To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, [(3)H] labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C(8), C(12), C(14), C(16), C(18), C(22), C(24), C(18:1), C(22:1), or C(24:1) fatty acids (FAs), or C20 sphingosine acetylated or acylated with C(8) or C(18) FA were prepared and added to culture media. GM1s uptake by HL-60 cells was affected by the structure of their ceramides. Resistance to removal with trypsin and the stoichiometry of [(125)I] cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides. The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion. The GM1s with sphingosine acylated with C(14), C(16), C(18) C(22), or C(24) FAs were similarly abundant in DRMs. GM1s acylated with C(18:1), C(22:1), or C(24:1) were less abundant than those acylated with saturated FA of the same length. GM1s acetylated or acylated with C(8) FA were detected in DRMs in the lowest proportion. Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GM1s acetylated or acylated with C(8) or unsaturated FAs but not of GM1 acylated with C(18), C(22), or C(24) FAs. After cross-linking with CT B subunit, all GM1s were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion. DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions. The GM1s with sphingosine acetylated or acylated with C(18) or C(18:1) FAs occurred in these fractions in different proportions.

An experimental method of measuring intercellular adhesion based on cell interactions in Poiseuille flow is presented. Hydrodynamic conditions of suspension flow, concentration of the suspension, and the size of cells allows determination of the capture efficiency, which is a measure of cell adhesiveness. The measurements of cell aggregation were performed with thymus cells and lectin from Ricinus communis. On the ground of theoretical description of the process of cell aggregation, it was possible to estimate the diffusion constant of the receptors in the cell membrane, which is equal to 5.6 X 10(-11) cm2/s. This value is in good agreement with the results of direct measurements of the diffusion constant of the lectin receptor. In the case of formalin-treated cells, the diffusion constant of the lectin receptors is equal to 6.8 X 10(-12) cm2/s. The total number of the receptor sites on the thymus cell surface for lectin R. communis and the affinity constant were also estimated.

Mouse lymphatic leukemia L1210 cells were characterized with polyclonal and monoclonal antibodies and lectins. The cells were found to have the phenotype IgG-, Thy-1.2-, Lyt-1-, Lyt-2-, asialo-GM1-, TL-, I-Ad-, IL-2R+, peanut agglutinin+, and Helix pomatia lectin +/-. They retained expression of H-2Kd and H-2Dd. Thus, these cells resemble "null" cells.

Lymphatic leukemia L 1210 cells were treated in vitro with various concentrations of Mafosfamide--a stabilized active derivative of cyclophosphamide (4-hydroxycyclophosphamide). L 1210 cells treated with Mafosfamide (L 1210-MAF cells) were used for vaccination of semisyngeneic CD2F1 mice against L 1210 leukemia. These cells do not grow in vivo but are viable in the test with trypan blue. L 1210-MAF cells, obtained by treatment of L 1210 cells two times with 50 micrograms/ml or 100 micrograms/ml of Mafosfamide, and injected into the mice induced resistance against L 1210 leukemia in these animals. L 1210 cells treated two times with higher concentration of Mafosfamide (200 micrograms/ml or 400 micrograms/ml) did not give this effect.

Balb/c x DBA/2 F1 mice (CD2F1 mice) bearing L1210 lymphatic (10 L1210 cells i.p. injected on day 0) were subjected to chemoimmunotherapy. They received 100 mg/kg of cyclophosphamide i.p. on day +8 and 10(6) or 10(7) immunogenic L1210 cells treated in vitro with mafosfamide - ASTA Z7654 (L1210-Maf cells) i.p. or i.p. + s.c. on days 0, +3, +6, +9, +12 after the leukemia implantation. About 30% of leukemia-bearing mice receiving cyclophosphamide and L1210-Maf cells after L1210 inoculation were able to reject the leukemia (as compared with 0% after injection of L1210-Maf cells only or 5% after cyclophosphamide administration). Better results (54% of cured mice) were obtained if 10(7) L1210-Maf cells were injected i.p. +s.c. beside cyclophosphamide. Biological response modifiers (BRM's): levamisole, BCG, bestatin did not improve these results in the doses used in the experiment. Augmentation of anti-L1210 therapeutic response is dependent on the administration of cyclophosphamide and L1210-Maf cels. Cyclophosphamide not only reduces the tumor burden but probably can potentiate the L1210-Maf dependent antitumor immunity as well.