Apoptosis of murine BW 5147 thymoma cells induced by cold shock.

Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation.     

The cellular apoptosis of murine BW5147 thymoma during cold shock]

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.     

Apoptosis of murine BW 5147 thymoma cells induced by dexamethasone and gamma-irradiation

The mode and the kinetics of the death of T-thymoma cells upon dexamethasone treatment and gamma-irradiation (10Gy) have been studied using flow cytometry and biochemical analysis. It has been shown that the hormone and gamma-irradiation induce cell death by apoptosis. In both cases the cells are initially blocked in G2/M and die only after overcoming the blockage and cytokinesis. A short exposure to dexamethasone results in a cytostatic effect, whereas a cytotoxic effect is absent. Reducing serum concentration to 2% causes more rapid death both following gamma-irradiation and dexamethasone. These results are discussed in relation to cell death and proliferation.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

The programmed cell death of murine BW5147 thymoma under the action of dexamethasone]

By flow cytometry, imitation modelling and biochemical analysis, the mode and kinetics of dexamethasone-treated T-lymphoma cell death were studied. The hormone was shown to induce delays in pre- and postsynthetic phases of the cell cycle and the death of part of cells. A short exposure to dexamethasone reveals its cytostatic rather than cytolytic effect. Following G2/M delay and cytokinesis, part of cells dies. A reduced serum concentration (2%) causes shorter delays in the cell cycle and a more rapid cell death. Dexamethasone stimulates apoptosis which is indicated by internucleosomal DNA fragmentation, and by a coincidence in time of the processes of DNA degradation and increase in the other membrane permeability. These results are discussed in relation to the cell death and proliferation.     

Suppressor factor produced by neonatal mouse spleen cells

Spleen cells from 5- to 6-day- old BDF₁ mice produce a soluble suppressor factor (SF) when cultured for 2–4 days in the presence of 10% fetal calf serum. This suppressor factor inhibits the mixed lymphocyte reactivity of adult mouse spleen cells as well as the in vitro generation of cytotoxic cells. The SF which is not H₂-restricted or antigen specific is most effective when added in the early phase of the culture period. The SF is resistant to heat and uv treatment and appears to consist of a large and small component. It is resistant to treatment with pronase or trypsin. The SF appears to be produced by neonatal spleen cells which are not adherent to plastic or Sephadex G-10 and are insensitive to treatment with anti-Thy 1.2 and complement. Incubation of SF with peritoneal exudate cells reduces suppressor activity.     

Reproductive death of the apoptosis type in irradiated cells of the BW5147 thymoma

The method of flow cytofluorometry and biochemical analysis were used to study the pattern and kinetics of the postirradiation death of proliferating BW5147 lymphoid cells. Irradiation with a dose of 10 Gy was shown to induce thymoma cell death by apoptosis. Radiation-induced synchronous transfer of part of cells from G1 to S-stage and blocking of all cells at G2/M stages of the cell cycle preceded the cell death. Decreasing of the growth factor content in a medium through its depletion or cultivation in conditions of low serum content accelerated cell death. A possible relationship between cell death and proliferation is discussed.     

Uptake and nature of the intracellular binding of cyclosporin A in a murine thymoma cell line, BW5147

In a survey of malignant cell lines including a variety of leukemias and lymphomas, BW5147, a T lymphoma from the spontaneous virus-associated thymoma in AKR mice, was found to be the most sensitive to growth inhibition by cyclosporin A (Cs A). Inhibition of growth was cell cycle phase-independent and inhibition of macromolecular precursor uptake was relatively nonspecific. Uptake of radiolabeled Cs A by these cells was characterized by two components: one that appeared saturable at low drug concentrations (0.03 to 1.0 microgram/ml), and another that was nonsaturable at higher drug concentrations (1.0 microgram/ml or higher). Most of the drug concentrated by cells (70 to 80%) was located in the cytosol (100,000 X G supernatant of lysed cells). The apparent m.w. of the drug-macromolecule complex was 15,000 to 20,000 as determined by m.w. exclusion columns. This complex could also be formed by adding drug to cytosol prepared from unexposed cells. The low m.w. complex migrated on a preparative isoelectric focusing column to form two peaks with isoelectric points of 6.8 and 8.5. A method was developed to assay for the binding component, and a sequence of m.w. exclusion columns and isoelectric focusing was used to effect partial purification of the Cs A binding component.     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

Production of thymocyte-stimulating factor by murine spleen lymphocytes: cellular and biochemical mechanisms.

