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In humans, the aryl hydrocarbon receptor (AHR) gene battery constitutes a set of contaminant-responsive genes, which have been recently shown to be involved in the regulation of several patho-physiological conditions, including tumorigenesis. As the domestic dog represents a valuable animal model in comparative oncology, mRNA levels of cytochromes P450 1A1, 1A2 and 1B1 (CYP1A1, 1A2 and 1B1), AHR, AHR nuclear translocator (ARNT), AHR repressor (AHRR, whose partial sequence was here obtained) and cyclooxygenase-2 (COX2) were measured in dog control tissues (liver, skin, mammary gland and bone), in 47 mast cell tumors (MCTs), 32 mammary tumors (MTs), 5 osteosarcoma (OSA) and related surgical margins. Target genes were constitutively expressed in the dog, confirming the available human data. Furthermore, their pattern of expression in tumor biopsies was comparable to that already described in a variety of human cancers; in particular, both AHR and COX2 genes were up-regulated and positively correlated, while CYP1A1 and CYP1A2 mRNAs were generally poorly expressed. This work demonstrated for the first time that target mRNAs are expressed in neoplastic tissues of dogs, thereby increasing the knowledge about dog cancer biology and confirming this species as an useful animal model for comparative studies on human oncology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

Subcutaneous in vivo injections of cells of the mastocytoma line P815 in syngenic DBA/2 mice induce locally fast growing solid tumors. These have been used extensively as a cancer model to analyze and manipulate the relationship between tumor cells and host's immune defenses. We report that progression of P815 tumors in vivo was accompanied by a burst (Days 5-7) of local inflammatory cells recruitment and angiogenesis observed histologically, corroborated in vivo by MRI with gadolinium, overtranscription of macrophage activation marker genes, secretion of TNF-alpha by regional lymph node cells and concomitant systemic inflammation. No substantial overtranscriptions of either VEGF or IL-10 or TGF-beta genes were observed. Induction of COX-2 gene was a late event. To establish a possible relationship between the tumor-induced local, regional and systemic increase of pro-inflammatory mediators and progression of tumors in vivo, we carried out experiments deliberately modulating the inflammatory status of the recipient animals. Pretreatment of recipient animals by i.p. injection of thioglycolate accelerated P815 tumor growth. At the opposite, treatment of mice with either a COX-1 + COX-2 inhibitor (aspirin, 1 mg/day/mouse) or a specific COX-2 inhibitor (celecoxib, 0.13 mg/day/mouse) for 2 weeks after injection of tumor cells, significantly reduced the size and growth rate of tumors compared to control mice. Experiments carried out in vitro indicated that peritoneal macrophages from untreated animals were strongly activated by live P815 cells and by P815 membrane preparations. The tumor-induced inflammatory reaction could establish a local micro environment favoring tumor progression. The P815 tumor model might be helpful to recognize important factors controlling host/tumor relationship., (Copyright 2002 Wiley-Liss, Inc.)

Objective: To evaluate splenic mast cell tumors (MCT) of cats for activating mutations in the proto-oncogene c-kit., Sample Population: 10 formalin-fixed, paraffin-embedded splenic MCT from cats in the pathology database of the Veterinary Medical Teaching Hospital at the University of California, Davis., Procedure: Genomic DNA was isolated from tumor specimens, and the polymerase chain reaction (PCR) procedure was performed for exons 11, 12, and 17. The PCR products were analyzed by use of agarose gel electrophoresis and then directly sequenced., Results: We did not identify mutations in the juxtamembrane domain (encoded by exons 11 and 12) or catalytic domain (encoded by exon 17) of c-kit in any of the splenic MCT specimens., Conclusions and Clinical Relevance: Although mutations in the proto-oncogene c-kit occur frequently in naturally developing MCT in dogs and aggressive mastocytosis in humans, the data reported here documented that dysregulation of Kit function through activating mutations is unlikely in splenic MCT of cats. Therapeutic strategies aimed at inhibiting Kit signaling (ie, kinase inhibitors such as imatinib [STl571]) may not be of benefit for the treatment of this disease in cats.

