Cytostatic effect of gangliosides present in the membrane of macrophages

Stimulated macrophages are known to inhibit the growth of certain tumor cells. Using mouse peritoneal exudates as a source of macrophages and the mastocytoma cell line P815 as the target, the inhibition was found to depend on direct contact between the macrophages and the growing cells. Cytostatic activities were detected in extracts of macrophages as well as in membranes of macrophages bound to substances of low molecular weight. Physical and biochemical characteristics of the cytostatic activity hint toward N-acetylneuraminic acid containing glycosphingolipids (gangliosides). The different macrophage gangliosides were separated by thin-layer chromatography. All types showed cytostatic activity, but the most effective gangliosides were identified as monosialoganglioside GM1 and disialoganglioside GD3.     

Oxygen consumption by tumor cells during membrane vesicle shedding

The incubation of mastocytoma P815 cells at low temperature (0 degrees C/1-2 hr), with a subsequent shift to greater than or equal to 20 degrees C results in the formation and shedding of membrane vesicles from the tumor cell surfaces. This process, when occurring at physiologic temperature (37 degrees C), mimics the morphological and membrane permeability changes occurring during T-lymphocyte mediated cytolysis of tumor cells. The latter is an oxygen dependent event, but it is not known whether this requirement is at the effector T cell or at the tumor cell level. The present study investigated the oxygen consumption rates of mastocytoma P815 cells induced to shed membrane vesicles by a temperature shift (0 degrees C/1-2 hrs----greater than or equal to 20 degrees C). Results showed that cells undergoing the membrane vesicle shedding process had significantly higher oxygen requirements than control non-shedding cells. Inhibition of the shedding process with deuterium oxide and hexylene glycol, reduced the oxygen consumption rates of low temperature treated cells to the level of control cells. The oxygen consumption rates of the latter were unaffected by these microtubule stabilizing agents. These data indicate that the oxygen required for immune T-cell mediated lysis of tumor cells may be at the target tumor cell level.     

Adherence of tumor cells to endothelial monolayers: inhibition by lymphokines

Tumor cells can adhere to endothelial cell monolayers in vitro. The kinetics of this reaction are rapid; 50% of maximal binding occurs by 30 min of incubation. In the case of the P815 mastocytoma, the maximal percentage of binding is approximately 70%, suggesting that there are both binding and nonbinding tumor cell populations. Binding is independent of tumor cell dose over a 200-fold range of cell concentrations. Lymphokine-containing preparations were found to markedly suppress the binding of either P815 mastocytoma or Ehrlich ascites cells to endothelium. This effect appeared to be due to both diminished attachment and enhanced dissociation. The activity is found in the same molecular weight range as tumor migration inhibition factor (TMIF), and is not found in preparations lacking TMIF activity. Thus, the factor may prove to be TMIF itself or a lymphokine related to it. Of equal interest is the possibility that it represents a previously undescribed factor.     

Intracamerally induced concomitant immunity: mice harboring progressively growing intraocular tumors are immune to spontaneous metastases and secondary tumor challenge

Intracameral inoculation of allogeneic P815 mastocytoma cells (DBA/2) into BALB/c mice resulted in progressively growing intraocular tumors. Intraocular tumor cells disseminated rapidly to the spleen and cervical lymph nodes, yet extraocular nests of tumor cells never developed into fulminant tumors. Further experiments showed that tumor cells were continuously seeded from the primary intraocular tumor and were rapidly cleared from extraocular sites. Hosts harboring intraocular P815 mastocytomas rejected tumorigenic doses of P815 cells inoculated subcutaneously or even into the contralateral anterior chamber. This systemic tumor immunity was found to be radiosensitive and T cell dependent. Spleen cells from animals with progressively growing intraocular tumors protected recipient mice challenged with intracamerally inoculated tumor cells and thus suggests that a cell-mediated mechanism is the underlying basis for this form of tumor immunity. The data indicate that mice harboring progressively growing intraocular tumors develop a potent state of "concomitant immunity," that prevents the development of metastases, yet is ineffective in controlling the primary tumor.     

The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

Pretreatment of P815 mastocytoma cells with inhibitors of protein synthesis reduces their susceptibility to lysis by cytotoxic thymus-derived lymphocytes

Protein synthesis inhibitors like cycloheximide, emetine, and puromycin diminish the ability of P815, a mastocytoma of DBA/2 mice, to react with anti-H-2^d cytotoxic thymus-derived lymphocytes (CTLs). Compared to untreated P815, tumor cells incubated with the protein synthesis inhibitors exhibited a reduced sensitivity to lysis and a reduced ability to inhibit lysis of untreated P815 cells. Consistent with this reduced reactivity of cycloheximide-treated P815 cells with CTLs was the inability of anti-H-2^d CTLs to form T cell-target cell conjugates with treated P815 cells. As evaluated by the binding of an anti-H-2^d serum, treated P815 cells expressed the same amount of H-2 membrane antigen as untreated cells. However, treated cells were still lysed by CTLs in the presence of the agglutinator, concanavalin A (Con A).     

