Introduction
The use of thymidine (TdR) and thymidine analogs such as 3′-fluoro-3′-deoxythymidine (FLT) as positron emission tomography (PET)-based proliferation markers can provide information on tumor response to treatment. Studies on another TdR analog, 4'-thiothymidine (4DST), suggest that it might be a better PET-based proliferation tracer than either TdR or FLT. 4DST is resistant to the catabolism that complicates analysis of TdR in PET studies, but unlike FLT, 4DST is incorporated into DNA.
Methods
To further evaluate 4DST, the kinetics of 4DST transport and metabolism were determined and compared to FLT and TdR. Transport and metabolism of FLT, TdR and 4DST were examined in the human adenocarcinoma cell line A549 under exponential-growth conditions. Single cell suspensions were incubated in buffer supplemented with radiolabeled tracer in the presence or absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENT). Kinetics of tracer uptake was determined in whole cells and tracer metabolism measured by high performance liquid chromatography of cell lysates.
Results
TdR and 4DST were qualitatively similar in terms of ENT-dependent transport, shapes of uptake curves, and relative levels of DNA incorporation. FLT did not incorporate into DNA, showed a significant temperature effect for uptake, and its transport had a significant NBMPR-resistant component. Overall 4DST metabolism was significantly slower than either TdR or FLT.
Conclusions
4DST provides a good alternative for TdR in PET and has advantages over FLT in proliferation measurement. However, slow 4DST metabolism and the short half-life of the 11C label might limit widespread use in PET.
1. The L and MI isozymes ofpyruvate kinase were purified to homogeneity from rat liver and muscle respectively and their specific antibodies were employed to quantify the isozyme concentration in rat liver during development.2. Total enzyme activity decreases towards birth and reaches a minimum on the 3rd postnatal day, but the activity increases dramatically after weaning.3. Immunoprecipitation revealed that the M, type predominates in the prenatal period but decreases sharply just before birth.4. The L isozyme contribution is augmented upon weaning and is sustained until the rat is adult and a L/M ratio of 9: 1 is maintained.5. By means of incorporation studies with [3H]leucine followed by immunoprecipitation, the increase in L-type activity when approaching term and after weaning is explained by a twofold increase in its rate of synthesis coupled with a concomitant reduction of the M,-type synthesis.It has been recognized that pyruvate kinase exists in multiple molecular forms in various animal tissues [I -61. In adult rat liver over 90% of the total enzyme activity is attributed to the L isozyme and the remaining activity due to the M2 type [7]. Changes in total enzyme activity during foetal and postnatal development have been reported by various workers [8, 91. Differences in their physical and kinetic properties enable the differentiation of the isozymes and thus facilitate the subsequent estimation of individual isozyme activity. Methods for separation of the isozymes by electrophoresis [lo], ion-exchange chromatography [7] and adsorption to a hydroxyapatite column [l I] have been reported. Middleton and Walker [I 21 further demonstrated that the respective contributions of the isozymes to the total activity could be assessed by virtue of their differential behaviour towards phosphoennlpyruvate and inhibition by Cu2 +.
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