Intracellular distribution of N4-behenoyl-1-beta-D-arabinofuranosylcytosine in blood cells.

In order to clarify differences in mode of action between N4-behenoyl-1-beta-D-arabinofuranosylcytosine (behenoyl-ara-C) and 1-beta-D-arabinofuranosylcytosine (ara-C), comparative studies on both agents were undertaken. When human erythrocytes incubated with behenoyl-ara-C[acyl-1-14C] were fractionated into stroma and stroma-free lysate, a marked accumulation of radioactivity in stroma was observed. In contrast, ara-C[cytosine-2-14C] was rapidly incorporated into the stroma-free lysate. Thin-layer chromatography of the extracts of L1210 cells incubated with behenoyl-ara-C[acyl-1-14C] or behenoyl-ara-C[cytosine-2-14C] at 37 degrees for 60 min revealed that most of the incorporated drug remained as unmetabolized behenoyl-ara-C. After incubation of 20 microM behenoyl-ara-C or ara-C with L1210 cells at 37 degrees for 60 min, subcellular fractionation of the cell suspension was performed; behenoyl-ara-C was accumulated markedly in the membrane, mitochondria and microsome fractions. In contrast, most of the ara-C was found in the 105,000g supernatant fraction. The accumulation of behenoyl-ara-C in membrane structures may result from the lipophilic nature of the agent, which may have a prolonged inhibitory action on leukemic cell proliferation.

Immobilized adriamycin: a tool for separating cell surface from intracellular mechanisms.

Pharmacologic agents may exert their biological activity at the level of the cell membrane. Of particular interest is the anticancer agent adriamycin. This drug has previously been considered to act by intercalation with nuclear DNA, but recent evidence suggests the possibility that the cell surface membrane may represent an alternative target. To test this hypothesis, adriamycin was attached to insoluble supports, and conditions suggesting that the drug was actively cytotoxic without entering cells were demonstrated.

Ultrastructural changes in the mitochondria of intestinal epithelium of rodents treated with methylglyoxal-bis(guanylhydrazone).

Ultrastructural studies of rats or mice treated for 24 hr with a toxic dose (100 mg/kg) of methylglyoxal-bis(guanylhydrazone) revealed the presence of damaged mitochondria in the crypt cells of the intestinal epithelium. Mitochondria were severely swollen and electron lucent, and appeared to be similar to those observed previously in a variety of cell types treated in vitro and in vivo with methylglyoxal-bis(guanylhydrazone). Since thymidine incorporation into the intestine was not found to be decreased until after 24 hr, it is concluded that the mitochondrial damage of methylglyoxal-bis(guanylhydrazone) could be responsible for the antiproliferative toxicities of the drug.

An inhibitor of (Na+, K+)-ATPase produced by Streptomyces pseudovenezuelae MF722-02; purification and properties.

A culture product of Streptomyces pseudovenezuelae MF722-02, with a molecular formula of C29H32N2O7, was isolated as yellow needles from culture broths and mycelia of the organism by means of a series of solvent extraction, column chromatography and crystallization. The antibiotic is active against some Gram-positive bacteria, inhibits growth in vitro of cells of mouse leukemia L-1210, prolongs the life span of mice inoculated with the leukemia cells, enhances deoxycholate-induced hemolysis in vitro and inhibits (Na+, K+)-ATPase in vitro.

Electron spin resonance studies on intact cells and isolated lipid droplets from fatty acid-modified L1210 murine leukemia.

