Mechanism of the elevation in cardiolipin during HeLa cell entry into the S-phase of the human cell cycle

2008 ◽  
Vol 417 (2) ◽  
pp. 573-582 ◽  
Author(s):  
Kristin Hauff ◽  
Dorota Linda ◽  
Grant M. Hatch
Keyword(s):  
Cell Cycle ◽  
Human Cell ◽  
Hela Cells ◽  
Thymidine Incorporation ◽  
De Novo ◽  
S Phase ◽  
Barth Syndrome ◽  
Nucleotide Synthesis ◽  
De Novo Biosynthesis ◽  
Elevated Activity

CL (cardiolipin) is a key phospholipid involved in ATP generation. Since progression through the cell cycle requires ATP we examined regulation of CL synthesis during S-phase in human cells and investigated whether CL or CL synthesis was required to support nucleotide synthesis in S-phase. HeLa cells were made quiescent by serum depletion for 24 h. Serum addition resulted in substantial stimulation of [methyl-3H]thymidine incorporation into cells compared with serum-starved cells by 8 h, confirming entry into the S-phase. CL mass was unaltered at 8 h, but increased 2-fold by 16 h post-serum addition compared with serum-starved cells. The reason for the increase in CL mass upon entry into S-phase was an increase in activity and expression of CL de novo biosynthetic and remodelling enzymes and this paralleled the increase in mitochondrial mass. CL de novo biosynthesis from D-[U-14C]glucose was elevated, and from [1,3-3H]glycerol reduced, upon serum addition to quiescent cells compared with controls and this was a result of differences in the selection of precursor pools at the level of uptake. Triascin C treatment inhibited CL synthesis from [1-14C]oleate but did not affect [methyl-3H]thymidine incorporation into HeLa cells upon serum addition to serum-starved cells. Barth Syndrome lymphoblasts, which exhibit reduced CL, showed similar [methyl-3H]thymidine incorporation into cells upon serum addition to serum-starved cells compared with cells from normal aged-matched controls. The results indicate that CL de novo biosynthesis is up-regulated via elevated activity and expression of CL biosynthetic genes and this accounted for the doubling of CL seen during S-phase; however, normal de novo CL biosynthesis or CL itself is not essential to support nucleotide synthesis during entry into S-phase of the human cell cycle.

scholarly journals The de novo centriole assembly pathway in HeLa cells

2005 ◽  
Vol 168 (5) ◽  
pp. 713-722 ◽  
Author(s):  
Sabrina La Terra ◽  
Christopher N. English ◽  
Polla Hergert ◽  
Bruce F. McEwen ◽  
Greenfield Sluder ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
De Novo ◽  
Variable Number ◽  
S Phase ◽  
Cycle Progression ◽  
Assembly Pathway ◽  
Centriole Assembly

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547–1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous “precentrioles” become morphologically recognizable centrioles before mitosis. De novo–assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo–formed centrioles do not mature if they are assembled in S phase–arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.

DE NOVO PURINE NUCLEOTIDE SYNTHESIS DURING CELL CYCLE

1981 ◽  
Vol 9 (2) ◽  
pp. 300P-300P
Author(s):  
L. THUILLIER ◽  
F. GARREAU ◽  
P. CARTIER
Keyword(s):  
Cell Cycle ◽  
De Novo ◽  
Purine Nucleotide ◽  
Nucleotide Synthesis

scholarly journals Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jan Wisniewski ◽  
Bassam Hajj ◽  
Jiji Chen ◽  
Gaku Mizuguchi ◽  
Hua Xiao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Elevated Temperatures ◽  
De Novo ◽  
Histone H3 ◽  
S Phase ◽  
Nuclear Accumulation ◽  
Histone H3 Variant ◽  
Functionally Impaired ◽  
Cell Cycle Dynamics ◽  
Cell Lethality

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.

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 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.

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