Biomedical subjects

D Wisniewski

Publications and source records attributed to D Wisniewski.

At least 19 recordsLinked to original sources

PubMed2005

Anthrax lethal factor inhibition.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Animals↗

PubMed2003

Chronic myelogenous leukemia as a paradigm of early cancer and possible curative strategies.

The chronological history of the important discoveries leading to our present understanding of the essential clinical, biological, biochemical, and molecular features of chronic myelogenous leukemia (CML) are first reviewed, focusing in particular on abnormalities that are responsible for the massive myeloid expansion. CML is an excellent target for the development of selective treatment because of its highly consistent genetic abnormality and qualitatively different fusion gene product, p210(bcr-abl). It is likely that the multiple signaling pathways dysregulated by p210(bcr-abl) are sufficient to explain all the initial manifestations of the chronic phase of the disease, although understanding of the circuitry is still very incomplete. Evidence is presented that the signaling pathways that are constitutively activated in CML stem cells and primitive progenitors cooperate with cytokines to increase the proportion of stem cells that are activated and thereby increase recruitment into the committed progenitor cell pool, and that this increased activation is probably the primary cause of the massive myeloid expansion in CML. The cooperative interactions between Bcr-Abl and cytokine-activated pathways interfere with the synergistic interactions between multiple cytokines that are normally required for the activation of stem cells, while at the same time causing numerous subtle biochemical and functional abnormalities in the later progenitors and precursor cells. The committed CML progenitors have discordant maturation and reduced proliferative capacity compared to normal committed progenitors, and like them, are destined to die after a limited number of divisions. Thus, the primary goal of any curative strategy must be to eliminate all Philadelphia positive (Ph+) primitive cells that are capable of symmetric division and thereby able to expand the Ph+ stem cell pool and recreate the disease. Several highly potent and moderately selective inhibitors of Bcr-Abl kinase have recently been discovered that are capable of killing the majority of actively proliferating early CML progenitors with minimal effects on normal progenitors. However, like their normal counterparts, most of the CML primitive stem cells are quiescent at any given time and are relatively invulnerable to the Bcr-Abl kinase inhibitors as well as other drugs. We propose that survival of dormant Ph+ stem cells may be the most important reason for the inability to cure the disease during initial treatment, while resistance to the inhibitors and other drugs becomes increasingly important later. An outline of a possible curative strategy is presented that attempts to take advantage of the subtle differences in the proliferative behavior of normal and Ph+ stem cells and the newly discovered selective inhibitors of Bcr-Abl. Leukemia (2003) 17, 1211-1262. doi:10.1038/sj.leu.2402912

Antineoplastic Agents↗

PubMed2000

Characterization of two novel sublines established from a human megakaryoblastic leukemia cell line transfected with p210(BCR-ABL).

Disease progression in chronic myelogenous leukemia (CML) is usually accompanied by chromosomal abnormalities such as an additional Ph chromosome, trisomies of chromosome 8 or 19, or i(17) in addition to the standard translocation t(9;22) (q34;q11). However, detailed studies of the various steps involved during this evolution are difficult to perform, thereby making the study of cell lines that contain the transposed genes BCR-ABL, especially those of human origin, an important focus. In this analysis we investigated the human megakaryoblastic cell line MO7e and its subline transfected with BCR-ABL, MO7e/p210. Initial studies demonstrated that the phenotype of the MO7e line was consistent with a megakaryocytic lineage as originally described and was growth factor dependent in liquid culture. The MO7e/p210 subline, however, was growth factor independent and could be further separated into two distinct sublines based on expression of glycophorin A using the monoclonal antibody R10. The subline R10 negative (R10-) was similar to the parent line MO7e but R10 positive (R10+) cells had a distinct erythroid phenotype. In addition, the R10- and R10+ sublines demonstrated strikingly different colony morphology when cultured in semisolid medium. Furthermore, R10+ cells had additional chromosomal abnormalities not detected in the R10- population. These results demonstrate that the insertion of the BCR-ABL in this human leukemia cell line resulted in two distinct subpopulations of cells, each now growth factor independent, but one with a phenotype and karyotype identical to the parent cell line and the other with a different phenotype and additional chromosomal abnormalities. These two subpopulations derived from the MO7e/p210 transfected cell line may prove useful in further understanding the multistep events that occur in the progression of this disease.

