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1.
Proc Natl Acad Sci U S A ; 98(13): 7152-7, 2001 Jun 19.
Article in English | MEDLINE | ID: mdl-11416200

ABSTRACT

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.


Subject(s)
Cell Degranulation/physiology , Mast-Cell Sarcoma/physiopathology , Phospholipids/metabolism , Animals , Cell Membrane Permeability , Fourier Analysis , Mass Spectrometry/methods , Phosphatidylcholines/metabolism , Phospholipase D/metabolism , Phospholipases A/metabolism , Phospholipases A2 , Phospholipids/chemistry , Rats , Spectrometry, Mass, Electrospray Ionization/methods , Substrate Specificity , Tumor Cells, Cultured , Type C Phospholipases/metabolism
2.
J Leukoc Biol ; 48(1): 1-6, 1990 Jul.
Article in English | MEDLINE | ID: mdl-2162899

ABSTRACT

Murine bone-marrow-culture-derived-macrophages can be differentially activated to lyse either vesicular stomatitis virus infected BALB/c3T3 cells or the tumor target P815. Macrophages were activated in a manner so that they could lyse both targets. The ability of this activated population to lyse either target type was differentially inhibited by varying the assay conditions. The lysis of P815 targets was more sensitive to inhibition by the proteinase inhibitor N-p-tosyl-L-lysine chloromethyl ketone than was the lysis of virally infected cells. On the other hand, reduction of the concentration of glucose in the assay medium, which inhibits the production of oxygen metabolites by the hexose monophosphate shunt, or the addition of anti-tumor necrosis factor (anti-TNF) serum were able to decrease the lysis of virally infected targets but not P815 targets. Thus, the observed differences in the lysis of these two targets were due to both the activation state of the macrophages and the differential susceptibility of the targets to different effector mechanisms.


Subject(s)
Fibroblasts/physiology , Macrophage Activation/physiology , Macrophages/physiology , Mast-Cell Sarcoma/physiopathology , Sarcoma, Experimental/physiopathology , Animals , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fibroblasts/microbiology , Glucose/metabolism , Mast-Cell Sarcoma/microbiology , Mast-Cell Sarcoma/pathology , Mice , Mice, Inbred BALB C , Protease Inhibitors/pharmacology , Sarcoma, Experimental/microbiology , Sarcoma, Experimental/pathology , Stomatitis/pathology , Stomatitis/physiopathology , Tosyllysine Chloromethyl Ketone/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/microbiology , Tumor Cells, Cultured/pathology , Tumor Necrosis Factor-alpha/pharmacology , Vesicular stomatitis Indiana virus/isolation & purification
3.
J Cell Biol ; 110(6): 2109-16, 1990 Jun.
Article in English | MEDLINE | ID: mdl-1693622

ABSTRACT

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.


Subject(s)
Ion Channels/drug effects , Membrane Glycoproteins , Membrane Proteins/pharmacology , Aminoquinolines , Animals , Calcium/metabolism , Cell Line , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cell Membrane Permeability/physiology , Electric Conductivity/physiology , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/physiology , Humans , Ion Channels/physiology , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Erythroblastic, Acute/pathology , Leukemia, Erythroblastic, Acute/physiopathology , Lymphoma/metabolism , Lymphoma/pathology , Lymphoma/physiopathology , Mast-Cell Sarcoma/metabolism , Mast-Cell Sarcoma/pathology , Mast-Cell Sarcoma/physiopathology , Membrane Potentials/physiology , Mice , Perforin , Pore Forming Cytotoxic Proteins , Sarcoma, Experimental/metabolism , Sarcoma, Experimental/pathology , Sarcoma, Experimental/physiopathology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , T-Lymphocytes/physiology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Cytotoxic/pathology , T-Lymphocytes, Cytotoxic/physiology
4.
Biochim Biophys Acta ; 1007(3): 295-300, 1989 Apr 12.
Article in English | MEDLINE | ID: mdl-2539190

ABSTRACT

Extracts of K21 murine mastocytoma cells contain a factor that enhances formation of amsacrine-induced topoisomerase II-DNA complexes (PDCs) when added to isolated K21 nuclei. The PDC-enhancing activity is reduced in extracts from 2 or 6 h cycloheximide or cordycepin-treated cells, implying that continuous protein synthesis is required to maintain the factor. The factor is heat-labile, proteinase-sensitive and has other properties that distinguish it from the two known classes of topoisomerases. The data suggest that the factor is a labile protein with a molecular weight in excess of 50,000. This appears to be the first direct evidence of a protein factor that modulates drug-induced topoisomerase II action.


