Tryptophan hydroxylase from mouse mastocytoma P-815. Reversible activation by ethylenediaminetetraacetate

Tryptophan hydroxylase from mouse mastocytoma P-815 is activated by ethylenediaminetetraacetate (EDTA). The activation proceeds in a pH-, temperature-, and time-dependent manner and leads to a 30-fold higher activity at maximum. The optimal EDTA concentration is 10 microns. The activation requires solely EDTA with desalted crude enzyme solution in the absence of any cellular metabolites. There are no indications that the activation is due to proteolysis, modification of protein-bound sulfhydryl groups or other covalent modifications such as phosphorylation and methylation. In the presence of appropriate stabilizing agents, the activated state is maintained after the removal of EDTA by gel-filtration. The activation is reversible under conditions in which bound metal(s) is dissociated from the complex with EDTA. These results imply that the role of EDTA is metal chelation. A model is proposed in which the enzyme has at least two interconvertible states, the activated state and ground state, corresponding to the free and metal-bound forms, respectively. The metal is probably derived from the cell. The assay method for tryptophan hydroxylase utilized a rapid and sensitive (5 pmol/injection) determination of 5-hydroxytryptophan by high performance liquid chromatography.     

5'-N-ethylcarboxamido [3H] adenosine binding sites of mouse P815 mastocytoma cell membranes: solubilization and partial purification by affinity chromatography

A 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding site of mouse mastocytoma P815 cell membranes has been purified approximately 100-fold by affinity chromatography. This adenosine binding site, which has a similar specificity to that of the A2 adenosine receptor, was absorbed on NECA-linked Sepharose 6B and eluted with NECA. The adsorption of the [3H]NECA binding site to the affinity matrix was specifically blocked by NECA. The [3H]NECA binding site bound on the affinity matrix was also specifically eluted by NECA. This affinity matrix adsorbed approximately 90% of the digitonin-solubilized [3H]NECA binding activity applied, and after the gel was washed, 30-50% of the adsorbed binding activity could be eluted with 500 microM NECA with specific binding activity of 50-70 pmol/mg of protein. The affinity-purified [3H]NECA binding site retained the same ligand binding specificities as the original membrane preparation. The results indicate that the NECA-Sepharose Sepharose 6B should provide a powerful tool for the eventual purification of [3H]NECA binding sites of P815 cell membranes.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

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.     

5'-N-ethylcarboxamide[3H]adenosine binding sites of mouse mastocytoma P815 cell membranes: characterization and solubilization

Mouse mastocytoma P815 cell membranes were found to possess adenosine binding sites as assessed by using the adenosine agonist [3H]5'-N-ethylcarboxamideadenosine (NECA). The Kd and Bmax for the [3H]NECA binding at 0 degrees C were 380 nM and 17 pmol/mg of protein, respectively. The rank order of potency for inhibition of [3H]NECA binding was NECA greater than 5'-N-cyclopropylcarboxamideadenosine greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine greater than theophylline greater than N6-[(R)-1-methyl-2-phenylethyl]adenosine = N6-[(S)-1-methyl-2- phenylethyl]adenosine. Thermodynamic analyses of the adenosine receptor agonist and antagonist binding showed that all such ligands displayed negative values of both enthalpy and entropy which suggested that the driving force for the binding was enthalpic. [3H]NECA binding sites of P815 cell membranes were solubilized with sodium cholate and retaining the same ligand-binding characteristics as those of the membrane-bound form. By gel filtration on a Sepharose CL-6B column, the adenosine binding site was estimated to have a Stokes radius of approximately 6.7 nm.     

Identification of a second major tumor-specific antigen recognized by CTLs on mouse mastocytoma P815

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.     

