Thymidine, radiolabelled

Measured end points are photosynthesis as the incorporation of radiolabelled H C03 ( C) and bacterial activity as the incorporation of radiolabelled thymidine (thym) fluorometric measurements basal fluorescence (Fo) and photon yield (Y) chlorophyll-a concentration (chl-a) species composition (spp) and the biovolume of algae obtained after algal counting (biovolume)... 

Both H-thymidine incorporation and radiolabeled leucine incorporation techniques have been recently used to determine bacterial activity and growth in the rhizosphere of barley seedling (28), Bacteria were initially released from the rhizosphere using homogenization and centrifugation before adding the labeled substrates. The cell incorporation rate was twice as high in the rhizosphere than in bulk soil. In addition, both the leucine and thymidine incorporation rates increased with the distances from the root tip (28). 

Bacterial protein synthesis is directly correlated to bacterial activity and can be determined by incorporation of 3H or 14C leucine, as this amino acid is incorporated into proteins only. The method for leucine incorporation (Baath 1994) is the same as for thymidine incorporation in case of DNA synthesis and the incorporation of both precursors can be carried out in a single assay if different radiolabels are used. 

With the exception of the effect on microtubules described in the foregoing paragraph, the bisindole alkaloids have little or no effect on macro-molecular synthesis at subtoxic concentrations (21,22). In experiments utilizing radiolabeled precursors ([ H]leucine, -uridine, or -thymidine) cells cultured in the presence of vinblastine showed no differential incorporation of radioactivity. Furthermore, there is no indication that treatment of cells with vinblastine or vincristine produces alterations in cellular DNA (23,24). 

Fig. 13. Phytohemagglutinin (PHA) dose response curves of 10 patients with multiple myelomatosis and 10 controls. Peripheral blood lymphocytes were cultured and stimulated with PHA in increasing concentrations. Transformation was assessed by counting the uptake of radiolabeled thymidine.

The use of radi olabeled nucleic acid precursors, primarily tritiated thymidine or (deoxy)uridine, however, is subject to numerous drawbacks. Nevertheless, the use of radiolabeled precursors have provided some promising clinical correlation data [174]. 

Nearly all of the studies mentioned above compared assimilation of radiolabeled monomers with bacterial biomass production as estimated by leucine or thymidine incorporation. To examine the total flux of monomers and DOM we must consider respiration. 

Effect of Sarin on DNA and Protein Content Kassa et al (2000) showed that not only symptomatic level 3 but also asymptomatic levels 1 and 2 of sarin were able to significantly decrease the incorporation of radiolabeled thymidine without changing total concentrations of DNA or... 

The deoxyuridine suppression test measures the effect of prior addition of deoxyuridine on the uptake of radiolabeled thymidine into the DNA of cultured bone marrow cells, peripheral blood lymphocytes, or whole blood. Normal samples that contain vitamin B12 can convert deoxyuridine to thymidine and therefore do not take up as much thymidine. Samples from patients who are deficient in vitamin B12 show less suppression than in normal patients. Because it is relatively time consuming, the deoxyuridine suppression test is not widely available for use as a diagnostic test. ... 

For preparation of radiolabeled plasmid, tritiated thymidine is available from DuPont/NEN (Markham, Ontario, Canada), with a specific activity of approx 6-7 Ci/mmol and a purity >95%. 

Fig. 5.9 Mode of action of radiolabeled thymidine analogues used for imaging cellular proliferation. The radiotracers are transported and phosphory-lated by thymidine kinase (salvage pathway) to the corresponding monophosphate that is subsequently phosphorylated to the triphosphate and...

Similarly, when cells in Gi were fused to cells in S phase and the fused cells exposed to radiolabeled thymidine, the label was incorporated into the DNA of the Gi nucleus as well as the S-phase nucleus, indicating that DNA synthesis began in the Gi nucleus shortly after fusion. However, when cells in G2 were fused to S-phase cells, no incorporation of labeled thymidine occurred In the G2 nuclei. Thus diffusible factors in an S-phase cell can enter the nucleus of a Gi cell and stimulate DNA synthesis, but these factors cannot In-... 

Two early sets of experiments pointed to interactions of cisplatin with DNA, rather than the many other possible cellular receptors, as an essential target responsible for cytotoxicity and antitumor properties.Monitoring the uptake of radiolabeled precursors for synthesizing DNA, RNA, and proteins, showed that [ H]thymidine incorporation was most affected by therapeutic levels of cisplatin for both cells in culture and Ehrlich ascites cells in mice. Since independent studies showed that di-DDP binding to DNA polymerase does not alter its ability to synthesize DNA, it was concluded that platination of the template and not the enzyme was responsible for the inhibition of replication. 

