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Regulation of MCP-1 chemokine transcription by p53.

Hacke K, Rincon-Orozco B, Buchwalter G, Siehler SY, Wasylyk B, Wiesmüller L, Rösl F - Mol. Cancer (2010)

Bottom Line: In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-alpha treatment.In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-alpha addition, directly confirming a crosstalk between p53 and MCP-1.These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Infektion und Krebs, Abteilung Virale Transformationsmechanismen, Heidelberg, Germany.

ABSTRACT

Background: Our previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. In silico analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process.

Results: The proposed p53 binding side could be confirmed in vitro by electrophoretic-mobility-shift assays and in vivo by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-alpha can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-alpha treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-alpha addition, directly confirming a crosstalk between p53 and MCP-1.

Conclusion: These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

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p53 binds to the enhancer region of MCP-1 in vivo. (A) DNA sequence of the 5'-regulatory region of the MCP-1 gene (-2550 to -2250) indicating MCP-1 specific primer pairs used for the chromatin immunoprecipitation (ChIP) assay by arrows. The p53 binding site is marked with bold letters. (B) 4Bv cells were maintained at 37°C or shifted to 32°C for 5 h and stimulated with TNF-α for additional 5 h. Cells were harvested for chromatin immunoprecipitation (ChIP) assay as described in Materials and Methods. Samples were subjected to immunoprecipitation without antibody (no ab), with p53 antibody or rabbit IgG (IgG); total lysate was used as a control for PCR amplification (input). (B, left panel) p53 binding was tested by using MCP-1 specific primers; (B, right panel) p21 primers were used as a positive control. Untreated control cells (-); cells treated with 250 U/ml of TNF-α for 5 h (+).
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Figure 5: p53 binds to the enhancer region of MCP-1 in vivo. (A) DNA sequence of the 5'-regulatory region of the MCP-1 gene (-2550 to -2250) indicating MCP-1 specific primer pairs used for the chromatin immunoprecipitation (ChIP) assay by arrows. The p53 binding site is marked with bold letters. (B) 4Bv cells were maintained at 37°C or shifted to 32°C for 5 h and stimulated with TNF-α for additional 5 h. Cells were harvested for chromatin immunoprecipitation (ChIP) assay as described in Materials and Methods. Samples were subjected to immunoprecipitation without antibody (no ab), with p53 antibody or rabbit IgG (IgG); total lysate was used as a control for PCR amplification (input). (B, left panel) p53 binding was tested by using MCP-1 specific primers; (B, right panel) p21 primers were used as a positive control. Untreated control cells (-); cells treated with 250 U/ml of TNF-α for 5 h (+).

Mentions: To investigate whether p53 interacts with its binding site within the MCP-1 enhancer also under in vivo conditions, chromatin immunoprecipitation (ChIP) experiments were carried out. For this purpose, we took again advantage of the temperature-sensitive cell system shown in Fig. 2A and 2B. 4Bv cells maintained at 37°C or shifted to 32°C were incubated with TNF-α. After 5 hours, treated and untreated controls were fixed with formaldehyde and DNA sheared samples were immunoprecipitated with antibodies directed against p53. Controls without antibodies or with antibodies against rabbit IgG were included. The origin of the PCR-primers flanking the p53 binding site is outlined in Fig. 5A. As shown in Fig. 5B, MCP-1 chromatin was specifically immunoprecipitated with anti-p53 antibodies only from TNF-α treated cells cultivated at 32°C. In contrast, PCR amplification covering the p53 binding site at the p21 promoter shows a signal after shifting to 32°C, independently of whether TNF-α was added or not. This indicates that, in contrast to p21, p53 binds to the MCP-1 enhancer in vivo in a TNF-α dependent manner.


Regulation of MCP-1 chemokine transcription by p53.

Hacke K, Rincon-Orozco B, Buchwalter G, Siehler SY, Wasylyk B, Wiesmüller L, Rösl F - Mol. Cancer (2010)

p53 binds to the enhancer region of MCP-1 in vivo. (A) DNA sequence of the 5'-regulatory region of the MCP-1 gene (-2550 to -2250) indicating MCP-1 specific primer pairs used for the chromatin immunoprecipitation (ChIP) assay by arrows. The p53 binding site is marked with bold letters. (B) 4Bv cells were maintained at 37°C or shifted to 32°C for 5 h and stimulated with TNF-α for additional 5 h. Cells were harvested for chromatin immunoprecipitation (ChIP) assay as described in Materials and Methods. Samples were subjected to immunoprecipitation without antibody (no ab), with p53 antibody or rabbit IgG (IgG); total lysate was used as a control for PCR amplification (input). (B, left panel) p53 binding was tested by using MCP-1 specific primers; (B, right panel) p21 primers were used as a positive control. Untreated control cells (-); cells treated with 250 U/ml of TNF-α for 5 h (+).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864217&req=5

Figure 5: p53 binds to the enhancer region of MCP-1 in vivo. (A) DNA sequence of the 5'-regulatory region of the MCP-1 gene (-2550 to -2250) indicating MCP-1 specific primer pairs used for the chromatin immunoprecipitation (ChIP) assay by arrows. The p53 binding site is marked with bold letters. (B) 4Bv cells were maintained at 37°C or shifted to 32°C for 5 h and stimulated with TNF-α for additional 5 h. Cells were harvested for chromatin immunoprecipitation (ChIP) assay as described in Materials and Methods. Samples were subjected to immunoprecipitation without antibody (no ab), with p53 antibody or rabbit IgG (IgG); total lysate was used as a control for PCR amplification (input). (B, left panel) p53 binding was tested by using MCP-1 specific primers; (B, right panel) p21 primers were used as a positive control. Untreated control cells (-); cells treated with 250 U/ml of TNF-α for 5 h (+).
Mentions: To investigate whether p53 interacts with its binding site within the MCP-1 enhancer also under in vivo conditions, chromatin immunoprecipitation (ChIP) experiments were carried out. For this purpose, we took again advantage of the temperature-sensitive cell system shown in Fig. 2A and 2B. 4Bv cells maintained at 37°C or shifted to 32°C were incubated with TNF-α. After 5 hours, treated and untreated controls were fixed with formaldehyde and DNA sheared samples were immunoprecipitated with antibodies directed against p53. Controls without antibodies or with antibodies against rabbit IgG were included. The origin of the PCR-primers flanking the p53 binding site is outlined in Fig. 5A. As shown in Fig. 5B, MCP-1 chromatin was specifically immunoprecipitated with anti-p53 antibodies only from TNF-α treated cells cultivated at 32°C. In contrast, PCR amplification covering the p53 binding site at the p21 promoter shows a signal after shifting to 32°C, independently of whether TNF-α was added or not. This indicates that, in contrast to p21, p53 binds to the MCP-1 enhancer in vivo in a TNF-α dependent manner.

Bottom Line: In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-alpha treatment.In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-alpha addition, directly confirming a crosstalk between p53 and MCP-1.These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Infektion und Krebs, Abteilung Virale Transformationsmechanismen, Heidelberg, Germany.

ABSTRACT

Background: Our previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. In silico analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process.

Results: The proposed p53 binding side could be confirmed in vitro by electrophoretic-mobility-shift assays and in vivo by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-alpha can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-alpha treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-alpha addition, directly confirming a crosstalk between p53 and MCP-1.

Conclusion: These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.

Show MeSH
Related in: MedlinePlus