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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

Western blot analysis of Sp3 expression in R 443 and LS 174 cells.Control R443 and LS174 cells (C) or their derivatives expressing Sp3 (S for R443 and S25 and S27 for LS174) were cultivated in the presence or absence of tetracycline for 24 hours and analysed for the presence of Sp3 by using Sp3- (Ab Sp3, D20 Santa Cruz, CA) and Myc-directed antibodies (Ab Myc, 9E10). The Sp1 levels were also analysed by using a Sp1 specific antibody (PEP-2, Santa Cruz, CA). Total Erk2 is shown as a loading control. This experiment is representative of three independent experiments.
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pone-0004478-g001: Western blot analysis of Sp3 expression in R 443 and LS 174 cells.Control R443 and LS174 cells (C) or their derivatives expressing Sp3 (S for R443 and S25 and S27 for LS174) were cultivated in the presence or absence of tetracycline for 24 hours and analysed for the presence of Sp3 by using Sp3- (Ab Sp3, D20 Santa Cruz, CA) and Myc-directed antibodies (Ab Myc, 9E10). The Sp1 levels were also analysed by using a Sp1 specific antibody (PEP-2, Santa Cruz, CA). Total Erk2 is shown as a loading control. This experiment is representative of three independent experiments.

Mentions: The role of Sp3 in apoptosis regulation was analysed in Chinese hamster lung fibroblasts (R443) and colon carcinoma cells (LS174) conditionally expressing full-length Myc-tagged Sp3 [11]. Both cells were chosen in order to determine the effect of Sp3 over-expression in a normal and a tumour cell line. Figure 1 shows that exogenous Sp3 was induced by tetracycline stimulation in the different cell types. Two independent LS174 clones (clone 25 (S25) and clone 27 (S27)) and one R443 clone conditionally expressing Sp3 were tested. Western blot experiments with an anti-Sp3 antibody showed that exogenous Sp3 was over-expressed compared to the endogenous form in both cell lines. Moreover, endogenous Sp3 was more highly expressed in LS174 than in R443 cells. Note that following over-expression, extra bands were detected at 60 and 35 kDa with both anti-Sp3 and anti-Myc antibodies. These bands were also observed in extracts from control LS174 cells after a longer exposure. Figure 1 also shows that overexpression of Sp3 do not significantly affect the Sp1 endogenous levels. Figure 2A shows that tetracycline induction of Sp3 resulted in decreased accumulation of both R443 and LS174 cells in a time dependent manner. Figure 2B shows that lower concentrations of tetracycline still induce cell death in R443 and S27 cells. The same effects were observed at concentrations of 1 or 0.1 µg/ml tetracycline since induction of Sp3 is equivalent at such concentrations as already described [11]. At a tetracycline concentration of 0.01 µg/ml Sp3 levels are significantly decreased while inexistent at 0.001 µg/ml.


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)

Western blot analysis of Sp3 expression in R 443 and LS 174 cells.Control R443 and LS174 cells (C) or their derivatives expressing Sp3 (S for R443 and S25 and S27 for LS174) were cultivated in the presence or absence of tetracycline for 24 hours and analysed for the presence of Sp3 by using Sp3- (Ab Sp3, D20 Santa Cruz, CA) and Myc-directed antibodies (Ab Myc, 9E10). The Sp1 levels were also analysed by using a Sp1 specific antibody (PEP-2, Santa Cruz, CA). Total Erk2 is shown as a loading control. This experiment is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004478-g001: Western blot analysis of Sp3 expression in R 443 and LS 174 cells.Control R443 and LS174 cells (C) or their derivatives expressing Sp3 (S for R443 and S25 and S27 for LS174) were cultivated in the presence or absence of tetracycline for 24 hours and analysed for the presence of Sp3 by using Sp3- (Ab Sp3, D20 Santa Cruz, CA) and Myc-directed antibodies (Ab Myc, 9E10). The Sp1 levels were also analysed by using a Sp1 specific antibody (PEP-2, Santa Cruz, CA). Total Erk2 is shown as a loading control. This experiment is representative of three independent experiments.
Mentions: The role of Sp3 in apoptosis regulation was analysed in Chinese hamster lung fibroblasts (R443) and colon carcinoma cells (LS174) conditionally expressing full-length Myc-tagged Sp3 [11]. Both cells were chosen in order to determine the effect of Sp3 over-expression in a normal and a tumour cell line. Figure 1 shows that exogenous Sp3 was induced by tetracycline stimulation in the different cell types. Two independent LS174 clones (clone 25 (S25) and clone 27 (S27)) and one R443 clone conditionally expressing Sp3 were tested. Western blot experiments with an anti-Sp3 antibody showed that exogenous Sp3 was over-expressed compared to the endogenous form in both cell lines. Moreover, endogenous Sp3 was more highly expressed in LS174 than in R443 cells. Note that following over-expression, extra bands were detected at 60 and 35 kDa with both anti-Sp3 and anti-Myc antibodies. These bands were also observed in extracts from control LS174 cells after a longer exposure. Figure 1 also shows that overexpression of Sp3 do not significantly affect the Sp1 endogenous levels. Figure 2A shows that tetracycline induction of Sp3 resulted in decreased accumulation of both R443 and LS174 cells in a time dependent manner. Figure 2B shows that lower concentrations of tetracycline still induce cell death in R443 and S27 cells. The same effects were observed at concentrations of 1 or 0.1 µg/ml tetracycline since induction of Sp3 is equivalent at such concentrations as already described [11]. At a tetracycline concentration of 0.01 µg/ml Sp3 levels are significantly decreased while inexistent at 0.001 µg/ml.

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