Chromosome Aberrations↗

PubMed2000

The phosphatidylinositol polyphosphate 5-phosphatase SHIP1 associates with the dok1 phosphoprotein in bcr-Abl transformed cells.

The initial phase of chronic myelogenous leukemia (CML) is triggered by constitutive protein tyrosine kinase activity of the chimeric kinase p210(bcr-abl) (Bcr-Abl). A major substrate of Bcr-Abl was recently identified as the RasGAP-associated 62 kDa docking protein Dok1. Here, we report complex formation between endogenous Dok1 and the SH2 domain-containing phosphatidylinositol polyphosphate 5-phosphatase SHIP1 in hematopoietic cells expressing Bcr-Abl. Expression of Bcr-Abl induced tyrosine phosphorylation of both Dok1 and SHIP1 and the formation of a Dok1/SHIP1 complex. Tyr(P) SHIP1 was also bound to Shc in Bcr-Abl expressing cells. A small amount of Shc/SHIP1/Dok1 trimolecular complex was detected and this was due to binding of Dok1 to SHIP1 that was bound to Shc. In contrast, association of Dok1 with SHIP1 or RasGAP was mutually exclusive. Both the SH2 domain of SHIP1 and the PTB domain of Dok1 were required for complex formation between the two proteins. Neither the specific activity of SHIP1 as an inositol phosphate 5-phosphatase nor the subcellular localization of SHIP1 appeared to be altered by tyrosine phosphorylation. However, the Dok1/SHIP1 complex was only detected in the cytosolic fraction of Bcr-Abl transformed hematopoietic cells. We propose that interaction between Dok1 and SHIP1 modulates the ability of these two proteins to interact with other cytosolic binding partners.

Animals↗

PubMed1999

Assay for IkappaB kinases using an in vivo biotinylated IkappaB protein substrate.

IkappaB kinases (IKK)-1 and -2 are related kinases that are induced by stimuli such as TNF or IL-1 to phosphorylate serines 32 and 36 of IkappaBalpha, the regulatory subunit of the transcription factor NF-kappaB. A procedure for an IKK protein kinase assay is described that uses an in vivo biotinylated IkappaB protein substrate, [gamma-(33)P]ATP, and capture onto a streptavidin membrane. Residues 1-54 of the IkappaBalpha substrate were expressed as a fusion with glutathione S-transferase (GST) and a short (22 amino acid) biotinylation sequence that allowed modification during bacterial expression. Using the streptavidin capture assay the phosphorylation activities of recombinant IKK-1 and -2 were characterized. The assay provided a convenient way to compare IKK protein and peptide substrate preferences; biotinylated GST-IkappaBalpha(1-54) was more readily phosphorylated by both IKK-1 and IKK-2 compared to biotinylated myelin basic protein or a 20-mer biotinylated peptide containing serines 32 and 36 of IkappaBalpha. IKK-1 had 83-fold less activity than IKK-2, and the IKK-1+2 complex had approximately 2-fold more activity than IKK-2. IKK-1+2 and IKK-2 had similar K(m) values for ATP and GST-biotin-IkappaB(1-54) and were similarly inhibited by staurosporine and two of its analogues K252a and K252b, suggesting that most of the IkappaBalpha kinase activity in the IKK-1+2 complex may be attributed to IKK-2. Several features of the assay including the broad linear binding range of the streptavidin membranes for the protein substrate GST-biotin-IkappaB(1-54) (1-4000 pmol of protein/cm(2)), the low background, and its capacity for both biotinylated peptides and proteins make it a useful tool for quantitating IKK activity. These factors and the ease of expressing in vivo biotinylated GST fusions will make this assay approach suitable for a wide variety of protein kinases.

Biotin↗

PubMed1999

A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells.

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.

Collagen↗

PubMed1998

Molecular cloning and characterization of p56dok-2 defines a new family of RasGAP-binding proteins.

Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210(bcr-abl), a chimeric protein with tyrosine kinase activity. Substrates for p210(bcr-abl) are likely to be involved in the pathogenesis of CML. Here we describe the purification, cDNA cloning, and characterization of a 56-kDa tyrosine phosphorylated protein, p56(dok-2) (Dok-2), from p210(bcr-abl) expressing cells. The human dok-2 cDNA encodes a 412-amino acid protein with a predicted N-terminal pleckstrin homology domain as well as several other features of a signaling molecule, including 13 potential tyrosine phosphorylation sites, six PXXP motifs, and the ability to bind to p120(RasGAP). Dok-2 was shown to be 35% identical to p62(dok-1), a recently identified RasGAP binding protein from CML cells, and analysis of the expressed sequence tag data base revealed the presence of at least four additional proteins containing a Dok homology sequence motif. Dok mRNAs were primarily expressed in tissues of hematopoietic origin. These findings strongly suggest that a family of Dok-related proteins exists that bind to RasGAP and may mediate the effects of p210(bcr-abl) in CML.

Adaptor Proteins, Signal Transducing↗

PubMed1997

p62(dok): a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells.

Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

CSK Tyrosine-Protein Kinase↗

PubMed1997

New understanding of the pathogenesis of CML: a prototype of early neoplasia.

The 9;22 chromosomal translocation characteristic of CML results in a fused bcr/abl gene and an abnormal fusion protein, p210bcr/abl. Relative to normal c-abl, p210bc1/abl has elevated tyrosine kinase activity that is essential for its transforming activity. We recently reported a prominent 62 kDa GAP-associated P-tyr protein and five additional consistent but less prominent P-tyr proteins as well as five more minor P-tyr proteins that are constitutively tyrosine phosphorylated in primary primitive lineage negative (lin-) chronic phase CML blasts but not in comparable primary lin- normal blasts. The GAP-associated p62 protein has now been purified, sequenced and its gene has been cloned; it is a previously unidentified protein and is currently being characterized. In analyzing P-tyr proteins in primary lin- normal blasts in response to various hematopoietic cytokines, we found a striking similarity in the tyrosine phosphorylation of four major and three minor proteins after stimulation with c-kit ligand (KL) and the P-tyr proteins that are constitutively phosphorylated in primary primitive lin- chronic phase CML blasts. Other cytokines tested (ie GM-CSF, G-CSF, IL-3, FLT3 ligand, TPO, EPO) were much less active or stimulated phosphorylation of other proteins. KL/c-kit and bcr/abl have some similar activities including enhancing survival and expansion of hematopoietic progenitor cells, probably acting primarily on early progenitors at the time of lineage commitment rather than on self-renewing stem cells. Activation of growth factor receptors promote a cascade of protein phosphorylations that can ultimately result in a wide range of cellular responses. Sustained activation of discrete signaling pathways in some types of cells results in differentiation, whereas transient activation instead causes a proliferative response; in other cell types, the converse is true. It may be postulated that stem cells and primitive progenitors are at a particularly susceptible stage of development that renders them especially responsive to sustained bcr/abl-induced phorphorylation of a number of signaling proteins that are components of critical regulatory pathways, including c-kit. The affected pathways control and coordinate multiple diverse cell processes including proliferation, differentiation, maturation and apoptosis, processes that are normally tightly regulated and integrated. Perturbation of these key pathways in primitive progenitors would be expected to seriously disrupt orderly hematopoiesis and could also explain the multiple subtle pleiotropic biological abnormalities characteristically observed in later maturing CML compartments that we have collectively designated 'discordant maturation'. The true situation is undoubtedly very complex and involves interaction of multiple cytokines and signaling pathways that we are now trying to define. Constitutive downstream activation of critical pathways in susceptible early progenitors that normally require KL or other factors for activation could explain most if not all features of the disease.

Adolescent↗

PubMed1996

Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

Amino Acid Sequence↗

PubMed1996

The utility of FK506-binding protein as a fusion partner in scintillation proximity assays: application to SH2 domains.

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor-ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Amino Acid Sequence↗

PubMed1996

Variable subcellular localization of a neuron-specific protein during NTera 2 differentiation into post-mitotic human neurons.