It has been suggested that the formation of cytoplasmic lipid droplets may produce an artifact and be responsible for the differences in membrane physical properties detected in lipid-modified cells using fluorescence polarization or spin label probes. To investigate this, the electron spin resonance spectra of lipid droplets isolated from the cytoplasm of L1210 leukemia cells were compared with spectra obtained from the intact cell. Mice bearing the L1210 leukemia were fed diets containing either 16% sunflower oil or 16% coconut oil in order to modify the fatty acid composition of the tumor. A microsome-rich fraction prepared from L1210 cells grown in animals fed the sunflower oil-rich diet contained more polyenoic fatty acids (52 versus 29%), while microsomes from L1210 cells grown in animals fed the coconut oil-rich diets contained more monoenoic fatty acids (37 versus 12%). The order parameter calculated for lipid droplets labeled with the 5-nitroxystearic acid spin probe was only about one-half that of intact cells, whereas it was similar to that obtained for pure triolein droplets suspended in buffer. Order parameters of the inner hyperfine splittings calculated from the spectra of cells grown in the sunflower oil-fed animals [0.543 +/- 0.001 (S.E.)] were lower than those from the cells grown in animals fed the coconut oil diets (0.555 +/- 0.002) (p less than 0.005). In contrast, the order parameters of the lipid droplets isolated from the cells grown in animals fed sunflower oil (0.303 +/- 0.029) or coconut oil (0.295 +/- 0.021) were not significantly different, indicating that motion of a spin label probe in the highly fluid cytoplasmic lipid droplets is not affected by these types of modifications in cellular fatty acid composition. Therefore, the electron spin resonance changes that are observed in the intact cells cannot be due to localization of the probe in cytoplasmic lipid droplets. These results support the conclusion that the electron spin resonance changes observed with the 5-nitroxystearic acid spin probe are due to changes in membrane fluidity produced by the modification in cellular lipid composition.

A comparison of the response to hyperthermia of murine haemopoietic stem cells (CFU-S) and L1210 leukaemia cells: enhanced killing of leukaemic cells in presence of normal marrow cells.

When the clonogenic survival of mouse haemopoietic stem cells (CFU-S) and leukaemia L1210 cells growth as ascites tumours are compared after being heated in vitro and assayed in vivo by spleen-colony assay, there is no significant difference in the terminal slopes of the survival curves. The shoulders of the survival curves differ, but this may be explained by differences in cell kinetics. By contrast, L1210 leukaemic marrow cells are considerably more susceptible to the lethal effects of hyperthermia (43 degrees C) than either normal marrow stem cells or L1210 leukaemic cells grown as ascites tumours. Moreover, the killing of L1210 ascites cells by hyperthermia can be enhanced by heating L1210 ascites cells with an equal number of normal marrow cells, or as upernatant removed from heated marrow cells. Most cells in lukaemic marrow are normal, and it is postulated that the increased thermal sensitivity of L1210 cells in leukaemic marrow is caused by diffusible factors (e.g. lysosomal enzymes) released by heating normal marrow cells.

Effects of the L isomer (+)-1,2-bis(3,5-dioxopiperazine-1-yl)propane on cell survival and cell cycle progression of cultured mammalian cells.

The effects of the L isomer (+)-1,2-bis(3,5-dioxopiperazine-1-yl)propane (ICRF 159; NSC 169780) on cell viability, growth, and progression through the cell cycle were investigated in suspension cultures of murine leukemia (Friend leukemia and L1210) cells and normal human lymphocytes stimulated with phytohemagglutinin and in adherent cultures derived from human neuroblastoma and Chinese hamster ovary (CHO) cells. CHO cell colony formation was inhibited by 50% following either an 8.5-hr exposure of exponentially growing cells to 10 micrograms ICRF 159 per ml or a 24-hr exposure to 3 micrograms ICRF 159 per ml. This effect was cell cycle phase specific; early G1- and G2-phase cells were more sensitive than were late-G1- or early and mid-S-phase CHO cells. Stationary-phase CHO cells were unaffected by the drug at concentrations up to 500 micrograms/ml. Incubation of L1210 cells with 3 micrograms ICRF 159 per ml for 24 hr or with 10 micrograms ICRF 159 per ml for 6 hr inhibited cell growth by 50%. In contrast, 24-hr incubation of human lymphocytes with up to 50 micrograms ICRF 159 per ml had no effect on their viability or on their ability to be stimulated by phytohemagglutinin. Constant exposure of Friend leukemia, L1210, human neuroblastoma, and phytohemagglutinin-stimulated human lymphocytes to 10.0 to 50 micrograms ICRF 159 per ml resulted in inhibition of cell division which led to cell growth at higher ploidy levels. Thus, proliferating human cells of normal or tumor origin and murine leukemic cell lines all had a similar sensitivity to the drug. Detailed analysis of cell cycle progression in L1210 cells in the presence of the drug determined that cell progression through G1 phase (G1A to G1B transition) was slowed by approximately 50%. The rate of traverse of cells through S phase was also slowed. However, the most pronounced effect was the accumulation of cells in G2 phase occurring almost immediately after addition of the drug. The data suggest that the L isomer has a range of cytotoxicity and identical cytokinetic effects similar to that of the clinically tested racemate (+/-)-ICRF 159 (NSC 129943) and, therefore, that the more soluble L isomer may have increased clinical applicability.

