title: The stability of endogenous tyrosine hydroxylase protein in PC-12 cells differs from that expressed in mouse fibroblasts by gene transfer.
abstract: Previous studies have used recombinant retroviruses encoding the tyrosine hydroxylase (TH) gene to transduce various cell lines, including fibroblasts (NIH-3T3), a pituitary tumor cell line (AtT20), and a pancreatic endocrine line (RIN). These genetically modified cells, synthesizing either 3,4-dihydroxyphenylalanine, dopamine, or both, are potential donors for treatment of Parkinson's disease. However, the levels of TH protein in such transduced cells have been low and heterogeneous. Using several modified versions of retrovirus vectors encoding TH, we demonstrated that protein stability is an important factor governing levels of TH in NIH-3T3 fibroblasts. Whereas low levels of TH protein were observed in infected NIH-3T3 cells, high levels of a TH-beta gal fusion protein were found. This difference was due to a significantly longer half-life of the TH-beta gal fusion protein relative to TH alone. However, the TH-beta gal fusion protein was found to be enzymatically inactive. We also found that the half-life of the endogenous TH protein in PC-12 cells is sevenfold longer than the TH protein in transduced fibroblasts, implying that a cell-type specific regulator or mechanism may stabilize TH in catecholaminergic cells.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: D K
authors.initials: DK
authors.lastname: Wu
authors.forename: C L
authors.initials: CL
authors.lastname: Cepko
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D006224:Cricetinae; D004355:Drug Stability; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007158:Immunologic Techniques; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016716:PC12 Cells; D012190:Retroviridae; D013194:Staining and Labeling; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
chemical_list: D015345:Oligonucleotide Probes; D014446:Tyrosine 3-Monooxygenase