Thymidine incorporation into DNA

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Confluent Swiss 3T3 cells were serum-starved by incubation for 48 hr in DME containing 0.1% PCS. Cells were then incubated with the indicated compound at 37 C in the presence of H-thymidine, 0.1 or 10% PCS for 19.5 hr, washed, and then assayed for H-thymidine incorporation into DNA as described in Methods. 

To establish whether rifaximin, like the other members of the rifamycin family [36, 58], specifically inhibits bacterial RNA synthesis the effect of this antibiotic as well as that of rifampicin and chloramphenicol on RNA (via 3H-uridine incorporation), DNA (via 3H-thymidine incorporation) and protein (via 35S-methionine incorporation) synthesis was studied in growing cultures of Escherichia coli [59], While chloramphenicol reduced protein synthesis, both rifaximin and rifampicin inhibited RNA synthesis in a concentration-dependent fashion. In contrast, none of them affected 3H-thymidine incorporation into DNA. These data suggest that rifaximin, like rifampicin, inhibits RNA synthesis by binding the (3 subunit of the bacterial DNA-dependent RNA polymerase [60],... 

Antiproliferation activity shown by the inhibition of [3H]thymidine incorporation into DNA in the proliferation of rat vascular smooth muscle cells. The effect was expressed as % of control 45 0-7.8,46 0-0.4. kActivity found as a constituent of a plant other than a Broussonetia species. 

The measurement of incorporation of radioactive metabolites is frequently used as a response to short and intermediate duration cytotoxicity. Measurements of pH]-thymidine incorporation into DNA and [3H]-uridine incorporation into RNA are the two most common methods of quantifying drug cytotoxicity. Furthermore, [125I]-iododesoxyuridine, a specific and stable label for DNA synthesis, is also employed, as well as measurements of [32P]-phosphate release into medium or incorporation into nucleotides, in addition to the incorporation of [14C]-glucose, [3H]-amino acid, and 45calcium. 

Fig. 12.7. Time course of [3H]thymidine incorporation into DNA of UV irradiated, hydroxyurea-treated lymphocytes. 3 X106 lymphocytes from actinic keratosis patients ( ) or age-matched normal individuals (o) were incubated with [3H]thymidine (5/iCi/ml, 18.5 Ci/mmol) and hydroxyurea (1.5 mM) at 37°C immediately after irradiation (20 J-m 2). After incubating for different times cells were fixed in Camoy s fixative and washed with 5% trichloracetic acid and ethanol before counting. (Reproduced from Abo-Darub et al., 1978, with kind permission of the authors and...

Among the first cell viability assays developed for HTS was the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymidine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced solution, added to cells in culture, and incubated for approximately 4 hr. Viable cells convert MTT into an intensely colored formazan product that can be quantitated by recording changes in absorbance at specific wavelengths. 

Our own very preliminary work on this factor indicates the presence in human epidermis of a tissue specific, species nonspecific, partially heat labile, nondialyzable, factor which inhibits thymidine incorporation into DNA and does not depend on epinephrine. Epidermal extracts from psoriatic lesions did contain the factor in amounts equal to or greater than normal (M8). 

In vitro testing of delayed-type chemical hypersensitivity is based on the detection of chemical-specific IgG antibodies and/or T cells. Chemical-specific IgG antibodies are detected in solid-phase assays where the chemical is bound to various carriers, such as nitrocellulose or sepharose. These methods are controversial and are not recommended for the routine diagnosis of chemical hypersensitivity. Chemical-specific T cell responses are measured in the so-called LTT. This test reveals a sensitization of T cells by an enhanced proliferative response of peripheral blood mononuclear cells to a certain chemical. Generally, lymphocyte transformation is measured by [3H]thymidine incorporation into DNA, but alternative,... 

As a result, S phase was prolonged in these cells (Figure 2). Following G1 treatment with cisplatin, synchronized Hela cells also showed a decrease in the amount of thymidine incorporation into DNA during S phase, but the effect was not immediately manifested in these cells (63) (Figure 3). Thus cells differ in the way in which their replication machinery responds to cispla-tin-induced daimage. Such differences might account for variations in cellular sensitivity (see below) and further studies in this area are warranted. 

