RESUMO
Gelatinolytic metalloproteinases implicated in connective tissue remodeling and tumor invasion are secreted from several types of cells in the form of inactive zymogens. In this report, characterization of gelatinase activity secreted by the BR line of dog mastocytoma cells reveals a phorbol-inducible, approximately 92-kD, Ca2+ - and Zn2+ -dependent proenzyme cleaved over time to smaller, active forms. Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products. The NH2-terminal 13 amino acids of the purified mastocytoma progelatinase are 50-67% identical to those of human, mouse, and rabbit 92-kD progelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore A23187 greatly accelerated proenzyme cleavage, suggesting that a serine protease present in secretory granules hydrolyzed the progelatinase to active fragments. To identify the activating protease, cells were coincubated with ionophore and a panel of selective serine protease inhibitors. Soybean trypsin inhibitor and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mast cell chymase, prevented progelatinase activation. Inhibitors of tryptase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-amidino-2-benzimidazolyl) methane (BABIM), did not. In further experiments using highly purified enzymes, mastocytoma cell chymase activated 92-kD progelatinase in the absence of other enzymes or cofactors; tryptase and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinase B that is directly activated outside of the cell by exocytosed chymase, and provide the first demonstration of a cell that activates a matrix metalloproteinase it secretes by cosecreting an activating enzyme. In mastocytomas, this pathway may facilitate tumor invasion of surrounding tissues, and in normal mast cells, it could play a role in tissue remodeling and repair.
Assuntos
Gelatinases/metabolismo , Sarcoma de Mastócitos/enzimologia , Sequência de Aminoácidos , Animais , Quimases , Cães , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Gelatinases/genética , Humanos , Mastócitos/enzimologia , Sarcoma de Mastócitos/genética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Coelhos , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/farmacologia , Células Tumorais CultivadasRESUMO
Synthesis of histamine in hematopoietic progenitor cells may be one of the earliest events in mastopoiesis. We therefore asked whether the key enzyme involved in histamine production, histidine decarboxylase (HDC), can be used as an immunohistochemical marker for the detection of immature neoplastic mast cells (MC) in patients with MC-proliferative disorders. To address this question, we examined bone marrow biopsy specimens in a cohort of 102 patients with mastocytosis using an antibody against HDC. Independent of the maturation stage of MC, the anti-HDC antibody produced clear diagnostic staining results in all patients with systemic MC disease examined including those with MC leukemia and MC sarcoma, in which MCs are particularly immature. In these patients, expression of HDC was reconfirmed at the messenger RNA level by reverse transcriptase polymerase chain reaction analyses performed with RNA of highly enriched CD117(+) MC. In summary, HDC is expressed in neoplastic MC in patients with systemic mastocytosis independent of the maturation stage of cells or the variant of disease. Histidine decarboxylase should therefore be considered as a new MC marker in the screen panel of antigens used to diagnose high-grade MC malignancies.
Assuntos
Histidina Descarboxilase/metabolismo , Imuno-Histoquímica/métodos , Leucemia de Mastócitos/enzimologia , Mastócitos/enzimologia , Sarcoma de Mastócitos/enzimologia , Mastocitose Sistêmica/enzimologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Feminino , Histidina Descarboxilase/análise , Histidina Descarboxilase/genética , Humanos , Leucemia de Mastócitos/complicações , Leucemia de Mastócitos/patologia , Masculino , Mastócitos/patologia , Sarcoma de Mastócitos/complicações , Sarcoma de Mastócitos/patologia , Mastocitose Sistêmica/etiologia , Mastocitose Sistêmica/patologia , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD) mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors. METHODS: In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16-20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations. RESULTS: No mutations or polymorphisms were identified in exons 16-20 of any of the MCTs examined. CONCLUSION: In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs.