Mutations in the proto-oncogene c-kit, including point mutations, deletions, or duplications in the negative regulatory juxtamembrane (JM) domain or point mutations in the catalytic domain, have been observed in human and canine cancers and often result in constitutive activation of Kit in the absence of ligand binding. To identify a receptor tyrosine kinase (RTK) inhibitor capable of blocking the function of mutant Kit, we evaluated 3 indolinones (SU11652, SU11654, and SU11655) that act as competitive inhibitors of adenosine triphosphate binding to several members of the split kinase family of RTKs, including VEGFR, FGFR, PDGFR, and Kit. Mast cell lines expressing either wild-type (WT) Kit, a point mutation in the JM domain, a tandem duplication in the JM domain, or a point mutation in the catalytic domain were used for these studies. All 3 indolinones inhibited phosphorylation of WT Kit in the presence of stem cell factor at concentrations as low as 0.01 microM. Autophosphorylation of both JM mutants was inhibited at 0.01 to 0.1 microM, resulting in cell cycle arrest within 24 hours, whereas autophosphorylation of the catalytic domain mutant was inhibited at 0.25 to 0.5 microM, resulting in cell death within 24 hours. poly(ADP-ribose) polymerase (PARP) cleavage was noted in all Kit mutant lines after indolinone treatment. In summary, SU11652, SU11654, and SU11655 are effective RTK inhibitors capable of disrupting the function of all forms of mutant Kit. Because the concentrations of drug necessary for receptor inhibition are readily achievable and nontoxic in vivo, these compounds may be useful in the treatment of spontaneous cancers expressing Kit mutations.

Mast cell proteinases are important inflammatory mediators in man and other species, but until now there has been no investigation of the nature of equine mast cell proteinases. These studies describe the purification and characterisation of two proteolytic components from equine mastocytoma tissue, detected using chromogenic substrates for trypsin and chymotrypsin. Following chromatographic purification, the trypsin-like component was found to be equine mast cell tryptase by N-terminal amino acid sequencing, showing a close similarity with human tryptase-beta (85% identity over 20 residues). It also had similar subunit molecular size (34-36kDa by SDS-PAGE) and substantially similar cleavage specificity to human tryptase-beta with the substrates tested. A 32kDa chymotrypsin-like component was also purified from mastocytoma extract, and termed equine mast cell proteinase-1 (eqMCP-1). The N-terminal amino acid sequence of eqMCP-1 was very similar to human granzyme H (95% over 19 residues). Rabbit antisera directed against tryptase and eqMCP-1 both detected equine mast cells by immunohistochemistry, and will be of use in future clinical studies of the relevance of mast cell proteinases in equine allergic disease.

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).

We previously reported that mast cell alpha-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. Alpha-chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1's alpha-chymase-mediated cleavage products reveals hydrolysis at Phe(12)-Cys(13) and Phe(23)-Val(24) in loop 1 and Phe(101)-Val(102) and Trp(105)-Asn(106) in loop 3 of the NH(2)-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to alpha-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, alpha-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with alpha-chymase increases gel B activity 2- to 5-fold, suggesting that alpha-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of alpha-chymase blocks degranulation-induced TIMP-1 processing (absent in alpha-chymase-deficient HMC-1 cells). Purified alpha-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell alpha-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.

Proliferation of mastocytoma P-815 cells in vitro was accompanied by a rise in cathepsin D, elastase- and trypsin-like proteinase activity during 6 hours of culturing and a decline by hour 24. Yet alpha 1-proteinase inhibitor activity was inversely proportional to proteinase concentration. Antiproliferative action of actinomycin D disrupted phase variation of proteinase activity and, consequently, the level of alpha 1-proteinase inhibitor rose after 6 hours of cell culturing while that of alpha 2-macroglobulin--after 48 hr. Antiproliferative effect of actinomycin D was eliminated by reduced inhibitor level brought about under the influence of exogenous trypsin. When trypsin was added cathepsin D activity reached its peak 6 hr later while that of alpha 1-proteinase inhibitor declined. That effect and the actomycin D-proteinase inhibitor mechanism were retained when trypsin and actomycin D were present together. It is suggested that cathepsin D and alpha 1-proteinase inhibitor activity plays a key role in realizing the proliferative potential of mastocytoma P-815 cells.

Presence of matrix metalloproteinases has been associated with tumor invasion and metastasis in human neoplasia. The presence of matrix metalloproteinase 2 and matrix metalloproteinase 9 was determined in canine mast cell tumor tissue and normal stromal tissue from 24 dogs with spontaneously occurring cutaneous mast cell tumors. Seventeen of the mast cell tumors were of histologic grade 2, and 7 were of histologic grade 3. Gelatin zymography and computer assisted densitometry image analysis were used to quantify matrix metalloproteinase concentration. Bands from canine tissues migrated in the same location as human proenzyme and active enzyme matrix metalloproteinase 2 and matrix metalloproteinase 9 standards. A semiquantitative value for each patient sample was obtained by comparing the optical assessment density of each unknown band to the optical density of the human standard. The presence of matrix metalloproteinase 2 and matrix metalloproteinase 9 in histologic grade 2 mast cell tumors and histologic grade 3 mast cell tumors was compared, as was presence of matrix metalloproteinases in tumor and stromal tissue. There was dramatically more proenzyme matrix metalloproteinase 9 activity in histologic grade 3 mast cell tumors when compared to grade 2 tumors (P = .03). There was also dramatically more active enzyme matrix metalloproteinase 2 and active enzyme matrix metalloproteinase 9 activity in tumor tissue compared to stromal tissue (P = .02, P < .0001). This study demonstrates that the proenzyme and active enzyme forms of matrix metalloproteinase 2 and matrix metalloproteinase 9 are present in canine mast cell tumors. This appears to be related to the degree of histologic malignancy, although histologic grade 1 tumors were not evaluated.

