Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

scholarly journals An advanced cell cycle tag toolbox reveals principles underlying temporal control of structure-selective nucleases

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia Bittmann ◽  
Rokas Grigaitis ◽  
Lorenzo Galanti ◽  
Silas Amarell ◽  
Florian Wilfling ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Regulation ◽  
Cell Cycle Control ◽  
S Phase ◽  
Specific Cell ◽  
Protein Functionality ◽  
M Phase ◽  
Cell Cycle Phases ◽  
Cycle Regulation

Cell cycle tags allow to restrict target protein expression to specific cell cycle phases. Here, we present an advanced toolbox of cell cycle tag constructs in budding yeast with defined and compatible peak expression that allow comparison of protein functionality at different cell cycle phases. We apply this technology to the question of how and when Mus81-Mms4 and Yen1 nucleases act on DNA replication or recombination structures. Restriction of Mus81-Mms4 to M phase but not S phase allows a wildtype response to various forms of replication perturbation and DNA damage in S phase, suggesting it acts as a post-replicative resolvase. Moreover, we use cell cycle tags to reinstall cell cycle control to a deregulated version of Yen1, showing that its premature activation interferes with the response to perturbed replication. Curbing resolvase activity and establishing a hierarchy of resolution mechanisms are therefore the principal reasons underlying resolvase cell cycle regulation.

scholarly journals Maximal binding levels of an H1 histone gene-specific factor in S-phase correlate with maximal H1 gene transcription.

1988 ◽  
Vol 8 (10) ◽  
pp. 4576-4578 ◽  
Author(s):  
S Dalton ◽  
J R Wells
Keyword(s):  
Cell Cycle ◽  
Nucleic Acids ◽  
Gene Transcription ◽  
S Phase ◽  
Histone Gene ◽  
Specific Factor ◽  
Synchronized Cells ◽  
Phase Regulation ◽  
G2 Phase ◽  
Binding Component

Levels of trans-acting factor (H1-SF) binding to the histone H1 gene-specific motif (5'-AAACACA-3' [L. S. Coles and J. R. E. Wells, Nucleic Acids Res. 13:585-594, 1985]) increase 12-fold from G1 to S-phase in synchronized cells and decrease again in G2 phase of the cell cycle. Since the H1 element is required for S-phase expression of H1 genes (S. Dalton and J. R. E. Wells, EMBO J. 7:49-56, 1988), it is likely that the increased levels of H1-SF binding component play an important role in S-phase regulation of H1 gene transcription.

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.

Maximal binding levels of an H1 histone gene-specific factor in S-phase correlate with maximal H1 gene transcription

1988 ◽  
Vol 8 (10) ◽  
pp. 4576-4578
Author(s):  
S Dalton ◽  
J R Wells
Keyword(s):  
Cell Cycle ◽  
Nucleic Acids ◽  
Gene Transcription ◽  
S Phase ◽  
Histone Gene ◽  
Specific Factor ◽  
Synchronized Cells ◽  
Phase Regulation ◽  
G2 Phase ◽  
Binding Component

Levels of trans-acting factor (H1-SF) binding to the histone H1 gene-specific motif (5'-AAACACA-3' [L. S. Coles and J. R. E. Wells, Nucleic Acids Res. 13:585-594, 1985]) increase 12-fold from G1 to S-phase in synchronized cells and decrease again in G2 phase of the cell cycle. Since the H1 element is required for S-phase expression of H1 genes (S. Dalton and J. R. E. Wells, EMBO J. 7:49-56, 1988), it is likely that the increased levels of H1-SF binding component play an important role in S-phase regulation of H1 gene transcription.

scholarly journals Mammalian Rad9 Plays a Role in Telomere Stability, S- and G2-Phase-Specific Cell Survival, and Homologous Recombinational Repair

2006 ◽  
Vol 26 (5) ◽  
pp. 1850-1864 ◽  
Author(s):  
Raj K. Pandita ◽  
Girdhar G. Sharma ◽  
Andrei Laszlo ◽  
Kevin M. Hopkins ◽  
Scott Davey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Survival ◽  
Cycle Phase ◽  
Specific Cell ◽  
Damage Repair ◽  
Homologous Recombinational Repair ◽  
Mammalian Homologue ◽  
G2 Phase ◽  
Checkpoint Genes ◽  
Kinetics Of

ABSTRACT The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1 +, rad3 +, rad9 +, rad17 +, rad26 +, and hus1 +) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of γ-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

scholarly journals Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.