Assuntos
Doenças do Cão/enzimologia , Sarcoma de Mastócitos/veterinária , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-kit/genética , Neoplasias Cutâneas/veterinária , Animais , Análise Mutacional de DNA , DNA de Neoplasias/genética , Cães , Éxons/genética , Feminino , Masculino , Sarcoma de Mastócitos/enzimologia , Sarcoma de Mastócitos/genética , Proteínas de Neoplasias/química , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-kit/química , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genéticaRESUMO
Immune responses to sheep erythrocytes were enhanced in mice bearing small mastocytomas soon after injection of a few tumor cells. In contrast, mice with larger tumors after transfer of a greater number of mastocytoma cells and those in the later stages of tumor development after transfer of small numbers of tumor cells showed moderately suppressed immune responses. Transfer of spleen cells from mastocytoma-bearing mice to irradiated recipients resulted in more antibody-forming cells as compared to transfer of splenocytes from normal donor mice. The addition of graded numbers of mastocytoma cells to a constant amount of normal spleen cells transferred to irradiated mice also resulted in enhanced responses and increased spleen weights in the recipients. This increase, in direct proportion to the number of mastocytoma cells transferred, also occurred when Escherichia coli lipopolysaccharide (a T-cell independent antigen) was used to immunize animals given spleen cells from normal mice and mastocytoma cells. Mastocytoma cell-free homogenates or X-irradiated tumor cells also heightened immune responses in recipient mice, which indicated that viable cells were not needed for the effect. Such homogenates, as well as the tumor cells per se, stimulated increased lactate dehydrogenase (LDH) activity in the sera of recipient mice. However, tumor cells passaged in tissue culture for several months, those derived from mice bearing a mastocytoma cell line with a low LDH-stimulatory activity, or UV-irradiated mastocytoma cells with a high LDH-stimulatory activity did not induce enhanced plaque-forming cell responses.
Assuntos
Sarcoma de Mastócitos/imunologia , Animais , Células Cultivadas , Feminino , Técnica de Placa Hemolítica , Imunização Passiva , Sarcoma de Mastócitos/enzimologia , Camundongos , Camundongos Endogâmicos DBA , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/imunologia , Tamanho do Órgão , Polissacarídeos Bacterianos , Baço/imunologiaRESUMO
The Kit receptor tyrosine kinase is critical for the growth and development of hematopoietic cells, germ cells, and the interstitial cells of Cajal. Gain-of-function mutations in codon 816 of the catalytic domain of human Kit [codon 814 of murine Kit (mKit)] are found in patients with mastocytosis, leukemia, and germ cell tumors. There are no drugs that inhibit the activity of Kit catalytic domain mutants to a greater extent than wild-type Kit. The objective of this study was to understand the biochemical mechanisms mediating mast cell transformation by this Kit mutant to identify molecular targets for pharmacological intervention. To this end, we examined signaling pathways activated in the murine mast cell line IC2 infected with either wild-type (IC2-mKit) or mutant mKit (IC2-mKit(D814Y)). In this study, we show that mKit(D814Y) is constitutively phosphorylated on tyrosine 719, and this likely results in constitutive association with activated phosphatidylinositol 3'-kinase (PI3K). In vitro growth of IC2-mKit(D814Y) cells is more sensitive to inhibition of PI3K than SCF-induced growth of IC2-mKit cells. s.c. injection of IC2-mKit(D814Y) in syngeneic mice results in mast cell tumors. To determine whether inhibition of PI3K could reduce mKit(D814Y)-mediated tumorigenicity, mice were treated with 1.5 mg/kg wortmannin three times a week. Five weeks after injection of tumor cells, a 75% reduction in tumor weight was observed when wortmannin treatments were initiated 2 days after inoculation with tumor cells. A 66% reduction occurred when treatment was initiated 2 weeks after inoculation. Treatment with wortmannin increased necrosis in the tumors, and this was associated with apoptosis. Interestingly, there was no effect on tumor vasculature. Thus, PI3K is required for survival and growth of the IC2-mKit(D814Y) mast cell line both in vitro and in vivo. These findings may provide insight into designing strategies for treatment of mastocytosis and other diseases associated with mutations in the Kit catalytic domain.