The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.

Quantitative aspects of the endogenous pathway of Ag processing and presentation by MHC class I molecules to CD8+ CTL were analyzed over a wide range of Ag expression in recombinant vaccinia virus-infected cells expressing beta-galactosidase as model Ag. Only the amount of starting Ag was varied, leaving other factors unaltered. Below a certain level of Ag synthesis, increasing protein amounts led to a sharp rise in recognition by CTL. Higher levels of Ag expression led to a saturation point, which intracellularly limited the number of naturally processed peptides bound to MHC and thereby also CTL recognition. The rate-limiting step was located at the binding of the antigenic peptide to MHC inside the vaccinia virus-infected cell or before this event.

Our prior work shows that cultured BR cells derived from dog mastocytomas secrete the 92-kDa proenzyme form of gelatinase B. We provided a possible link between mast cell activation and metalloproteinase-mediated matrix degradation by demonstrating that alpha-chymase, a serine protease released from secretory granules by degranulating mast cells, converts progelatinase B to an enzymatically active form. The current work shows that these cells also secrete gelatinase A. Furthermore, gelatinases A and B both colocalize to alpha-chymase-expressing cells of canine airway, suggesting that normal mast cells are a source of gelatinases in the lung. In BR cells, gelatinase B and alpha-chymase expression are regulated, whereas gelatinase A expression is constitutive. Progelatinase B mRNA and enzyme expression are strongly induced by the critical mast cell growth factor, kit ligand, which is produced by fibroblasts and other stromal cells. Induction of progelatinase B is blocked by U-73122, Ro31-8220, and thapsigargin, implicating phospholipase C, protein kinase C, and Ca2+, respectively, in the kit ligand effect. The profibrotic cytokine TGF-beta virtually abolishes the gelatinase B mRNA signal and also attenuates kit ligand-mediated induction of gelatinase B expression, suggesting that an excess of TGF-beta in inflamed or injured tissues may alter mast cell expression of gelatinase B, which is implicated in extracellular matrix degradation, angiogenesis, and apoptosis. In summary, these data provide the first evidence that normal mast cells express gelatinases A and B and suggest pathways by which their regulated expression by mast cells can influence matrix remodeling and fibrosis.

Murine mastocytoma P815 induces CTL responses against at least four distinct Ags (AB, C, D, and E). Recent studies have shown that the main component of the CTL response against the P815 tumor is targeted against Ags P815AB and P815E. The gene P1A has been well characterized. It encodes the P815AB Ag in the form of a nonameric peptide containing two epitopes, P815A and P815B, which are recognized by different CTLs. Here, we report the identification of the P815E Ag. Using a cDNA library derived from tumor P815, we identified the gene coding for P815E. We also characterized the antigenic peptide that anti-P815E CTLs recognize on the MHC class I molecule H-2Kd. The P815E Ag results from a mutation within an ubiquitously expressed gene encoding methionine sulfoxide reductase, an enzyme that is believed to be important in the protection of proteins against the by-products of aerobic metabolism. Surprisingly, immunizing mice i.p. with syngeneic tumor cells (L1210) that were constructed to express B7-1 and P815E did not induce resistance against live P815, even though a strong anti-P815E CTL response was observed with splenocytes from immunized animals.

Mast cell accumulations are generally considered to arise almost exclusively from the recruitment of non-granulated, bone-marrow-derived, precursor cells, with the stem cell factor (SCF) reported to play a crucial role in the growth, development and maturation of granulated mast cells within specific tissue sites. In this study dog mastocytoma specimens have been examined by both immunohistochemical and ultrastructural techniques, to demonstrate that fully granulated mast cells are capable of mitotic activity. Observations showing the formation of mitotic spindles, chromosome separation and cytokinesis all support the concept that granulated mast cells are capable of proliferative activity. The ability of mature granulated mast cells to replicate provides an alternative process for local increases in mast cell numbers, at least in canine mast cell tumours. Such observations suggest the possibility that normal or neoplastic human mast cells, fully granulated, have the potential to proliferate in specific tissue sites.

