GRSL Cells Research Articles

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal–isobaric conditions (310.15K and 1atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes.

Phorbol ester-inducible T-cell-specific expression of variant mouse mammary tumor virus long terminal repeats

Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, we made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. In XC rat fibrosarcoma cells, neither normal nor variant LTRs gave rise to detectable CAT activity, either in the presence or in the absence of TPA, but dexamethasone strongly stimulated CAT activity driven by N and L42 LTRs. The L13 LTR was considerably less active, probably caused by the deletion of the distal part of the glucocorticoid responsive element. We conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.

Rapid detection of leukemia cells by use of a complement-mediated cytolytic reaction and an imaging sensor system.

We describe a system for detection of leukemia cells involving complement-mediated cytotoxic reaction and an image processing system, consisting of a charge-coupled-device image sensor, an image memory board, a personal computer, and a phase-contrast microscope. Then added to a cell suspension, monoclonal antibody specific to the fetal thymus antigen-1 of the mouse leukemia GRSL cell produced cytolysis of only GRSL cells. This cytolysis decreased the brightness of the cells observed by phase-contrast microscopy. The remaining brightness was subtracted from that of the phase-contrast image of the cells before cytolysis, which had been converted to a digital signal and stored in computer memory. Measurement time is 2 s. The time course for complete GRSL cytolysis, as measured with this system, is 12 min; overall measurement time, including reaction time, is approximately 15 min. GRSL cells in a suspension of mixed cells were determined specifically by the system.

Catalytic hydrogenation of fatty acyl chains in plasma membranes; effect on membrane lipid fluidity and expression of cell surface antigens

Optimal reaction conditions were established for hydrogenation of plasma membranes of living murine GRSL leukemia cells, using the water-soluble catalyst Pd(QS) 2 (QS, sulphonated alizarine; C 14H 6O 7NaS). Under these conditions more than 80% of the cells remained viable. Analysis by gas chromatography revealed that hydrogenation occurred predominantly in the 18:2, 20:4 and 22:6 fatty acyl chains of the membrane phospholipids. Under the same conditions hydrogenation was also performed in purified plasma membranes from GRSL cells and from rat liver, and in liposomes prepared from the total lipid extracts of these membranes. Hydrogenation increased the lipid structural order parameter in the membranes, as measured by fluorescence polarization. This increase was more pronounced in the liposomes (46%) than in the plasma membranes (17–25%). Hydrogenation increased the expression of a 15 kDa antigen on the surface of viable GRSL cells, as measured in a Fluorescence Activated Cell Sorter, using monoclonal antibodies. The expression of four other antigens, among which H-2 k , was not or much less affected by this treatment.

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16.

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.

Antigenic modulation of mammary tumour virus envelope antigen or GR thymic lymphoma cells in relation to expressions of H-2, TL cell-surface antigens and THY1.

The MLr antigen, a mammary tumour virus-induced antigen on the surface of GR thymic lymphoma cells (GRSL) can be modulated from the cell surface upon incubation with specific antiserum for 1-2 h at 37 degrees C, followed by washing the cells. In contrast, a number of other cell-surface antigens on these GRSL cells cannot be modulated under similar conditions. These antigens include histocompatibility antigens of the H-2 complex (H-2.8 of the K-end and H-2dx(D) of the H-2dx haplotype) and two thymic markers, TL1.2 and Thy1.2. Antigenic modulation of MLr as tested by trypan-blue exclusion and by chromium51 release does not lead to a measurable change in the expression of H-2K, H-2D, TL and Thy1.2 antigens. These results could be confirmed by absorption analysis. The latter analysis showed that the number of antigenic sites per cell are about the same for MLr and the two H-2 antigens, while TL antigens are scarcer and Thy1.2 antigens are more abundant. The procedure of antigenic modulation showed that the MLr antigen resides on MTVgp52, the major protein of the envelope. There was no evidence of internal proteins, such as MTVp27, on the surface of GRSL cells.

Impaired Maturation of Mouse Mammary Tumor Virus Precursor Polypeptides in Lymphoid Leukemia Cells, Producing Intracytoplasmic A Particles and No Extracellular B-Type Virions

Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.

Membrane fluidity, capping of cell-surface antigens and immune response in mouse leukaemia cells

Transplantation of primary GRSL cells in the ascitic form led to a decrease in membrane microviscosity as measured by the fluorescence polarization technique. The transplanted GRSL ascitic cells showed a markedly lower ability to form caps with respect to both virus-related (MLr, GIX) and normal (H-2.7(G), H-2.8(K) and TL1.2) cell-surface antigens and their appropriate antisera in the indirect membrane immunofluorescence tests, than did primary GRSL cells, transplanted GRSL cells growing in solid form, and thymocytes, which all exhibited significantly higher membrane microviscosities. Transplantation of primary GRSL cells into syngeneic mice pre-irradiated with 400 rad did not lead to a fall in membrane microviscosity. It is suggested that the host immune response in intact mice leads to a selective survival of ascitic tumour cells with low membrane microviscosity.

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, η , of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, η was also determined in isolated plasma cell supernatants. For isolated plasma membranes of thymocytes and GRSL cells η values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively. Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane. Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types η (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.