Three-dimensional reconstructions of nucleolus-organizing regions in PHA-stimulated human lymphocytes

Ultrastructure and three-dimensional distribution of nucleolus-organizing regions have been studied on ultrathin serial sections of PHA-stimulated human lymphocytes. During the 48 hr of activation the size of fibrillar centers (FCs) decreased from 0.6-0.9 microns to 0.2-0.3 microns and the number of FCs increased rapidly from one to 75-107 per cell. The number of fibrillar complexes (i.e. associations of a different number of FCs connected by the dense fibrillar component) also increased but did not reach the maximum number of nucleolar organizers presented here. Three-dimensional computer reconstructions of fibrillar complexes showed that lymphocyte activation was accompanied by early (2-4 hr) changes in the shape of the primary fibrillar center. Invagination of the dense fibrillar component on its surface occurred and division into two or more smaller FCs followed. Gradually, the typical structure of the nucleolus with several fibrillar complexes and many FCs was formed. These results confirm the hypothesis of fibrillar complex-nucleolar organizer correlation published recently.     

Analysis of ring-shaped nucleoli in serially sectioned human lymphocytes

Summary Serial section analysis has demonstrated that ring-shaped nucleoli of mature human lymphocytes are spherical structures consisting of a peripheral ribonucleoprotein shell that surrounds one large fibrillar center. The shell exhibits usually one or, less frequently, two openings. The fibrillar center is in contact with the nucleoplasm and perinucleolar condensed chromatin, which frequently appears as a pedicle-like structure. Several chromocenters are associated with the ring-shaped nucleolus.     

Effect of the tumor promoter TPA on the distribution of PHA-stimulated human lymphocytes by cell cycle phases

Patterns of the cell cycle distribution in human peripheral blood lymphocytes, stimulated by PHA alone and PHA plus 12-o-tetradecanoylphorbol-13-acetate (TPA), were studied using DNA cytometry in different times after PHA stimulation. In the first period (nearly 3 days after PHA stimulation) TPA induces no significant differences in the characters under consideration, but in the later period, when the proliferation of the cultures stimulated by PHA alone is reducing, in other cultures stimulated by PHA plus TPA the percentage of cells in S-phase does not reduce, whereas the percentage of cells in G2-phase is rising, which may suggest that this phase is blocked. Concurrently the tetraploid cells are appearing. Accumulation of cells in G2-phase can be overcome by the application of chlorpromazine, which is known to inhibit the membrane-associated protein kinase C.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

Quantitative studies on RNA accumulation in human PHA-stimulated lymphocytes during blast transformation

The net RNA accumulation in PHA-stimulated individual lymphocytes during blast transformation was assayed. The RNA content of the cells was measured by UV microspectrophotometry and the concentration of ribosomes in the cytoplasm of non-stimulated and transforming lymphocytes was determined by counting RNP particles in electronmicrographs.The mean RNA content of non-stimulated lymphocytes was 1.9 ± 0.8 × 10⁻¹²g/cell. During transformation the RNA content increased considerably, the maximal increase being about ten-fold. This increase was proportional to the increase in cellular dry mass. In fact, the RNA concentration was constant during transformation and approximately the same in non-stimulated and transforming lymphocytes. Furthermore the ribosome concentration was approximately the same in the cytoplasm of non-stimulated and transforming lymphocytes. However, a pronounced transition of single ribosomes to ribosomal clusters occurred during transformation and was one of the earliest structural signs of stimulation observed.     

Small nuclear RNAs synthesis in PHA-stimulated and nonstimulated human peripheral blood lymphocytes

Five fractions of small nuclear RNAs (snRNAs) were identified in nuclei of human peripheral blood lymphocytes. They have been designated as: H, G', D, C and A (nomenclature according to Weinberg) in order to decreasing electrophoretic mobility. Their synthesis was observed from the first day of treatment with mitogen and was continued with different intensity during four days of culture. In nonstimulated lymphocytes exclusively A and G' fractions were detected but no (14C)-uridine incorporation was observed.     