Heparan sulfate proteoglycans, attached to cell surfaces or in the extracellular matrix, interact with a multitude of proteins via their heparan sulfate side chains. Degradation of these chains by limited (endoglycosidic) heparanase cleavage is believed to affect a variety of biological processes. Although the occurrence of heparanase activity in mammalian tissues has been recognized for many years, the molecular characteristics and substrate recognition properties of the enzyme(s) have remained elusive. In the present study, the substrate specificity and cleavage site of heparanase from human hepatoma and platelets were investigated. Both enzyme preparations were found to cleave the single beta-D-glucuronidic linkage of a heparin octasaccharide. A capsular polysaccharide from Escherichia coli K5, with the same (-GlcUAbeta1,4-GlcNAcalpha1,4-)n structure as the unmodified backbone of heparan sulfate, resisted heparanase degradation in its native state as well as after chemical N-deacetylation/N-sulfation or partial enzymatic C-5 epimerization of beta-D-GlcUA to alpha-L-IdceA. By contrast, a chemically O-sulfated (but still N-acetylated) K5 derivative was susceptible to heparanase cleavage. O-Sulfate groups, but not N-sulfate or IdceA residues, thus are essential for substrate recognition by the heparanase(s). In particular, selective O-desulfation of the heparin octasaccharide implicated a 2-O-sulfate group on a hexuronic acid residue located two monosaccharide units from the cleavage site, toward the reducing end.

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell alpha-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by alpha-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2. 3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog alpha-chymase yielded an approximately 84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89-98 and 92-101 of the cDNA predicted sequence, respectively. Thus, alpha-chymase cleaves the catalytic domain of gelatinase B at the Phe88-Gln89 and Phe91-Glu92 bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by alpha-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its proform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a approximately 15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by approximately 5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell alpha-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O -Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAbeta1-4GlcNAcalpha1-4]2-GlcAbeta1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides.

Tryptophan hydroxylase (EC 1.14, 16.4) was purified from yellowfin tuna liver and properties of this enzyme were compared with those of tryptophan hydroxylase from some other species (mouse mastocytoma and rat brain-stem). The molecular weight of the yellowfin tuna enzyme was estimated to be about 280,000 Da. This value is similar to that for the enzymes from mouse mastocytoma and rat brain-stem. On SDS-polyacrylamide gel electrophoresis analysis, yellowfin tuna enzyme was estimated to be about 96,000 Da. This value is different from that for the enzymes from mouse mastocytoma (53,000 Da) and rat brain-stem (59,000 Da) and suggests that yellowfin tuna enzyme may be a dimer of identical subunits of Mr 96,000 Da.

In this study, we demonstrated that NH2-terminal Ser and Ile residues of pRL1b (SI-pRL1a) (SIIPGLPLSL) are not involved in the recognition by RLmale 1-specific cytotoxic T lymphocyte. The sensitization activity observed with pRL1b (SI-pRL1a) was not greater than that of peptides substituted with irrelevant amino acids at these positions. In serum-free medium, pRLla retained sensitization activity, but pRL1b (SI-pRL1a) did not. Furthermore, addition of bestatin to serum-containing medium blocked sensitization by pRL1b (SI-pRL1a). On the other hand, the addition of captopril enhanced it, probably by inhibiting the degradation of pRL1a by ACE. pRL1a-D peptide with D-Ile in place of the L-Ile residue of pRL1a (IPGLPLSL) showed sensitization, but SI-pRLla-2,3D peptide, which has D-Iles in place of the L-Ile residues of pRLlb (SI-pRL1a), and which was not cleaved between the two D-Iles, did not. The findings suggest that pRL1a is the antigenic peptide bound to L(d) molecules and pRL1b (SI-pRL1a) peptide is its natural precursor, which generates pRL1a via proteolysis.

Objective and Design: Several kinds of flavonoids, widely distributed natural products of the vegetable kingdom which possess anti-inflammatory activity, were examined for inhibitory effects on the acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) acetyltransferase activity., Methods: Acetyl-CoA:lysoPAF acetyltransferase activity was determined using homogenates of a rat mucosal-type mastocytoma cell line, RBL-2H3 as an enzyme source. The production of platelet-activating factor (PAF) in rat peripheral white blood cells stimulated with the calcium ionophore A23187 was studied., Results: Of the flavonoids tested, luteolin and quercetin exhibited significant inhibitory effects (IC50, 45 microM and 80 microM, respectively), whereas other structurally-related flavonoids failed to affect the lysoPAF acetyltransferase activity. Luteolin did not suppress the activity of choline acetyltransferase, suggesting that the inhibition observed here was specific. Luteolin also inhibited the production of PAF in rat peripheral white blood cells., Conclusions: These results indicate that luteolin could become a leading compound for developing a novel type of anti-inflammatory, anti-allergic drugs that target lysoPAF acetyltransferase.

