The synergism of the action of gamma radiation and cardiovascular preparations on lymphoid cells in culture

In experiments with lymphoid human cells Raji synergism of the effects of gamma radiation and cardiovascular drugs (e. g. valocordin, valerian, ouabain, and digoxin), administered in nontoxic doses to culture medium 15 min after irradiation (0.5, 1, and 2.5 Gy) was displayed by the inhibition of cell proliferation.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Structural properties of leukemic and normal lymphoid cells

DNA synthesis intensity and spectral and fluorescent properties of leucemic, PHA-induced and intact normal mouse spleen cells and of nuclei isolated from these cells were investigated. The cell electrophoretic mobility and DNA-protein interaction in the nuclei were studied. Similarity in cell and nuclei fluorescence, fluorescence of the probe ANS conjugated with the cells, the electrophoretic mobility and tightness of DNA--protein interaction for leucemic and PHA--induced cells and also the similarity of the tightness of DNA--protein interaction for leucemic and normal intact cells were found inspite of the differences in DNA synthesis intensity and cell functional peculiarities.     

Increase in histone poly (ADP-ribosylation) in mitogen-activated lymphoid cells

Poly (ADP-ribosylated) histones appear to be intermediates in nuclear processes that involve DNA strand breaks. We have studied histone ADP-ribosylation in cellular lysates from activated human lymphoid cells in culture. Modified histones differing in the number of ADP-ribose groups gave separate bands upon two-dimensional gel electrophoresis. Cellular lysates from control cells contained histones modified with 1 to 15 ADP-ribose groups. Stimulation of the cells during culture with phytohemagglutinin (PHA) or a phorbol ester (TPA) as well as combinations of these two reagents led to a significant increase in the upper limit number of ADP-ribose groups attached to histones in the presence of divalent metal ions. Hyper (ADP-ribosylated) H2B carrying at least 32 ADP-ribose groups gave a distinctly characteristic pattern on two-dimensional gels showing that highly ordered enzymatic steps are followed for its synthesis. Moreover, it was found that PHA and/or TPA induces branching of the poly (ADP-ribose) on H2B. The increase in histone poly (ADP-ribosylation) following lymphocyte activation was less dramatic during incubation of cellular lysates in the absence of divalent metal ions. The increased histone modification observed in this study may result from an increase in cell proliferation during activation of lymphoid cells. The finding that the number of ADP-ribose groups on H4 equals or exceeds by one the number of acetyl groups suggests that the two modifications may share common functions.     

Effect of thyrotropin on the properties of messenger RNA from cultured human thyroid cells

When added to the culture medium of thyroid cells isolated from diffuse nontoxic goiter, thyrotropin increased the poly(adenylic acid) content and the template activity of the unfractionated RNA. This increase was correlated with higher thyroglobulin messenger activity, as demonstrated by specific immunoprecipitation of the labeled peptides synthesized in two heterologous cell-free systems. When RNAs were separated in a sucrose gradient, thyrotropin was shown to enhance the poly(adenylic acid) content and template activity of fractions with sedimentation coefficients of 34, 23 and 15 S. Specific immunoprecipitation showed that a thyroglobulin messenger activity was present in these three fractions. Another way by which thyrotropin regulates the thyroid protein synthesis is suggested by the shift of poly(adenylic acid)-containing RNA to large polysomes when thyroid cells were cultured in the presence of the hormone.     

Minimum estimate of the lifetime of histone messenger RNA of HeLa cells

The lifetime of histone mRNA of HeLa cells has been studied by its kinetic of approach to steady-state labeling. Cells preincubated with low concentrations of actinomycin d to inhibit rRNA synthesis, were incubated with (³H)uridine. Linear incorporation of uridine was observed for only two hours under the conditions chosen. Polyribosomes were isolated from cells incubated overnight with trace amounts of (¹⁴C)uridine and for 30 to 150 min with (³H)uridine. RNA was extracted from polyribosomes and fractionated by polyacrylamide gel electrophoresis. Histone mRNA was identified as a peak migrating in a characteristic position, which was absent in gels of RNA obtained from cells treated with the inhibitor of DNA synthesis cytosine arabinoside. The kinetic of labeling of histone mRNA was linear up to 150 min, which represents a minimum estimate of the lifetime of this mRNA.Abbreviations MPB 2,mercapto-1 (-pyridethyl)benzimidazole - EDTA ethylene diamino tetracetic acid - SDS sodium dodecyl sulphate     

RNA extracts of lymphoid cells sensitized to DNP-oligolysines convert nonresponder lymphoid cells to responder cells which release migration inhibition factor

Strain 13 nonresponder peritoneal exudate cells were converted to responder status to α or ?,DNP-oligolysines after incubation of the cells with RNA extracts prepared from responder guinea pigs skin test sensitive to these synthetic antigens. The conversion of nonresponder strain 13 cells was assessed by the direct cell migration inhibition correlate of delayed hypersensitivity. Nonresponder cells were not converted by RNA extracts prepared from unimmunized responder guinea pigs or from non-responder strain 13 guinea pigs previously injected with DNP-oligolysines. Thus, it seems possible to correct immunological unresponsiveness in vitro in spite of a specific genetically determined deficiency of the immune response related to the Ir gene.     

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.     

Chlorambucil-sensitive and -resistant lymphoid cells display different responses to the histone deacetylase inhibitor, sodium butyrate

Clinical chemoresistance is a frequent complication of alkylating agent treatment of malignant tumours. Chromatin remodelling using histone deacetylase inhibitors (e.g., sodium butyrate, NaBu) may increase target cell chemosensitivity. Apoptotic responses and expression of chromatin modifying enzymes in lymphoid cell lines, LP-1 and NCI-H929, to chlorambucil (CLB) and/or NaBu were examined in this study. NaBu augmented the apoptotic response in CLB-resistant LP-1 cells but antagonised it in CLB-sensitive NCI-H929 cells. CLB increased expression of methyltransferase I and histone acetyltransferase I in both cell lines while NaBu had only small effect. CLB-induced increased gene expression was attenuated by NaBu in CLB-sensitive NCI-H929 cells but not in resistant LP-1 cells. These results suggest that chromatin modifying agents may have differential effects on cells depending on their chemosensitivity.