Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.     

Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor

Diphtheria fusion proteins are chimeric proteins consisting of the catalytic and translocation domains of diphtheria toxin (DT(388)) linked through an amide bond to one of a variety of peptide ligands. The ligand targets the molecule to cells and the toxin enters the cell, inactivates protein synthesis and induces cell death. Diphtheria fusion proteins directed to human myeloid leukemic blasts are a novel class of therapeutics for patients with chemotherapy refractory myeloid leukemia. Because of the presence of interleukin-3 (IL3) receptors on myeloid leukemic progenitors and its absence from mature myeloid cells, we synthesized four bacterial expression vectors encoding DT(388) fused to human IL3. Different molecules were engineered to assess the effects of modifications on yield, purity and potency of product. The constructs differed in the size of the linker peptide between the DT(388) and IL3 domains and in the presence or absence of an oligohistidine tag on the N- or C-terminus. Escherichia coli were transformed and recombinant protein induced and purified from inclusion bodies. Similar final yields of 3-6 mg of purified protein per liter of bacterial culture were obtained with each of the four molecules. Purity ranged from 70 to 90% after partial purification by anion-exchange, size-exclusion chromatography and/or nickel affinity chromatography. Proteins were soluble and stable at 4 degrees C and -80 degrees C in phosphate-buffered saline at 0.03-0.5 mg/ml. The fusion proteins showed predicted molecular weights by SDS-PAGE, HPLC and tandem mass spectrometry and had full ADP-ribosylating activities. Each was immunoreactive with antibodies to DT(388) and IL3. Each of the fusion proteins with the exception of the one with an N-terminal oligohistidine tag showed full IL3 receptor binding affinity (K:(d) = 3 nM) and potent and selective cytotoxicity to IL3 receptor positive human myeloid leukemia cell lines (IC(50) = 5-10 pM). In contrast, the N-terminal histidine-tagged fusion protein bound IL3 receptor with a 10-fold lower affinity and was 10-fold less cytotoxic to IL3 receptor positive blasts. Thus, we report a series of novel, biologically active DT(388)IL3 fusion proteins for potential therapy of patients with receptor positive myeloid leukemias.     

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.     

Restoration of energy metabolism in leukemic mice treated by a siddha drug--Semecarpus anacardium Linn. nut milk extract

Chronic myeloid leukemia (CML) is a clonal disorder characterized by proliferation of hematopoietic cells that possess the BCR-ABL fusion gene resulting in the production of a 210 kDa chimeric tyrosine kinase protein. CML, when left untreated, progresses to a blast phase during which the disease turns aggressive and shows poor response to known treatment regimens. We have studied a Siddha herbal agent, Semecarpus anacardium Linn. nut milk extract (SA) for its antileukemic activity and its effect on the changes in energy metabolism in leukemic mice. Leukemia was induced in BALB/c mice by tail vein injection of BCR-ABL(+) 12B1 murine leukemia cell line. This resulted in an aggressive leukemia, similar to CML in blast crisis, myeloid subtype, confirmed by histopathological study and RT-PCR for the p210 mRNA in the peripheral blood, spleen and liver. Leukemia-bearing mice showed a significant increase in lipid peroxides, glycolytic enzymes, a decrease in gluconeogenic enzymes and significant decrease in the activities of TCA cycle and respiratory chain enzymes as compared to control animals. SA treatment was compared with standard drug imatinib mesylate. SA administration to leukemic animals resulted in clearance of the leukemic cells from the bone marrow and internal organs on histopathological examination and this was confirmed by RT-PCR for the p210 mRNA. Treatment with SA significantly reversed the changes seen in the levels of the lipid peroxides, the glycolytic enzymes, the gluconeogenic enzymes and the mitochondrial enzymes. These effects are probably due to the flavonoids, polyphenols and other compounds present in SA which result in total regression of leukemia and correction of the alterations in energy metabolism. Study of animals treated with SA alone did not reveal any adverse effects. On the basis of the observed results, SA can be considered as a readily accessible, promising and novel antileukemic chemotherapeutic agent.     

GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells

DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.     

Increased PRAME-Specific CTL Killing of Acute Myeloid Leukemia Cells by Either a Novel Histone Deacetylase Inhibitor Chidamide Alone or Combined Treatment with Decitabine

As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8⁺ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201⁺ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME^100–108 or PRAME^300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

鼠粒细胞集落刺激因子受体的纯化及鉴定

粒细胞集落刺激因子受体(G-CSFR)在鼠NFS-60细胞中有较高的含量,通过对NFS-60细胞的大规模培养,用CHAPS及超速离心抽提G-CSFR, 经G-CSF亲和层析纯化获得G-CSFR, 采用ABC-ELISA进行鉴定.     

