scholarly journals Ultrastructural cytochemical studies of plasma membrane phosphatase activities during the HeLa S3 cell cycle.

1979 ◽  
Vol 27 (12) ◽  
pp. 1596-1603 ◽  
Author(s):  
A Vorbrodt ◽  
T W Borun
Keyword(s):  
Cell Cycle ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
S Phase ◽  
Cell Nuclei ◽  
Membrane Fluctuations ◽  
Hela S3 ◽  
G1 Cells

Alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg2+-activated ATPase (Mg-ATPase) and Ca2+-activated ATPase (Ca-ATPase) were studied in sychronized HeLa S3 cells with cytochemical methods and electron microscopy. It was found that AP activity, as determined by the deposition of lead phosphate reaction product (r.p.) was most active in mitotic (M), early and middle G1 cells, less active in late G1 and almost undetectable in S phase cells. Most AP enzyme activity was found to be associated with undulations (mainly microvilli) of the plasma membrane. Fluctuations and the redistribution of 5'N were also observed; the reaction for 5'N was positive in all phases of the cell cycle studied, it was strongest in M cells and in the majority of middle G1 cells. Mg-ATPase activity was present in the plasma membranes of cells throughout the cell cycle, but did not show noticeable fluctuations in activity and distribution. Ca-ATPase activity appeared in plasma membranes and in limited areas of cell nuclei but was evident only in S phase cells. The results of the present study confirm and extend previous biochemical observations and indicate that changes in membrane phosphate activities are associated with enzyme activity redistributions within the plasma membrane during the HeLa S3 cell cycle.

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.

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 Further characterization of HeLa S3 plasma membrane ghosts

1978 ◽  
Vol 77 (2) ◽  
pp. 448-463 ◽  
Author(s):  
E Costantino-Ceccarini ◽  
PM Novikoff ◽  
PH Atkinson ◽  
AB Novikoff
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Simple Procedure ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Major Band ◽  
Plasma Membrane Fraction ◽  
Coomassie Blue ◽  
Hela S3

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.

scholarly journals Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists

1997 ◽  
Vol 322 (3) ◽  
pp. 823-828 ◽  
Author(s):  
Irma ROMERO ◽  
Ana M. MALDONADO ◽  
Pilar ERASO
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Glucose Regulation ◽  
Wild Type ◽  
Calmodulin Antagonists ◽  
Dependent Protein Kinase ◽  
Yeast Plasma Membrane ◽  
Dependent Protein

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitroeffect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of ≈1 μM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

Raft Localization and Nucleotidase Activity of CD39 Depends on Internal Proteolytic Cleavage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3925-3925
Author(s):  
Kim E Olson ◽  
Joan HF Drosopoulos ◽  
Ashley E Olson ◽  
Marinus Johan Broekman ◽  
Aaron J Marcus
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Sucrose Gradient ◽  
Proteolytic Cleavage ◽  
High Density ◽  
Full Length ◽  
Low Density ◽  
293 Cells ◽  
Discontinuous Sucrose Gradient