Assuntos
Transformação Celular Neoplásica/metabolismo , Mastócitos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Androstadienos/farmacologia , Animais , Células COS , Domínio Catalítico , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Transformação Celular Neoplásica/patologia , Chlorocebus aethiops , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Mastócitos/citologia , Mastócitos/metabolismo , Sarcoma de Mastócitos/enzimologia , Sarcoma de Mastócitos/genética , Sarcoma de Mastócitos/patologia , Camundongos , Camundongos Endogâmicos DBA , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/genética , Fator de Células-Tronco/farmacologia , Transfecção , WortmaninaRESUMO
Both adult (I) and embryonic (II) forms of uridine kinase have been identified in the transplantable EL-4 leukemia of C57BL/6 mice and in the P815Y mastocytoma of DBA/2 mice. Only Species I is found in primary tumor cells of lymphoid orgin (virus-induced feline lymphosarcoma, human acute and chronic lymphocytic leukemia) and in normal calf thymocytes and porcine peripheral blood lymphocytes; Species I was induced 4-fold upon stimulation of the normal blood lymphocytes with phytohemagglutinin. The level of uridine kinase activity in the feline lymphosarcoma of thymus-dependent lymphocyte orgin and childhood lymphocytic leukemia of possible thymus-dependent lymphocyte or null-cell origin was similar to the induced level in phytohemagglutinin-stimulated normal lymphocytes, i.e., thymus-dependent lymphocytes. In contrast lymphocytes of a patient with chronic lymphocytic leukemia of thymus-independent lymphocyte origin had a level of uridine kinase activity comparable to that of the unstimulated normal lymphocytes or thymocytes. The uridine kinase activity in the EL-4 tumor cells was repressed by acute treatment of the mice with 5-azacytidine.
Assuntos
Isoenzimas/metabolismo , Neoplasias/enzimologia , Fosfotransferases/metabolismo , Uridina Quinase/metabolismo , Animais , Azacitidina/farmacologia , Linfócitos B/enzimologia , Carcinoma Hepatocelular/enzimologia , Gatos , Indução Enzimática/efeitos dos fármacos , Humanos , Leucemia Experimental/enzimologia , Leucemia Linfoide/enzimologia , Neoplasias Hepáticas/enzimologia , Ativação Linfocitária , Linfócitos/enzimologia , Linfoma não Hodgkin/enzimologia , Masculino , Sarcoma de Mastócitos/enzimologia , Camundongos , Pessoa de Meia-Idade , Neoplasias Experimentais/enzimologia , Ratos , Linfócitos T/enzimologia , Uridina Quinase/biossínteseRESUMO
DNA methyltransferase was isolated as a single polypeptide of 190 kDa from mouse P815 mastocytoma cells by immunoaffinity chromatography. This polypeptide seems to be highly susceptible to proteolytic degradation resulting in additional polypeptides in the size range of 150 to 190 kDa. A polypeptide of 190 kDa was immunoprecipitated by monoclonal anti-DNA methyltransferase antibodies from extracts of two different human cell lines, Raji and K562. The 190 kDa polypeptide was synthesized in rapidly proliferating cells and, albeit at a much lower rate, also in cells grown to saturating density. DNA methyltransferase polypeptides smaller than 190 kDa were synthesized neither in log phase nor in stationary phase cells.
Assuntos
DNA (Citosina-5-)-Metiltransferases/isolamento & purificação , Sarcoma de Mastócitos/enzimologia , Animais , Linhagem Celular , Humanos , Camundongos , Peso Molecular , Fragmentos de Peptídeos/análiseRESUMO
Cultured mouse mastocytoma P-815 cells were treated with 1 mM sodium n-butyrate for 40 h. The treated cell homogenate showed high activities in synthesizing prostaglandin D2, E2, and F2 alpha. Such activities were virtually absent in untreated cell homogenate. Direct addition of sodium n-butyrate to the homogenate showed no effects. Pre-exposure of cells to acetylsalicylic acid did not diminish the effect of the subsequent treatment with sodium n-butyrate. These data suggest that sodium n-butyrate induces fatty acid cyclooxygenase in P-815 cells.
