Trypsinization severely perturbs radioiodide transport via membrane Na/I symporter proteolysis: implications for reporter gene imaging.
BACKGROUND
Cell preparation procedures injurious to Na/I symporters (NIS) could deter their usefulness for reporter gene assays and in vivo cell imaging. In this study, we investigated the effects of cell collection by trypsinization on radioiodide transport and in vivo cell imaging results.


METHODS
The influence of trypsinization procedures on (125)I transport was evaluated using Huh-7/NIS hepatoma cells. The effects of graded concentrations of trypsin and EDTA were assessed on Huh-7/NIS and A431/NIS lung cancer cells. Trypsin-induced NIS proteolysis was investigated by immunoblots of plasma membrane prepared from adenovirus-infected mouse liver tissue. (99m)Tc-O(4)(-) scintigraphy was performed in Balb/C nude mice at 1 and 4 h following administration of Huh-7/NIS cells collected with and without trypsin.


RESULTS
(125)I Transport ability of Huh-7/NIS cells was severely impaired within minutes of standard trypsinization and further deteriorated up to 24 h after termination of treatment. This perturbation was caused by trypsin, which dose- and time-dependently induced substantial reductions of (125)I uptake in Huh-7/NIS and A431/NIS cells. Immunoblot analysis revealed significant dose- and time-dependent losses of membrane NIS protein by trypsin. NIS proteolysis was completely blocked by soybean trypsin inhibitor, and partial protection was offered by the substrates iodide and perchlorate. On (99m)Tc-O(4)(-) scintigraphy of mice, cells prepared by trypsinization were poorly visualized, whereas those collected with a nonenzymatic method showed significantly better uptake and contrast.


CONCLUSIONS
Trypsinization leads to serious perturbations in iodide accumulating capacity through tryptic degradation of membrane NIS protein. Hence, NIS-based reporter assays and in vivo cell imaging studies may benefit from better-optimized cell cultivation and harvesting procedures.
Jung
Kyung-Ho
KH
Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. jkhope.jung@sbri.co.kr
Paik
Jin-Young
JY
Lee
You La
YL
Lee
Yong Jin
YJ
Lee
Jaetae
J
Lee
Kyung-Han
KH
D006528:Carcinoma, Hepatocellular / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008175:Lung Neoplasms / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D015336:Molecular Probe Techniques; D010447:Peptide Hydrolases / Q000378:metabolism; D049268:Positron-Emission Tomography / Q000379:methods*; D027981:Symporters / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014357:Trypsin / Q000008:administration & dosage*; D000099209:Sodium-Iodide Symporters
D010447:Peptide Hydrolases; D027981:Symporters; D014357:Trypsin; D000099209:Sodium-Iodide Symporters