scholarly journals Inhibition of cell division by interferons. The relationship between changes in utilization of thymidine for DNA synthesis and control of proliferation in Daudi cells

1983 ◽  
Vol 214 (3) ◽  
pp. 983-990 ◽  
Author(s):  
D R Gewert ◽  
G Moore ◽  
M J Clemens
Keyword(s):  
Cell Proliferation ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Thymidine Incorporation ◽  
De Novo ◽  
Culture Conditions ◽  
Interferon Treatment ◽  
Daudi Cells ◽  
And Control ◽  
The Relationship

Inhibition of the proliferation of Daudi cells by exposure to human lymphoblastoid interferons is associated with an early and marked decrease in the incorporation into DNA of exogenous [3H]thymidine when cells are incubated with trace amounts of this precursor. In contrast, incorporation of exogenous deoxyadenosine into DNA is unchanged under the same conditions. Interferon treatment results in a lowering of thymidine kinase activity, an effect which may be largely responsible for the inhibition of incorporation of labelled thymidine into DNA. At higher concentrations of exogenous thymidine, which minimize the contribution of intracellular sources to the dTTP pool, the inhibition of thymidine incorporation is abolished. Under conditions in which exogenous thymidine is rigorously excluded from the medium or, conversely, in which cells are entirely dependent on exogenous thymidine for growth, the magnitude of the inhibition of cell proliferation by interferons is the same as under normal culture conditions. We conclude that, even though cell growth is impaired, the rate of DNA synthesis is not grossly inhibited up to 48 h after commencement of interferon treatment. Furthermore, changes in neither the utilization of exogenous thymidine nor the synthesis of nucleotides de novo are responsible for the effect on cell proliferation.

ANF-C-receptor-mediated inhibition of aortic smooth muscle cell proliferation and thymidine kinase activity

1994 ◽  
Vol 266 (1) ◽  
pp. R194-R203 ◽  
Author(s):  
P. A. Cahill ◽  
A. Hassid
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Receptor Binding ◽  
Kinase Activity ◽  
Thymidine Kinase Activity ◽  
Aortic Smooth Muscle ◽  
Inhibition Of Dna Synthesis ◽  
Type Receptor

We have investigated the inhibition of DNA synthesis and cell proliferation by rat atrial natriuretic factor [rANF-(99-126)] and several synthetic peptides that bind selectively to the ANF-C-type clearance receptors in subcultured aortic smooth muscle cells. These peptides decreased serum-induced 1) [3H]thymidine incorporation, 2) cell proliferation, and 3) thymidine kinase activity without altering basal or elevated cAMP or cGMP levels. In contrast, another ANF-C-receptor-binding peptide, des[Gln116,Ser117,Gly118,Leu119,Gly120] rANF-(102-121)-NH2 (cANF), failed to decrease serum-induced mitogenesis, yet 100 nM cANF reversed the inhibition of DNA synthesis and cell proliferation and the decrease of thymidine kinase activity elicited by other C receptor-binding peptides, including rANF-(99-126), rANF-(103-125), and porcine C-type natriuretic peptide [pCNP-(1-22)]. Delayed addition experiments indicated that atrial peptides influence a relatively late event (or events) during the G1 phase of the cell cycle. The inhibition of DNA synthesis by C-receptor-binding atrial peptides appeared to be selective for aortic smooth muscle cells, inasmuch as a potent inhibitory agonist peptide, Cys116-rANF-(102-116), was without significant influence on the incorporation of thymidine in cultured rat mesangial cells or bovine pulmonary artery endothelial cells. These results indicate that atrial natriuretic peptide analogues decrease vascular smooth muscle cell mitogenesis and proliferation by a cyclic nucleotide-independent mechanism involving the C-type receptor. Moreover the inhibition of DNA synthesis by rANF-(99-126) and the neuropeptide pCNP-(1-22) appears to be mediated by the ANF-C-type receptor and is associated with inhibition of thymidine kinase activity.

