In vivo imaging of proteasome inhibition using a proteasome-sensitive fluorescent reporter.
A proteasome degrades numerous regulatory proteins that are critical for tumor growth and is therefore recognized as a promising anticancer target. Determining proteasome activity in the tumors of mice bearing xenografts is essential for the development of novel proteasome inhibitors. We developed a system for in vivo imaging of proteasome inhibition in the tumors of living mice, using a proteasome-sensitive fluorescent reporter, ZsProSensor-1. This reporter consists of a green fluorescent protein, ZsGreen, fused to mouse ornithine decarboxylase, which is degraded by the proteasome without being ubiquitinated. In stably transfected cells expressing ZsProSensor-1, the fluorescent reporter was rapidly degraded under steady-state conditions, whereas it was stabilized in the presence of proteasome inhibitors. Subcutaneous inoculation of the transfected cells into nude mice resulted in tumor formation. When the proteasome inhibitor bortezomib was intravenously administered to mice bearing these tumors, the ZsProSensor-1 protein accumulated in the tumors and emitted a fluorescent signal in a dose-dependent manner. Robust fluorescence was sustained for 3 days and then gradually decreased to baseline levels within 15 days. Intravenous administration of bortezomib also showed potent antitumor activity. In contrast, oral administration of bortezomib did not result in fluorescent protein accumulation in tumors or exhibit any antitumor activity. These results indicate that in vivo imaging using the ZsProSensor-1 fluorescent protein can be used as an indicator of antitumor activity and will be a powerful tool for the development of novel proteasome inhibitors.
Institute of Microbial Chemistry, Numazu, Shizuoka, Japan. imomose@bikaken.or.jp
Isao
I
Momose
Daisuke
D
Tatsuda
Shun-ichi
S
Ohba
Tohru
T
Masuda
Daishiro
D
Ikeda
Akio
A
Nomoto
D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D001897:Boronic Acids / Q000008:administration & dosage / Q000494:pharmacology*; D000069286:Bortezomib; D002460:Cell Line; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000008:administration & dosage / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D001897:Boronic Acids; D061988:Proteasome Inhibitors; D011719:Pyrazines; D049452:Green Fluorescent Proteins; D000069286:Bortezomib; D046988:Proteasome Endopeptidase Complex