Studies on satellite nucleoli in human normal and leukemic lymphocytes

Lymphocytes from normal and leukemic patients, and phytohemagglutinin-stimulated lymphocytes were investigated by means of immunofluorescence procedures and a silver reaction for the demonstration of proteins characteristic of nucleolus organizer regions in interphasic cells to provide basic information on the presence of satellite nucleoli in these cells. The results clearly indicated that satellite nucleoli are present in a limited but constant percentage of peripheral lymphocytes. An increased percentage of lymphocytes with satellite nucleoli was found only in leukemic patients and after silver staining. In contrast, a decreased percentage of satellite nucleoli was found 24 h after stimulation with phytohemagglutinin vitro. In leukemic patients, the discrepancy in the percentage of lymphocytes with satellite nucleoli between immunostained and silver-stained preparations may suggest that the silver reaction demonstrates the presence of an additional argyrophilic protein besides proteins B23 and C23, or altered forms of these proteins, which does not react with the specific antibodies.     

Mechanism of the formation of micronuclei in PHA-stimulated lymphocytes from human peripheral blood

The micronuclei of PHA-stimulated human lymphocytes were assayed by comparison of electron microscopy and light microscopy data. The length of the cell cycle was checked by the flow cytofluorimetry method. Both the variability of the apoptosis stages and the increase in (G2+M) period in gamma-irradiated cultures were found. It is concluded that at least part of the micronuclei results from the cell nucleus "partition" during the apoptosis type cell death.     

Incorporation of hypoxanthine by PHA-stimulated HPRT-deficient lymphocytes

Phytohemagglutinin (PHA) markedly stimulates ³H-hypoxanthine incorporation by lymphocytes of normal subjects as revealed by radioautography. There is no corresponding increase in activity of hypoxanthine phosphoribosyltransferase (HPRT) in lysates but the level of phosphoribosylpyrophosphate (PRPP), the cosubstrate for the reaction, is higher. Lymphocytes from a patient with partial HPRT deficiency responded to PHA as did the normals, whereas the response in Lesch-Nyhan (LN) subjects was variable. PHA-stimulated lymphocytes from two LN patients showed some increase in ³H-hypoxanthine incorporation, while two others failed to respond. The observations provide further evidence of genetic heterogeneity among LN patients.     

Uptake of selenium-75 by PHA-stimulated lymphocytes

To determine which of a variety of inorganic and organic selenium compounds could best stimulate glutathione peroxidase, human lymphocytes were cultured with a number of selenium sources. The phytohemagglutinin-transformed lymphocytes were cultured in the presence of⁷⁵Se bound to serum proteins (25% v/v) or 10^?7 M concentrations of [⁷⁵Se]-selenite, [⁷⁵Se]-selenate, [⁷⁵Se]-selenocystine, and [⁷⁵Se]-selenomethionine. Organic forms of selenium were taken up in preference to inorganic forms. Control cultures, from which exogenous selenium had been omitted, showed a decreased level of glutathione peroxidase activity at the end of a 4 d culture period. Of the Se sources tested, [⁷⁵Se]-selenocystine and [⁷⁵Se]-labeled fetal calf serum proteins increased enzyme activity significantly, 79 and 47%, respectively, but selenite increased activity only by 7%. These results indicate that selenium from the two organic sources is most readily available for glutathione peroxidase synthesis.     

