Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The knowledge about the differentiation of basophilic leukocytes is fragmentary. This report discusses a detailed phenotypic characterization of molecular markers for hematopoietic differentiation in a basophilic leukemia cell line, KU812. The expression of markers for lymphoid, erythroid, neutrophil, eosinophil, monocytic, megakaryocytic, mast cell and basophil differentiation was analyzed at the mRNA level by Northern blots in the KU812 cells, and for reference, in a panel of human cell lines representative of the different hematopoietic differentiation lineages. KU812 was found to express a number of mast cell and basophil-related proteins, i.e. mast cell tryptase, mast cell carboxypeptidase A, high-affinity immunoglobulin (IgE) receptor alpha and gamma chains and the core protein for heparin and chondroitin sulphate synthesis. We found no expression of a number of monocyte/-macrophage or neutrophil leukocyte markers except for lysozyme. From earlier studies, it has been shown that lysozyme is not expressed in murine mucosal mast cell lines. This finding, together with the expression of the mast cell carboxypeptidase in KU812 might distinguish the phenotype of this cell line from that typical of mucosal mast cell lines in rodents. We found a low level of expression of the eosinophil and basophil marker, major basic protein, which might indicate a relationship between basophils and eosinophils. No expression is, however, detected with the eosinophil-specific markers eosinophil cationic protein, eosinophil-derived neurotoxin or eosinophil peroxidase. We also report an extensive screening for inducers of basophilic differentiation of the KU812 cells. The most efficient protocol of induction included serum starvation which led to a dramatic increase in a number of markers specific for mast cells and basophils such as tryptase, carboxypeptidase A and the heparin core protein. Finally, diisopropylfluorophosphate analysis of total protein extracts from KU812 show four labeled protein bands with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that this cell line expresses at least three previously undescribed serine proteases of which one or more could be a potential basophil-specific marker(s).
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PMID:Phenotypic characterization of KU812, a cell line identified as an immature human basophilic leukocyte. 163 3

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.
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PMID:Establishment and characterization of hybrid rat mast cells. 182 10

We report here that extended culture of purified rat peritoneal mast cells (RpMC), typical of the connective tissue-type (CTMC), gives rise to continuously proliferative cell lines without the requirement of exogenous growth factors such as IL-3 and IL-4 or accessory cells. Two of the cell lines established, RCMC1 and RCMC2, are described here. Both cell lines have been maintained in continuous culture in vitro for over a year. Although these cell lines were derived from CTMC, they exhibit phenotypic characteristics of mucosal-type mast cells, i.e., they contain rat mast cell protease II (RMCP II), low levels of histamine and stain alcian blue+/safranin-. Previous studies have identified both high and low affinity receptors for IgE, designated Fc epsilon RI and Rc epsilon RII, respectively, on RpMC and rat basophilic leukemia (RBL) cells. At the early stages of cell culture, RCMC1 expressed predominantly Fc epsilon RI and a gradual increase in the expression of Fc epsilon RII has been observed with time in culture. By comparison, RCMC2 expressed predominantly Fc epsilon RII throughout its entire period of cell culture.
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PMID:Factor-independent tissue cultured mast cells: establishment from rat peritoneal mast cells. 245 19

Granules that are potently cytolytic in vitro can be obtained from cytotoxic lymphocytes that kill virally infected cells and tumor cells. These granules contain pore-forming proteins and several serine proteases. Here we indicate that at least two different proteases participate in the lysis mediated by granule proteins from RNK-16 rat leukemia cells. We report twelve different mechanism-based or "suicide" isocoumarin serine protease inhibitors which have different 3- and 7-substituents that confer selectivity and reactivity towards either the chymotrypsin- ("chymase") or trypsin-like ("tryptase") protease activities of RNK-16 cells. Second order inhibition rates of inactivation (kobsd/[I]) for the RNK-16 granule proteases ranged between 164 and 22,640 M-1s-1. These new, specific and highly reactive isocoumarin serine protease inhibitors also abrogated the cytolysis mediated by lymphocytes granule proteins. The eight inhibitors with large hydrophobic or basic substituents that conferred chymase or tryptase specificities were more effective at inactivating lytic function than the four elastase-directed inhibitors with smaller substituents. All twelve new isocoumarin inhibitors blocked cytolysis at lower concentrations than 3,4-dichloroisocoumarin, a potent general mechanism-based serine protease inhibitor that also blocks RNK-16 granule protease activities and lysis.
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PMID:Selective isocoumarin serine protease inhibitors block RNK-16 lymphocyte granule-mediated cytolysis. 281 73

