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PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein.

Stuber G, Flaberg E, Petranyi G, Otvös R, Rökaeus N, Kashuba E, Wiman KG, Klein G, Szekely L - Mol. Cancer (2009)

Bottom Line: The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET.Our results suggest that mutant p53 is not the sole target of PRIMA-1MET.We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden. Gyorgy.Stuber@ki.se

ABSTRACT
The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

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PRIMA-1MET-induced translocation of virus encoded endogeneous EBNA-5 in the Epstein-Barr virus transformed human lymphoblastoid cell line LSsp that carries virus encoded EBNA-5 and harbors wild type p53. 24 hours treatment with 50 uM PRIMA-1MET leads to the disappearance of EBNA-5 from the PML bodies and to relocation to the nucleolus.
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Figure 1: PRIMA-1MET-induced translocation of virus encoded endogeneous EBNA-5 in the Epstein-Barr virus transformed human lymphoblastoid cell line LSsp that carries virus encoded EBNA-5 and harbors wild type p53. 24 hours treatment with 50 uM PRIMA-1MET leads to the disappearance of EBNA-5 from the PML bodies and to relocation to the nucleolus.

Mentions: We found that 12–24 hours treatment with 50 uM PRIMA-1MET induced nucleolar translocation of EBNA-5 in all cell lines such as the lymphoblastoid cells LSsp expressing virus encoded endogenous EBNA-5 (Figure 1) and transfected tumor cell lines such as the colon carcinoma line SW480 (endogenous mutant p53; Figure 2), the breast carcinoma line MCF7 (wt p53) and the lung adeno-carcinoma line H1299 (p53 cells) as well as its transfected variant expressing mutant p53 (His175). The identity of the nucleolus was ascertained by B23 staining and/or phasecontrast imaging in parallel.


PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein.

Stuber G, Flaberg E, Petranyi G, Otvös R, Rökaeus N, Kashuba E, Wiman KG, Klein G, Szekely L - Mol. Cancer (2009)

PRIMA-1MET-induced translocation of virus encoded endogeneous EBNA-5 in the Epstein-Barr virus transformed human lymphoblastoid cell line LSsp that carries virus encoded EBNA-5 and harbors wild type p53. 24 hours treatment with 50 uM PRIMA-1MET leads to the disappearance of EBNA-5 from the PML bodies and to relocation to the nucleolus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667484&req=5

Figure 1: PRIMA-1MET-induced translocation of virus encoded endogeneous EBNA-5 in the Epstein-Barr virus transformed human lymphoblastoid cell line LSsp that carries virus encoded EBNA-5 and harbors wild type p53. 24 hours treatment with 50 uM PRIMA-1MET leads to the disappearance of EBNA-5 from the PML bodies and to relocation to the nucleolus.
Mentions: We found that 12–24 hours treatment with 50 uM PRIMA-1MET induced nucleolar translocation of EBNA-5 in all cell lines such as the lymphoblastoid cells LSsp expressing virus encoded endogenous EBNA-5 (Figure 1) and transfected tumor cell lines such as the colon carcinoma line SW480 (endogenous mutant p53; Figure 2), the breast carcinoma line MCF7 (wt p53) and the lung adeno-carcinoma line H1299 (p53 cells) as well as its transfected variant expressing mutant p53 (His175). The identity of the nucleolus was ascertained by B23 staining and/or phasecontrast imaging in parallel.

Bottom Line: The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET.Our results suggest that mutant p53 is not the sole target of PRIMA-1MET.We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden. Gyorgy.Stuber@ki.se

ABSTRACT
The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

Show MeSH
Related in: MedlinePlus