Subject(s)
Amsacrine/pharmacology , DNA, Neoplasm/metabolism , Mast-Cell Sarcoma/physiopathology , Topoisomerase II Inhibitors , Animals , Cell Nucleus/physiology , Cell-Free System , Cytoplasm/physiology , Hot Temperature , Macromolecular Substances , Mice , Peptide Hydrolases/pharmacology , Protein Binding/drug effects
5.
Article in English | MEDLINE | ID: mdl-2888233

ABSTRACT

Electron microscopic observations of an originally established mouse mastocytoma cell line (BSP-MST-2) revealed that the cytoplasm of many of the MST-2 cells contained small and low osmiophilic granules and a few mature electron-dense granules. Fluorescent- and immuno-histochemical examinations also suggested the immaturity of granules as the cytoplasmic reaction for serotonin (5-HT) was weak. Induction of further maturation of granules was investigated by administration of various chemical agents. Among the chemicals examined, sodium butyrate and hydrocortisone were effective. In the presence of 1 mM sodium butyrate for 24 h, the cytoplasmic granules contained an abundant dense matrix. MST-2 cells incubated with hydrocortisone at 5 micrograms/ml for 24 h showed a somewhat different granulopoietic pattern from those incubated with sodium butyrate, including numerous electron-dense progranules. Fluorescent- and immuno-histochemical studies showed increased reactions of cytoplasmic 5-HT of both butyrate- and hydrocortisone-treated MST-2 cells. The specificity of these morphological and cytochemical changes was confirmed by treatment with reserpine, a drug which depletes cellular 5-HT; electron-dense materials were virtually diminished and cytochemical reactions were significantly decreased. The mode of induced production of 5-HT in mastocytoma granules is discussed, in relation to mastocyte differentiation.


Subject(s)
Granulocytes/physiology , Hematopoiesis , Mast-Cell Sarcoma/metabolism , Serotonin/biosynthesis , Animals , Cell Differentiation , Cells, Cultured , Cytoplasmic Granules/ultrastructure , Histocytochemistry , Mast-Cell Sarcoma/physiopathology , Mast-Cell Sarcoma/ultrastructure , Mice
6.
Am J Physiol ; 251(3 Pt 1): C387-94, 1986 Sep.
Article in English | MEDLINE | ID: mdl-3092676

ABSTRACT

We examined the interaction between mast cell-derived mediators and the electrical and ion transport properties of canine tracheal epithelium. We compared the effect of mediators released by immunologic challenge of sensitized lung parenchyma with that of mediators released from canine mastocytoma cells challenged with calcium ionophore A23187. Short-circuit current (Isc) increased by 19.2 +/- 3.0 microA/cm2 in response to mediators released from sensitized lung fragments challenged with ragweed antigen. This effect was not due to histamine. When the epithelial tissues were pretreated with indomethacin, the same mediator supernatant increased Isc by only 3.8 +/- 4.3 microA/cm2. The mediators released from 10(7) mastocytoma cells challenged with calcium ionophore increased Isc by 25.1 +/- 13.6 microA/cm2. In the presence of indomethacin, the Isc increased by 2.0 +/- 0.4 microA/cm2. Mastocytoma-derived mediators produced an increase in net chloride secretion without a significant effect on net sodium absorption. This study provides direct evidence that mast cell-derived mediators can stimulate epithelial ion transport in canine trachea and suggests that the effect is indirect and dependent on intact cyclooxygenase pathways in the tracheal epithelium.


Subject(s)
Mast Cells/physiology , Trachea/physiology , Animals , Biological Transport/drug effects , Calcimycin/pharmacology , Dogs , Electric Conductivity , Electrophysiology , Epithelium/physiology , Indomethacin/pharmacology , Ions , Lung/immunology , Mast-Cell Sarcoma/physiopathology , Mice , Mice, Nude , Pollen/immunology , Prostaglandin-Endoperoxide Synthases/physiology
7.
J Immunol ; 136(4): 1490-6, 1986 Feb 15.
Article in English | MEDLINE | ID: mdl-3080524