Assuntos
Butiratos/farmacologia , Sarcoma de Mastócitos/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Aspirina/farmacologia , Células Cultivadas , Indução Enzimática , CamundongosRESUMO
Histidine decarboxylase (EC 4.1.1.22) from a mouse mastocytoma has been purified by chromatography on charged and non-charged n-alkyl derivatives of agarose. The former was represented by the coupling product of CNBr-activated agarose and alkylmonoamines (alkylamino-agarose), the latter by the coupling of agarose and alkylglycidyl ehters (alkyl agarose). The choice of fractionation medium was restricted by the enzyme stability; excessively high ionic strength media could not be used. Under the conditions investigated, the best result was obtained with the non-charged ocytl agarose. The enzyme was adsorbed to this gel at a relatively high ionic strength, and on stepwise decrease in ionic stength of the eluting buffer it was desorbed with a total recovery of 80%. There was an approx. 10-fold increase in specific activity. The histidine decarboxylase, thus purified, retained 90-100% of its activity for 10 days or more at 6-8 degrees C. Some general comments on protein fractionation on charged and non-charged alkyl derivatives of agarose are given. The complexity of protein interaction with the charged alkyl derivatives is illustrated by experiments with a colored protein, phycoerythrin.
Assuntos
Carboxiliases/isolamento & purificação , Histidina Descarboxilase/isolamento & purificação , Sarcoma de Mastócitos/enzimologia , Animais , Cromatografia de Afinidade , Histidina Descarboxilase/metabolismo , Camundongos , Neoplasias Experimentais/enzimologia , SefaroseRESUMO
We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.
Assuntos
Doenças do Cão/enzimologia , Leucócitos/enzimologia , Sarcoma de Mastócitos/veterinária , Peptídeo Hidrolases/sangue , Serina Endopeptidases/isolamento & purificação , Neoplasias Cutâneas/veterinária , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimases , Cães , Humanos , Cinética , Sarcoma de Mastócitos/enzimologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases/metabolismo , Neoplasias Cutâneas/enzimologiaRESUMO
Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.
Assuntos
Citarabina/farmacologia , DNA/análogos & derivados , DNA/metabolismo , Animais , Linhagem Celular , DNA Bacteriano/metabolismo , DNA-Citosina Metilases/metabolismo , Cinética , Sarcoma de Mastócitos/enzimologia , Metilação , Camundongos , Polidesoxirribonucleotídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Nuclei from K21 murine mastocytoma cells do not form topoisomerase II-DNA adducts in response to amsacrine in the absence of a cytoplasmic factor tentatively identified as a type of casein kinase (Darkin, S.J. and Ralph, R.K. (1991) Biochim. Biophys. Acta 1088, 285-291). The stimulatory activity was present in extracts from cells grown in horse serum but not in calf serum. Activity was lost following growth arrest by serum deprivation. In contrast, topoisomerase II activity in isolated nuclei did not decline during growth arrest. These results suggest that the resistance of some non-cycling tumour cells to anti-cancer drugs may result from decreased activation of topoisomerase II.
Assuntos
Amsacrina/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Sarcoma de Mastócitos/enzimologia , Animais , Sangue , Divisão Celular , Meios de Cultura , Meios de Cultura Livres de Soro , DNA/efeitos dos fármacos , DNA/metabolismo , Cinética , Camundongos , Células Tumorais CultivadasRESUMO
Tryptophan hydroxylase (tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4) purified from the neoplastic murine mast cells by hydroxylapatite chromatography following ammonium sulfate fractionation showed maximum activity at pH 6.0 in the presence of 2-mercaptoethanol, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine and Fe2+, and pH 7.6 to 8.0 in the absence of addED Fe2+. The Km values were 38.5 muM and 22.2 muM for tryptophan, 298 muM and 204 muM for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine, and 6.45% for oxygen in either presence or absence of added Fe-2+, respectively. From kinetic data the reaction mechanism of tryptophan hydroxylation appears to be of the sequential, rather than the ping-pong, type. Tryptophan hydroxylase from mast cells was considerably inhibited by o-phenanthroline like phenylalanine hydroxylase as well as tyrosine hydroxylase from other sources, and its Ki was between 1.2 muM and 4.53 muM. It was found that the inhibition by o-phenanthroline was competitive with respect to both tryptophan and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not molecular oxygen under the assay conditions employed.
