scholarly journals Intracellular free Ca2+ in the cell cycle in human fibroblasts: transitions between G1 and G0 and progression into S phase.

1993 ◽  
Vol 4 (3) ◽  
pp. 293-302 ◽  
Author(s):  
M Wahl ◽  
E Gruenstein
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Dose Response ◽  
Human Fibroblasts ◽  
S Phase ◽  
Free Calcium ◽  
Response Curves ◽  
Serum Stimulation ◽  
Inhibition Of Dna Synthesis ◽  
Stimulation Of

Intracellular free calcium ([Ca2+]i) has been proposed to play an important part in the regulation of the cell cycle. Although a number of studies have shown that stimulation of quiescent cells with growth factors causes an immediate rise in [Ca2+]i (Rabinovitch et al., 1986; Vincentini and Villereal, 1986; Hesketh et al., 1988; Tucker et al., 1989, Wahl et al., 1990), a causal relationship between the [Ca2+]i transient and the ability of the cells to reenter the cell cycle has not been firmly established. We have found that blocking the mitogen-induced elevation of [Ca2+]i with the cytoplasmic [Ca2+]i buffer dimethyl BAPTA (dmBAPTA) also blocks subsequent entry of cells into S phase. The dose response curves for inhibition of serum stimulation of [Ca2+]i and DNA synthesis by dmBAPTA are virtually identical including an anomalous stimulation observed at low levels of dmBAPTA. Reversal of the [Ca2+]i buffering effect of dmBAPTA by transient exposure of the cells to the Ca2+ ionophore ionomycin also reverses the inhibition of DNA synthesis 20-24 h later. Ionomycin by itself does not stimulate DNA synthesis. These data are consistent with the conclusion that a transient increase in [Ca2+]i occurring shortly after serum stimulation of quiescent fibroblasts is necessary but not sufficient for subsequent entry of the cells into S phase. This study is the first to show a direct relationship between early serum stimulated Cai2+ increase and subsequent DNA synthesis in human cells. It also goes beyond recent studies on BALB/3T3 cells by providing dose response data and demonstrating reversibility, which are strong indications of a cause and effect relationship.

scholarly journals The jun and fos protein families are both required for cell cycle progression in fibroblasts.

1991 ◽  
Vol 11 (9) ◽  
pp. 4466-4472 ◽  
Author(s):  
K Kovary ◽  
R Bravo
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Serum Stimulation ◽  
Fos Protein ◽  
Stimulation Of

The expression of different members of the Jun and Fos families of transcription factors is rapidly induced following serum stimulation of quiescent fibroblasts. To determine whether these proteins are required for cell cycle progression, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, c-Jun, JunB, and JunD, and antibodies that recognize either the Fos or the Jun family of proteins, into Swiss 3T3 cells and determined their effects in cell cycle progression by monitoring DNA synthesis. We found that microinjection of anti-Fos and anti-Jun family antibodies efficiently blocked the entrance to the S phase of serum-stimulated or asynchronously growing cells. However, the antibodies against single members of the Fos family only partially inhibited DNA synthesis. In contrast, all three Jun antibodies prevented DNA synthesis more effectively than did any of the anti-Fos antibodies.

The jun and fos protein families are both required for cell cycle progression in fibroblasts

1991 ◽  
Vol 11 (9) ◽  
pp. 4466-4472
Author(s):  
K Kovary ◽  
R Bravo
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Serum Stimulation ◽  
Fos Protein ◽  
Stimulation Of

The expression of different members of the Jun and Fos families of transcription factors is rapidly induced following serum stimulation of quiescent fibroblasts. To determine whether these proteins are required for cell cycle progression, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, c-Jun, JunB, and JunD, and antibodies that recognize either the Fos or the Jun family of proteins, into Swiss 3T3 cells and determined their effects in cell cycle progression by monitoring DNA synthesis. We found that microinjection of anti-Fos and anti-Jun family antibodies efficiently blocked the entrance to the S phase of serum-stimulated or asynchronously growing cells. However, the antibodies against single members of the Fos family only partially inhibited DNA synthesis. In contrast, all three Jun antibodies prevented DNA synthesis more effectively than did any of the anti-Fos antibodies.

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1670-1676 ◽  
Author(s):  
K T Riabowol ◽  
R J Vosatka ◽  
E B Ziff ◽  
N J Lamb ◽  
J R Feramisco
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Antisense Rna ◽  
Proliferating Cells ◽  
Rna Transcripts ◽  
Serum Stimulation ◽  
Inhibition Of Growth ◽  
Nonspecific Immunoglobulins ◽  
3T3 Cell Lines ◽  
Stimulation Of

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

scholarly journals Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.

1986 ◽  
Vol 6 (10) ◽  
pp. 3373-3381 ◽  
Author(s):  
R N Johnston ◽  
J Feder ◽  
A B Hill ◽  
S W Sherwood ◽  
R T Schimke
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Dna Content ◽  
S Phase ◽  
Phase Cell ◽  
Drug Inhibition ◽  
Specific Proteins ◽  
Inhibition Of Dna Synthesis

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

scholarly journals Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells.

1988 ◽  
Vol 8 (4) ◽  
pp. 1670-1676 ◽  
Author(s):  
K T Riabowol ◽  
R J Vosatka ◽  
E B Ziff ◽  
N J Lamb ◽  
J R Feramisco
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Antisense Rna ◽  
Proliferating Cells ◽  
Rna Transcripts ◽  
Serum Stimulation ◽  
Inhibition Of Growth ◽  
Nonspecific Immunoglobulins ◽  
3T3 Cell Lines ◽  
Stimulation Of

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

scholarly journals Murine Coronavirus Replication Induces Cell Cycle Arrest in G0/G1 Phase

2004 ◽  
Vol 78 (11) ◽  
pp. 5658-5669 ◽  
Author(s):  
Chun-Jen Chen ◽  
Shinji Makino
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Hepatitis Virus ◽  
Cyclin E ◽  
S Phase ◽  
Cyclin D3 ◽  
Cyclin D2 ◽  
Serum Stimulation ◽  
Infected Cells ◽  
Cellular Dna

ABSTRACT Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G0/G1 phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G0 phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G1 and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21Cip1, p27Kip1, and p16INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G1 cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G1 cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G0/G1 phase.

Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell

1986 ◽  
Vol 6 (10) ◽  
pp. 3373-3381
Author(s):  
R N Johnston ◽  
J Feder ◽  
A B Hill ◽  
S W Sherwood ◽  
R T Schimke
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Dna Content ◽  
S Phase ◽  
Phase Cell ◽  
Drug Inhibition ◽  
Specific Proteins ◽  
Inhibition Of Dna Synthesis

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

scholarly journals Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

2005 ◽  
Vol 280 (16) ◽  
pp. 15773-15785 ◽  
Author(s):  
Julie K. Horton ◽  
Donna F. Stefanick ◽  
Jana M. Naron ◽  
Padmini S. Kedar ◽  
Samuel H. Wilson
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Cycle Arrest ◽  
Signaling Pathways ◽  
Dna Polymerase ◽  
S Phase ◽  
Dna Polymerase Β ◽  
Cycle Arrest ◽  
Parp Activity ◽  
Inhibition Of Dna Synthesis

Mouse fibroblasts, deficient in DNA polymerase β, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase β null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2′-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G2/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.

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.

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