Measurement of Protein Synthesis: In Vitro Comparison of ⁶⁸Ga-DOTA-Puromycin, [³H]Tyrosine, and 2-Fluoro-[³H]tyrosine

Abstract

Aim: Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using ⁶⁸Ga-DOTA-puromycin (⁶⁸Ga-DOTA-Pur), [³H]tyrosine, and 2-fluoro-[³H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. Methods: DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. ⁶⁸Ga was obtained from a ⁶⁸Ge/⁶⁸Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741–1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [³H]Tyrosine and 2-fluoro-[³H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). ⁶⁸Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20–25 MBq ⁶⁸Ga-DOTA-Pur. Results: No significant differences in the behavior of [³H]tyrosine and 2-fluoro-[³H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45–55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, ⁶⁸Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. ⁶⁸Ga-DOTA-Pur μPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors. Summary: Whereas the metabolic pathway of ⁶⁸Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [³H]tyrosine nor 2-fluoro-[³H]tyrosine can be considered useful for determination of protein synthesis.Research Support: This work has been supported by the Ministry of Education, Youth, and Sports of the Czech Republic and The Grant Agency of the Czech Republic.