The effect of ionizing radiation on lipid metabolism in lymphoid cells

Lipid metabolism was studied in lymphoid tissues of rats after whole body irradiation with doses producing damage of different degrees to lymphoid cells (4-10 Gy). The content of free cholesterol, cholesterol esters, and total phospholipids was determined in peripheral blood lymphocytes and thymocytes 1-2 h after exposure. Simultaneously, the rate of in vitro incorporation of 2 14C-acetate into total lipids, phospholipids, and cholesterol of lymphoid cells was estimated. It was shown that exposure of rats to ionizing radiation caused activation of lipogenesis. Cholesterol synthesis was activated after a dose of 4 Gy and decreased with increasing dose.     

Effect of membrane fatty acid changes on the radiation sensitivity of human lymphoid cells

To test the influence of changes in membrane fatty acid composition on the radiation response of mammalian cells, human LDV cells were cultured in medium containing delipidated serum supplemented with either oleic or linoleic acid. Analysis of lipid extracts of the cells by gas liquid chromatography showed that, after 3 or more days growth in oleic or linoleic acid supplemented media, there were substantial overall increases in the proportion of oleate and linoleate, respectively, in the cellular lipid. Smaller absolute changes were measured in nuclear phospholipid, which was found to be low in unsaturated phospholipid compared to the rest of the cell. Fluorescence polarization measurements using diphenylhexatriene indicated an increased membrane fluidity in cells grown in the presence of excess linoleic acid and to a lesser extent for oleic acid. The clonogenic capacity of the cells after irradiation in air or nitrogen was not altered by any of these membrane compositional changes. The lack of effect on radiation sensitivity, in contrast to that reported for bacteria (E. coli K1060), is consistent with the fact that little or no change was brought about in the nuclear membrane composition, since evidence from partial cell irradiation experiments indicates that the cell nucleus is the sensitive target for cell killing by ionizing radiation. The ability of the cell to maintain a low level of unsaturated phospholipids in its nuclear membrane may be an important general defence mechanism against free radical damage to chromatin mediated by lipid peroxidation.     

Plasma membrane sterol distribution resembles the surface topography of living cells

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8-2.5 microm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1--a putative raft marker--did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane.     

Human autologous rosette-forming cells. I. Expression of cell surface antigens in relation to age and lymphoid organ distribution

Autologous rosette-forming cells (auto-RFC) were characterized with monoclonal antibodies to various cell surface antigens using a technique combining immunofluorescence and rosette formation. In peripheral blood, auto-RFC were T cells (Leu 1+/OKT3+) the majority being derived from the helper/inducer subset (Leu 3a+/OKT4+). A small proportion of the circulating auto-RFC were Leu 2a+/OKT8+ and virtually none of them bore T10, T6, and DR antigens or peanut agglutinin (PNA) receptors. In the elderly, the percentages of Leu 3a+ auto-RFC increased significantly along with the augmentation of the Leu 3a+ circulating pool. After Con A stimulation of peripheral blood lymphocytes the autorosette population was expanded and therefore their phenotype was again that of helper cells. In the thymus, high levels of autorosettes are found (30 to 50%). Simple or double labeling of the rosetting cells with various monoclonal antibodies permitted the confirmation of the existence of distinct thymocyte subpopulations and moreover to identify the location of the auto-RFC in the intrathymic differentiation scheme. Nearly 70% of the rosetting cells were derived from common thymocytes, those cells defined by the coexpression of T10, T6, T4, and T8 antigens whether or not they were also stained by OKT3 antibodies. The remaining auto-RFC were found with similar frequency among the T4+ and T8+ mature thymocytes. In the spleen low percentages of auto-RFC were found and the majority resided in the Leu 3a+/OKT4+ population, similarly to peripheral blood autorosettes. Taken together, these data suggest that the expression of autologous erythrocyte receptors is acquired in the thymus and is gradually lost during T-cell maturation.     

Molecular mechanisms of radiation death of lymphoid cells. Radioprotective effect of cysteamine on a subpopulation of thymocytes differing in radiation sensitivity

The radioprotective effect of cysteamine, incubated with thymocytes for 20 min prior to gamma-irradiation, was much more manifest with regard to subpopulations of radiosensitive small cortical cells (DMF = 1.8) than large thymocytes (DMF = 1.2). Radioprotective concentrations of cysteamine relaxed completely superhelical DNA of thymocytes. The relaxation was not due to the formation of single-strand breaks. A partial recovery of superhelical conformation of DNA of small thymocytes before irradiation reduced the radioprotective effect of cysteamine.     

Demonstration of a surface antigen on Epstein-Barr virus genome-carrying lymphoid cells: distinction from the virus-determined membrane antigen.

