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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1940 ◽

1989 ◽

Vol 9 (5) ◽

pp. 1940-1945 ◽

Cited By ~ 14

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4466 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4466-4472 ◽

Cited By ~ 260

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.4.3.293 ◽

1993 ◽

Vol 4 (3) ◽

pp. 293-302 ◽

Cited By ~ 28

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1940-1945.1989 ◽

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4466-4472.1991 ◽

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.

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RB Reversibly Inhibits DNA Replication via Two Temporally Distinct Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5404-5420.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5404-5420 ◽

Cited By ~ 25

Author(s):

Steven P. Angus ◽

Christopher N. Mayhew ◽

David A. Solomon ◽

Wesley A. Braden ◽

Michael P. Markey ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

Cellular Proliferation ◽

Negative Regulator ◽

Proliferating Cells ◽

Factor C ◽

Replication Factors ◽

Multiple Replication

ABSTRACT The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the “chronic” RB-mediated arrest state and the “acute” arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

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