Secondary induction of cytotoxic T lymphocytes with solubilized syngeneic tumor cell plasma membranes.

Secondary induction of in vitro cytotoxic T lymphocytes in a syngeneic system has been achieved with plasma membrane, both in the particulate and solubilized forms. Both the induction and the lytic phases were shown to be immunologically specific. The effector cells generated were completely susceptible to treatment with anti-theta antibody and complement, suggesting that they are T lymphocytes.

Proliferation of mitochondria during the cell cycle of the human cell line (HL-60).

Using rhodamine 123 to stain mitochondria of the human cell line HL-60, we have followed their increase over the cell cycle by flow cytometry. A near-linear synthesis of mitochondrial mass was shown to occur over the cell cycle. A comparison with the cell's DNA synthesis pattern obtained by the same technique established a common time-base. The mitochondrial synthesis curve changes with culture age. As a control, thd dye was tested for its binding specificity and for its use to resolve mitochondria microscopically. Its stoichiometric range was established and, above 0.25 microgram/ml, it was shown to reduce growth rate and cell viability in culture.

Quantitative reflection contrast microscopy of living cells.

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.

Contact-induced redistribution of specific membrane components: local accumulation and development of adhesion.

We have used a model system to explore the importance of long-range lateral diffusion of membrane proteins in specific membrane-membrane adhesion. Single, cell-size phospholipid vesicles containing a dinitrophenyl (DNP)-lipid hapten were maneuvered into contact with rat basophilic leukemia (RBL) cells carrying fluorescent anti-DNP IgE in their cell-surface Fc epsilon receptors. Upon cell-vesicle contact the antibody molecules underwent a marked lateral redistribution, accumulating at the site of contact and becoming significantly depleted from noncontacting membrane. As assayed with a micropipette suction method, there was a time-dependent increase in the strength of cell-vesicle adhesion. This development of adhesion paralleled the kinetics of accumulation of the adhesion-mediating antibody molecules at the zone of membrane-membrane contact. Both adhesion and redistribution were absolutely dependent upon a specific interaction of the IgE with the hapten: No redistribution occurred when vesicles lacking the DNP hapten were pushed against IgE-armed RBL cells, and on cells bearing a 1:1 mixture of nonimmune rat IgE and anti-DNP mouse IgE, only the latter underwent redistribution. Vesicles containing DNP-lipids bound to RBL cells carrying anti-DNP IgE but not to cells carrying nonimmune rat IgE. Measurable nonspecific binding did not develop even after 15 min of pushing DNP-bearing vesicles against RBL cells sensitized with nonimmune IgE. Neither redistribution nor adhesion was blocked by metabolic poisons such as NaN3 and NaF. Both redistribution and adhesion occurred in plasma membrane blebs previously shown to lack cytoskeletal filaments. The above observations are consistent with contact-induced redistribution of the IgE being a result of passive diffusion-mediated trapping rather than active cellular responses. Thus, long-range diffusion of specific proteins can in some cases contribute to the formation of stable adhesion between membranes.

Antibody-induced modulation and shedding of mammary tumor virus antigens on the surfaces of GR ascites leukemia cells as compared with normal antigens.

The distribution, antibody-induced redistribution, and shedding of murine mammary tumor virus (MuMTV) antigens and the surfaces of GR mouse ascites leukemia (GRSL) cells were studied by the immunoferritin technique and compared with the same activities of thy 1.2 and H-2.8 antigens. MuMTV antigens were redistributed easily and then largely shed from the cell surface; in contrast, H-2.8 antigen moved easily and probably was partially released from the plasms membrane and Thy 1.2 antigen moved slowly and was some what interiorized. The complement-dependent cytotoxicity test was used to study the possibility of antigenic modulation for these cell-surface antigens from the surface of the GRSL cells could be modulated by preincubation with anti-MuMTV serum, in contrast to H-2.8 and Thy 1.2 antigens. The results obtained with the immunoferritin technique and the cytotoxicity test correlated well and sug-ested that the shedding of MuMTV antigens from the cell surfaces may occur in vivo, providing the tumor a way to escape from the immune defense of the host. Thy 1.2 and H-2.8 antigens were present on the envolope of B and C particles, which suggested that these viruses do not select a Thy 1.2 or H-2.8-negative area of the GRSL cell surface as amaturation site.

Redistribution and modulation of Gross murine leukemia virus antigens induced by specific antibodies.

