Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor

1984 ◽  
Vol 4 (9) ◽  
pp. 1807-1814
Author(s):  
J Campisi ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Transcriptional Control ◽  
S Phase ◽  
G1 Phase ◽  
Insulin Like Growth Factor ◽  
Igf I ◽  
Translational Inhibition

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.

scholarly journals Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor.

1984 ◽  
Vol 4 (9) ◽  
pp. 1807-1814 ◽  
Author(s):  
J Campisi ◽  
A B Pardee
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Transcriptional Control ◽  
S Phase ◽  
G1 Phase ◽  
Insulin Like Growth Factor ◽  
Igf I ◽  
Translational Inhibition

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.

scholarly journals Amiloride added together with bumetanide completely blocks mouse 3T3-cell exit from G0/G1-phase and entry into S-phase

1986 ◽  
Vol 239 (3) ◽  
pp. 745-750 ◽  
Author(s):  
R Panet ◽  
D Snyder ◽  
H Atlan
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Dna Synthesis ◽  
Serum Insulin ◽  
S Phase ◽  
G1 Phase ◽  
G1 Arrest ◽  
Inhibitory Effects ◽  
Cell Transition ◽  
Mitogenic Signal

In this study we tested the hypothesis that stimulation of univalent-cation fluxes which follow the addition of growth factors are required for cell transition through the G1-phase of the cell cycle. The effect of two drugs, amiloride and bumetanide, were tested on exit of BALB/c 3T3 cells from G0/G1-phase and entry into S-phase (DNA synthesis). Amiloride, an inhibitor of the Na+/H+ antiport, only partially inhibited DNA synthesis induced by serum. Bumetanide, an inhibitor of the Na+/K+ co-transport, only slightly suppressed DNA synthesis by itself, but when added together with amiloride completely blocked cell transition through G1 and entry into S-phase. Similar inhibitory effects of the two drugs were found on the induction of ornithine decarboxylase (ODC) (a marker of mid-G1-phase) in synchronized cells stimulated by either partially purified fibroblast growth factor (FGF) or serum. To test this hypothesis further, cells arrested in G0/G1 were stimulated by serum, insulin or FGF. All induced similar elevations of cellular K+ content during the early G1-phase of the cell cycle. However, serum and FGF, but not insulin, released the cells from the G0/G1 arrest, as measured by ODC enzyme induction. This result implies that the increase in cellular K+ content may be necessary but not sufficient for induction of early events during the G1-phase. The synergistic inhibitory effects of amiloride and bumetanide on the two activities stimulated by serum growth factors, namely ODC induction (mid-G1) and thymidine incorporation into DNA (S-phase), suggested that the amiloride-sensitive Na+/H+ antiport system together with the bumetanide-sensitive Na+/K+ transporter play a role in the mitogenic signal.

Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats

1996 ◽  
Vol 150 (1) ◽  
pp. 121-127 ◽  
Author(s):  
C G Prosser ◽  
J Schwander
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Plasma Clearance ◽  
Binding Protein ◽  
Plasma Concentrations ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Lactating Goats ◽  
Igf I ◽  
Insulin Like Growth Factors

Abstract Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n=4) was determined following a single intravenous injection of the corresponding 125I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of 125I-IGFBP-2 from circulation was 0·37 ± 0·06 ml/min/kg, which is markedly greater than that of 125I-IGF-I or -II (0·11 ± and 0·12 ± 0·01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 ± 6 min) and -II (254 ± 8 min) compared with IGFBP-2 (110 ± 9 min). Three hours after injection of the 125I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1·54 ±0·04, 3·3 ±0·6 and 4·1 ±0·4 for IGFBP-2, IGF-I and -II respectively. The form of 125I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 ± 8 and 198 ±7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2. Journal of Endocrinology (1996) 150, 121–127

Insulin and insulin-like growth factor I (IGF I) in early mouse embryogenesis

Development ◽  
1990 ◽  
Vol 108 (3) ◽  
pp. 491-495
Author(s):  
R. Spaventi ◽  
M. Antica ◽  
K. Pavelic
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Indirect Evidence ◽  
Mouse Embryos ◽  
Insulin Like Growth Factor ◽  
Embryonic Cells ◽  
Mouse Development ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I

Growth factors have an important role in the regulation of cell growth, division and differentiation. They are also involved in the regulation of embryonic growth and differentiation. Insulin and insulin-like growth factor I (IGF I) play an important part in these events in the later stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that insulin and IGF I are also secreted by embryonic tissues during the prepancreatic stage of mouse development. We found measurable amounts of insulin and IGF I in 8- to 12-day-old mouse embryos. We also showed that embryonic cells derived from 8-, 9- and 10-day-old mouse embryos secrete insulin, IGF I and/or related molecules. Furthermore, the same growth factors, when added to the culture of 9-day-old mouse embryonic cells, stimulate their proliferation. These results lead to the conclusion that insulin can stimulate the growth of embryonic cells during the period when pancreas is not yet formed, which is indirect evidence for a paracrine (or autocrine) type of action.

