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 EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

1973 ◽  
Vol 59 (3) ◽  
pp. 669-676 ◽  
Author(s):  
Helmut M. Maul ◽  
Betty Yee Li Hsu ◽  
Thaddeus M. Borun ◽  
Gerd G. Maul
Keyword(s):  
Cell Cycle ◽  
Pore Formation ◽  
Atp Synthesis ◽  
Nuclear Size ◽  
Size Increase ◽  
Metabolic Inhibitors ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Etching Technique ◽  
Hela S3

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S3 cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G1 phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.

scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

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 Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

1997 ◽  
Vol 139 (5) ◽  
pp. 1077-1087 ◽  
Author(s):  
Li Yang ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Nuclear Lamina ◽  
Normal Function ◽  
S Phase ◽  
Nuclear Volume ◽  
G1 Phase ◽  
Binding Region ◽  
Recombinant Polypeptide ◽  
Volume Increase

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1–398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it also is caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298–373) but not by the chromatin-binding domain (residues 1–88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Moreover, injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume.

scholarly journals Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroaki Shimono ◽  
Atsushi Kaida ◽  
Hisao Homma ◽  
Hitomi Nojima ◽  
Yusuke Onozato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Time Lapse ◽  
S Phase ◽  
G1 Phase ◽  
M Phase ◽  
G2 Phase ◽  
The Difference ◽  
Time Lapse Imaging ◽  
First Time

AbstractIn this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library

1993 ◽  
Vol 71 (7-8) ◽  
pp. 372-380 ◽  
Author(s):  
Gin Wu ◽  
Shiawhwa Su ◽  
Tzyy-Yun Tzeng Kung ◽  
R. Curtis Bird
Keyword(s):  
Cell Cycle ◽  
Cdna Library ◽  
Light Chain ◽  
Hela Cells ◽  
Subtractive Hybridization ◽  
S Phase ◽  
G1 Phase ◽  
Chain Gene ◽  
Centrifugal Elutriation ◽  
Light Chain Gene

Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71–95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.Key words: cell cycle, G1 phase, subtractive hybridization, cDNA cloning, ribosomal proteins, apoferritin light chain, HeLa cells.

Bioinformatics Analysis of Gefitinib or Rapamycin on Inhibiting the Survival of Hela in the Low Glucose and High Lactic Acid Environment

2019 ◽  
Vol 9 (2) ◽  
pp. 319-323 ◽  
Author(s):  
Li Ping ◽  
Li Mingzhu ◽  
Lü Yuchun
Keyword(s):  
Cell Cycle ◽  
Lactic Acid ◽  
Hela Cells ◽  
Annexin V ◽  
S Phase ◽  
Normal Glucose ◽  
Acid Environment ◽  
G0 Phase ◽  
High Lactate ◽  
Glucose Group

Objective: To explore on the antitumor effect of gefitinib and rapamycin and possible mechanism in normal glucose and high lactic acid microenvironment. Methods: Hela cells are cultured in six conditions: the normal glucose group (NG, glucose 3 mmol/L); the normal glucose + gefitinib group (NGG, glucose 3 mmol/L, gefitinib 2.67 μmol/L); the normal glucose + rapamycin group (NGR, glucose 3 mmol/L, rapamycin 2.67 μmol/L); the high lactate group (NGHL, glucose 3 mmol/L, lactic acid 2.5 mmol/L); the normal glucose + high lactate + gefitinib group (NGHLG, glucose 3 mmol/L, lactic acid 2.5 mmol/L, gefitinib 2.67μmol/L); the normal glucose + high lactate + rapamycin group (NGHLG, glucose 10 mmol/L, lactic acid 2.5 mmol/L, rapamycin 2.67μmol/L). Growth inhibitory rate of Hela cell is determined by CCK-8; Flow cytometry (FCM) is performed to evaluate the cell cycle; The annexin V-phycoerythrin/Propidium Iodide (annexin V-PE/PI) staining combined with flow cytometry is used to examine the cell cycle and apoptosis of Hela cells. Results: Under normal glucose with gefitinib or rapamycin environment, the apoptosis rate of Hela cells is higher than that of the normal glucose group. But the cell apoptosis rate of the gefitinib or rapamycin group decreases in high lactic acid and normal glucose, which is lower than that of the normal glucose and high lactate. Combined with the results of cell cycle, compared with the normal glucose group, percentage of Hela cells in G1/G0 phase increases significantly, the proportion of S phase cells decreases significantly in high lactic acid environment. In the normal glucose and gefitinib environment, Hela cells in G1/G0 phase and S phase are slightly higher than the proportion of normal glucose group, and G2/M phase cells are mild lower than the proportion of normal glucose group. Under the environment of high lactate and normal glucose, the percentage of G1/G0 and S phase cells in the gefitinib increase. As for rapamycin, normal glucose and high lactic acid environment makes cells stay in G1/G0 phase. The presence of rapamycin in the environment of normal sugar and high lactate makes more cells stay in G1/G0 or G2/M phase. Conclusion: Normal glucose and high lactic acid environment is conducive to Hela cell survival, and can promote the expression of EGFR and mTOR. Gefitinib is an antagonist of EGFR and rapamycin is an inhibitor of mTOR.

scholarly journals Apoptosis and S Phase of the Cell Cycle in BeWo Trophoblastic and HeLa Cells are Differentially Modulated by Toxoplasma gondii Strain Types

Placenta ◽  
2009 ◽  
Vol 30 (9) ◽  
pp. 785-791 ◽  
Author(s):  
M.B. Angeloni ◽  
N.M. Silva ◽  
A.S. Castro ◽  
A.O. Gomes ◽  
D.A.O. Silva ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Toxoplasma Gondii ◽  
Hela Cells ◽  
S Phase

scholarly journals Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

2010 ◽  
Vol 21 (19) ◽  
pp. 3421-3432 ◽  
Author(s):  
Donna Garvey Brickner ◽  
Jason H. Brickner
Keyword(s):  
Cell Cycle ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Pore ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation ◽  
Inducible Genes ◽  
Active Genes

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

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