Oleic acid (pM) [3H]Thymidine incorporation into DNA ofLLC cells (xlO3 dpm/well) Capillary-like tube formation (mm length/field Adherence of LLC cells to HMVEC (xlO3 LLC cells/well)... 

Biosynthesis and catabolism of DNA and RNA altered Thymidine incorporation into DNA reduced DNA polymerase activity reduced - E.coli P incorporation into RNA reduced... 

CDAA (N-N-diallyl-2-chloroacetamide) was reported to reduce mitosis in barley (Hordeum vulgare L.) roots nearly 90% after 96 h at 57 yM (38). Propachlor (2-chloro-N-isopropylacetanilide) totally inhibited mitosis in onion (Allium cepa L.) root tips after an 18 h treatment with 75 yM (3 ). At 20 yM cell division was reduced approximately 50% and cell enlargement was reduced 40% in oat coleoptiles (39). After 24 h, 100 yM ioxynil (4-hydroxy-3,5-diiodobenzonitrile) reduced the mitotic index in broad bean (Vicia faba L. ) and pea root tips ( ). Few herbicides that inhibit cell division have been studied in adequate detail to locate the site of the block. A notable exception is the herbicide chlorsulfuron (DPX 4189, 2-chloro-N- [(4-methoxy-6-methyl-l,3,5-triazin-2-yl)amino]carbonyl -benzenesulfonamide) ( ). Ray reported a 50% reduction in corn growth 3 h after treatment with 28 yM chlorsulfuron. Mitosis in broad bean root tips was significantly reduced by 2.8 yM, whereas in three different tests, cell enlargement was not influenced with concentrations of 28 yM. Thymidine incorporation into DNA was inhibited in corn root tips after a 1 h treatment with... 

EDjq (pM) for inhibiting thymidine incorporation into DNA FEAU FMAU FEA ifMAU... 

Compounds 27-30 were evaluated for in vitro antitumour activity by means of their inhibitory effect on [ H] thymidine incorporation into DNA in HL-60 cells. Their inhibition was showed in Table 1. 

In S phase the catabolism increases when the total amount of guanosine taken up by the cells exceeds 600 nmol/10 cells, which coincides with a decrease of thymidine incorporation into DNA. The greater importance of catabolic route in Gi cells may be an expression of the lower guanine phosphoribosy1 transferase activity in this stage of the cell cycle. ... 

Fig. 5.16. (A) Labelling index of thymidine incorporation into DNA as a function of root length following emergence from the quiescent Allium cepa seed. Each point represents one root, approximately 1000 cells counted per root. (B) Mitotic index as a function of root length. Each point represents a random count of approximately 1000 cells from one root. After Bryant, 1969 [24]...

The in vivo effect of chronic lead exposure on the acquisition of neural cells in the developing rat brain was assessed by measuring the rate of pH]thymidine incorporation into DNA. Wistar rats were used throughout the entire study and were maintained at 20°C and in a 12 h light/dark cycle. The animals had ad libitum access to food and water at all times. The pups were exposed to lead from their time of conception to the required postnatal period via their dams, who had ad libitum access to drinking water containing 400 mg PbCl2/l. Food and water intake was constantly monitored over exposure periods, and no difference in food and water intake was observed between control and lead-exposed animals. 

Duque A, Rakic P (2011) Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fete. J Neurosci 31 15205-15217... 

Sphingosine and sphingosine 1-phosphate can induce DNA synthesis in growth-arrested Swiss 3T3 cells. FBj incubated with the cells elevated sphingosine and induced an increase in [ H] thymidine incorporation into DNA (59). Further studies showed that sphinganine was required for stimulation of DNA synthesis. Studies with LLC-PK renal cells showed a close correlation between fumonisin-induced cytotoxicity and inhibition of de novo sphingolipid biosyntheses (82). However, studies with primary rat hepatocytes have not shown any relationship between cytotoxicity and elevation of free sphingoid bases (38). 

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