The role of Fas-associated phosphatase 1 in leukemia stem cell persistence during tyrosine kinase inhibitor treatment of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3ß (Gsk3ß), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of ß-catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and ß-catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased ß-catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.

Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis.

Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.

Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells.

Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.

SHP1 protein-tyrosine phosphatase inhibits gp91PHOX and p67PHOX expression by inhibiting interaction of PU.1, IRF1, interferon consensus sequence-binding protein, and CREB-binding protein with homologous Cis elements in the CYBB and NCF2 genes.

The CYBB and NCF2 genes encode the phagocyte respiratory burst oxidase proteins, gp91PHOX and p67PHOX. Previously, we identified homologous CYBB and NCF2 cis elements that are necessary for lineage-specific transcription during late myeloid differentiation. We determined that these homologous cis elements are activated by PU.1, IRF1, interferon consensus sequence-binding protein (ICSBP), and the CREB-binding protein (CBP). Since expression of PU.1 and ICSBP is lineage-restricted, our investigations identified a mechanism of lineage-specific CYBB and NCF2 transcription. Since PU.1, IRF1, ICSBP, and CBP are expressed in undifferentiated myeloid cells, our investigations did not determine the mechanism of differentiation stage-specific CYBB and NCF2 transcription. In the current investigations, we determine that SHP1 protein-tyrosine phosphatase (SHP1-PTP) inhibits gp91PHOX and p67PHOX expression, in undifferentiated myeloid cell lines, by decreasing interaction of PU.1, IRF1, ICSBP, and CBP with the CYBB and NCF2 genes. We also determine that IRF1 and ICSBP are tyrosine-phosphorylated during interferon gamma differentiation of myeloid cell lines, and we identify IRF1 and ICSBP tyrosine residues that are necessary for CYBB and NCF2 transcription. Therefore, these investigations identify a novel mechanism by which SHP1-PTP antagonizes myeloid differentiation and determine that tyrosine phosphorylation of IRF1 and ICSPB mediates stage-specific transcriptional activation in differentiating myeloid cells.

Tyrosine phosphorylation of HoxA10 decreases DNA binding and transcriptional repression during interferon gamma -induced differentiation of myeloid leukemia cell lines.

The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. The CYBB gene, which encodes the respiratory burst oxidase component gp91(phox), is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon gamma (IFN-gamma)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-gamma treatment. However, IFN-gamma-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreases in vitro DNA binding to Pbx-HoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation.

Recruitment of CREB-binding protein by PU.1, IFN-regulatory factor-1, and the IFN consensus sequence-binding protein is necessary for IFN-gamma-induced p67phox and gp91phox expression.

Activation of the phagocyte respiratory burst oxidase requires interaction between the oxidase components p47phox, p67phox, p22phox, and gp91phox. IFN-gamma induces transcription of the genes encoding p67phox (the NCF2 gene) and gp91phox (the CYBB gene) during monocyte differentiation, and also in mature monocytes. In these studies, we identify an NCF2 cis element, necessary for IFN-gamma-induced p67phox expression, and determine that this element is activated by cooperation between the transcription factors PU.1, IFN regulatory factor 1 (IRF1), and the IFN consensus-binding protein (ICSBP). Previously, we identified a CYBB cis element, necessary for IFN-gamma-induced gp91phox expression, and also activated by this transcription factor combination. In these investigations, we determine that recruitment of a coactivator protein, CBP (the CREBbinding protein), to the CYBB or NCF2 promoter is the molecular mechanism of transcriptional activation by PU.1, IRF1, and ICSBP. Also, we determine that the multiprotein interaction of CBP with PU. 1, IRF1, and ICSBP requires either the CYBB- or NCF2--binding site. Because IFN-gamma induces simultaneous expression of p67phox and gp91phox, these investigations identify a molecular event that coordinates oxidase gene transcription during the inflammatory response. Also, these investigations identify CBP recruitment by cooperation between PU.1, IRF1, and ICSBP as a novel molecular mechanism for IFN-gamma-induced activation of myeloid genes that are involved in the system of host defense.