Cultures of murine spleen lymphocytes treated with Thy 1.2 antiserum plus complement do not produce thymocyte-stimulating factor (TSF). The population of thymocytes composed of immunocompetent, low-density cells produces only small amounts of TSF. Experiments with cyclophosphamide-injected mice and with spleen cells treated in vitro with antiserum to the murine B lymphocyte antigen plus complement and experiments using spleen cells stimulated in vitro with Sepharose-bound phytohemagglutinin indicate that B lymphocytes neither cooperate with T lymphocytes for the production of TSF nor produce TSF. Some lectins (pokeweed mitogen, Lens culinaris hemagglutinins A and B) have been found to induce the production of TSF by spleen cells. Other lectins (wheat germ agglutinin, Agaricus bisporus agglutinin) and sodium periodate do not. Spleen cells of mice immunized in vivo with keyhole limpet hemocyanin bound to bentonite particles or with BCG produce TSF when challenged in vitro with the specific antigen. Experiments using inhibitors of the macromolecular metabolism showed that DNA synthesis is not required for the production of TSF by spleen lymphocytes, whereas RNA and protein synthesis are required. Resolution of spleen lymphocytes on a discontinuous albumin gradient into six subpopulations showed that the TSF activity was rather uniformly distributed among the various subpopulations of cells.     

Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

NBxFO factor--a novel suppressor factor produced by fusion of neonatal spleen and a nonsecreting myeloma

Spleens from 1-day-old DBA/2J mice were fused to the nonsecreting myeloma, FO. Dialyzed supernates of these cells were found to suppress the antigen-specific proliferative response of cloned helper or alloreactive T cells at a final concentration of less than or equal to 5%. The same supernate-containing factor did not suppress the response to IL-2 of an IL-2-addicted T cell line. The factor was found not to suppress the production of either IL-2 or antibody, following stimulation of spleen cells with LPS. Absorption analysis revealed that the target of the factor was the accessory cell population. Further experiments indicated that the factor blocked the proliferation of thymocytes due to IL-1. Biochemical analysis revealed a molecular weight for the factor of about 90,000 and a pI of approximately 4.5.     

Isolation of a colony-stimulating factor produced by L 1210 murine leukemia cells

Murine L1210 leukemia cells spontaneously produce very low amounts of colony stimulating factor (CSF). CSF production was markedly increased by stimulating L1210 cells with lipopolysaccharide, lectins, and sheep red blood cells. From the conditioned medium of phytohemagglutinin-stimulated L1210 cells we isolated a CSF with an apparent molecular weight of approximately 27,000. This CSF promoted the proliferation and the differentiation of murine GM-CFU showing a weak differentiation-inducing activity on WEHI-3 D (+) cells.     

Biochemical characterization of murine leukaemia inhibitory factor produced by Krebs ascites and by yeast cells

A cDNA library was constructed using mRNA from Krebs ascites tumor cells that was shown by Northern blot hybridization to contain mRNA for murine leukemia inhibitory factor (LIF). This library was screened with an oligonucleotide corresponding to the 3' end of a partial LIF cDNA clone, and an overlapping cDNA clone isolated. Nucleotide sequence analysis of this latter clone allowed the complete sequence of LIF to be derived. A cDNA molecule encoding the entire mature LIF protein was installed in a yeast expression vector, and LIF produced up to about 100 ng/ml in the growth medium. The LIF produced by yeast cells has the same biologic properties as native LIF and competes with native 125I-LIF for binding to specific cellular receptors. Two forms of native LIF, distinguishable by their chromatographic behavior on DEAE-Sepharose, were converted by neuraminidase treatment to a form with similar chromatographic behavior, suggesting that the major difference between these two species is the content of sialic acid on the carbohydrate portion. Moreover, yeast-derived recombinant LIF appears to display a different pattern of glycosylation to both forms of native LIF. From in vitro experiments, we conclude that the nature of the glycosylation is not crucial to biologic activity.     

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.     

Properties and purification of a colony-stimulating factor of granulocytes and macrophages produced by mouse spleen cells

Mouse splenocytes are induced by pokeweed mitogen to secrete a factor that stimulates mouse hemopoetic (spelling per Nomina Histologica in the Nomina Anatomica, 5th edition, 1983, Williams and Wilkins, Baltimore) progenitor cells to undergo proliferation and differentiation into granulocytes and macrophages in a semi-solid culture system. The granulocyte and macrophage colony-stimulating factor (GM-CSF) was purified with a four-step procedure that includes ultrafiltration, chromatography on DEAE-agarose, Sephacryl S-200, and chromatofocusing gel. The isoelectric point (pI) of 4.2 of the GM-CSF was determined by analytical isoelectrofocusing gel electrophoresis. The sensitivity of the biological activity of GM-CSF to digestion by trypsin and neuraminidase suggests that GM-CSF is a glycoprotein with its sugar moieties at the active site. The GM-CSF is also sensitive to heat denaturation at 60 degrees C or higher suggesting that a three-dimensional conformation is required for its biological activity. The molecular weight of GM-CSF is approximately 57,000 Daltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.