Effects of low concentrations of actinomycin D on the initiation of DNA synthesis in rapidly proliferating and stimulated cell cultures

Epifanova, O. I.; Abuladze, Marina; Zosimovskaya, A.I.

doi:10.1016/0014-4827(75)90632-1

Cited by 34 publications

(9 citation statements)

References 22 publications

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“…Enhanced actin synthesis therefore appears to be a marker specific for the Go to S transit, while enhanced synthesis of the 57,000 and 33,000 dalton proteins appears to be a marker specific to cycling as opposed to quiescent cells (37). In addition, the early peak in actin synthesis during the Go to S transit lends support to the notion that the first part of the Go to S transit is different from the G, phase of proliferating cells (38,39).…”

Section: Discussionmentioning

confidence: 52%

“…In order to obtain quiescent Go cultures, 2 X 106 cells were plated into Lux 150 X 15 mm culture dishes in 25 ml of medium. On the following day the cultures were shifted into medium containing 0.5% serum and the cells were allowed to grow in this medium for [38][39][40][41][42][43][44][45][46][47][48] hr. Cells were stimulated to transit from Go to S by replacing the 0.5% serum medium with medium containing 20% serum.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

Riddle¹,

Dubrow²,

Pardee³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons. This protein also showed the greatest change in synthesis during the transit from Go to S phase. Its synthesis rose to a maximum 4-6 hr after stimulation and then declined as cells entered S phase. The protein was present in both nuclear and cytoplasmic extracts. It was identified as actin on the basis of its mobility on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels. Other proteins synthesized preferentially by stimulated cells had molecular masses of 57,000 daltons (cytoplasmic), 33,000 daltons (cytoplasmic and nuclear), and 15,000 daltons (nuclear). The synthesis of the 57,000 and 33,000 dalton proteins increased gradually after stimulation and remainec high during S phase. The 15,000 dalton proteins began to be synthesized as cells entered S phase. The preferential synthesis of these proteins provides biochemical markers for the transition from quiescence to proliferation.Changes in biochemical properties during progression of the cell cycle have been widely described (1-3). However, the detection of marker proteins specific for different phases of the cell cycle is sorely lacking. Such markers would greatly facilitate more detailed analysis of biochemical events associated with the cell cycle.Crucial X 15 mm culture dishes in 25 ml of medium. On the following day the cultures were shifted into medium containing 0.5% serum and the cells were allowed to grow in this medium for [38][39][40][41][42][43][44][45][46][47][48] hr. Cells were stimulated to transit from Go to S by replacing the 0.5% serum medium with medium containing 20% serum. In order to monitor the entry of cells into S, cells were plated in 150-mm culture dishes containing coverslips and arrested in Go by serum deprivation as described above.[3H]-Thymidine at 0.5 ,uCi/ml (1 Ci = 3.7 X 1010 becquerels) was then included in the 20% serum medium used to stimulate the Go to S transit. Coverslips were processed for autoradiography (12) at the indicated times.Cultures were labeled for 2-hr periods with [3H]isoleucine (105 Ci/mmol; 20 or 40 ,uCi/ml) or ['4C]isoleucine (296 mCi/ mmol; 5 ,Ci/ml) in medium containing 1% (1 mg/liter) of the normal Dulbecco's medium isoleucine concentration. The cultures were then washed three times with cold phosphatebuffered saline and the cells were scraped off the plate with a rubber policeman. Cells were fractionated into cytoplasmic and nuclear fractions by using the method of Becker and Stanners (13). The nuclei were pelleted by a 2-min centrifugation at 600 X g. The supernatant was centrifuged at 250,000 X g for 30 min in a Beckman L5-65 ultracentrifuge (SW 50....

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Section: Discussionmentioning

confidence: 52%

mentioning

confidence: 99%

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

Riddle¹,

Dubrow²,

Pardee³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…3 and 6) are almost identical, 9-10 hr. These similar progression times suggest that PT-18 cells in the absence of CSF are not in a Go state but rather at a very early stage of the G1 phase, since it takes longer for cells in Go than for cells in G1 to progress to the S phase (13,14).…”

Section: Discussionmentioning

confidence: 72%

Colony-stimulating factor (CSF) controls proliferation of CSF-dependent cells by acting during the G1 phase of the cell cycle.

Pluznik¹,

Cunningham²,

Noguchi³

1984

Proc. Natl. Acad. Sci. U.S.A.

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Proliferation of granulocyte/macrophage (GM) progenitor cells in soft agar cultures is dependent on the continuous presence of colony-stimulating factors (CSF). To elucidate this dependency we studied the effect of deprivation and readdition of CSF on the cell cycle kinetics of a GM-CSFdependent murine mast/basophil cell line (PT-18). These findings indicate that deprivation of GM-CSF does not move PT-18 cells out of the cycle to a Go phase but rather arrests them at early G, phase. Finally, we demonstrate that, for the cells to progress through the cell cycle, the requirement for the presence of GM-CSF is limited to the first 6 hr of the 10-hr duration of the G, phase.

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“…Defining the cell cycle parameters in maximally stimulated thyrocytes In resting cells a prereplicative phase longer than the GI phase is generally taken as indicating that the cells are in a out-of-cycle Go stage prior to stimulation (Baserga, 1976;Epifanova et al, 1975;Martin and Stein, 1976). Therefore, we have compared the duration of the prereplicative phase initiated by the addition of the combination of EGF, TSH, and serum, and the GI phase duration determined from the cell cycle Darameters of Collagenase (type I, 150 U/mg) was purchased from exponentially growing thyroid cells.…”

Section: Methodsmentioning

confidence: 99%

Induction of DNA synthesis in dog thyrocytes in primary culture: Synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

Roger¹,

Servais²,

Dumont³

1987

Journal Cellular Physiology

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We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.

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Effects of low concentrations of actinomycin D on the initiation of DNA synthesis in rapidly proliferating and stimulated cell cultures

Cited by 34 publications

References 22 publications

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

Colony-stimulating factor (CSF) controls proliferation of CSF-dependent cells by acting during the G1 phase of the cell cycle.

Induction of DNA synthesis in dog thyrocytes in primary culture: Synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

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