ABSTRACT

The binding of tumor cells by macrophages activated with Bacillus Calmette-Guerin is a necessary step toward destruction of those cells. Although several characteristics of the interaction have been defined, little is known of how the actual binding process develops. We used a technique to quantify the forces required to disrupt cell-cell interactions. Over a range of applied relative centrifugal forces, the majority of targets that bound to the activated macrophages fell on two distinct plateaus. Approximately 90% of added targets were bound to the monolayers of macrophages over the range of 1 to 100 X G; 25 to 30% remained bound from 1200 X G to 1500 X G. Two strengths of binding, termed weak and strong binding, respectively, were thus defined on the basis of these curves. Strong binding developed only between activated macrophages and tumor cells. By contrast, weak interactions occurred between either activated or nonactivated macrophages and neoplastic or non-neoplastic target cells. The strong binding required time (60 to 90 min), metabolic activity by the macrophages, and trypsin-sensitive surface structures on the macrophages for development, whereas the weak interaction occurred rapidly and required none of these. Additional evidence indicated the weak binding developed into strong when activated macrophages bound neoplastic cells. This stabilization increased the strength of force to separate tumor cells from the macrophages at least approximately 15 fold (i.e., from approximately 16 mu dynes/cell to approximately 240 mu dynes/cell). Of note, the development of strong binding of antibody-coated targets had distinct requirements for establishment. Taken together, the data suggest the stabilization of binding (i.e., the development of weak into strong binding) leading to effective cell-cell interaction is a complex and dynamic process that may vary depending upon the recognition system involved.


Subject(s)
Cell Communication , Macrophage Activation , Macrophages/physiology , Mast-Cell Sarcoma/immunology , Animals , BCG Vaccine/pharmacology , Cell Adhesion/drug effects , Cell Communication/drug effects , Cell Line , Cytotoxicity Tests, Immunologic/methods , Kinetics , Macrophage Activation/drug effects , Macrophages/immunology , Macrophages/metabolism , Mast-Cell Sarcoma/physiopathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Tetradecanoylphorbol Acetate/pharmacology
8.
J Immunol ; 136(4): 1504-9, 1986 Feb 15.
Article in English | MEDLINE | ID: mdl-3080525

ABSTRACT

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.


Subject(s)
Antigens, Ly , Cytotoxicity Tests, Immunologic , Mast-Cell Sarcoma/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, Ly/immunology , Cell Division , Cell Line , Cytotoxicity Tests, Immunologic/methods , Leukocyte Count , Lymphatic Metastasis , Lymphocyte Activation , Mast-Cell Sarcoma/mortality , Mast-Cell Sarcoma/physiopathology , Mast-Cell Sarcoma/therapy , Mice , Mice, Inbred DBA , Phenotype , Splenic Neoplasms/immunology , Splenic Neoplasms/secondary , T-Lymphocytes, Cytotoxic/classification
9.
C R Seances Soc Biol Fil ; 180(1): 121-7, 1986.
Article in French | MEDLINE | ID: mdl-2943365

ABSTRACT

The effect of a transplantation of mastocytoma cells in the abdominal cavity on the sensitivity of mice to a systemic hyperthermia was studied. The systemic hyperthermia was induced by exposing whole-body of animals to 2,450 MHz waves under anesthesia. Core body temperature was raised up to 42.0 +/- 0.2 degrees C in 15 min and maintained constant at the temperature for variable length of time. Thermosensitivity of animal was expressed with LD50, 42 degrees which was the length of heating time at the temperature of 42 degrees C lethal for 50% of the animals examined. The transplants were mastocytoma FMA3 cells. They were transplanted at a dose of 10(5) cells per mouse. The LD50, 42 degrees observed 3, 12 hrs, 1, 2, 3 and 6 days after the transplantation was 33, 23, 17, 24 and 35 min, respectively. In mice without tumor it was 43 min.


Subject(s)
Hyperthermia, Induced , Mast-Cell Sarcoma/physiopathology , Animals , Body Temperature , Female , Mice , Mice, Inbred Strains , Neoplasm Transplantation , Species Specificity
10.
Cell Immunol ; 95(2): 247-57, 1985 Oct 15.
Article in English | MEDLINE | ID: mdl-3930072

ABSTRACT

Tumor cells can adhere to endothelial cell monolayers in vitro. The kinetics of this reaction are rapid; 50% of maximal binding occurs by 30 min of incubation. In the case of the P815 mastocytoma, the maximal percentage of binding is approximately 70%, suggesting that there are both binding and nonbinding tumor cell populations. Binding is independent of tumor cell dose over a 200-fold range of cell concentrations. Lymphokine-containing preparations were found to markedly suppress the binding of either P815 mastocytoma or Ehrlich ascites cells to endothelium. This effect appeared to be due to both diminished attachment and enhanced dissociation. The activity is found in the same molecular weight range as tumor migration inhibition factor (TMIF), and is not found in preparations lacking TMIF activity. Thus, the factor may prove to be TMIF itself or a lymphokine related to it. Of equal interest is the possibility that it represents a previously undescribed factor.


