scholarly journals Lymphocyte cell-cycle analysis by flow cytometry. Evidence for a specific postmitotic phase before return to G0.

1980 ◽  
Vol 85 (2) ◽  
pp. 459-465 ◽  
Author(s):  
D P Richman
Keyword(s):  
Cell Cycle ◽  
Human Lymphocytes ◽  
Late Phase ◽  
S Phase ◽  
Cycle Phase ◽  
Thymidine Uptake ◽  
Large Numbers ◽  
Distinct Cell ◽  
Lymphocyte Cell ◽  
Supravital Staining

We studied the cell cycle of lectin-stimulated human lymphocytes, making use of a flow cytometer. The RNA and DNA content of large numbers of individual cells was determined by supravital staining with acridine orange. The present study confirmed previous observations by others of a progression from G0 through G1 and S phase to G2/mitosis during the first 3 d in culture. It was also found that on subsequent days stimulated cells, before their return to G0, remained stationary in a state in which they contained the G0 complement of DNA and approximately twice the G0 complement of RNA. Cell-cycle manipulation with vinblastine and 5-bromo-2-deoxyuridine (BUdR) revealed that previous passage through both S phase and mitosis was required for entry into this newly observed late phase. In addition, there was high correlation (r = 0.973, P less than 0.001) between the number of cells in the late phase and measured [3H]thymidine uptake. It therefore appears that, in this system, stimulated cells remain in a distinct cell-cycle phase for a number of hours before their return to the resting state.

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum

1993 ◽  
Vol 105 (4) ◽  
pp. 1121-1130 ◽  
Author(s):  
S. Lang ◽  
T. Decristoforo ◽  
W. Waitz ◽  
P. Loidl
Keyword(s):  
Cell Cycle ◽  
Nuclear Matrix ◽  
Physarum Polycephalum ◽  
Nuclear Periphery ◽  
S Phase ◽  
Cycle Phase ◽  
Electron Microscopic ◽  
Nuclear Lamins ◽  
Synchronous Cell ◽  
Matrix Bound

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

scholarly journals Inhibition of human lymphocyte proliferation by monoclonal antibody to transferrin receptor

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 821-826 ◽  
Author(s):  
J Mendelsohn ◽  
I Trowbridge ◽  
J Castagnola
Keyword(s):  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Lymphocyte Proliferation ◽  
Human Lymphocytes ◽  
Nitrilotriacetic Acid ◽  
S Phase ◽  
Inhibitory Effects ◽  
Mitogenic Response ◽  
Cell Cycle Inhibition ◽  
Partial Reversal

Abstract A monoclonal antibody, 42/6, which blocks the binding of transferrin to its receptor on the cell membrane, inhibits proliferation of human lymphocytes stimulated by phytohemagglutinin. Anti-receptor antibody B3/25, which does not block transferrin binding, does not alter the mitogenic response. Addition of soluble iron, in the form of ferric nitrilotriacetic acid, results in partial reversal of inhibition. Lymphocytes in the quiescent phase of the cell cycle at the time of 42/6 antibody addition are unable to traverse S phase, whereas cells actively proliferating when antibody is added are sensitive to its inhibitory effects throughout all phases of the cell cycle. Inhibition is static rather than cidal, since it can be reversed by removal of antibody after up to 48 hr of exposure.

scholarly journals TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

1972 ◽  
Vol 55 (2) ◽  
pp. 433-447 ◽  
Author(s):  
Gerd G. Maul ◽  
Helmut M. Maul ◽  
Joseph E. Scogna ◽  
Michael W. Lieberman ◽  
Gary S. Stein ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Hela Cells ◽  
Pore Formation ◽  
Human Lymphocytes ◽  
Time Sequence ◽  
S Phase ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Nuclear Pores

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ2 and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ∼2000 in G1 to ∼4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.

scholarly journals Precise detection of S phase onset reveals decoupled G1/S transition events

2018 ◽  
Author(s):  
Gavin D. Grant ◽  
Katarzyna M. Kedziora ◽  
Juanita C. Limas ◽  
Jeremy E. Purvis ◽  
Jeanette Gowen Cook
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Phase ◽  
Reporter System ◽  
Phase Boundaries ◽  
Fluorescent Reporter ◽  
Molecular Events ◽  
Cell Cycle Phases

AbstractThe eukaryotic cell division cycle is the process by which cells duplicate their genomes and proliferate. Transitions between sequential cell cycle phases are tightly orchestrated to ensure precise and efficient cell cycle progression. Interrogating molecular events at these transitions is important for understanding normal and pathological cell proliferation and mechanisms that ensure genome stability. A popular fluorescent reporter system known as “FUCCI” has been widely adopted for identifying cell cycle phases. Using time-lapse fluorescence microscopy, we quantitatively analyzed the dynamics of the FUCCI reporters relative to the transitions into and out of S phase. Although the original reporters reflect the E3 ubiquitin ligase activities for which they were designed, SCFSkp2 and APCCdh1, their dynamics are significantly and variably offset from actual S phase boundaries. To precisely mark these transitions, we generated and thoroughly validated a new reporter containing a PCNA-interacting protein degron whose oscillations are directly coupled to the process of DNA replication itself. We combined this reporter with the geminin-based APCCdh1 reporter to create “PIP-FUCCI.” PIP degron reporter dynamics closely correlate with S phase transitions irrespective of reporter expression levels. Using PIP-FUCCI, we made the unexpected observation that the apparent timing of APCCdh1 inactivation frequently varies relative to the onset of S phase. We demonstrate that APCCdh1 inactivation is not a strict pre-requisite for S phase entry, though delayed APCCdh1 inactivation correlates with longer S phase. Our results illustrate the benefits of precise delineation of cell cycle phase boundaries for uncovering the sequences of molecular events at critical cell cycle transitions.

