Reactive chemiluminescence of human lymphocytes during phytohemagglutinin stimulation

The luminol-mediated chemiluminescence of highly purified human peripheral blood lymphocytes has been studied. The incubation of lymphocytes with phytohemagglutinin has lead to dose-dependent chemiluminescence, most pronounced by the end of the first minute. The kinetics of the reaction does not depend on the dose of the stimulant, the concentration of lymphocytes or the presence of mononuclear phagocytes. Monocytes (adhering cells) are not stimulated with phytohemagglutinin though their presence in the total fraction of mononuclear blood cells enhances the chemiluminescence of lymphocytes.     

Interchromatin granules during phytohemagglutinin stimulation of human lymphocytes

Summary We studied the formation of interchromatin granules (IGs) in phytohemagglutinin (PHA)-stimulated lymphocytes. The bismuth staining method was used for the visualization of IGs, and we also applied high-resolution autoradiography after incubating cells in the presence of³H-leucine during different stages of lymphocyte activation. The disaggregation of chromatin and the enlargement of interchromatinic areas in stimulated lymphocytes were found to be accompanied by an increase in the number of IGs, and it was shown that IGs were formed during all of the investigated stages of lymphocyte stimulation.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Protein synthesis in resting and stimulated human lymphocytes

Summary The ribosomal profiles in lysates from resting and phytohemagglutinin stimulated human lymphocytes have been analyzed by sucrose gradient centrifugation. The percentage of polyribosomes increased during lymphocyte transformation reaching a maximal value of 60 to 70% of the total ribosomes after 72 hours of mitogen addition. This time period coincides with maximalin vivo protein synthesis. On the other hand, in nonstimulated lymphocytes, about 25% of the ribosomal particles appeared as aggregates, independently of the incubation period.Experiments performed with homologous cell free systems containing ribosomes and supernatant fluids prepared from unstimulated or activated lymphocytes demonstrate that the mixtures containing both components from stimulated lymphocytes are several fold more active in polypeptide synthesis than the systems which contain ribosomal particles and cell sap from resting cells. Assays carried out with mixtures combining the components from both sources indicate that the increased activity depends on ribosomes as well as on the supernatant fractions.Dedicated to Professor LUIS F. LELOIR on the occasion of his 70^th birthday.     

Protein phosphorylation in human peripheral lymphocytes - stimulation by phytohemagglutinin and N6 monobutyryl cyclic AMP.

Protein phosphorylation was studied in human peripheral lymphocytes. The cells were preincubated with

, exposed to phytohemagglutinin (PHA), N⁶ monobutyryl cAMP (MBcAMP) or 8 bromo cGMP (BcGMP), homogenized and analyzed by SDS-polyacrylamide gel electrophoresis. Both PHA and MBcAMP produced early increases in the [³²P]content of multiple proteins in the 30,000–100,000 molecular weight range. After further incubation with PHA there was a shift in the phosphorylation response to smaller molecular weight proteins. BcGMP (1 mM-10 pM) had no effect on protein phosphorylation. These results suggest a role for cAMP in the early action of PHA on human peripheral lymphocytes.     

Activation of c-myb expression by phytohemagglutinin stimulation in normal human T lymphocytes.

The expression of c-myb in normal human T lymphocytes directly derived from a normal subject and not adapted to continuous growth in culture was found to be markedly increased after phytohemagglutinin stimulation. In the same cells, the expression of c-myc mRNA is a much earlier event compared with the appearance of c-myb mRNA, which takes place soon after that of histone H3 mRNA. The increase in c-myb expression was not due to a particular T-lymphocyte subset, as shown by in situ hybridization assays.     

Interchromatin granules during phytohemagglutinin stimulation of human lymphocytes. A cytochemical study

We studied the formation of interchromatin granules (IGs) in phytohemagglutinin (PHA)-stimulated lymphocytes. The bismuth staining method was used for the visualization of IGs, and we also applied high-resolution autoradiography after incubating cells in the presence of 3H-leucine during different stages of lymphocyte activation. The disaggregation of chromatin and the enlargement of interchromatinic areas in stimulated lymphocytes were found to be accompanied by an increase in the number of IGs, and it was shown that IGs were formed during all of the investigated stages of lymphocyte stimulation.     

Metabolism of proliferating and resting cells]

This review concerns the modern trends and experimental approaches to the study of cell cycle (cell population kinetics, cell structure and functions at various steps of the cycle,, etc.) and their input into the current views of cell proliferation controls. The resting state of the cell is considered and metabolic features of proliferating and resting cells are compared. Evidence is presented that resting cells are metabolically active and less resistant to the damaging factors that it has been previously supposed. The importance of this finding for biology and medicine, especially for cancer chemotherapy, is discussed.     

Unique acceptors for poly(ADP-ribose) in resting,proliferating and DNA-damaged human lymphocytes

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.     