Assuntos
Sarcoma de Mastócitos/enzimologia , Oxigenases de Função Mista/metabolismo , Triptofano Hidroxilase/metabolismo , Animais , Ligação Competitiva , Catalase , Ativação Enzimática , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Cinética , Mercaptoetanol/farmacologia , Camundongos , Camundongos Endogâmicos DBA , Nitrogênio , Oxigênio , Fenantrolinas/farmacologia , Pteridinas/farmacologia , Triptofano Hidroxilase/antagonistas & inibidores , Triptofano Hidroxilase/isolamento & purificaçãoRESUMO
Rat brain homogenates incubated with exogenous [32-P] phosphatidylcholine liberated: LYSO[32-P] phosphatidylcholine, sn-glycero-3-[32-P] phosphorylcholine, [32-P] phosphorylcholine, sn-gleycero-3-[32-P] phosphate and 32-Pi. Further investigation showed that [32-P] phosphorylcholine was released exclusively from sn-glycero-3-[32-P] phosphorylcholien by a novel diesterase activity. We propose that the enzyme be termed L-3-glycerylphosphinicocholine cholinephosphohydrolase (EC 3.1.4.-). Parallel experiments on rat liver homogenates and a P815Y mouse mastocytoma cell-lysate, revealed no diesterase activity.
Assuntos
Encéfalo/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Linhagem Celular , Glicerilfosforilcolina , Fígado/enzimologia , Lisofosfatidilcolinas , Sarcoma de Mastócitos/enzimologia , Camundongos , Fosfolipases/metabolismo , Diester Fosfórico Hidrolases/metabolismo , RatosRESUMO
Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for 'wild-type' K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3-4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of 'wild-type' cells. In 'wild-type' K 21 cells incubated at 39.5 degrees C, thymidine kinase activity was approx. 30% of that at 33 degrees C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5 degrees C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5 degrees C to 33 degrees C was inhibited by actinomycin D and cycloheximide.
Assuntos
Sarcoma de Mastócitos/genética , Mutação , Timidina Quinase/genética , Animais , Ciclo Celular , Linhagem Celular , Temperatura Baixa , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Temperatura Alta , Cinética , Sarcoma de Mastócitos/enzimologia , Sarcoma de Mastócitos/fisiopatologia , Camundongos , Fosforilação , Timidina Quinase/metabolismoRESUMO
The possibility of post-translational processing of mouse mastocytoma histidine decarboxylase (HDC; EC 4.1.1.22) was investigated. The molecular mass of the recombinant HDC expressed in Sf9 cells using HDC cDNA from mouse mastocytoma cells was determined to be 74 kDa by SDS-PAGE. In contrast to the native HDC from mastocytoma cells, the recombinant 74 kDa HDC was essentially inactive and precipitable in Sf9 cells. On the other hand, deletion mutants of the recombinant HDC lacking a C-terminal region equivalent to 10 (64 kDa) or 20 kDa (54 kDa) in size were present as active forms in the soluble fraction of Sf9 cells. To examine the C-terminal deletion of the 74 kDa species yielding the 53 kDa species by means of the immunoblotting analysis, two peptides (corresponding to residues 323-337 and 572-586 of the recombinant 74 kDa HDC peptide) were synthesized, and rabbit antiserum specific for each peptide was prepared. On immunoblotting analysis, anti-peptide 323-337 antiserum recognized both the recombinant 74 kDa and native enzyme subunit peptides, but anti-peptide 572-586 antiserum recognized only the recombinant 74 kDa peptide, i.e., not the native enzyme subunit peptide. Furthermore, HDC activity in the crude extract from Sf9 cells was not precipitable with antipeptide 572-585 antiserum. These results strongly suggest that the 53 kDa subunit peptide of native mastocytoma HDC is derived from the unidentified inactive 74 kDa HDC peptide, probably by post-translational processing of HDC in its C-terminal region.
