Tag Archives: Nr2f1

Although the p53 tumor suppressor/transcription factor often accumulates in the cytoplasm

Although the p53 tumor suppressor/transcription factor often accumulates in the cytoplasm of healthy cells, limited information is available on the cytoplasmic function of p53. Systems (Promega) according to manufacturer protocols. Protein binding was analyzed by co-immunoprecipitation. Where indicated, the expressed proteins were incubated with GST or GST-coupled proteins (Abnova), followed by precipitation with glutathione-conjugated Sepharose (Amersham). The precipitates were analyzed by Western blotting. Expression constructs and mutagenesis Expression constructs were prepared using pcDNA3, pCMV/myc/mito, pEGFP-C1, and pTRE-Tight vectors. The former two vectors were obtained from Invitrogen, while the latter was from Clontech. These vectors were used for the following purposes: pEGFP-C1, for confocal microscopy and intravasation assays; pTRE-Tight, for the expression of pro-apoptotic Bcl-2 members (Bax and Bak); pCMV/myc/mito, for the expression of ND5 and ND5G13289A; and pcDNA3, for all other purposes. p53R175H, p53K305N, p53K305N/R175H, Bcl-wG94A, and ND5G13289A were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) [43]. Animals Female BALB/cAnNCrj-nu/nu mice (6 wks old) were purchased from Charles River. All animal experiments were performed under approved protocols of our Institutional Animal Care and Use Committee. Intravasation assay H460 cells stably transfected with pEGFP-C1 vectors encoding the indicated genes were subcutaneously injected into the hind legs of mice (107/mouse) to form xenograft tumors. Tumor volumes were calculated as described [44]. After 2 weeks, mice were anesthetized, blood was obtained via cardiac puncture, and 0.1 mL of blood was mixed with 2 mL RBC-lysis buffer (Intron Biotech). Cells were collected by centrifugation (350 < 0.05, which was determined by a Student's test or one-way ANOVA using GraphPad software. SUPPLEMENTARY MATERIAL AND FIGURES Click here to view.(863K, pdf) Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2012M2A2A7010459, 2012R1A2A2A01045978, 2008-0062611). Footnotes CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES 1. Muller PA, Vousden KH, Norman JC. p53 and its mutants in tumor cell migration and invasion. J Cell Biol. 2011;192:209C218. [PMC free article] [PubMed] 2. Riley T, Sontag E, Chen Nr2f1 P, Levine A. Transcriptional control of human p53- regulated genes. Nat Rev Mol Cell Biol. 2008;9:402C412. [PubMed] 3. Moll UM, LaQuaglia M, Bnard J, Riou G. Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc Natl Acad Sci USA. 1995;92:4407C4411. [PMC free article] [PubMed] 4. Jansson A, Gentile M, Sun XF. p53 Mutations are present in colorectal cancer with cytoplasmic p53 accumulation. Int J Cancer. 2001;92:338C341. [PubMed] 5. Sembritzki O, Hagel C, Lamszus K, Deppert W, Bohn W. Cytoplasmic localization of wild-type p53 in glioblastomas correlates with expression of vimentin and glial fibrillary acidic protein. Neuro Oncol. 2002;4:171C178. [PMC free article] [PubMed] 6. Moll UM, Riou G, Levine AJ. Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad Sci USA. 1992;89:7262C7266. [PMC free article] [PubMed] 7. Cory S, Bedaquiline (TMC-207) Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002;2:647C656. [PubMed] 8. Bae Bedaquiline (TMC-207) IH, Park MJ, Yoon SH, Kang SW, Lee SS, Choi KM, Um HD. Bcl-w promotes gastric cancer cell invasion by inducing matrix metalloproteinase-2 expression via phosphoinositide 3-kinase, Akt, and Sp1. Cancer Res. 2006;66:4991C4995. [PubMed] 9. Bae IH, Yoon SH, Lee SB, Park JK, Ho JN, Um HD. Signaling components involved in Bcl-w-induced migration of gastric cancer cells. Cancer Lett. 2009;277:22C28. [PubMed] 10. Kim EM, Kim J, Park JK, Hwang SG, Kim WJ, Lee WJ, Kang SW, Um HD. Bcl-w promotes cell invasion by blocking the invasion-suppressing action of Bax. Cell Signal. 2012;24:1163C1172. [PubMed] 11. Weiler M, B?hr O, Hohlweg U, Naumann U, Rieger J, Huang H, Tabatabai G, Krell HW, Ohgaki H, Weller M, Wick W. BCL-xL: time-dependent dissociation between modulation of apoptosis and invasiveness in human malignant glioma cells. Cell Bedaquiline (TMC-207) Death Differ. 2006;13:1156C1169. [PubMed] 12. Ho JN, Kang GY, Lee SS, Kim J, Bae IH, Hwang SG, Um HD. Bcl-XL and STAT3 mediate malignant actions of gamma-irradiation in lung cancer cells. Cancer Sci. 2010;101:1417C1423. [PubMed] 13. Choi J, Choi K, Benveniste EN, Rho SB, Hong YS, Lee JH, Kim J, Park K. Bcl-2 promotes invasion and lung metastasis by inducing matrix metalloproteinase-2. Cancer Res. 2005;65:5554C5560. [PubMed] 14. Zuo J, Ishikawa T, Boutros S, Xiao Z, Humtsoe JO, Kramer RH. Bcl-2 overexpression induces a partial epithelial to mesenchymal transition and promotes squamous carcinoma cell invasion and metastasis. Cancer Res. 2010;8:170C182. [PubMed] 15. Del Bufalo D, Biroccio A, Leonetti C, Zupi G. Bcl-2 overexpression Bedaquiline (TMC-207) enhances the metastatic.