scholarly journals DNA damage checkpoint dynamics drive cell cycle phase transitions

2017 ◽  
Author(s):  
Hui Xiao Chao ◽  
Cere E. Poovey ◽  
Ashley A. Privette ◽  
Gavin D. Grant ◽  
Hui Yan Chao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Phase Transitions ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cell Cycle Phase ◽  
Cycle Phase ◽  
Dna Damage Checkpoint ◽  
Cycle Progression ◽  
Dna Damage Checkpoints

ABSTRACTDNA damage checkpoints are cellular mechanisms that protect the integrity of the genome during cell cycle progression. In response to genotoxic stress, these checkpoints halt cell cycle progression until the damage is repaired, allowing cells enough time to recover from damage before resuming normal proliferation. Here, we investigate the temporal dynamics of DNA damage checkpoints in individual proliferating cells by observing cell cycle phase transitions following acute DNA damage. We find that in gap phases (G1 and G2), DNA damage triggers an abrupt halt to cell cycle progression in which the duration of arrest correlates with the severity of damage. However, cells that have already progressed beyond a proposed “commitment point” within a given cell cycle phase readily transition to the next phase, revealing a relaxation of checkpoint stringency during later stages of certain cell cycle phases. In contrast to G1 and G2, cell cycle progression in S phase is significantly less sensitive to DNA damage. Instead of exhibiting a complete halt, we find that increasing DNA damage doses leads to decreased rates of S-phase progression followed by arrest in the subsequent G2. Moreover, these phase-specific differences in DNA damage checkpoint dynamics are associated with corresponding differences in the proportions of irreversibly arrested cells. Thus, the precise timing of DNA damage determines the sensitivity, rate of cell cycle progression, and functional outcomes for damaged cells. These findings should inform our understanding of cell fate decisions after treatment with common cancer therapeutics such as genotoxins or spindle poisons, which often target cells in a specific cell cycle phase.

scholarly journals Intrinsic S phase checkpoint enforced by an antiproliferative oncosuppressor cytokine

2021 ◽  
Author(s):  
Livio Mallucci ◽  
Valerie Wells
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Self Regulation ◽  
Cyclin A ◽  
S Phase ◽  
Cycle Phase ◽  
Control Mechanisms ◽  
Checkpoint Control ◽  
Normal Cells ◽  
Orderly Transition

AbstractThe cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the βGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.

scholarly journals Expression of mRNA for Adenosine A1, A2a, A2b, and A3 Receptors in HL-60 Cells: Dependence on Cell Cycle Phases

2011 ◽  
pp. 913-920 ◽  
Author(s):  
M. HOFER ◽  
L. DUŠEK ◽  
Z. HOFEROVÁ ◽  
L. STIXOVÁ ◽  
M. POSPÍŠIL
Keyword(s):  
Cell Cycle ◽  
Mrna Expression ◽  
S Phase ◽  
Cell Cycle Phase ◽  
Cycle Phase ◽  
Block Method ◽  
Physiological Mechanisms ◽  
Adenosine A1 ◽  
Adenosine A2a ◽  
Adenosine A2b

The present studies investigated changes in expression of mRNA for adenosine A1, A2a, A2b, and A3 receptors in samples of HL-60 promyelocytic cells differing in the actual presence of cells in various phases of the cell cycle induced by the double thymidine block method. Real-time PCR technique was used for obtaining data on mRNA expression. Statistical analysis of the data revealed that the mRNA expression of adenosine A1, A2a, and A3 receptors is dependent on the cell cycle phase. G0/G1 and G2/M phases were characterized by a higher mRNA expression of adenosine A1 receptors and a lower one of adenosine A2a and A3 receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A2b receptors was independent on the cell cycle phase. The results indicate the plasticity of mRNA expression of adenosine receptors in the investigated promyelocytic cells and its interaction with physiological mechanisms of the cell cycle.

Effect of near ultraviolet and visible light on amoeba

1975 ◽  
Vol 19 (1) ◽  
pp. 117-126
Author(s):  
S. Chatterjee ◽  
S.K. Bhattacharjee
Keyword(s):  
Cell Cycle ◽  
Visible Light ◽  
S Phase ◽  
Nuclear Transplantation ◽  
Phase Cell ◽  
Cycle Phase ◽  
Leucine Incorporation ◽  
Repair System ◽  
Near Ultraviolet ◽  
G2 Phase

The near ultraviolet and visible light (VL) impinging at an intensity of 2–5 × 10(2) J s-1 m-2 for 2–5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.

DNA Damage Induced by DNA Topoisomerase I- and Topoisomerase II- Inhibitors Detected by Histone H2AX Phosphorylation in Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4240-4240
Author(s):  
Dorota H. Halicka ◽  
Xuan Huang ◽  
Fevzi M. Ozkaynak ◽  
Karen Seiter ◽  
Frank Traganos ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Topoisomerase I ◽  
Caspase 3 ◽  
S Phase ◽  
Cell Cycle Phase ◽  
Cycle Phase ◽  
Dna Topoisomerase I ◽  
H2ax Phosphorylation ◽  
Dna Topoisomerase ◽  
Histone H2ax

Abstract Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (γH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of γH2AX with: (a) cell cycle phase; (b) caspase-3 activation; and (c) apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of γH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of γH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of γH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in γH2AX was apparent at a concentration range between 0.4 and 1.6 μM TPT. Thus, the intensity of γH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in γH2AX immunofluorescence. On the basis of our laboratory results, the present clinical study is evaluating ex vivo the feasibility of assessing DNA damage induced by treatment with topoisomerase inhibitors in patients with acute leukemias.

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