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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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Related in: MedlinePlus

Temperature shifting does not affect the cell cycle or TNF-α signaling. (A) 4Bv cells were maintained at 37°C or 32°C overnight, followed by treatment with or without TNF-α for 6 h. Flow-cytometric analysis was performed on DAPI/SR 101 stained cells to determine the percentage of 4Bv cells in different cell cycle phases (G1, S, G2/M). Mean values (columns) and standard deviations (bars) are given for three independent experiments. Steady state expression (B) and phosphorylation (C) of p38 MAP kinase was analyzed after treatment of 4Bv cells (at 37°C or 32°C) with TNF-α as indicated. Total cellular protein (50 μg) was separated in a 12% SDS-PAGE gel. After electrotransfer, the filter was consecutively incubated with antibodies as indicated and re-probed with anti-actin antibody as loading-controls. (-): untreated control cells; (15')/(30')/(60'): cells treated with 250 U/ml of TNF-α for the indicated time.
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Figure 3: Temperature shifting does not affect the cell cycle or TNF-α signaling. (A) 4Bv cells were maintained at 37°C or 32°C overnight, followed by treatment with or without TNF-α for 6 h. Flow-cytometric analysis was performed on DAPI/SR 101 stained cells to determine the percentage of 4Bv cells in different cell cycle phases (G1, S, G2/M). Mean values (columns) and standard deviations (bars) are given for three independent experiments. Steady state expression (B) and phosphorylation (C) of p38 MAP kinase was analyzed after treatment of 4Bv cells (at 37°C or 32°C) with TNF-α as indicated. Total cellular protein (50 μg) was separated in a 12% SDS-PAGE gel. After electrotransfer, the filter was consecutively incubated with antibodies as indicated and re-probed with anti-actin antibody as loading-controls. (-): untreated control cells; (15')/(30')/(60'): cells treated with 250 U/ml of TNF-α for the indicated time.

Mentions: Although activation of threshold levels of wild-type p53 and p21 does not induce growth arrest in 4Bv cells [37], we wanted to exclude the possibility that increased MCP-1 expression may simply result from a kind of synchronization effect, accumulating more cells in cell cycle phases that can re-express MCP-1 upon TNF-α addition than a non-synchronized cell population. However, flow cytometric analyses of cells growing at different temperatures revealed no significant changes in their percentage distributed in different phases of the cell cycle, indicating that synchronization cannot account for the stronger MCP-1 expression (Fig. 3A).


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)

Temperature shifting does not affect the cell cycle or TNF-α signaling. (A) 4Bv cells were maintained at 37°C or 32°C overnight, followed by treatment with or without TNF-α for 6 h. Flow-cytometric analysis was performed on DAPI/SR 101 stained cells to determine the percentage of 4Bv cells in different cell cycle phases (G1, S, G2/M). Mean values (columns) and standard deviations (bars) are given for three independent experiments. Steady state expression (B) and phosphorylation (C) of p38 MAP kinase was analyzed after treatment of 4Bv cells (at 37°C or 32°C) with TNF-α as indicated. Total cellular protein (50 μg) was separated in a 12% SDS-PAGE gel. After electrotransfer, the filter was consecutively incubated with antibodies as indicated and re-probed with anti-actin antibody as loading-controls. (-): untreated control cells; (15')/(30')/(60'): cells treated with 250 U/ml of TNF-α for the indicated time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Temperature shifting does not affect the cell cycle or TNF-α signaling. (A) 4Bv cells were maintained at 37°C or 32°C overnight, followed by treatment with or without TNF-α for 6 h. Flow-cytometric analysis was performed on DAPI/SR 101 stained cells to determine the percentage of 4Bv cells in different cell cycle phases (G1, S, G2/M). Mean values (columns) and standard deviations (bars) are given for three independent experiments. Steady state expression (B) and phosphorylation (C) of p38 MAP kinase was analyzed after treatment of 4Bv cells (at 37°C or 32°C) with TNF-α as indicated. Total cellular protein (50 μg) was separated in a 12% SDS-PAGE gel. After electrotransfer, the filter was consecutively incubated with antibodies as indicated and re-probed with anti-actin antibody as loading-controls. (-): untreated control cells; (15')/(30')/(60'): cells treated with 250 U/ml of TNF-α for the indicated time.
Mentions: Although activation of threshold levels of wild-type p53 and p21 does not induce growth arrest in 4Bv cells [37], we wanted to exclude the possibility that increased MCP-1 expression may simply result from a kind of synchronization effect, accumulating more cells in cell cycle phases that can re-express MCP-1 upon TNF-α addition than a non-synchronized cell population. However, flow cytometric analyses of cells growing at different temperatures revealed no significant changes in their percentage distributed in different phases of the cell cycle, indicating that synchronization cannot account for the stronger MCP-1 expression (Fig. 3A).

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