scholarly journals Arginine deprivation in KB cells: I. Effect on cell cycle progress

1977 ◽  
Vol 75 (3) ◽  
pp. 881-888 ◽  
Author(s):  
AS Weissfeld ◽  
H Rouse
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
Experimental Period ◽  
Cell Multiplication ◽  
Starvation Period ◽  
Kb Cells ◽  
Insoluble Material ◽  
Cell Cycle Inhibition ◽  
Cellular Dna

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.

scholarly journals Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

1985 ◽  
Vol 5 (11) ◽  
pp. 2936-2942 ◽  
Author(s):  
H T Liu ◽  
R Baserga ◽  
W E Mercer
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Histone H3 ◽  
Adenovirus Type ◽  
3T3 Cells ◽  
Cycle Dependent ◽  
Cellular Dna ◽  
Adenovirus Type 2

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

scholarly journals Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.

1977 ◽  
Vol 73 (1) ◽  
pp. 200-205 ◽  
Author(s):  
A S Weissfeld ◽  
H Rouse
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Multiplication ◽  
Ovary Cells ◽  
Cell Cycle Phases

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum

1985 ◽  
Vol 5 (11) ◽  
pp. 2936-2942
Author(s):  
H T Liu ◽  
R Baserga ◽  
W E Mercer
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Histone H3 ◽  
Adenovirus Type ◽  
3T3 Cells ◽  
Cycle Dependent ◽  
Cellular Dna ◽  
Adenovirus Type 2

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

Mediators of the mitogenic action of human V1vascular vasopressin receptors

2000 ◽  
Vol 279 (5) ◽  
pp. H2529-H2539 ◽  
Author(s):  
Marc Thibonnier ◽  
Doreen M. Conarty ◽  
Christine L. Plesnicher
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Tritiated Thymidine ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Thymidine Uptake ◽  
Mitogenic Action

Arginine vasopressin (AVP) activation of V1 vascular receptors (V1Rs) stimulates cell growth and proliferation in different tissues via cellular signaling pathways that remain to be identified. To explore the intracellular mediators of the mitogenic action of V1R, Chinese hamster ovary (CHO) cells were stably transfected with the human V1R cDNA clone we isolated previously. We assessed AVP effects on kinase activation (immunoblotting with phosphospecific antibodies), DNA synthesis (tritiated thymidine uptake), cell cycle progression (flow cytometry analysis after nuclear labeling with propidium iodide), and cell proliferation (conversion of the colorimetric reagent MTS) in the presence or absence of various pathway inhibitors. AVP stimulation of V1Rs leads to the phosphorylation of several kinases, an increase in DNA synthesis, a progression through the S and G2–M phases of the cell cycle, and an increase in cell proliferation. The mediators of the mitogenic action of V1R activation included calcium mobilization, coupling to a Gq protein, and the simultaneous and parallel activation of several kinases, mainly calcium/calmodulin-dependent kinase II, phosphatidylinositol 3 kinase, protein kinase C, and p42/p44 mitogen-activated protein kinase.

scholarly journals SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

1973 ◽  
Vol 58 (2) ◽  
pp. 340-345 ◽  
Author(s):  
Kenneth D. Ley ◽  
Marilyn M. Murphy
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Time Course ◽  
Nuclear Dna ◽  
Tritiated Thymidine ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
In Cells

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals Prespore Cell Fate Bias in G1 Phase of the Cell Cycle in Dictyostelium discoideum

2005 ◽  
Vol 4 (10) ◽  
pp. 1755-1764 ◽  
Author(s):  
Guokai Chen ◽  
Adam Kuspa
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Fate ◽  
Developmental Cycle ◽  
Morphological Development ◽  
Cell Type ◽  
Chromosomal Dna ◽  
Prespore Cell ◽  
Cellular Dna ◽  
Spore Cell

ABSTRACT By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.

scholarly journals Inhibition of Cellular DNA Synthesis by the Human Cytomegalovirus IE86 Protein Is Necessary for Efficient Virus Replication

2006 ◽  
Vol 80 (8) ◽  
pp. 3872-3883 ◽  
Author(s):  
Dustin T. Petrik ◽  
Kimberly P. Schmitt ◽  
Mark F. Stinski
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Single Amino Acid ◽  
Cycle Progression ◽  
Artificial Chromosomes ◽  
Early Genes ◽  
Cellular Dna

ABSTRACT Human cytomegalovirus (HCMV) expresses several proteins that manipulate normal cellular functions, including cellular transcription, apoptosis, immune response, and cell cycle control. The IE2 gene, which is expressed from the HCMV major immediate-early (MIE) promoter, encodes the IE86 protein. IE86 is a multifunctional protein that is essential for viral replication. The functions of IE86 include transactivation of cellular and viral early genes, negative autoregulation of the MIE promoter, induction of cell cycle progression from G0/G1 to G1/S, and arresting cell cycle progression at the G1/S transition in p53-positive human foreskin fibroblast (HFF) cells. Mutations were introduced into the IE2 gene in the context of the viral genome using bacterial artificial chromosomes (BACs). From these HCMV BACs, a recombinant virus (RV) with a single amino acid substitution in the IE86 protein was isolated that replicates slower and to lower titers than wild-type HCMV. HFF cells infected with the Q548R RV undergo cellular DNA synthesis and do not arrest at any point in the cell cycle. The Q548R RV is able to negatively autoregulate the MIE promoter, transactivate viral early genes, activate cellular E2F-responsive genes, and produce infectious virus. This is the first report of a viable recombinant HCMV that is unable to inhibit cellular DNA synthesis in infected HFF cells.

scholarly journals THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

1966 ◽  
Vol 31 (1) ◽  
pp. 1-9 ◽  
Author(s):  
David M. Prescott
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Total Protein ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Protein Labeling ◽  
Constant Rate ◽  
Tritiated Amino Acids ◽  
Histone Synthesis ◽  
Macronuclear Division

The syntheses of histone, total protein, and DNA during the cell cycle were measured in the macronucleus of Euplotes eurystomus by assaying the incorporation of tritiated amino acids and tritiated thymidine in groups of 800 to 1000 synchronized cells. The synthesis of DNA begins at 30% completion of the cell cycle, proceeds at a constant rate, and ends very shortly before the beginning of macronuclear division. Histone labeling is absent during G1, begins in phase with DNA synthesis, continues at an unchanging rate during the S phase, and ends with the completion of DNA synthesis. The results support the view that the syntheses of histone and DNA are closely coupled events. Label in total protein accumulates at a constant rate during G1 and appears to shift to a slightly higher rate when histone synthesis begins. At division, radioactive DNA, histone, and total protein are distributed equally between the daughter macronuclei without loss of radioactivity. Radioautographic analysis showed that protein labeling occurs throughout the macronucleus during the entire life cycle. There was no clear difference in the degree of protein labeling between replicated and unreplicated regions of the macronucleus. The distribution of label suggests that most of macronuclear protein labeling during the cell cycle is concerned with the events of transcription rather than replication.

scholarly journals INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS

1973 ◽  
Vol 57 (2) ◽  
pp. 359-372 ◽  
Author(s):  
J. B. Rattner ◽  
Stephanie G. Phillips
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Temporal Relationship ◽  
Nuclear Dna ◽  
S Phase ◽  
Centriole Duplication ◽  
And Migration ◽  
Cellular Dna ◽  
Arabinosyl Cytosine ◽  
Mitotic Cells

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G1 nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G1 or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.

Sign in / Sign up

Export Citation Format

Share Document