The biosynthesis of heparin and heparan sulfate involves a series of polymer-modification reactions that is initiated by N-deacetylation and subsequent N-sulfation of N-acetylglucosamine residues. These reactions are catalysed by a combined N-deacetylase/N-sulfotransferase. Proteins expressing both activities have previously been purified from mouse mastocytoma, which generates heparin, and from rat liver, which produces heparan sulfate. In the present study, the mouse mastocytoma enzyme has been expressed in the human kidney cell line, 293, to investigate whether it could promote modification of the endogenous heparan sulfate precursor polysaccharide into a heparan-like molecule. The N-deacetylase activity of the stably transfected cell clones as approximately 8-fold higher, on a cell-protein basis, than that of control cells, while the N-sulfotransferase activity was increased approximately 2.5 fold. The amounts of glycosaminoglycans synthesized were the same in control and transfected cells, measured as incorporation of [3H]-glucosamine, whereas 35S-labeled glycosaminoglycans were approximately 50% increased in transfected cells, with an increased relative content of heparin sulfate. Structural analysis demonstrated the the glucosamine units of the "heparan sulfate" from transfected cells were almost exclusively N-sulfated, as expected for heparin, whereas more than half of the glucosamine units of the control polysaccharide remained N-acetylated. Notably, the increased N-sulfation was not accompanied by increased O-sulfation, not by C-5 epimerization of D-glucuronic to L-iduronic acid units. The implications of these findings are discussed with regard to the regulation of the biosynthetic process.

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.

A peculiar point mutation results in constitutive activation of c-kit receptor tyrosine kinase (KIT) in three different tumor mast cell lines; ie, the HMC-1, P-815, and RBL-2H3. Because constitutive activation of KIT was also observed in the FMA3 mouse mastocytoma cell line, we investigated the molecular mechanism. Sequencing of the whole coding region of the c-kit showed that the point mutation found in HMC-1, P-815, and RBL-2H3 cells was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs (bp) encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at codons 573 to 579 at the juxtamembrane domain. The FMA3-type c-kit cDNA with 21 bp deletion was introduced into the IC-2 cell line, which was derived from murine cultured mast cells. IC-2 cells were dependent on interleukin (IL)-3 and did not express KIT on the surface. In IC-2 cells introduced with the FMA3-type c-kit cDNA, KIT was constitutively phosphorylated on tyrosines and activated. Moreover, the FMA3-type KIT was dimerized without the stimulation by stem cell factor (SCF), a ligand for KIT. The spontaneously dimerized FMA3-type KIT without SCF binding was not internalized even after the activation. IC-2 cells expressing the FMA3-type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. The deletion of seven amino acids at the juxtamembrane domain appeared to be a new activating mutation of KIT that might be involved in neoplastic growth of mast cells.

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.

We examined three tissue samples from each of four cows with non-lesional skin, tissue samples from a cow with multiple cutaneous mast cell tumors, and samples from another cow in which mast cells were infiltrating multiple lymphosarcomas of the skin, for the presence of tryptase and chymase by enzyme cytochemical and immunohistological methods. The enzyme activities of tryptase and chymase were tested using N-carbobenzoxy-glycilglycil-L-arginine-2-naphthylamide (Z-Gly-Gly-Arg-NA) and naphthol-AS-D-chloroacetate (N-AS-D-CA) as substrates, respectively. Tryptase reactivity could be demonstrated in frozen and Carnoy-fixed paraffin sections. Chymase reactivity was seen in neither frozen nor paraffin sections of formalin- or Carnoy-fixed skin tissues. Antibody linkage with a polyclonal rabbit anti-human skin tryptase antibody was highly specific in bovine normal cutaneous, infiltrating, and tumor mast cells. More than 90% of the tumor mast cells were distinctly tryptase-positive. With alcian blue, only slightly more than 10% of the mast cells stained clearly positive and with methylene blue hardly any staining of mast cell granules could be demonstrated. No antibody labeling of mast cell granules in any of the tissue sections was detected by the use of rabbit anti-dog chymase antiserum. These results indicate that there is a striking antigenic similarity of bovine tryptase to its canine and human equivalents. The demonstration of tryptase is an important tool in confirming the diagnosis of undifferentiated mast cell tumors. In contrast to other species, chymase appears to be completely absent in bovine skin mast cells.

Cutaneous mast cell tumours of a 16-year-old female Indian lion (Panthera leo) were studied histologically and ultrastructurally. The proliferation index detected with an antibody against the nuclear antigen Ki-67 was 16.5%. A cytochemical test for chymase activity was negative. Mast cell tumours are well known in domestic animals and in cats are of two distinct histological types. The present paper is the first report of poorly differentiated cutaneous mast cell tumours in a lion. In their histological and ultrastructural appearance, and in their lack of chymase activity, the neoplastic mast cells resembled tumour mast cells in cats.

Levels of canine tryptase from various tissues were quantified using a competition enzyme-linked immunosorbent assay (ELISA). The assay utilises an affinity-purified rabbit anti-tryptase antibody in the solid phase and alkaline-phosphatase conjugated tryptase together with unlabelled tryptase in the fluid phase. The assay will rapidly quantify 40-5000 ng ml-1 of tryptase in tissue extracts. Tissues from the skin, gut, liver and lung were studied, of which canine gut appeared to contain the highest levels of tryptase per milligram wet weight, which may suggest an important role for this enzyme at this site. This assay may prove valuable in assessing the role of mast cells in various disease states in the dog.

