cytochalasin-b and Lymphoma

C57BL mice bearing EL4 tumors and C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p-boronophenylalanine-(10)B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-(10)B (BSH) or l-p-boronophenylalaninol (BPA-ol), a newly developed (10)B-containing alpha-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and / or tirapazamine (TPZ), the tumors were heated at 40 degrees C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the (10)B uptake of all (10)B-compounds into both tumor cells. BPA-ol has good potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Boranes; Boron Neutron Capture Therapy; Bromodeoxyuridine; Carcinoma, Squamous Cell; Combined Modality Therapy; Cytochalasin B; Drug Screening Assays, Antitumor; Fluorescent Antibody Technique, Indirect; Hindlimb; Hyperthermia, Induced; Injections, Intraperitoneal; Interphase; Lymphoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Micronucleus Tests; Molecular Structure; Neutrons; Phenylalanine; Radiation-Sensitizing Agents; Radiometry; Tirapazamine; Triazines

Response of quiescent (Q) and total tumor cells in solid tumors to reactor neutron beam irradiation with two different cadmium (Cd) ratios was examined in terms of micronucleus (MN) frequency and apoptosis frequency, using four different tumor cell lines.. C57BL mice bearing EL4 tumors, C3H/He mice bearing SCC VII or FM3A tumors, and Balb/c mice bearing EMT6/KU tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty min after i.p. injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutron beams. The tumors without 10B-compound administration were irradiated with neutron beams or gamma-rays. This neutron beam irradiation was performed using neutrons with two different Cd ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the MN frequency in cells without BrdU labeling (=Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, for apoptosis assay, 6 h after irradiation, tumor cell suspensions obtained in the same manner were fixed, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequencies in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU.. Without 10B-compounds, the sensitivity difference between total and Q cells was reduced by neutron beam irradiation. Under our particular neutron beam irradiation condition, relative biological effectiveness (RBE) of neutrons was larger in Q cells than in total cells, and the RBE values were larger for low Cd-ratio than high Cd-ratio neutrons. With 10B-compounds, both frequencies were increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH did. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of Q cells treated with BSH. Whether based on the MN frequency or the apoptosis frequency, similar results concerning the sensitivity difference between total and Q cells, the values of RBE, and the enhancement effect by the use of 10B-compound were obtained.. Apoptosis frequency, as well as the MN frequency, can be applied to our method for measuring the Q cell response to reactor neutron beam irradiation within solid tumor in which the ratio of apoptosis to total cell death is relatively high, as in EL4 tumor. The absolute radiation dose required to achieve the same endpoint for Q cells is much higher than that for total cells when combined with 10B-compound, especially with BPA.

Topics: Animals; Apoptosis; Borohydrides; Boron Compounds; Boron Neutron Capture Therapy; Bromodeoxyuridine; Cadmium; Carcinoma, Squamous Cell; Cytochalasin B; Fluorescent Antibody Technique; Lymphoma; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Micronucleus Tests; Neoplasms; Neutrons; Phenylalanine; Radiation Tolerance; Radiation-Sensitizing Agents; Radiobiology; Radioisotopes; Relative Biological Effectiveness; Sarcoma, Experimental; Sulfhydryl Compounds; Tumor Cells, Cultured

The retro-retinoids, metabolites of vitamin A (retinol), belong to a family of lipophilic signalling molecules implicated in regulation of cell growth and survival. Growth-promoting properties have been ascribed to 14-hydroxy-retro-retinol (14HRR), while anhydroretinol (AR) was discovered to act as a natural antagonist triggering growth arrest and death by apoptosis. Based on morphological studies and inhibition of apoptosis by the kinase blocker, herbimycin A, it has been suggested that retro-retinoids exhibit their function in the cytosolic compartment. F-actin emerged as a functional target for retro-retinoid action. By FACS analysis and fluorescence microscopy of phalloidin-FITC labeled cells we demonstrated that F-actin reorganization was an early event in AR-triggered apoptosis. Fluorescence images of AR-treated fibroblasts displayed short, thick, stick-like and punctate structures, and membrane ruffles at the cell periphery along with an increased diffuse staining pattern. Reversal of the AR effect by 14HRR or retinol indicates that F-actin is a common site for regulation by retro-retinoids. Inhibition of both cell death and actin depolymerisation by bcl-2 implies that cytoskeleton reorganization is downstream of bcl-2-related processes. Furthermore, stabilization of microfilaments by jasplakinolide increased the survival potential of AR treated cells, while weakening the cytoskeleton by cytochalasin B abetted apoptosis. Thus the cytoskeleton is an important way station in a communication network that decides whether a cell should live or die.