PU.1, interferon regulatory factor 1, and interferon consensus sequence-binding protein cooperate to increase gp91(phox) expression.

gp91(phox) is a subunit of the phagocyte respiratory burst oxidase catalytic unit. Transcription of CYBB, the gene encoding gp91(phox), is restricted to terminally differentiated phagocytic cells. An element in the proximal CYBB promoter binds a protein complex, referred to as hematopoiesis-associated factor (HAF1), that is necessary for interferon-gamma (IFNgamma)-induced gp91(phox) expression. In these investigations, we determined that HAF1 was a multiprotein complex, cross-immunoreactive with the transcription factors PU.1, interferon regulatory factor 1 (IRF-1), and interferon consensus sequence-binding protein (ICSBP). In electrophoretic mobility shift assay, the HAF1 complex was reconstituted by either in vitro translated PU.1 with IRF-1 or PU.1 with ICSBP, but not by IRF-1 with ICSBP. HAF1a, a slower mobility complex with the same binding site specificity as HAF1, was also investigated. Similar to the HAF1 complex, the HAF1a complex was cross-immunoreactive with PU. 1, IRF-1, and ICSBP. Unlike the HAF1 complex, reconstitution of the HAF1a complex required in vitro translated PU.1 with both IRF-1 and ICSBP. An artificial promoter construct containing the HAF1/HAF1a binding site was modestly activated in the myelomonocytic cell line U937 by co-transfection either with PU.1 and IRF-1 or with PU.1 and ICSBP, but it was strongly activated by co-transfection with PU.1, IRF-1, and ICSBP. This activation required serine 148-phosphorylated PU.1. These studies describe a novel mechanism for PU.1 transcriptional activation via interaction with both IRF-1 and ICSBP, a target gene for the interaction of IRF-1 with ICSBP, and a novel activation function for ICSBP as a component of a multiprotein complex.

Identification and characterization of TF1(phox), a DNA-binding protein that increases expression of gp91(phox) in PLB985 myeloid leukemia cells.

The CYBB gene encodes gp91(phox), the heavy chain of the phagocyte-specific NADPH oxidase. CYBB is transcriptionally inactive until the promyelocyte stage of myelopoiesis, and in mature phagocytes, expression of gp91(phox) is further increased by interferon-gamma (IFN-gamma) and other inflammatory mediators. The CYBB promoter region contains several lineage-specific cis-elements involved in the IFN-gamma response. We screened a leukocyte cDNA expression library for proteins able to bind to one of these cis-elements (-214 to -262 base pairs) and identified TF1(phox), a protein with sequence-specific binding to the CYBB promoter. Electrophoretic mobility shift assay with nuclear proteins from a variety of cell lines demonstrated binding of a protein to the CYBB promoter that was cross-immunoreactive with TF1(phox). DNA binding of this protein was increased by IFN-gamma treatment in the myeloid cell line PLB985, but not in the non-myeloid cell line HeLa. Overexpression of recombinant TF1(phox) in PLB985 cells increased endogenous gp91(phox) message abundance, but did not lead to cellular differentiation. Overexpression of TF1(phox) in myeloid leukemia cell lines increased reporter gene expression from artificial promoter constructs containing CYBB promoter sequence. These data suggested that TF1(phox) increased expression of gp91(phox).

Characterization of three promoter elements and cognate DNA binding protein(s) necessary for IFN-gamma induction of gp91-phox transcription.

The cytochrome b558 heavy chain (gp9l-phox) is expressed in terminally differentiated myelomonocytic cells. Three cis-elements located between -450 and -100 bp of the gp91-phox promoter are required for IFN-gamma induced transcription. Mutations that disrupt individual cis-elements incrementally decrease gp9l-phox promoter activity, and one of the two proximal elements must be present for an IFN-gamma response. The DNA-binding activities that interact with each of the cis-elements exhibit similar gel mobility and binding site specificity, although a consensus binding site common to the three elements is not apparent. An increased level of each DNA/protein complex is observed in myeloid cells following treatment with PMA, retinoic acid/dimethylformamide, or IFN-gamma, but not in similarly treated HeLa cells. The myeloid-specific increase in the intensity of each complex is delayed 12 to 24 h following IFN-gamma treatment, and the complexes are not immunoreactive with antisera directed against IFN-responsive factors such as IRF-1, IRF-2, IFN consensus sequence binding protein, Stat1, and IFN-stimulated gene factor-3 gamma, although IRF-2 is additionally detected as binding to the middle cis-element. These results reveal cis-elements and a DNA-binding factor(s) that participate in a common pathway in response to various stimuli that induce gp9l-phox transcription.

Characterization of a gp91-phox promoter element that is required for interferon gamma-induced transcription.

The cytochrome b558 heavy chain (gp91-phox) is expressed nearly exclusively in terminally differentiating myelomonocytic cells, thereby providing a model to study the events of late myeloid differentiation. We describe a tissue culture assay for studying interferon gamma induction of gp91-phox expression and a cis-element in the gp91-phox promoter that is necessary but not sufficient for this activity. In vitro assays reveal two DNA-binding proteins that interact with this cis-element. One factor is restricted to hematopoietic cells, is required for an interferon gamma response, and binds to an element similar to the Ets protein family consensus, although it does not correspond to known family members. The second factor is the ubiquitous CCAAT-binding protein CP1, which is dispensable for an interferon gamma response. Single base pair mutations in the gp91-phox promoter that specifically abolish the binding of the hematopoietic-associated factor have previously been identified in chronic granulomatous disease patients (Newburger, P. E., Skalnik, D. G., Hopkins, P. J., Eklund, E. A., and Curnutte, J. T. (1994) J. Clin. Invest. 94, 1205-1211). The data reported here directly demonstrate the functional significance of the hematopoietic-associated factor for gp91-phox promoter activity and reveal the binding properties and tissue distribution of this novel DNA-binding protein.