O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modification and the formation of antithrombin binding regions in the biosynthesis of heparin/heparan sulfate. The resulting GlcNSO3(3-OSO3) units are largely restricted to heparin chains with high affinity for antithrombin (HA heparin). Low affinity (LA) heparin fails to serve as a substrate in the 3-O-sulfotransferase reaction yet contains potential 3-O-sulfate acceptor sites (Kusche, M., Torri, G., Casu, B., and Lindahl, U. (1990) J. Biol. Chem. 265, 7292-7300), as verified in the present study using a novel sequencing procedure. O-Desulfated, re-N-sulfated LA heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of LA heparin, both yielded labeled HA components following incubation with solubilized mouse mastocytoma microsomal enzymes and [35S]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfo-transferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region. Indeed, the addition of LA heparin precluded enzymatic 3-O-sulfation of a synthetic pentasaccharide substrate. The Km for the pentasaccharide was determined to approximately be 6 microM. Incubations of mixed pentasaccharide substrate and saccharide inhibitors revealed Ki values for intact LA heparin and for a heparin octasaccharide fraction of approximately 1.3 and approximately 0.7 microM, respectively. Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate and GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-sulfotransferase. By contrast, chondroitin sulfate or dermatan sulfate showed no significant inhibitory activity. It is proposed that the regulation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan sulfate depends on the topological organization of the membrane-bound enzyme machinery in the intact cell.

O-Sulfotransferases involved in heparin biosynthesis were purified > or = 10,000-fold from detergent extracts of mouse mastocytoma tissue by sequential chromatographies on DEAE-Sephacel, heparin-agarose, blue Sepharose, and 3',5'-ADP-Sepharose. The resultant preparation catalyzed the transfer of 35S from 3'-phosphoadenosyl-5'-phospho-[35S]sulfate into N,O-desulfated, re-N-sulfated heparin. Anion-exchange high performance liquid chromatography of disaccharides obtained by deaminative cleavage of the 35S-labeled polysaccharide product revealed O-35S-sulfation at C-2 of L-iduronic acid and at C-6 of D-glucosamine units. SDS-polyacrylamide gel electrophoresis of semipurified enzyme followed by extraction of gel segments and renaturation of proteins consistently showed two distinct fractions of O-sulfotransferase activity, corresponding to proteins of approximately 20 and approximately 60 kDa. The approximately 60-kDa enzyme(s) catalyzed both the 2-O- and 6-O-sulfotransferase reactions, whereas the approximately 20-kDa fraction promoted iduronosyl 2-O-sulfation only. These results are discussed in relation to previous findings, indicating that some of the enzymes involved in heparin biosynthesis catalyze more than one reaction.

A 110-kDa protein involved in heparin biosynthesis in mouse mastocytoma cells was previously shown to express both glucosaminyl N-deacetylase and N-sulfotransferase activity. In this study, the complete nucleotide sequence corresponding to this protein is reported. The mRNA, estimated to contain 3.9 kilobases encodes a protein with an M(r) of 101,092. The predicted domain structure of the protein resembles those of previously characterized Golgi proteins with an N-terminal cytoplasmic tail, a single membrane-spanning domain, and a large catalytic domain linked to the transmembrane domain through a "stem region." Comparison of the deduced amino acid sequence of the mouse mastocytoma protein and a previously cloned similar enzyme from rat liver demonstrated that while large portions of the proteins, corresponding essentially to the putative catalytic domains, were closely related, other portions, in particular in the N-terminal parts, were markedly different. The divergence was not due to species differences since two separate mouse transcripts could be identified that hybridized with probes specific for the two proteins. Also, functional differences were noted since the mastocytoma enzyme, contrary to the liver enzyme, requires a polycation cofactor for expression of N-deacetylase activity. The results are discussed in relation to the structural properties of heparin and heparan sulfate.

The E. coli K5 capsular polysaccharide is composed of 4)GlcpA(beta 1-4)GlcpNAc(alpha 1-disaccharide units. A partially N-deacetylated/N-sulfated heptasaccharide, derived from this polymer and having a nonreducing terminal GlcNAc unit, was used as acceptor for a mastocytoma microsomal GlcA-transferase involved in heparin biosynthesis. An octasaccharide with nonreducing-terminal GlcA similarly served as acceptor for the microsomal GlcNAc-transferase. Analysis of the labeled octa- and nona-saccharides formed by transfer of monosaccharide units from UDP-[14C]GlcA and UDP-[3H]GlcNAc, respectively, showed that both glycosyltransferases could utilize partially N-sulfated acceptors. The GlcA-transferase showed a marked preference for a terminal GlcNAc-GlcA-GlcNSO3-sequence, particularly when this sequence was followed by an additional N-sulfated disaccharide unit. Enzymes catalyzing the same GlcA and GlcNAc transfer reactions were solubilized from E. coli K5 membranes. The K5 capsular polysaccharide, like the heparin/heparan sulfate precursor polysaccharide, thus probably grows by stepwise, alternating addition of the two constituent monosaccharide units, from the corresponding UDP-sugars, to the nonreducing ends of the chains. Moreover, the bacterial glycosyltransferases utilized the same partially N-sulfated oligosaccharide substrates as the mammalian enzymes, and with similar preference for N-sulfate groups in certain positions.