Ontogeny of cell proliferation and DNA synthesis in rat colon: role of glucocorticoids

1983 ◽  
Vol 244 (5) ◽  
pp. G469-G474 ◽  
Author(s):  
J. P. Buts ◽  
R. De Meyer ◽  
J. Kolanowski
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Dna Synthesis ◽  
Mitotic Index ◽  
Dna Content ◽  
Thymidine Incorporation ◽  
Rat Colon ◽  
Epithelial Cell Proliferation ◽  
New Findings

This study was undertaken to determine whether the rat colon exhibits ontogenic changes in epithelial cell proliferation and DNA synthesis during growth. DNA synthesis was measured at intervals after birth in four colonic segments by the incorporation rates of [3H]thymidine. The labeled crypt cell index was determined by radioautography. New findings from our study are that 1) in each colonic segment of suckling rats, [3H]thymidine incorporation rate overshot the adult levels (49-119%) with a peak occurring at day 14 postpartum, 2) between days 14 and 20, the incorporation rates declined sharply to adult values and remained thereafter unchanged until adulthood; during the same period, the labeled and mitotic index decreased, respectively, from 52 to 19% and from 3.58 to 1.43%, 3) the decrease in DNA synthesis and in cell proliferation rates was concomitant with an upsurge in plasma total corticosterone initiated on day 14, and 4) treatment of 10-day-old sucklings with physiological doses of hydrocortisone for 4 consecutive days significantly depressed (P less than 0.01) colonic DNA content and DNA synthesis rates to levels about 45-67% of the control values. These data indicate that growth of the colon may be under the control of glucocorticoid secretion at the weaning period.

Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis

1990 ◽  
Vol 68 (12) ◽  
pp. 1364-1371 ◽  
Author(s):  
Jim A. Wright ◽  
Arthur K. Chan ◽  
Bob K. Choy ◽  
Robert A. R. Hurta ◽  
Grant A. McClarty ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Drug Resistance ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Cell Biology ◽  
De Novo ◽  
Mutant Cell ◽  
Resistance Mechanisms ◽  
M2 Protein ◽  
Iron Storage

Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible-for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurea-resistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin. Several regulatory models are presented, including a model showing the relationships between M2 protein levels, deoxyribonucleotide pools, and DNA synthesis, and a model demonstrating a linkage between M2 and ferritin proteins in regulating DNA synthesis in normal and hydroxyurea-resistant mammalian cells.Key words: DNA synthesis, cell proliferation, ribonucleotide reductase, drug resistance.

scholarly journals HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism.

1997 ◽  
Vol 8 (8) ◽  
pp. 1247-1256
Author(s):  
N I Neverov ◽  
G A Kaysen ◽  
R Nuccitelli ◽  
R H Weiss
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Mesangial Cell ◽  
Thymidine Incorporation ◽  
Mesangial Cells ◽  
Mitogenic Effect ◽  
Kinase C ◽  
Mesangial Cell Proliferation

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.

Novel siRNA Expressing Lymphoma Model Identify Distinct Molecular Mechanisms Regulating NFkB1 and NFkB2 Induced Cell Proliferation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2372-2372
Author(s):  
Leon Bernal-Mizrachi ◽  
John Harding ◽  
Ratner Lee
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Dna Synthesis ◽  
Cell Line ◽  
Cell Lines ◽  
Thymidine Incorporation ◽  
Tumor Development ◽  
Nfkb Pathway ◽  
Pol Iii

