scholarly journals INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE

1974 ◽  
Vol 60 (1) ◽  
pp. 249-257 ◽  
Author(s):  
Jeffrey E. Froehlich ◽  
Martin Rachmeler
Keyword(s):  
Cell Growth ◽  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Fresh Medium ◽  
G1 Phase ◽  
Cyclic Monophosphate ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G1 phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N6, O2-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.

scholarly journals EFFECT OF ADENOSINE 3'-5'-CYCLIC MONOPHOSPHATE ON CELL PROLIFERATION

1972 ◽  
Vol 55 (1) ◽  
pp. 19-31 ◽  
Author(s):  
Jeffrey E. Froehlich ◽  
Martin Rachmeler
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Dna Synthesis ◽  
Contact Inhibition ◽  
Fresh Medium ◽  
Cyclic Monophosphate ◽  
Human Diploid Fibroblasts ◽  
Dependent Inhibition ◽  
Camp Metabolism ◽  
Inhibition Of Dna Synthesis

Secondary cultures of human diploid fibroblasts, which demonstrate density-dependent inhibition of cell growth, were used to study the effect of adenosine 3'-5'-cyclic monophosphate (cAMP) on cell proliferation. DNA synthesis in nonconfluent cultures and in contact-inhibited cultures stimulated to grow by refeeding with fresh medium was found to be inhibited by exogenous cAMP. The properties of this inhibition of DNA synthesis, together with the alterations in cAMP metabolism observed in confluent cultures of cells stimulated with fresh medium to resume growth, strongly suggest that cAMP is involved in contact-inhibition of cell proliferation.

scholarly journals Cell population heterogeneity in the inducibility of DNA synthesis in human diploid fibroblasts.

1981 ◽  
Vol 89 (2) ◽  
pp. 194-197 ◽  
Author(s):  
M V Rao
Keyword(s):  
Dna Synthesis ◽  
Cytochalasin B ◽  
Nuclear Dna ◽  
S Phase ◽  
Population Heterogeneity ◽  
Diploid Fibroblasts ◽  
Binucleate Cells ◽  
Human Diploid Fibroblasts ◽  
Significant Difference ◽  
Sister Cells

The initiation of nuclear DNA synthesis has been studied in cytochalasin B (CB)-induced binucleate human diploid fibroblasts (WI-38 cells). Mitotic cells from different passage levels were rendered binucleate by a brief pulse of CB. The cells were then washed free of the drug, and DNA synthesis was studied by [3H]thymidine labeling. The results showed that, in a small percentage of binucleate cells, one nucleus was labeled (S phase) and the other nucleus was unlabeled (G1 phase). There was no significant difference in the percentage of these cells with increasing passage levels. The results of this study suggest that some WI-38 cells retire from the cell cycle at different passage levels, and thereby become refractory to inducers of nuclear DNA synthesis generated by sister cells in S phase.

scholarly journals Inducers of DNA synthesis: levels higher in transformed cells than in normal cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 571-576 ◽  
Author(s):  
P N Rao ◽  
K L Satya-Prakash
Keyword(s):  
Dna Synthesis ◽  
Sendai Virus ◽  
Transformed Cells ◽  
G1 Phase ◽  
Normal Cells ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Tumor Viruses ◽  
Latex Beads ◽  
Normal Human

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.

scholarly journals The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts

1991 ◽  
Vol 100 (4) ◽  
pp. 869-876 ◽  
Author(s):  
I.R. Kill ◽  
J.M. Bridger ◽  
K.H. Campbell ◽  
G. Maldonado-Codina ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Indirect Immunofluorescence ◽  
Laser Scanning ◽  
S Phase ◽  
Nuclear Antigen ◽  
In Vitro Techniques ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts

The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.

Timing of the phases of the cell cycle with tritiated thymidine and Feulgen cytophotometry during the period of synchronous division in Lymnaea

Development ◽  
1971 ◽  
Vol 26 (3) ◽  
pp. 351-366
Author(s):  
J. A. M. van den Biggelaar
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Energy Requirements ◽  
G1 Phase ◽  
Cell Stage ◽  
Cell Cycles ◽  
Feulgen Cytophotometry

The duration of the phases of the cell cycle during the 1-, the 2- and the 4-cell stage of the Lymnaea egg were determined with [3H]thymidine and with Feulgen cytophotometry. The M, S and G2 phases occupy 48, 27 and 25% of the first three cell cycles. A G1 phase cannot be observed. Only from the 4-cell stage was [3H]thymidine readily incorporated into DNA. The theory that an increase in respiration during the S phase of the 4-cell stage is connected with the energy requirements of DNA synthesis is discussed.

Regulation of the p21Sdi1/Cip1/Waf1 DNA Synthesis Inhibitor in Senescent Human Diploid Fibroblasts

1996 ◽  
Vol 15 (2) ◽  
pp. 315-329
Author(s):  
Ryan S. Robetorye ◽  
James R. Smith
Keyword(s):  
Dna Synthesis ◽  
Kinase Activity ◽  
Human Fibroblasts ◽  
Large Body ◽  
S Phase ◽  
G1 Phase ◽  
Protein Kinase Activity ◽  
Proliferating Cells ◽  
Synthesis Inhibitor ◽  
Human Diploid Fibroblasts

ABSTRACTA large body of evidence has demonstrated that normal human fibroblasts have a limited division potential in culture and underwent senescence, a process whereby cells became arrested in the G1 phase of the cell cycle and overexpressed a DNA synthesis inhibitor(s). Cyclin-dependent kinase two (Cdk2) is required for the promotion of the Gi-to-S phase transition in human cells. Senescent fibroblasts contain intact cyclin-Cdk2 complexes but cannot induce Cdk2 protein kinase activity in response to mitogen stimulation. Recently, we cloned p21Sdi1, a potent inhibitor of DNA synthesis and Cdk2 kinase activity, from a senescent cell cDNA library and demonstrated that it was expressed at significantly higher levels in senescent cells than actively proliferating cells. In contrast to actively dividing cells, mitogen-stimulated senescent cells do not down-regulate the expression of p21Sdi1 and do not express late G1 phase gene products that are required for entry into S phase. We suggest that the inability of mitogen-stimulated senescent cells to down-regulate p21Sdi1 levels contributes to the resulting lack of late Gi gene expression and failure to traverse the G1/S phase boundary.

Growth factor-deprived BALB/c 3T3 murine fibroblasts can enter the S phase after induction of c-myc gene expression

1987 ◽  
Vol 7 (10) ◽  
pp. 3554-3560
Author(s):  
F Cavalieri ◽  
M Goldfarb
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Dna Synthesis ◽  
S Phase ◽  
3T3 Cells ◽  
Glucocorticoid Treatment ◽  
Plasmid Construct ◽  
Murine Fibroblasts ◽  
Myc Gene

Induction of quiescent BALB/c 3T3 murine fibroblasts by platelet-derived growth factor (PDGF) or fibroblast growth factor (FGFs) is accompanied by induction of c-myc gene expression. To study the role of c-myc in cell growth, we transfected BALB/c 3T3 cells with a plasmid construct containing a glucocorticoid-inducible c-myc gene. When these transfected cells were growth arrested in PDGF-FGF-freedefined medium, glucocorticoid treatment induced S-phase DNA synthesis. This induction of DNA synthesis was inefficient, and cell proliferation was not evident, suggesting that growth factors act through stimulation of c-myc expression together with other intracellular events.

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