Anomalous reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leukemia viruses.

Certain alloantisera prepared in mice against H-2 region membrane antigens were found to be unexpectedly cytotoxic for murine sarcoma and leukemia cells in culture. This anomalous cytotoxicity was shown to be the result of antibody in these alloantisera directed against the p15 and gp70 envelope proteins of Mu LV which were present on the surface of the tumor target cells. Sera from aged unimmunized mice of strains used for the preparation of alloantisera also contained antibodies against MuLV protein p15 and gp70 that were cytotoxic for sarcoma and leukemia cells, which indicates that these antibodies occurred naturally in mice. These results independently confirm earlier findings of the widespread occurrence in mouse serum of antibodies reactive with MuLV. The presence of antibody against MuLV in mouse serum which can cause cytotoxic reactions with tumor cells points to the fact that particular caution should be used during the typing of murine sarcomas or leukemias for cell surface antigens, since mouse antisera may yield cytotoxicity (or other serologic reactions) based on anti-MuLV specificities, rather than on anticipated antigens.     

125I-fibrin deposition in IgE-dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells

We investigated the clotting associated with IgE-dependent immediate hypersensitivity reactions in the mouse by injecting monoclonal mouse anti-dintrophenyl IgE antibodies i.d. and, the next day, administering 125I-guinea pig fibrinogen i.v. 10 to 30 min before i.v. antigen (2,4-dinitrophenylated human serum albumin) challenge. In normal mice, 2-hr passive cutaneous anaphylaxis (PCA) reactions were associated with substantial leakage of 125I-fibrinogen and deposition of 125I-fibrin. Thus, ears injected with IgE contained up to six times the total cpm of 125I and up to 30 times the cross-linked 125I-fibrin-associated cpm of 125I than did control ears. Several lines of evidence indicated that the 125I-fibrin deposition associated with the PCA reactions was dependent on the activity of mast cells: 1) Mast cell degranulation occurred at sites of PCA reactions. 2) Antigen-induced influx of 125I-fibrinogen and deposition of 125I-fibrin were virtually abolished by heating the IgE (56 degrees C, 1 hr) before i.d. injection. 3) Little or no IgE-dependent 125I-fibrinogen influx or 125I-fibrin deposition occurred in mast cell-deficient WBB6F1-W/Wv or WCB6F1-S1/S1d mice X 4) Adoptive transfer of cutaneous mast cell populations into WBB6F1-W/Wv mice (by each of three approaches: i.v. transplantation of normal bone marrow cells or local i.d. injection of cultured, growth factor-dependent mast cells 2 days or 9 to 10 wk before antigen challenge) conferred on the recipients the ability to express the 125I-fibrinogen influx and 125I-fibrin deposition associated with PCA reactions. These data demonstrate that 125I-fibrinogen influx and 125I-fibrin deposition occurs in association with PCA reactions in the mouse, and that the reaction is largely or entirely dependent on the function of cutaneous mast cells. The experiments also demonstrate the utility of a novel model system for the analysis of mast cell function in vivo: WBB6F1-W/Wv mice locally reconstituted with mast cells by the injection of mast cell populations generated in vitro.     

Infection of cultured rat hepatoma cells by mouse mammary tumor virus.

A continuous line of buffalo rat hepatoma (HTC) cells has been successfully infected with mouse mammary tumor virus (MMTV) produced by the GR mammary tumor cell line. Uniform infection required initial exposure of the HTC cells to greater than 10(5) MMTV particles per cell. The resultant chronically infected cell population was found to have stably acquired 20-30 copies of MMTV DNA. The infected cells contain viral RNA and express viral antigens; however, very few MMTV particles are released into the culture medium. In spite of the biochemical evidence for infection, we have not detected any alterations in the morphology or growth properties of the infected HTC cells. As is the case in mammary tumor cells, the intracellular concentration of viral RNA is strongly stimulated (50-150 fold) by the synthetic glucorcorticoid, dexamethasone. Thus it appears that the mechanisms by which glucorticoids regulate MMTV gene expression in mouse cells are maintained when this virus infects nonmurine cells.     

Intraspecies identification of cultured murine cells by using H-2 antigen typing

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.     