We previously reported that cultured mast cells expressed three discrete phospholipases A2 (PLA2S), one of which showed a remarkable preference for phospholipids bearing an arachidonoyl residue at the sn-2 position [M. Murakami, et al., J. Biochem., 111, 175 (1992)]. In the present study, we have purified and characterized this enzyme using rat mastocytoma RBL-2H3 as an enzyme source. The elution profiles of the arachidonoyl-preferential PLA2 activity of rat mastocytoma RBL-2H3 cells on several column chromatographies were indistinguishable from those of 85-kDa cytosolic PLA2 (cPLA2) characterized so far. The molecular mass of the partially purified PLA2 was estimated to be about 90 kDa by gel filtration and it hydrolyzed arachidonate-containing phospholipids preferentially in the presence of submicromolar Ca2+ concentrations. Furthermore, it was immunoprecipitated with an anti-rabbit cPLA2 antibody almost completely. From these observations, we have concluded that the arachidonoyl-preferential PLA2 in mast cells belongs to the "cPLA2" family.

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.

A mitogen-inducible prostaglandin G/H synthase (PGHS-2 or cyclooxygenase-2) has recently been cloned from chicken and mouse fibroblasts. This protein is distinct from classic prostaglandin G/H synthase (PGHS-1 or cyclooxygenase-1) but has a similar enzymatic activity. Because PGHS-1 is a rate-limiting enzyme in the synthesis of prostaglandins, PGHS-2 may also play an important role in prostaglandin production. To examine whether PGHS-2 is induced by phorbol ester in mast cells, we studied mRNA expression of PGHS-2 and also measured prostaglandin D2 (PGD2) production when canine mastocytoma cells were incubated with phorbol myristate acetate (PMA). PGHS-2 mRNA was induced by PMA, with a maximal induction after 4 h of incubation with 10 nM PMA. There was concentration-dependent production of PGD2 after incubation with PMA. In contrast, PGHS-1 mRNA was expressed in resting cells, and the expression of PGHS-1 mRNA was down-regulated by PMA. Dexamethasone inhibited PMA-induced mRNA expression of PGHS-2 and PGD2 production. Aspirin had no effect on mRNA expression of PGHS-2 but inhibited PGD2 production. In conclusion, PGHS-2 is induced by phorbol ester in canine mast cells. We speculate that PGHS-2 may be important in airway inflammation in which mast cells are activated.

Poly(ADP-ribose)polymerase, a chromatin-bound enzyme, actively participates in processes such as cell proliferation, differentiation, and DNA repair and replication. This enzyme is also implicated in cell transformation, and its inhibition has been proposed to potentiate anti-cancer drug activity. Since cells prepared from tumor biopsies and established tumor cell lines are commonly used to evaluate the efficiency of anticancer therapies, we have compared poly(ADP-ribose)polymerase activity in animal tumor cells growing in vivo and in cell culture. Three tumor types were tested: a mastocytoma (P815), a lymphoma (RDM4), and a glioma (C6). Our results show that cell culture alters poly(ADP-ribose)polymerase levels and activity. Endogenous poly(ADP-ribose) activity was several fold higher in exponentially growing cells than in cells freshly recovered from solid or ascitic tumors. Moreover, polymerase activity increased with culture time, reaching a maximum when cells became confluent. Measurements of poly(ADP-ribose)polymerase gene expression and protein amount indicate that lower enzyme activity in tumors grown in vivo are sustained by decreases in poly(ADP-ribose)polymerase mRNA and protein amount. In contrast, the increase in endogenous poly(ADP-ribose)polymerase activity observed in cultured cells was due to enzyme activation and not to de novo protein synthesis. Such differences must be considered when assessing the applicability of cell-culture results to in vivo situations.

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.

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.

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.

