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Dual role of Sp3 transcription factor as an inducer of apoptosis and a marker of tumour aggressiveness.

Essafi-Benkhadir K, Grosso S, Puissant A, Robert G, Essafi M, Deckert M, Chamorey E, Dassonville O, Milano G, Auberger P, Pagès G - PLoS ONE (2009)

Bottom Line: The ambiguous role of transcription factor Sp3 for tumour progression is still debated since it was described as a transcriptional repressor or activator.Progression coincides with re-accumulation of the full length form of Sp3.The presence of high levels of the full-length form of Sp3 indicates a poor prognosis for overall survival of patients with head and neck tumours.

View Article: PubMed Central - PubMed

Affiliation: University of Nice-Sophia Antipolis, Institute of Developmental Biology and Cancer Research UMR CNRS 6543, Centre Antoine Lacassagne, Nice, France.

ABSTRACT

Background: The ambiguous role of transcription factor Sp3 for tumour progression is still debated since it was described as a transcriptional repressor or activator. Here we tried to decipher the molecular mechanisms implicated in Sp3 accumulation observed in aggressive tumours.

Methodology: We generated normal and tumour cell lines conditionally expressing Sp3. Cell growth was analyzed in vitro and after inoculation in nude mice. Apoptosis was assessed by pan- caspase activity assays, by counting fragmented nuclei and by determination of caspase 9 cleavage. Gene expression was determined by quantitative PCR. Cleavage by different caspases was performed after in vitro translation of the Sp3 cDNA in the presence of [S(35)] labelled methionine. Different tumour cell lines and head and neck tumour samples were tested for the presence of Sp3 by western blots. Correlation between Sp3 expression and overall survival has been statistically determined.

Principal findings: Conditional over-expression of Sp3 induces apoptosis and modifies expression of genes implicated in the regulation of cell cycle and pro and anti apoptotic genes. Sp3 over-expression strongly reduces the development of tumours in nude mice confirming its pro-apoptotic potential in vivo. However, cells can survive to apoptosis through selective Sp3 cleavage by caspase. Sp3 induction in established tumours resulted in transient regression then progression. Progression coincides with re-accumulation of the full length form of Sp3. Sp3 is over-expressed in tumour cell lines of different origins. The presence of high levels of the full-length form of Sp3 indicates a poor prognosis for overall survival of patients with head and neck tumours.

Conclusions: Full length Sp3 accumulation highlights bypass of tumour cell apoptotic capacities and is indicative of head and neck tumours aggressiveness.

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Related in: MedlinePlus

Sp3 over-expression induces apoptosis.Cells described in Figure 1 were tested for Sp3-mediated apoptosis by different methods. A) Pan-caspase activity was measured in R443 and LS174 cells expressing Sp3 (S27 for LS174) in the presence or absence of 50 µM Z-VAD-FMK as indicated in materials and methods. Results are the mean of three independent experiments performed in duplicate and significant modifications (* p<0.05) are indicated. B) Control or Sp3 expressing LS174 (S27) cells were cultivated in the absence (−Tet) or presence (+Tet) of tetracycline then stained with DAPI. At least ten photographs for each condition (twenty nuclei per photograph) were examined for the presence of fragmented nuclei. The percentage of cells with fragmented nuclei is plotted. The results are representative of three independent experiments. A significant increase of cell number with fragmented nuclei (* p<0.05) is indicated. C) Western blot analysis of caspase 9 (sc-7885, Santa Cruz, CA antibody) and PARP (C2-10, Santa Cruz, CA antibody) in LS174 or LS174 cells expressing Sp3 (S27) treated with (+) or without (−) tetracycline. The caspase 9 and PARP cleaved fragments (cC9 and cPARP respectively) are indicated by arrows. Erk2 is shown as a loading control. The results are representative of three independent experiments. D) LS174 cells expressing Sp3 (S27) were incubated overnight in the presence or absence of tetracycline (1 µg/ml) supplemented or not with 50 µM of Z-VAD-FMK. Cell number in absence of tetracycline and Z-VAD-FMK was taken as the reference value (100%). The percentage of cells remaining in the absence (−) or presence (+) of tetracycline supplemented or not with Z-VAD-FMK is plotted. These results are the mean of two independent experiments. Significant modifications (* p<0.05) are indicated.
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pone-0004478-g003: Sp3 over-expression induces apoptosis.Cells described in Figure 1 were tested for Sp3-mediated apoptosis by different methods. A) Pan-caspase activity was measured in R443 and LS174 cells expressing Sp3 (S27 for LS174) in the presence or absence of 50 µM Z-VAD-FMK as indicated in materials and methods. Results are the mean of three independent experiments performed in duplicate and significant modifications (* p<0.05) are indicated. B) Control or Sp3 expressing LS174 (S27) cells were cultivated in the absence (−Tet) or presence (+Tet) of tetracycline then stained with DAPI. At least ten photographs for each condition (twenty nuclei per photograph) were examined for the presence of fragmented nuclei. The percentage of cells with fragmented nuclei is plotted. The results are representative of three independent experiments. A significant increase of cell number with fragmented nuclei (* p<0.05) is indicated. C) Western blot analysis of caspase 9 (sc-7885, Santa Cruz, CA antibody) and PARP (C2-10, Santa Cruz, CA antibody) in LS174 or LS174 cells expressing Sp3 (S27) treated with (+) or without (−) tetracycline. The caspase 9 and PARP cleaved fragments (cC9 and cPARP respectively) are indicated by arrows. Erk2 is shown as a loading control. The results are representative of three independent experiments. D) LS174 cells expressing Sp3 (S27) were incubated overnight in the presence or absence of tetracycline (1 µg/ml) supplemented or not with 50 µM of Z-VAD-FMK. Cell number in absence of tetracycline and Z-VAD-FMK was taken as the reference value (100%). The percentage of cells remaining in the absence (−) or presence (+) of tetracycline supplemented or not with Z-VAD-FMK is plotted. These results are the mean of two independent experiments. Significant modifications (* p<0.05) are indicated.

