scholarly journals Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling.

1980 ◽  
Vol 86 (3) ◽  
pp. 810-819 ◽  
Author(s):  
R B Cornell ◽  
A F Horwitz
Keyword(s):  
Cell Cycle ◽  
Cholesterol Synthesis ◽  
Cultured Cells ◽  
De Novo ◽  
Lipid Synthesis ◽  
Lipid Classes ◽  
Sterol Synthesis ◽  
G1 Arrest ◽  
Proliferating Cells ◽  
Cell Cycling

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts (WI-38 and CEF). Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold. 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline. The inhibition of sterol synthesis leads to a threefold reduction in the sterol:phospholipid ratio that parallels the development of proliferative and G1 cell cycle arrests and alterations in cellular morphology. All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol. A comparison of the timing of these responses with respect to the development of the G1 arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G1 arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol. In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G1 arrest but may in fact give rise to it. Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G1 arrests caused by confluence or differentiation. In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled.

S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells

2001 ◽  
Vol 359 (2) ◽  
pp. 361-367 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Stephanie C. WRIGHT
Keyword(s):  
Cell Cycle ◽  
Cellular Proliferation ◽  
Cultured Cells ◽  
S Phase ◽  
Proliferating Cells ◽  
Specific Expression ◽  
Related Function ◽  
Erythroleukemia Cells ◽  
A Cell ◽  
Pass Through

The Myc/Max/Mad transcription factor network plays a central role in the control of cellular proliferation, differentiation and apoptosis. In order to elucidate the biological function of Mad3, we have analysed the precise temporal patterns of Mad3 mRNA expression during the cell cycle and differentiation in cultured cells. We show that Mad3 is induced at the G1/S transition in proliferating cells; expression persists throughout S-phase, and then declines as cells pass through G2 and mitosis. The expression pattern of Mad3 is coincident with that of Cdc2 throughout the cell cycle. In contrast, the expression of Mad3 during differentiation of cultured mouse erythroleukemia cells shows two transient peaks of induction. The first of these occurs at the onset of differentiation, and does not correlate with the S-phase of the cell cycle, whereas the second is coincident with the S-phase burst that precedes the terminal stages of differentiation. Our results therefore suggest that Mad3 serves a cell-cycle-related function in both proliferating and differentiating cells, and that it may also have a distinct role at various stages of differentiation.

scholarly journals DNA replication during acute MEK inhibition drives acquisition of resistance through amplification of the BRAF oncogene

2021 ◽  
Author(s):  
Prasanna Channathodiyil ◽  
Anne Segonds-Pichon ◽  
Paul D Smith ◽  
Simon J Cook ◽  
Jonathan Houseley
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
De Novo ◽  
Combined Treatment ◽  
Mek Inhibitor ◽  
G1 Arrest ◽  
Cell Cycle Entry ◽  
Genes Encoding ◽  
Pathway Inhibition

Mutations and gene amplifications that confer drug resistance emerge frequently during chemotherapy, but their mechanism and timing is poorly understood. Here, we investigate BRAFV600E amplification events that underlie resistance to the MEK inhibitor selumetinib (AZD6244/ARRY-142886) in COLO205 cells. We find that de novo focal BRAF amplification is the primary path to resistance irrespective of pre-existing amplifications. Although selumetinib causes long-term G1 arrest, we observe that cells stochastically re-enter the cell cycle during treatment without reactivation of ERK1/2 or induction of a normal proliferative gene expression programme. Genes encoding DNA replication and repair factors are downregulated during G1 arrest, but many are transiently induced when cells escape arrest and enter S and G2. Nonetheless, mRNAs encoding key DNA replication factors including the MCM replicative helicase complex, PCNA and TIPIN remain at very low abundance, which likely explains previous reports of replication stress and mutagenesis under long-term RAF-MEK-ERK1/2 pathway inhibition. To test the hypothesis that DNA replication in drug promotes de novo BRAF amplification, we exploited the combination of palbociclib and selumetinib to reinforce the G1 arrest. Using a palbociclib dose that suppresses cell cycle entry during selumetinib treatment but not during normal proliferation, we show that combined treatment robustly delays the emergence of drug resistant colonies. Our results demonstrate that acquisition of MEK inhibitor resistance can occur through de novo gene amplification events resulting from DNA replication in drug, and is suppressed by drug combinations that impede cell cycle entry.

scholarly journals Plk4 triggers autonomous de novo centriole biogenesis and maturation

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Catarina Nabais ◽  
Delphine Pessoa ◽  
Jorge de-Carvalho ◽  
Thomas van Zanten ◽  
Paulo Duarte ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
De Novo ◽  
Cell Types ◽  
Proliferating Cells ◽  
Cycle Progression ◽  
Controlled System ◽  
Pericentriolar Material ◽  
Centriole Assembly ◽  
Critical Components

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.

scholarly journals Mouse spleen lymphoblasts generated in vitro. Recovery in high yield and purity after floatation in dense bovine plasma albumin solutions.

