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Hsp72 mediates TAp73α anti-apoptotic effects in small cell lung carcinoma cells.

Nyman U, Muppani NR, Zhivotovsky B, Joseph B - J. Cell. Mol. Med. (2011)

Bottom Line: However, the precise mechanism by which TAp73α exerts its pro-survival effect is yet unclear.Finally, we revealed that TAp73β counteracts the anti-apoptotic effect of TAp73α by preventing Hsp72 induction.Our results thus provide additional evidence for the potential oncogenic role of TAp73α, and extend the understanding of the mechanism for its anti-apoptotic effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institutet, Stockholm, Sweden.

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Hsp72 is required for TAp73α anti-apoptotic effect. (A) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the presence of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (B) H82 cells were co-transfected with EGFP and asHsp72 vector (green). Samples were stained with anti-Hsp72 antibody (red) and nuclei counterstained with Hoechst (blue). Images are representatives of three independent experiments. (C) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the expression of HSF1, Hsp90 and Hsc70. G3PDH was used as protein loading control and numbers indicate the ratio between protein (HSF1, Hsp90 or Hsc70) and G3PDH band intensity. H82 cells were co-transfected with EGFP and asHsp72 vector (green) and the effect of asHsp72 expression on Hsp27 protein levels were detected using immunofluorescent staining for Hsp27 (red) and counterstaining of nuclei with Hoechst (blue). (D) H82 cells were co-transfected with EGFP, asHsp72 vector and empty vector or p73 expression vectors (TAp73α, TAp73β, ΔNp73α). Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. (E) H82 cells were transfected with siRNAs pool designed to interfere specifically with the expression of human Hsp72 mRNA and total protein cell extracts were analysed by immunoblotting for the expression of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (F) H82 cells were co-transfected with EGFP, Hsp72 siRNAs pool and empty or TAp73α expression vectors. Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.
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fig02: Hsp72 is required for TAp73α anti-apoptotic effect. (A) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the presence of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (B) H82 cells were co-transfected with EGFP and asHsp72 vector (green). Samples were stained with anti-Hsp72 antibody (red) and nuclei counterstained with Hoechst (blue). Images are representatives of three independent experiments. (C) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the expression of HSF1, Hsp90 and Hsc70. G3PDH was used as protein loading control and numbers indicate the ratio between protein (HSF1, Hsp90 or Hsc70) and G3PDH band intensity. H82 cells were co-transfected with EGFP and asHsp72 vector (green) and the effect of asHsp72 expression on Hsp27 protein levels were detected using immunofluorescent staining for Hsp27 (red) and counterstaining of nuclei with Hoechst (blue). (D) H82 cells were co-transfected with EGFP, asHsp72 vector and empty vector or p73 expression vectors (TAp73α, TAp73β, ΔNp73α). Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. (E) H82 cells were transfected with siRNAs pool designed to interfere specifically with the expression of human Hsp72 mRNA and total protein cell extracts were analysed by immunoblotting for the expression of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (F) H82 cells were co-transfected with EGFP, Hsp72 siRNAs pool and empty or TAp73α expression vectors. Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.

Mentions: We suggested that since TAp73α is a potent inducer of Hsp72 expression, the anti-apoptotic effect of TAp73α might depend on the induction of Hsp72 protein. To investigate whether depletion of Hsp72 affects the anti-apoptotic activity of TAp73α we took advantage of an antisense Hsp72 vector (asHsp72). Transfection of cells with asHsp72 lead to a reduction in Hsp72 protein levels as shown both by Western blot (Fig. 2A) and confocal imaging (Fig. 2B). However, expression of the asHsp72 vector did not affect levels of Hsp72-related proteins HSF1, Hsp90, Hsc70 (as shown by Western blot, Fig. 2C) or Hsp27 (as shown by immunofluorescent staining, Fig. 2C). Subsequently, H82 cells were co-transfected with EGFP, p73 isoforms and asHsp72 vector, and treated with VP16 for 24 hrs. TAp73α and ΔNp73α are able to repress drug-induced apoptosis, whereas TAp73β enhances it (Fig. 2D, and as previously reported [16]). Upon co-transfection of ΔNp73α with asHsp72, ΔNp73α still exhibits an anti-apoptotic effect upon VP16 treatment (Fig. 2D, ΔNp73α black and grey bars). These data were further confirmed using a siRNA targeting HSP72/HSPA1A mRNA (Fig. 2E and F). This indicates the anti-apoptotic effect of ΔNp73α being independent on induction of Hsp72, consistent with the data described above (Fig. 1) where, in H82 cells, ΔNp73α do not show any induction of Hsp72, neither on the level of the promoter nor on mRNA and protein levels. The pro-apoptotic activity of TAp73β was unaffected by the co-expression of asHsp72. However, co-transfection of TAp73α together with asHsp72 significantly reduced the anti-apoptotic effect of TAp73α (Fig. 2D, TAp73α black and grey bars). Hence, the anti-apoptotic activity of TAp73α in SCLC H82 cells treated with chemotherapeutic drugs seems to depend on the induction of Hsp72.


Hsp72 mediates TAp73α anti-apoptotic effects in small cell lung carcinoma cells.