Mentions: Prevention of cell accumulation can be attributed to cell cycle arrest or induction of apoptosis. We carefully addressed the apoptosis mechanisms by three independent methods: measurement of pan-caspase activity, evaluation of the number of fragmented nuclei and cleavage of caspase 9. Figure 3A shows that induction of Sp3 resulted in increased pan-caspase activity. The specific pan-caspase inhibitor Z-VAD-FMK inhibits both basal and Sp3-dependent caspase activation which was stronger in LS174 cells (clone S27), probably reflecting the higher basal amounts of Sp3 in the absence of tetracycline. We have then more carefully addressed the apoptotic potential of Sp3 in the LS174 tumour cells. A quantitative analysis of fragmented nuclei upon Sp3 induction presented in Figure 3B illustrates that more than 50% cells were apoptotic following 72 hours of Sp3 induction and that the increased number of apoptotic cells paralleled the cleavage/activation of caspase 9 and PARP cleavage (Figure 3C), two substrates for active caspase 3 [12]. Sp3 effects on cell accumulation are partially inhibited by the pan-caspase inhibitor Z-VAD-FMK indicating that Sp3 induces a caspase-dependent apoptosis. Such reversion is coherent with the results of Deniaud et al who has observed the same effects of Z-VAD-FMK on Sp1-induced apoptosis [7]. Also note that Z-VAD-FMK treatment by itself results in cell accumulation suggesting that the basal caspase activity observed in figure 3A has been inhibited. Altogether our data clearly show that Sp3 over-expression is pro-apoptotic.


Dual role of Sp3 transcription factor as an inducer of apoptosis and a marker of tumour aggressiveness.

Essafi-Benkhadir K, Grosso S, Puissant A, Robert G, Essafi M, Deckert M, Chamorey E, Dassonville O, Milano G, Auberger P, Pagès G - PLoS ONE (2009)

Sp3 over-expression induces apoptosis.Cells described in Figure 1 were tested for Sp3-mediated apoptosis by different methods. A) Pan-caspase activity was measured in R443 and LS174 cells expressing Sp3 (S27 for LS174) in the presence or absence of 50 µM Z-VAD-FMK as indicated in materials and methods. Results are the mean of three independent experiments performed in duplicate and significant modifications (* p<0.05) are indicated. B) Control or Sp3 expressing LS174 (S27) cells were cultivated in the absence (−Tet) or presence (+Tet) of tetracycline then stained with DAPI. At least ten photographs for each condition (twenty nuclei per photograph) were examined for the presence of fragmented nuclei. The percentage of cells with fragmented nuclei is plotted. The results are representative of three independent experiments. A significant increase of cell number with fragmented nuclei (* p<0.05) is indicated. C) Western blot analysis of caspase 9 (sc-7885, Santa Cruz, CA antibody) and PARP (C2-10, Santa Cruz, CA antibody) in LS174 or LS174 cells expressing Sp3 (S27) treated with (+) or without (−) tetracycline. The caspase 9 and PARP cleaved fragments (cC9 and cPARP respectively) are indicated by arrows. Erk2 is shown as a loading control. The results are representative of three independent experiments. D) LS174 cells expressing Sp3 (S27) were incubated overnight in the presence or absence of tetracycline (1 µg/ml) supplemented or not with 50 µM of Z-VAD-FMK. Cell number in absence of tetracycline and Z-VAD-FMK was taken as the reference value (100%). The percentage of cells remaining in the absence (−) or presence (+) of tetracycline supplemented or not with Z-VAD-FMK is plotted. These results are the mean of two independent experiments. Significant modifications (* p<0.05) are indicated.
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Related In: Results  -  Collection

