The Ultrastructure of Synchronized Hela Cells

1971 ◽  
Vol 8 (2) ◽  
pp. 353-397
Author(s):  
R. A. ERLANDSON ◽  
E. DE HARVEN
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Rna Synthesis ◽  
S Phase ◽  
Nuclear Bodies ◽  
Daughter Cells ◽  
Protein And Rna Synthesis ◽  
G2 Cells ◽  
G1 Cells

Synchronous populations of mitotic HeLa cells were obtained by the controlled agitation method, and a detailed morphological study of the cells in all phases of the cell cycle was undertaken to correlate variations in cell structures to known coexisting biochemical events. Autoradiographic techniques using tritiated thymidine (3H-TdR) were used to detect S cells, and colcemid was added to some G2 samples to prevent the cells from going into the next cycle, thus preventing contamination with G1 cells. The approximate duration (in hours) of the 4 phases were as follows: M = 1.4, G1 = 8-9, S = 7, G2 = 4, and the generation time (T) = 21 ± 2 h. Randomization of the cell populations became apparent in the G2 phase (contaminated with S and M cells) and was most likely a result of the genetic make-up of the individual (mixoploid) HeLa cells, nutritional factors (serum lots used), temperature shock, and handling effects. Polyribosomes shifted to monomeric ribosomes during late prophase, at which time nucleoli also break down. These changes are correlated with the drop in protein and RNA synthesis reported for mitotic mammalian cells. The Golgi apparatus persisted in a modified form throughout mitosis. The mid-body forms from the anaphase stem-body and may interfere with the separation of daughter cells. Our studies suggest that the mid-body goes to one of the daughter cells where remnants of this structure were seen in early G1 cells. Large numbers of polyribosomes and the presence of well-developed nucleoli (many attached to the nuclear envelope) characterized G1. These structures, which play a major role in protein and RNA synthesis, persist with slight variations throughout interphase. Dense fibrillar nuclear bodies were prominent in late G1 cells. Centrioles separate during G1, and replicate by orthogonal budding during the S phase. Reproducible labelling patterns which reflect the asynchronous multireplicon nature of DNA synthesis in mammalian cells were characteristic of the various stages of the S phase. Granular nuclear bodies, which were prominent in S and G2 cells, may correspond to the larger species of heterogeneous nuclear RNA found in HeLa cells. G2 cells were similar in appearance to S cells. As heterochromatin areas increased in late G2 and prophase, perichromatin granules (of unknown significance) became prominent. Mitochondria behaved as independent cell organelles throughout the cell cycle. Hypertrophied RER, SER, and annulate lamellae, characterized the cytoplasm of colcemidtreated cells. The above changes are indicative of increased metabolic activity, and these structures may function in the production of colcemid-detoxify enzymes in a manner analogous to that of drug-treated hepatocytes, such as those treated with phenobarbital.

scholarly journals The use of conventional and zonal centrifugation to study the life cycle of mammalian cells. Phospholipid and macromolecular synthesis in neoplastic mast cells

1970 ◽  
Vol 119 (3) ◽  
pp. 493-499 ◽  
Author(s):  
A. M. H. Warmsley ◽  
C. A. Pasternak
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Rna Synthesis ◽  
Gene Dosage ◽  
Gradient Centrifugation ◽  
Mean Cell Volume ◽  
Protein And Rna Synthesis ◽  
G2 Phase ◽  
The Mean ◽  
Phospholipid Degradation

1. Conventional gradient centrifugation has been used to separate cells according to their position in the cell cycle, and to obtain synchronously growing cells. Analysis of prelabelled cells by gradient centrifugation confirms that phospholipid, protein and RNA synthesis is continuous throughout the cell cycle and shows that the rate of synthesis begins to increase already during the G1 phase. The pattern of phospholipid degradation follows that of synthesis. 2. The limitations of conventional gradient centrifugation have been overcome by use of a zonal rotor. Analysis of prelabelled cells confirms the results obtained by conventional centrifugation and in addition shows that the rates of phospholipid, protein and RNA synthesis decrease during the G2 phase. The mean cell volume and the net amount of phospholipid, protein and RNA, unlike that of DNA, are found to increase continuously throughout the intermitotic period. 3. These results show that the synthesis of macromolecules, and probably that of membranes also, is controlled by a mechanism other than that of gene dosage.