Abstract The NFkB pathway has been implicated in tumorigenesis of several lymphoid malignancies. Most aggressive lymphomas have a constitutively active NFkB. Activation of NFkB results in processing of p105 and p100 to produce p50 and p52, respectively, and degradation of IkB. As a result, heterodimers of p65 and p50 or p52 and REL-B are formed and translocated to the nucleus where they induce transcription. Previously, it has been suggested that NFkB regulation of cyclin D and Myc expression is important for cell proliferation. However, these studies fail to explain all the NFkB regulatory effects, as well as the role of each individual pathway. To address these questions we tested the effect of NFkB on proliferation in two virally mediated murine lymphoma cell lines over-expressing Tax/HTLV-1 oncoprotein (SC and BLA) after knocking down (KD) p105 or p100, using a lentivirus expressing siRNAs. After synchronization, cells were released for 24 hrs and the rate of proliferation and cell cycle analysis were measured by thymidine incorporation and PI staining (4,8,16,24 hrs), respectively. As it is seen below, KD p100 or p105 cell lines had a reduced rate of thymidine incorporation compare to controls (Luciferase-siRNA). However, minimal changes were observed between cell lines in cell cycle analysis. To confirm these findings, xenograft experiments with siRNA expressing cell lines were developed. These experiments demonstrated that KD p100 or p105 in xenografts, prevented (in some) or delayed tumor formation, reduced tumor size and prolonged disease free survival. We then investigated NFkB regulation of cell proliferation by assessing gene expression in each cell line. Our results revealed a distinct proliferation-related gene profile between pathways: KD p105 dependent genes include: Cell cycle: Cyclin D2, B2 and G, CDK-8, P27, RB6, Transcription factor Dp2 and AKT-substrate-1, Growth factors: Platelet derived growth factor and RhoG, RNA synthesis: RNA Pol III. On the other hand KD p100 dependent genes include: Cell cycle: Cyclin D1,B2, retinoblastoma-like 1 (p107), p16, ring box-1 and histone deacetylase 2, Growth factors: HRAS and RhoG, RNA/DNA synthesis: RNA pol III and DNA pol (p17), histone 1 and 2, and thymidine kinases. Interestingly, both KD cell lines share many over-expressed genes such as TGFB, p21, p53, RB7 and BRCA1. In conclusion, our novel experimental model demonstrates: the main effect of both NFkB pathways involves DNA replication and early S phase. KD of either p100 or p105 reduces tumor development. The mechanism of cell proliferation regulated by each NFkB pathway is more complex than what was previously suspected; we suggest that RNA, DNA synthesis and regulation of p21, BRCA-1 and other cell cycle regulatory proteins also play an important role. Effect of Knocking Down p100 or p105 in Tumor Development Luciferase siRNA P100 siRNA P105 siRNA NR=Non reached. *Total 58 days f/u. Ten animals per cell line. H3 Thymidine SC 1 ± 0.09 0.38 ± 0.16 0.68 ± 0.37 H3 Thymidine BLA 1 ± 0.09 0.53 ± 0.07 0.69 ± 0.21 Tumor weight (mg) 617.18 ± 251 257.1± 285.1 258.6 ± 195.7 Tumor initiation (day) 32 ± 11 51 ± 11 43 ± 10 Tumor (+) animals (%) 100% 45% 78% Median DFS (day)* 30 NR 37

scholarly journals Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

1991 ◽  
Vol 115 (5) ◽  
pp. 1275-1282 ◽  
Author(s):  
T Usui ◽  
M Yoshida ◽  
K Abe ◽  
H Osada ◽  
K Isono ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
S Phase ◽  
Continuous Treatment ◽  
Protein Kinase Activity

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia

1987 ◽  
Vol 62 (1) ◽  
pp. 10-14 ◽  
Author(s):  
A. F. Junod ◽  
H. Petersen ◽  
L. Jornot
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Thymidylate Synthase ◽  
Kinase Activity ◽  
Primary Cultures ◽  
Thymidine Kinase Activity ◽  
Aortic Endothelial Cells ◽  
Key Enzyme

To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells.

scholarly journals A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells

1996 ◽  
Vol 318 (3) ◽  
pp. 965-971 ◽  
Author(s):  
Pamela H SCOTT ◽  
Christopher M BELHAM ◽  
Jenan AL-HAFIDH ◽  
Edwin R. CHILVERS ◽  
Andrew J. PEACOCK ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Dna Synthesis ◽  
Airway Smooth Muscle ◽  
Kinase Activity ◽  
Fold Increase ◽  
Platelet Derived Growth Factor ◽  
Airway Smooth Muscle Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
S6 Kinase

In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation.

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