Electron microscopic study of Ag-protein localization in the nucleoli of human lymphocytes

The present study was undertaken to provide more information on the ultrastructural localization of a silver reaction in normal resting and stimulated lymphocytes as well as leukaemic resting lymphocytes. The results obtained indicated that in the ring-shaped nucleoli of normal mature lymphocytes silver stained proteins (SSPs) were present mostly within single fibrillar centers. In the nucleoli of lymphocyte cultures, being in the presence of phytohemagglutinin (PHA) for 6--72 hours, SSPs formed finger or loop-like protrusions from fibrillar centers towards the adjacent areas of the nucleoli. In the ring-shaped nucleoli of mature leukaemic lymphocytes SSPs are present not only within fibrillar centers, but also in protrusions diverging from fibrillar centers into the surrounding peripheral nucleolar ring. In this respect the nucleoli of leukaemic mature lymphocytes were similar to normal lymphocytes shortly after mitogen stimulation.     

A comparative study of the micronucleus test and chromosome aberrations in PHA-stimulated human lymphocytes

A dose dependence of the number of cells with chromosome aberrations was studied in PHA-stimulated donor's peripheral blood lymphocytes irradiated in vitro with doses of 10-400 cGy. In studying the number of chromosome aberrations and percentage of cells with micronuclei in parallel cultures no correlation was found between these indices within the groups exposed to a similar radiation dose.     

Basic types and vital forms of human peripheral and PHA-stimulated lymphocytes studied in vitro

Natural diversity in peripheral and PHA-stimulated lymphocytes seen in the same donors was studied using digitized streak photo of living cells in observational camera. Cells were monitored for 5-8 h at the superior limit of optical resolution by means of phase-contrast microscopy. Intact lymphocytes were observed in autological blood plasma, and PHA-stimulated lymphocytes were examined in self-conditioned centrifuged growth medium. The majority of intact cells were small- and middle-sized floating lymphocytes with microvilli, and middle-sized caudate lymphocytes capable of stick-slip motion. The lesser part consisted of "spread-eagle" or movable forms of both large granular lymphocytes and middle-sized lymphocytes of several types: narrow-plasm lymphocytes with lamellipodia, wide-plasm lymphocytes without cytoplasmic processes, lymphocytes with single pseudopodia, and lymphocytes with single lobopodia of complex shape. On the contrary, the minor fraction of PHA-stimulated lymphocytes of 3 day old cultures contained floating cells with microvilli or floating cells with microvilli and two pseudopodia, whereas the majority of these lymphocytes were spread-eagle or movable forms of cells of different type. These substrate-adhesive PHA-stimulated lymphocytes had well defined apical and basal cell surfaces, but upon mechanical stress are easily pinched off to become ball-shaped. At least 6 different cell types were distinguished among substrate-adhesive PHA-stimulated lymphocytes, with more than half of these being heavily vacuolated spheroid lymphocytes prone to forming cell clusters. The rest PHA-stimulated lymphocytes were represented by signet-ring lymphocytes with dark or light cytoplasm, narrow-plasm lymphocytes with large prolonged nuclei and lamellipodia, lymphocytes with single lobopodia, and lymphocytes with single spiral structures in the cytoplasm. The spiral structure is 10-11 microns in length and 0.5-0.7 micron in width, being presumably a mitochondrion or a group of butt-joined mitochondria. Since some of the caudate middle-sized lymphocytes also contain this structure, these may be regarded as putative precursors of respective type of PHA-stimulated lymphocytes. Under the conditions of observation, interphase nuclei of all live PHA-stimulated lymphocytes were seen to contain numerous globular or fiber structures of condensed chromatin made of 0.3-0.8 micron beads. These beads are doubtless interphase chromomeres.     

Deoxynucleotide pools and DNA synthesis in resting and PHA-stimulated human lymphocytes treated with mutagens.

In resting and PHA-stimulated PBL treated with uv light or MMS we measured the sizes of the dTTP and dATP pools and the variation of ATP content taken as an indicator of cytotoxicity. The effects on DNA synthesis were examined by measuring DNA repair (unscheduled DNA synthesis) in resting PBL and the inhibition of DNA replication in stimulated PBL. While both treatments affected DNA synthesis, only MMS perturbed dNTP pools and decreased the intracellular concentration of ATP. All the effects were more evident in cycling than in resting lymphocytes.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.