Characterization of a cell cycle-dependent nuclear autoantigen

Analysis of reactivity to nuclear antigens in autoimmune sera revealed a serum that produced a previously undescribed cell cycle-dependent immunofluorescence staining pattern. By indirect immunofluorescence using HEp-2 cells as substrate, the serum generated a speckled and nucleolar pleomorphic staining pattern. This characteristic immunofluorescence pattern was detected in different cell lines from various species indicating that the antigen was highly conserved. This serum immunoprecipitated a 85 kDa protein using an extract from [³⁵S]-labeled HeLa cells. Indirect immunofluorescence of proliferating mouse 3T3 cells displayed the characteristic pleomorphic staining which was not observed in serum-starved cells. Resting human and mouse peripheral blood lymphocytes were negative in immunofluorescence while mitogen-stimulated lymphocytes were positive. Germinal centers of mice two weeks after immunization with 2-phenyl-oxazolone showed speckled immunofluorescence staining in the dark zones whereas unimmunized mice were completely negative. Cell synchronization experiments showed a characteristic sequence of locations of the antigen during the cell cycle. In G₁, cells were completely negative. In late G₁, G₁/S and S phase, speckles were visible. In early G₂, speckles were visible, and later in G₂, the nucleoli were positive. During mitosis chromosomes were stained. Further characterization of this antibody specificity and cloning of cDNA are in progress.     

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.     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.     

DNA polymerase δ mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol α and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol α ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages

Summary X-ray induced chromosomal aberrations in peripheral blood lymphocytes as well as in skin fibroblasts from ataxia telangiectasia patients, and from normal individuals were studied. At all stages of cell cycles—namely G₀, G₁, and G₂, more aberrations were induced in AT cells than in normal cells. In addition, AT cells were sensitive to induction of chromosomal aberrations by tritium beta rays from incorporated radioactive thymidine. Possible reasons for the increased sensitivity of AT cells for induction of chromosomal aberrations by ionizing radiations are discussed.     

Author index for volume 49

Using mouse thymocytes, mitogen-induced [³H]thymidine incorporation was compared with a recently developed flow-cytometric technique, based on acridine orange staining of cells, which differentiates the G₀ and G₁ phase of thymocytes. PHA induces a transient but considerable G₀-G₁ shift without any substantial proliferation. On the other hand, crude supernatants derived from Con A-stimulated human peripheral blood mononuclear cells induce only a minor G₀-G₁ shift and no proliferation. However, PHA in the presence of this supernatant induced an increased [³H]thymidine uptake in thymocytes and a shift from G₁ to S. These results support the current hypothesis that a factor present in Con A-activated supernatants in conjunction with PHA stimulation indeed facilitates the entrance of G₁ cells into the S phase. The flow-cytometric technique might be used in the study of the interaction of endogenous mediators with exogenous mitogenic agents in activating lymphocytes to proceed through the initial G₀-G₁ phases of the cell cycle.     

The plaque-forming cell response of human blood lymphocytes. I. PFC response of lymphocytes to formalin-treated staphylocci.

The plaque-forming cell and proliferative responses of human peripheral blood lymphocytes induced by formalin-treated Staphylococcus aureus of the Cowan strain were studied in vitro. Human blood mononuclear cells were incubated for 6 days with staphylococci in culture medium RPMI 1640 supplemented with 10% human AB serum. The number of anti-sheep erythrocyte plaque-forming cells was determined by the Jerne technique. Lymphocyte proliferation was measured by [³H]thymidine incorporation. Individual lymphocyte donors could be classified as high or low responders to staphylococci. Lymphocyte proliferation appeared necessary for the generation of plaque-forming cells. The plaque-forming cell response was greatly influenced by the source of the human AB serum used in the culture medium. The addition of hydrocortisone to the culture medium augmented the plaque-forming cell response. Human B lymphocytes prepared by passage through a column containing Sepharose 4B conjugated to anti-human F(ab)₂ generated plaque-forming cells when incubated with staphylococci. However, the addition of T lymphocytes to these B-lymphocyte preparations augmented the plaque-forming cell response to staphylococci.     