Subject(s)
Carcinoma, Ehrlich Tumor/physiopathology , Endothelium/physiology , Immunosuppressive Agents/physiology , Lymphokines/physiology , Mast-Cell Sarcoma/physiopathology , Animals , Binding, Competitive , Carcinoma, Ehrlich Tumor/immunology , Cattle , Cell Adhesion , Endothelium/immunology , Guinea Pigs , Humans , Kinetics , Mast-Cell Sarcoma/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred DBA
11.
Invest Ophthalmol Vis Sci ; 26(10): 1368-76, 1985 Oct.
Article in English | MEDLINE | ID: mdl-3930419

ABSTRACT

The authors have studied histopathologically the growth, invasion, and regression of allogeneic P815 mastocytoma cells placed in the anterior chambers of eyes of three different strains of inbred mice. Previous clinical observations had suggested that the degree of immunogenetic disparity between tumor and recipient governed the intensity and specificity of the host's immune response to the intraocular neoplasm. Our histopathologic studies confirm this conclusion. BALB/c mice, which are H-2 syngeneic with P815 cells, develop progressively growing tumors around which no evidence of host immune or inflammatory response can be found. P815 cells confront A/J mice with a single class I MHC disparity; early intraocular tumor growth is vigorous in this strain, and when the tumor rejection takes place, it is followed by marked fibrovascular scar tissue formation which destroys intraocular structures, resulting in atrophia bulbi. C57BL/6 mice recognize immunologically three categories of allogeneic class I antigens on P815 cells. In these hosts, early tumor growth within the anterior chamber is feeble and rejection is attended by only minimal inflammation; as a consequence, scar formation is trivial and the eye remains anatomically intact. Based on certain histopathologic features, the authors suggest that two distinctly different immune effector mechanisms are involved in intraocular tumor rejection in A/J and C57BL/6 hosts. One immune rejection process results in extensive innocent bystander destruction of normal host tissues (A/J mice) while the other immune mechanism is more precise and does not damage host ocular structures (C57BL/6 mice).


Subject(s)
Anterior Chamber/pathology , Eye Neoplasms/pathology , Mast-Cell Sarcoma/pathology , Animals , Eye Neoplasms/immunology , Eye Neoplasms/physiopathology , Female , Graft Rejection , Graft Survival , Mast-Cell Sarcoma/immunology , Mast-Cell Sarcoma/physiopathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Transplantation
12.
Mol Cell Biochem ; 67(1): 31-8, 1985 May.
Article in English | MEDLINE | ID: mdl-2991742

ABSTRACT

A substantial increase in cyclic AMP-dependent protein kinase activity occurred in nuclei of PY815 mastocytoma cells during G1 phase growth arrest by DB cyclic AMP and the increased nuclear protein kinase was accompanied by changes in nuclear protein phosphorylation. However, there was no obligatory association between the rise in nuclear cyclic AMP-dependent protein kinase in G1 phase and growth arrest because nuclear cyclic AMP-dependent protein kinase also increased during G1 phase in cycling PY815 cells synchronized with amethopterin. These observations suggest that maintenance of high cyclic AMP levels during G1 phase may cause growth arrest by activating a cyclic AMP-dependent protein kinase that normally increases in PY815 cell nuclei during G1 phase.


