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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 is a caspase substrate.A) Analysis by western blot of the exogenous form of Sp3 in tetracycline-induced S27 cells (24 hours) and in clones chronically cultivated in the presence of tetracycline (1 to 9) with the Myc antibody. Arrows indicate the different reactive bands corresponding to the full-length protein (FL) or cleavage products (CP 1 and CP 2). Erk2 is shown as a loading control. This result is representative of two independent experiments. B) In vitro cleavage by caspase 3, 6 or 7 (BD Biosciences, Pharmingen) in the presence or absence of 50 µM Z-VAD-FMK of in vitro-translated Sp3. Cleavage products are indicated by asterisks. C) In vitro cleavage by caspase 3 in the presence or absence of 50 µM Z-VAD-FMK of Sp3 from tetracycline-stimulated LS174 cells (S27). The presence of cleaved products was analysed by western blot using an anti-Sp3 antibody. Cleaved products are indicated by asterisks. Erk2 is shown as a loading control.
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pone-0004478-g005: Sp3 is a caspase substrate.A) Analysis by western blot of the exogenous form of Sp3 in tetracycline-induced S27 cells (24 hours) and in clones chronically cultivated in the presence of tetracycline (1 to 9) with the Myc antibody. Arrows indicate the different reactive bands corresponding to the full-length protein (FL) or cleavage products (CP 1 and CP 2). Erk2 is shown as a loading control. This result is representative of two independent experiments. B) In vitro cleavage by caspase 3, 6 or 7 (BD Biosciences, Pharmingen) in the presence or absence of 50 µM Z-VAD-FMK of in vitro-translated Sp3. Cleavage products are indicated by asterisks. C) In vitro cleavage by caspase 3 in the presence or absence of 50 µM Z-VAD-FMK of Sp3 from tetracycline-stimulated LS174 cells (S27). The presence of cleaved products was analysed by western blot using an anti-Sp3 antibody. Cleaved products are indicated by asterisks. Erk2 is shown as a loading control.

Mentions: After extensive apoptosis, a few cells became progressively resistant to Sp3 induction. Figure 5A shows the two main Sp3 degradation products of 60 and 35 kDa (cleaved product 1 and 2, (CP1, CP2)) detected with the Myc antibody in all the resistant clones. This suggests a cleavage mechanism at specific proteolytic sites. Note that both fragments were observed in control cells at a lower intensity, probably reflecting the high basal caspase activity of LS174 cells (Figure 1). In vitro cleavage assays on in vitro translated protein (Figure 5B) or on cell extracts from tetracycline-induced cells (Figure 5C) demonstrated that Sp3 is cleaved by caspase 3 and/or caspase 6 but not caspase 7. Such cleavage generates two fragments equivalent to those detected by western blot in control and tetracycline-resistant clones. Our results strongly suggest that Sp3 is a caspase substrate.


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 is a caspase substrate.A) Analysis by western blot of the exogenous form of Sp3 in tetracycline-induced S27 cells (24 hours) and in clones chronically cultivated in the presence of tetracycline (1 to 9) with the Myc antibody. Arrows indicate the different reactive bands corresponding to the full-length protein (FL) or cleavage products (CP 1 and CP 2). Erk2 is shown as a loading control. This result is representative of two independent experiments. B) In vitro cleavage by caspase 3, 6 or 7 (BD Biosciences, Pharmingen) in the presence or absence of 50 µM Z-VAD-FMK of in vitro-translated Sp3. Cleavage products are indicated by asterisks. C) In vitro cleavage by caspase 3 in the presence or absence of 50 µM Z-VAD-FMK of Sp3 from tetracycline-stimulated LS174 cells (S27). The presence of cleaved products was analysed by western blot using an anti-Sp3 antibody. Cleaved products are indicated by asterisks. Erk2 is shown as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636865&req=5

pone-0004478-g005: Sp3 is a caspase substrate.A) Analysis by western blot of the exogenous form of Sp3 in tetracycline-induced S27 cells (24 hours) and in clones chronically cultivated in the presence of tetracycline (1 to 9) with the Myc antibody. Arrows indicate the different reactive bands corresponding to the full-length protein (FL) or cleavage products (CP 1 and CP 2). Erk2 is shown as a loading control. This result is representative of two independent experiments. B) In vitro cleavage by caspase 3, 6 or 7 (BD Biosciences, Pharmingen) in the presence or absence of 50 µM Z-VAD-FMK of in vitro-translated Sp3. Cleavage products are indicated by asterisks. C) In vitro cleavage by caspase 3 in the presence or absence of 50 µM Z-VAD-FMK of Sp3 from tetracycline-stimulated LS174 cells (S27). The presence of cleaved products was analysed by western blot using an anti-Sp3 antibody. Cleaved products are indicated by asterisks. Erk2 is shown as a loading control.
Mentions: After extensive apoptosis, a few cells became progressively resistant to Sp3 induction. Figure 5A shows the two main Sp3 degradation products of 60 and 35 kDa (cleaved product 1 and 2, (CP1, CP2)) detected with the Myc antibody in all the resistant clones. This suggests a cleavage mechanism at specific proteolytic sites. Note that both fragments were observed in control cells at a lower intensity, probably reflecting the high basal caspase activity of LS174 cells (Figure 1). In vitro cleavage assays on in vitro translated protein (Figure 5B) or on cell extracts from tetracycline-induced cells (Figure 5C) demonstrated that Sp3 is cleaved by caspase 3 and/or caspase 6 but not caspase 7. Such cleavage generates two fragments equivalent to those detected by western blot in control and tetracycline-resistant clones. Our results strongly suggest that Sp3 is a caspase substrate.

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