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.     

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.     

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.     

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.     

Natural occurrence of an inhibitor of mammalian cell growth in human and mouse cells of normal and tumor origin.

N6-(delta2-isopentenyl)adenosine was found both as a component of tRNA and as the cytoplasmic mononucleotide in human leukemic lymphoblasts and myeloblasts from peripheral blood and bone marrow samples. This hypermodified nucleotide was also found in the tRNA and as a mononucleotide in human (MRC-5 and KB) and mouse (A9, FLV, LM, and RAG) cell lines. The relative amounts of this hypermodified nucleotide in the tRNA of the cell lines and the human leukemias were similar (the mean value being 0.06 +/- 0.03 mole % of the total tRNA nucleotide content); whereas the amounts occurring as the free cytoplasmic mononucleotide were more varied but still comparable (the mean value being 0.53 +/- .09 mole % of all cytoplasmic nucleotides) for all cells investigated with the notable exception of all normal, diploid cell lines under study (0.04 mole%). A possible relationship of the free cytoplasmic mononucleotide with the nucleotide in the tRNA for control of mammalian cell protein synthesis in vivo was investigated by addition of N6-(delta2-isopentenyl)adenosine to the culture medium. The exogenously added nucleoside caused inhibition of cell growth within 3 h and cell death within 36 h at concentrations as low as 0.4 muM. No comparable effects were seen when adenosine, adenine, or N6-(delta2-isopentenyl)-adenine were added to the cultures. The simultaneous presence of adenosine in cultures containing N6-(delta2-isopentenyl)adenosine did not alter the detrimental effects of the hypermodified nucleoside on cell growth even when the concentration of adenosine was 50-fold that of N6-(delta2-isopentenyl)adenosine. Addition of N6-(delta2-isopentenyl)adenosine to cell cultures caused within the first 6 h a significant reduction in the rates of RNA and protein synthesis; whereas DNA synthesis continued at a rate comparable to control and adenosine-treated cells for 18 h before decreasing.     

In vivo hybridisation of cultured melanoma cells and isogenic normal mouse cells.

Somatic cell hybrids were derived by fusing tumourigenic and melanogenic melanoma (PAZG) cells with normal diploid male mouse cells in vivo. Their chromosomal composition was equivalent to the sum of both parental genomes and included a Y chromosome lacking in the melanoma parent. Our study showed that in PAZG X C57BL hybrids (MP), tumourigenicity was suppressed but pigmentation was expressed.     

Synthesis and secretion of transferrin by cultured mouse hepatoma cells.

The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [3H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.