1978 ◽  
Vol 147 (2) ◽  
pp. 279-296 ◽  
Author(s):  
R M Steinman ◽  
B G Machtinger ◽  
J Fried ◽  
Z A Cohn
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Mouse Spleen ◽  
High Yield ◽  
Cell Cycle Distribution ◽  
Low Density ◽  
Proliferating Cells ◽  
M Phase ◽  
Yield And Purity

Mouse spleen lymphoblasts, stimulated to divide in vitro, acquired a low cell density and could be separated by isopycnic techniques. Cultured cells were suspended in BPA columns, rho = 1.080, and spun to equilibrium. The method was simple, fast, accomodated large numbers of cells, and was reproducible. It provided lymphoblasts in high yield and purity (at least 80% of the low density cells were blasts). It allowed for the recovery of proliferating cells in their first cell cycle, and did not alter the subsequent ability of cells to proliferate when recultured in vitro. Certain properties of mouse spleen lymphoblasts were analyzed in detail. Lymphoblasts induced by LPS, FCS, con A (tetravalent and succinylated), and MLC were very similar except in the absolute numbers that were induced. The blasts exhibited the classic cytologic features of enlarged nucleoli and abundant cytoplasmic polyribosomes (basophilia). As a population, they were enlarged in size relative to nondividing cells, but this seemed to apply primarily to cells in the S and G2+ M phase of the cell cycle rather than G1. The cell cycle distribution of lymphoblasts was analyzed by flow microfluorometry. By analyzing low density cells obtained at varying intervals after mitogen stimulation, FMF indicated that lymphoblasts enter the S phase of their first cell cycle beginning at 20-24 h after stimulation.

scholarly journals Plk4 triggers autonomous de novo centriole biogenesis and maturation

2020 ◽  
Author(s):  
Catarina Nabais ◽  
Delphine Pessoa ◽  
Jorge de-Carvalho ◽  
Thomas van Zanten ◽  
Paulo Duarte ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
De Novo ◽  
Cell Types ◽  
Proliferating Cells ◽  
Cycle Progression ◽  
Controlled System ◽  
Centriole Assembly ◽  
Critical Components ◽  
Kinetics Of

AbstractCentrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell-types, centrioles assemble de novo, yet by poorly understood mechanisms. Here, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at high Plk4 concentration, centrioles form de novo, mature and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the kinetics of centriole assembly. Moreover, our results suggest Plk4 operates in a switch-like manner to control the onset of de novo centriole formation, and that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and reveal that proteins of the pericentriolar matrix (PCM) promote biogenesis, likely by locally concentrating critical components.

Dolichol delays G1-arrest for one cell cycle in human fibroblasts subjected to depletion of serum or mevalonate

1992 ◽  
Vol 103 (4) ◽  
pp. 1065-1072 ◽  
Author(s):  
O. Larsson ◽  
J. Wejde
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Human Fibroblasts ◽  
Video Recording ◽  
Time Lapse ◽  
G1 Arrest ◽  
Proliferating Cells ◽  
Serum Factors

It is well-established that some product(s) or metabolite(s) of mevalonate is (are) critical for growth of mammalian cells. In the search for this (these) compound(s) it seems meaningful to distinguish between compounds needed for cell cycle progression in proliferating cells and compounds needed for growth activation of arrested cells. By using time-lapse video recording we have studied the possible regulatory role of cholesterol, dolichol and mevalonate in the cell cycle of human diploid fibroblasts (HDF). HDF, which are serum-dependent, were rapidly growth-arrested in the first part of G1 upon removal of serum factors. They also responded to mevinolin (an HMG CoA reductase inhibitor) by a similar G1-block, indicating that a mevalonate-derived product is involved in the G1-located cell cycle control of HDF. Interestingly, dolichol counteracted the G1-block caused by both types of treatment. Hence, the early G1-cells could traverse the remainder of the cell cycle and divide despite depletion of serum or mevalonate. We also demonstrated that addition of dolichol resulted in a significant decrease in the rate of protein degradation. This protein stabilizing effect may constitute the mechanism by which dolichol delays the G1-arrest of HDF.

scholarly journals Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin

2021 ◽  
Author(s):  
Vanessa Byles ◽  
Yann Cormerais ◽  
Krystle Kalafut ◽  
Victor Barrera ◽  
James E Hughes Hallett ◽  
...  
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Metabolic Response ◽  
Lipid Synthesis ◽  
Global Changes ◽  
Wild Type ◽  
Primary Hepatocytes ◽  
Proliferating Cells ◽  
Amino Acid Synthesis ◽  
Mtorc1 Signaling