The current report describes the molecular characterization of the human (the D4S234 locus) and mouse (the m234) homologs of a gene that was isolated during our genomic analysis of the Huntington disease gene region. Sequence comparisons of full-length cDNA clones revealed that the mouse and human homologs encoded evolutionarily conserved 21-kDa proteins with greater than 90% amino acid sequence identity. Extensive sequence identity between the D4S234 gene and the rat p1A75 gene (a previously identified rat neuron-specific gene) showed that these genes are interspecies homologs. Furthermore, the D4S234 protein exhibited significant amino acid similarity to a 19-kDa mouse protein that localizes to the Golgi apparatus of embryonic neurons. However, nonconservative sequence differences suggested that these genes are independent members of a multigene family. Northern analyses revealed that rodent D4S234 expression occurred predominantly in the brain and included all brain regions. Neuron-specific expression was demonstrated using Northern analysis of cultured glial cells and quinolinic acid-treated rat brain samples. Minimal amounts of the rodent D4S234 mRNA were detected prenatally; however, elevated adult levels were detected within 1 month of birth. Sequence analyses of the human and mouse D4S234 proteins identified an evolutionarily conserved hydrophobic sequence and a consensus nuclear localization signal in both genes. Immunofluorescence microscopy, using an antipeptide antibody, established that the human D4S234 protein preferentially localized to the nucleus of mitotic cultured cells. Since the rat p1A75 protein was previously mapped to the neuronal cytoplasm by in situ hybridization, the subcellular localization of the D4S234 protein was subsequently examined during differentiation of the NTera 2 (NT2) cell line. Following differentiation into postmitotic NT2-N neurons, the D4S234 protein demonstrated cytoplasmic staining and reduced or undetectable nuclear staining in many cells. The variation in the intracellular localization of the D4S234 protein in mitotic and nonmitotic cells suggests that the subcellular localization of this protein is developmentally regulated and provides clues about the biochemical function of this protein.

Amino Acid Sequence↗

PubMed1996

c-kit ligand stimulates tyrosine phosphorylation of a similar pattern of phosphotyrosyl proteins in primary primitive normal hematopoietic progenitors that are constitutively phosphorylated in comparable primitive progenitors in chronic phase chronic myelogenous leukemia.

Characteristic of Philadelphia (Ph)+ chronic myelogenous leukemia (CML) is the presence of the chimeric BCR/ABL (p210) protein possessing elevated protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Our previous studies demonstrated subtle differences in the growth, phenotypic and morphologic characteristics of the most primitive subpopulations of primary lin-Ph+ chronic phase CML blasts and comparable primary lin- normal blasts. Recently, in comparing proteins phosphorylated on tyrosine in these cell populations, we reported a prominent 62 kDa phosphotyrosyl (P-tyr) protein constitutively present in primary primitive lin- CML chronic phase blasts which was virtually undetectable in primary primitive lin- normal blasts. In the present studies, we demonstrate that this P-tyr p62 from primary primitive lin- chronic phase CML blasts co-immunoprecipitates with ras-GAP. Furthermore, in addition to the p210 protein, we show in whole cell lysates the presence of other clearly consistent but less prominent P-tyr proteins with molecular weights of approximately 155, 140, 110, 55 and 45 kDa as well as more minor P-tyr proteins of approximately 190, 85, 52, 42 and 39 kDa constitutively present in primary primitive lin- chronic phase CML blasts. In analyzing proteins tyrosine phosphorylated in primary primitive lin- normal blasts in response to various hematopoietic growth factors, we found a striking similarity in the phosphorylation of four major (approximately 140, 110, 62 and 56 kDa) and three minor (approximately 51, 45 and 42 kDa) P-tyr proteins after stimulation with c-kit ligand and the P-tyr proteins constitutively phosphorylated in primary primitive lin- chronic phase CML blasts. Other growth factors tested (ie GM-CSF, G-CSF, IL-3, FLT3 ligand and EPO) were much less active or stimulated phosphorylation of other proteins. It is provocative that at least seven proteins rapidly and transiently phosphorylated on tyrosine in the c-kit ligand signal transduction pathway in lin- normal blasts may be constitutive substrates for the p210 activated tyrosine kinase in comparable lin- chronic phase CML blasts. In addition, it is intriguing that some of the biological effects on hematopoietic progenitors attributed to the c-kit ligand may be similar to some of the observed biological consequences of the p210 protein, including survival and expansion of a more mature stem cell population, probably at the time of lineage commitment rather than at the level of the earliest self-renewing stem cell.