1997 ◽  
Vol 8 (8) ◽  
pp. 1587-1601 ◽  
Author(s):  
M R Young ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
S Phase ◽  
Specific Cell ◽  
Replication Origins ◽  
Proliferating Cells ◽  
M Phase ◽  
Mcm Proteins ◽  
And Function

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.

scholarly journals A UV-responsive G2 checkpoint in rodent cells.

1995 ◽  
Vol 15 (7) ◽  
pp. 3722-3730 ◽  
Author(s):  
D K Orren ◽  
L N Petersen ◽  
V A Bohr
Keyword(s):  
Cell Cycle ◽  
Uv Irradiation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Chinese Hamster Ovary Cells ◽  
S Phase ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Synchronized Cells ◽  
G2 Phase

We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.

scholarly journals Transforming growth factor β decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase

1994 ◽  
Vol 299 (1) ◽  
pp. 227-235 ◽  
Author(s):  
D J Grainger ◽  
P R Kemp ◽  
C M Witchell ◽  
P L Weissberg ◽  
J C Metcalfe
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Cyclic Amp ◽  
Vascular Smooth Muscle Cells ◽  
Transforming Growth Factor ◽  
S Phase ◽  
Tgf Beta ◽  
M Phase ◽  
G2 Phase

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.

The comparison of epirubicin-treated MCF-7 mammosphere cells to the treated monolayer cells

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e13542-e13542
Author(s):  
M. Huang ◽  
F. Zhang ◽  
Y. Xu ◽  
H. Wang ◽  
S. Lin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
S Phase ◽  
G1 Phase ◽  
Cell Cycle Distribution ◽  
Inhibitory Effects ◽  
Lower Effect ◽  
G2 Phase ◽  
Cell Inhibition ◽  
Mcf 7

e13542 Objective: To explore the different effects of epirubicin on the MCF-7 mammosphere cells and the monolayer cells. Methods: MCF-7 cells were cultured in suspension to generate primary mammospheres. The inhibitory effects of epirubicin on MCF-7 mammosphere cells and the monolayer cells by were measured by MTT assay. The change of CD44+CD24- expression and cell cycle distribution in MCF-7 mammosphere cells and the monolayer cells under epirubicin condition was analyzed by flow cytometry. Results: The cell inhibition was lower in MCF-7 mammosphere cells than that in the monolayer cells when induced by the same concentration of epirubicin (>100 ng/ml),(P<0.01). The CD44+CD24- expression was significantly higher in MCF-7 mammosphere cells than that in the monolayer cells under 400 ng/μl epirubicin for 72 h, (22.8% ± 4.8% Vs 3.3% ± 0.8%),(P<0.01). The cell cycle indicated that MCF-7 mammosphere cells had higher proportion of G0/G1 phase than the monolayer cells, (74.33% ± 3.20% Vs 53.40% ± 3.45%) (P<0.01). Epirubicin had little effect on the G0/G1 phase of MCF-7 mammosphere cells and the monolayer cells, but the S phase and G2 phase was not the case. Conclusion: Epirubicin had lower inhibitory effects on MCF-7 mammosphere cells and it can be used to enrich breast cancer stem cell. Epirubicin had lower effect on the G0/G1 phase of MCF-7 mammosphere cells as compared with control. No significant financial relationships to disclose.

scholarly journals The Paramyxovirus Simian Virus 5 V Protein Slows Progression of the Cell Cycle

2000 ◽  
Vol 74 (19) ◽  
pp. 9152-9166 ◽  
Author(s):  
Grace Y. Lin ◽  
Robert A. Lamb
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Simian Virus ◽  
S Phase ◽  
V Protein ◽  
Simian Virus 5 ◽  
C Terminus ◽  
M Phase ◽  
Infected Cells ◽  
Affect Cell Cycle Progression

ABSTRACT Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G1 to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G2 or M phase. The levels of p53 and p21CIP1were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VΔC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.

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