Changes in proteolytic activities of human leukemic promyelocytes (HL-60 cells) during maturation

Proteolytic activity was measured in human leukemic promyelocytic cell line (HL-60) grown in culture, before and after the addition of agents which promote differentiation. The 36000 X g soluble fraction of the cells degraded [14C]globin with maximal activity at pH 3.6, while the insoluble fraction had a pH optimum at 8.0. This pattern did not change upon differentiation. The acid protease activity of the soluble fraction increased following differentiation. After 4 days in the presence of dimethyl sulfoxide, the differentiated cells exhibited 4-fold higher specific activity as compared with 4 day-old control cells. In contrast, the alkaline activity of the insoluble fraction of the differentiated cells was 4-fold lower than that of the undifferentiated cells. It is suggested that the changes in enzyme activities may serve the new functions acquired by the mature granulocytes.     

Properties and activities of phosphoribomutase in the human leukemic cells

1. The activities and properties of phosphoribomutase were determined in human leukemic leukocytes and normal polymorphonuclear leukocytes. 2. Leukemic leukocytes had markedly increased phosphoribomutase activity. 3. The Km value of leukemic cells to R-1-P was identical to normal cells. However, the Km value to glucose-1-6-diphosphate in leukemic cells had been shown to be low. 4. Inhibition rates by 2-3-DPG and Pi were lower in leukemic cells. 5. Leukemic cells enzyme was unstable by heating. 6. There were no significant differences in isoelectric points between normal cells and leukemic cells.     

Higher tyrosine protein kinase activity in resting lymphocytes than in proliferating normal or leukaemic blood cells

We have studied tyrosine phosphorylation in particulate fractions from 11 leukaemic cell lines by using as substrate either a synthetic tyrosine containing peptide or the endogenous proteins. The results were compared with those obtained using similar fractions from normal lymphocytes and bone marrow cells. Particulate fractions from all the leukaemic cell lines and normal bone marrow cells exhibited lower levels of tyrosine protein kinase activity compared to normal lymphocytes. When the phosphorylation of endogenous substrates was assayed, proteins were phosphorylated on tyrosine residues (rather than serine or threonine residues) to a larger extent in normal lymphocytes than in leukaemic cell lines. Separation of labelled endogenous substrates on sodium dodecyl sulfate-polyacrylamide gels showed a number of phosphorylated alkali-resistant bands in the range 14-175kd in the lymphoid cell lines; normal lymphocytes exhibited a smaller number of strongly phosphorylated bands. Normal lymphocytes from different individuals showed reproducible patterns of phosphorylated substrates. Normal bone marrow cells and myeloid leukaemia lines showed weak, if any, phosphorylation. Among the leukaemic cell lines no particular pattern of phosphorylated substrates common to cells of similar phenotype could be detected. We suggest that the level of overall tyrosine protein kinase activity in these fractions reflects their position in the cell cycle rather than their normal or malignant status.     

Lymphokine regulation of human lymphocyte proliferation: Formation of resting G0 cells by removal of interteukin 2 in cultures of proliferating T lymphocytes

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G₀ phase and stay ready for a new activation (G₀-G₁ transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G₀) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G₀ T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G₀ phase, supporting the concept of direct S/G₂/M-G₁ transition. However, when IL-2 was removed from the cultures, the [³H]thymidine incorporation per 10⁴ cells and correspondingly the number of cells in the S/G₂/M and G₁ phases were reduced drastically and during the following 72-hr period, the number of G₀ cells increased markedly. Restimulation of such in vitro formed G₀ cells, under conditions permitting observation of their shift from the G₀ to G₀ phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G₀ phase of the cell cycle where they remain functional.     

Kinase activities in the non-histone chromosomal proteins of resting and proliferating BHK21 C13 cells.

The in vitro polymerization of bovine brain tubulin in the presence of Zn²⁺ has been studied. Zn²⁺, at concentrations higher than 5 × 10^?5 M caused the formation of sheets, sometimes consisting of up to 50–60 protofilaments oriented in parallel. The sheets were stable towards colchicine, Ca²⁺ and cold treatment. The induction of sheets cannot solely be ascribed to the sulfhydryl blocking properties of Zn²⁺, since other SH reagents (NEM, PCMBS, Cd²⁺ and Hg²⁺) failed to cause similar effects. There are reasons to believe that Zn²⁺ interferes with the closing process of microtubules so that more protofilaments are added in the assembly process than during normal conditions. The system described herein offers a favourable preparation for revealing the ultrastructural organization of microtubules.     

Effect of serotonin on proliferating and resting cells in culture

The influence of exogenous serotonin on cell division of L929 and L-41 cell strains has been investigated under various conditions of cell growth in culture (the incubation either in 10% serum medium without changing the medium, or in the medium with 0.5% serum). The data obtained show that serotonin in physiological concentration (10(-7) M) stimulates proliferation of resting cells. In proliferating cells, compared to resting ones, the sensitivity to exogenous amine appeared statistically non-significant. Exogenous serotonin is suggested to be a proliferating stimulus for resting cells.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry.
Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.     

Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes

Abstract. The expression of the 170-kDa (α) and the 180-kDa ( β ) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phyto-haemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo II β -associated immunofluorescence was about 2.5 times significantly higher ( P< 0.001) than that associated with the a isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G₂+M phases was found, the levels of topo IIα and β increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96–120 h), topo IIα immunofluorescence was not significantly changed, while that relative to topo II β declined to about 50% of the peak value (P<0.02). At this time however, topo IIα-associated immunofluorescence was not significantly different from that related to the β isozyme. These results suggest that in resting PBL topo IIα is required at levels lower than topo II β , while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.