Subject(s)
Cyclic AMP/metabolism , Interphase , Mast-Cell Sarcoma/physiopathology , Animals , Bucladesine/pharmacology , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Interphase/drug effects , Mast-Cell Sarcoma/metabolism , Mice , Neoplasm Proteins/biosynthesis , Phosphoproteins/biosynthesis , Phosphorylation , Protein Kinases/metabolism
13.
Cancer Res ; 44(9): 3870-2, 1984 Sep.
Article in English | MEDLINE | ID: mdl-6430556

ABSTRACT

We have previously described a noncytotoxic lymphokine, tumor migration inhibition factor, with the capacity of inhibiting the in vitro migration of a variety of tumor cells maintained by animal passage in ascitic form. In the present study, we demonstrate that it is possible to prepare viable, motile tumor cell suspensions from solid tumors and that those cells migrate better than cells that had been propagated in ascitic form. Such preparations derived from solid tumors are inhibited by tumor migration inhibition factor to a degree comparable to that achieved with cells from ascitic tumors. Comparative studies utilizing a methylcholanthrene-induced fibrosarcoma as well as solid and ascitic forms of P815 mastocytoma and Ehrlich tumor demonstrate that responsiveness to tumor migration inhibition factor is not merely a property conferred upon tumor cells by prior animal passage in suspension. These results provide a further suggestion that the capacity for lymphokine-induced migration inhibition is a general property of tumor cells. This in turn raises the possibility that this capacity might vary in a predictable manner with malignant potential.


Subject(s)
Cell Migration Inhibition , Fibrosarcoma/physiopathology , Lymphokines/physiology , Animals , Carcinoma, Ehrlich Tumor/physiopathology , Cell Line , Cell Movement/drug effects , Humans , Leukemia, Lymphoid/physiopathology , Male , Mast-Cell Sarcoma/physiopathology , Mice , Mice, Inbred C57BL
14.
Cancer Res ; 44(6): 2561-6, 1984 Jun.
Article in English | MEDLINE | ID: mdl-6327018

ABSTRACT

The antitumor cytotoxic mechanisms of Adriamycin-elicited peritoneal exudate cells were investigated. Peritoneal exudate cells from mice collected 1 day after an i.p. injection of Adriamycin (10 mg/kg) displayed enhanced cytotoxicity against P815 (natural killer-insensitive, macrophage-sensitive) but not YAC-1 (natural killer-sensitive) tumor cell lines. These cells contained a sufficient concentration of the drug to be cytotoxic for P815 tumor cells in 18-hr chromium release assays. Freeze-thaw lysates of these peritoneal exudate cells were found to be as cytotoxic to P815 as their corresponding whole cells. The lytic activity of these lysates was removed by centrifugation at 100,000 X g, indicating the insolubility of the effector moiety. These cells were also shown to produce significant amounts of superoxide anion and H2O2 in response to phorbol myristate acetate. A catalase-inhibitable augmentation of the cytotoxicity of these cells against P815 was observed when phorbol myristate acetate was added to the assay. Neutrophils and not macrophages were likely responsible for this effect. Peritoneal lymphocytes from mice given injections of Adriamycin 5 to 7 days previously were cytotoxic to YAC-1 tumor cells in 4-hr assays. Finally, peritoneal macrophages harvested 5 to 7 days after Adriamycin administration were cytotoxic to P815 in the absence of detectable Adriamycin. The addition of phorbol myristate acetate inhibited the lysis of P815 by these cells.


Subject(s)
Doxorubicin/therapeutic use , Leukemia, Experimental/drug therapy , Mast-Cell Sarcoma/drug therapy , Animals , Cell Adhesion , Cell Line , Cell Survival/drug effects , Cytotoxicity, Immunologic , Leukemia, Experimental/physiopathology , Macrophages/drug effects , Macrophages/immunology , Mast-Cell Sarcoma/physiopathology , Mice , Mice, Inbred Strains , Superoxides/metabolism
15.
Biol Cell ; 50(1): 1-7, 1984.
Article in English | MEDLINE | ID: mdl-6234039

ABSTRACT

Sodium butyrate induced adhesion of cultured mastocytoma p-815 cells to the surface of a standard tissue culture grade petri dish. The ratio of the number of adherent cells to that of total cells (adherent plus floating cells) was dependent on the serum concentration and on the dose of sodium butyrate. During approximately the first 6 hr after the addition of sodium butyrate, no cells adhered. The optimum conditions for adhesion were provided by 2 mM sodium butyrate and 15% fetal calf serum, 44 hr after addition of this compound. Morphologically, adherent cells consisted of spindle-shaped and round cells: the latter clustered to the former. Low concentrations of actinomycin D (0.005 microgram/mL) and of cycloheximide (0.5 microgram/mL) inhibited cell adhesion. Adherent cells were easily detached by 0.25% trypsin-0.02% EDTA but not by EDTA alone. Adherent mastocytoma cells which were cultured in the presence of 2 mM sodium butyrate, re-adhered to the surface of the dish. The ratio of adhesion in the second dish, however, was very low (35% after 2 hr incubation). Radioactive iodinated surface proteins of butyrate-treated adherent cells showed two new bands (70,000 and 92,000 D) which were not detected in control cells, but there was no difference in the extent of labeling of high molecular weight protein (250,000 D) between butyrate-treated and control cells.


