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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

Expression of MCP-1 in Li Fraumeni fibroblasts and after p53 knockdown in A172 cells. (A) RT-PCR of MCP-1 and GAPDH in Li Fraumeni cells (MDAH041) in passage 8 (p:8, p53 mut/wt) and passage 160 (p:160, p53 mut/mut), respectively. Cells were treated with 250 U/ml of TNF-α for 6 h (+). Untreated control cells: (-). (B) Western blot analysis of p53 in MDAH041 cells (p:8) and (p:160) treated with TNF-α (250 U/ml) (+) for 6 h. (-): untreated control. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Actin confirms equal loading. (C) A172 cells were transiently transfected with pSUPER-p53 or with the empty pSUPER vector. After 24 h, cells were stimulated with TNF-α for additional 5 h. Cells were harvested and Western blot analysis was performed. Filters were probed with anti-p53 (DO-1) (see also panel B). (D) 4 μg of total RNA were separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a MCP-1 cDNA probe. Hybridization of the same filter with a cDNA probe coding for the housekeeping gene β-actin confirmed equal loading. The positions of the 28S and 18S ribosomal RNA are indicated.
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Figure 4: Expression of MCP-1 in Li Fraumeni fibroblasts and after p53 knockdown in A172 cells. (A) RT-PCR of MCP-1 and GAPDH in Li Fraumeni cells (MDAH041) in passage 8 (p:8, p53 mut/wt) and passage 160 (p:160, p53 mut/mut), respectively. Cells were treated with 250 U/ml of TNF-α for 6 h (+). Untreated control cells: (-). (B) Western blot analysis of p53 in MDAH041 cells (p:8) and (p:160) treated with TNF-α (250 U/ml) (+) for 6 h. (-): untreated control. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Actin confirms equal loading. (C) A172 cells were transiently transfected with pSUPER-p53 or with the empty pSUPER vector. After 24 h, cells were stimulated with TNF-α for additional 5 h. Cells were harvested and Western blot analysis was performed. Filters were probed with anti-p53 (DO-1) (see also panel B). (D) 4 μg of total RNA were separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a MCP-1 cDNA probe. Hybridization of the same filter with a cDNA probe coding for the housekeeping gene β-actin confirmed equal loading. The positions of the 28S and 18S ribosomal RNA are indicated.

Mentions: To further prove a more general role of p53 in MCP-1 regulation, early passage immortalized cells from a Li-Fraumeni patient were used. These fibroblasts initially carry a germ-line mutation in one p53 allele [51]. After longer in vitro cultivation, however, they spontaneously immortalize at a high rate, with frequent somatic mutations in the remaining wild-type allele of p53 [34,35]. As demonstrated in Fig. 4A, cell cultures at passage 8 still showed a significant MCP-1 induction after TNFα treatment, which was strongly reduced when immortalized cells at passage 160 were examined. The usage of the p53 specific DO-1 antibody illustrates that wt-p53 can still be detected and induced by TNF-α in early, but not in late passage cells (Fig. 4B), carrying a frameshift mutation at amino acid position 175 (changing arginine to histidine) [36,52,53].


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)

Expression of MCP-1 in Li Fraumeni fibroblasts and after p53 knockdown in A172 cells. (A) RT-PCR of MCP-1 and GAPDH in Li Fraumeni cells (MDAH041) in passage 8 (p:8, p53 mut/wt) and passage 160 (p:160, p53 mut/mut), respectively. Cells were treated with 250 U/ml of TNF-α for 6 h (+). Untreated control cells: (-). (B) Western blot analysis of p53 in MDAH041 cells (p:8) and (p:160) treated with TNF-α (250 U/ml) (+) for 6 h. (-): untreated control. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Actin confirms equal loading. (C) A172 cells were transiently transfected with pSUPER-p53 or with the empty pSUPER vector. After 24 h, cells were stimulated with TNF-α for additional 5 h. Cells were harvested and Western blot analysis was performed. Filters were probed with anti-p53 (DO-1) (see also panel B). (D) 4 μg of total RNA were separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a MCP-1 cDNA probe. Hybridization of the same filter with a cDNA probe coding for the housekeeping gene β-actin confirmed equal loading. The positions of the 28S and 18S ribosomal RNA are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Expression of MCP-1 in Li Fraumeni fibroblasts and after p53 knockdown in A172 cells. (A) RT-PCR of MCP-1 and GAPDH in Li Fraumeni cells (MDAH041) in passage 8 (p:8, p53 mut/wt) and passage 160 (p:160, p53 mut/mut), respectively. Cells were treated with 250 U/ml of TNF-α for 6 h (+). Untreated control cells: (-). (B) Western blot analysis of p53 in MDAH041 cells (p:8) and (p:160) treated with TNF-α (250 U/ml) (+) for 6 h. (-): untreated control. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Actin confirms equal loading. (C) A172 cells were transiently transfected with pSUPER-p53 or with the empty pSUPER vector. After 24 h, cells were stimulated with TNF-α for additional 5 h. Cells were harvested and Western blot analysis was performed. Filters were probed with anti-p53 (DO-1) (see also panel B). (D) 4 μg of total RNA were separated in a 1% agarose gel and transferred to a Gene screen Plus membrane. The filter was subsequently hybridized with a MCP-1 cDNA probe. Hybridization of the same filter with a cDNA probe coding for the housekeeping gene β-actin confirmed equal loading. The positions of the 28S and 18S ribosomal RNA are indicated.
Mentions: To further prove a more general role of p53 in MCP-1 regulation, early passage immortalized cells from a Li-Fraumeni patient were used. These fibroblasts initially carry a germ-line mutation in one p53 allele [51]. After longer in vitro cultivation, however, they spontaneously immortalize at a high rate, with frequent somatic mutations in the remaining wild-type allele of p53 [34,35]. As demonstrated in Fig. 4A, cell cultures at passage 8 still showed a significant MCP-1 induction after TNFα treatment, which was strongly reduced when immortalized cells at passage 160 were examined. The usage of the p53 specific DO-1 antibody illustrates that wt-p53 can still be detected and induced by TNF-α in early, but not in late passage cells (Fig. 4B), carrying a frameshift mutation at amino acid position 175 (changing arginine to histidine) [36,52,53].

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