435 REDUCTION OF CELL CYCLE LENGTH AND INCREASE IN S-PHASE BY GROWTH FACTORS IN PIG FETAL FIBROBLASTS

2010 ◽  
Vol 22 (1) ◽  
pp. 374
Author(s):  
S. Waghmare ◽  
B. Mir
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Cycle Length ◽  
S Phase ◽  
Proliferation Rate ◽  
Fetal Fibroblasts ◽  
Cell Cycle Length ◽  
Number Of Cells

Gene targeting in primary somatic cells is inefficient compared with embryonic stem cells. This is because of a slow rate of cell proliferation, fewer cells in S-phase at a given time point under normal culture conditions, and low rate of homologous recombination. Homologous recombination occurs mainly in late S-phase and increase in gene targeting efficiency has been reported in S-phase synchronized cells in bovine and rhesus macaque fetal fibroblasts. In this study we tested several growth factors: platelet-derived growth factor (PDGF), tumor necrosis factor a (TNFα), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor β1 (TGFβ1), insulin-like growth factor 1 (ILGF-1) and insulin-like growth factor II (ILGF-II) individually and in various combinations to see the effect on cell proliferation rate. Each experimental set consisted of 3 replicates. TGFβ1-, ILGF1-, ILGFII-, and FGF-treated cells grew very slowly compared with untreated cells. However, a combination of 3 growth factors: PDGF (15 ng mL-1), EGF (50 ng mL-1) and TNFa (100 pg mL-1), herein referred to as the cocktail, accelerated cell proliferation rate and reduced cell cycle length on average from 24.5 ± 0.2 to 20.4 ± 0.5 h with no significant change in number of cells in S-phase. Further, cells grown in the presence of the cocktail showed changes in morphology. The cells became spindle-shaped and occupied less surface area per cell compared with untreated cells. Importantly, cocktail-treated cells maintained a normal karyotype without any chromosomal abnormality. Thymidine has been used successfully to block various cell types in S-phase but it failed to synchronize these cells in S-phase in the concentration range of 2 to 10 mM for 24 to 48 h. However, serum starvation (0.2% fetal bovine serum) for 48 h blocked the cell proliferation rate effectively and synchronized cells in G0 phase (80-82% cells). After releasing from the block, cells were grown in the absence or presence of cocktail and cell cycle analysis was done at different time points by flow cytometry. Each time point was repeated 3 times. We observed the maximum number of cells in S-phase at 22 to 23 h (61.33% ± 7.77 in cocktail-treated cells v. 41.7% ± 3.28 in untreated cells). In summary, the cocktail-treated cells showed changes in cell morphology, higher proliferation rate, reduction in cell cycle length by 16.7%, and maximum percentage of cells in S-phase following serum starvation but maintained normal karyotypes. This high proliferation rate, reduction in cell cycle length, and maximum number of cells in S-phase should be very helpful in increasing the efficiency of gene-targeting in pig fetal fibroblasts.

scholarly journals Insulin Growth Factor-I and Epidermal Growth Factor Receptors Recruit Distinct Upstream Signaling Molecules to Enhance AKT Activation in Mammary Epithelial Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (12) ◽  
pp. 6027-6035 ◽  
Author(s):  
Jodie M. Fleming ◽  
Gwenaëlle Desury ◽  
Tiffany A. Polanco ◽  
Wendie S. Cohick
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Egf Receptor ◽  
Mammary Epithelial ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Mapk Activation ◽  
Igf I ◽  
Epidermal Growth

IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth and tumorigenesis. Whereas both growth factors increase DNA synthesis in MECs, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. In the present study, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5–100 ng/ml). IGF-I activated AKT via insulin receptor substrate-1 and p85, the regulatory subunit of phosphatidylinositol-3 kinase. Treatment with EGF had no effect on insulin receptor substrate-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2 and Gab2 with phospho-SHC, phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

2002 ◽  
Vol 80 (8) ◽  
pp. 790-795 ◽  
Author(s):  
Shirley C Paski ◽  
Zhaoming Xu
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Number ◽  
3T3 Cells ◽  
Intracellular Pool ◽  
Igf I ◽  
Epidermal Growth ◽  
Number Counts

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I) - stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I) - stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.Key words: PDGF, EGF, IGF-I, labile intracellular pool of zinc, cell proliferation, DNA synthesis, 3T3 cells.

The Effect of 2-Phenyl Ethanol on the DNA Synthesis Cycle of Schizosaccharomyces Pombe

1970 ◽  
Vol 7 (2) ◽  
pp. 523-530
Author(s):  
C. J. BOSTOCK
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Normal Growth ◽  
S Phase ◽  
G1 Phase ◽  
Synchronous Cultures ◽  
Number Of Cells

The effect of different concentrations of 2-phenyl ethanol (PE) on growth and DNA synthesis of Schizosaccharomyces pombe is described. o.3% PE inhibits the entry of cells into S phase, but allows a doubling in the number of cells in the culture. The effect of o.2% PE on random and synchronous cultures of S. pombe shows that, in the continued presence of the inhibitor, the S phase is moved to a different point in the cell cycle. Cells continue to grow in the presence of o.2% PE with a G1 phase occupying a significant portion of the cell cycle. This differs from normal growth when the G1 phase is absent.

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