Topics: 3T3 Cells; Actins; Animals; Antineoplastic Agents; Apoptosis; Benzoquinones; Cell Survival; Cytochalasin B; Cytosol; Depsipeptides; Diterpenes; DNA Damage; Fibroblasts; Flow Cytometry; Kinetics; Lactams, Macrocyclic; Lymphoma; Mice; Peptides, Cyclic; Quinones; Retinoids; Rifabutin; Tumor Cells, Cultured; Vitamin A

Binding of the lectin Con A to its ligand on the cell surface of normal circulating lymphocytes induces capping in 28-32% of these cells. This Con A cap formation is markedly decreased in malignant cells from the human hematopoietic system. Among others, the human lymphoma Daudi cell line exhibit a cap formation with Con A in only 5-10% of the cells. In this study, we found that inactivated influenza viruses induced changes in the cell surface membrane of Daudi cells resulting in an increased percentage of Con A cap forming cells (30-40%). This phenomenon occurred independently of viral replication and was initiated by adsorption of inactivated viral particles or isolated hemagglutinin and neuraminidase viral glycoproteins. This phenomenon may be due to the binding of Con A molecules to viral receptors and to cell receptors leading to crosslinking of Con A receptors that will induce their mobility and the formation of a cap. Alternatively, experiments performed with cytochalasin B and colchicine suggest that the viral interaction with the cell membrane may have induced changes in the cytoskeleton at the level of microtubules. These changes induced increased lateral movement of the Con A receptors resulting into formation of a cap.

Topics: Adsorption; Cell Membrane; Colchicine; Concanavalin A; Cytochalasin B; Cytoskeleton; Humans; Lymphoma; Orthomyxoviridae; Receptors, Concanavalin A; Tumor Cells, Cultured; Virus Replication

A novel type of cell membrane movement was characterized in B lymphocytes. Local submicron cell membrane displacements, within the frequency range 0.3-15 Hz, were registered in a murine lymphoma B cell line by a novel optical method based on point dark field microscopy. The cell membrane displacements were measured by monitoring changes in light scattering from very small illuminated areas (0.25 microns2) at the edge of the cell surface. B lymphocytes manifest a relative change in light scattering of 7.7 +/- 1.3% (mean +/- SD) which corresponds to cell membrane transverse displacement of 131 +/- 22 nm. The confinement of cell membrane displacements to microdomains (less than or equal to 0.2 microns2) emerged from the observed dependence of the displacement amplitude on the area size from which it is monitored. Colchicine (1 microM) decreased membrane fluctuations down to a value of 88 +/- 14 nm, whereas dihydrocytochalasin B (2 microM) increased the amplitude of membrane displacements up to 184 +/- 31 nm. These findings demonstrate the existence of a dynamic mechanical interaction between the cytoskeleton and the cell membrane in the frequency range of 0.3-15 Hz. The modulation of these interactions by the disruption of microfilaments or or microtubules is explained in terms of the induced strain changes imposed on the cell membrane.

Topics: Animals; B-Lymphocytes; Cell Line; Cell Membrane; Cell Movement; Colchicine; Cytochalasin B; Lymphoma; Mice