Characterization and use of neuraminidase-modified L1210 plasma membranes for protection against tumor growth.

Purified plasma membranes were prepared from L1210 ascites tumor cells and analyzed for their protein and carbohydrate composition. Conditions were developed for treating the isolated plasma membranes with Vibrio cholerae neuraminidase (VCN) so that 88% of the N-acetylneuraminic acid was removed without changing membrane proteins or other membrane carbohydrate constituents. The VCN-induced modifications were characterized by labeling VCN-treated and untreated L1210 cells by the galactose oxidase:sodium [3H]borohydride procedure. This showed that N-acetylneuraminic acid is the predominant saccharide at the nonreducing terminus of plasma membrane glycoproteins and that galactose and/or N-acetylgalactosamine residues are penultimate to these. VCN modification exposed the penultimate residues and was not limited to any single plasma membrane glycoprotein. DBA/2J mice were given i.p. injections of VCN-treated or untreated membranes and were challenged 3 weeks later with 10(4) viable L1210 cells. Mice pretreated with VCN-treated membranes resisted the tumor challenge; those receiving untreated membranes or no treatment succumbed to the tumor. Our results demonstrate that appropriately modified plasma membranes can be used to induce resistance to tumor growth. They also suggest that tumor cell membrane carbohydrate structures have an important role in this phenomenon.

Immunotherapy of L1210 leukemia using neuraminidase-modified plasma membranes combined with chemotherapy.

Purified L1210 plasma membranes treated with Vibrio cholerae neuraminidase (VCN) were used for active immunotherapy of L1210 tumors in DBA/2J mice. Immunotherapy with VCN-treated membranes was effective only when combined with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU). Successful therapy was a function of the dose of MeCCNU, the dose of VCN-treated membranes, and the time after MeCCNU treatment when VCN-treated membranes were administered. Optimum conditions for treating animals with tumors initiated with 10(4) cells were MeCCNU (20/kg) given 3 days after tumor inoculation and 0.25 mg VCN-treated membranes given 1 day after chemotherapy. Control membranes, not treated with VCN, that were administered 1 day after MeCCNU were ineffective; when given 4 days after chemotherapy, the caused accelerated mortality, suggesting immunological enhancement of tumor growth. Our results indicate that VCN-treated plasma membranes can be used for active immunotherapy of established tumors and underscore the importance of carefully designing immunotherapy protocols to achieve optimum desirable effects.

OTO method for preservation of actin filaments in electron microscopy.

Osmium tetroxide, commonly used as a fixative in electron microscopy, can destroy actin filaments. Thiocarbohydrizide (TCH) is a bipolar substance that binds to the osmium. By sandwiching TCH between two phases of osmium treatment, tissue exposure to osmium could be minimized without destroying actin filaments. The contrast of osmophilic components of cells was also enhanced.

G-banding patterns in mouse lymphoblastic leukemia L1210.

The karyotype of murine lymphoblastic leukemia L1210 was studied with the use of the trypsin-Giemsa technique. The line was hypodiploid with a modal number of 38 chromosomes, including three biarmed chromosomes. All cells examined contained at least one copy of each normal chromosome with the exception of chromosome #1, in which a whole copy along with parts of the second copy was found in the marker chromsome's composition. In 65% of the cells, trisomy of chromosome #15 was observed. Nearly all cells contained only one X-chromosome. The 13 marker chromosomes that frequently occurred are described, and the presumed origin of seven of these chromosomes is described in detail. Comparison of the karyotype of murine L1210 leukemia with the karyotypes of other luekemias of the mouse showed the existence of similar chromosome aberrations. Among these aberrations, the trisomy of chromosome #15 and monosomy of the X-chromosome may be of special importance. The possible role of karyotypic changes in the genesis of the malignant phenotype of the cells is discussed.