白喉毒素-白细胞介素2重组嵌合毒素的克隆表达及特异性细胞毒作用的研究

应用PCR技术分别扩增出编码白喉毒素氨基端 389个氨基酸 (DT3 89)的基因片段及人IL 2全基因 ,将两基因串连插入 pET3a载体 ,构建成含有DT3 89 IL 2融合基因的表达载体 ,转化大肠杆菌BL2 1,经表达、纯化后 ,用3 H Leucine掺入法测定其对HUT 10 2细胞的蛋白合成抑制作用。SDS PAGE电泳分析表明 ,表达产物分子质量 (Mr)约为 5 8kD ;重组嵌合毒素能够特异性地抑制高表达IL 2受体的HUT 10 2细胞的蛋白生物合成 ,且有一定的剂量反应关系 ,其细胞半数抑制浓度 (IC50 )约为 3 3× 10 -11mol/L。为进一步研制特异性的抗IL 2受体高表达肿瘤和相关疾病的药物打下了基础。     

Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.     

Cytotoxicity of IL6-PE40 and derivatives on tumor cells expressing a range of interleukin 6 receptor levels

The chimeric toxin IL6-PE40, which is composed of interleukin 6 (IL6) fused to a mutant form of Pseudomonas exotoxin (PE) devoid of its native cell recognition domain, can kill myeloma and hepatoma cells which express high levels of IL6 receptors. To enhance the usefulness of IL6-PE40 on potential target cells, we have attempted to develop more potent IL6-PE derivatives. We have developed nine new IL6-PE derivatives and assessed their cytotoxicity on human myeloma cells. Two of these new forms, IL6-domain II-PE40 and IL6-PE664Glu were more toxic to myeloma cells bearing IL6 receptors than was IL6-PE40. These two chimeric toxins were compared with IL6-PE40 for cytotoxicity toward a variety of tumor cell lines. We found that most tumor cell lines which are sensitive to IL6-PE40 are more sensitive to IL6-domain II-PE40 and IL6-PE664Glu. Cells with as few as 200-600 IL6 receptors/cell could be killed. The specificity of these chimeric toxins was shown through competition with recombinant IL6. Toxicity studies in mice demonstrated that the two new molecules had an LD50 of 10-20 micrograms/mouse. This compares to an IL6-PE40 LD50 of 20 micrograms/mouse. The new IL6-toxins could be detected in the serum up to 8 h after intraperitoneal administration with a peak at 1 h. These data suggest that IL6-domain II-PE40 and IL6-PE664Glu may be more useful than IL6-PE40 in killing IL6 receptor-bearing tumor cells in animals.     

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.     

Interaction of granulocyte colony-stimulating factor (G-CSF) with its receptor. Evidence that Glu19 of G-CSF interacts with Arg288 of the receptor.

Granulocyte colony-stimulating factor (G-CSF) forms a tetrameric complex with its receptor, comprising two G-CSF and two receptor molecules. The structure of the complex is unknown, and it is unclear whether there are one or two binding sites on G-CSF and the receptor. The immunoglobulin-like domain and the cytokine receptor homologous module of the receptor are involved in G-CSF binding, and Arg288 in the cytokine receptor homologous module is particularly important. To identify residues in G-CSF that interact with Arg288, selected charged residues in G-CSF were mutated to Ala. To clarify whether there are two binding sites, a chimeric receptor was created in which the Ig domain was replaced with that of the related receptor gp130. This chimera bound G-CSF but could not transduce a signal, consistent with failure of dimerization and loss of one binding site. The G-CSF mutants had reduced mitogenic activity on cells expressing wild-type receptor. When tested with the chimeric receptor, all G-CSF mutants except one (E46A) showed reduced binding, suggesting that Glu46 is important for interaction with the Ig domain. On cells expressing R288A receptor, all the G-CSF mutants except E19A showed reduced mitogenic activity, indicating that Glu19 of G-CSF interacts with Arg288 of the receptor.     