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.

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3443-3450
Author(s):  
J M Leeds ◽  
M B Slabaugh ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Ribonucleotide Reductase ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Cell Activity ◽  
S Phase ◽  
Whole Cell ◽  
Ribonucleotide Reductase Activity

Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle.

Cell cycle variations of dinucleoside polyphosphates in synchronized cultures of mammalian cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2444-2450
Author(s):  
G Orfanoudakis ◽  
M Baltzinger ◽  
D Meyer ◽  
N Befort ◽  
J P Ebel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
S Phase ◽  
Serum Deprivation ◽  
Specific Cell ◽  
Culture Cells ◽  
Mouse Embryo Fibroblasts ◽  
Atp Pool ◽  
Dinucleoside Polyphosphates ◽  
Synchronized Cultures

Zajdela hepatoma culture cells (ZHC) and mouse embryo fibroblasts (Swiss 3T3) were synchronized in G1 or S phase by serum deprivation and aphidicolin treatment, respectively, to study the variations in adenylyl nucleotide (Ap4X) pool size during the progress of the cell cycle. Only minor variations, which never exceeded a factor of 2, were observed when the Ap4X concentrations were expressed on a cellular basis. The variations were found to be strictly parallel to the ATP variations. Upon release from an aphidicolin block, the minor variations of Ap4X followed DNA synthesis and preceded cytokinesis. When the nucleotide content was compared with the amount of proteins, the faint specific cell cycle changes were almost completely damped when the cells were synchronized by serum deprivation, but remained practically unchanged in the case of aphidicolin synchronization. These results suggest that the observed variations could reflect the accumulation of some nucleotides before cell division. It is not clear yet whether the variation in Ap4X concentration is significant by itself or is simply a phenomenon resulting from changes in the ATP pool.

scholarly journals Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

1997 ◽  
Vol 17 (3) ◽  
pp. 1425-1433 ◽  
Author(s):  
S E Lee ◽  
R A Mitchell ◽  
A Cheng ◽  
E A Hendrickson
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Strand Break ◽  
S Phase ◽  
Dependent Manner ◽  
Severe Combined Immune Deficiency ◽  
Dsb Repair ◽  
G2 Checkpoint ◽  
G2 Phase

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.

scholarly journals EXTH-56. MEDULLOBLASTOMAS RESPOND TO CDK4/6 INHIBITION WITH NANOPARTICLE-DELIVERED PALBOCICLIB WITH ALTERED S-PHASE PROGRESSION

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi94-vi94
Author(s):  
Taylor Dismuke ◽  
Chaemin Lim ◽  
Timothy Gershon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Flow Cytometric Analysis ◽  
Pediatric Brain Tumors ◽  
S Phase ◽  
Flow Cytometric ◽  
Phase Progression ◽  
Reduced Rate ◽  
G1 Cells ◽  
Pediatric Brain

Abstract CDK4/6 inhibition is a promising therapy for medulloblastoma, one of the most common malignant pediatric brain tumors. To improve pharmacokinetics, we developed a polyoxazoline nanoparticle-encapsulated formulation of the FDA-approved CDK4/6 inhibitor palbociclib (POx-palbo). We then administered POx-palbo to transgenic medulloblastoma-prone GFAP-Cre/SmoM2 mice, to determine the efficacy and mechanisms of action and resistance. We found that POx-palbo slowed tumor progression, but consistently failed to be curative. Further analysis showed that while CDK4/6 inhibition acutely blocked G1 cells from re-entering the cell cycle, this effect wore off within hours of drug administration. However, flow cytometric analysis of EdU uptake hours after palbociclib demonstrated aberrant S-phase with reduced rate of DNA synthesis. This POx-palbociclib-induced alteration of S-phase progression seems to remain true at later time points even when we observed that palbociclib G1/S inhibition began to decrease. Based on these data, we propose that the combinational therapy of POx-palbociclib and S-phase targeting agents will further improve treatment. Faulty tumor cell cycle progression in the presence of Pox-palbociclib may give increased window to target the S-phase for irreversible cell-cycle exit.

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.

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