S49 mouse lymphoma cells respond to swelling deformation with rapid increases in intracellular calcium and cAMP. Experiments demonstrate that these increases in calcium and cAMP concentrations are not coupled in a regulatory manner. Direct inhibition of adenylate cyclase in wild type cells with miconazole prevented swelling-induced accumulation of cAMP. No effect of swelling was observed on the activity of cAMP phosphodiesterase. Additionally, complete inhibition of cAMP phosphodiesterase did not prevent swelling-induced cAMP accumulation. Experiments involving cyc- mutants (lacking the Gs-alpha protein) and 2',5'-dideoxyadenosine indicate that increased adenylate cyclase activity with swelling is not mediated by Gs. No evidence was found for attenuation of Gi-mediated inhibition of adenylate cyclase activity following swelling. In addition, exposure to pertussis toxin or phorbol ester, which disrupts Gi inhibition of adenylate cyclase did not prevent cAMP accumulation following swelling. Disruption of the actin membrane skeleton resulted in a significant accumulation of cAMP which was not further increased by swelling. Disruption of the microtubular cytoskeleton also increased cAMP content in S49 cells which could be further increased by swelling. It is concluded that S49 cell-adenylate cyclase responds directly to mechanical forces transmitted through the actin membrane skeleton.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Calcimycin; Calcium; Cell Line; Colchicine; Cyclic AMP; Cytochalasin B; Ionomycin; Kinetics; Lymphoma; Mice; Miconazole; Papaverine; Pertussis Toxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Microcell production by means of Colcemid-induced micronucleation and subsequent enucleation with the density gradient technique was adjusted for use with the murine T-lymphoma line ESb-M. Modification of the standard protocol for a cell type on which no experiments had previously been performed required careful monitoring of the multiple steps in the procedure in order to optimize the final microcell yield. Traditional microscopic verification may sometimes be ambiguous, due to the lack of a clear cutoff point between small whole cells and cell fragments; in these conditions, the level of variability increases, thus impairing quantitative estimations. Flow cytometric (FCM) analysis of DNA content and size of donor cells and microcells was therefore applied in parallel to provide additional quantitative information. The FCM results supplemented the microscopic data in assessing which fraction recovered from the gradient has the lowest percentage of contaminant whole cells; however, FCM analysis may provide more statistically significant data due to the large size of the sample examined. Moreover, FCM is of prospective use in providing the basis for subsequent sorting of either pure microcells or specific subpopulations of defined DNA content and size.

Topics: Animals; Cell Line; Cell Nucleus; Cytochalasin B; Demecolcine; DNA; Ficoll; Flow Cytometry; Lymphoma; Mice; T-Lymphocytes; Tumor Cells, Cultured

Known synergism between forskolin and hormones in adenylate cyclase activation leads to the supposition that hormone might stimulate forskolin binding. That possibility was tested using intact wild type S49 cultured lymphoma cells. Using 40 nM [3H]-forskolin it was shown that the extent of forskolin binding using a filtration technique increased with the concentration of epinephrine or isoproterenol (INE). Moreover, the hormone-dependent forskolin binding was stereospecific (requiring l- rather than d-epinephrine), it was not observed in the cyc- variant and it was not inhibited by cytochalasin B. These observations lead to the conclusion that the binding is specifically associated with the adenylate cyclase system and requires a functional Gs unit. Epinephrine-stimulated forskolin binding did not correlate exactly with forskolin activation of adenylate cyclase in the presence of similar concentrations of epinephrine. It was concluded from that observation that there is not a one to one correspondence between binding and activation.

Topics: Animals; Binding, Competitive; Colforsin; Cytochalasin B; Epinephrine; Isoproterenol; Ligands; Lymphoma; Propranolol; Radioimmunoassay; Stereoisomerism; Tumor Cells, Cultured

Little information is available on the interaction between lymphocytes and fibronectin (fn). To gain a better understanding on this issue we examined the adhesion of 12 lymphoid cell lines, each exhibiting different phenotypic characteristics, to fn-coated substratum. Of the cell lines tested, five that adhered to fn possessed B-cell characteristics, while neither the T-cell lines nor the pre-B-cell line adhered. The physiology and biochemistry of adhesion of a B-cell line, MOPC 315, were examined in detail. Our results indicated that (1) the adhesion was a specific and time-dependent process, (2) the adhesion was temperature-dependent and inhibited by metabolic inhibitors, such as KCN and 2-deoxyglucose, (3) the presence of cycloheximide and pretreatment of cells with trypsin inhibited adhesion, (4) a 140-kDa surface protein was immunoprecipitated by anti-fn receptor antibodies, (5) the presence of divalent cations was essential for adhesion, (6) the presence of colchicine had no effect on adhesion, while cytochalasin B partially inhibited adhesion, and (7) the treatment of cells by both phorbol 12-myristate 13-acetate and calcium ionophore A23187 enhanced adhesion. In this study, we have established the interaction between lymphoid cell lines and fn. Such an interaction might play an important role in the behavior of lymphocytes in tissues.

