DNA synthesis in cultures of calf aortic endothelial cells treated with corpus luteum extract: Its inhibition by 3H-thymidine

Adenosine stimulation of proliferation of breast carcinoma cell lines: Evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro

“…We performed a series of control experiments to examine whether the apparent effect of adenosine on [ 3 H]thymidine incorporation represented an authentic stimulation of DNA synthesis, or might have been compromised by the known ability of [ 3 H]thymidine to perturb DNA replication, cell cycle progression, and proliferation (Jakob et al, 1981; Hoy et al, 1990; Hu et al, 2002), or by interference with the assay due to adenosine.…”

“…2b). Thirdly, we diluted out the radioactive [ 3 H]thymidine with concentrations of ‘cold’ thymidine up to 100 μM, which has been shown to ameliorate the effect of radioactive thymidine (Jakob et al, 1981; Solary et al, 1992). Again, there was no significant change to the form of the adenosine response, even with the addition of 100 μM unlabeled thymidine (Fig.…”

“…It has long been known that in certain cell lines and primary cell cultures, incubation with [ 3 H]thymidine can interfere with DNA replication, cell cycle progression, and proliferation (Jakob et al, 1981; Hoy et al, 1990; Hu et al, 2002). This effect has been most clearly attributed to the internal beta radiation emitted by the tritium radiolabel (Jakob et al, 1981; Hu et al, 2002), which produces DNA fragmentation in some cell lines (Solary et al, 1992). Indeed, other low‐energy beta‐emitting radioisotopes such as 35 S also inhibit cell cycle progression, proliferation, and survival (Hu and Heikka, 2000).…”

“…Indeed, other low‐energy beta‐emitting radioisotopes such as 35 S also inhibit cell cycle progression, proliferation, and survival (Hu and Heikka, 2000). The artifact due to [ 3 H]thymidine is protocol‐dependent (Hoy et al, 1990) and is ameliorated by the addition of ‘cold’ (unlabeled) thymidine (Jakob et al, 1981; Solary et al, 1992). Our standard protocol in fact includes addition of ‘cold’ thymidine to lower the specific activity of the radioisotope, making this problem unlikely.…”

“…Our experiments showed that: (i) the tritiated compound (without dilution of the specific radioactivity) had no effect on the proliferation or viability of either cell line at radioactive concentrations up to 10 μCi/ml (10‐fold higher than we use in our incorporation assay); (ii) decreasing or increasing the radioactive concentration of [ 3 H]thymidine to 0.1 or 10 μCi/ml did not change the form of the adenosine response; (iii) diluting out the [ 3 H]thymidine with ‘cold’ thymidine at concentrations as high as 100 μM to ameliorate potential effects of radioactive thymidine (Jakob et al, 1981; Solary et al, 1992) did not change the results (other than reducing the net incorporation of label); and (iv) delaying the addition of [ 3 H]thymidine to the point at which we had determined by cell cycle analysis (Fig. 4) that the cells were traversing S phase (thereby minimizing the exposure to the radiolabel) again produced the same form of dose‐response curve to adenosine.…”