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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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The PRIMA-1MET-induced nucleolar translocation of DSRed-EBNA-5 is reversible in the p53 -/- H1299 cells. The translocation was induced by 12 hours drug treatment followed by extensive washing with drug free medium. Time-lapse movie was recorded for 6 hours, one frame per minute. The fluorescence images are combined with phase contrast pictures to demonstrate the intact cellular morphology of the cells during the entire length of the experiment.
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Figure 8: The PRIMA-1MET-induced nucleolar translocation of DSRed-EBNA-5 is reversible in the p53 -/- H1299 cells. The translocation was induced by 12 hours drug treatment followed by extensive washing with drug free medium. Time-lapse movie was recorded for 6 hours, one frame per minute. The fluorescence images are combined with phase contrast pictures to demonstrate the intact cellular morphology of the cells during the entire length of the experiment.

Mentions: PRIMA-1MET regularly induces apoptosis in mutant p53 expressing cells. To explore the possibility whether the protein aggregation phenomenon is a feature advanced stage cellular agony we have treated DSRed-EBNA-5 expressing, p53 -/-, H1299 cells with PRIMA-1MET for 12 hours. These cells are much less sensitive to PRIMA-1MET induced apoptosis than its mutant p53 expressing derivatives. The drug treatment induced nucleolar accumulation of the protein in most nucleoli. Removing PRIMA-1MET by repeated washing with drug free medium led to the complete dissolution of the aggregates as it could be demonstrated by combined fluorescent/phase contrast time lapse microscopy. No cytopathic effects were detected at any time during the experiment (Figure 8).


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)

The PRIMA-1MET-induced nucleolar translocation of DSRed-EBNA-5 is reversible in the p53 -/- H1299 cells. The translocation was induced by 12 hours drug treatment followed by extensive washing with drug free medium. Time-lapse movie was recorded for 6 hours, one frame per minute. The fluorescence images are combined with phase contrast pictures to demonstrate the intact cellular morphology of the cells during the entire length of the experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: The PRIMA-1MET-induced nucleolar translocation of DSRed-EBNA-5 is reversible in the p53 -/- H1299 cells. The translocation was induced by 12 hours drug treatment followed by extensive washing with drug free medium. Time-lapse movie was recorded for 6 hours, one frame per minute. The fluorescence images are combined with phase contrast pictures to demonstrate the intact cellular morphology of the cells during the entire length of the experiment.
Mentions: PRIMA-1MET regularly induces apoptosis in mutant p53 expressing cells. To explore the possibility whether the protein aggregation phenomenon is a feature advanced stage cellular agony we have treated DSRed-EBNA-5 expressing, p53 -/-, H1299 cells with PRIMA-1MET for 12 hours. These cells are much less sensitive to PRIMA-1MET induced apoptosis than its mutant p53 expressing derivatives. The drug treatment induced nucleolar accumulation of the protein in most nucleoli. Removing PRIMA-1MET by repeated washing with drug free medium led to the complete dissolution of the aggregates as it could be demonstrated by combined fluorescent/phase contrast time lapse microscopy. No cytopathic effects were detected at any time during the experiment (Figure 8).

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