Humoral mechanisms of in vitro inhibition of mouse lymphocyte immunoreactivity by mastocytoma P815 cells

The inhibitory capacity of mastocytoma cell line P815 and its cultural supernatant (CS) was studied in the reaction of blast transformation (RBT) and mixed lymphocyte culture (MLC). An addition of both P815 cells and CS resulted in dose-dependent inhibition of lymphocyte proliferation in RBT and MLC. The treatment of DBA/2 spleen cells with CS for 2 h at 37 degrees C resulted in a significant decrease in proliferative activity and induction of supressor cells.     

cDNA-derived amino acid sequence of L-histidine decarboxylase from mouse mastocytoma P-815 cells

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.     

Thapsigargin induces mitochondrial dysfunction and apoptosis in the mastocytoma P815 cell line and in mouse thymocytes

Thapsigargin is a plant-derived inhibitor of the endoplasmic reticulum Ca(2+)-ATPase.Treatment with thapsigargin leads to a rapid, large and prolonged increase in the intracellular calcium ion concentration ([Ca(2+)](i)). Previously thapsigargin has been shown to inhibit proliferation and induce apoptosis. Here we report the results of thapsigargin treatment in thymocytes harvested from 10-day-old mice and in the P815 mastocytoma cell line. In thapsigargin-treated cells we observed enlarged mitochondria with disrupted cristae structure. These mitochondria closely resembled those observed after the induction of phase transition. To determine if the mitochondria were functioning normally the cells were stained with rhodamine 123 (R123) and analysed with flow cytometry. After thapsigargin treatment the R123 staining decreased, indicative of a loss of mitochondrial membrane potential. Furthermore intracellular ATP concentrations were also found to be reduced in cells treated with thapsigargin. Taken together these results indicate an increase in the [Ca(2+)](i) caused by thapsigargin treatment results in dysfunctional mitochondria and reduced ATP. We propose that this decrease in the concentration of ATP provokes the onset of thapsigargin-induced apoptosis. To investigate the effect of thapsigargin treatment on the cell cycle, rapidly cycling P815 cells were sorted into populations enriched for either G(0)/G(1) or S/G(2)/M phases, and these populations were then treated with thapsigargin. Thapsigargin treatment induced a cell cycle block before S phase. We propose that the block in the cell cycle induced by thapsigargin was a result of the decreased intracellular ATP concentration interfering with the energy requiring processes of DNA replication. The block could also be related to the high intracellular calcium ion concentration that would interfere with the subtle calcium transients involved in the cell's preparations for replication and mitosis. Apoptosis occurred to an equal extent in both populations of cells.     

Cyclic AMP and c-myc gene expression in PY815 mouse mastocytoma cells

The possibility was examined that inhibition of growth of PY815 mouse mastocytoma cells by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (DB cyclic AMP) results from inhibition of c-myc gene expression. Temporary increases in c-myc RNA which occurred soon after DB cyclic AMP treatment and upon removal of the drug were not consistent with direct inhibition of c-myc gene expression by DB cyclic AMP. The increases in c-myc RNA coincided with the passage through, or accumulation of cells in late G1-early S phase. It is proposed that cyclic AMP may stimulate c-myc gene expression which normally occurs only in late G1-early S phase in PY815 cells and that cyclic AMP prevents c-myc expression in cells at other phases of the cell cycle by inhibiting their progression past a cyclic AMP-sensitive restriction point in early G1 phase.     

Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells.

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Effect of tunicamycin on functions of PGE1 receptors from mouse mastocytoma P-815 cells