Gross murine leukemia virus (G-MuLV)-induced rat leukemia cells in tissue culture replicate G-MuLV, express strong virus-associated membrane antigenicity, and are consistently killed by specific antibodies and complement in cytotoxicity tests. To explore the effect of specific antibodies, rat anti-G-MuLV antisera were added to the cultures of leukemia cells for variable periods of time. Redistribution of virus particles as well as of membrane virus antigens in the form of polar patches and caps was observed by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy. Substantial decreases in cytotoxicity indexes accompanied these changes. The antigen modulation induced by anti-G-MuLV antibodies in vitro paralleled similar changes obtained in vivo by transplanttion of leukemia cells in rats with high anti-G-MuLV antibody titers. The importance of antigen modulation in this system resides in its direct relationship with the malignant potential of the leukemia cells.

Ultrastructural study of liver sinusoids of mice during invasion by leukemic myelocytes.

Results of an ultrastructural study of liver of RF mice during invasion by leukemic myelocytes were reported. In early stages of infiltration, leukemia cells adhered to the endothelial cells of the sinusoidal wall; gaps 1-4 mu in diameter then developed in the endothelium, and leukemia cells passed through the gaps to enter the extravascular space. Other sinusoids became occluded by leukemic myelocytes, the endotheilium disintegrated, and the occluding cells thus became extravascular. In late stages of infiltration, when leukemia cells migrated back into the sinusoids, the endothelium was continuous and leukemia cells passed through temporary pores located within endothelial cells.

Uropod-bearing lymphocytes (hand mirror cells) in a virus-induced murine lymphoma.

A Moloney murine leukemia virus-induced T-cell preleukemic thymic lymphoma tissue culture from an inbred C3H/HeJ mouse contained numerous hand mirror cells (HMC). The cells were studied by light and phase-contrast microscopy, special stains, indirect immunofluorescence for terminal deoxynucleotidyl transferase, and scanning and transmission electron microscopy. The uropods of the mouse and human HMC were similar. In contrast, viruses were noted on the tip of the mouse HMC uropod by transmission electron microscopy. These observations, reported for the first time in an animal model, will enable investigators to study the HMC under controlled conditions.

Quantitation of tumor antigen expression on the surface of malignant line Ib lymphocytes by immuno-electron microscopy.

Malignant line Ib lymphocytes obtained from strain C58/J mice moribund with the syngeneic, transplantable leukemia were examined by transmission electron microscopy. A large, multilobulated nucleus containing a prominent nucleolus was typically viewed. In addition to normal organelles, cytoplasmic elements included frequent intracisternal A-type virions. The cell membrane possessed pseudopodia, and occasionally C-type viruses were noted budding from the plasma membrane. On the basis of complement-mediated antibody cytotoxicity assays with the use of either antiserum against mouse IgM, antiserum against mouse IgG, or antiserum against Thy 1.2 antigen, Ib cells were characterized as T-cells. Similarly, leukemia lymphocytes were lysed by a highly specific rabbit anti-Ib tumor-associated surface antigen (TASA) in the presence of complement. The density and distribution of TASA on malignant lymphocytes were analyzed by immuno-electron microscopy with the use of a bridging technique in conjunction with antiserum against Ib TASA. The following observations were reported after the examination of more than 100 randomly selected Ib lymphocytes: a) The density of the TASA was low, b) the distribution of the TASA was random, and c) no evidence indicated cell cycle-dependent expression of the TASA.

Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. I. High-yield purification of plasma membrane fragments.

In this first paper of a series comparing the membranes of normal lymphocyte populations from male outbred Syrian hamsters with those of neoplastic transformants (GD 248) induced by simian virus 40, a method is described for the isolation of representative plasma membrane (PM) fragments from both cell types. Multiple criteria were used to monitor the purity and yield of PM material after cell disruption by nitrogen cavitation and after membrane fractionation by a combination of differential centrifugation and isopyknic ultracentrifugation in dextran density gradients. Lactoperoxidase-catalyzed radioiodination before cell disruption was used as an extrinsic surface marker; Na+,K+-activated ATPase, as well as alkaline phosphatase, was used as intrinsic functional PM markers. The distribution of nuclei, mitochondria, lysosomes, and endoplasmic reticulum (ER) during fractionation was monitored by the measurement of DNA, succinate dehydrogenase and monoamine oxidase, beta-glucuronidase and glucose-6-phosphatase, and NADH:lipoamide oxidoreductase, respectively. According to the three PM markers employed, a 15- to 20-fold purification (over homogenate) and a PM yield of about 65% were obtained for both cell categories, with negligible contamination by DNA, mitochondria, lysosomes, and er. The procedure also allowed recovery of 60% of the mitochondria free of other cell elements.