GROWTH INHIBITION OF MURINE TUMOR CELLS, IN VITRO, BY PUROMYCIN, [6N]O2''-DIBUTYRYL 3'',5''-ADENOSINE MONOPHOSPHATE, OR ADENOSINE : Evidence of Commitment for Cell Division

The cytostatic effects of puromycm, [⁶N]O^2'-dibutyryl 3',5'-adenosine monophosphate, and adenosine on asynchronous and synchronous cultures of the murine mastocytoma, P815Y, have been studied. Cell growth was arrested after a minimum of one further division. A model is proposed for the inhibition of cell division in which the periods of inhibition and growth arrest are separated in time by one cell cycle.     

Activation of phospholipase hydrolysis in the process of oxidative cell damage]

Effect of tert-butyl hydroperoxide toxic action on phospholipase A2 activity and the changes in phospholipid composition from mastocytoma P815 cells were investigated. Oxidative damage of tumor cell membranes was accompanied by the release of arachidonic acid from membrane phospholipids and the accumulation of lysophosphatidylcholine, the product of phospholipase A2 reaction and a potent detergent. Tert-butyl hydroperoxide also increased relative contents of sphingomyelin, phosphatidylserine and phosphatidic acid in tumor cell membranes. It is possible that phospholipase A2 activation and the changes of phospholipid molecular species contents may cause the damage of cell membrane stability.     

The preparation of xenoantiserum specific for a murine mastocytoma cell line

Summary A method is described whereby a xenoantiserum directed toward membrane components of DBA/2 murine mastocytoma P815 cells was raised. This antiserum was found to be specific for tumor cell extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation. The antiserum was capable of killing P815 cells in the presence of guinea-pig complement but had no reactivity with L1210 leukemia cells or a variety of normal DBA/2 cell preparations. When mixed with varying numbers of tumor cells and injected IP to either DBA/2 or B6D2 Fl mice, the antiserum demonstrated a protective effect by either prolonging survival time or, when low numbers of cells were injected, apparently facilitating complete removal from the body of tumor cells. When administered following resection of SC grown tumors in B6D2 Fl mice, the antiserum prevented tumor recurrence in 50% of treated mice in comparison to animals treated with normal rabbit serum, in which recurrence occurred in all test animals.     

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.     

Tumor vaccine based on cell surface expression of DcR3/TR6

DcR3/TR6, a secreted protein belonging to the TNF receptor superfamily, interacts with lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entrance mediator (LIGHT), Fas ligand (FasL), and TL1A, all members of the TNF superfamily. Solid-phase TR6 can trigger reverse signaling of LIGHT and FasL expressed on T cells, and lead to T cell costimulation. In this study, we engineered tumor cells to express cell surface TR6 and used these cells as a tumor vaccine. We demonstrated that mastocytoma P815 cells expressing surface TR6 (TR6-P815) effectively augmented the T cells response in vitro and ex vivo in terms of proliferation, as well as IL-2 and IFN-gamma secretion. TR6-P815 cells had reduced tumorigenicity compared with parental P815 cells. When inactivated TR6-P815 cells were employed as a vaccine, they protected the mice from challenge with live parental P815 cells, and eliminated established P815 tumors. The cell surface TR6-based tumor vaccine was also effective against low antigenicity tumors, such as B16 melanoma; co-administration of bacillus Calmette-Guérin further enhanced the vaccine's efficacy. Thus, cell surface TR6 expression is a useful addition to our tumor vaccine arsenal.     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

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.     

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.     

Properties of syngeneic and allogeneic antisera raised to tumor-specific suppressor factor from DBA/2J mice

Summary Tumor-specific suppressor factor was prepared by injecting soluble membrane extracts of the syngeneic mastocytoma, P815, into DBA/2J mice 4 days prior to sacrifice. The suppressor factor was purified by passage of spleen extracts over an immunoadsorbent containing P815 membrane components. Antisera raised in syngeneic and allogeneic (C57Bl/6) mice by repeated injections of suppressor factor were tested. It was found that these antisera, but not their controls, were capable of absorbing out the suppressor factor. The antisera were also capable, in the presence of complement, of eliminating suppressor cells from suppressive spleen cell populations. However, the antisera were not capable of eliminating syngeneic tumor-specific in vitro-generated killer cells, indicating that the receptor molecules on suppressor and effector cells in this system are distinct from each other.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.     

Cellular damage and altered carbohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis

Treatment with direct electric current (DC) can inhibit tumor growth in several systems. To evaluate the cellular reactions generated by this treatment, we stimulated mouse mastocytoma P815 cells with DC and examined their viability and ultrastructural characteristics, as well as the effect of DC on surface carbohydrate expression. DC treatment affected cell viability and caused marked alterations in vital structures of P815 cells. Alterations varied depending on the duration of stimulation and polarity of electrode. Anodic and cathodic treatments caused decrease in cell viability, although the latter was more effective in generating cell lysis. DC stimulation also induced changes such as membrane damage, alterations in cell shape and chromatin organization, mitochondrial swelling and condensation, cytoplasmic swelling, and matrix rarefaction. Stimulation of P815 cells without contact with electrodes produced no alterations, suggesting that this contact might be essential for the occurrence of the cellular modifications. DC treatment also altered the membrane distribution of anionic sites of P815 cells, as well as the surface carbohydrate exposition, involving a diminished binding of Concanavalin A to the cell surface after cathodic stimulation, and an increased binding of sialic acid- and fucose-specific lectins after anodic treatment. In this work we describe important cellular targets for the action of DC, which may contribute to the understanding of the mechanisms by which DC supresses several kinds of tumors.     

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.