The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells

Background aimsCytokine-induced killer (CIK) cells may serve as an alternative approach to adoptive donor lymphocyte infusions (DLI) for patients with acute leukemia relapsing after haplo-identical hematopoietic stem cell transplantation (HSCT). We investigated the feasibility of enhancing CIK cell-mediated cytotoxicity by interleukin (IL)-15 against acute myeloid and lymphoblastic leukemia/lymphoma cells.MethodsCIK cells were activated using IL-2 (CIK_IL-2) or IL-15 (CIK_IL-15) and phenotypically analyzed by fluorescence-activated cell sorting (FACS). Cytotoxic potential was measured by europium release assay.ResultsCIK_IL-2 cells showed potent cytotoxicity against the T-lymphoma cell line H9, T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT-4 and subtype M4 acute myeloid leukemia (AML) cell line THP-1, but low cytotoxicity against the precursor B (pB)-cell ALL cell line Tanoue. IL-15 stimulation resulted in a significant enhancement of CIK cell-mediated cytotoxicity against acute lymphoblastic leukemia/lymphoma cell lines as well as against primary acute myeloid and defined lymphoblastic leukemia cells. However, the alloreactive potential of CIK_IL-15 cells remained low. Further analysis of CIK_IL-15 cells demonstrated that the NKG2D receptor is apparently involved in the recognition of target cells whereas killer-cell immunoglobulin-like receptor (KIR)-HLA mismatches contributed to a lesser extent to the CIK_IL-15 cell-mediated cytotoxicity. In this context, CD3 ⁺ CD8 ⁺ CD25 ⁺ CD56^? CIK_IL-15 cell subpopulations were more effective in the lysis of AML cells, in contrast with CD56 ⁺ CIK_IL-15 cells, which showed the highest cytotoxic potential against ALL cells.ConclusionsThis study provides the first evidence that CIK_IL-15 cells may offer a therapeutic option for patients with refractory or relapsed leukemia following haplo-identical HSCT.     

Induction of hepatocyte growth factor/scatter factor by interferon-γ in human leukemia cells

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-γ remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-α and IFN-β were as effective as IFN-γ in RPMI-8226 cells, but less than IFN-γ in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-γ. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1β, and tumor necrosis factor (TNF)-α, the former three were as effective as IFN-γ in KG-1 cells, but only TNF-α stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-α, IFN-β, and IFN-γ. The effect of IFN-γ in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-γ in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-γ is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-γ is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis. J. Cell. Physiol. 174:107–114, 1998. © 1998 Wiley-Liss, Inc.     

A proper amino terminus of diphtheria toxin is important for cytotoxicity

A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.     

Identification of the differentiation-associated p93 tyrosine protein kinase of HL-60 leukemia cells as the product of the human c-fes locus and its expression in myelomonocytic cells

A differentiation-associated 93-kDa tyrosine kinase (p93) was purified previously from the human promyelocytic leukemia cell line HL-60. The present study conclusively identifies p93 as the c-fes proto-oncogene product and shows that expression of p93c-fes and its associated tyrosine kinase activity are marked in mature granulocytes, monocytes, and human myeloid leukemia cell lines. Antisera to peptides obtained by expression of c-fes cDNA fragments in Escherichia coli reacted strongly with p93 purified from HL-60 cells. Western blots using one of these antisera demonstrated high levels of p93c-fes protein in normal human granulocytes and monocytes, as well as the cell lines KG-1, THP-1, HEL, and U-937, all of which can be induced to differentiate along the myelomonocytic pathway. Conversely, in cell lines resistant to myeloid differentiation, p93c-fes expression was either very low or absent. Expression of immunoreactive p93c-fes in these cell lines showed a strong positive correlation with p93c-fes tyrosine kinase activity, which was measured in cell extracts using a nondenaturing gel assay. Finally, the expression of p93c-fes, its tyrosine kinase activity, and the binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) were all coordinately increased in HL-60 cells treated with the granulocytic differentiation inducer dimethyl sulfoxide, while all three parameters were low in untreated or differentiation-resistant HL-60 cells. These results suggest that expression of p93c-fes tyrosine kinase activity may be an essential component of myeloid differentiation and responsiveness to granulocyte-macrophage colony-stimulating factor.     

Localization of the human G-CSF gene to the region of a breakpoint in the translocation typical of acute promyelocytic leukemia

Summary The colony-stimulating factors regulate growth, differentiation, and function of blood cells. The effect of granulocyte colony-stimulating factor (G-CSF) on myeloid leukemias is unique among colony-stimulating factors in driving the leukemic cells from a self-renewing malignant state to a mature differentiated phenotype with the concomitant loss of tumorigenicity. This property of G-CSF has led to suggestions that its absence is responsible for lack of differentiation of leukemic cells and that the therapeutic administration of G-CSF could reverse this defect and result in a cure for leukemia. Here we show that the gene coding for human G-CSF is localized to chromosome 17, bands q11.2-21. The translocation of the long arm of chromosome 17 at q12-21 to chromosome 15 is a specific abnormality occurring in a high proportion of, if not all, patients with acute promyelocytic leukemia, a disease characterized by undifferentiated myeloid cells and a dismal prognosis. Abnormalities of the regulation of a specific differentiation factor gene mediated by a specific chromosomal rearrangement may be directly implicated in the pathogenesis of human leukemia.