Cell Cycle Arrest during Measles Virus Infection: a G0-Like Block Leads to Suppression of Retinoblastoma Protein Expression

One of the major mechanisms by which measles virus (MV) infection causes disease and death is suppression of the immune response. The nonresponsiveness of MV-infected human lymphocytes to mitogens and a partial block in the G₀/G₁ phase of the cell cycle observed in vitro is thought to reflect in vivo immunosuppression. In order to molecularly dissect MV-induced immunosuppression, we analyzed expression of surface activation markers and cell cycle-regulatory proteins in MV-infected human T lymphocytes. MV Edmonston (MV-Ed) could induce and maintain a high level of the early activation marker CD69 in the absence of proliferation. Expression of cyclins D3 and E, which positively control entry into S phase, was also significantly decreased. Analysis of inhibitors of progression into S phase showed that a high level of p27 was maintained in the G₀/G₁-blocked subpopulation of MV-Ed-infected cells compared to the proliferating MV-infected cells. Furthermore, cell cycle-related upregulation of retinoblastoma (Rb) protein synthesis did not occur in the MV-Ed-infected lymphocytes. Acridine orange staining, which distinguishes cells in G₀ from cells in G₁, showed that RNA levels were not upregulated following activation, which is consistent with cells remaining in a G₀ state. Although expression of surface activation markers indicated entry into the cycle, intracellular Rb and RNA levels suggested a quiescent state. These results indicate that MV can uncouple activation of T lymphocytes from transition of G₀ to G₁.     

The influence of T lymphocyte subsets and humoral factors on colony formation by human bone marrow and blood megakaryocyte progenitor cells in vitro

Cellular and humoral influences of T lymphocytes on human megakaryocyte colony formation in vitro were assessed by using a microagar system. Megakaryocyte colony formation from nonadherent low density T lymphocyte-depleted (NALDT-) bone marrow cells was increased significantly after the addition of aplastic anemia serum (AAS) or purified megakaryocyte colony-stimulating factor (Meg-CSF). The addition of conditioned medium obtained from phytohemagglutinin-stimulated T lymphocytes replaced, at least partially, the requirement for AAS or purified Meg-CSF for the growth of megakaryocyte colonies. The cellular influence of T lymphocytes and T lymphocyte subsets on megakaryocyte colony formation was assessed by removing either T cells from nonadherent peripheral blood mononuclear cells with monoclonal OKT4, OKT8, or OKT3 antibodies plus complement, or by adding back populations of bone marrow or blood T4+ or T8+ lymphocytes, isolated by means of fluorescence-activated cell sorting, respectively, to NALDT--bone marrow or -blood cells. When sorted T cell subpopulations were added to a fixed number of NALDT--bone marrow or -peripheral blood cells in the presence of AAS or Meg-CSF, T4+ cells enhanced megakaryocyte colony formation and T8+ cells decreased it. These studies demonstrate that although the stimulation of megakaryocytic progenitor cells by Meg-CSF may not require the presence of monocytes or T lymphocytes, T4+ lymphocytes enhance and T8+ lymphocytes down-regulate megakaryocyte colony formation induced by Meg-CSF. These observations suggest that the immune system is capable of modulating the proliferative response of human megakaryocytic progenitor cells to Meg-CSF.     

Effects of chlorambucil on human chromosomes

No significant amount of chromosomal damage was found in the 48-h cultures of lymphocytes of 18 patients who had been treated with the bifunctional alkylating agent chlorambucil (CBC). However, there was suggestive evidence of chromatid damage (i.e. of types attributable to damage during or after DNA synthesis in the cell cycle). In marrow cells of 3 patients given a single large dose of chlorambucil (equivalent to 2 days' normal treatment) there was also suggestive evidence of induced chromatide-type damage.Extensive series of in vitro experiments yielded evidence that (a) exposure of human lymphocytes over the whole period of culture showed chromatid-type damage; (b) this damage increased sharply from concentrations of 0.5 μg/ml to3.0 μg/ml; (c) although chromatide-type damage always predominated, there was suggestive evidence also of chromosome-type aberrations attributable to damage occuring in the G₀/G₁ period, although some or all of this could be attributed to “derived” chromatid damage; (d) even if lymphocytes were only exposed during the G₀ or G₁ periods of the cycle, damage was found in the subsequent metaphases and it was almost entirely of the chromatid type; (e) much more damage occurred in lymphocytes exposed for varying periods to the drugs after stimulation by phytohaemagglutinins than in those exposed in whole blood, or in medium before stimulation; (f) damaged occurred in lymphocytes exposed to the drug while in S but not exposed only when in G₂; (g) no evidence was found that unschaduled DNA synthesis during G₀ or G₁ was induced by the drug; (h) there appeared to be no delay caused by the drug in the time at which cells reached the first “S” phase in culture but there was some evidence consistent with prolongation of “S” in cells exposed in culture; (i) there was evidence that CBC alone could stimulate lymphocyte tto DNA synthesis, and that a few cells proceeded in the cycle to prophase, or even metaphase. However, there was a considerable amount of cell-killing during CBC-stimulated DNA synthesis.     