Objective: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. Methods: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. Results: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver in response to feeding and in a hepatocyte-intrinsic manner by insulin. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of non-essential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. Conclusion: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

scholarly journals Gene Expression in Proliferating Cells of the Dinoflagellate Alexandrium catenella (Dinophyceae)

2010 ◽  
Vol 76 (13) ◽  
pp. 4521-4529 ◽  
Author(s):  
Eve Toulza ◽  
Mi-Sun Shin ◽  
Guillaume Blanc ◽  
Stéphane Audic ◽  
Mohamed Laabir ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Algal Blooms ◽  
Cultured Cells ◽  
Significant Proportion ◽  
Regulation Of Transcription ◽  
Toxic Dinoflagellate ◽  
Proliferating Cells ◽  
Alexandrium Catenella ◽  
Toxic Blooms

ABSTRACT Understanding the conditions leading to harmful algal blooms, especially those produced by toxic dinoflagellate species, is important for environmental and health safety. In addition to investigations into the environmental conditions necessary for the formation of toxic blooms, we postulate that investigating gene expression in proliferating cells is essential for understanding bloom dynamics. Expressed sequence tags were produced from cultured cells of the toxic dinoflagellate Alexandrium catenella sampled during the initiation phase of growth using Sanger's method and by 454 pyrosequencing. A significant proportion of identified genes (ca. 25%) represented enzymes and proteins that participate in a variety of cellular regulatory mechanisms that may characterize proliferating cells, e.g., control of the cell cycle and division, regulation of transcription, translation and posttranslational protein modifications, signaling, intracellular trafficking, and transport. All of the several genes selected for gene expression assays due to their involvement in metabolism and the cell cycle were overexpressed during exponential growth. These data will be useful for investigating the mechanisms underlying growth and toxin production in toxic Alexandrium species and for studying and monitoring the development of toxic blooms.

Lipid metabolism in the embryos of diabetic rats during early organogenesis: modulatory effect of prostaglandin E2

2003 ◽  
Vol 15 (1) ◽  
pp. 75 ◽  
Author(s):  
Debora Sinner ◽  
J. Matías Caviglia ◽  
Alicia Jawerbaum ◽  
R. Ariel Igal ◽  
Elida Gonzalez
Keyword(s):  
Lipid Metabolism ◽  
Prostaglandin E2 ◽  
Lipid Profile ◽  
De Novo ◽  
Lipid Synthesis ◽  
Diabetic Rats ◽  
Lipid Classes ◽  
De Novo Synthesis ◽  
Lipid Species ◽  
Early Organogenesis

The purpose of this work was to evaluate de novo lipid biosynthesis and the lipid profile, and to study the effect of prostaglandin E2 (PGE2; prostaglandin has previously been found to be involved in diabetes embryopathy) on lipid metabolism in embryos from control and streptozotocin-induced diabetic rats during organogenesis. Increased levels of triacylglycerols were found in embryos of diabetic rats compared with controls, whereas no differences were detected in the levels of cholesterol, cholesterylester, phosphatidylcholine and phosphatidylethanolamine. When the de novo synthesis of lipids in the embryo was studied using [14C]acetate as a tracer, a diminished rate of incorporation of [14C]acetate into the evaluated lipid classes was detected in the diabetic embryo compared with controls. Addition of PGE2 did not modify the incorporation of [14C]acetate into any of the lipid species of control embryos, but enhanced the incorporation of [14C]acetate into triacylglycerol, cholesterylesters, phosphatidylcholine and phosphatidylethanolamine of embryos from diabetic rats. The study’s results show alterations in both synthesis and concentrations of lipids in the embryos of diabetic rats. Interestingly, the results demonstrate that the addition of PGE2, a prostaglandin that reverses the embryonic morphological abnormalities induced by diabetes, prevents disturbances in embryo lipid synthesis caused by diabetes.

scholarly journals Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells

2007 ◽  
Vol 176 (2) ◽  
pp. 173-182 ◽  
Author(s):  
Yumi Uetake ◽  
Jadranka Lončarek ◽  
Joshua J. Nordberg ◽  
Christopher N. English ◽  
Sabrina La Terra ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
De Novo ◽  
S Phase ◽  
Human Cells ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Normal Human ◽  
Centrosomal Proteins

How centrosome removal or perturbations of centrosomal proteins leads to G1 arrest in untransformed mammalian cells has been a mystery. We use microsurgery and laser ablation to remove the centrosome from two types of normal human cells. First, we find that the cells assemble centrioles de novo after centrosome removal; thus, this phenomenon is not restricted to transformed cells. Second, normal cells can progress through G1 in its entirety without centrioles. Therefore, the centrosome is not a necessary, integral part of the mechanisms that drive the cell cycle through G1 into S phase. Third, we provide evidence that centrosome loss is, functionally, a stress that can act additively with other stresses to arrest cells in G1 in a p38-dependent fashion.

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