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. 2008 Feb 28;14(8):1175-81.
doi: 10.3748/wjg.14.1175.

Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901

Yan Tao  1 , Yong-Chang Chen, Yue-Ying Li, Shu-Qin Yang, Wen-Rong Xu
Affiliations

Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901

Yan Tao et al. World J Gastroenterol. .

Abstract

Aim: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Localization of endogenous RhoA in SGC-7901 cells. RhoA was immunofluorescently stained with a specific mAb and it was localized in the cytoplasm, the membrane, and the nucleus (× 400).
Figure 2
Figure 2
Distribution of RhoA in membrane, cytosol, and nucleus in SGC-7901 cells. SGC-7901 cells were lysed and membrane (A), cytosol (B) and nuclear (C) fractions were obtained. Expression of RhoA in each fraction was detected by Western blotting using a specific anti-RhoA mAb.
Figure 3
Figure 3
Distribution of endogenous and exogenous RhoA in membrane, cytosol, and nucleus in SGC-7901 cells. SGC-7901 cells were transfected with three types of exogenous EE-tagged RhoA plasmids, and 24 h later, cells were lysed and membrane (A), cytosolic (C) and nuclear (B) fractions were obtained. The expression of different RhoA proteins (lane 1 mock, lane 2 wild type, lane 3 63L, lane 4 N19) in each fraction was detected by Western blotting with mAb against RhoA.
Figure 4
Figure 4
Localization of RhoA in the nucleolus of interphase SGC-7901 cells. RhoA in SGC-7901 cells of interphase was immunofluorescently stained with monoclonal antibody against RhoA (A). The chromosomes were stained with Hoechst 33342 (B) (× 1000).
Figure 5
Figure 5
RhoA could be detected in nucleolus in SGC-7901 cells. SGC-7901 cells were lysed and the nucleolus fraction was obtained. Expression of RhoA in the nucleolus fraction (B) was detected by Western blotting with a mAb directed against RhoA, and total protein obtained from SGC-7901 cells (A) served as the positive control.
Figure 6
Figure 6
The expression of RhoA protein was higher in interphase than in mitosis phase in HeLa cells. HeLa cells were treated with Taxol (5 μg/mL, 12 h). Control (attached) (A) or mitotic (“shake off”) (B) cells were lysed and the expressions of RhoA and GAPDH were detected by Western blotting with mABs directed against RhoA and GAPDH.
Figure 7
Figure 7
The mRNA expression of RhoA gene was higher in interphase than in mitosis phase in HeLa cells. HeLa cells were treated with Taxol (5 μg/mL, 12 h). Total mRNA was isolated from control (attached) and mitotic (“shake off”) cells, and the expression of RhoA gene was detected by real-time quantitative PCR. Each bar represents mean ± SE obtained from five independent experiments. aP < 0.05.
Figure 8
Figure 8
LPA-induced RhoA translocation from cytosol towards membrane and nucleus in SGC-7901 cells. SGC-7901 cells were treated with or without LPA (1 μmol/L, 15 min). Control or LPA -treated cells were lysed and the membrane (A), cytosolic (C), and nuclear (B) fractions were obtained. Expressions of RhoA and GAPDH proteins in each fraction were detected by Western blotting, and the representative band images were shown in the left panels. Densitometric analysis of the bands was shown in the right panels. Each bar represents mean ± SE obtained from five independent experiments. aP < 0.05.
Figure 9
Figure 9
cAMP-induced RhoA translocation from membrane and nucleus towards cytosol in SGC-7901 cells. SGC-7901 cells were treated with or without CPT-cAMP (250 μmol/L, 30 min). Control or CPT-cAMP -treated cells were lysed, and membrane (A), cytosolic (C), and nuclear (B) fractions were obtained. Expressions of RhoA and GAPDH proteins in each fraction were detected by Western blotting, and the representative band images were shown in the left panels. Densitometric analysis of the bands was shown in the right panels. Each bar represents mean ± SE obtained from five independent experiments. aP < 0.05.
Figure 10
Figure 10
cAMP could block LPA-induced RhoA translocation in SGC-7901 cells. SGC-7901 cells were pretreated with CPT-cAMP (250 μmol/L, 30 min), and then with or without LPA (1 μmol/L, 15 min) treatment. LPA or CPT-cAMP+LPA -treated cells were lysed and membrane (A), cytosolic (C), and nuclear (B) fractions were obtained. Expressions of RhoA and GAPDH proteins in each fraction were detected by Western blotting, and the representative band images were shown in the left panels. Densitometric analysis of the bands was shown in the right panels. Each bar represents mean ± SE obtained from five independent experiments. aP < 0.05.
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References

    1. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992;70:389–399. - PubMed
    1. McCormick F. ras GTPase activating protein: signal transmitter and signal terminator. Cell. 1989;56:5–8. - PubMed
    1. Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990;348:125–132. - PubMed
    1. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990;249:635–640. - PubMed
    1. Takai Y, Kaibuchi K, Kikuchi A, Kawata M. Small GTP-binding proteins. Int Rev Cytol. 1992;133:187–230. - PubMed

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