Cell Lineage↗

PubMed1996

c-kit ligand stimulates tyrosine phosphorylation of the c-Cbl protein in human hematopoietic cells.

c-kit ligand (KL) is a hematopoietic growth factor that plays a major role in the survival, expansion and differentiation of hematopoietic progenitor cells of various lineages. The biological actions elicited by KL are initiated by binding to its cognate receptor, c-kit, which is a transmembrane tyrosine kinase. The resulting ligand/receptor complex rapidly activates the intrinsic kit receptor tyrosine kinase and subsequent phosphorylation of specific intracellular substrates that are involved in downstream signaling events. In the present studies, we demonstrate that KL stimulates the rapid tyrosine phosphorylation of the proto-oncogene, c-Cbl, in two KL-responsive human hematopoietic cell lines, MO7e and TF-1. In both these cell lines we found a constitutive in vivo association between c-Cbl and the adaptor protein Grb2 and demonstrate (in vitro) that c-Cbl binds primarily to the N-terminal SH3 domain of Grb2. Furthermore, the stoichiometry of this association was not significantly affected upon c-kit receptor activation. We also provide evidence that c-Cbl is not stably associated with the kit receptor either prior to or following KL stimulation. Our findings suggest that c-Cbl is an important component in the KL signaling pathway in human hematopoietic progenitor cells.

Adaptor Proteins, Signal Transducing↗

PubMed1995

High-yield expression, refolding, and purification of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus strain 27R.

The mecA-27R gene, which encodes PBP2a from methicillin-resistant Staphylococcus aureus strain 27R, was modified to remove the putative N-terminal membrane-spanning region, cloned into the T7 RNA polymerase expression vector pET11d, and used to transform Escherichia coli strain BL21(DE3). The majority of PBP2a was expressed in the form of inclusion bodies, which were extracted, denatured, and refolded. The protein was then purified by anion-exchange and size-exclusion chromatography. A 6-liter culture of induced E. coli provided 37 mg of purified PBP2a which was greater than 99% pure. Binding affinities for [3H]benzylpenicillin, imipenem, and L-695,256 (a beta-lactam with high affinity for PBP2a) were shown to be comparable to PBP2a found in membrane preparations of S. aureus strain 27R. A direct binding assay, using 14C-labeled L-695,256 was developed and used to show stoichiometric binding to the refolded, soluble PBP2a. In addition, electrospray mass spectrometry showed that 100% of the refolded PBP2a was covalently bound to the beta-lactam in a stoichiometric fashion. Finally, two mutations of the putative active-site serine showed the predicted loss of covalent binding of the beta-lactam to the PBP2a, demonstrating the high specificity of the soluble binding assay.

Amino Acid Sequence↗

PubMed1994

A 62-kilodalton tyrosine phosphoprotein constitutively present in primary chronic phase chronic myelogenous leukemia enriched lineage negative blast populations.

Ph+ chronic myelogenous leukemia (CML) is associated with the reciprocal translocation between chromosomes 9 and 22 culminating in the production of the chimeric p210bcr/abl protein possessing elevated protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Our recent studies have revealed subtle differences in the growth, phenotypic and morphologic characteristics of subpopulations of primary lin- Ph+ chronic phase CML blasts and comparable primary normal blasts. In an attempt to correlate these biologic abnormalities and the presence of the p210bcr/abl protein, we initiated studies to identify differences in proteins constitutively phosphorylated on tyrosine in whole cell lysates of comparable primary early blast subpopulations derived from normal and Ph+ chronic phase CML marrows. Immunoblotting with anti-P-tyr Abs demonstrated a prominent 62 kDa phosphotyrosyl protein (pp62) constitutively present in 11/11 Ph+ chronic phase linblasts while being virtually undetectable in equivalent amounts of protein derived from 15/15 and 2/2 comparable normal and Ph-negative chronic phase blast populations, respectively. Immunoblotting with an Ab reportedly specific for the ras GTPase activating protein (GAP) associated p62 protein revealed that the pp62 present in CML blasts is not immunologically related to the former protein. Although the identity of the pp62 is presently not known, its prominent presence in chronic phase CML blasts, in which the only known molecular abnormality is putatively the p210bcr/abl protein, strongly suggests that it may be a critical p210bcr/abl substrate involved in an early stage of expansion of the Ph+ clone.