In the experiment shown in Figure 9.17, the SV40-infected cells were treated with cisplatin. After 24 h, [ H]thymidine was added and, after 24 more hours, the cells were harvested, and SV40 DNA was isolated the amount of DNA synthesis was recorded by comparing incorporated radiolabel with results from control experiments where no platinum was present. The data show that, when 25 platinum was present, SV40 DNA replication was reduced to about 5 percent of control. Quantitation reveals that, at 2 platinum atoms bound per thousand nucleotides (drug-per-nucleotide, or (DIN)h, — 0.002), synthesis is only 10 percent that of control. 

Cytosolic thymidine kinase salvages exogenous thymidine extremely efficiently. Experiments with radiolabeled precursors show that dTTP derived from salvage synthesis is usually incorporated into DNA in preference to thymidine nucleotides generated by de novo synthesis. 

Cemilton , a product obtained from rye pollen, is an effective agent in the treatment of prostatic inflammatory disease and benign prostatic hyperplasia [139]. In 1995, DIBOA was identified as a constituent of the water soluble pollen extract [66]. The purified active fraction of Cemilton showed dose-dependent effects on DNA-synthesis in epithelial cells. At low concentrations, DNA synthesis was stimulated, indicated by a 300% increase in radiolabelled thymidine incorporation. Concentrations larger than 1 pg/ml resulted in an inhibition up to 80% after 5 days of exposure. Fibroblast cells react in a similar way and prostate DU 145 cells showed the same inhibition after treatment with the active fraction as they do after exposure to synthetic DIBOA. From the studies is was clear, that DIBOA must be causative for the effects of Cemilton. Cell growth inhibition was thought to be due to the chelating properties of the compound [67]. As demonstrated MCF-7 breast cancer-and COS-7-cells were inhibited as well [140]. From the morphology of treated DU-145 cells the authors concluded DIBOA-induced cell death. 

In P. infestans [17], cymoxanil when applied at concentrations up to 100 pg mL did not inhibit the uptake of radiolabeled precursors of DNA (thymidine), RNA (uridine) or proteins (phenylalanine). However, after less than 2-hours treatment, although uptake was not affected thymidine incorporation was considerably reduced while uridine incorporation was slightly affected and phenylalanine incorporation was insensitive. In contrast, cymoxanil at 10 pg mL did inhibit thymidine or uridine incorporation weakly while inhibition of mycelial growth was complete, suggesting that DNA and RNA synthesis inhibition is a secondary effect. RNA polymerase activity in isolated nuclei was not inhibited by cymoxanil. 

The role of lipoxygenase in these events is still unknown. Mitogens cause release of arachidonic acid from the phospholipids [330] and generation of thromboxane Bj and lipoxygenase products [154]. Arachidonic acid, but not other fatty acids, stimulates blast transformation and incorporation of radiolabeled thymidine and uridine [331], and this effect is inhibited by lipoxygenase inhibition [232]. Thus, it is proposed that the lipoxygenase pathway produces factor(s) that promote mitogene-sis. 

Following UV irradiation (254 nm, 45 J/m2) of cultured hepatocytes [6] repair patches were radiolabeled with [methyl- HJthymidine for either 20 min (pulse period), 120 min (20 min pulse followed by a 100 min chase period with unlabeled thymidine), or 180 min (20 min pulse followed by 160 min chase). At the end of these treatments, repair incorporated radioactivity was quantified in MOPS-DNA and random DNA... 

The use of radiolabeled nucleosides, taken up into proliferating cells and incorporated into DNA, is a well-known approach for the determination of cell proliferation, e.g., utilizing [ H] thymidine or fluorinated compounds (Shields et al. 1996). Nucleosides are taken up into cells by facilitated diffusion controlled by equilibrative and concentrative transporters. From the two groups of equilibrative transporters (es and is), the es-transporter is mainly expressed in human cell lines investigated so far. 

Haubner R, Avril N, Hantzopoulos PA, Gansbacher B, Schwaiger M. In vivo imaging of herpes simplex virus type 1 thymidine kinase gene expression early kinetics of radiolabelled FIAU. Eur J Nucl Med 2000 27 283-291. 

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