Phenotypic stability of mouse mammary tumor cells cultured on collagen gels

Summary This study demonstrates that phenotypic characteristics of androgen-responsive (AR) Shionogi mouse mammary tumors and androgen-independent (AI) derivatives can be maintained in culture. Cells were seeded onto collagen gels in medium containing 2% dextran-characoal-treated fetal bovine serum with or without 0.01 μg/ml dihydrotestosterone (DHT). Androgen-responsive tumors grew more rapidly than AI tumors in vivo and consequently, cells from AR tumors cultured in DHT-containing medium grew faster than cells in DHT-deprived medium and cells from AI tumors. Androgen-responsive tumors had a sheetlike growth pattern; AI tumors formed clumps or irregular cords of cells. Cells from AR tumors cultured in the presence of DHT formed confluent pavements, whereas cells maintained in the absence of DHT and cells from AI tumors formed clusters or cords of cells. Ultrastructurally, cells of AR tumors were elongated; cells of AI tumors were smaller and rounder. These cellular morphologies persisted in culture. Tumorigenicity of cells was assayed by injecting cells s.c. into host mice. Tumors arising from cells of freshly dissociated AR tumors and cells of AR tumors cultured in the presence of DHT appeared more rapidly and grew faster in intact males than in castrated males and intact females. Tumors arising from cells cultured in the absence of DHT and from freshly dissociated or cultured cells of AI tumors had identical rates of appearance and growth in all hosts. This culture system permits these cells to retain their state of malignant progression in vitro and should be a useful model for studying the origin of heterogeneity within tumors and its role in tumor behavior. This work was supported by a grant from the National Cancer Institute of Canada. J. T. E. is a Research Scholar of the National Cancer Institute of Canada.     

Migration capacity of tumor cells treated with immune sera

The action of antitumour sera on the adhesive properties of the L-cells and the cells of Ehrlich's ascitic carcinoma. It was shown that after the treatment with immune sera the tumour cells lost their capacity to form rosettes with sheep red blood cells, to adhere to plastics and glass; when administered into the mouse organism these cells were worse retained in the lungs, spleen and the liver. The data obtained indicated that the antibodies could play a significant role in the tumour cell metastasis.     

In vitro reaggregation of dissociated mouse cerebellar cells : I. Demonstration of different aggregation mechanisms

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca²⁺ concentrations, T and MT cells aggregated better at high Ca²⁺ concentration. Their aggregation rates were reduced by approx. 50% at low Ca²⁺ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca²⁺ concentration, being half maximal at approx. 0.1 mM Ca²⁺.The ability of M cells to aggregate at low or high Ca²⁺ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca²⁺ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca²⁺ concentrations were reduced to 35%. Variation of the Ca²⁺ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca²⁺-independent binding mechanism, whereas T or MT cells aggregate using a Ca²⁺-dependent one which may be functionally silent in M cells.     

Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells.

Using the S49 T-cell lymphoma system for the study of immunodeficiency diseases, we characterized several variants in purine salvage and transport pathways and studied their responses to the cytotoxic action of adenosine (5-20 micron) in the presence of adenosine deaminase (ADA) inhibitors. Both an adenosine transport deficient mutant and a mutant lacking adenosine (ado) kinase activity are resistant to the cytotoxic effects of adenosine up to 15 micron. Variants lacking hypoxanthine-guanine phosphoribosyl transferase or adenine phosphoribosyltransferase are sensitive to the killing action of adenosine. We monitored the intracellular concentrations of purine and pyrimidine nucleotides, orotate, and PPriboseP in mutant and wild-type cells following the addition of adenosine and an ADA inhibitor. We conclude that at low concentrations, adenosine must be phosphorylated to deplete the cell of pyrimidine nucleotides and PPriboseP and to promote the accumulation of orotate. These alterations account for one mechanism of adenosine toxicity.     

The occurrence of the Golgi apparatus in mouse oocytes. Demonstration of thiamine pyrophosphatase activity

Location of thiamine pyrophosphatase activity as a marker of the Golgi apparatus was studied ultracytochemically in mouse oocytes with germinal vesicle (OGV), oocytes at metaphase I (OMI) and oocytes at metaphase II (OMII), and further in cells of the respective cumulus oophorus serving as comparative objects. TPPase activity in cumulus oophorus cells and in OGV was found exclusively in the Golgi apparatus. In OMI the reaction product of TPPase activity was observed in isolated smooth vesicles, and in only one case in structures identifiable as the Golgi apparatus. In OMII the occurrence of TPPase activity was also recorded in isolated smooth vesicles in cortical cytoplasm and further, exceptionally, in smooth concentrically arranged vesicles or tubules. The TPPase activity was not present in vesicular complexes. The results have shown that after the resumption of meiosis the occurrence of the reaction product of TPPase activity drops abruptly due to the reduction of the Golgi apparatus. Changes affecting the Golgi apparatus after the resumption of meiosis are related to the loss of the nucleus after the germinal vesicle breakdown.     