Blast Crisis↗

PubMed1993

Characterization of lineage-negative blast subpopulations derived from normal and chronic myelogenous leukemia bone marrows and determination of their responsiveness to human c-kit ligand.

Lineage-negative (lin-) normal and chronic myelogenous leukemia (CML) marrow blast populations were obtained by negative selection and subsequently separated on the basis of size by velocity sedimentation. The three subpopulations of lin- blasts obtained were enriched for F8 (the more primitive small blasts), F11 (blasts intermediate in size), and F13 (the more mature large blasts). We examined the morphological and phenotypic characteristics and cell cycle status of the subpopulations and determined the responsiveness of granulocyte-monocyte progenitors (colony-forming units/granulocyte-macrophage) derived from each subpopulation to mast cell growth factor in combination with granulocyte (G-CSF) or granulocyte-macrophage (GM-CSF) colony-stimulating factors alone and in combination. Morphological assessment revealed that an increased proportion of CML lin- blasts exhibited early cytoplasmic maturation as evidenced by the appearance of azurophilic (nonspecific) granules in the cytoplasm. Although the percentages of CML and normal small blasts expressing CD34 were similar, the proportion of CML lin- blasts expressing CD34 declined in the intermediate and more mature large lin- blast subpopulations by about 50%, whereas the percentage of CD34+ normal blasts remained essentially the same, indicating an earlier loss of CD34 expression by CML lin- blasts. In addition, the percentages of CML small blasts expressing CD33 were higher than normal (26-61% versus 0-16%, respectively), indicating that a higher proportion of CML small lin- blasts had a more mature phenotype. Mast cell growth factor addition to cultures stimulated by G-CSF, GM-CSF, or G-CSF plus GM-CSF, exerted the greatest synergistic effect (increased colony number and size) in the normal small and intermediate lin- blast cultures, but mast cell growth factor had considerably less effect, or no effect, in cultures of comparable CML subpopulations, indicating that CML lin- progenitors had a somewhat lower requirement for multiple growth factors. The findings suggest that the differences observed between normal and CML marrow subpopulations are proportional differences and that a greater proportion of CML lin- blast subpopulations exhibit characteristics associated with a more advanced stage of maturation than comparable normal lin- blast subpopulations.

Bone Marrow↗

PubMed1993

Differences in the composition and in the efficiency of red cell production of normal and CML erythroid progenitor populations are highlighted by response to human c-kit ligand.

Previous studies have suggested that erythroid progenitors derived from patients with chronic myelogenous leukemia (CML) in chronic phase may have reduced proliferative capacity. Considering recent evidence that mast cell growth factor (MGF) enhances the proliferative capacity of normal erythroid burst-forming units (BFU-E), we examined whether MGF could increase the proliferative potential of CML erythroid progenitors to normal capacity. To evaluate the total proliferative capacity achieved, the BFU-E were divided into four subpopulations (XL = extra large, L = large, M = medium, S = small) and colonies were aspirated to determine the cellularity of BFU-E from each subpopulation. MGF alone or in combination with MoT cell line conditioned medium (MoCM) or granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-3 (IL-3) significantly increased the proliferative capacity of erythropoietin (EPO) dependent CML and normal BFU-E. Although the total number of BFU-E generated were similar, the number of BFU-E with high proliferative potential were considerably less in CML BFU-E populations. BFU-E designated XL (129,000-431,000 cells) were only found in MGF cultures and only normal BFU-E had this proliferative capacity. BFU-E designated L were increased in both normal and CML BFU-E populations but less CML BFU-E had this proliferative capacity (mean number 25% of normal) and CML L BFU-E from 2/3 CML patients comprised fewer cells than normal L BFU-E. Normal BFU-E populations comprised 16-24% high proliferative BFU-E (XL + L) in contrast to 4-5% high proliferative BFU-E (L only) comprising CML BFU-E populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