A surface antigen (SA) was detected on Epstein-Barr virus (EBV)-carrying lymphoid cell lines by indirect membrane immunofluorescence with an antiserum from a rabbit immunized with Raji cells; the antiserum had been extensively absorbed with normal human blood and tonsil cells. The SA was not detected on normal human umbilical cord and adult peripheral blood lymphocytes or EBV-negative cell lines. Incidences of the SA and EBV-determined membrane antigen (MA) on certain EBV-carrying cell lines were not compatible. Antibody against SA was differentially absorbed by the SA-positive MA-negative cell lines whereas MA antibody was absorbed by MA-positive SA-negative cell lines. The results of cross-absorption tests of antiserum against Raji cells or P3HR-1 cells suggested that SA may contain more than one antigenic determinant.     

Differential distribution of calpain in human lymphoid cells

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both Calpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of Calpain (55–80%) whereas the monocytic line (U-937) showed prodominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.     

The subcellular distribution of adenylate and guanylate cyclases in murine lymphoid cells.

Membrane vesicles can be prepared from murine lymphoid cells by nitrogen cavitation and fractionated by sedimentation through nonlinear sucrose density gradients. Two subpopulations of membrane vesicles, PMI and PMII, can be distinguished on the basis of sedimentation rate. The subcellular distribution of adenylate and guanylate cyclases in these membrane subpopulations have been compared with the distribution of a number of marker enzymes. Approximately 20-30% of the total adenylate and guanylate cyclase activity is located at the top of the sucrose gradient (soluble enzyme), the remainder of the activity being distributed in the PMI and PMII fractions (membrane-bound enzyme). More than 90% of the 5'-nucleotidase and NADH oxidase activities detected in lymphoid cell homogenates are located in PMI and PMII fractions, whereas succinate cytochrome c reductase activity is detected only in the PMII fractions. In addition, beta-galactosidase activity is distributed in the soluble and PMII fractions of the sucrose density gradients. On the basis of the fractionation patterns of these various enzyme activities, it appears that PMI fractions contain vesicles of plasma membrane and endoplasmic reticulum, whereas PMII fractions contain mitochondria, lysomes, and plasma membrane vesicles. Approximately 30-40% of the adenylate and guanylate cyclase activities in PMII can be converted to a PMI-like form following dialysis and resedimentation through a second nonlinear sucrose gradient. Adenylate and guanulate cyclases can be distinguished on the basis of sensitivity to nonionic detergents.     

Abscopal action of gamma radiation in the death of lymphoid cells

In the in vitro experiments with lymphoid cells Raji and X63-Ag8.653 gamma-irradiated in a synthetic medium, it was shown that a cessation of division (reproductive death) and lysis (interphase death) of cells were evoked by the abscopal effect of long-lived quinoid radiotoxins enhancing the direct effect of radiation to make it not additive but synergistic: a synergism coefficient was 1.3.     

Changes in the organization and biosynthesis of cell surface acidic sugars during the phytohemagglutinin-induced blast formation of human T-lymphocytes

Use of cell electrophoresis combined with specific enzymes and varying ionic strength revealed a topological change of acidic sugars in lymphocyte membrane treated with a T-cell mitogen, phytohemagglutinin (PHA). The suggested alterations were an early translocation of hyaluronic acid to the cell periphery within 15 min of PHA addition and, 4 h later, the appearance of chondroitin sulphate in T-lymphocytes, but not in B-lymphocytes. As the contribution of chondroitin sulfate to the electrophoretic mobility increased with time up to 24 h, that of sialic acid decreased conversely. Several agents which block blast formation (2 mM ethylene glycol bis-β-aminoethylethyl-N,N,N′,N′-tetraacetic acid, 2 × 10⁻⁷ M ouabain, 0.1 μg/ml colchicine and 1 μg/ml cytochalasin B) also blocked the translocation of hyaluronic acid at the same concentrations. Chemical analysis of [¹⁴C]glycosaminoglycans by means of gel filtration followed by paper chromatography revealed a four-fold enhancement of the biosynthesis of chondroitin sulfate C after PHA stimulation. The presence of chondroitin sulfate in the cell periphery was also detected electrophoretically in T-cell type leukemia cells (MOLT-4B). These results suggest that the reorganization of glycosaminoglycans may be one of the membrane changes associated with blast formation of lymphocytes.     