Cloning of a cDNA encoding a human DNA-binding protein similar to ribosomal protein S1.

We report the cloning of a human complementary DNA that encodes a protein which exhibits 36% identity and 62% similarity to Escherichia coli ribosomal protein S1 (rpS1), including conservation of four copies of an RNA-binding domain. This clone was obtained by ligand-screening a lambda gt11 expression library with a DNA probe derived from the CYBB gene promoter. Electrophoretic mobility shift and Southwestern blot assays confirm DNA binding activity of the protein, which exhibits preferential binding to single-stranded and double-stranded DNA and a low binding affinity for RNA. Hence, the rpS1 protein domain previously identified as an RNA-binding motif can also serve as a DNA-binding domain.

Mutations in the promoter region of the gene for gp91-phox in X-linked chronic granulomatous disease with decreased expression of cytochrome b558.

We examined the molecular defect in two kindreds with "variant" X-linked chronic granulomatous disease (CGD). Western blots of neutrophil extracts showed decreased immunoreactive cytochrome b558 components gp91-phox and p22-phox. Analysis of mRNA demonstrated reduced gp91-phox transcripts, with relative preservation of an alternative mRNA species created by transcription initiation in the third exon of the gene. Single strand conformation polymorphism analysis of the 5' flanking region of the patients' gp91-phox genes revealed an electrophoretic abnormality not detected in 40 other gp91-phox genes. Genomic sequencing demonstrated a single base change associated with CGD in each kindred: in one, adenine to cytosine at base pair-57 and in the other, thymidine to cytosine at -55. These mutations are located between the "CCAAT" and "TATA" box consensus sequences involved in eukaryotic gene transcription. Gel shift assays revealed two specific DNA-protein complexes formed between phagocyte nuclear extracts and an oligonucleotide probe representing bases -31 to -68 of the gp91-phox promoter region; the faster-migrating complex could not be formed with oligonucleotides containing either of the promoter mutations. Thus, these promoter region mutations appear to be causally related to the loss of association of a DNA-binding protein and lead to diminished gp91-phox expression, abnormal transcription initiation, and the development of CGD.

Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein.

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.

A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.

The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.

Growth and erosion of thin solid films.

Thin films that are grown by the process of sputtering are, by and large, quite unlike the smooth, featureless structures that one might expect. In general, these films have a complicated surface morphology and an extended network of grooves and voids in their interiors. Such features can have a profound effect on the physical properties of a thin film. The surface irregularities and the bulk defects are the result of a growth instability due to competitive shadowing, an effect that also plays a role in geological processes such as erosion. For amorphous thin films, the shadow instability can be described by a remarkably simple model, which can be shown to reproduce many important observed characteristics of thin film morphology.

Purification and characterization of a lipid thiobis ester from human neutrophil cytosol that reversibly deactivates the O2- -generating NADPH oxidase.

Intact neutrophils possess a cellular mechanism that efficiently deactivates the microbicidal O2-generating NADPH oxidase during the respiratory burst (Akard, L. P., English, D., and Gabig, T. G. (1988) Blood 72, 322-327). The present studies directed at identifying the molecular mechanism(s) involved in NADPH oxidase deactivation showed that a heat- and trypsin-insensitive species in the cytosolic fraction from normal unstimulated neutrophils was capable of deactivating the membrane-associated NADPH oxidase isolated from opsonized zymosan- or phorbol 12-myristate 13-acetate-stimulated neutrophils. This cytosolic species also deactivated the cell-free-activated oxidase. Deactivation by this cytosolic species occurred in the absence of NADPH-dependent catalytic turnover and was reversible, since NADPH oxidase activity could be subsequently reactivated in the cell-free system. The sedimentable particulate fraction from unstimulated neutrophils did not demonstrate deactivator activity. Deactivator activity was demonstrated in the neutral lipid fraction of neutrophil cytosol extracted with chloroform:methanol. Following complete purification of cytosolic deactivator activity by thin layer chromatography and reversed phase high performance liquid chromatography, the deactivator species was shown to be a lipid thiobis ester compound by mass spectroscopy. Cellular metabolism of this compound in human neutrophils may reveal a unique mechanism for enzymatic control of the NADPH oxidase system and thereby play an important role in regulation of the inflammatory response.