Cell cycle-related morphological changes of feline lymphoid cells as revealed by electron microscopy.

The aim of this study was to correlate morphological changes in cells to known phases of the cell cycle. For this purpose, feline lymphoblastoid cells (FL74) chronically infected with feline leukemia virus were synchronized and examined by scanning and transmission electron microscopy. Scanning electron microscopy revealed that the cell surface underwent distinct changes which were found to be cell cycle specific. Morphological changes shown by transmission electron microscopy were used as ultrastructural markers to confirm the findings made by scanning electron microscopy relating to the various phases of the cell cycle. The results of these experiments suggest that the presence of morphologically mixed populations of cells in many lymphoproliferative disorders, which had been described previously, may be explained by normal cell cycle-dependent variations.

Ultrastructural, cell membrane, and cytogenetic characteristics of B-cell leukemia, a murine model of chronic lymphocytic leukemia.

A murine model of a spontaneous, transplantable BALB/c B-cell leukemia (BCL1) is described. Extreme leukemia and splenomegaly develop in H-2d-compatible recipients of tumor cells. Tumor cells are medium to large lymphocytes that can be transformed into plasmacytoid cells following in vitro stimulation with lipopolysaccharide. Karyotypic analysis of transformed tumor cells reveals 36 chromosomes with several monosomies and 7 markers chromosomes. The ultrastructure of the tumor cells was studied using transmission and scanning electron microscopy. Although the appearance of tumor cells seems normal by morphological criteria, an impaired capping ability was documented using the fluorescein-conjugated concanavalin A-binding test. Impaired capping ability was documented before leukemia was overt as early as 1 to 3 days following inoculation of tumor cells. The B-cell leukemia (BCL1) provides a useful murine model for the study of various aspects of human bone marrow-derived malignant disorders.

Isolation of fluoropyrimidine-resistant murine leukemic cell lines by one-step mutation and selection.

The effectiveness of the clinically useful fluoropyrimidines in the treatment of human cancer is often limited by the development of resistance to the drugs by the tumor. In order to systematically study the mechanisms of resistance to 5-fluorouracil and its nucleoside derivatives, several cell lines resistant to these drugs have been derived from murine leukemia cells by a one-step mutation and selection procedure. Logarithmically growing suspension cultures of L1210 and P388 cells were treated with ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, or ICR-191 at concentrations which result in 20 to 30% cell survival. After a 10-day expression time, mutagenized cells were plated into soft-agarose medium that contained 10(-5) M 5-fluorouracil, 10(-5) M 5-fluoro-2'-deoxyuridine, or 10(-6) M 5-fluorouridine. Twenty stable clones were isolated and found to be 5- to 28-fold resistant to growth inhibition by 5-fluorouracil, 4,000- to 25,000-fold resistant to 5-fluoro-2'-deoxyuridine, or 8- to 220-fold resistant to 5-fluorouridine. The clones retain their drug-resistant phenotype after repeated passaging in the absence of selection. Since the biochemical changes responsible for resistance to one drug can render the cells collaterally sensitive to other drugs, the growth-inhibitory effects of antimetabolites that inhibit other steps in pyrimidine metabolism were examined in the wild-type cells and in the fluoropyrimidine-resistant sublines. Although cross-resistance to 5-azacytidine was found in L1210 cells selected for resistance to 5-fluorouridine, none of the cell lines tested demonstrated collateral sensitivity to methotrexate, 1-beta-D-arabinofuranosylcytosine, 5-azacytidine, or N-(phosphonacetyl)-L-aspartate.

Isolation and characterization of plasma membranes from Friend erythroleukaemic cells. A study with sphingomyelinase C.

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (I) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6-7% of phospholipid, 8-9% of cholesterol and 12-15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15-20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C (S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes.

EL4 cels were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membranes phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the P value of both probes compared to the P value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, P values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in P values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization from monitoring physical properties of membranes (such as 'fluidity') is discussed.

Fluorescence polarization measurements on normal and tumour cells and their corresponding plasma membranes.