Rat RNK-16 leukemia cells kill YAC-1, which are the cells lysed by rodent natural killer lymphocytes. We found chymotrypsin-like proteinase ('chymase') activity in the RNK-16 dense granules that also contain cytolytic activity. The chymase activity hydrolyzed the thiobenzyl peptide substrate Suc-Phe-Leu-Phe-SBzl and, in comparison to RNK-16 tryptase activity, was selectively inhibited by three different types of serine proteinase inhibitors. The selective inhibitors were the fungal aldehyde chymostatin, the chloromethylketone Z-Gly-Leu-Phe-CH2Cl, and the mechanism-based or 'suicide' inhibitor 7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin. These proteinase inhibitors also blocked RNK-16 granule-mediated cytolysis. Chymostatin, a reversible inhibitor, delayed granule-mediated cytolysis, whereas the irreversible chloromethylketone and isocoumarin proteinase inhibitors completely abrogated granule-mediated cytolysis. The two irreversible inhibitors displayed biphasic inhibition of the chymase activity, indicating that at least two chymases are present in the granules. By Northern blot analysis, we found that RNK-16 mRNA hybridized strongly with a cDNA probe of CCPI, a mouse cytotoxic T lymphocyte serine proteinase gene. These data imply that chymase activity in the cytotoxic granules is important for cytolytic function and is likely to belong to a new subfamily of serine proteinases.
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PMID:Localization, implications for function, and gene expression of chymotrypsin-like proteinases of cytotoxic RNK-16 lymphocytes. 326 87

One mechanism by which cytotoxic T lymphocytes and natural killer cells inflict target cell death depends upon secreting the contents of their specialized cytoplasmic granules, containing a pore-forming protein, perforin, and a family of homologous serine proteases ("granzymes") with various enzyme activities. We used a granzyme B-specific mouse anti-human monoclonal antibody 2C5 and Western blotting to demonstrate that nuclear extracts of human interleukin-2-stimulated peripheral blood mononuclear cells, the human NK leukemia cell line YT, and the rat NK leukemia cell line RNK-16 contain abundant granzyme B. In interleukin-2-activated peripheral blood mononuclear cells, more than 50% of the total cellular granzyme B was present in the nuclear lysate. Nuclear granzyme B had an apparent molecular mass of approximately 32 kDa in human cells and approximately 30 kDa in RNK-16 and was eluted from immobilized heparin at the same NaCl concentration as granzyme B from cytoplasmic granules. Granzyme B that was affinity-purified with 2C5 from the nuclei of YT or human LAK cells was capable of efficiently cleaving synthetic peptide thiobenzyl ester substrates with the same specificity (peptide cleavage after aspartic acid) as granule-localized granzyme B. By contrast perforin, which colocalizes with granzymes in cytotoxic granules, was not detectable in nuclear lysates. Granzyme B was also demonstrated to be present in the nucleus and cytoplasmic granules of YT by immunohistochemical staining with monospecific anti-granzyme B antisera. Other protease activities (tryptase and peptide cleavage after methionine) were also readily detectable in nuclear and cytoplasmic lysates of YT, RNK-16, and LAK cells, as determined by the cleavage of the synthetic substrates N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) and Boc-Ala-Ala-Met-S-benzyl, except that BLT-esterase activity was absent from the nucleus of YT. The localization of serine proteases in the nucleus was restricted to lymphocytes with cytotoxic capacity, as non-cytotoxic cell lines expressed high levels of peptide cleavage after methionine and tryptase activities in their cytoplasm, but possessed no nuclear serine protease activity. Furthermore, non-cytotoxic monkey kidney COS-7 cells transfected with an SV40-driven expression plasmid incorporating full-length human granzyme B cDNA contained abundant cytoplasmic granzyme B, but demonstrated minimal nuclear granzyme B accumulation. We conclude that serine proteases of NK cells are not restricted to cytolytic granules and, further, that their capacity to access the nucleus may have implications for the role of these enzymes in eliciting target cell death.
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PMID:Granule serine proteases are normal nuclear constituents of natural killer cells. 803 81