Flow cytometric analysis of double-stranded RNA content distributions.

A staining procedure is described for quantitative analysis of cellular RNA content distributions by flow cytometry (FCM). Cells were fixed in ethanol, treated with DNAse and stained with propidium iodide. FCM analysis showed that more than 90% of the fluorescence resulted from the double-stranded RNA (dsRNA) bound fluorochrome. The dsRNA content distributions were measured in HeLa S3 cells in culture 24-120 hr after plating, in L1210 ascitic leukemic cells 1-7 days after transplantation, and in regenerating bone marrow 3-7 days after injection of cyclophosphamide. In all three cell populations, maximal dsRNA content was observed during the period of most active cell proliferation, as determined by the S-phase index derived from the DNA histograms. In the dsRNA distributions in L1210 leukemia cell populations 1-2 days after transplantation, two separate peaks were evident, representing weakly fluorescent, presumably nontumor cells, and intensively fluorescent tumor cells. The amount of dsRNA-bound fluorochrome was significantly greater in L1210 cells than in normal and regenerating bone marrow cells and also greater in spontaneous thymic lymphoma cells than in normal thymic cells.

Ultrastructral changes induced by 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea in L1210 lymphocytic leukemia cells in mice.

Ultrastructural changes in L1210 leukemic cells infiltrated in the sinusoid of mouse liver after treatment with a single dose of a newly developed antitumor agent, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride, were observed by electron microscopy. In the earliest stage after injection of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride in tumor-bearing mice, marked changes were observed in both nucleolar and cytoplasmic ultrastructures. At 48 hr after administration, numerous Golgi complexes were observed in the cytoplasm, and lysosome-like granules also increased. The most striking change after treatment with 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride, however, occurred in the nucleolus. The chromatin was condensed, tending to collect near the nuclear membrane. Segregation of the nucleolar constituents was observed in the nucleus. Many necrobiotic cells were also observed within the liver sinusoid at this stage.

Adherence of L1210 murine leukemia cells to sephacryl-aminopropylcobalamin beads treated with transcobalamin-II.

Sephacryl beads containing an immobilized aminopropylcobalamin-transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.

A selective effect of methylglyoxal-bis(guanylhydrazone) on the synthesis of mitochondrial DNA of cultured L1210 leukemia cells.

Methylglyoxal-bis(guanylhydrazone) (MGBG) is a polycationic drug which is useful in the chemotherapy of lymphoid and myeloid proliferative disorders. The drug has recently been shown to produce selective ultrastructural damage to the mitochondria of proliferating cell populations. It is important to understand the molecular basis for this action, since it may be related to the known ability of MGBG to block polyamine biosynthesis. Accordingly, the effect of MGBG treatment on the incorporation of [3H]thymidine into both mitochondrial and nuclear DNA has been examined. Exponentially growing L1210 leukemia cells were prelabeled with [14C]thymidine, treated with MGBG for 1.5 to 16 hr, and then pulse labeled with [3H]-thymidine. Incorporation of [3H]thymidine into mitochondrial DNA was selectively inhibited at 5 hr with concentrations of 1 to 10 microM MGBG. Nuclear DNA, however, was not similarly affected until 8 to 11 hr of drug treatment. Dye-CsCl gradients of mitochondrial DNA indicated that the inhibition of synthesis occurred in replicative forms of circular DNA. Uptake studies excluded the possibility of drug interference with cellular uptake of thymidine. Ultrastructural studies revealed a very close correlation between the dose-response curve for mitochondrial damage and that for MGBG inhibition of mitochondrial DNA synthesis. This correlation suggests a direct cause-and-effect relationship between inhibition of mitochondrial DNA synthesis and ultrastructural damage, but the possibility of both phenomena being related to another action by the drug, such as inhibition of polyamine biosynthesis, or a drug effect on mitochondrial function, must also be considered.