We tested the effects of four eosinophil granule cationic proteins: major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), on guinea pig tracheal epithelium in vitro. Examination by inverted microscopy revealed that MBP, both the form stabilized by alkylation of sulfhydryl groups as well as the native form of the molecule, ECP, EPO by itself, as well as EPO + H2O2 + halide, but not EDN, cause dose-related damage to the tracheal epithelium. The lowest concentrations of MBP and ECP causing damage were 10 and 100 micrograms/ml, respectively. In contrast, EDN, although biochemically similar to ECP, did not damage the tracheal epithelium in concentrations of up to 200 micrograms/ml. MBP caused exfoliation, as well as bleb formation and ciliostasis. EPO in the presence of the H2O2-producing enzyme glucose oxidase (GO), Cl-, 0.11 M, and iodide caused ciliostasis, bleb formation, and exfoliation of epithelial cells at concentrations as low as 1 U/ml (3.9 micrograms/ml). EPO + GO in the presence of Cl-, 0.11 M, alone or with Cl- and l-, 10(-4) M, or Cl- and Br-, 5 x 10(-5) M, were all toxic to epithelium. Surprisingly, EPO by itself caused partial ciliostasis, bleb formation, and exfoliation of epithelial cells in a dose-related manner at concentrations as low as 10 to 30 U/ml (39 to 121 micrograms/ml). These results confirm prior observations showing the toxicity of MBP to tracheal epithelium and indicate that ECP and EPO alone, as well as EPO + GO + halide, cause damage. Thus, several eosinophil granule proteins are able to damage respiratory epithelium.

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) were isolated from lysates of human eosinophil granules by gel filtration and ion exchange chromatography on heparin-Sepharose. Radioimmunoassay, using monoclonal antibodies, of fractions from the heparinSepharose chromatography showed one peak of EDN activity and two peaks of ECP activity (termed ECP-1 and ECP-2).EDN, ECP-1, and ECP-2 each exhibited heterogeneity in charge and molecular weight when analyzed by two-dimensional nonequilibrium pH gradient electrophoresis and NaDodSO4/PAGE. Digestion of EDN with endoglycosidase F (endo F) decreased its molecular weight and charge heterogeneity. Thus, EDN likely contains a single complex oligosaccharide. Endo F digestion of ECP-1 and ECP-2 decreased the molecular weight of both polypeptides, indicating that both likely contain at least one complex oligosaccharide. Amino acid sequence analyses showed that ECP-1 and ECP-2 are identical from residue 1 through residue 59 and that the sequences of EDN and ECP are highly homologous (37 of 55 residues identical). Both EDN and ECP NH2-termnu*al sequences showed significant homology to RNase, especially in regions of the RNase molecule involved in ligand binding. EDN, ECP-1, and ECP-2 had neurotoxic activity, causing the Gordon phenomenon at doses down to 0.15 ,ug when injected into the cisterna magna; the proteins were comparable in their activities. These results indicate that EDN and ECP are related proteins and suggest that they derived from genes associated with the RNase family.The human eosinophil granule contains several cationic proteins including the major basic protein (1-3), the eosinophil cationic protein (ECP) (4,5), the eosinophilderived neurotoxin (EDN) (6, 7), and the eosinophil peroxidase (EPO) (8). Major basic protein and ECP are potent helminthotoxins (9-11), and major basic protein is toxic to mammalian cells (9, 12). When injected intrathecally into rabbits or guinea pigs, both EDN and ECP, but not major basic protein or EPO, produce the Gordon phenomenon, a neurologic syndrome characterized in the rabbit by stiffness, ataxia, muscle weakness, and muscle wasting (6,7,13 MATERIALS AND METHODSEosinophil Granules. From nine patients with marked peripheral blood eosinophilia, eosinophils were collected by cytapheresis with hydroxyethyl-starch (15). The mean percentage of eosinophils in the concentrate was 79% (range, 64-94%); between 2 x 1010 and 2 x 1011 eosinophils were collected from a single patient. For purification of the eosinophil granules from the large numbers of cells obtained, we used procedures described previously (1-3, 7, 16). The enriched granule fractions were transferred to 2-ml freezing vials (Nunc) and stored in liquid nitrogen for up to 3 years.EDN and ECP Isolation. Frozen granule pellets were thawed, adjusted to pH 3 with 0.1 M HCl, and centrifuged at 40,000 x g for 20 min. Supernatants from the granule extracts were applied to a 1.2 x 48 cm Sephadex G-50 column equilibrated with 0.025 M NaOAc/0.15 M Na...

