Green fluorescent protein tag for studies of drug-induced translocation of nucleolar protein RH-II/Gu.
We have constructed a human osteogenic sarcoma cell line, U-2 OS/GFP-Gu, that expresses nucleolar RNA helicase RH-II/Gu tagged with green fluorescent protein (GFP). The presence of a GFP tag does not inhibit RNA helicase, RNA folding and ATPase activities of RH-II/Gu protein. The derived cell line responds to cytotoxic agents like the parental cell line U-2 OS. In the presence of either actinomycin D or toyocamycin, the GFP-RH-II/Gu fusion protein translocates from the nucleolus to the nucleoplasm in the same way as the translocation of endogenous RH-II/Gu. The drug-induced translocation of GFP-RH-II/Gu is easily monitored by direct observation of live cells in vivo. This cell line can be used to screen cytotoxic drugs and to study the mechanisms of drug-induced translocation of RH-II/Gu. The cellular localization of RH-II/Gu during the cell cycle-dependent formation of the nucleolus is readily monitored. Real-time results are obtained more quickly without the disadvantages associated with cell fixation and immunofluorescence-based staining.
Valdez
B C
BC
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA. bvaldez@bcm.tmc.edu
Perlaky
L
L
Cai
Z J
ZJ
Henning
D
D
Busch
H
H
D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002466:Cell Nucleolus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012516:Osteosarcoma; D050828:Protein Inhibitors of Activated STAT; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins; D014127:Toyocamycin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D002352:Carrier Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; C106529:PIAS1 protein, human; D050828:Protein Inhibitors of Activated STAT; D025841:Small Ubiquitin-Related Modifier Proteins; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases; D014127:Toyocamycin