Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901.
OBJECTIVE
To elucidate the localization of RhoA in gastric SGC-7901 cancer cells and its translocation by lysophosphatidic acid (LPA) and/or 8-chlorophenylthio-cAMP (CPT-cAMP).


METHODS
Immunofluorescence microscopy was used to determine the localization of RhoA. Western blotting was used to detect both endogenous and exogenous RhoA in different cellular compartments (membrane, cytosol, nucleus) and the translocation of RhoA following treatment with LPA, CPT-cAMP, or CPT-cAMP + LPA.


RESULTS
Immunofluorescence staining revealed endogenous RhoA to be localized in the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus to be the nucleolus. Western blotting identified both endogenous and exogenous RhoA within different cellular compartments (membrane, cytosol, nucleus, nucleolus). After stimulation with LPA, the amount of RhoA within membrane and nuclear extracts increased, while it decreased in the cytosol fractions. After treatment with CPT-cAMP the amount of RhoA within the membrane and the nuclear extracts decreased, while it increased within the cytosol fraction. Treatment with a combination of both substances led to a decrease in RhoA in the membrane and the nucleus but to an increase in the cytosol.


CONCLUSIONS
In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus. Within the nucleus its precise localization could be demonstrated to be the nucleolus. Stimulation with LPA caused a translocation of RhoA from the cytosol towards the membrane and the nucleus; treatment with CPT-cAMP caused the opposite effect. Furthermore, pre-treatment with CPT-cAMP was found to block the effect of LPA.
Tao
Yan
Y
School of Medicine, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, Jiangsu Province, China.
Chen
Yong-Chang
YC
Li
Yue-Ying
YY
Yang
Shu-Qin
SQ
Xu
Wen-Rong
WR
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D021381:Protein Transport; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology; D013873:Thionucleotides / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis*
D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D013873:Thionucleotides; C066737:RHOA protein, human; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D020742:rhoA GTP-Binding Protein; C032881:lysophosphatidic acid