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pone-0004478-g003: Sp3 over-expression induces apoptosis.Cells described in Figure 1 were tested for Sp3-mediated apoptosis by different methods. A) Pan-caspase activity was measured in R443 and LS174 cells expressing Sp3 (S27 for LS174) in the presence or absence of 50 µM Z-VAD-FMK as indicated in materials and methods. Results are the mean of three independent experiments performed in duplicate and significant modifications (* p<0.05) are indicated. B) Control or Sp3 expressing LS174 (S27) cells were cultivated in the absence (−Tet) or presence (+Tet) of tetracycline then stained with DAPI. At least ten photographs for each condition (twenty nuclei per photograph) were examined for the presence of fragmented nuclei. The percentage of cells with fragmented nuclei is plotted. The results are representative of three independent experiments. A significant increase of cell number with fragmented nuclei (* p<0.05) is indicated. C) Western blot analysis of caspase 9 (sc-7885, Santa Cruz, CA antibody) and PARP (C2-10, Santa Cruz, CA antibody) in LS174 or LS174 cells expressing Sp3 (S27) treated with (+) or without (−) tetracycline. The caspase 9 and PARP cleaved fragments (cC9 and cPARP respectively) are indicated by arrows. Erk2 is shown as a loading control. The results are representative of three independent experiments. D) LS174 cells expressing Sp3 (S27) were incubated overnight in the presence or absence of tetracycline (1 µg/ml) supplemented or not with 50 µM of Z-VAD-FMK. Cell number in absence of tetracycline and Z-VAD-FMK was taken as the reference value (100%). The percentage of cells remaining in the absence (−) or presence (+) of tetracycline supplemented or not with Z-VAD-FMK is plotted. These results are the mean of two independent experiments. Significant modifications (* p<0.05) are indicated.
Mentions: Prevention of cell accumulation can be attributed to cell cycle arrest or induction of apoptosis. We carefully addressed the apoptosis mechanisms by three independent methods: measurement of pan-caspase activity, evaluation of the number of fragmented nuclei and cleavage of caspase 9. Figure 3A shows that induction of Sp3 resulted in increased pan-caspase activity. The specific pan-caspase inhibitor Z-VAD-FMK inhibits both basal and Sp3-dependent caspase activation which was stronger in LS174 cells (clone S27), probably reflecting the higher basal amounts of Sp3 in the absence of tetracycline. We have then more carefully addressed the apoptotic potential of Sp3 in the LS174 tumour cells. A quantitative analysis of fragmented nuclei upon Sp3 induction presented in Figure 3B illustrates that more than 50% cells were apoptotic following 72 hours of Sp3 induction and that the increased number of apoptotic cells paralleled the cleavage/activation of caspase 9 and PARP cleavage (Figure 3C), two substrates for active caspase 3 [12]. Sp3 effects on cell accumulation are partially inhibited by the pan-caspase inhibitor Z-VAD-FMK indicating that Sp3 induces a caspase-dependent apoptosis. Such reversion is coherent with the results of Deniaud et al who has observed the same effects of Z-VAD-FMK on Sp1-induced apoptosis [7]. Also note that Z-VAD-FMK treatment by itself results in cell accumulation suggesting that the basal caspase activity observed in figure 3A has been inhibited. Altogether our data clearly show that Sp3 over-expression is pro-apoptotic.

Bottom Line: The ambiguous role of transcription factor Sp3 for tumour progression is still debated since it was described as a transcriptional repressor or activator.Progression coincides with re-accumulation of the full length form of Sp3.The presence of high levels of the full-length form of Sp3 indicates a poor prognosis for overall survival of patients with head and neck tumours.

View Article: PubMed Central - PubMed

Affiliation: University of Nice-Sophia Antipolis, Institute of Developmental Biology and Cancer Research UMR CNRS 6543, Centre Antoine Lacassagne, Nice, France.

ABSTRACT

Background: The ambiguous role of transcription factor Sp3 for tumour progression is still debated since it was described as a transcriptional repressor or activator. Here we tried to decipher the molecular mechanisms implicated in Sp3 accumulation observed in aggressive tumours.

Methodology: We generated normal and tumour cell lines conditionally expressing Sp3. Cell growth was analyzed in vitro and after inoculation in nude mice. Apoptosis was assessed by pan- caspase activity assays, by counting fragmented nuclei and by determination of caspase 9 cleavage. Gene expression was determined by quantitative PCR. Cleavage by different caspases was performed after in vitro translation of the Sp3 cDNA in the presence of [S(35)] labelled methionine. Different tumour cell lines and head and neck tumour samples were tested for the presence of Sp3 by western blots. Correlation between Sp3 expression and overall survival has been statistically determined.

Principal findings: Conditional over-expression of Sp3 induces apoptosis and modifies expression of genes implicated in the regulation of cell cycle and pro and anti apoptotic genes. Sp3 over-expression strongly reduces the development of tumours in nude mice confirming its pro-apoptotic potential in vivo. However, cells can survive to apoptosis through selective Sp3 cleavage by caspase. Sp3 induction in established tumours resulted in transient regression then progression. Progression coincides with re-accumulation of the full length form of Sp3. Sp3 is over-expressed in tumour cell lines of different origins. The presence of high levels of the full-length form of Sp3 indicates a poor prognosis for overall survival of patients with head and neck tumours.

Conclusions: Full length Sp3 accumulation highlights bypass of tumour cell apoptotic capacities and is indicative of head and neck tumours aggressiveness.

Show MeSH
Related in: MedlinePlus