Using 1,6-diphenyl-1,3,5-hexatriene as a probe, the degree of fluorescence polarization (P) at 25 degrees C of intact and disrupted cells and isolated plasma membranes were compared for a variety of systems. 1. Human erythrocytes, mouse thymocyte and leukemia cells, rat liver and hepatoma cells, and human and mouse milk fat globules displayed P values ranging from 0.300 to 0.120. 2. P values or probe labelling rates of intact and disrupted cells were similar. 3. As compared with whole or disrupted cells, the higher to much higher P values of plasma membranes isolated from the corresponding cells showed only a limited mutual variation. 4. delta P values, being the difference in P values between plasma membranes and whole cells were attributed to the extent to which endomembranes and non-membrane lipids contributed. Among these, triglycerides had the greatest relative effect. 5. Though a particular isolation procedure for plasma membranes may select for more rigid fragments, this effect is by far not sufficient to account for the observed delta P values. It is concluded that the fluorescence polarization technique with a lipophilic probe applied to whole cells represents a measure of the average fluidity of all lipids being present in a cell and thus does not exclusively monitor the cell surface membrane.

Morphology of lysosomes in different phases of growth of lymphoblasts of L5178Y murine leukemia.

Parallel studies were carried out on the morphology of lysosomes, activity of DNA synthesis measured by the rate of 3 H-thymidine incorporation, and proliferative activity on the basis of values of the mitotic index of leukemia L5178Y lymphoblasts under varying growth conditions. Morphology of lysosomes was studied by the method of intravital staining with euchrysine. Observations were made on cells growing from the logarithmic phase to saturation density and synchronized by double blocking of DNA synthesis by hydroxyurea. The results showed some variation of the morphology of lysosomes related to growth phase of the cells.

AKR mouse leukemia cells in tissue culture. I. Establishment of leukemic cell line AKR 87 and its morphologic and biologic characteristics.

A line of AKR leukemic mouse cells cultured in vitro, designated AKR 87, was established and characterized. Two types of cells were distinguished in this line fibroblasts and lymphoblasts. The lymphoblast-like cells resembled the original tumor cells spontaneous leukemia of AKR mice. In the XC test, cells of this line reacted specifically with XC cells, forming syncytia characteristic of cells infected with mouse leukemia viruses. Ultrathin sections of cells of this line examined on the electron microscope showed presence of type C viral particles.

Effect of verapamil on rhodamine 123 mitochondrial damage in adriamycin resistant cells.

Mitochondrial damage was found in Friend leukemia cells treated with rhodamine 123 (Rho 123). In contrast, when cells resistant to the drug were similarly treated, mitochondria were unaffected. These results correlated with higher levels of Rho 123 in sensitive as compared to resistant cells. However, when resistant cells were co-treated with verapamil, intracellular Rho 123 levels reached those of sensitive cells. At these levels mitochondrial damage and subsequent cytotoxicity in resistant cells were the same as in sensitive cells. These data suggest that differences in Rho 123 mitochondrial damage and subsequent cytotoxicity in sensitive and resistant cells result entirely from increased intracellular drug levels and not from differences in mitochondrial sensitivity or other mechanisms.

Antitumor agents. XXXV: Effects of brusatol, bruceoside A, and bruceantin on P-388 lymphocytic leukemia cell respiration.

Brusatol, a quassinoid with potent antineoplastic activity against P-388 lymphocytic leukemia cell proliferation, significantly inhibited P-388 cell hexokinase, phosphofructokinase, malic dehydrogenase, and succinic dehydrogenase. Mitochondrial oxidative phosphorylation, basal, and adenosine diphosphate-stimulated respiration, utilizing succinate and alpha-ketoglutarate as the substrate, was suppressed significantly by in vivo treatment with brusatol. However, brusatol treatment had no effect on liver oxidative phosphorylation. Brusatol greatly increased P-388 cyclic AMP levels but had no effect on liver cyclic nucleotides. Similar inhibitory effects on P-388 cell oxidative phosphorylation were found in vitro with brusatol, bruceoside A, and bruceantin. Brusatol had no effect on adenosine triphosphatase activity or on uncoupling of oxidative phosphorylation. Rather, brusatol appeared to increase the concentration of reduced mitochondrial electron-transport cofactors, thereby blocking aerobic respiration. A proposed mechanism of action is discussed.