Prostaglandin E1 (PGE1) receptors from mouse mastocytoma P-815 cells were found to bind to a wheat germ agglutinin (WGA)-Agarose column, suggesting that the receptors are glycoproteins. To further elucidate the role of carbohydrate moieties in the PGE1 receptors for their binding activity to ligand, the P-815 cells were treated with tunicamycin, swainsonine or monensin. Tunicamycin, an inhibitor of N-glycosylation, dose- and time-dependently inhibited the binding of PGE1 to mastocytoma P-815 cells. Neither swainsonine, an inhibitor of Golgi mannosidase II, nor monensin, an inhibitor of processing beyond the high mannose stage, altered PGE1 binding properties of the cells. The inhibition of PGE1 binding by tunicamycin was observed when incorporation of [3H]glucosamine into macromolecules was inhibited. The inhibitory effect was not on their affinity but on their number of binding sites. Subcellular distributions of [3H]PGE1-binding activity showed that decreases in the binding activity by tunicamycin were highest in plasma membrane fractions. Treatment of membranes with various endo- and exoglycosidases did not affect PGE1 binding. PGE1-stimulated cyclic AMP accumulation in the cells was also inhibited by tunicamycin. These results suggest that PGE1 receptors of mastocytoma P-815 cells are glycoproteins and that inhibition of N-glycosylation of PGE1 receptors by tunicamycin results in the arrest of the translocation of newly synthesized receptors to the surface of mastocytoma P-815 cells.     

DNA-cytosine-5-methyltransferase from P815 mouse mastocytoma cells: "maintenance" and "de novo" activities are carried out by the same enzyme molecule

DNA-5-methyltransferase has been purified (about 1400-fold) from rapidly proliferating mouse P815 mastocytoma cells by chromatographies on DEAE cellulose, hydroxyapatite and a heparine-agarose affinity step. The isolated enzyme has an isoelectric point of 7.3 and in neutral 10-30% glycerol gradient it bands in an area corresponding to molecular weight of 135,000 dalton. During the enzymatic reaction, the enzyme first interacts with DNA and then accomplishes a series of methyl group transfers without being detached. The formation of the initial DNA-enzyme complexes is probably random and independent of the cofactor, S-adenosyl-L-methionine, as well as the sequences recognized as methylation sites. The "maintenance" and "de novo" types of activity have been monitored using hemimethylated and completely unmethylated DNA as methyl group accepting polymers. Both these activities copurify in three different chromatographic procedures. This, together with the fact that the enzyme purified near to homogeneity possesses both types of activities suggests that "de novo" and "maintenance" DNA methyltransferase activities are exercised by the same enzyme molecule.     

Tumor cell surface expression of granulocyte-macrophage colony-stimulating factor elicits antitumor immunity and protects from tumor challenge in the P815 mouse mastocytoma tumor model.

A novel membrane-bound form of GM-CSF (mbGM-CSF) was expressed on the surface of the mouse mastocytoma cell line P815 to target tumor cell-associated Ags to epidermal Langerhans cells. Transfected clones stimulated the proliferation of syngeneic bone marrow cells, indicating that mbGM-CSF is biologically active. We evaluated the in vivo effects of mbGM-CSF by comparing the growth of mbGM-CSF cells (termed 1D6.1E5) to that of wild-type P815 cells in DBA/2 mice. The growth rates of tumors initiated by P815 and 1D6.1E5 were similar until day 12, after which P815 tumors grew to large sizes while 1D6. 1E5 tumors were rejected. In contrast, the growth of both tumors was unimpeded when injected into nude mice, suggesting that a T cell-dependent antitumor response was induced by 1D6.1E5 in normal mice. Lymphocytes from 1D6.1E5-vaccinated mice were able to kill 51Cr-labeled P815 cells in a dose-dependent fashion that was inhibited by anti-CD8 Abs, suggesting that the antitumor response involved CD8+ CTL. We then tested whether vaccination with these cells would elicit a protective antitumor response by injecting mice with either irradiated 1D6.1E5 or P815 cells and challenging them with nonirradiated P815 cells. 1D6.1E5-treated mice grew small tumors that soon disappeared in all animals. In contrast, the majority of animals receiving the irradiated wild-type tumor vaccine grew large tumors, and 50% died. These data demonstrate that mbGM-CSF expressed on the surface of tumor cells is biologically active and elicits protective antitumor immunity.     

Distribution pattern and enzymic hypermethylation of inverted repetitive DNA sequences in P815 mastocytoma cells.

A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L-[Me-3H]methionine and [14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary intermediatry sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.