Human monocyte-mediated tumor cytotoxicity. I. Demonstration of an oxygen-dependent myeloperoxidase-independent mechanism

The cytolytic capacity of monocytes per se and stimulated monocytes has been documented to only a limited extent, and when observed has been ascribed to the generation of a variety of cytolytic molecular entities. In the present study we have examined de novo human monocyte-mediated tumor cytotoxicity and that induced by the agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Cytolytic function was analyzed by reference to the release of [111In] oxine from two prelabeled tumor cell lines, K562 and U937, in a 16-hr assay in the presence of serum to more closely mimic in vivo circumstances. Observed cytolysis was clearly related to TPA concentration and effector cell number. Maximal cytolysis was obtained with TPA at 5 ng/ml, at which specific releases were 43% +/- 6 and 18% +/- 5 (mean +/- 1 SEM) at an effector cell to target cell (E:T) ratio of 2.5:1 and 65% +/- 6, and 41% +/- 12 at an E:T ratio of 20:1, for K562 and U937, respectively. In contrast, unstimulated monocytes expressed minimal cytolytic activity, or at best a low cytotoxic effect at high cellular ratios. When TPA-stimulated monocyte-mediated cytolysis was examined, catalase (2750 U/ml) inhibited K562 and U937 cytolysis by 92% and 84%, respectively; superoxide dismutase (300 U/ml) only inhibited cytotoxicity by 17% and 24%, respectively, implicating a central role of H2O2 rather than superoxide ions. Sodium azide (1 mM), an inhibitor of myeloperoxidase, did not diminish cytolysis; in contrast, it increased K562 and U937 cytolysis by 34% and 57%. This increased cytotoxicity was observed for K562 at low levels of cytotoxicity. These data tend to dismiss an essential role of the H2O2-halide-myeloperoxidase pathway of cytolysis. The OH scavengers, histidine (20 mM) and ethanol (40 mM), did not affect K562 killing; mannitol (50 mM), another OH scavenger, had only a slight inhibitory effect (23%). Finally, H2O2 generated by a glucose-glucose oxidase system directly mediated K562 killing and, to a lesser extent, U937 lysis. These results point strongly towards the role of: 1) a myeloperoxidase-independent mechanism of cytotoxicity, with 2) H2O2 as a key mediator of the cytolytic mechanism, and 3) a limited role of O2.- in synergy with H2O2 in the cytolytic activity of monocytes, and suggest that significant cytolytic function requires an inductive event.     

Induction by synthetic polyribonucleotide poly(I) of differentiation of cultured mouse myeloid leukemic cells.

The effects of some synthetic polyribonucleotides on induction of differentiation of mouse myeloid leukemic M1 cells were examined. Poly(I) was found to be a potent inducer; on treatment with 100--200 microgram/ml of poly(I) for 2--4 days, M1 cells differentiated into cells resembling macrophages and granulocytes and developed phagocytosis and locomotive activities, Fc receptors and lysozyme activity. Poly(C) was less effective than poly(I) for induction of phagocytic activity, while the other single-stranded RNAs, poly(U) and poly(A), had no effect. Double-stranded RNAs, such as poly(I) . poly(C) and poly(A) . poly(U), were cytotoxic to M1 cells, and differentiation of the cells could not be detected even at the highest tolerable concentrations of these double-stranded RNAs.     

Interaction of phospholipid vesicles with cultured mammalial cells. I. Characteristics of uptake

The interaction of monolayer cultures of Chinese hamster V79 cells with artificially generated, unilamellar lipid vesicles (approximately 500 A diameter) was examined. Vesicles prepared from a variety of natural and synthetic radiolabeled phosphatidyl cholines (lecithins) were incubated with V79 cells bathed in a simple balanced salt solution. After incubation, the cells were analyzed for exogenous lipid incorporation. Large quantities (approximately 10(8) molecules/cell/h) of lecithin became cell associated without affecting cell viability. The effects of pH, charged lipids, and the influence of the vesicle lipid phase transition on the uptake process were examined. Glutaraldehyde fixation of cells before vesicle treatment, or incubation in the presence of metabolic inhibitors, failed to reduce the lecithin uptake by more than 25-50%, suggesting that the lipid uptake is largely energy independent. Cells in sparse culture took up about ten times more lipid than dense cultures. Prolonged incubation (greater than 15 h) of sparse cell cultures with lecithin vesicles resulted in significant cell death while no deleterious effect was found in dense cultures, or with 1:1 lecithin/cholesterol vesicles. When vesicle-treated cells were homogenized and fractionated, about 20-30% of the exogenous lipid was found in the plasma membrane fraction, with the remainder being distributed into intracellular fractions. Electron microscope radioautography further demonstrated that most of the internalized lipid was present in the cytoplasm, with little in the nucleus. These results are discussed in terms of possible modification of cell behavior by lipid vesicle treatment.     

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.