No abstract

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.Gemcitabine (2Ј,2Ј-difluoro 2Ј-deoxycytidine), a pyrimidine-based antimetabolite, is currently licensed for the treatment of pancreatic cancer. Recent clinical studies have also demonstrated extensive activity of this agent against a variety of additional neoplasms, including carcinomas of the ovary, lung, and breast, acute leukemias, and refractory lymphomas (Carmichael, 1998;Nabhan et al., 2001). Because of its widespread use, there is considerable interest in understanding factors that affect sensitivity and resistance to this agent. Earlier studies demonstrated that gemcitabine is taken into cells on concentrative nucleoside transporter 1 and phosphorylated to gemcitabine 5Ј-monophosphate by deoxycytidine kinase (Plunkett et al., 1996). Subsequent addition of 5Ј-phosphates results in the formation of gemcitabine diphosphate and gemcitabine triphosphate, both of which contribute to the antiproliferative effects of gemcitabine (Plunkett et al., 1996). Gemcitabine diphosphate inhibits ribonucleotide reductase, thereby depleting deoxyribonucleotide levels. Gemcitabine triphosphate is a substrate for replicative DNA polymerases and causes chain termination one base pair beyond the site of incorporation.Because gemcitabine inhibits replication, this drug is predicted to activate the S-phase checkpoint, a series of reactions that inhibit DNA synthesis and enhance survival when cells experience replication stress. According to current understanding, the kinases ATR and Chk1 play critical roles in this checkpoint. When replicati...

Human and animal eosinophils contain a pow-

IntroductionThe nucleoside analog cytarabine, an effective and widely used agent for the treatment of acute myelogenous leukemia (AML), 1 induces remissions when administered on various schedules either as a single agent or in combination with other antileukemic drugs. 2,3 Unfortunately, despite the inclusion of cytarabine in a variety of induction and consolidation regimens, most patients with AML ultimately relapse and die with drug-resistant disease. [3][4][5][6] Accordingly, there is considerable interest in understanding the mechanisms of resistance to cytarabine and devising strategies for overcoming them. 6,7 Earlier studies identified a number of mechanisms of cytarabine resistance, including diminished uptake on nucleoside transporters, 8 increased degradation of cytarabine to uracil arabinoside, 9 diminished formation or retention of cytosine arabinoside triphosphate, [10][11][12] and reduced incorporation into DNA resulting from decreased passage of cells through S phase. 13 Strategies for overcoming several of these mechanisms have been successfully implemented in clinical trials. [14][15][16] Recent observations suggest that signaling by checkpoint kinase Chk1 might also contribute to cytarabine resistance. Chk1 is activated by a number of replication inhibitors. [17][18][19][20][21] According to current understanding, these inhibitors cause DNA polymerases to stall but allow DNA helicases to continue advancing. 22,23 The resulting single-stranded DNA then binds replication protein A, which recruits 2 protein complexes, one consisting of the ataxia telangiectasia mutated-and Rad3-related (ATR) kinase and its binding partner ATR-interacting protein (ATRIP) and another consisting of the Rad9-Rad1-Hus1 clamp. The Rad9-Rad1-Hus1 complex facilitates ATR-mediated phosphorylation and activation of Chk1. Once activated, Chk1 phosphorylates the phosphatase Cdc25A. [24][25][26][27] The resulting protease-mediated degradation of Cdc25A contributes to S-phase slowing by preventing phosphatasemediated activation of cyclin E/cyclin dependent kinase 2 complexes (reviewed by Sagata 28 ). In addition, Chk1-mediated phosphorylation stabilizes stalled replication forks until replication can resume. 18,21 The potential importance of these events in drug resistance is highlighted by the observation that Chk1 gene deletion or pharmacologic Chk1 inhibition sensitizes cells to replication inhibitors. 21,[29][30][31] Collectively, these observations have raised the possibility that disrupting Chk1 signaling might enhance nucleoside analog cytotoxicity and overcome Chk1-mediated drug resistance.Heat shock protein 90 (Hsp90) is currently receiving considerable attention as a potential anticancer drug target. 32 The Hsp90 complex is a chaperone that facilitates the initial folding and/or stabilization of a variety of polypeptides, which are known as Akt,37,38 and Bcr/abl, 39,40 play important roles in leukocyte biology and leukemogenesis. The ability of the Hsp90 complex to stabilize these clients is inhibited by the benzoqui...

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC 50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 Mmol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser 473 . At doses that inhibited proliferation, the compound also caused a G 0 -G 1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo. (Cancer Res 2006; 66(1): 362-71)