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The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, Saxena S, Gietz RD, Greenberg AH - J. Exp. Med. (1997)

Bottom Line: Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria.After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death.In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

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Nip3 sensitizes Rat-1  cells to drug-induced apoptosis.  Rat-1 cells were transfected with  T7-tagged Nip3 then 12 h later  treated with increasing amounts  of etoposide, camptothecin or  granzyme B and perforin. Cells  expressing Nip3 were identified  by anti-T7 antibody and the nucleus stained with Hoechst dye.  Apoptotic cells expressing Nip3  (Nip3 +) or cells not expressing  Nip3 (Nip3 −) were enumerated. This is representative of  three experiments showing similar results.
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Figure 5: Nip3 sensitizes Rat-1 cells to drug-induced apoptosis. Rat-1 cells were transfected with T7-tagged Nip3 then 12 h later treated with increasing amounts of etoposide, camptothecin or granzyme B and perforin. Cells expressing Nip3 were identified by anti-T7 antibody and the nucleus stained with Hoechst dye. Apoptotic cells expressing Nip3 (Nip3 +) or cells not expressing Nip3 (Nip3 −) were enumerated. This is representative of three experiments showing similar results.

Mentions: To determine whether Nip3 can influence apoptosis induced by other cell death signals, Rat-1 cells were transiently transfected with T7-Nip3 for 12 h then treated with increasing doses of the cytotoxic lymphocyte protease granzyme B, or the topoisomerase I and II inhibitors etoposide or camptothecin. The 12-h transfection was chosen because of minimal effects of Nip3 on cell survival at this time. Apoptotic cells were then quantitated by Hoechst dye in cells either expressing or not expressing T7-Nip3 protein within the same samples, using immunofluorescence staining by anti-T7 antibody to detect Nip3. The frequency of apoptotic cells was clearly higher in Nip3-expressing cells (Fig. 5). A 5–10-fold increase in sensitivity to the drugs and a 2–3-fold increase in granzyme B apoptosis was observed in three independent experiments.


The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, Saxena S, Gietz RD, Greenberg AH - J. Exp. Med. (1997)

Nip3 sensitizes Rat-1  cells to drug-induced apoptosis.  Rat-1 cells were transfected with  T7-tagged Nip3 then 12 h later  treated with increasing amounts  of etoposide, camptothecin or  granzyme B and perforin. Cells  expressing Nip3 were identified  by anti-T7 antibody and the nucleus stained with Hoechst dye.  Apoptotic cells expressing Nip3  (Nip3 +) or cells not expressing  Nip3 (Nip3 −) were enumerated. This is representative of  three experiments showing similar results.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199165&req=5

Figure 5: Nip3 sensitizes Rat-1 cells to drug-induced apoptosis. Rat-1 cells were transfected with T7-tagged Nip3 then 12 h later treated with increasing amounts of etoposide, camptothecin or granzyme B and perforin. Cells expressing Nip3 were identified by anti-T7 antibody and the nucleus stained with Hoechst dye. Apoptotic cells expressing Nip3 (Nip3 +) or cells not expressing Nip3 (Nip3 −) were enumerated. This is representative of three experiments showing similar results.
Mentions: To determine whether Nip3 can influence apoptosis induced by other cell death signals, Rat-1 cells were transiently transfected with T7-Nip3 for 12 h then treated with increasing doses of the cytotoxic lymphocyte protease granzyme B, or the topoisomerase I and II inhibitors etoposide or camptothecin. The 12-h transfection was chosen because of minimal effects of Nip3 on cell survival at this time. Apoptotic cells were then quantitated by Hoechst dye in cells either expressing or not expressing T7-Nip3 protein within the same samples, using immunofluorescence staining by anti-T7 antibody to detect Nip3. The frequency of apoptotic cells was clearly higher in Nip3-expressing cells (Fig. 5). A 5–10-fold increase in sensitivity to the drugs and a 2–3-fold increase in granzyme B apoptosis was observed in three independent experiments.

Bottom Line: Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria.After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death.In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

Show MeSH
Related in: MedlinePlus