responses to toxic shock syndrome (TSS) toxin-1 in menstrual and nonmenstrual TSS.

Toxic shock syndrome toxin-1 (TSST-1) is implicated as the major exotoxin associated with menstrual toxic shock syndrome. The role of TSST-1 in nonmenstrual TSS is less certain. We examined serum IgG responses to TSST-1 in 16 nonmenstrual (9 female, 7 male) and 14 menstrual TSS patients, and in 87 women and 66 men as age-matched healthy controls, using an enzyme-linked immunosorbent assay (ELISA). Relative ELISA titers were expressed as percent activity of a mid level serum standard tested concurrently. Based on 95% confidence estimates for predicting a negative titer (20.6 +/- 8.2%) using sera in which TSST-1 specific IgG was demonstrated to be absent by western blot, 24% of control women and 9% of control men lacked TSST-1 specific IgG in the random survey (p less than 0.05, Fisher's exact test). Relative titers in acute sera of menstrual TSS women (26.2 +/- 5.2%, mean +/- S.E.), but not nonmenstrual TSS women (71.8 +/- 18.6%), were significantly lower than those of control women (78.9 +/- 7.3%, p less than 0.01, Mann-Whitney test). Acute titers from male TSS patients (37.0 +/- 15.6%) were also significantly lower than those in control men (114.6 +/- 11.0% (p less than 0.05). Antibody titers from menstrual TSS women and TSS men remained low during convalescence. Nevertheless, seroconversion to TSST-1 was demonstrated by western blot in 7 of 10 patients in whom TSST-1 positive S. aureus was isolated, but in neither of two patients without toxigenic S. aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and quantitation of toxic shock syndrome toxin 1 in vitro and in vivo by noncompetitive enzyme-linked immunosorbent assay.

Toxic shock syndrome toxin 1 (TSST-1), an exotoxin produced by many Staphylococcus aureus strains, is implicated as the prime causal agent of toxic shock syndrome (TSS). A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxins including A, B, C1, C2, C3, D, and E. Possible interactions with protein A were readily eliminated by pretreatment of test samples with 10% normal rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro and for detection of TSST-1 in vaginal washings of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST positive by Ouchterlony immunodiffusion and 59 of 60 isolates confirmed to be TSST-1 negative gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated from TSS patients quantitatively produced significantly more TSST-1 in vitro compared with toxigenic control strains (P less than 0.05, Mann-Whitney rank sum test). TSST-1 could be detected by ELISA in three of four vaginal washings collected within 3 days of hospitalization from three women with acute menstrual TSS, compared with 0 of 17 washings from nine TSS patients hospitalized longer than 3 days (P = 0.003, Fisher's exact test) and 1 of 15 washings from 14 healthy control women (P = 0.016). This noncompetitive ELISA should be particularly useful for rapid screening of TSST-1 production by S. aureus isolates, for the purification and biochemical characterization of TSST-1, and for human and animal studies of the pathogenesis of TSS.

Migration of pollutants in groundwater. II. adsorbable pollutants and numerical dispersion reduction.

We discuss here the partial differential equations governing the migration of a decomposing pollutant adsorbing according to a Langmuir isotherm and undergoing 2-dimensional flow in a saturated aquifer. The equation governing the mass transfer of the pollutant to the surfaces within the aquifer are solved in closed form, permitting the use of larger values of the time increment Δt in the numerical integration of the dispersion-advection equation governing the behavior of the dissolved pollutant. In this numerical integration transverse numerical dispersion is eliminated by using conformal coordinates (velocity potential and stream function), and longitudinal numerical dispersion is very substantially reduced by use of an asymmetrical 4-point formula to represent the advection term. Some representative results are given as contour maps. The mass transfer rate coefficient is estimated as the least positive eigenvalue of a diffusion problem.