Thymidine tritiated

HTdR Tritiated thymidine 5-HT 5-Hydroxyttyptamine also known as Serotonin HTLV-1 Human T-cell leukaemia virus-1... 

Kandell and Bernstein published one of the earliest reports to suggest that bile acids also demonstrate DNA-damaging effects in eukaryotic cells. They showed that human foreskin fibroblasts underwent unscheduled DNA synthesis (indicating DNA repair), as measured by tritiated thymidine incorporation when cells were treated with increasing concentrations of sodium deoxycholate or chenodeoxycholate. Utilising mutant Chinese hamster ovary cells deficient in strand rejoining (EM9), the authors were able to demonstrate that the repair of deoxycholate-induced DNA damage was dependent on strand break repair capacity. 

Maleuric acid (A -carbamoylmaleamic acid, XVIII), when injected into mice bearing Ehrlich ascites tumours, can produce cytoplasmic abnormalities in all phases of mitosis. This acid also inhibits the incorporation of tritiated thymidine into DNA, and prevents the progression of premitotic cells into mitosis [217]. This substance, which is an open-chain analogue of orotic acid, may possibly be an antimetabolic of this pyrimidine or related compounds. 

Measurements of the rate and extent of cell proliferation are essential for understanding the effects of cell growth factors and inhibitors. In the absence of aneuploidy, the DNA content is an invariant characteristic of each particular cell type, and typically the incorporation of tritiated thymidine into DNA is a useful measure of cell prolifera-... 

Cell number frequency distribution of nuclear DNA content of cell population, protein content, protein synthesis (e.g., 14C-labeled methionine incorporation), DNA synthesis (e.g., tritiated thymidine incorporation), DNA stains (e.g., Hoechst 33 342 4, 6-diamidino-2-phe-nylindole, DAPI picogreen) mass tracker dyes (e.g., LysoTracker green for lysosomes, MitoTracker deep red for mitochondria, ER-Tracker blue-white DPX for endoplasmic reticulum)... 

The normal cell cycle consists of a definable sequence of evenfs fhaf characferize fhe growfh and division of cells and can be observed by morphological and biochemical means. The cell cycle is depicfed in Fig. 55.1. Two of the four phases of the cell cycle can be studied directly the M-phase, or mitosis, is easily visible using light microscopy because of chromosomal condensation, spindle formation, and cell division. The S-phase is the period of DNA synthesis and is observed by measuring the incorporation of tritiated thymidine into cell nuclei. 

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]. 

Chloro-orr/2o-toluidine given orally to male mice at a dose of 200 mg/kg bw inhibited testicular DNA synthesis, as measured by tritiated thymidine incorporation (Seiler, 1977). 

Cell kinetics is defined as the measurement of time parameters m biological systems. Traditionally, this has involved the use of radioactive precursors of DNA, such as tritiated thymidine (3HTdR), and autoradiography to detect their incorporation into DNA. This technique has provided detailed knowledge of cell kinetics in both in vitro and in vivo experimental systems. The technique, however, is time consuming and arduous and is not readily applicable to human tumor research because of ethical problems involved in incorporation of a radioisotope into DNA. 

Foley J., Ton T., Maronpot R., Butterworth B, and Goldsworthy T. L (1993) Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ Health Persp. 101(Suppl. 5), 199-206... 

Thymidine Uptake Studies. Tritiated thymidine (52 mCi/ anole 0.1 /rCi/ml) was added to cells for 60 min at 37°C. Cells were then washed with PBS, incubated at 4°C for 15 min in the presence of ice cold 5% TCA, rinsed with TCA and scraped from the flasks with a rubber policeman. Cells were again washed with PBS, and solubilized in 0.1N NaOH overnight. Aliquots were assayed for protein (62) and radioactivity (scintillation fluid 100 ml Biosolve (Beckman, Fullerton, CA.), 7g of PPO and 0.6 g of POPOP per liter of toluene). 

Effects of Minor Human Brain Ganglioside on Tritiated Thymidine Incorporation (CPMxlO ) in Mixed Leukocyte Cultures... 

Unscheduled DNA Synthesis in Mammalian Cells. The principle of this test is that it measures the repair that follows DNA damage and is thus a reflection of the damage itself. It depends on the autoradiographic measurement of the incorporation of tritiated thymidine into the nuclei of cells previously treated with the test chemical. 

In vitro lymphocyte proliferative responses to mitogens such as phytohemagglutinin, concanavalin A, and pokeweed mitogen or to specific antigens of interest. The response may be measured by tritiated thymidine incorporation or by cytokine production (IFN-r, IL-2, TNF, and others). 

