scholarly journals THYMIDINE TRANSPORT BY CULTURED NOVIKOFF HEPATOMA CELLS AND UPTAKE BY SIMPLE DIFFUSION AND RELATIONSHIP TO INCORPORATION INTO DEOXYRIBONUCLEIC ACID

1972 ◽  
Vol 55 (1) ◽  
pp. 161-178 ◽  
Author(s):  
Peter G. W. Plagemann ◽  
John Erbe
Keyword(s):  
Thymidine Incorporation ◽  
Initial Rate ◽  
Hepatoma Cells ◽  
Novikoff Hepatoma ◽  
Simple Diffusion ◽  
Transport Reaction ◽  
Rate Limiting Step ◽  
Low Concentrations ◽  
Thymidine Transport ◽  
Inhibition Of Dna Synthesis

The initial rate of thymidine-3H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent Km of 0.5 µM, and the Vmax values increased with an average Q10 of 1.8 with an increase in temperature. The intracellular acid-soluble 3H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 µM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent Km and Ki values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 µM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.

Stimulatory effect of dimethylsulfoxide on [3H]thymidine incorporation into DNA in Novikoff hepatoma cells

1978 ◽  
Vol 9 (6) ◽  
pp. 389-394 ◽  
Author(s):  
Rosemary Barra ◽  
Herbert Hicks ◽  
Michael R. Koch ◽  
Michael A. Lea
Keyword(s):  
Thymidine Incorporation ◽  
Hepatoma Cells ◽  
Novikoff Hepatoma

scholarly journals Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells.

1975 ◽  
Vol 64 (1) ◽  
pp. 29-41 ◽  
Author(s):  
P G Plagemenn ◽  
D P Richey ◽  
J M Zylka ◽  
J Erbe
Keyword(s):  
Cell Cycle ◽  
De Novo ◽  
Hepatoma Cells ◽  
S Phase ◽  
Transport Processes ◽  
Transport Systems ◽  
Thymidine Transport ◽  
Inhibition Of Dna Synthesis ◽  
Rat Hepatoma ◽  
Rat Hepatoma Cells

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.

scholarly journals Inhibition of DNA synthesis in adrenocortical cells by cytochalasin B.

1980 ◽  
Vol 86 (1) ◽  
pp. 129-134 ◽  
Author(s):  
M A McPherson ◽  
J Ramachandran
Keyword(s):  
Dna Synthesis ◽  
Cytochalasin B ◽  
Thymidine Incorporation ◽  
Cell Types ◽  
Mouse Tumor ◽  
Adrenocortical Cells ◽  
Normal Cells ◽  
Normal Rat ◽  
Thymidine Transport ◽  
Inhibition Of Dna Synthesis

ACTH inhibits DNA synthesis in normal rat and mouse tumor Y-1 adrenocortical cells within the same concentration range that it stimulates steroidogenesis. These processes can be independently regulated as demonstrated by the divergent actions of cytochalasin B on these cells. In the normal cells, cytochalasin B does not increase steroidogenesis in serum-free or serum-containing media, and it decreases the stimulation produced by ACTH. In the absence of serum, the Y-1 cells respond in a similar way. However, in serum-containing media, cytochalasin B increases steroidogenesis in these cells and does not inhibit the response to ACTH. In both cell types, cytochalasin B inhibits [3H]thymidine incorporation into DNA by a mechanism different than that of ACTH. In the Y-1 cells, this inhibition is caused by a decreased uptake of [3H]thymidine into the cell, which probably reflects a decreased transport across the cell membrane. In the normal cells, cytochalasin B, like ACTH, does not affect [3H]thymidine transport, but it decreases DNA synthesis much more rapidly than does ACTH. This inhibition may be the result of the disruption of microfilaments by cytochalasinB, because our evidence indicates that it is not caused by a decrease in glucose uptake by the cells.

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

scholarly journals K+/H+-antiporter nigericin arrests DNA synthesis in Ehrlich ascites carcinoma cells

1989 ◽  
Vol 86 (17) ◽  
pp. 6626-6629 ◽  
Author(s):  
L B Margolis ◽  
Y u Novikova I ◽  
I A Rozovskaya ◽  
V P Skulachev
Keyword(s):  
Membrane Potential ◽  
Dna Synthesis ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Low Concentrations ◽  
Ehrlich Ascites ◽  
Inhibition Of Dna Synthesis ◽  
Dna Synthesis Inhibition

Acidification of the cytoplasm of Ehrlich ascites carcinoma cells to pH 6.3 arrests DNA synthesis in these cells. Such an effect can be achieved by incubating the cells at pH 6.2 or by adding low concentrations of the K+/H+ antiporter, the antibiotic nigericin, at neutral pH. Glucose and anaerobiosis potentiate the nigericin effect. The inhibition of DNA synthesis by nigericin occurs without any significant decrease in the ATP concentration and in the mitochondrial membrane potential. The DNA synthesis inhibition is caused neither by a decrease in the intracellular [K+] nor by an increase in the intracellular [Na+] accompanying the nigericin effect (at least at low concentrations of the antibiotic). Nigericin should thus be regarded as a type of a cytostatic primarily affecting intracellular pH.

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