Hexokinase isozyme distribution and regulatory properties in lymphoid cells

The glycolytic enzyme hexokinase is studied in cultured leukemic lymphoblasts, in normal lymphocytes and in lymphoblasts obtained by stimulation of normal lymphocytes with phytohaemagglutinin.Hexokinase activity levels in cultured lymphoblasts and in normal lymphocytes are identical, but somewhat higher levels are found in stimulated lymphocytes. Cultured leukemic lymphoblasts differ in isozyme content in comparison to the other lymphoid cells. Besides hexokinase I, which is detected in all the lymphoid cells, they are characterized by the presence of hexokinase II. The concentration of type II increases during cell growth. Another difference between leukemic lymphoblasts and mature and stimulated lymphocytes is found in the regulatory properties of hexokinase I. Hexokinase I from both normal and stimulated lymphocytes is inhibited by glucose-1,6-diphosphate. This inhibition is decreased in part by addition of inorganic phosphate. Hexokinase I from leukemic lymphocytes, however, is inhibited to a lesser extent by glucose-1,6-diphosphate. Inorganic phosphate has no effect at all on this inhibition.In accordance with these findings a different pattern in the hexokinase I region was detected in electrophoresis with several cell types. The subisozyme hexokinase Ib, which appears to be the phosphate-regulated form, is predominant in lymphocytes, whereas it is present in a minor fraction in the cultured leukemic lymphoblasts. In these cells hexokinase Ic predominates.     

Phenotypic heterogeneity for cells bearing surface immunoglobulin in human lymphoid lines.

Long-term cultures of human lymphoid lines, even after subcloning, were found to contain a mixture of surface immunoglobulin (SIg) positive and negative cells using three different techniques. Cell culture (TM) was mixed with anti-Ig antisera plus complement to lyse SIg positive cells. The remaining SIg negative viable cells were separated on a bovine serum albumin gradient, cultured, and sampled at different time points. Within 24–48 hr the same percentage of cells were SIg(+) as prior to treatment with antisera plus complement. Culturing the same cells with frequent additions of fresh or “spent” culture media failed to alter the ratio of Ig positive/ negative cells. Mechanisms for in vitro regulation of SIg are discussed.     

Detection of thymic surface antigens and radioactive labeling of mouse lymphoid cells.

A method for the permanent and simultaneous detection of tissue-specific surface antigens and internal radioactive labeling of mouse lymphoid cells is described. Target cells were first reacted with a mouse-derived "antithymocyte serum", incubated with peroxidase-conjugated rabbit serum against mouse immunoglobulins and placed in a substrate solution that leads to staining of the antigen-positive cells. Radioactive labeling was demonstrated by autoradiography performed after the antigen assay. More than 90% of antigen-positive thymocytes could be specifically stained in the assay without staining of similarly treated antigen-negative cells. Autoradiographic grains could be detected over both antigen-positive and antigen-negative cells.     

Biosynthesis of membrane bound Ig and secretion of Ig by chicken lymphoid cells.

The metabolic turnover of membrane proteins of chicken lymphoid cells is studied, using a double isotope labeling technique (i.e., [14C]amino acid pulse and [3H]leucine chase). Compared with other membrane proteins, the metabolic turnover of membrane bound immunoglobulins (M-Ig) is very slow. There was no difference in the turnover between M-Ig and specific antigen binding receptor immunoglobulins. Immunoglobulins appear to be a stable constituent of the lumphocyte membrane. Cellular kinetic experiments show that the rate of biosynthesis of secreted immunoglobulins (S-Ig) is nearly ten times as much as that of M-Ig, suggesting that metabolic pathway leading to M-Ig are distinct from those leading to S-Ig. The difference in 3H/14C ratios between S-Ig and M-Ig reflects the rate of biosynthesis of these immunoglobulins by two types of bursa derived lymphoid cells.     

Abnormalities in the erythrocyte membrane in acute lymphoid leukaemia.

Erythrocytes from patients suffering from acute lymphoid leukaemia (ALL) show decreased proportions of spectrin tetrameters and altered spatial distribution of band 4.1 and ankyrins. These abnormalities of the cytoskeleton are probably responsible for altered membrane fluidity and transbilayer distribution of phosphatidylethanolamine in ALL. ALL is associated with severe anaemia and usually, but not always, with overproduction of lymphocytes. To our knowledge, this is the first report of abnormalities in the erythrocyte membrane in ALL which may, in part, be responsible for the observed anaemia.     

The effects of radiation and alkylating agents on chromatin degradation in normal and tumour lymphoid cells.

The dynamic of chromatin degradation was studied in thymocytes and LS/BL tumour cells. In permeabilised LS/BL cells, the rate of DNA degradation induced by endogenous calcium and magnesium-dependent endonuclease was approx. 25 times slower than in thymocytes. In LS/BL cells irradiation does not induce chromatin degradation. The alkylating agent TS 160 induced chromatin degradation in both LS/BL lymphosarcoma cells and thymocytes.