Abstract We have previously shown that CD39 undergoes limited cleavage and that inhibition of proteolysis results in a decrease in ATPase activity. The reduction in enzymatic activity correlated with a decrease in the fraction of full-length CD39 present in active membrane raft-localized oligomeric complexes. We exploited N-and C-terminal VP16-and V5-tagged CD39, both transiently and stably expressed in 293 cells, to further elucidate the role of cleavage in the regulation of CD39 processing and activity. To characterize the complexes generated by cross-linking, N-terminal VP16-tagged and C-terminal V5-tagged CD39 were co-expressed in 293 cells. Following crosslinking of membranes with DTSSP and immunoprecipitation with anti-V5, DTT-cleaved species were visualized by Western Blot using VP16 antibody. Interestingly, both VP16-tagged full-length and N-terminal fragments (30 kDa) were immunoprecipitated by anti-V5. This indicates that both full-length CD39 and the N-terminal cleavage fragment are present in raft-localized complexes. The composition of raft-localized CD39 complexes was studied by separating membrane fractions on a discontinuous sucrose gradient using a non-detergent method. When overexpressed, CD39 and its C-terminal fragment distribute across the gradient as visualized by Western with anti-VP16. Importantly, specific activity (expressed as ATPase activity divided by total CD39 content) was 8 times greater in low-density raft-enriched fractions than in high density raft-free fractions. In addition, relative ADPase activity was higher in fractions containing a higher proportion of C-terminal CD39 relative to full-length CD39. Thus, CD39 forms oligomeric complexes and possesses optimal enzyme activity in lipid rafts. The relationship between CD39 cleavage, ATPase activity and raft localization was further studied in 293 cells transfected with C-or N-terminal VP16-tagged CD39. Subcellular fractionation on a discontinuous sucrose gradient yielded membrane fractions enriched in endoplasmic reticulum (ER), early endosomes (EE) and plasma membrane/Golgi (PM-Golgi). Importantly, the EE fraction contained both full-length and C-terminal (or N-terminal) CD39 at the same level as seen in the PM-Golgi fraction, suggesting that near 50% of CD39 resides in the EE compartment. Furthermore, EE-expressed CD39 exhibited an ATPase and ADPase activity equivalent to that seen in Golgi-PM fractions. This led us to examine effects of NH4Cl and bafilomycin (which block acidification of EE), and chloroquine (blocks EE maturation) on CD39 cleavage, activity and raft localization. Each treatment inhibited CD39 cleavage and correspondingly decreased ATPase activity. A shift of ~50% of full-length CD39 from raft fractions to high density membrane fractions was observed upon sucrose gradient fractionation following chloroquine treatment of cells transfected with N-terminal VP16 tagged CD39. This redistribution of CD39 in the membrane correlated with a 40% decrease in ATPase activity and a striking inhibition of CD39 cleavage. Here, at a lower level of expression than cited above, ATPase activity in low-density raft fractions was ~100-fold greater than in high density fractions. Thus, cleavage of a portion of CD39 molecules is required for both raft localization of full-length CD39 and optimal enzyme activity. Regulated proteolytic cleavage of CD39 would allow for rapid upregulation of CD39 activity in response to alterations in cell environment. This would occur via cycling of CD39 between plasma membrane and endosomal compartments, the proposed site of CD39 cleavage and assembly of fully active oligomeric complexes.

scholarly journals Stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in mouse uterine epithelial cells by oestradiol-17 β

1984 ◽  
Vol 218 (3) ◽  
pp. 849-855 ◽  
Author(s):  
P A Wilce ◽  
L Leijten ◽  
L Martin
Keyword(s):  
Cell Cycle ◽  
Enzyme Activity ◽  
Epithelial Cells ◽  
Time Course ◽  
Hormone Treatment ◽  
S Phase ◽  
Uterine Epithelial Cells ◽  
Coa Reductase ◽  
Two Peaks ◽  
Stimulation Of

The characteristics of 3-hydroxy-3-methylglutaryl-CoA reductase from mouse uterine epithelial cells were studied. Preliminary experiments showed that enzyme activity was stimulated approx. 10-fold 18h after administration of 100ng of oestradiol-17 beta. This activity was associated with all particulate fractions of the uterine luminal cell. The Km for D-3-hydroxy-3-methylglutaryl-CoA was 5.54 +/- 1.12 microM. The detailed time-course of oestrogen stimulation showed two peaks of activity, 9 and 15h after hormone treatment. The DNA content of the epithelial cells doubled between 6 and 12h after hormone treatment, whereas the protein content increased linearly over the 18h period. The peak of enzyme activity at 9h is associated with early S phase of the epithelial cells; the peak at 15h may be associated with a second S phase or with mitosis. Pretreatment with progesterone for 3 days before injection of oestradiol-17 beta (a treatment which inhibits uterine epithelial DNA synthesis) reduced the oestrogenic stimulation of enzyme activity by 63%; progesterone treatment alone did not stimulate enzyme activity. These data suggest that uterine epithelial 3-hydroxy-3-methylglutaryl-CoA reductase may play an important role in the cell cycle in this tissue.

scholarly journals Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase

1977 ◽  
Vol 162 (3) ◽  
pp. 473-482 ◽  
Author(s):  
D E Snider ◽  
C W Parker
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Prostaglandin E1 ◽  
Plasma Membranes ◽  
Subcellular Fractions ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Marker Enzyme ◽  
Discontinuous Sucrose Gradient

Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.

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.

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