Assuntos
Histidina Descarboxilase/metabolismo , Sarcoma de Mastócitos/enzimologia , Sequência de Aminoácidos , Animais , Baculoviridae , Sequência de Bases , Linhagem Celular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Histidina Descarboxilase/biossíntese , Histidina Descarboxilase/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Mariposas , Mutagênese , Oligonucleotídeos Antissenso , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Deleção de Sequência , TransfecçãoRESUMO
Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.
Assuntos
Histidina Descarboxilase/metabolismo , Elastase Pancreática/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Cromatografia em Agarose , Endopeptidases/metabolismo , Histidina Descarboxilase/química , Histidina Descarboxilase/isolamento & purificação , Immunoblotting , Sarcoma de Mastócitos/enzimologia , Camundongos , Peso Molecular , Pâncreas/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , SuínosRESUMO
Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.
Assuntos
Fibroblastos/enzimologia , Mastócitos/enzimologia , Colagenase Microbiana/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Cromatografia em Gel , Cães , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Esterases/metabolismo , Gengiva/enzimologia , Macrófagos/enzimologia , Sarcoma de Mastócitos/enzimologia , Peso Molecular , Concentração Osmolar , Temperatura , alfa-Macroglobulinas/farmacologiaRESUMO
12-O-Tetradecanoylphorbol-13-acetate (TPA) markedly enhanced the increase in L-histidine decarboxylase (HDC) activity induced by dexamethasone in mouse mastocytoma P-815 cells, even with a concentration of the latter that had the maximal effect, whereas it induced a rapid and transient increase in HDC activity, which peaked after 3 h in the absence of dexamethasone. The synergistic effect of TPA on HDC activity induced by dexamethasone was detected after 4 h, a plateau level being reached by 6 h, which was similar to the time course with dexamethasone alone. TPA enhanced the induction of HDC activity by various glucocorticoids, but had no effect on the induction by dibutyryl cAMP, prostaglandin E2 or sodium butyrate. Both 1-oleoyl-2-acetylglycerol, a protein kinase C activator, and okadaic acid, a protein phosphatase inhibitor, enhanced the increase in HDC activity induced by dexamethasone, but 4 alpha-phorbol-12,13-didecanoate, an inactive derivative of TPA, did not. Protein kinase C inhibitors, such as staurosporin, H-7 and K255a, suppressed the increase in HDC activity induced by TPA with or without dexamethasone. The enhancement of HDC activity by dexamethasone was completely suppressed by cycloheximide or actinomycin D. Furthermore, TPA markedly enhanced the accumulation of HDC mRNA due to dexamethasone (5 to 10-fold, from 6 to 12 h after). TPA did not cause a significant increase in the level of either [3H]dexamethasone binding capacity or preformed HDC activity in cells. These results taken together suggest that dexamethasone-induced de novo synthesis of HDC in mastocytoma P-815 cells is up-regulated by TPA-activated protein kinase C through the mechanism involving an increased rate of transcription.
Assuntos
Dexametasona/farmacologia , Histidina Descarboxilase/biossíntese , Sarcoma de Mastócitos/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Animais , Carcinógenos , Diglicerídeos/farmacologia , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Éteres Cíclicos/farmacologia , Histidina Descarboxilase/efeitos dos fármacos , Isoquinolinas/farmacologia , Sarcoma de Mastócitos/metabolismo , Camundongos , Ácido Okadáico , Ésteres de Forbol/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Piperazinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.