Background

[PubMed]

Puromycin (Pur) is an aminonucleoside antibiotic isolated from the bacteria Streptomyces alboniger. This antibiotic is a structural analog of aminoacyl-tRNA, and the insertion of Pur at the C-terminus of the protein inhibits further elongation of the protein chain in the ribosomes (at the translational level) in all organisms (prokaryotic and eukaryotic) (1). This phenomenon leads to accumulation of truncated and mis-folded proteins and culminates in cell death. The exact mechanism of action of Pur has been discussed by Starck and Roberts (2). Because cells in cancerous tumors have a very high rate of protein synthesis compared with normal cells, amino acids labeled with radionuclides have often been used to monitor anti-cancer therapy. The amino acids, however, can be utilized in the cells for protein synthesis through several different pathways; as a result, observations made with these tracers may not represent the actual anti-cancer efficacy of a drug (3). Protein synthesis is controlled primarily at the translation level, and it has been suggested that imaging the synthesis of nascent proteins may help researchers understand the relationships between the various normal and abnormal (diseased) physiological pathways in vivo (4). In this regard, fluorescence-labeled deoxy-cytidine derivatives of Pur and other fluorescent dyes have been used to investigate the synthesis of proteins in cells (5) and in cell-free systems (6), respectively. However, this approach has limited application under in vivo conditions because fluorescence cannot penetrate deep tissues and generates very low signal/noise ratios (4). Investigators have also used anti-Pur antibodies to study protein synthesis in the cell, but these reagents are most suited for use with immunoblots and produce inconsistent results at the cellular level (4).

In an effort to improve the visualization of protein synthesis under in vivo conditions, a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-labeled derivative of Pur was prepared for labeling with radionuclides such as ⁶⁸Ga or ⁴⁴Sc, which can be used with positron emission tomography (PET) (7). Although ⁶⁸Ga is useful in some clinical applications, the short half-life of this radionuclide (half-life = ~68 min) limits its use to the imaging of only those biological processes that occur rapidly (7). Because protein synthesis is a slow process, it requires the use of a tracer that has a longer half-life, such as ⁴⁴Sc (half-life = ~4 h) (7). Eigner et al. labeled DOTA-Pur with ⁴⁴Sc ([⁴⁴Sc]-DOTA-Pur) and evaluated the use of this radiolabeled antibiotic for the visualization of AT1 cell tumors (derived from rat prostate carcinoma) and Walker carcinoma 256 cell tumors (rat breast carcinoma cell line) in rats (7). The biodistribution of [⁴⁴Sc]-DOTA-Pur was also studied in these animals (7).

Synthesis

[PubMed]

DOTA-Pur was synthesized and labeled with ⁴⁴Sc as described by Eigner et al. (7). The radiochemical yield of [⁴⁴Sc]-DOTA-Pur was 59.0 ± 6.0%, with a radiochemical purity of >97% as determined with thin-layer chromatography. The specific activity of purified [⁴⁴Sc]-DOTA-Pur was 1.5 ± 0.1 GBq/μmol (40.5 ± 2.7 mCi/μmol). Purified [⁴⁴Sc]-DOTA-Pur was formulated in 0.9% sodium chloride at a concentration of 30 MBq/ml (810 μCi/ml).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The uptake of [⁴⁴Sc]-DOTA-Pur was investigated in DU145 cells and BJ cells in the presence or absence of increasing concentrations (10 pmol to ≥200 pmol) of either Pur (irreversible competitive inhibitor) or cyclohexamide (non-competitive inhibitor of Pur), as described elsewhere (7). The uptake of radioactivity by the DU145 cells and BJ cells in the absence of any inhibitor was 2.0 ± 0.1% of the applied dose per 1 × 10⁶ cells and 0.2 ± 0.1% of the applied dose per 1 × 10⁶ cells, respectively. Both cell lines were reported to incorporate 93% of the applied dose of radioactivity into the protein fraction (7).

In another study, unlabeled Pur (≥200 pmol) was shown to decrease the accumulation of label from [⁴⁴Sc]-DOTA-Pur in the DU145 cells to <8.2 ± 0.2% of the initial uptake value (without Pur). Similarly, unlabeled cyclohexamide (≥200 pmol) decreased the incorporation of radioactivity in these cells to <12.1 ± 0.2% of the initial uptake value (7).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No information is currently available.

References

1.

Croons V., Martinet W., Herman A.G., De Meyer G.R. Differential effect of the protein synthesis inhibitors puromycin and cycloheximide on vascular smooth muscle cell viability. J Pharmacol Exp Ther. 2008;325(3):824–32. [PubMed: 18322149]

2.

Starck S.R., Roberts R.W. Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition. RNA. 2002;8(7):890–903. [PMC free article: PMC1370306] [PubMed: 12166644]

3.

Haubner R. PET radiopharmaceuticals in radiation treatment planning - synthesis and biological characteristics. Radiother Oncol. 2010;96(3):280–7. [PubMed: 20724013]

4.

Liu J., Xu Y., Stoleru D., Salic A. Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc Natl Acad Sci U S A. 2012;109(2):413–8. [PMC free article: PMC3258597] [PubMed: 22160674]

5.

Starck S.R., Green H.M., Alberola-Ila J., Roberts R.W. A general approach to detect protein expression in vivo using fluorescent puromycin conjugates. Chem Biol. 2004;11(7):999–1008. [PubMed: 15271358]

6.

Blower M.D., Feric E., Weis K., Heald R. Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules. J Cell Biol. 2007;179(7):1365–73. [PMC free article: PMC2373496] [PubMed: 18166649]

7.

Eigner S., Vera D.R., Fellner M., Loktionova N.S., Piel M., Lebeda O., Rosch F., Ross T.L., Henke K.E. Imaging of Protein Synthesis: In Vitro and In Vivo Evaluation of (44)Sc-DOTA-Puromycin. Mol Imaging Biol. 2013;15(1):79–86. [PubMed: 22565849]