Recently, in collaboration with R. J. Kuchler and D. Cryan of the Bureau of Biological Research, Rutgers University, we have observed that Balb/c 3T3 fibroblasts when cultured for prolonged periods with 25-400 Ug/ml of purified W. floribunda agglutinin accumulated massive amounts of vacuoles. This treatment with lectin is apparently not toxic to the cells as measured by the ability of treated cells to incorporate tritiated thymidine into cellular DNA. Figure 4 shows the effect on cells of treatment for 48 hours with 150 lig/ml of lectin as compared to cells treated in... 

UNSCHEDULED DNA SYNTHESIS (UDS) DNA synthesis that occurs at a stage in the cell cycle other than S incorporation of precursors (e.g., tritiated thymidine) into ENA in the absence of semiconservative replication a manifestation of genetic repair, whose occurrence has been used as an indicator of induced DNA damage. 

Bone marrow is placed into a balanced salt solution containing preservative-free heparin and a single cell suspension prepared by passing the marrow through a fine wire mesh. Alternatively, a small Potter-Elvehjem tissue homogenizer (Hll) may be used. The nucleated cell count is adjusted to 5-10 X 106 ml of balanced salt solution and deoxyuridine added. Following incubation, 5 p,Ci of tritiated thymidine is added and the mixture incubated for an additional hour. The cells are then washed and the nucleated cell count determined. A 0.1 ml aliquot is then placed on a filter paper disk and dried. The activity of the dried disks is then measured in a scintillation... 

K3. Killman, S. A., Effect of deoxyuridine on incorporation of tritiated thymidine Difference between normoblasts and megaloblasts. Acta Med. Scand. 175, 483-497 (1964). 

Kenny (1973) recommends a Soy peptone-yeast dialysate medium (Appendix 4) for culture of mycoplasma. Inclusion of arginine (16 mM) and 0.4 mg% phenol red indicates the presence of arginine deaminase by formation of alkali (purple coloration). Alternatively, incubation with tritiated thymidine and analysis of the culture medium for tritiated thymine can be used to detect thymidine phosphorylase (House and Waddell, 1967). 

Autoradiography of contaminated cultures labelled with tritiated thymidine shows grains over the whole cell rather than localised to the nucleus. This is a result of degradation of the thymidine to thymine followed by incorporation into mycoplasma DNA and other cell constituents. 

Fig. 10.2. Mitotic stages. Mouse L929 cells, pulse labelled with tritiated thymidine, were processed for autoradiography. Among the S-phase cells (covered with grains) are cells in other stages of interphase and several mitotic cells. A metaphase cell, two anaphase cells and a late telophase cell are distinguishable.

If cells are synchronised at the G1 /S boundary and then released, the rate of DNA synthesis is initially slow but accelerates to reach a maximum at about 3h and then decelerates until S-phase is essentially complete in 6-7 h (Stubblefield and Mueller, 1962 Adams, 1969b). As replication occurs different numbers of replicons are active at any one time, and so it is not surprising that more careful labelling reveals bursts of tritiated thymidine incorporation throughout S-phase rather than a steady even progression (Klevecz, 1969 Lett and Sun, 1970 Klevecz et al., 1974). 

This was shown clearly by Stubblefield and Mueller (1962), who demonstrated focalised synthesis of DNA by pulse-labelling a random population of cells with tritiated thymidine and then after varied time intervals visualised autoradiographically the regions of metaphase chromosomes where incorporation has occurred. 

Detailed analysis using several different methods (Pulse and continuous label with tritiated thymidine, DNA fluorescence per cell using the fluorescent Feulgen assay and flow microfluorography (Van Dilla et al., 1969), and DNA specific fluorescence using the diaminobenzoic acid assay (Kissane and Robins, 1958) reveals that the DNA content of a cell increases in a saltatory fashion, and that the early portion of S-phase is a period of low net DNA synthesis which may be mistaken for G1 if insensitive methods of measurement are used (Klevecz et al., 1975). 

Tritiated thymidine pulse method (Howard and Pelc, 1953)... 

Fig. 10.7. Fraction of labelled mitoses. The solid line represents the theoretical change in the fraction of mitotic cells after labelling for 100 min with tritiated thymidine. The dotted line follows data obtained with mouse L cells. The hatched areas represent the duration of the exposure to tritiated thymidine. (Reproduced from Cleaver, 1967, with kind permission of the author.)...

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