The basophilic leukaemia cell line KU812 can be induced to differentiate into basophil-like cells in vitro when exposed to supernatant from the Mo T-cell line. KU812 cells express affinity receptors for IgE, produce histamine and tryptase and have the capacity for IgE-mediated histamine release. In this study we have examined the cytokines, produced by the Mo cell line, which are responsible for the observed differentiation-inducing effect in the KU812 cell line. It was shown that interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) induced differentiation in the KU812 cells and that these cytokines were responsible for the differentiation-inducing effect of the Mo supernatant. Other cytokines tested, IL-1 beta, IL-2, IL-4, IL-5, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and nerve growth factor (NGF) were without effect on the KU812 cells. KU812 was also shown to express receptors for both TNF-alpha and IL-6 after 3 days cultivation with conditioned media from the Mo T-cell line. Untreated cells showed no detectable levels of TNF-alpha or IL-6 receptors indicating induction of these receptors during differentiation. Spontaneous differentiation was shown to occur under serum-free conditions which may be the result of endogenous IL-6 production through an autocrine loop. The activity of TNF-alpha and IL-6 could be blocked by specific monoclonal antibodies (mAb) to the respective cytokine.
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PMID:TNF-alpha and IL-6 induce differentiation in the human basophilic leukaemia cell line KU812. 813 23

We have biochemically purified a 27-kDa serine protease (designated RNK-Tryp-2) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) which has tryptase activity. Utilizing molecular sieve chromatography and reverse-phase HPLC, we purified RNK-Tryp-2 to homogeneity and sequenced 33 NH2-terminal amino acids. Oligonucleotide primers were used in the PCR to generate a 528-bp cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate an 884-bp RNK-Tryp-2 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 233 amino acids which does not have potential sites for N-linked glycosylation. The cDNA encodes a leader peptide of at least 25 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the N-terminus, and the His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, are conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Tryp-2 is distinct protease. Southern blot analysis suggests the existence of one or more related genes. A single 1.3-kb mRNA transcript was detected by Northern blot analysis of total cellular RNA from the in vivo passaged RNK-16, rat splenocytes, lung and liver nonparenchymal cells, as well as in highly purified rat LGL and T cells. RNK-Tryp-2 is a novel serine protease that is expressed in the granules of large granular lymphocytes.
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PMID:Purification and cloning of a novel serine protease, RNK-Tryp-2, from the granules of a rat NK cell leukemia. 813 42

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Both heparin and chondroitin sulfate were found to be present in approximately equal amounts. HMC-1 lacked surface expression of the high-affinity IgE receptor, which was confirmed by the absence of mRNA of the alpha- and beta-chains of the IgE-receptor complex. However, a strong expression of the gamma-chain of the IgE-receptor complex was detected. A positive staining of the monocyte/macrophage marker CD68 was obtained, as well as a strong hybridization signal for the eosinophilic/basophilic-related differentiation marker the Charcot-Leyden crystal. Treatment of HMC-1 with conditioned media from the human T-cell line Mo, retinoic acid or DMSO induced only moderate changes in the surface or intracellular expression of the studied markers. The agents tested neither induced any of the monocyte/granulocyte markers examined, nor expression of the Fc epsilon RI alpha-chain.
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PMID:Phenotypic characterization of the human mast-cell line HMC-1. 819 Dec 24

A cDNA encoding rat mast cell tryptase (rMCT) was successfully cloned, and sequenced, from peritoneal cells of Lewis rats infected with Nippostrongylus brasiliensis by the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. The cDNA was 1,097 base-pairs long, and included 822 base-pairs of an open reading frame. As judged from the deduced amino acid sequence, rMCT is highly homologous to mouse mast cell protease-6, and is considered to be translated as a prepro-enzyme with a 19-amino acid signal peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The rMCT mRNA was not detected in peritoneal cells of mast cell-deficient Ws/Ws rats, though it was strongly detected in ones of littermate +/+ and Lewis rats. In addition to in peritoneal mast cells, the rMCT mRNA was detected in the tongue. However, mRNA signals were not detected in the small intestine regardless of N. brasiliensis infection. Nor were mRNA signals detected in RBL2H3 rat basophilic leukemia cells. In the lung, the rMCT mRNA was strongly detected after infection with N. brasiliensis, though it was only faintly detected before infection. These results suggest that the rMCT is basically specific for connective tissue mast cells, but not for mucosal mast cells and that it is up-regulated in the lung during the inflammatory process of a parasitic infection.
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PMID:cDNA sequencing and expression of rat mast cell tryptase. 853 14


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