The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in GO and non-random in G1 human lymphocytes

The nuclear topography of pericentromeric DNA of chromosome 11 was analyzed in G₀ (nonstimulated) and G₁ [phytohemagglutinin (PHA) stimulated] human lymphocytes by confocal microscopy. In addition to the nuclear center, the centrosome was used as a second point of reference in the three-dimensional (3D) analysis. Pericentromeric DNA of chromosome 11 and the centrosome were labeled using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence. To preserve the 3D morphology of the cells, these techniques were performed on whole cells in suspension. Three-dimensional images of the cells were analyzed with a recently developed 3D software program (Interactive Measurement of Axes and Positioning in 3 Dimensions). The distribution of the chromosome 11 centromeres appeared to be random during the G₀ stage but clearly non-random during the G₁ stage, when the nuclear center was used as a reference point. Further statistical analysis of the G₁ cells revealed that the centromeres were randomly distributed in a shell underlying the nuclear membrane. A topographical relationship between the centrosome and the centromeres appeared to be absent during the G₀ and G₁ stages of the cell cycle.     

Existence of a commitment program for mitosis in early G1 in tumour cells

The proliferation of normal non-tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G₁ (which we have designated G₁ pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G₁ pm and enter G₀. The G₁ pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G₁ or pre S phase (G₁ ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA 82 , 5365). As shown in the present study two tumour-transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25-hydroxycholesterol (an inhibitor of 3-hydroxy-3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [³H]-thymidine uptake. Time-lapse analysis revealed that the cells were blocked specifically in early G₁ (3-4h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G₁ amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25-hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a ‘G₁ pm program’, which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour-transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G₁ pm-like stage. Our conclusion is that G₁ pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.     

Gi-Coupled GPCR Signaling Controls the Formation and Organization of Human Pluripotent Colonies

Background

Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G_i-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.

Methodology/Principal Findings

Specific and irreversible inhibition of G_i-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G_s-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.

Conclusions/Significance

Experiments with pertussis toxin suggest that G_i signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G_i-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications.     

Requirement of the expression of 3-phosphoglycerate dehydrogenase for traversing S phase in murine T lymphocytes following immobilized anti-CD3 activation

Murine resting (G₀) T lymphocytes contained no detectable mRNA of 3-phosphoglycerate dehydrogenase (PHGDH) catalyzing the first step in the phosphorylated pathway of l-serine biosynthesis. Immobilized anti-CD3 activation of G₀ T cells expressed the PHGDH mRNA in G₁ with a maximum level in S phase. G₀ T cells activated with either immobilized anti-CD3 plus CsA or PBu₂, which failed to drive the activated T cells to enter S phase, did not express the PHGDH mRNA unless exogenous rIL-2 was added. Blocking of IL-2R signaling by adding anti-IL-2 and anti-IL-2Rα resulted in no expression of the PHGDH mRNA during immobilized anti-CD3 activation of G₀ T cells. Deprivation of l-serine from culture medium or addition of antisense PHGDH oligonucleotide significantly reduced [³H]TdR incorporation of activated T cells. These results indicate that the PHGDH gene expression, dictated by IL-2R signaling, is a crucial event for DNA synthesis during S phase of activated T cells.     

Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth

The proliferation rate of mammalian cells is regulated normally in the G₁ phase of the cell cycle. During this phase, it is convenient to assign positive and negative roles to the molecular programs that regulate the duration of G₁ and the phase transition from G₁ to S phase. Density-dependent inhibition of cellular proliferation results in an increase in the duration of G₁. This form of regulation is due to both secreted factors and cell—cell contact. Serum is mitogenic to a variety of mammalian cell types. Because quiescent cells enter S phase as a result of serum addition to culture media, serum is usually regarded as a source of positive regulatory growth factors. We have measured the length of the G₁, S and G₂+ M phases of NIH 3T3 cells during exponential growth as a function of cell density and serum concentration. The G₁ length increases during exponential growth as a function of density while S and G₂+ M are relatively constant. Further, this increase in G₁ phase time, or density mediated negative regulation, is inhibited by increasing serum concentration. This phenotype is saturable between 10% to 20% serum. Serum concentrations above 2.5% are able to increase the rate of cell cycling (decrease the G₁ phase time) by inhibiting density dependent negative regulation of NIH 3T3.     

Regulation of human ornithine decarboxylase expression following prolonged quiescence: Role for the c-Myc/Max protein complex

WI-38 cells can remain quiescent for long periods of time and still be induced to reenter the cell cycle by the addition of fresh serum. However, the longer these cells remain growth arrested, the more time they require to enter S phase. This prolongation of the prereplicative phase has been localized to a point early in G₁, after the induction of “immediate early” G₁ genes such as c-fos and c-jun but before maximal expression of “early” G₁ genes such as ornithine decarboxylase (ODC). Understanding the molecular basis for ODC mRNA induction can therefore provide information about the molecular events which regulate the progression of cells out of long-term quiescence into G₁ and subsequently into DNA synthesis. Studies utilizing electrophoretic mobility shift assays (EMSA) of nuclear extracts from short- and long-term quiescent WI-38 cells identified a region of the human ODC promoter at ?491 bp to ?474 bp which exhibited a protein binding pattern that correlated with the temporal pattern of ODC mRNA expression. The presence of a CACGTG element within this fragment, studies with antibodies against c-Myc and Max, the use of purified recombinant c-Myc protein in the mobility shift assay, and antisense studies suggest that these proteins can specifically bind this portion of the human ODC promoter in a manner consistent with growth-associated modulation of the expression of ODC and other early G₁ genes following prolonged quiescence. These studies suggest a role for the c-Myc/Max protein complex in regulating events involved in the progression of cells out of long-term quiescence into G₁ and subsequently into S. © 1995 Wiley-Liss, Inc.     

Human lymphocyte cell cycle: Studies with the use of BrUdR

Summary The chronological distributions of human blood lymphocytes in first, second and third mitosis following PHA stimulation in vitro are presented. The first G₁ phase is shown to be of variable length resulting in some first mitoses appearing only about 150 h after stimulation. The serum or plasma used in the culture medium influences cell cycle time. A total cell cycle time of 10.6 h was estimated for cultures with autologous donor plasma and of 14.7 h for cultures with fetal calf serum. It was further calculated that following PHA stimulation 90% of the lymphocytes divide once, about 65% divide for a second and about 40% divide for a third time.     

The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus-dependent

tsAF8, ts13, tsHJ-4, and TK^?ts13 cells are G₁-specific temperature-sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°-40.6°). TK^?ts13 are, in addition, defective in thymidine kinase. Different G₁ functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons. We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G₁ functions during the transition of cells from the non-proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G₀) or serum-stimulated (S) tsAF8 cells with G₀-ts13, G₀-tsHJ-4, and G₀-TK^?ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G₁ functions. Cytoplasts from S-tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ-4 recipient cells entered S at the nonpermissive temperature, and TK^?ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G₀-tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ-4 and TK^?ts13 cells. Cytoplasts from 6-day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6-day starved tsAF8 cells were fused with G₀-ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6-day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells. These results demonstrate that three functions required in G₁ cannot be detected in the cytoplasm of serum-starved cells, although they are present in the cytoplasm of S-phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G₁ functions in serumstimulated cells.     

The repair of x-ray-induced chromosome aberrations in stimulated and unstimulated human lymphocytes

The repair time for chromosome breaks induced by X-irradiation of unstimulated (G₀) and stimulated (G₁) human lymphocytes has been determined by dose fractionation studies. In both types of cells repair time was approx. 4–5 h. Treatment with hydroxyurea, a DNA synthesis inhibitor, did not prevent or delay the rejoining of broken chromosomes, whereas treatment with cycloheximide, a potent protein synthesis inhibitor, did. Thus, the repair of radiation-induced chromosome breaks in human lymphocytes is similar to the repair observed with plant cells.