Topics: Animals; Calcimycin; Cations, Divalent; Cell Adhesion; Cell Differentiation; Cell Line; Cycloheximide; Cytochalasin B; Fibronectins; Humans; Lymphocytes; Lymphoma; Multiple Myeloma; Receptors, Fibronectin; Receptors, Immunologic; Tetradecanoylphorbol Acetate; Trypsin

We have covalently attached a monoclonal antibody (11-4.1) against the murine major histocompatibility antigen, H-2Kk, on the surface of liposomes. The interaction of these antibody-coated liposomes (immunoliposomes) with target cells, RDM-4 lymphoma (H-2Kk), was investigated. About 90% of the immunoliposomes taken up by target cells at 4 degrees C could be removed by a mild protease treatment of the cells, whereas only 30% of the uptake at 37 degrees C was labile to the same treatment. Furthermore, the uptake of immunoliposomes at 37 degrees C was inhibitable by cytochalasin B or by a combination of 2-deoxyglucose and NaN3. These results suggest that immunoliposome binding to the target cell surface is the primary uptake event at 4 degrees C and that the surface-bound liposomes are rapidly internalized by the cells at 37 degrees C, probably via an endocytic pathway. Studies with fluorescence microscopy of target cells treated with immunoliposomes containing carboxyfluorescein also supported this conclusion. If endocytosis is the mechanism by which immunoliposomes gain entry into target cells, the efficacy of a cytotoxic drug encapsulated would depend on the resistance of the drug to lysosomal inactivation and its ability to escape from the lysosomal system. Consistent with this notion, we observed that methotrexate encapsulated in liposomes bearing 11-4.1 antibody specifically inhibited deoxy[6-3H]uridine incorporation into DNA in target RDM-4 cells but not in P3-X63-Ag8 myeloma cells (H-2Kd) at the same doses. The observed cytotoxic effect of encapsulated methotrexate could be reversed by the treatment of cells with a lysosomotropic amine, chloroquine, which has been shown to increase the intralysosomal pH of mammalian cells. On the other hand, cytosine-beta-D-arabinofuranoside encapsulated in immunoliposomes showed no target-specific killing, probably because the drug is readily inactivated in the lysosomal system. These results are discussed in terms of the drug carrier potential of immunoliposomes.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cell Survival; Colchicine; Cytochalasin B; Deoxyglucose; Fluorescent Antibody Technique; H-2 Antigens; Kinetics; Liposomes; Lymphoma; Methotrexate; Mice

Topics: Adenylyl Cyclases; Alprostadil; Animals; Cell Line; Cholera Toxin; Cyclic AMP; Cytochalasin B; Cytoskeleton; Genetic Variation; Isoproterenol; Lymphoma; Neoplasms, Experimental; Prostaglandins E; Protein Kinases; Receptors, Adrenergic; Receptors, Adrenergic, beta