Heparin-derived pentasaccharides with the general structures GlcN-GlcA/IdoA-GlcN-GlcA/IdoA-GlcN (where GlcA represents D-glucuronic acid and IdoA represents L-iduronic acid) and GlcNSO3-GlcA/IdoA-GlcNSO3-GlcA/IdoA- GlcNSO3 (where -NSO3 represents an N-sulfate group) were tested as exogenous sulfate acceptors in incubations with adenosine 3'-phosphate 5'-[35S]phosphosulfate and microsomal enzymes from a heparin-producing mouse mastocytoma. No transfer occurred to the N-unsubstituted pentasaccharide containing only L-iduronic acid, but the other three isomers incorporated various amounts of 35S, which was totally present in N-sulfate groups. After complete chemical N-sulfation, all four pentasaccharides served as acceptors in O-sulfotransferase reactions and incorporated from 20 to greater than 200 times as much radioactivity as did the nonsulfated parent compounds. The C-6 position of the internal glucosamine unit was labeled preferentially, irrespective of the structures of the adjacent hexuronic acid units. Significant 2-O-35S-sulfation of IdoA units occurred in both -IdoA-Glc-NSO3-GlcA- and -GlcA-GlcNSO3-IdoA- sequences, whereas no significant sulfation of GlcA residues was detected. The pentasaccharide GlcNSO3-GlcA-Glc-NSO3-GlcA-GlcNSO3 thus can be used as a selective substrate in assays for glucosaminyl-6-O-sulfotransferase activity. The antithrombin-binding region, essential for the blood anticoagulant activity of heparin, has been identified as a pentasaccharide sequence with the predominant structure GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-++ +IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R represents either a sulfate or an acetyl group and -OSO3 represents an O-sulfate/ester sulfate group, with locations of O-sulfate groups indicated in parentheses) (Lindahl U., Thunberg, L., Bäckström, G., Riesenfeld, J., Nordling, K., and Björk, I. (1984) J. Biol. Chem. 259, 12368-12376). The products of [35S]sulfate transfer to the pentasaccharide GlcNSO3-GlcA-GlcNSO3-IdoA-GlcNSO3 contained molecules with high affinity for antithrombin, corresponding to 0.3-0.5% of the total label. Structural analysis suggested the occurrence of O-[35S]sulfate groups at both C-6 of the nonreducing terminal glucosamine unit and C-3 of the internal glucosamine unit. No products with high affinity for antithrombin were formed from the pentasaccharides that had a different monosaccharide sequence than the binding region; and moreover, these oligosaccharides appeared unable to incorporate glucosaminyl 3-O-sulfate groups. These findings point to the importance of the uronic acid sequence in the generation of the antithrombin-binding region of heparin.

A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)----(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3'-phosphate 5'-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.

The primary structure of L-histidine decarboxylase (HDC: L-histidine carboxy-lyase, EC 4.1.1.22) from mouse mastocytoma P-815 cells has been determined by parallel analysis of the amino acid sequence of the protein and the nucleotide sequence of the corresponding cDNA. HDC contains 662 amino acid residues with a molecular mass of 74017, which is larger by about 21,000 Da than that of the previously purified HDC subunit (53 kDa), suggesting that HDC might be posttranslationally processed. The HDC cDNA hybridized to a 2.7 kilobase mRNA of mastocytoma cells. Homology was found between the sequences of mouse mastocytoma HDC and fetal rat liver HDC.

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.

The phospholipids from murine mastocytoma FMA3 and P-815 clone cells were quantitatively analyzed, and the major glycerophospholipids were examined for their fatty acyl chain distribution. In these cells, the content of histamine was less than 1/100 of normal mouse mast cells, and FMA3 cells had 1.5-fold as much histamine content as P-815 cells. The predominant phospholipid species of both mastocytoma FMA3 and P-815 were choline-containing glycerophospholipids (48%) and ethanolamine-containing glycerophospholipids (29%). The remaining minor constituents were sphingomyelin (6%, 7%), phosphatidylinositol (7%, 5%), phosphatidylserine (2%, 5%), cardiolipin (4%, 3%), and phosphatidic acid (2%, 1% for FMA3 and P-815, respectively). The choline-containing glycerophospholipids consisted of high amounts of 1-O-alkyl-2-acyl type (31%, 25%) and 1,2-diacyl type (63%, 66%) and a smaller amount of 1-O-alk-1'-enyl-2-acyl type (7%, 8%). In contrast, ethanolamine-containing glycerophospholipids were characterized by high contents of 1-O-alk-1'-enyl-2-acyl type (36%, 31%) and 1,2-diacyl type (55%, 58%), and a lower level of 1-O-alkyl-2-acyl type (12% and 11% for FMA3 and P-815, respectively). Unlike choline-containing glycerophospholipids and sphingomyelin that were rich in palmitic acid, ethanolamine-containing glycerophospholipids, phosphatidylserine and phosphatidylinositol showed a high proportion of stearic acid in the overall fatty acid composition. The content of arachidonic acid was highest in phosphatidylinositol. Sphingomyelin had a large amount of long chain and polyunsaturated fatty acids. In both choline- and ethanolamine-containing glycerophospholipids, the predominant fatty acids in the sn-1-position were palmitic, stearic, and oleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)