Subject(s)
Butyrates/pharmacology , Mast-Cell Sarcoma/physiopathology , Animals , Butyric Acid , Cell Adhesion/drug effects , Cell Line , Cell Membrane/physiology , Culture Media , Edetic Acid/pharmacology , Kinetics , Membrane Proteins/analysis , Mice , Molecular Weight
16.
Biochim Biophys Acta ; 741(1): 77-85, 1983 Oct 13.
Article in English | MEDLINE | ID: mdl-6412755

ABSTRACT

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for 'wild-type' K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3-4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of 'wild-type' cells. In 'wild-type' K 21 cells incubated at 39.5 degrees C, thymidine kinase activity was approx. 30% of that at 33 degrees C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5 degrees C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5 degrees C to 33 degrees C was inhibited by actinomycin D and cycloheximide.


Subject(s)
Mast-Cell Sarcoma/genetics , Mutation , Thymidine Kinase/genetics , Animals , Cell Cycle , Cell Line , Cold Temperature , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Hot Temperature , Kinetics , Mast-Cell Sarcoma/enzymology , Mast-Cell Sarcoma/physiopathology , Mice , Phosphorylation , Thymidine Kinase/metabolism
17.
Chromosoma ; 88(3): 241-8, 1983.
Article in English | MEDLINE | ID: mdl-6414787

ABSTRACT

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60-75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.


Subject(s)
Cell Nucleus/physiology , Chromosomes/physiology , Mast-Cell Sarcoma/physiopathology , Nuclear Envelope/physiology , Animals , Cell Line , Cell Nucleus/ultrastructure , Centrifugation, Density Gradient/methods , Cricetinae , Cricetulus , Female , Flow Cytometry , Mast-Cell Sarcoma/genetics , Metaphase , Mice , Nuclear Envelope/ultrastructure , Osmolar Concentration , Ovary
18.
J Immunol Methods ; 55(1): 135-9, 1982 Nov 26.
Article in English | MEDLINE | ID: mdl-6818280

ABSTRACT

Transplantation of ascitic P-815 mastocytoma cells intradermally in the ears of syngeneic DBA/2J mice allows a simple and precise quantitation of tumor growth by ear swelling. Tumor growth was retarded and then arrested in allogeneic DBA/1J mice, whereas syngeneic hosts died as a result of tumor dissemination in draining lymph nodes, liver and spleen.


Subject(s)
Cell Transformation, Neoplastic , Ear Neoplasms/physiopathology , Mast-Cell Sarcoma/physiopathology , Animals , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , Dose-Response Relationship, Immunologic , Female , Graft Rejection , Hypersensitivity, Delayed/diagnosis , Mast-Cell Sarcoma/pathology , Mice , Mice, Inbred DBA , Mitomycins/pharmacology , Neoplasm Transplantation
20.
Can J Physiol Pharmacol ; 60(7): 920-4, 1982 Jul.
Article in English | MEDLINE | ID: mdl-6812935

ABSTRACT

The steady-state transmembrane potentials of P815 mastocytoma cells were recorded when the cells were bathed in salines of different compositions. In the normal growth medium (RPMI 1640 with added fetal calf serum) the mean membrane potential was -8.7 mV (SEM +/- 0.4, n = 22). A family of Tris-buffered salines (TBS), modeled from Dulbecco's modified PBS (289 mosmol, 169 milliionic strength units, pH 7.5), having different K+ and different C1- concentrations, were designed and used to bathe the tumor cells. All of the TBS solutions had constant, but reduced levels of ionized Ca2+. In the absence of external C1-, an increase of external K+ from 2 to 20 mM results in a 5.7 mV depolarization. In the presence of external C1- the same increase in external K+ results in a 2.1 mV depolarization. The presence of 145 mM C1- resulted in a steady-state depolarization (for either level of K) of about 50%. One explanation for these results would be the presence of an inward-going active C1- transport.


Subject(s)
Chlorides/pharmacology , Mast-Cell Sarcoma/physiopathology , Potassium/pharmacology , Cell Line , Humans , Membrane Potentials/drug effects
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