Antibodies to a cytoplasmic myosin, rat lymphoma myosin, and to rat skeletal myosin were prepared in rabbits and shown to be specific for their corresponding antigens. The two antibodies did not cross-react. The skeletal myosin antibody was directly labeled with rhodamine, and the cytoplasmic myosin antibody was detected by indirect immunofluorescence with fluorescein-labeled goat anti-rabbit antibody. The two antibodies were used to examine developing rat muscle cultures for the presence and location of the antigens. The antibody to cytoplasmic myosin reacted with multinucleated myotubes and with all the mononucleated cells in the culture. The antibody to skeletal myosin reacted with myotubes and with a small fraction of the mononucleated cells. In the myotubes, the cytoplasmic myosin appeared to be localized primarily in two structures: fine stress fibers, often visible also by phase microscopy and present predominantly in the ends of the cells, and in a submembranous rim all along the cell's border. In addition, a diffuse fluorescence within the cells was observed. The skeletal myosin was localized in the central part of the myotubes in sarcomeres or in fibers without periodicities and was excluded from the ends of the myotubes. When the same cells were doubly stained with the two antibodies, the complementary distribution of the two isozymes was very clear. There was also a narrow region of overlap of staining, with cytoplasmic myosin present in some stress fibers that appeared to be continuous with fibrous elements containing skeletal myosin. Myotubes that rounded up with cytochalasin B or with trypsin displayed a diffuse distribution of both isozymes. When these cells were allowed to respread into extended configurations, the location of the two myosins were essentially the same as in untreated cells. The ability of myotubes to adhere to the surface and to move in culture may be related to the presence of cytoplasmic myosin. Our results show that in myotubes and myoblasts the two isozymes differ sufficiently to be localized in distinct regions of the cell and to be sorted out into different structures, even after the cytoplasmic contents have been reshuffled. The cell can, by some unknown mechanism, distinguish the two myosins.

Topics: Cell Differentiation; Cytochalasin B; Cytoplasm; Fluorescent Antibody Technique; Isoenzymes; Lymphoma; Muscles; Myosins

Topics: Animals; Cytochalasin B; Cytotoxicity, Immunologic; Epitopes; Histocompatibility Antigens; Isoantigens; Lymphoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Spleen; Subcellular Fractions; T-Lymphocytes

Lymphoid cells from many normal W/Fu rats reacted in a 51Cr release cytotoxicity assay against (C58NT)D, a syngeneic Gross leukemia virus-induced tumor. The reactivity was maximal in young rats at 5-8 weeks of age and rapidly declined thereafter. Within reactive rats, the cytotoxicity was widely distributed among the various lymphoid organs. Since immunization of W/Fu rats with (C58NT)D was shown to elicit specific cell-mediated cytotoxic reactivity, studies were done to compare the characteristics of the natural reactivity with those of the immune reactivity. The specificity of both types of reactivity was analyzed in detail by an inhibition assay. The natural and the immune reactivities appeared directed against antigens associated with rat endogenous type-C viruses. The major differences between the natural reactivity and immune reactivity were the nature of the effector cells. Whereas immune reactivity was T-cell dependent, normal reactivity was not affected by treatment with antisera against T cells plus complement. Natural effector cells were not adherent and did not have macrophage properties. The active cells also did not have receptors for Ig or complement. The absence of detectable cell-surface markers on the natural effector cells was seen in studies of natural cytotoxic reactivity of mice, and it is proposed that the natural cytotoxicity in both systems is mediated by a unique subpopulation of lymphoid cells, tentatively designated "N" cells.

Topics: Age Factors; AKR murine leukemia virus; Animals; Cell Line; Cytochalasin B; Cytotoxicity Tests, Immunologic; Germ-Free Life; Lymphocytes; Lymphoma; Male; Phagocytes; Rats; Rats, Inbred Strains; Receptors, Antigen, B-Cell; T-Lymphocytes

Topics: Animals; Antilymphocyte Serum; Bucladesine; Cell Adhesion; Cell Separation; Chromium Radioisotopes; Colchicine; Cytochalasin B; Cytotoxicity Tests, Immunologic; Deoxyuridine; Edetic Acid; Female; Histamine; Immunization; Iodine Radioisotopes; Lymphoma; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred DBA; Neoplasms, Experimental; Phagocytosis; Trypsin

Topics: Adult; Aged; Blood Bactericidal Activity; Carcinoma; Cytochalasin B; Female; Humans; Iodoacetates; Lymphoma; Male; Middle Aged; Monocytes; Neoplasms; Staphylococcus; Tuberculosis

Topics: Animals; Antibodies, Neoplasm; Antibody Specificity; Ascitic Fluid; Chromium Isotopes; Complement System Proteins; Cytochalasin B; Cytotoxicity Tests, Immunologic; Depression, Chemical; Fluorides; Leukemia, Experimental; Lymphocytes; Lymphoma; Macrophages; Neoplasm Transplantation; Phagocytosis; Rats; Transplantation, Homologous