Nyman U, Muppani NR, Zhivotovsky B, Joseph B - J. Cell. Mol. Med. (2011)

Hsp72 is required for TAp73α anti-apoptotic effect. (A) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the presence of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (B) H82 cells were co-transfected with EGFP and asHsp72 vector (green). Samples were stained with anti-Hsp72 antibody (red) and nuclei counterstained with Hoechst (blue). Images are representatives of three independent experiments. (C) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the expression of HSF1, Hsp90 and Hsc70. G3PDH was used as protein loading control and numbers indicate the ratio between protein (HSF1, Hsp90 or Hsc70) and G3PDH band intensity. H82 cells were co-transfected with EGFP and asHsp72 vector (green) and the effect of asHsp72 expression on Hsp27 protein levels were detected using immunofluorescent staining for Hsp27 (red) and counterstaining of nuclei with Hoechst (blue). (D) H82 cells were co-transfected with EGFP, asHsp72 vector and empty vector or p73 expression vectors (TAp73α, TAp73β, ΔNp73α). Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. (E) H82 cells were transfected with siRNAs pool designed to interfere specifically with the expression of human Hsp72 mRNA and total protein cell extracts were analysed by immunoblotting for the expression of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (F) H82 cells were co-transfected with EGFP, Hsp72 siRNAs pool and empty or TAp73α expression vectors. Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.
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fig02: Hsp72 is required for TAp73α anti-apoptotic effect. (A) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the presence of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (B) H82 cells were co-transfected with EGFP and asHsp72 vector (green). Samples were stained with anti-Hsp72 antibody (red) and nuclei counterstained with Hoechst (blue). Images are representatives of three independent experiments. (C) H82 cells were transfected with asHsp72 vector and total protein cell extracts were analysed by immunoblotting for the expression of HSF1, Hsp90 and Hsc70. G3PDH was used as protein loading control and numbers indicate the ratio between protein (HSF1, Hsp90 or Hsc70) and G3PDH band intensity. H82 cells were co-transfected with EGFP and asHsp72 vector (green) and the effect of asHsp72 expression on Hsp27 protein levels were detected using immunofluorescent staining for Hsp27 (red) and counterstaining of nuclei with Hoechst (blue). (D) H82 cells were co-transfected with EGFP, asHsp72 vector and empty vector or p73 expression vectors (TAp73α, TAp73β, ΔNp73α). Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. (E) H82 cells were transfected with siRNAs pool designed to interfere specifically with the expression of human Hsp72 mRNA and total protein cell extracts were analysed by immunoblotting for the expression of Hsp72. G3PDH was used as protein loading control. Quantification of protein band-intensity was made using ImageJ software. (F) H82 cells were co-transfected with EGFP, Hsp72 siRNAs pool and empty or TAp73α expression vectors. Cells were treated with VP16 for 24 hrs, nuclei counterstained with Hoechst and apoptosis was assayed by scoring of EGFP transfected cells presenting condensed or fragmented nuclei. Figures are mean ± S.D. of three independent experiments, where *P < 0.05, **P < 0.01 and ***P < 0.001.
Mentions: We suggested that since TAp73α is a potent inducer of Hsp72 expression, the anti-apoptotic effect of TAp73α might depend on the induction of Hsp72 protein. To investigate whether depletion of Hsp72 affects the anti-apoptotic activity of TAp73α we took advantage of an antisense Hsp72 vector (asHsp72). Transfection of cells with asHsp72 lead to a reduction in Hsp72 protein levels as shown both by Western blot (Fig. 2A) and confocal imaging (Fig. 2B). However, expression of the asHsp72 vector did not affect levels of Hsp72-related proteins HSF1, Hsp90, Hsc70 (as shown by Western blot, Fig. 2C) or Hsp27 (as shown by immunofluorescent staining, Fig. 2C). Subsequently, H82 cells were co-transfected with EGFP, p73 isoforms and asHsp72 vector, and treated with VP16 for 24 hrs. TAp73α and ΔNp73α are able to repress drug-induced apoptosis, whereas TAp73β enhances it (Fig. 2D, and as previously reported [16]). Upon co-transfection of ΔNp73α with asHsp72, ΔNp73α still exhibits an anti-apoptotic effect upon VP16 treatment (Fig. 2D, ΔNp73α black and grey bars). These data were further confirmed using a siRNA targeting HSP72/HSPA1A mRNA (Fig. 2E and F). This indicates the anti-apoptotic effect of ΔNp73α being independent on induction of Hsp72, consistent with the data described above (Fig. 1) where, in H82 cells, ΔNp73α do not show any induction of Hsp72, neither on the level of the promoter nor on mRNA and protein levels. The pro-apoptotic activity of TAp73β was unaffected by the co-expression of asHsp72. However, co-transfection of TAp73α together with asHsp72 significantly reduced the anti-apoptotic effect of TAp73α (Fig. 2D, TAp73α black and grey bars). Hence, the anti-apoptotic activity of TAp73α in SCLC H82 cells treated with chemotherapeutic drugs seems to depend on the induction of Hsp72.

Bottom Line: However, the precise mechanism by which TAp73α exerts its pro-survival effect is yet unclear.Finally, we revealed that TAp73β counteracts the anti-apoptotic effect of TAp73α by preventing Hsp72 induction.Our results thus provide additional evidence for the potential oncogenic role of TAp73α, and extend the understanding of the mechanism for its anti-apoptotic effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institutet, Stockholm, Sweden.

Show MeSH
Related in: MedlinePlus